Title of Invention	"A STABILIZED IMMUNOSTIMULATORY COMPLEX"
Abstract	The present invention relates to a stabilized immunostimulatory complex comprising a cationic synthetic peptide immunogen comprising a target B cell or a CTL epitope, and a T helper cell epitope and anionic CpG oligonucleotide wherein the cationic synthetic peptide immunogen has a net positive charge at a pH in the range of 5.0 to 8.0 calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H), a-1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for all other amino acids in the peptide immunogen and wherein the anionic CpG oligonucleotide has a net negative charge at a pH in the range of 5.0-8. 0 and is a single-stranded DNA comprising 8 to 64 nucleotide bases with a repeat of a cytosine-guanidine motif and the number of repeats of the CpG motif is in the range of 1 to 10 wherein the CpG oligonucleotide is modifed with a phosphorothiorate or a phosphodiester, and said anionic CpG oligonucleotide is preferably selected from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO : 2, a 24 base length oligomer plus an phosphorothioate group designated as n or a combination thereof.

Title of Invention

"A STABILIZED IMMUNOSTIMULATORY COMPLEX"

Abstract

The present invention relates to a stabilized immunostimulatory complex comprising a cationic synthetic peptide immunogen comprising a target B cell or a CTL epitope, and a T helper cell epitope and anionic CpG oligonucleotide wherein the cationic synthetic peptide immunogen has a net positive charge at a pH in the range of 5.0 to 8.0 calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H), a-1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for all other amino acids in the peptide immunogen and wherein the anionic CpG oligonucleotide has a net negative charge at a pH in the range of 5.0-8. 0 and is a single-stranded DNA comprising 8 to 64 nucleotide bases with a repeat of a cytosine-guanidine motif and the number of repeats of the CpG motif is in the range of 1 to 10 wherein the CpG oligonucleotide is modifed with a phosphorothiorate or a phosphodiester, and said anionic CpG oligonucleotide is preferably selected from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO : 2, a 24 base length oligomer plus an phosphorothioate group designated as n or a combination thereof.

Full Text	Stabilized Synthetic Immunogen Delivery Systems FIELD OF THE INVENTION [0001] The present invention relates to a stabilized immunostimulatory complex and a method for preparing the stabilized immunostimulatory complex. More specifically, the present invention provides stabilized synthetic immunostimulatory complexes that are useful in vaccine delivery systems with improved immune responses in vivo. These immunostimulatory complexes are also useful for preparing vaccine formulations designed to function as a depot for controlled release of the immunostimulatory complex. The immunostimulatory complex may also be incorporated in formulations designed to target specific cell types to synergistically improve the quality of the immune responses elicited. BACKGROUND OF THE INVENTION [0002] Vaccines have been successfully employed for many years in prophylactic compositions for the prevention of infectious disease and more recently in therapeutic compositions for the treatment of cancers and non-infectious diseases. [0003] Traditionally vaccines have been derived from attenuated or killed viral or bacterial pathogens and have proven to be very effective against diseases such as polio virus and BordeteJIa pertussis. In spite of these successes, there are growing concerns over the safety of such vaccines. This has led to the development of subunit vaccines derived from components of these pathogens or fully synthetic peptide immunogens. [0004] Examples of subunit vaccines include Tetanus toxoid and hepatitis B surface antigen. These antigens are often poorly immunogenic and require adjuvants to improve the immune responses obtained. Well-characterized biologically active compounds such as synthetic peptides are preferred substrates for inducing biological responses, for safety and regulatory purposes. However, these immunogens are not optimal, and induce partial or negligible protective responses in animal' models. The synthetic peptides require both stabilization and adjuvantation for the induction of an effective immune response in vivo. [0005] Various methods have been employed to protect synthetic peptide immunogens against degradation in vitro and in vivo, mediated by various processes including chemical and physical pathways.1 (The superscript numbers refers to publications, which more fully describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference. The citation of each reference is found at the end of this section). [0006] Various methods have been employed to improve peptide solubility or protect a peptide against degradation in vivo.2 These generally include simple procedures like modifying the salt concentration and/or the pH of the solution. Peptides have also been chemically modified by conjugation with water soluble compounds like polyethylene glycol (PEG) or polyethylene oxide (PEG) both to improve their aqueous solubility and circulation time in vivo.3 It has been documented that adjuvants derived from PEG or PEO can down regulate the immune system.4 Thus, PEG or PEO modified peptides would not be expected to function effectively as adjuvants. The, addition of multiple lysines to add charge to a peptide can improve its aqueous solubility but does not generally result in improved immunogenicity. [0007] The objective of these various strategies is to improve circulation time in vivo or minimize or eliminate immunogenicity problems associated with the physical conditions (e.g. salt, pH, temperature, buffer type) and/or chemical incompatibilities when peptides are employed in a vaccine formulation. [0008] Polyether block copolymers, comprising polycationic polymers, were disclosed by Kabanov et al., U.S. Patent No. 5,656,6115 for stabilizing polynucleotides or oligonucleotides. The polyether block copolymer-polynucleotide complexes are employed to facilitate the transport of the polynucleotide across a cell membrane for improved biological activity. However, these polynucleotide-polyether block copolymers are not immunogenic and are not suitable as vaccines. [0009] Allcock et al. US patent No. 5,562,9096 describes an immunoadjuvant derived from phosphazene polyelectrolytes. The immunoadjuvant was admixed directly with an antigen in solution and may be prepared as microparticles by spray drying a solution of the polymer and the antigen or by a process described by Cohen in US patent No. 5,149,543.7 Although, increased adjuvanticity was shown for these systems, there are difficulties in preparing the microparticular compositions due to the cumbersome mechanical processes employed, which would be difficult to scale up for commercial production. Furthermore, the stability of the polymer-antigen complex so formed is highly dependent on salt concentration and pH conditions. [0010] A different approach is described in Moss et al. W091/040528, wherein a solid vaccine composition is prepared from an antigen, which may be a peptide, a saponin and a polycationic adjuvant such as OEAE-dextran. Vaccines formulated from this combination provided improved longevity, making such combinations suitable for use as implants. However, the antigen must first be chemically conjugated to a carrier molecule and exhaustively purified. The purified antigen-carrier was then combined with a saponin and a polycationic adjuvant to provide a solid composition. This process provides no control over the physical properties, such as particle size, of the product. [0011] Numerous adjuvants and/or depot-based parenteral, mucosal or transdermal delivery systems destined for use with human or veterinary vaccines have been developed to enhance the immune response. These include the use of mineral salts, water-in-oil (w/o)-emulsions, liposomes, polymeric microparticles, nanoparticles and gels/hydrogels.9 A large number of clinical trials employing various (w/o)-emulsion compositions have been conducted. [0012] In spite of this vast body of clinical research, typical parenteral formulations, administered subcutaneously or intramuscularly, are prepared with adjuvants derived from aluminum salts, such as aluminum phosphate or aluminum hydroxide. Alum salts are suitable and effective for many vaccines based on attenuated pathogens, killed pathogens and subunit antigens derived from biological agents. However, the aluminum-based adjuvants are often totally ineffective for synthetic peptide-based immunogens because of the large dose of peptide required and the need of much stronger adjuvantation. The combination of a large dose of immunogen with a weakly adjuvanting alum in a vaccine composition is not ideal as it can lead to immunogen tolerance and reactogenicity, i.e., undesired side reactions, such as swelling and redness at the site of injection. [0013] Freund's complete adjuvant (FCA), a suspension of heatkilled Mi tuberculosis mycobacteria in mineral o-il containing a surfactant, has been recognized as one of the most powerful adjuvants. However, severe adverse reactions, ranging from minor irritation to lesions and sterile abscesses at the site of injection have been documented. Due to these adverse reactions, FCA has been banned from human and veterinary applications. [0014] Thus, there is a clear need to develop adjuvants which are safe without the toxicological and/or reactogenic problems associated with alum or FCA and can effectively enhance immunogenicity and prolong the effectiveness of peptide immunogens to avoid the problem of tolerance associated with alum. It is also most desirable to develop compositions and methods, which can both, stabilize a peptide immunogen and adjuvant the immune responses in a single composition. [0015] Jones et al.10 have disclosed two specific CpG oligonucleotides that may be co-administered with a peptidebased malaria vaccine in Aotus monkeys to enhance immune responses. In the Jones study, the 4-onization point (IP) of the peptide used is 5.96. This corresponds to the pH at which the peptide will have a theoretical zero charge.11 By virtue of its araino acid composition, the peptide used would be effectively uncharged at physiological pH in the aqueous solvent selected. Thus, no complexation can take place with the two CpG oligomers. The resultant mixture when formulated in a w/o-emulsion is expected to be transiently adjuvanted. To achieve a useful level of immunogenicity, multiple injections and a large quantity of adjuvant would be required. Further, the long-term stability of such a composition is questionable. In fact, Jones et al. disclosed that it was necessary to employ a large dose of CpG oligonucleotide, 500 p.g per injection. Furthermore, the methods, employed to prepare the w/o-emulsions, cannot be easily scaled up for commercial .applications. It is to be noted that Jones et al. taught that different CpG oligomers are useful for different mammalian species. For example, a CpG oligomer, CpG ODN 1826 is mitogenic for mice and a lower primate, but not for chimpanzees or humans and the effect is not predictable. [0016] Krieg et al., U.S. patent No. 6,194,388 Bl12 describes unmethylated CpG oligonucleotides particularly useful for therapeutic applications based on their ability to stimulate immune responses when mixed with an antigen. Krieg et al., US 6,207,646 Bl13 further describes the use of unmethylated CpG oligonucleotides to redirect a Th2 response to a Thl response'. In both, the effectiveness of the CpG oligomers were shown by B-cell stimulation wherein B-cells were cultured with phosphorothioate modified CpG oligomers. There is no disclosure or suggestion on how the CpG oligomers can be used to provide a stabilized immunostimulatory complex or a vaccine. [0017] Another area of intense interest and research has been focused on methods to formulate synthetic immunogens for alternate delivery routes, such as mucosally, transdermally, or orally. Mucosal immunity is mediated by the induction of secretory immunoglobulin (slgA) found in external secretions (e.g., intestinal, bronchial or nasal washings). It is believed that transdermal or mucosal delivery of vaccines would be effective against a majority of pathogenic organisms, which gain entry via mucosal surfaces. For example, an orally administered cholera vaccine has been shown to be far superior to the parenterally administered analog.14 [0018] Friede et al., W099/5254915 teaches that vaccine compositions intended for mucosal use can be derived from a combination of an antigen with a polyoxyethylene ether or polyoxyethylene ester as the primary adjuvant. It was suggested that the target antigen might be a synthetic peptide. Friede et ?.l. also suggests the addition of CpG oligonucleotides into the vaccine composition to provide improved responses. They showed that a combination of a polyoxyethylene ether or polyoxyethylene ester with a CpG oligonucleotide could improve mucosal responses when co- administered with an antigen. However, the results showed a lack of any adjuvanticity from simple mixtures of CpG oligonucleotides with antigen described. [0019] Transdermally administered vaccines represent an area of recent interest. Ideally, devices, i.e., patches or needle-free jet injectors can be employed to target the intradermal Langerhan cells, i.e., dendritic cells. These specialized cells are responsible for the effective processing and presentation of an immunogen and can be used to directly induce systemic humoral and cellular responses. In some cases, intramuscular immunization was achieved by transdermal methods.16 For example, a recent paper described a diptheria vaccine administered as a patch. Systemic antibodies to diptheria toxoid were found for a variety of compositions when co-administered with adjuvants.17 [0020] Although the prior art has illustrated the potential of various vaccine formulations, there are a number of practical limitations for the development of synthetic peptide-based vaccine formulations for mucosal or transdermal delivery. These include: 1) immunogen degradation by mucosal fluids or secretions and/or proteolytic enzymes at the mucosal surface or within the intradermis; 2) negligible adsorption across the mucosal epithelium or through the intradermal layers; and 3) dilution of the immunogen to a concentration below that required to induce a suitable level of immune responses. [0021] Few strategies exist which both stabilize and adjuvant a synthetic peptide-based immunogen in a single vaccine composition. Such a composition would be essential for the development of highly efficacious parenteral, mucosal or transdermal peptide-based vaccines. [0022] It is also desirable to prolong the duration of immunogenic responses in order to reduce the number of administrations required. This would result in improved compliance and reduce the overall cost for vaccination. [0023] Various methods may be employed to adjuvant synthetic peptide-based immunogens, but normally a carrier or depot system is required for effective long-term immunogenic responses. Notable examples include adsorbing the immunogen onto a mineral salt or gel. For example, encapsulating a pepti.de immunogen within a polymeric matrix (monolithic matrix) or gel, or layering a polymeric material around a peptide immunogen (core-shell) may be an effective strategy. Or, an immunogen may be incorporated in a liposome or vesicular type of formulation, with the immunogen either embedded in the lipid matrix or physically entrapped in the internal aqueous phase. Another strategy may employ a mineral-based, vegetable-based or animal-based oil, with an aqueous solution of the immunogen in various proportions, to prepare a water-in-oil (w/o)-emulsion or a water-in-oil-inwater (w/o/w)-double emulsion18. [0024] Diverse particle sizes, morphologies, surface hydrophobicity and residual surface charge are possible formulation dependent variables for consideration. Control of these parameters is known to be important for the phagocytosis of micron-sized particulates via parenteral administration19' 20 and for the uptake of particulates at specialized M-cells of the Peyers Patches within the intestinal tract21' 22 for oral delivery. Similarly, these parameters have been shown to be important for access to the nasal-associated lymphoid tissue of the nasalpharyngeal tract, a target of intranasal delivery.23' 2" [0025] Krone et al., U.S. Patent No. 5,700,45925 describes the use of polyelectrolyte complexes in microparticulate form derived from polyacids and polybases, in which the complexing agent is a polymer. Various uses for these complexes are described and include vaccine compositions comprising antigens or antigenic peptides. Some of the compositions are controlled release formulations employing potentially biodegradable materials. In one of the examples, a method of incorporating an antigen in polyelectrolyte complex microparticles is described. However, the mechanical process described for preparing microparticles by grinding the mixture of 100 uM size particles to about 1-4 uM, is cumbersome. This would not be easily scaled up for commercial production. [0026] Eldridge et al.26 developed polymeric biodegradable microspheres manufactured from poly-D,L-lactide-co-glycolide copolymers for the controlled release of an antigen in vivo. The polymers disclosed to be useful for encapsulating an antigen into microparticles include poly-D,L-lactide, polyglycolide, polycaprolactone, polyanhydrides, polyorthoesters and poly(a-hydroxybutyric acid). [0027] Although the controlled release of an antigen was achieved in the prior art, difficulties were encountered when microparticles were manufactured by methods described. The methods described are difficult to scale-up. Moreover, the exposure of biological materials to organic solvents and mechanical processing can lead to denaturation and low to modest encapsulation efficiencies. Furthermore, hydrophilic antigens are inefficiently encapsulated in the processes described. [0028] Henry, et al., U.S. Pat. Nos. 5,126,141 and 5,135,75127' 28 described aqueous, thermally reversible gel compositions formed from a polyoxyalkylene polymer and an ionic polysaccharide for application to injured areas of the body to prevent adhesion. Rosenberg, et al., WO93/0128629 described the use of the same type of polyoxyalkylene polymers for the local delivery of antisense oligonucleotides to surgically exposed surface of blood vessels for treatment of restenosis. Neither Henry et al. nor Rosenberg et al. taught or suggest the use of a gel composition as a vaccine. [0029] Dunn et al., U.S. Patent Nos. 4,938,763 and 5,702,71630/ 31 describe polymeric compositions useful for the controlled release of biologically active materials. A biocompatible solvent was used to prepare solutions or suspensions of antigen for direct parenteral injection, whereupon in-situ gelling results in implant formation. Utility for a variety of antigens including small synthetic peptide-based immunogens was claimed. However, Dunn et al., US 5,702,71631, stated that the controlled release compositions require up to 15% by weight of a gel rate-retarding agent. The retarding agents were added to modulate the gelling rate and were needed for higher entrapment efficiencies for antigens, which are easily extracted in vivo. As the solvent extraction is governed largely by diffusion, this presents more of a problem for small synthetic immunogens than for larger sub-unit or protein-based antigens. [0030] Neither US 4,938,76330 nor US 5,702,71631 taught nor suggested synthetic peptide-based immunogen stabilized as an immunostimulatory complex suspended within a biocompatible solvent. Furthermore, neither US 4,938,76330 nor US 5,702,71631 taught nor suggested compositions which are self-adjuvanting and can upregulate immune responses in both the priming and boosting phases. [0031] It is an object of this invention to develop stable immunostimulatory complexes from synthetic peptide immunogens and stabilizing molecules, which possess self-adjuvanting properties in vivo. It is a further object of the present invention to provide a simple method to stabilize a synthetic peptide immunogen in vitro and in vivo. [0032] It is a still further object of the present invention to provide sustained or controlled release delivery vehicles compatible with these stabilized synthetic peptide-based immunostimulatory complexes. [0033] It is a still further object of the invention to develop formulations using a combination of stabilized synthetic peptide-based immunostimulatory complexes and uncomplexed immunogens in a controlled release delivery system to achieve a synergistic enhancement of the immune response including protective responses. References Cited 1. Manning MC, et al. Pharmaceutical Research, 1989, 6:903- 918. 2. Monfardini C, et al., Bioconjugate Chem., 1998, 9:418- 450. 3. Roberts MJ, et al., J Pharm Sci, 1998, 87:1440-1445. 4. Hilbert AK, et al., Vaccine, 1999, 17:1065-1073. 5. Kabanov AV, et al., U.S. 5,656,611, 1997. 6. Allcock HR, et al., U.S. Patent No. 5,562,909, 1996. 7. Cohen S, et al., U.S. Patent No. 5,149,543, 1992. 8. Moss BA, et al., WO 91/04052, 1991. 9. Cox JC, et al. Vaccine, 1997, 15:248-256. 10. Jones TR, et al., Vaccine, 1999, 17:3065-3071. 11. Bjellqvist B, et al., Electrophoresis, 1993, 14:1023- 1031. 12. Kreig AM, et al., U.S. Patent No. 6,194,388 Bl, 2001. 13. Kreig AM, et al., US Patent No. 6,207,646 Bl, 2001. 14. Suharyono, et al. Lancet, 1992, 340:689-694. 15. Freide M, Hermand P., WO 99/52549, 1999. 16. Aguiar JC, et al. Vaccine, 2002, 20:275-280. 17. Scharton-Kersten T, et al. Infect Immun, 2000, 68:5306- 5313. 18. Powell MF, et al., Pharmaceutical Biotechnology, Vol. 6, Plenum Press, New York, 1995. 19. Ikada Y, et al., J Bioactive Compat Polym, 1986, 1:32-46. 20. Kreuter J, et al. Vaccine, 1986, 4:125-129. 21. Jepson MA, et al. J Drug Targeting, 1993, 1:245-249. 22. Moldoveanu Z, et al., J' Infect Pis, 1993, 167:84-90. 23. Matsuo K, et al. Vaccine, 2000, 18:1344-1350. 24. Higaki M, et al., Vaccine, 1998, 16:741-745. 25. Krone V, et al., U.S. Patent No. 5,700,459, 1997. 26. Eldridge JH, et al., Mol Immunol, 1991, 28:287-297. 27. Henry RL, U.S. Patent No. 5,126,141,1992. 28. Henry RL, U.S. Patent No. 5,135,751, 1992. 29. Rosenberg RD, et al., WP 93/01286, 1993. 30. Dunn RL, et al., U.S. Patent No. 4,938,763, 1990. 31. Dunn RL, et al., U.S. Patent No. 5,702,716, 1997. 32. Papisov IM, et al., Advances in Polymer Science, 1988, 90, 1988,139-177. 33. Chu RS, et al., J Exp Med, 1997, 186:1623-1631. 34. Akasaka T, 'et al., Bioconjugate Chem., 2001, 12:776-785. 35. Ballico M, et al., Bioconjug Chem, 2001, 12:719-725. 36. Klinman DM, et al., Vaccine,1999, 17:19-25. 37. Krieg AM, et al., Nature 1995, 374:546-549. 38. Klinman DM, et al., Infect Immun, 1999, 67:5658-5663. 39. Nagel KM, et al., Pharmacotherapy, 1993, 13:177-188. 40. Weeratna RD, et al., Vaccine, 2000, 18:1755-1762. 41. McCluskie MJ, et al., Vaccine, 2000, 18:231-237. 42. LiCalsi C, et al., Vaccine, 1999, 17:1796-1803. 43. Romera SA, et al., Vaccine, 2001, 19:132-141. 44. Wright JC, et al., J Controlled Release, 2001, 75:1-10. 45. Graham PD, et al., J Controlled Release, 1999, 58:233- 245. 46. DesNoyer JR, et al., J Controlled Release, 2001, 70:285- 294. 47. Aguado MT, et al., Imrounobiol, 1992, 184:113-125. 48. Visscher GE, et al., J Biomed Mater Res, 1985, 19:349- 365. 49. Forbes RT, et al., J Pharm Sci, 1998, 87:13161321. 50. Overcashier DE, et al., J Pharm Sci, 1999, 88:688-695. 51. Wang CY, et al., WO 99/67293, 1999. 52. Wang CY, U.S. Patent No. 5,763,160, 1998. 53. Wang CY, U.S. Patent No. 6,090,388, 2000. 54. Ladd AE, et al., U.S. Patent No. 5,759,551, 1998. 55. Wang CY, U.S. 6,025,468, 2000. 56. Wang CY, USSN 09/865,294, 2001. 57. Wang CY, et al., U.S. Patent No. 6,107,021, 2000. 58. Wang CY, USSN 09/747802, 2001. 59. Wang CY, WO 99/66957, 1999. 60. Wang CY, WO 99/66950, 1999. 61. Mascotti DP, et al., Proc Nat head Sci, USA, 1990, , 87:3142-3146. 62. Kabanov AV, et al., Bioconjuq Chem, 1995, 6:7-20. 63. MacDonald RC, et al., Biochim Biophys Acta, 1991, 1061:297-303. 64. Shen F, et al., Vaccine, 1999, 17:3039-3049. 65. Wang CY, et al., Proc. Nat. Acad. Sci., USA, 1999, 96:10367-10372. 66. Hanson CV, et al., J. Clin Microbiol, 1990, 28:2030-2034. 67. Park TG, et al., J Controlled Release, 1995, 33:211-222. 68. www.expasy.ch/tools/pi_tool.html 69. Hakim, I, et al. J Immunol, 1996, 157:5503-5511. 70. Zeng, XY, et al., Theriogenology, 2002, 58:1315-1326. 71. Office International des epizootics (OIE). Foot-in-mouth disease. Manual of standards for diagnostic tests and vaccines. Paris(France): OIE, 1996, p. 47-56 [chapter 2.1.1] 72. Proietti, E, et al., J. of Immunol, 2002, 375-383. SUMMARY OF THE INVENTION [0034] The present invention is directed to a stabilized immunostimulatory complex comprising a cationic peptide and anionic molecule or oligonucleotide or polynucleotide and a method for stabilizing a cationic peptide by complexation with an anionic molecule or oligonucleotide or polynucleotide via electrostatic association. The stabilized immunostimulatory complex may be incorporated into a pharmaceutical composition as an immunogen delivery system. [0035] A "cationic peptide" as described herein refers to a peptide, which is positively charged at a pH in the range of 5.0 to 8.0. The net charge on the peptide or peptide cocktails is calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H), a -1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for the other amino acid within the sequence. The charge contributions from the N-terminal amine (+1) and C-terminal carboxylate (-1) end groups of each peptide effectively cancel each other when unsubstituted. The charges are summed for each peptide and expressed as the net average charge. A suitable peptide immunogen has a net average positive charge of +1. Preferably, the peptide inununogen has a net positive charge in the range that is larger than +2. [0036] The peptide immunogens comprise B-cell epitopes and Th epitopes. The Th epitopes may be intrinsic to the peptide or added thereto as described in the prior art. Suitable peptide immunogens are described herein below. [0037] An "anionic molecule" as described herein refers to molecules, which are negatively charged at a pH in the range of 5.0-8.0. The net negative charge on the oligomer or polymer is calculated by assigning a -1 charge for each phosphodiester or phosphorothioate group in the oligomer. A suitable anionic oligonucleotide is a single-stranded DNA molecule with 8 to 64 nucleotide bases, with the number of repeats of the CpG motif in the range of 1 to 10. Preferably, the CpG immunostimulatory single-stranded DNA molecules contain 18-48 nucleotide bases, with the number of repeats of CpG motif in the range of 3 to 8. , [0038] More preferably the anionic oligonucleotide is represented by the formula: 5' X1CGX2 3' wherein C and G are unmethylated; and X1 is selected from the group consisting of A (adenine), G or T (thymine). Or, the anionic oligonucleotide is represented by the formula: 5' (X3) 2CG (X4) 2 3' wherein C and G are unraethylated; and X3 is selected from the group consisting of A, T or G,. and X4 is C or T. [0039] Most preferably, the CpG oligonucleotide is selected from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer, and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO: 2, a 24 base length oligomer plus a phosphorothioate group (designated as n at the 5' end). [0040] The resulting immunostimulatory complex is in the form of particles with a size typically in the range from 1-50 microns and is a function of many factors including the relative charge stoichiometry and molecular weight of the interacting species.32 The particulated immunostimulatory complex has the added advantage of providing adjuvantation and upregulation of specific immune responses in vivo. Additionally, the stabilized immunostimulatory complex is suitable for preparing vaccine formulations by various processes including water-in-oil emulsions, mineral salt suspensions and polymeric gels. [0041] The term "stabilize" as used herein may be accomplished by the use of any material, which protects the synthetic peptide immunogen against degradation in vitro or in vivo. This may be accomplished by virtue of chemical modification and/or physical association. A stabilizer may augment the physiological properties of a synthetic peptide immunogen, an oligosaccharide-modified glycopeptide or a lipidated peptide to provide a more efficacious formulation. [0042] The term "adjuvant" as described herein refers to any material, which can enhance or upregulate the immune responses elicited by an immunogen in humans or animals. The adjuvant itself may or may not induce an immunogenic response. [0043] The stabilizer may also preferably function in a vaccine as an adjuvant, effectively upregulating the immune responses. The stabilizer may act as an adjuvant by actively facilitating the presentation of the immunogen to professional processing cells of the immune system, such as macrophages and dendritic cells. In the present invention, the stabilized immunostimulatory complex ideally remains as an integral unit in solution when administered. [0044] The stabilized immunostimulatory complex may also be formulated for controlled release and remains as a complex in a concentrated form in a "depot" near the site of administration. These formulations synergistically combine the benefits of a stabilized adjuvanted immunogen coupled with a sustained local release of immunogen to immune effector cells. In some compositions the role of the adjuvant itself may also involve attracting cells of the immune system to the vicinity of the immunogen depot and stimulate such cells to elicit an immune response. [0045] In a second aspect of this invention, there is provided a method for preparing a vaccine composition containing an immunostimulatory complex. In a preferred embodiment the immunostimulatory complex has the added advantage of being a stabilized synthetic peptide-based immunogen in vitro and at the same time is self-adjuvanting with upregulation of specific immune responses in vivo. [0046] In a third aspect of this invention, there is provided a method for preparing a vaccine composition from the immunostimulatory complex. The immunostimulatory complex or a mixture of the immunostimulatory complex with the uncomplexed immunogen may be formulated as a suspension in solution, a water-in-oil emulsion, a suspension in combination with a mineral salt suspension or a reconstituted suspension in a biocompatible solution. The inununostimulatory complex alone or in a mixture with uncomplexed immunogens may also be coformulated in a biocompatible solvent in a polymeric gel. [0047] This invention is further directed to the production of useful immunogen delivery systems for administration by various routes, including parenteral, oral, intranasal, rectal, buccal, vaginal and transdermal routes. BRIEF DESCRIPTION OF THE DRAWINGS [0048] The present invention is further understood with reference to the drawings. [0049] Figure 1 is a schematic showing the complexation process of cationic peptide immunogens and CpG oligonucleotides. [0050] Figure 2 shows the typical size distribution for the stabilized immunostimulatory complexes prepared from LHRH peptide immunogens and CpGl oligonucleotides in various proportions as determined by laser diffraction measurements. [0051] Figure 3 is a schematic of the process for preparing a water-in-oil (w/o) emulsion employing homogenization or extrusion techniques. [0052] Figure 4 shows a typical photomicrograph for a w/o emulsion prepared via homogenization from ISA Montanide® 51 and LHRH:CpGl immunostimulatory complexes, wherein LHRH:CpGl is 4:1 at a fixed final total peptide concentration of 100 ug/mL. [0053] Figure 5 shows a typical ph6tomicrograph for a w/o emulsion prepared via extrusion from ISA Montanide® 72-0 and LHRH:CpGl immunostimulatory complexes, wherein LHRH:CpGl is 4:1, at a fixed final LHRH peptide concentration of 200 fig/mL. [0054] Figure 6 is a schematic detailing the in-situ polymer gel process employing reconstitution. [0055] Figure 7 shows the serum IgG responses in guinea pigs immunized intramuscularly (I.M.) in accordance with the immunization protocols described in Example 7. [0056] Figure 8 shows the serum IgG responses in guinea pigs immunized intramuscularly (I.M.) in accordance with the immunization protocols described in Example 7. No sera were obtained for the animals immunized with the immunostimulatory complex derived from CD4 peptides and CpG2 on week 17. This is indicated by an asterisk in Figure 8. [0057] Figure 9 shows the serum IgG responses in guinea pigs immunized intramuscularly (I.M.) in accordance with the immunization protocols described in Example 7. [0058] Figure 10 shows the serum IgG responses in guinea pigs immunized intramuscularly (I.M.) in accordance with the immunization protocols described in Example 7. No sera were obtained for the animals immunized with the immunostimulatory complex derived from CD4 peptides and CpG2 on week 17. This is indicated by an asterisk in Figure 10. [0059] Figure 11 shows the serum IgG responses in guinea pigs immunized intramuscularly (I.M.) in accordance with the immunization protocols described in Example 8. [0060] Figure 12 shows the serum IgG responses in guinea pigs immunized intramuscularly (I.M.) in accordance with the immunization protocols described in Example 8. [0061] Figure 13a shows the serum IgG responses and 13b shows the total serum testosterone in male rats immunized intramuscularly (I.M.) in accordance with immunization protocols as described in Example 10. [0062] Figure 14a shows the serum IgG responses and 14b and 14c shows the total serum testosterone in male baboons immunized intramuscularly (I.M.) in accordance with immunization protocols as described in Example 11. [0063] Figure 15a shows the serum IgG response, 15b shows the total serum testosterone and 15c shows the average weight gain per group in boars immunized intramuscularly (I.M.) over the trial period in accordance with immunization protocols as described in Example 13. [0064] Figure 16 is a schematic detailing the process of preparing a mixed suspension of immunostimulatory complex and a mineral salt. DETAILED DESCRIPTION OF THE INVENTION [0065] In accordance with a first aspect of the invention, a cationic peptide immunogen is complexed with an anionic single-stranded DNA to form a stable immunostimulatory complex. [0066] The cationic peptide immunogen is a peptide with a net positive charge at a pH in a range of 5.0 to 8.0. The net charge on the peptide or peptide cocktails is calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H), a -1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for the other amino acids in the sequence. The charge contributions from the N-terminal amine (+1) and C-terminal carboxylate (-1) end groups of each peptide effectively cancel each other when unsubstituted. The charges are summed for each peptide and expressed as the net average charge. Preferably, the net average charge of the peptide immunogen is at least +2. [0067] The cationic peptide immunogen may intrinsically have a net positive charge as calculated above based on its amino acid sequence. It may be made to have a positive charge by the addition of a lysine, an arginine or a histidine or a mixture of these amino acids to the N-terminal or C-terminal of the peptide immunogen. Other synthetic moieties, such as polyethyleneimine or polyamines, which provide a positive charge to the peptide immunogen in aqueous solution, may also be added. [0068] The cationic peptide immunogen comprises a Th epitope and a target B-cell epitope. The Th epitope may be intrinsic to the peptide "or may be synthetically added to a peptide, which functions as a target B-cell epitope. Suitable peptide immunogens include peptides that elicit protective or therapeutic immune responses and are derived from pathogens or proteins known to cause diseases. These include: human or animal IgE peptides for the immunotherapy of allergies, e.g., the IgE peptide immunogens described in WO 99/6729351; HIV peptides for protective immunity and immunotherapy for HIV infection described in US 5,763* 160s2; CD4 peptides for protective immunity from HIV and immunotherapy of HIV infection and immune disorders described in US 6,090,388s3; Luteinizing Hormone Releasing Hormone (LHRH) peptides for immunotherapy of androgen and estrogen-dependent tumors, contraception and inimunocastration, and removal of boar taint described in US 5,749,551s" and US 6,025,468SS; (3-amyloid peptides for prevention and immunotherapy of Alzheimer's Disease described in USSN 09/865,294s6; foot-and-mouth disease virus peptides for protective immunity against foot-and-mouth disease described in US 6,107,021s7; peptides from bacterial pili for protective immunity from urinary tract infection described in USSN 09/747802s8; -Plasmodium peptides for protective immunity from malaria described in WO 99/669S759; and somatostatin peptides for growth promotion in livestock described in WO 99/66950.60 The specific peptide immunogens named herein are examples for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. [0069] The "anionic single stranded DNA" is a polynucleotide or oligonucleotide that is negatively charged at a pH in the range of 5.0-8.0. The net negative charge on the polynucleotide or oligonucleotide is calculated by assigning a -1 charge for each phosphodiester or phosphorothioate group in the oligomer. A suitable anionic oligonucleotide is a singlestranded DNA molecule with 8 to 64 nucleotide bases, with a repeated CpG motif and the number of repeats of the CpG motif is in the range of 1 to 10. Preferably, the CpG immunostimulatory single-stranded DNA molecules contain 18-48 nucleotide bases, with the number of repeats of CpG motif in the range of 3 to 8. [0070] Preferably, the anionic oligonucleotide is represented by the formula: 5' XrCGX2 3' wherein C and G are unmethylated; and X1 is selected from the group consisting of A (adenine), G (guanine) and T (thymine); and X2 is C (cytosine) or T (thymine). Or, the anionic oligonucleotide is represented by the formula: 5' (X3)2CG(X4)2 3' wherein C and G are unmethylated; and X3 is selected from the group consisting of A, T or G.. and X4 is C or T. The CpG oligonucleotide may be modified at the 5' end with a phosphorothiorate or a thiolacetamido glycopolymer.34 [0071] Most preferably, the CpG oligonucleotide is selected from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer, and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO: 2, a 24 base length .oligomer plus an ph6sphorothioate bridging group {designated as n at the 5' end). [0001] Moreover, it is known that the DNA sequences derived from unmethylated cytosine-guanine (CpG) dinucleotides activate lymphocytes and can enhance a subject's immune responses, including IL-6, TNF-a, IL-12, IFN-yresponses33. These molecules represent preferred complementary substrates that can both stabilize the synthetic cationic peptide immunogens and provide a novel immunogen delivery system, based on these findings. The stabilizing immunostimulatory^ complexes of the present invention also provide selfadjuvantation of the immune responses in vivo without significant dilution at the site of injection. [0073] The formation of discrete immunostimulatory complexes derived from cationic peptide immunogens is principally a function of charge neutralization. It is expected that stable complexes may be formed from CpG-containing immunostimulatory DNA molecules derived from both natural or synthetically modified nucleotide sequences. Furthermore, improvements in the stability of an immunostimulatory complex may be realized by increasing the cationic charge residing on the peptide immunogen. These include extending the peptides with additional lysine, arginine or histidine or other synthetic moieties, which provides a positive charge to the modified peptide in an aqueous solution as described above. [0074] It is expected that non-CpG containing immunostimulatory sequences (ISS) will be identified and contemplated that these single-stranded DNA substrates would prove to be useful materials to form immunostimulatory complexes from when combined with synthetic cationic peptide immunogens in appropriate aqueous solvents. [0075] Modified CpG motifs are also envisioned, wherein a defined anionic single-stranded DNA has been chemically conjugated to another biologically functional molecule, such as lectins, sugars or lipids for enhancing cell-specific uptake and targeting, or polymers, copolymers and graft copolymers such as PEG for improved circulation in vivo. The chemically conjugated DNA.may be polyanionic and may subsequently be complexed with a cationic peptide immunogen to provide a modified immunostlmulatory complex with potentially novel physical or biological properties.34' 3S [0076] It is contemplated that block and graft co-polymers derived from polyanionic oligomers and polyethylene glycol ' represent another class of anionic molecules, which may also provide improved stability and improved adjuvanticity. [0077] In another aspect of this invention an inununostimulatory complex may be prepared from a modified CpG oligonucleotide, wherein an additional phosphorothioate or another bridging group has been added at the 5' end of the oligomer for improved complexation. [0078] Preferably, the inununostimulatory complexes have an average aggregate particle size distribution in the range of about 1 to 50 pM. More preferably, the immunostimulatory complexes have an average aggregate particle size distribution in the range of about 1 to 30 pM. Most preferably, the immunostimulatory complexes have an average aggregate particle size distribution in the range of about 1 to 15 uM. [0079] There is evidence that the number of CpG motif repeats influences the degree of intrinsic adjuvanticity and immune stimulation, with a minimum number of CpG repeats being required. Moreover, there is strong evidence that the selection of flanking nucleotide bases adjacent to the CpG is very important, as this appears to directly impact the adjuvanticity in a species-specific manner.10' 36 For example, it was demonstrated by Kreig et aJ.13 that enhanced immunostimulatory activity of human cells occurred when oligonucleotides containing CpG motifs are represented by the formula X1X2CGX3X4 where C and G were unmethylated, and XX2 were selected from the groups GpT, GpG, GpA and ApA and/or X3X4 were selected from the groups TpT, CpT and GpT. [0080] Although CpG oligonucleotides can function as B-cell mitogens37 and are useful adjuvants, it has been shown that the immune responses generally peak 2 weeks after administration for an antigen mixed with CpG oligonucleotides in a soluble form. This necessitates multiple repeat injections to maintain high antibody titers to ensure protection.38 Thus, a method for effectively delivering constructs with these oligonucleotides in a controlled release formulation is strongly desired. [0081] With respect to stability, the phosphodiester bonds in the CpG backbone are sensitive to degradation by nucleases in vivo39. Thus to improve the duration of the immune response, the phosphate groups may be modified to phosphorothioate groups. [0082] The immunostimulatory complex of the present invention may be formulated for delivery by numerous pathways including parenteral, mucosal and transdermal. The immunostimulatory complex of the present invention is particularly desirable for vaccine formulations in that the CpG oligonucleotides in the complex are useful adjuvants for upregulating both parenteral and mucosal responses in vivo.40' 41 [0083] The results of our experiments show that the aggregate particle size of the immunostimulatory complex varies based on the ratio of the peptide immunogen to the CpG oli-gonucleotide. The intrinsic stability of the immunostimulatory complex and the ability to control the size of the composition increases the potential for phagocytosis by a parenteral route.19 Mucosal immunization by targeting specific cells, such as Mcells located on Peyer's Patches via oral route21 or the nasalassociated lymphoid tissue (NALT) via intranasal route23 is similarly facilitated by the use of the stabilized immunogen of the present invention. [0084] The immunostimulatory complex of the present invention is prepared by a controlled self-assembling process wherein the anionic CpG oligonucleotide in aqueous solution is added to an aqueous solution of the cationic peptide immunogen. Suitable aqueous solutions for the preparation of an immunostimulatory complex is selected from the group consisting of distilled deionized water (DOW), normal saline (NS) or phosphate buffered saline (PBS). Distilled deionized water and normal saline typically exhibit a pH of about 5.5, whereas in PBS, the pH is controlled in a range of 7.2-7.4. The complexation process is a function of the charge ratios, the molecular weight of the interacting electrolytes, pH and ionic strength of the medium.32 [0085] Multiply charged anionic molecules, such as the short CpG oligomers possess a net negative charge when the pH is in the range 5.5-7.4 in aqueous solutions. The net charge on the peptide immunogen is dependent on the amino acid composition of the peptide and may be affected by the pH of the aqueous solution. Thus, the aqueous medium is selected to ensure a net positive charge for efficient complexation. An examination of the ionization point (IP) or point of zero charge for the individual peptides can guide the selection process. In general, the IP is determined by the motion of the molecule through a pH-gradient in an isoelectric focusing experiment.11 To ensure a peptide is positively charged, the pH of the selected aqueous medium should be less than the isoelectric point for the peptide in question. [0086] To prepare an immunostimulatory complex the following steps are followed. Firstly, the average molar positive charge contribution is determined for a desired peptide immunogen or for a cocktail of peptide immunogens based on the molar ratios of peptides mixed together and the charge contribution from each peptide component in the final vaccine composition. Secondly, the molar negative charge contribution is determined for the complexing oligonucleotide based on the molar ratio of oligomer and the charge contribution from this component in the final vaccine composition. Thirdly, the amount of peptide immunogen, based on total average molar positive charge, is dependent on the amount of oligonucleotide employed for complexation and the total molar negative charge thereof. This relationship is used to define the relative amounts of peptide immunogens and oligonucleotides to be combined in an aqueous solvent to form an immunostimulatory complex. An excess of the cationic immunogen peptide may be employed to provide a mixture of the immunostimulatory complex and an excess of the uncomplexed peptide. Or, an excess of the oligonucleotide may also be employed to provide an excess of the oligonucleotide. The relative amounts of the peptide immunogen and the oligonucleotide selected are based on the vaccine formulation desired. [0087] Finally, the calculated amount of anionic oligonucleotide in a compatible aqueous solvent is added with mixing to the calculated amounts of cationic peptide immunogens similarly dissolved in a compatible aqueous solvent. The amount in nmol of the cationic peptide immunogen used is generally in a range to provide 8 positive charges to 0.5 positive charge to the amount in nmol of the anionic oligonucleotide to provide one negative charge. This is referred to as the charge ratio. Where the charge ratio is 8:1, there is a large excess of the peptide immunogen. Where the charge ratio is 1:2, there is a moderate excess of the anionic oligonucleotide. The complex forms spontaneously in •the form of a suspension in solution. Estimation of the residual amounts of peptide immunogens or oligonucleotides can be made by separating the complex from the solution and assaying the supernatant solutions by ultraviolet spectrophotometry (UV) or by reverse phase high performance chromatography (RP-HPLC). [0088] The immunostimulatory complex as prepared as a suspension may be used as a vaccine composition. If the immunostimulatory complex is to be injected parenterally, t'he aqueous solvents are selected such that the final vaccine composition is isotonic and suitable for such a purpose. In cases where the complex is first formed in distilled deionized water, aqueous buffers of suitable salt concentration are added to ensure the final vaccine composition is isotonic. [0089] The immunostimulatory complex prepared as a suspension or solution may be lyophilized. The lyophilized composition may then be reconstituted and incorporated into different vaccine formulations in accordance with the desired mode of delivery. The immunostimulatory complex of the present invention may also be formulated as a water-in-oil emulsion, in combination with a mineral salt suspension or a biocompatible polymeric gel. [0090] In accordance with a further aspect of the invention, the invention describes a process for isolating the stabilized immunostimulatory complexes as stable particles via lyophilization. Reconstitution of the stabilized immunostimulatory complex as a suspension in aqueous solvents or biocompatible solvents shows essentially no changes in the particle size distribution or in vivo potency. This represents an important advantage over formulations requiring refrigeration to maintain efficacy, such as Alum-based vaccine compositions. This feature extends the potential utility of these systems to include direct reconstitution prior to immunization and alternative modes of delivery which require stable solid state dosage forms, such as a dry powder aerosol or nebulization for pulmonary or intranasal delivery.42 [0091] In accordance with a further aspect of the present invention, the invention provides various processes for preparing stable water-in-oil emulsions43 comprising the stabilized immunostimulatory complex of the present invention. In such an emulsion, preferably the aqueous phase comprises the immunostimulatory complex or a mixture of the immunostimulatory complex with the uncomplexed peptide immunogen; and the continuous oil phase comprises a synthetic, mineral, animal or vegetable oil. Additionally, the oil phase may also comprise an immunostimulatory emulsifier, a biocompatible or a metabolizable component. [0092] In particular, the oils useful for preparing the waterin- oil emulsions of the present invention include, but are not limited to, synthetic oils (e.g. isopropyl myristate), vegetable oils (e.g. peanut oil), mineral oils (e.g. Drakeol™ or Marcol™), metabolizable animal oils (e.g. squalene or squalane), and a mixture thereof. The mineral oils are preferred. The oil-based emulsifiers useful for stabilizing the emulsions include, but are not limited to, the family of mannide oleates and derivatives thereof. [0093] The relative amount of emulsifier required is a function of the hydrophile-lipophile balance (HLB) and the intrinsic stability of the water-in-oil emulsion produced under specified conditions. Methods for selecting oils and combinations of emulsifiers are well known to those skilled in the art. [0001] The w/o-emulsion may comprise between 10 v/v% and 80 v/v% water in the internal aqueous phase. For most purposes, the optimal water concentration is in the range of 30 v/v% and 50 v/v%. The internal aqueous phase is characteristically comprised of very fine droplets with sizes typically in the range of 1-10 jiM, preferably 1-5 uM. The preparations are stable when maintained at room temperature or refrigerated. [0095] Other stabilizing agents may also be used to prepare the emulsion. These include surfactants, colloidal particles, proteins, and other polymerizing and stabilizing agents knoton to those skilled in the art. [0001] The w/o-emulsion may further comprise at least one oilsoluble lipophilic adjuvant such as 3-0-desacyl-4'- monophosphoryl lipid A (MPL), W-acetyl-muramyl-L-alanyl-Disoglutamine (MDP), Dimethyldioctadecylammonium bromide (DDA), N, W-dioctadecyl-W, N' -bis(2-hydroxyethyl) propanediamine (Avridine), N- (2-Deoxy-2-l-leucylamino-p-D-glucopyranosyl) -Noctadecyl- dodecanoylamide hydroacetate (BAY-1005), 3p -[N- (N, W-dimethylaminoethane)-carbamoyl] cholesterol (DC-Choi), NAc-Mur-L-Thr-D-isoGln-sn-glycerol dipalmitoyl (Murapalmitine) and mixtures or derivatives thereof. The w/o-emulsion may also comprise in the dispersed phase at least one watersoluble adjuvant, e.g., poly(di(carboxylatophenoxy)] phosphazene(PCPP), Quillaja saponin (QS-21), Cholera Holotoxin (CT) or Cholera Toxin B subunit(CTB), heat labile Enterotoxin from E. Coli (LT) or heat labile Enterotoxin B subunit from E. Coli (LTB) and cytokines such as Interleukin-1 p (IL-lfi), Interleukin-2 (IL-2), Interleukin-12 (IL-12), interferon-y (IFN-y)and mixtures and derivatives thereof. The water-soluble adjuvant may be synthetic or natural. The presence of a water soluble adjuvant with film forming properties, such as an oligomer or polymer, can additionally serve to stabilize the emulsion. The w/o-emulsion can facilitate presentation of the immunogens to the immune system to provide a more efficacious vaccine. [0097] A water-in-oil emulsion comprising an immunostimulatory complex, or a mixture thereof with uncomplexed immunogen may be prepared as follows. Firstly, an immunostimulatory complex is prepared from a peptide immunogen and an oligonucleotide in a ratio to ensure the formation of the immunostimulatory complex alone or in a mixture with excess residual peptide immunogen in an aqueous solution. Secondly, the aqueous solution is mixed with an oil containing emulsifiers and homogenized to provide a water-in-oil emulsion wherein the aqueous phase is dispersed in a continuous oil phase. The water-in-oil emulsion as such is suitable for parenteral injection. [0098] Emulsification of the aqueous and oil phases can be accomplished by homogenization or by transfer between two syringes or by extruding the components through a membrane filter of a controlled pore size. The low-energy serai-manual methods are rapid. However, because there is considerably less shear than other processes, the emulsion produced is not as fine as that produced using high shear mechanical systems. Examples of high-shear systems include rotostators, microfluidizers, and sonifiers. Other devices similar to these high-shear systems that are well known for emulsification may also be employed. [0099] In accordance with a further aspect of the present invention, the invention provides various processes for preparing physiologically acceptable suspensions of mineral salts comprising the stabilized immunostimulatory complex of the present invention. In such a mixed system, the aqueous phase comprises a combination suspension of mineral salt and immunostimulatory complex, which may additionally contain residual, unbound peptide immunogens in solution. [00100] In particular, the mineral salts useful for preparing the full aqueous-based suspensions of the present invention include, but are not limited to, Aluminum hydroxide (e.g., Alhydrogel®, Rehydragel HPA®, Rehydragel LV®} , Aluminum phosphate (e.g., Adju-phos® or Rehyraphos®) or calcium phosphate (e.g., Calphos®), and mixtures thereof. [00101] Methods for selecting mineral salts and determining the preferred concentration of mineral salt to employ or combinations thereof are well known to those skilled in the art. [00102] Other stabilizing agents may also be used to prepare the mineral salt suspension. These include surfactants, antioxidants, physiological acceptable buffers, tonifiers, preservatives and other agents known to those skilled in the art. [0001] The mineral salt suspension may further comprise at least one additional adjuvant (e.g. MPL, MDP, DDA, N,NAvridine, BAY-1005, DC-Choi, Murapalmitine, PCPP, QS-21, CT or CTB, LT or LTB and cytokines such as IL-lli, IL-2, IL-12, IFNyand mixtures and derivatives thereof). The mineral salt can facilitate presentation of the immunogens to the immune system in the form of a depot or attract specific cells of the immune system by a process known as chemotaxis. [00104] A mineral salt suspension comprising an immunostimulatory complex, or a mixture of immunostimulatory complex in combination with residual uncomplexed immunogens may be prepared as follows. Firstly an immunostimulatory complex is prepared from a peptide immunogen and an oligonucleotide in a charge ratio to ensure the complete complexation of all peptide immunogens and the oligonucleotide in solution. Alternatively, an immunostimulatory complex is prepared from a peptide immunogen and an oligonucleotide in a charge ratio to ensure partial complexation of the peptide immunogens and oligonucleotide components in solution; Secondly, the aqueous suspension is combined with a mineral salt suspension with mixing to provide a full aqueous suspension of all components. The suspension combination as such is suitable for parenteral injection. [00105] In a complementary method, a mineral salt suspension comprising an immunostimulatory complex, or a mixture thereof with uncomplexed peptide immunogens may be prepared as follows. Firstly, peptide immunogen is mixed with a mineral salt suspension. Depending on the physical properties of the mineral salt, peptide immunogens and aqueous buffer various proportions of immunogen may be absorbed directly by the mineral salt at this stage; Secondly, to this suspension is added an oligonucleotide with stirring. Partial or full complexation of the residual unbound peptide immunogens in solution results. The suspension combination as such is suitable for parenteral injection. In Figure 16, both methods of preparation are shown. [00106] In accordance with another aspect of this invention, there is provided a process for preparing an in-situ gelling biodegradable polymer in which a stabilized immunostimulatory complex or a mixture of a stabilized immunostimulatory complex and uncomplexed immunogen is dispersed. The immunostimulatory complex may be dispersed either in solution or as a suspension within a biocompatible solvent. The biocompatible solvent may further comprise a soluble adjuvant that is synthetic or natural. The solution or suspension in the biodegradable gelling polymer is designed for the delivery of the immunogen to a host. The in-situ gelling polymer is biodegradable and is a copolymer of poly-D,L-lactide-co-glycolide {PLG) and poly-D,L-lactic acid-co-glycolic acid weight in the range of about 2,000 to about 100,000 daltons and an inherent viscosity of about 0.2 to 1.0 dl/g. The formula of the in-site gelling polymer is: (Figure Removed) wherein Rl is OH or alkoxy having 1 to 5 carbons and R2 is H; x:y is the ratio of each monomer unit of the copolymer with x+y=l. In the case of PLG, Rl is alkoxy and the monomer units are lactide and glycolide and in the case of PLGA, Rl is OH and the monomer units are lactic acid and glycolic acid. [00107] The stabilized inununostimulatory complex or a mixture thereof with the uncomplexed immunogen with the in~situ gelling polymer may be prepared as a single phase or as a suspension in a biocompatible solvent. [00108] The biocompatible solvent useful in the present invention is selected from the group consisting of dimethyl sulfoxide (DMSO), N-methyl pyrrolidine (NMP), triacetin and glycerin. DMSO is preferred. DMSO has a high capacity for solubilizing a large quantity in weight percent of the polymer. It has been widely used as a solvent for in-situ gelling of polymers. DMSO may be also be used to prepare a suspension of the stabilized immunostimulatory complex of the present invention. [00109] Importantly, it has been demonstrated in animal models that there is a high tolerance for DMSO when used in small amounts.1'1 Thus, toxicity concerns are minimal when compositions comprising DMSO are administered via a parenteral route. [00113] In accordance with the present invention, a controlled or delayed release vaccine preparation in stable form is provided together with a method of making such a vaccine preparation. The gel matrix of the controlled or delayed release vaccine composition comprise a biodegradable polymer selected from the group consisting of poly-D, L-lactide-co-' glycolide (PLG) and poly-D, L-lactic acid-co-glycolic acid (PLGA), polycaprolactones, polyanhydrides, polyorthoesters and poly(a-hydroxybutyric acid), a biocompatible solvent and a stabilized immunostimulatory complex. [00114] The polymeric gel may further comprise at least one additional adjuvant, e.g., MPL, MDP, DDA, A7, N- Avridine, BAY- 1005, DC-Choi, Murapalmitine, PCPP, QS-21, CT or CTB, LT or LTB or a cytokine such as IL-IJi, IL-2, IL-12, IFN-yand mixtures and a derivative thereof. [00115] Advantages of the controlled released composition of the present invention include: (a) a. fully biodegradable and biocompatible gel formulation; (b) a sustained release of the immunogen for presentation to the immune effector cells resulting in improved immunogenicity; (c) a high loading of the gel with a desired immunogen in a stable composition; and (d) a flexible mode of delivery including a suspension of a stabilized immunostimulatory complex that is self-adjuvanting. [00116] The molecular weight and crystallinity of the polymer directly impacts the entrapment efficiency in vivo. The polymeric gelling material is miscible in polar aprotic solvents such as DMSO. However, upon intramuscular or subcutaneous injection, DMSO is extracted into the surrounding body tissues with water reversibly penetrating the polymer rich solution. This process serves as the primary mechanism controlling in-situ gel formation. The rate at which this process proceeds directly affects the initial burst of rele'ase of the immunogen during the time interval when the biocompatible solvent is actively extracted into body tissue and exchanged with physiological solutions and before the entrapment of the immunogen by the formation of the gel.45 It is known that controlling the crystallization process is an important key mechanism by which the retention -of immunogens within the gel can be improved.46 This is intimately connected to the internal morphology of the gel formed which limits the diffusional pathways by which immunogens may be released. [00117] The entrapped or retained immunostimulatory complexes are subsequently released from the gel in limiting amounts in a sustained fashion with a larger boost released when the bulk of the matrix forming polymer is eroded. This varies depending on many of the same conditions that influence gellation, such as molecular weight, degree of crystallinity, constitution, hydrophobicity, and the presence of additives. [00118] The potential for an adverse toxicological response to the solvent DMSO has been addressed in a recent study44 wherein a device containing a peptide hormone suspended in DMSO was surgically implanted subcutaneously in a dog and in a human. The volume of DMSO employed in the study was 150uL. The implant was designed to release the peptide over the course of 1 year and surgically removed at the end of the study. In neither the dog or the human were any adverse tissue reactions observed. The controlled release of the peptide/DMSO mixture from the implant into physiological tissues is useful as a model for evaluating potential toxicity concerns for a fully biodegradable in-situ gelling polymeric composition. It is contemplated that the amount of DMSO useful in the present invention is essentially the same as that used in the study. [00119] It is expected that there is an initial extraction of the immunostimulatory complex in DMSO into body tissue before the solubility limit of the polymer is exceeded. Solidification takes place, retarding the release of the stabilized immunostimulatory complex. Subsequently, the stabilized immunostimulatory complex is released along with DMSO by diffusion-controlled pathways or retained in the gel with a release rate that is a function of the polymer properties. It is apparent that the diffusivity of these molecules within the gel is governed by several factors, such as the internal gel morphology and porosity, the degree of penetration by water into the gel and the hydrolysis of the bulk of the polymer.45 [00120] in the case of the stabilized immunostimulatory complex of the present invention, which is dispersed as a suspension throughout the gel, the initial extraction is largely that: of DMSO with a small quantity of the immunostimulatory complex located near the gelling front. Thus, the small amount of the immunostimulatory complex, not effectively entrapped during the initial gelling phase, would be responsible for the initial priming of the immune response in vivo. Thereafter, it is expected that DMSO would continue to be released by diffusion, with the bulk of the immunostimulatory complex remaining entrapped in the gel matrix until the bulk of the polymer has sufficiently been hydrolyzed to result in a full release of the encapsulated immunostimulatory complex. [00121] Specifically-, polymeric gels formulated from higher molecular weight and more crystalline polymers derived from ochydroxy acids degrade over a longer period of time than lower molecular weight amorphous analogs. This phenomenon, is known to be related to the accessibility of water to the hydrolytically unstable ester bonds.46 [00122] It has been established that random amorphous copolymers composed of 50% D,L-lactide and 50% glycolide with a molecular weight in the range of from 8,000-50,000 daltons, exhibit the highest degradation rates. 50% by weight or less of the polymer remains after approximately 6-8 weeks, when immersed in a PBS buffer.47 [00123] It is desirable to modify the parameters controlling the rate of gellation, the kinetics of immunogen release and the internal gel morphology. Specifically, use of various pore forming agents, plasticizers, and stabilizers such as surfactants, sugars, proteins, polymers and other excipients known to those skilled in the art are useful for this purpose. [00124] The preparations are stable when refrigerated or at room temperature. The vaccine composition of the present invention employing DMSO will freeze when refrigerated since the freezing point of DMSO is about 18°C. It has been found that thawing does not cause a change in the efficacy of the vaccine in vivo. [00125] In accordance with the present invention, the in-situ gelling biodegradable polymer and the stabilized immunostimulatory complex may be formulated separately. The in-situ gelling polymer may be solubilized in a biocompatible solvent in one vial, and the stabilized immunostimulatory complex in dry form in a separate vial. The immunostimulatory complex in dry form may be prepared by spray-drying, or preferably, lyophilization.48' 49 The dried immunostimulatory complexes can then be reconstituted in a biocompatible solvent, and dispensed by syringe either as a solution or as a suspension into the biodegradable polymer solution. The mixture is then used immediately or soon thereafter for immunization. [00126] Employing separate vials has an added advantage of minimizing any potential stability problems. These include polymer degradation in the presence of the peptide immunogeri, which may have reactive functional side chains, such as, free' amine groups or carboxylate groups,50 or the oxidation of selective amino acids, such as cysteine and tryptophan in the peptide immunogen in the presence of DMSO.1 [00127] To prepare a reconstitutable in-situ gelling polymer composition containing an immunostimulatory complex, an immunostimulatory complex is prepared as described above. Then, this aqueous solution is lyophilized. to form a dried composition. The dried composition is then reconstituted as a suspension in a biocompatible solvent containing a calculated weight percentage of a biodegradable gelling polymer. The final vaccine composition represents an in-situ gelling polymer and is suitable for parenteral injection. [00128] The compositions of the present invention are contemplated to be useful as vaccines or for therapeutic purposes. Other biological materials which may be modified to possess cationic charge at pH's generally ranging from 4.0-8.0 for efficient complexation with CpG oligonucleotides, may include proteins, protein mimetics, bacteria, bacterial lysates, viruses, virus-infected cell lysates, antigens, antibodies, pharmacological agents, antibiotics, carbohydrates, lipids, cytokines, chemokines, lipidated amino acids, glycolipids, haptens and combinations and mixtures thereof. [00129] These compositions can be administered parenterally via subcutaneous or intramuscular injection. When administered, parenterally, the immune response may be a cell-mediated response or a local or serum antibody response to provide neutralizing antibodies. For compositions administered mucosally the immune responses would additionally include a local secretory antibody response. [00130] It will be readily apparent to those skilled in the art that immunostimulatory complexes may also be admixed in oil- in-water encapsulation of stabilized immunostimulatory complexes within water-in-oil-in-water (w/o/w) double emulsions, biodegradable polymeric microparticles, lipid vesicles or liposome structures. Most of these delivery systems are attractive for the development of sustained release formulations. [00131] Preferably, the peptide immunostimulatory complex of the present invention may be employed in w/o-based emulsions employing SEPPIC's oil-based adjuvants, in combination with mineral salts such as Aluminum hydroxide, Aluminum phosphate and Calcium phosphate or in a single-dose controlled release in-situ gelling formulation based on biodegradable poly-D,Llactide- co-glycolide (PLG) or poly-D,L-lactic acid-co-glycolic acid (PLGA) copolymers. [00132] Most preferably, the vaccine compositions of the present invention comprise a stabilized immunostimulatory complex of the peptide immunogen with a CpG oligonucleotide admixed with the uncomplexed peptide immunogen. [00133] It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of medicine and in particular vaccination, diagnosis and treatment of infections with pathogens including bacteria and viruses. Further uses of the present invention are described below. Vaccine Preparation [00134] Immunogenic compositions, suitable to be used as vaccines, may be prepared from an immunostimulatory complex of the present invention, as a w/o emulsion, as a suspension in combination with a mineral salt suspension or as an in-situ gelling polymer or a combination of these systems as disclosed herein. The immunogenic composition containing the immunostimulatory complex is useful for eliciting an immune response by the host to which it has been administered. The immune response includes the production of antibodies by the host. [00135] The immunogenic composition may be prepared as injectables, as liquid solutions or suspensions, as lyophilized or spray-dried powders or emulsions. The composition comprising the immunostimulatory complex may be mixed with physiologically acceptable buffers or excipients, such as, water, saline, dextrose, glycerol, ethanol and combinations thereof. The vaccine may further contain additional substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants to further enhance the effectiveness thereof. The vaccine may further contain additional biocompatible substances, specifically in conjunction with the in-situ gelling polymers such as dimethyl sulfoxide (DMSO), N-methyl pyrrolidine (NMP), triacetin, glycerin, and poly vinyl pyrrolidone (PVP). [00136] The vaccine of the present invention may be administered parenterally, e.g., by injection subcutaneously, intramuscularly or transdermally. The vaccines of the present invention may be administered mucosally via oral, intranasal, rectal, vaginal or ocular routes. [00137] The vaccines are administered in a manner compatible with the formulation, and in such amount as to. be therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject or species to be treated, including, for example, the capacity of the subject's or species' immune system to synthesize antibodies, and if needed, to produce cell-mediated immune responses. [00138] Precise amounts of emulsifying oils, mineral salts or gelling polymers and material having biological activity required to be administered for effect depend on the judgement of the practitioner or veterinarian. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms to milligrams. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage of the vaccine may also depend on the route of administration and thus vary from one host or species to another. EXAMPLES [00139] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in the form and substitution of equivalents are contemplated as circumstances may suggest it as necessary to achieve a particular goal. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations. [00140] Methods of chemistry, organic chemistry, polymer chemistry, protein biochemistry and immunology used but not explicitly described in this disclosure and these Examples are amply reported in the scientific literature and are well within the ability, of those skilled in the art. Preparation of Immunostimulatory complex [00141] In general, an immunostimulatory complex of a synthetic peptide immunogen and a CpG oligonucleotide in aqueous solutions is prepared by the dropwise addition of a stock peptide solution in an appropriate aqueous solvent into a vial containing a gently stirred stock solution of CpG oligonucleotide dissolved in an appropriate aqueous solvent. The reverse mode of addition is equally effective. Compatible aqueous solvents include, but are not limited to, distilled ' deionized water, normal saline (NS =0.9% NaCl) or phosphate buffered saline (PBS = 10 mM Phosphate buffer, 0.9% NaCl) or mixtures thereof. The complexation process is largely unaffected by physiologic buffers, providing flexibility when selecting a compatible solvent system for both the synthetic peptide inununogen and the CpG oligonucleotide. [00142] The complex forms immediately and can be identified visually by the observation of a fine precipitate suspended in solution. The quantity of suspension so formed is a function of the relative amounts of the CpG oligonucleotide to the cationic peptide in solution. The precipitation process is controlled by the electrostatic neutralization of oppositely charged molecules. In a thermodynamically favourable process, the highly charged polyanionic single-stranded DMA binds with the positively charged cationic peptide immunogen.61 [00143] The CpG oligonucleotide is selected from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer, and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO: 2, a 24 base length oligomer plus an phosphorothioate group (designated as n at the 5' end). The CpG oligonucleotides were synthesized by Oligo's Etc. (Wilsonville, Oregon), and are obtained in a lyophilized dry state. These materials were reconstituted in the appropriate aqueous solvent prior to use. CpGl possesses a CpG motif sequestered within a sequence of 8 nucleotide bases and may provide stronger adjuvantation in vivo and improved stability by binding cationic peptides with higher affinities, than shorter oligonucleotides. A phosphorothioate modified group at the 5' end of CpG2 increases the molar negative charge density and potentially promotes improved binding. [00144] The peptide immunogens were synthesized and the appropriate aqueous buffer used to ensure that the peptide is cationic in solution. This is an important consideration in vaccines where complexation of the peptide immune-gen to the CpG oligonucleotide is desired. The ionization point or IP for each peptide immunogen and the pH of the medium is used to guide the selection of the appropriate buffer. The pH for an aqueous mixture of a stock peptide solution dissolved in distilled deionized water or normal saline (NS) was approximately 5.5, whereas in phosphate buffered saline (PBS) the pH of the stock peptide solutions was significantly higher at approximately 7.2. Careful selection of aqueous solvent systems is made to ensure full protonation for peptides derived from amino acids with weakly basic side chains, notably Histidine. [00145] Table 1 list the physical properties of the synthetic peptide immunogens and CpG oligonucleotides used to form immunostimulatory complexes. Three exemplary peptide immunogen targets are depicted in Table 1. A cocktail of two or three peptide immunogens or in one case a combinatorial library of peptides containing analogs of the peptide have been employed to prepare each vaccine. Each peptide immunogen comprises two segments, a B-cell target epitope and a T-helper epitope. The Th epitope is included to improve the immunogenicity of the peptide immu-riogen. [00146] The B-cell and T-help epitopes were selected after screening libraries of peptides in the appropriate animal models. Detailed information regarding the identification and composition of these constructs can be found by referring to US patent Nos. 6,090,38853, US patent No. 5,759,551s' and WO99/6729351 and US 6, 107, 021. 5V SEQ ID NOS: 7-9 in Table 1 comprise the LHRH immunogen peptides and are useful in a vaccine for prostate cancer immunotherapy, designed for hormone ablation treatment. SEQ ID NOS: 10-11 are useful In an anti-IgE immunotherapeutic vaccine for the treatment of allergy. SEQ ID NOS: 4-6 are useful in an anti-CD4 immunotherapeutic vaccine for the treatment of HIV infection. SEQ ID NOS: 12-13 comprise a combinatorial library of FMD peptides and are useful in an anti-FMD vaccine for protective immunity against foot-and-mouth disease. [00147] The immunostimulatory complex of the present invention may be prepared with various ratios of cationic peptides to CpG oligonucleotides to provide different physical properties, such as the size of the microparticulate complexes. Table 2 shows the calculated average molar positive charge and average formula weight for the peptide immunogen in the mixture. Table 2 also provides the calculated average molar negative charge contribution from CpGl (S.EQ ID NO: 1) and CpG2 NO: 2), respectively. Example 1: Preparation of Tmmunostimulatory complex of LHRH Immunogens and CpGl Oligonucleotides [00148] This Example illustrates the preparation of immunostimulatory complex from LHRH peptide immunogens and CpGl oligonucleotides in various proportions. A flow diagram of the process of complex formation as described herein is shown in Figure 1. [00149] All glassware, stir bars and pipette tips were autoclaved for 40 minutes at 121°C prior to use. -All reagents were weighed, dispensed, transferred or added to reaction vessels in a laminar flow hood to prevent contamination. [0001] A LHRH peptide immunogen stock solution was prepared by mixing a 1:1:1 molar ratio of peptides of SEQ ID NOS: 7-9 at a concentration of 3 mg/mL in distilled deionized water. 33 jaL of the stock solution (100 jig of the peptide immunogens) was added to each of a series of 2 mL vials equipped with micro stir bars. To this solution was added 0.5 mL of distilled deionized water as a diluent. A stock solution of 2.0 ng/\|iL of CpGl oligonucleotide was prepared in distilled deionized water. Various amount of the CpGl oligonucleotide stock solution was added to each vial to form the immunostimulatory complex. The amount of CpGl oligonucleotide added to each vial was determined by calculation to provide a charge ratio of LHRH:CpGl ranging from 8:1, with a large excess of LHRH, to 1:2 with an excess of CpGl. The respective amounts of CpGl used to prepare these compositions are shown in Table 3. It is to be noted that the ratio of LHRH:CpGl are represented as molar charge ratios and is based on the calculations shown in Table 3. [00151] The additions were made at room temperature with continuous stirring and equilibrated for 30 min. In all cases, an immediate clouding of the reaction mixture was observed upon addition of the CpG oligonucleotide stock solution. After complete addition of the CpGl oligonucleotide, a fine white particulate suspension was observed. The particles gradually settled and could be easily resuspended with gentle shaking. [0001] The solid microparticulate complexes can be essentially removed after settling and allows the separated supernatant solutions to be analyzed'by ultraviolet spectroscopy for residual uncomplexed peptide immunogens (at X = 280 nm) or for residual CpGl oligonucleotide (at A. = 260 nm) . For the immunostimulatory complexes prepared using an excess of LHRH, wherein LHRH:CpGl = 8:1, 4:1 or 2:1, excess amounts of peptide were detected. [00153] The result obtained by ultraviolet spectroscopy is an estimate and may be ±20% of the number obtained for the following reasons. The peptide chromophores have sizeably smaller extinction coefficients as compared to CpG oligonucleotides and the wavelength maxima used to detect the peptides and CpGl are fairly close to each other. Thus, the estimates for free residual peptide may possibly be exaggerated. Further, a small amount of nanoparticles of peptide CpG complex may be present in the supernatant. The interpretation of these results is further complicated by the observation that increasing the excess amount of the peptide immunogen relative to CpG generally results in complex aggregates with smaller average particle sizes. [0001] It is observable from Figure 2 that the ranking of LHRH/CpGl complexes with respect to average aggregate particle size distribution is in the order LHRH:CpGl 2:1 > 4:1 > 8:1. It is expected that the efficiency of the complexation process would vary based on the physical properties of the peptide immunogens and CpG oligonucleotides selected and the relative ratio of each in the vaccine composition. For the LHRH:CpGl system, the residual levels of uncomplexed peptide as determined by UV spectroscopy range from 60-90% (LHRH:CpGl = 8:1), 40-80% (LHRH:CpGl = 4:1} and 25-65% LHRH:CpGl = 2:1) over the background, respectively. For the LHRH:CpGl complex prepared at a 1:1 charge ratio there was very little detectable concentration of residual peptide immunogen, ~3%, or residual CpG oligonucleotide, r2%.' The large increase in the aggregate size of this complex coupled with the essentially complete complexation of immunogen is consistent with expected polyelectrolyte behavior at neutral charge. For the immunostimulatory complex LHRH:CpGl = 1:2 charge ratio, an excess of CpGl, 48% residual level of CpGl was found at X = 260 nm. This amount of residual CpGl approximates the quantity of CpGl expected if the first equivalent of CpGl was fully complexed with the peptide immunogen in solution. [00155] The results of the UV method demonstrate that immunostimulatory complex compositions prepared with a high excess of peptide to oligonucleotide (e.g. LHRHrCpGl =8:1 charge ratio) results in a significant amount of peptide free in solution. Similarly, immunostimulatory complexes prepared from a moderate excess of oligonucleotide to peptide (e.g. LHRHrCpGl = 1:2 charge ratio) result in compositions with excess free oligonucleotide. The presence of excess oligonucleotide can serve to stabilize smaller aggregates as shown in Figure 2. [00156] This example demonstrates that there may be no practical advantages to preparing immunostimulatory complex with a high excess of LHRH, LHRH:CpGl =8:1 charge ratio, wherein a significant amount of peptide remains free in solution. Similarly, there is no practical advantage for immunostimulatory complex prepared with a moderate excess of CpGl, LHRHrCpGl = 1:2 charge ratio, wherein it is reasonable to assume that after complete complexation at the point of electrical neutrality, excess oligonucleotide can serve only to stabilize the aggregates as shown in Figure 2. This result does reveal that compatible anionic molecules and/or polymers may be sequentially added to a preformed 1:1 electrically neutral complex in order to reduce the effective particle size of the composition. This presents a novel strategy for complete immunostimulatory complexation coupled with particle size control. [00157] It is an object of this invention to effectively bind the peptide immunogens in solution for certain applications to maximize the stability of the vaccine in vivo. Thus immunostimulatory complex prepared with charge ratios of peptide immunogens to CpG oligonucleotides ranging from 4:1 to 1:1 respectively are preferred. It is another object of this invention to maximize the adjuvanticity of the immunostimulatory complex in vivo by using smaller more discrete particles {~10 microns or less) for presentation to the immune system. [00158] It has been found that the presence of residual free and uncomplexed peptide is more desirable for more complex vaccine formulations such as water-in-oil emulsions or absorption on to mineral salts. In these formulations, adjuvantation of the immune responses may result from immunogens bound as immunostimulatory complex and also from uncomplexed immunogens dispersed within the w/o emulsion or adsorbed on the mineral salt directly. Thus, the immunostimulatory complexes prepared with charge ratios of peptide immunogens to CpG oligonucleotides ranging from 8:1 to 2:1 are found to be useful for these applications. [00159] More preferably, a combination of maximal peptide complexation for stability and small particle size for improved adjuvanticity is found for immunostimulatory complexes prepared with charge ratios of peptide immunogens to CpG oligonucleotides ranging from 4:1 to 2:1. [00160] The most preferred immunostimulatory complex are those prepared to possess physical properties, which make them suitable for alternative delivery modalities. Specifically, average particle sizes on the order of 10 microns or less are desirable in particular for rectal, vaginal, oral and nasal 'delivery. Example 2: Quantisation of Peptide Immunogen and Oligonucleotide Complexation Efficiency by RP-HPLC [0001] This Example.illustrates a preferable method for determining the complexation efficiency with respect to the peptide immunogen and oligonucleotide vaccine components employing reverse phase high performance liquid chromatography (RP-HPLC). This technique allows the quantitation of each residual uncomplexed individual peptide in a peptide cocktail mixture to be separated and identified (at X = 226 nm) and can be used to verify full complexation of the CpG oligonucleotide (at A. = 260 nm) . The solid microparticulate complexes can be separated from the supernatant solution by centrifuging followed by filtration. Two separate RP-HPLC programs are run on supernatant samples to identify and quantitate residual LHRH peptide immunogens and CpGl oligonucleotides in solution. [00162] The peptide(s) were resolved by high performance liquid chromatography (HPLC) using the Vydac 4.6 x 250 mm C-18 column, Cat. number 218TP54, with a gradient from 95% of solution A (0.05% TFA in HPLC grade water) and 5% solution B (0.05% TFA in HPLC grade acetonitrile) to 24% of solution A and 76% solution B in 40 minutes at the flow rate of 1 mL/minute. The UV wavelength absorbance was monitored at 226 nm. Peptide identity was determined by retention time, using standard peptides. [00163] The oligonucleotide(s) were resolved by high performance liquid chromatography (HPLC) using the PerSeptive Biosystem 4.6 x 100 mm Oligo R3 column, Cat. number R330-050, with a gradient from 95% of solution A ( 0.1 M TEAA in HPLC grade water, pH=8) and 5% solution B (HPLC grade acetonitrile) to 24% of solution A and 76% solution B in 40 minutes at the flow rate of I mL/minute. The UV wavelength absorbance was monitored at 260 nm. Peptide identity was determined by retention time, using standard oligonucleotide. [00164] Immunostimulatory complexes of LHRH and CpGl ranging from 8:1 to 1:1 were prepared for this study as described in Example 1 and Example 11. The respective amounts of CpGl used to prepare these compositions are shown in Table 3 and Table 9. It is to be noted that the ratio of LHRH:CpGl are represented as molar charge ratios and is based on the calculations shown in Table 3 and Table 9. [00165] For the immunostimulatory complexes prepared using an excess of LHRH, wherein LHRH:CpGl = 8:1, 4:1 or 2:1, nonequivalent amounts of residual peptide were detected by RPHPLC in the supernatant solutions indicating that the complexation process is selective. This technique enables a ranking of LHRH peptide immunogens with preferences for CpGl oligonucleotide in solution based on binding affinity. In all cases no residual uncomplexed CpGl oligonucleotide could be detected by RP-HPLC, indicating full complexation of this component. [00166] For the immunostimulatory complex prepared using an equivalent amount of LHRH to CpGl oligonucleotide based on charge ratio (LHRH:CpGl = 1:1), essentially no peptide and no CpGl was detected by RP-HPLC indicating full complexation of all components. [00167] The complete set of results for these analyses is depicted in Table 8. It is clear that the binding of LHRH peptide immunogens with CpGl oligonucleotide may be ranked as p607E > p667 > p500. The three peptides have near identical ionization points and all three are positively charged as calculated in Table 1. The preference of CpGl oligonucleotide for p607E (+4 charge) over p667 (-1-5 charge) both of which are preferred to p500 (+4 charge) is likely related to the molecular weight and the distribution of charges within these peptides. Example 3: Preparation of Dried Immunostimulatory Complex [00168] This Example illustrates the procedure used to prepaxe an immunostimulatory complex in a dry state. [00169] Suspensions of LHRH/CpGl complexes, prepared as described in Example 1, in 0.5-1.0 mL in aqueous solvent, distilled deionized water, normal saline or phosphate buffered saline, were placed in a dry ice/acetone bath and frozen for 15 minutes. The frozen samples were then placed on a freezedryer (Vertis 25LEZ) and the water removed by sublimation at 200 millitorr over three days. This procedure provided a near transparent glassy finished product in the vial. The appearance of the residual solid recovered depends on the aqueous solvent used and can range from a near transparent glass to a white fluffy solid. [00170] Reconstitution of the dried materials in- the same volume of aqueous solvent regenerated a suspension of discrete particles. The particle size distributions, determined as described in Example 2, showed essentially no change. [00171] This demonstrated that the drying and resuspension process does not effect the physical properties of the prepared immunostimulatory complexes. Thus, a vaccine composition comprising the immunostimulatory complexes of the present invention may be provided in the form of a suspension, a solid or a dried powder. Example 4a: Preparation of Water—in-Oil Emulsions Using High Shear Homogenization [00172] This Example illustrates the process of preparing a water-in-oil (w/o)-emulsion from cationic peptides derived from LHRH peptide immunogens (SEQ ID NOS: 7-9 in a 1:1:1 molar ratio in solution), IgE peptide immunogens (SEQ ID NO: 10-11 in a 2:1 molar ratio in solution) CD4 peptide immunogens (SEQ ID No: 4-6 in a 2:1:1 molar ratio in solution) or immunostimulatory complex derived from LHRH, IgE or CD4 immunogens and CpGl or CpG2 oligonucleotides in various proportions using homogenization techniques. A flow diagram' illustrating the process of emulsion formation via homogenization as described herein is shown in Figure 3. [00173] All glassware, stir bars and pipette tips were autoclaved for 40 minutes at 121°C prior to use. All reagents were weighed, dispensed, transferred or added to reaction vessels in a laminar flow hood to prevent contamination. [00174] The w/o-emulsions were optimized for stability with respect to the volume ratio of the aqueous to oil phases required. For compositions employing Montanide® ISA 720 oils (SEPPIC, Inc) the ratio of water to oil was 30:70 by volume. For compositions employing ISA-Montanide® 51 or ISA Montanide® 50v oils (SEPPIC, Inc.) the ratio of water to oil was 50:50 by volume. Example 4b: Preparation of Water-in-Oil Emulsions from ISA Montanide® 720 and Immunostimulatory complex [0001] To a 10 mL vessel, was added 3,333 ug of peptide immunogens dissolved in an appropriate aqueous buffer (1,111 uL, 3 mg/mL) or an immunostimulatory complex prepared from 3,333 \iq of peptide immunogens dissolved in an appropriate aqueous buffer (1,111 ^L, 3 mg/mL) and either CpGl or CpG2 oligonucleotides. Table 3 and Table 4, shows the calculations for determining relative amounts of each reagent employed, [00176] Specifically, to prepare an immunostimulatory complex from LHRH peptide immunogens at a 4:1 charge ratio of LHRH:CpGl, 244 ng CpGl oligonucleotide (122 uX, 2.0 were used. [00177] Specifically, to prepare an immunostimulatory complex of IgE peptide immunogens at a 4:1 charge ratio of IgE:CpGl, 387 jag of CpGl oligonucleotide (193.5 ^.L, 2.0 \|ig/^L) were used. To form a 1:1 neutral complex of IgE:CpGl, 1,548 \|ig of CpGl oligonucleotide (774.0 uX, 2.0 ng/uL) were used. [00178] Specifically, to prepare an immunostimulatory complex of CD4 peptide immunogens at a 2:1 charge ratio of CD4:CpG2, 402 ug of CpG2 oligonucleotide (201 ^L, 2.0 ug/u.L) were used. To form a 1:2 charge ratio of CD4:CpG2, 1608 fig of CpG2 oligonucleotide (804 ^iL, 2.0 ng/^L) were used. [00179J To each of the vessels additional diluent aqueous solvent was added so that the final volume of the aqueous phase was fixed at 3.0 mL for preparation of ISA Montanide® 720 w/o-emulsions respectively. [00180] For LHRH or IgE peptides normal saline or PBS was found to be suitable for complexation. The calculated IP for each peptide immunogen is greater than 9.0 (Table 1), far greater than the pH of the aqueous solvent selected. [00181] In the case of CD4 peptides the choice of aqueous solvent proved important. Upon dilution with either normal saline or PBS a solid precipitate was observed to quickly form in solution. This instability would preclude use of this immunogen combination by parenteral routes. An examination of the peptide immunogens revealed that peptide sequence ID No: 6 (Table 1) has a calculated ionization point of 6.91. In PBS (pH ~ 7.2), this peptide would tend to aggregate and be expected to exhibit instability. A solution to this problem was found by first preparing the immunostimulatory complex in distilled deionized water followed by dilution with saline or PBS of sufficient ionic strength to ensure that the suspension was isotonic and suitable for injection. [00182] This example demonstrates the advantages of stabilizing immunogens in solution in the form of an immunostimulatory complex of LHRH, IgE or CD4 peptides. [00183] The diluted aqueous solutions or suspensions were then slowly added to a dry 25 mL reaction vessel charged with 7.0 mL of ISA Montanide® 720. The additions were made while homogenizing (High Shear Laboratory Mixer, Sealed Unit, Silverson) the mixture at low speeds (2,000-3,000 rpm) to generate a coarse emulsion. This processing speed was maintained until the aqueous sample had been completely added and was continued a full 2 minutes to ensure uniform premixing of the aqueous and oil phases. The homogenization speed was then ramped up (5,000-8,000 rpm) and maintained for from 5 to 10 minutes further resulting in the formation of a homogeneous white finely dispersed w/o-emulsion. [0001] The final concentration of immunogens once formulated as suspensions or in water-in-oil emulsions as described above was 200 u.g/mL. Example 4c; Stability Evaluation for Water-in-Oil Emulsions Prepared by Homogenization Methods. [00185] The consistency and stability of the w/o-emulsions prepared by homogenization was checked by a variety of methods. To prove that the emulsion was water-in-oil (w/o) and not oil-in-water (o/w) or water-in-oil-in-water (w/o/w), a droplet of the composition was added to a beaker containing distilled deionized water. A droplet of a w/o-emulsion will float on the surface and not disperse into water. Conversely, a droplet from an o/w emulsion will instantly disperse into water and a droplet from a w/o/w double emulsion will disperse both on the surface and into the bulk of the aqueous phase. Droplets from the emulsions prepared from ISA Montanide® 720 were observed to float on the surface with minimal dispersal and droplets from the emulsions prepared from ISA Montanide® 51 oils were observed to float on the surface with essentially no dispersal. These results indicated that the emulsions were w/o and further that the tendency towards dispersal was higher for the w/o-emulsion prepared from ISA Montanide® 720. This is related to the initial viscosity of the oils themselves and the viscosity of the resultant emulsions. This is an important consideration for maximizing the depot potential of the resultant vaccine formulation. [00186] The apparent viscosity of the finished emulsions and oils were checked (Brookfield DV-1 + rotational viscometer) for lot to lot consistency and for long-term stability trials. ISA Montanide® 720 had a viscosity of -15 mPa at 25°C whereas the w/o emulsion prepared from ISA Montanide® 720 had a viscosity of ~45-50 mPa at 25°C. This provided a fairly fluid product, which is desirable for facilitating the handling, and dispensing of the vaccine with a syringe. [00187] In contrast, ISA Montanide® 51 had a viscosity of -50 mPa at 20°C whereas the w/o-emulsions prepared from ISA Montanide® had a viscosity of - 1,500-2,900 mPa at 25°C. The wide variation in viscosity was found to be a function of buffer selection. (PBS = -2,900 mPa, NS = -2,500 mPa and distilled deionized water = -1,500 mPa) Material of this high viscosity can present some difficulties with respect to transferring and dispensing with a syringe. However, the long-term stability of these compositions was improved. The long-term stability of the emulsion was evaluated by placing 1 mL of each emulsion in a 1.5 mL eppendorf vial and centrifuging the contents under high speed (5,000 rpm) for 10 minutes. These conditions do not simulate actual storage conditions but can be used to predict the resistance of the emulsion to separation. In the case of ISA Montanide® 720, 5- 10% of the volume separated out with a clear or straw yellow oil phase observed on the surface. In the case of ISA Montanide® 51, 0-2% of the volume separated out with a clear or straw yellow oil phase observed on the surface. The higher viscosity of the ISA Montanide® 51 emulsion products accounts for the greater stability to sedimentation and resistance to separation. [00188] The particle size and distribution for the w/o-emulsion was further characterized by optical microscopy (Nikon DIAPHOT 200) . A photomicrograph of each composition was obtained and an estimate of the size range of particles was made using a computer-generated scale. The scale itself is externally referenced against standards of known particle sizes (MIST traceable microparticles - Duke Scientific). For w/o emulsions prepared from either ISA Montanide® 720 or ISA Montanide® 51 and peptide immunogens, the particles sizes were essentially the same (c.a. 1-2 microns) with minimal aggregation or coalescence. For w/o-emulsions prepared from either ISA Montanide® 720 or ISA Montanide® 51 and immunostimulatory complex the particle sizes were slightly larger (c.a. 1-3 microns) with minimal aggregation or coalescence. Figure 4 is a photomicrograph obtained from a w/o-emulsion prepared via homogenization from ISA Montanide® 51 and an immunostimulatory complex derived from 100 ng of LHRH peptide immunogens, with LHRH:CpGl in a final charge ratio of 4:1. [00189] The initial average particle size was on the order of 10 microns. The process of homogenizing an aqueous suspension of immunostimulatory complexes under high shear resulted in a smaller average aggregate particle size. A stable w/o- emulsion with droplets in the size in the range of 1-3 microns was obtained. Example 5a: General Preparation of Water-in-Oil Emulsions Using Low Shear Extrusion [00190] This Example illustrates the process of w/o-emulsion formation from cationic peptides derived from LHRH, IgE or CD4 immunogens or immunostimulatory complex derived from LHRH, IgE or CD4 immunogens and CpGl or CpG2 oligonucleotides in various proportions using extrusion techniques. Table 3 and Table 4, shows the general calculations for determining the relative amounts of each reagent employed. A flow diagram illustrating the process of emulsion formation via extrusion as described herein is shown in Figure 3. 100191] All glassware, stir bars and pipette tips and the entire extruder mechanism were autoclaved for 40 minutes at 121°C prior to use. All reagents were weighed, dispensed, transferred or added to reaction vessels in a laminar flow hood to prevent contamination. [00192] The extrusion process involves repeatedly passing an aqueous phase loaded in one syringe into an oil phase loaded in a second syringe through a narrow bore tube joining the two syringes. Emulsification occurs as the fluids are driven through the narrow bore under pressure, typically 100 psi. By contrast, a homogenizer system typically operates at a pressure that is greater than 1,000 psi. The number of return passages necessary for the above extrusion process often exceeds 20 to 30 before a visually uniform w/o-emulsion is generated. This manual extrusion process cannot generate significant shear and the number of exchanges required to efficiently produce a w/o-emulsion is highly variable. The physical properties of the w/o-emulsions are inconsistent and the overall stability and consequently in vivo potency are typically highly irreproducible. In spite of these problems, there are a number of possible applications for products produced by this process. [0001] TO address these shortcomings an extruder mechanism has been developed(LiposoFast™ Basic, Avestin, Inc., Ottawa, Canada). The device consists of two syringes (0.5 mL or 1.0 mL) fitted via luer locks with a narrow bore passage connected to a holder with a polycarbonate membrane of a defined pore size placed in between the two syringes. The device as originally designed was for the preparation of liposomes of a controlled size.63 The application of such a device with a compatible oil-based product for the preparation of w/o emulsions appears not to have been contemplated. The membrane pore size can be selected (Whatman Nucleopore, 0.05 ^M - 10 fiM) . The smaller pore size allows for the extrusion of dispersions under increased shear. The larger pore size can be selected for formulating wherein the particulates are larger sized. Using this device, the efficiency of emulsification was increased. Fewer return passages were required to provide a more uniform and stable product. The in vivo potency of such a preparation would be predicted to be more reproducible. However, there are still limitations due to the small maximum volume, c.a. 1.0 mL, which can be practically employed and the practical restrictions on the choice for the oil component. [00194] The process works well for the preparation of w/oemulsions derived from ISA Montanide® 720. However, the higher viscosities of emulsions derived from ISA Montanide® 51 results in significant backpressure, precluding the use of this extrusion device. As such, this method can be best viewed as a process for preparing instant w/o emulsions from oils with apparent viscosities of less than 1,500 mPa. [00195] In particular, where the costs associated with storage and stability of vaccines are of concern or where patient compliance is an issue or application as a palliative medicine is involved, this method of delivery may be cost effective and practical. Ideally, trained practitioners such as doctors or pharmacists can be relied upon for preparing instant w/oformulations on-site for general, use. [00196] This device and protocol may be used for the preparation of instantaneous o/w, and w/o/w, microemulsions for which controlled shear and extrusion are required, or for the preparation of refined products. Example 5b: Preparation of Water-in-Oil Emulsions from ISA Montanide® 720 [0001] To a 1.0 mL glass syringe (gas tight), was added 333 fig of LHRH, IgE or CD4 peptide immunogens dissolved in an appropriate aqueous buffer (111 uL, 3 mg/mL) or an immunostimulatory complex (with a 4:1 charge ratio) prepared from 333 fig of LHRH or IgE peptide immunogens or an immunostimulatory complex (with a 2:1 charge ratio) prepared from 333 fig of CD4 peptide immunogens dissolved in an appropriate aqueous solvent (111 fiL, 3 mg/mL) and CpGl or CpG2 oligonucleotides in appropriate ratios as described in Tables 3 and Table 4, respectively. [00198] Specifically, to prepare immunostimulatory complex from LHRH peptide immunogens at a charge ratio of LHRH:CpGl of 4:1, 24.3 ug CpGl oligonucleotide (12.2 uL, 2.0 ug/mL) were added. [00199] To prepare immunostimulatory complex from IgE peptide immunogens at a charge ratio of IgE:CpGl of 4:1, 38.7 fig of CpGl oligonucleotide (19.4 uL, 2.0 ug/uL) were added or to form a 1:1 neutral complex of IgE:CpGl, 154.8 fig of CpGl oligonucleotide (77.4 fiL, 2.0 ug/uL) were added. [00200] To prepare immunostimulatory complex from CD4 peptide immunogens at a charge ratio of CD4:CpG2 of 2:1, 40 ng of CpG2 oligonucleotide (20 uX, 2.0 jig/fiL) were added or to form a complex at a charge ratio of CD4:CpG2 of 1:2, 160 \iq of CpG2 oligonucleotide (80 (iL, 2.0 ng/jxL) were added. [00201] Additional diluent aqueous solvent was added so that the final volume of the aqueous phase was 300 ui. [00202] To a second 1 . 0 mL glass syringe (gas tight), was added 700 u.L of ISA Montanide® 720. The syringes were connected via luer locks to an extrusion-housing unit containing a membrane holder and support for the polycarbonate membrane filter. Membrane filters with pore sizes 3 \iM or 5 ^iM were selected for w/o-emulsions to be prepared with peptide immunogens in the aqueous phase. Whereas, melnbrane filters with pore sizes 5 nM or 10 fiM were selected for the preparation of w/o emulsions with immunostimulatory complex suspended in the aqueous phase. The aqueous phase was then first passed through the membrane into the oil phase, typically with great ease. The subsequent exchanges require additional pressure with the increased backpressure generated during the emulsion process. After 8-12 passages the backpressure upon extrusion had equalized and a homogeneous white emulsion was typically obtained. [00203] The final concentration of immunogens once formulated as solutions or as immunostimulatory complex in water-in-oil emulsions as described above was 200 Example 5c: Stability Evaluation for Water-in-Oil Emulsions Prepared by Extrusion [0001] Stability tests similar to those conducted in Example 4c, prepared by the extrusion processes confirmed that these compositions were w/o-emulsion. Droplets placed on the surface of distilled deionized water were observed to float with minimal dispersal. The viscosities of the resultant emulsions were found to range from ~35-40 mPa, a slight reduction when compared to the high energy homogenized system in Example 4c. The size distribution of the emulsion droplets A was larger than the analogous homogenized systems. About 1-3 microns for w/o emulsions prepared from peptide immunogens or 2-5 microns for w/o emulsions prepared from immunostimulatory complex. For comparison, Figure 5 is a photomicrograph obtained from a w/o emulsion sample prepared via extrusion from ISA Montanide® 720 and an immunostimulatory complex derived from 200 jag of LHRH peptide immunogens and CpGl oligonucleotides in a final charge ratio of LHRH:CpGl of 4:1. [00205] In general, the size of the droplets obtained by the low energy extrusion process as shown in Figure 5 were larger and more highly aggregated than the high energy homogenized droplets as shown in Figure 4. Overall, these instant w/o emulsions are sufficiently stable for immediate or same day use. Example 6a: Preparation of In-situ Polymeric Gels [00206] This Example illustrates the process of in-situ gel matrix formation and encapsulation of immunostimulatory complex derived from IgE or CD4 immunogens and CpGl or CpG2 oligonucleotides in various proportions using direct reconstitution techniques. A flow diagram showing the process of preparing an in-situ gel formulation with either peptide immunogens or itnmunostimulatory complex via reconstitution as described herein is shown in Figure 6. [00207] All glassware, stir bars and pipette tips were autoclaved for 40 minutes at 121°C prior to use. All reagents were weighed, dispensed, transferred or added to reaction vessels in a laminar flow hood to prevent contamination. [00208] In general, varying weight percentages of PLG or PLGA copolymers (Boehringer Ingelheim) were dissolved in biocompatible solvents such as anhydrous dimethyl sulfoxide (DMSO, Aldrich). The solubilization process required vigorous agitation for higher molecular weight polymers with continuous stirring maintained for from 4-6 hours to ensure complete dissolution. The polymer solution was then filtered through an organic solvent compatible membrane of pore size 0.45 microns (Phenomenex, PTFE). To this was added a solution of cationic peptide immunogens or more preferably a suspension of immunostimulatory complex in an appropriate biocompatible solvent. The peptide immunogen or peptide/CpG complex or a mixture thereof was first lyophilized (as described in Example 3) and subsequently dissolved or resuspended in an appropriate biocompatible solvent, such as DMSO. [00209] The use of polar aprotic solvents, such as DMSO, may present some problems with respect to long-term peptide stability. DMSO is known as a powerful oxidizing agent and peptides containing sensitive amino acids such as cysteine and tryptophan may be chemically incompatible in these solutions. Thus, peptide immunogens containing these residues may have to be reconstituted on site for immediate use. [00210] The lyophilized peptide immunogens or immunostimulatory complex or a mixture thereof was reconstituted directly in the vial into a solution of polymer in DMSO at the time of use, thereby avoiding prolonged exposure of the peptide immunogens to DMSO. The dissolution of the peptide immunogen or the resuspension of immunostimulatory complex or a mixture of the immunostimulatory complex with peptide immunogens was rapid, requiring gentle shaking to ensure sample uniformity. [00211] There are minimal stability and manufacturing issues expected for these generic compositions. The polymer solutions in anhydrous DMSO are not as susceptible to hydrolytic degradation as compared with peptide immunogens dissolved or suspended in aqueous solutions. The polymer solution may be frozen (DMSO freezes at c.a. 18°C) with no detectable changes in the physical properties upon thawing. The peptide or immunostimulatory complex isolated in the lyophilized dry state would also be expected to exhibit increased stability in the absence of water. [00212] The mixture of polymer and peptide immunogens or immunostimulatory complex as such constitutes a single-phase solution or a suspension suitable for subcutaneous or intramuscular injection. [00213] Of importance for either system is the viscosity of the solution or suspension. This directly influences the ability to syringe and inject the compositions by a subcutaneous or intramuscular route. [00214] The apparent viscosity of these systems is a function of various factors including the constitution, molecular weight, crystallinity and intrinsic viscosity of the PLG or PLGA copolymers. These factors delimit the useful amount by weight for each polymer that can be dissolved while maintaining practical flow characteristics. Table 5 shows the physical properties for selected PLG or PLGA polymers and the corresponding amount in weight percentages that may be dissolved in DMSO to obtain solution viscosities of practical use. The apparent viscosity for these solutions was determined by a Brookfield DV-1+ rotational viscometer. [00215] 100 mPa was arbitrarily chosen as the upper desired limit for these compositions. In most cases a solution or suspension formulated as an in-situ gelling polymer solution with an apparent viscosity less than 200 mPa can be uniformly delivered through conventional syringes. hydrolytic degradation as compared with peptide immunogens dissolved or suspended in aqueous solutions. The polymer solution may be frozen (DMSO freezes at c.a. 18°C) with no detectable changes in the physical properties upon thawing. The peptide or immunostimulatory complex isolated in the lyophilized dry state would also be expected to exhibit increased stability in the absence of water. [00212] The mixture of polymer and peptide immunogens or immunostimulatory complex as such constitutes a single-phase solution or a suspension suitable for subcutaneous or intramuscular injection. [00213] Of importance for either system is the viscosity of the solution or suspension. This directly influences the ability to syringe and inject the compositions by a subcutaneous or intramuscular route. [00214] The apparent viscosity of these systems is a function of various factors including the constitution, molecular weight, crystallinity and intrinsic viscosity of the PLG or PLGA copolymers. These factors delimit the useful amount by weight for each polymer that can be dissolved while maintaining practical flow characteristics. Table 5 shows the physical properties for selected PLG or PLGA polymers and the corresponding amount in weight percentages that may be dissolved in DMSO to obtain solution viscosities of practical use. The apparent viscosity for these solutions was determined by a Brookfield DV-1+ rotational viscometer. [00215] 100 mPa was arbitrarily chosen as the upper desired limit for these compositions. In most cases a solution or suspension formulated as an in-situ gelling polymer solution with an apparent viscosity less than 200 mPa can be uniformly delivered through conventional syringes. was 200 ng/mL. The immunogen or immunostimulatory complex was immediately reconstituted in solution or suspension with gentle shaking to ensure content uniformity. [00219] The apparent viscosities of the solutions prepared from these polymers at the specified weight ratios were found to be close to 100 mPa. (see Table 5). The concentrations for Resomer® RG 503H, Resomer® RG 504H and Resomer® RG 756 for a solution viscosity of around 100 mPa were critical, Resomer® RG 503H and Resomer® RG 504H have much lower inherent viscosities and both are derived from PLGA with amorphous character, wherein the monomer composition was around 50% D,Llactide and 50% glycolide. These materials would be expected to degrade at a rate of 6-8 weeks for 50% of the polymer in vivo. Thus, the encapsulated components would have a twothree month release profile with gels prepared from Resomer® RG 503H or Resomer® RG 504 H. Gels prepared from these materials would be most suited for short-term single-dose controlled release applications. Conversely, Resomer® RG 756 is a more crystalline polymer composed of 75% D,L-lactide to 25% glycolide residues. It may require from 4-6 months for 50% of the polymer to degrade in vivo and the release rate for encapsulated components would consequently be more protracted. Resomer® RG 756 would be expected to be more desirable for long-term single-dose controlled release applications. Example 7: The Immunogenicity of IgE and CD4 Pep tide Immunogens Formulated as Immunostimulatory complex or as W/O Emulsion or in Combinations [00220] This Example illustrates the immunogenicity of IgE and CD4 peptide inununogens formulated as immunostimulatory complexes with CpGl or CpG2 oligonucleotides, as a w/oemulsion or as immunostimulatory complex dispersed in a w/oemulsion in guinea pigs, which were immunized intramuscularly. was 200 p.g/mL. The immunogen or immunostimulatory complex was immediately reconstituted in solution or suspension with gentle shaking to ensure content uniformity. [00219] The apparent viscosities of the solutions prepared from these polymers at the specified weight ratios were found to be close to 100 mPa. (see Table 5). The concentrations for Resomer® RG 503H, Resomer® RG 504H and Resomer® RG 15-6 for a solution viscosity of around 100 mPa were critical, Resomer® RG 503H and Resomer® RG 504H have much lower inherent viscosities and both are derived from PLGA with amorphous character, wherein the monomer composition was around 50% D,Llactide and 50% glycolide. These materials would be expected to degrade at a rate of 6-8 weeks for 50% of the polymer in vivo. Thus, the encapsulated components would have a twothree month release profile with gels prepared from Resomer® RG 503H or Resomer® RG 504H. Gels prepared from these materials would be most suited for short-term single-dose controlled release applications. Conversely, Resomer® RG 756 is a more crystalline polymer composed of 75% D,L-lactide to 25% glycolide residues. It may require from 4-6 months for 50% of the polymer to degrade in vivo and the release rate for encapsulated components would consequently be more protracted. Resomer® RG 756 would be expected to be more desirable for long-term single-dose controlled release applications. Example 7: The Immunogen i city of IgE and CD4 Pep tide Inununogens Formulated as Immunostimulatory complex or as W/O Emulsion or in Combinations [00220] This Example illustrates the immunogenicity of IgE and CD4 peptide immunogens formulated as immunostimulatory complexes with CpGl or CpG2 oligonucleotides, as a w/oemulsion or as immunostimulatory complex dispersed in a w/oemulsion in guinea pigs, which were immunized intramuscularly. The w/o-emulsion.s were prepared by homogenization as described in Example 4a/4b or by extrusion as described in Example 5a/5b. [00221] Groups of three, 6 to 8 week old female guinea pigs (Covance Research Products Inc., Denver, PA) were immunized' intramuscularly (I.M.) on week 0, .3 and 6 with the following compositions: 100 ng of the IgE peptides/CpGl immunostimulatory complex (4:1 charge ratio and 1:1 charge ratio) prepared as described in Table 4 suspended in a final volume of 250 fiL PBS, pH 7.4; CD4 peptides/CpG2 immunostimulatory complexes (2:1 charge ratio and 1:2 charge ratio) prepared as described in Table 4 in 75 ^L suspended in a final volume of 250 nL PBS' PH 7-4'"' I9E peptides in 75 fiL PBS, pH 7.4 emulsified with ISA Montanide® 720 (175 \iL) ; CD4 peptides in 75 fiL distilled deionized water emulsified with ISA Montanide® 720 (175 fiL) ; IgE peptide/CpGl immunostimulatory complex in 75 nL PBS, pH 7.4 prepared as described in Table 4 (4:1 charge ratio and 1:1 neutral charge ratio) emulsified with ISA Montanide® 720 (175 nL) , or CD4 peptides/CpG2 immunostimulatory complexes prepared as described in Table 4 in 75 \)iL distilled deionized water (2:1 charge ratio and 1:2 charge ratio) emulsified with ISA Montanide® 720 (175 [00222] The guinea pigs showed no gross pathologies or behavioral changes after receiving immunostimulatory complexes, w/o emulsions containing peptide immunogens or w/o emulsions containing the immunostimulatory complexes. Sera were obtained on weeks +3, + 5, +9, +11 and +17 and were evaluated for the presence of anti-IgE antibodies in the case of IgE immunogens or anti-CD4 antibodies in the case of CD4 immunogens, by immunogen-specif ic ELISAs. Measurement of anti-IgE antibodies [00223] Anti-IgE peptide titers were determined by IgE peptide ELISA and cross-reactivities to human IgE by human IgE ELISA. Peptide ELISAs for determination of anti-IgE peptide reactivity were conducted in microtiter plates coated with the target antigen site peptide without the T helper site, as described64. For determination of anti-human IgE crossreactivity, human IgE ELISAs were conducted in microtiter plates coated in a likewise fashion with a human IgE myeloma protein (American Biosystems, Inc. cat. no. A113) at 5 ug/ml. [00224] Captured anti-peptide or anti-IgE antibodies were detected by horseradish peroxidase-labeled anti-guinea pig IgG goat antibody. ELISA titers, expressed as logio of reciprocal dilution, were calculated based on linear regression analysis of the absorbances, with cutoff A value was rigorous, as the values for diluted normal guinea pig control samples run with each assay were less than 0.15. Hyperimmune guinea pig anti-IgE peptide immunogen antiserum was used as a positive control. Pre-immune sera were used as negative controls. Measurement of anti-CD4 Antibodies [00225] ELISAs for binding to recombinant soluble CD4 were done in 96 well microtiter plates coated with rsCD4 (American Bio- Technologies) at 0.25 pg/mL, using 100 pL per well in lOmM NaHC03 buffer, pH 9.5. Wells were blocked with 250 pL of 3% gelatin, washed with 0.05% TWEEN 20 in phosphate-bufferedsaline (PBS) and dried. Test wells were reacted with 100 pL of diluted immune sera for 1 hour at 37 C. Wells were washed with 0.05% TWEEN 20 in PBS, reacted with 100 pL of horseradish peroxidase-labeled goat anti-mouse IgG (Pierce) diluted 1:1000 in 1% goat serum, 0.05% TWEEN® 20 in PBS, and washed. 100 pL of orthophenylenediamine (OPD) substrate at 0.04% by weight (Pierce) and 0.12% H202 in sodium citrate buffer, pH 5.0, was added for 15 minutes. Reactions were stopped by addition of 100 uL 1.0 M H2SC>4 and £492 determined. Hyperimmune guinea pig anti-CD4 peptide immunogen antiserum was used as a positive control. Pre-immune sera were used as negative control. Measurement of Functional Antigenicity by Competitive ELISA [00226] In this competitive ELISA, functional antigenicity was quantitated for CD4 immunogens by testing the evoked antibodies for the capacity to competitively inhibit a functional monoclonal antibody, mAb B4, whose known specificity was for the CD4 complex on the host cell surface that binds HIV. This anti-binding site monoclonal antibody has been well characterized for its high affinity for the HIV binding complex, for binding to domain 1 of soluble recombinant CD4 (rsCD4), and for its ability to neutralize HIV-1 primary isolates.65 [0001] The anti-binding site monoclonal antibody was purified by protein A affinity chromatography and conjugated to horseradish peroxidase. The mAb B4-HRP conjugate was used in the assay as a tracer, at a concentration of 0.5 ng/ml. 96- well microtiter plates were coated with recombinant soluble CD4 protein, 1 ng/ml in 0.1 M sodium carbonate buffer, pH 9.5, with overnight incubation. Reactions were done in the microtiter wells in 100 fil total volume of PBS/goat serum/gelatin/TWEEN® 20, with serially diluted immune serum (guinea pig, swine, or baboon) and 30 \il of the mAb B4-HRP working stock. Diluted serum and mAb B4-HRP were preincubated prior to adding the mixture to the well. The positive control for the competition ELISA was 5 p.1 of unlabelled-anti-binding site mAb at 0.5 jag/ml in normal serum, the negative control is normal serum. The serum/antibody dilution mixture, 100 pi was added to a coated well and incubated for one hour at 37°. The plates were drained and washed and bound mAb B4-HRP was detected by reaction with a chromogen. The chromogen was 3,3',5,5'-tetramethylbenzidine (TMB) and TMB-bound mAb B4 conjugate was detected at h^. K calibration curve was obtained with purified mAb B4 serially diluted from 10 ug/ml into normal serum of the appropriate species so as to obtain an mAb equivalent value for the dilutions of immune sera which competitively inhibit the binding to human recombinant soluble CD4 of the mAb B4-HRP. Virus Neutralization Assay [00228] The MT-2 microplaque assay was carried out as described66 except that heat-inactivated sera were serially diluted in 50% high glucose DMEM with 15% FBS, antibiotics, 2% glutamine and bicarbonate buffer, and 50% pooled, defibrinated normal human plasma. In this assay, diluted serum is incubated with 20 pfu of HIV in microtiter wells. HIVsensitive MT-2 cells are added and formed into monolayers by centrifugal force under molten agarose. Residual virus infectivity is detected by the presence of propidium iodidestained plaques one week later. The endpoint is the serum dilution at which there was a 50% or 90% reduction in the plaque count. Immunogenicity Results [00229] The serum antibody titers following immunization of IgE immunogens are shown in Figure 7 (homogenized systems) and Figure 9 (extruded systems) and for CD4 immunogens are shown in Figure 8 (homogenized systems) and Figure 10 (extruded systems). Table 6 compares the competitive inhibition of a B4 monoclonal antibody assayed on sera obtained from the CD4 peptide study (homogenized and extruded w/o emulsion systems) on week 9, week 11 and week 17, respectively. Table 7 compares virus neutralization activity (50% and 90% inhibition respectively) assayed on sera obtained from the CD4 peptide study (homogenized and extruded w/o emulsion systems) on week 9 and week 11, respectively. Control experiments demonstrated that unadjuvanted peptide was non-immunogenic or weakly immunogenic in all cases. [00230] For both IgE and CD4 vaccines, the results of immunizations indicated that immunostimulatory complexes were adjuvanting and titers by week 9 were slightly less than or comparable to those obtained with w/o emulsions, irrespective of whether the emulsions were prepared by homogenized or extruded techniques as shown in Figures 7-10. [00231] The combination systems with inununostimulatory complex dispersed as w/o emulsions prepared by either method consistently provided the highest sustained immune responses. As depicted in Figures 7-10, the antibody titers elicited from week 11 through week 17, for both IgE and CD4 immunogens were found to be on the order of a log unit or more higher than antibody titers obtained with either the inununostimulatory complex alone or the w/o emulsions with peptide alone. The sole exception to this being the CD4 w/o emulsions prepared by homogenization, where this separation is not found until week 17. [00232] This observation is further supported by data obtained from the CD4 peptide competitive inhibition and virus neutralization studies highlighted in Tables 6 and 7, wherein the immune sera to the w/o emulsion combination systems with CD4 peptide/CpG oligonucleotides (charge ratio CD4:CpG2 = 2:1) competitively inhibited the highest level of B4 monoclonal antibody compared to the immune sera to the simple w/o emulsions or immunostimulatory complex with CD4 peptides alone. Moreover, the same formulations are shown to be the most effective at eliciting neutralizing activity against infectious virus. [00233] Small differences are noted between the homogenized or extruded preparations and include the rate with which the titers elicited to IgE or CD4 immunogens obtained by ELISA were observed to peak. The immune responses peaked earlier (wk 9) for the extruded w/o emulsions, although the duration of the responses obtained are good (essentially equivalent by week 11 and slightly reduced by week 17). The analogous homogenized system peaked a little later (week 11) and provided sustained responses as can be seen by the high titers persisting at week 17. [00234] This trend is also supported by data obtained from the CD4 peptide/mAb B4-HRP competitive inhibition assay and the" virus neutralization studies highlighted in Tables 6 and 7. [00235] By week 9, assays on sera obtained from animals immunized by the w/o emulsion system prepared from uncomplexed CD4 peptides via homogenization (87.2%) or by extrusion (88.6%) have been shown to competitively inhibit high levels of B4 monoclonal antibody. These were found to be the same order of magnitude (within experimental error) as for sera obtained from animals immunized with the w/o emulsion combination systems of CD4 peptide/CpG2 oligonucleotides (2:1) prepared via homogenization (70.1%) or by extrusion (94.9%). By week 17, assays on sera obtained from animals immunized by the w/o emulsion system prepared from uncomplexed CD4 peptides via homogenization (11.2%) or by extrusion (42.6%) competitively inhibited dramatically lower levels of B4 monoclonal antibody. The degree of inhibition were no longer generally comparable with that obtained for sera from animals immunized with the w/o emulsion combination systems of CD4 peptide/CpG2 oligonucleotides (2:1) prepared via homogenization (85.0%) or by extrusion (77.2%) processes. [00236] Moreover, the same trend is shown for sera obtained from formulations of C04 peptides when assayed for neutralization of infectious HIV-1 virus. [00237] By week 9, assays on sera obtained from animals immunized by the w/o emulsion system prepared from uncomplexed A CD4 peptides via homogenization (73% for 50% virus neutralization or 27% for 90% virus neutralization) or by extrusion (31% for 50% virus neutralization or 12% for 90% virus neutralization) neutralized essentially the same percentages of infectious HIV-1 virus (within experimental error). Assays on sera obtained from animals immunized with the w/o emulsion combination systems of CD4 peptide/CpG2 oligonucleotides (2:1) prepared via homogenization (12% for 50% virus neutralization or neutralization) or by extrusion (103% for 50% virus neutralization or 33% for 90% virus neutralization) has shown that the homogenized system is significantly less effective at generating neutralizing antibodies against infectious HIV-1 virus at this time point. By week 11, assays on sera obtained from animals immunized by the w/o emulsion system prepared from uncomplexed CD4 peptides via homogenization (57% for 50% virus neutralization or 17% for 90% virus neutralization) or by extrusion ( 90% virus neutralization) demonstrated that the capacity to neutralize infectious HIV-1 virus is reduced or eliminated (within experimental error). Assays on sera obtained from animals immunized with the w/o emulsion combination systems of CD4 peptide/CpG oligonucleotides (2:1) prepared via homogenization (40% for 50% virus neutralization or 30% for 90% virus neutralization) or by extrusion (120% for 50% virus neutralization or 39% for 90% virus neutralization) has clearly shown that both the homogenized and extruded systems were now effective at eliciting neutralizing antibody responses against infectious HIV. The response for the homogenized system was shown to be delayed relative to the extruded composition. [00238] One possible explanation for these trends may be that w/o emulsions prepared via low energy extrusion processes can interact or present complexed immunogens more efficiently at earlier time points to immune effector cells, whereas the high energy homogenization process, provides a more uniform emulsion and essentially acts as a more efficient depot. The immune responses obtained by such a system would be anticipated to be delayed and potentially more sustainable. [00239] The combination of the immunostimulatory complex and residual uncomplexed peptides dispersed in w/o emulsions were found to synergistically enhance the overall titers for both the IgE and CD4 immunogens. The method of preparation can influence the kinetics and duration of the responses, with both homogenization and extrusion shown to be compatible for preparing an efficacious vaccine. [00240] In addition, in a separate study we examined the effect of CpG dose on immune responses. For both IgE and CD4 immunogens, immunostimulatory complexes were prepared from varying doses of CpGl or CpG2 oligonucleotides (i.e. IgErCpGl = 4:1 or 1:1 and CD4:CpG2 = 2:1 or 1:2) and administered in either an aqueous buffer or dispersed in a w/o emulsion. All compositions were found to be immunogenic, however, in both studies the smaller relative dose of oligonucleotide {excess of synthetic peptide immunogen to oligonucleotide) elicited the superior immune responses in terms of absolute titer and of duration. In the case of IgE peptides/CpGl oligonucleotides the ranking follows the order 4:1 charge ratio > 1:1 neutral charge ratio whereas for CD4 peptides/CpG2 oligonucleotides the ranking follows the order 2:1 charge ratio > 1:2 charge ratio. Most often improved adjuvanticity can be correlated with increasing doses of adjuvants. In these Examples, we have demonstrated the unexpected advantages of co-formulating stabilized immunostimulatory complex and uncomplexed peptide for maximum synergy of immune responses. [00241] Thus, it can be concluded that immunostimulatory complex prepared from IgE or CD4 immunogens as described in this Example can effectively adjuvant immune responses in vivo. The responses obtained are comparable to those found for these same immunogens dispersed in w/o emulsions, however, it is anticipated that fewer reactogenicity issues would result from use of the fully aqueous dispersable immunostimulatory complex of synthetic immunogens as opposed to dispersing synthetic immunogens as w/o emulsions. [00242] In this Example we have demonstrated that the delivery of IgE or CD4 immunogens as immunostimulatory complexes in combination with uncomplexed peptides dispersed within a water-in-oil emulsion vehicle provided the most effective presentation of immunogen to the immune system. The immune responses obtained for the combined system are significantly greater than the sum of the immune responses obtained for each system independently. [00243] Furthermore, assays of sera obtained from animals immunized with combinations of immunostimulatory complex of CD4 immunogens and uncomplexed CD4 immunogens dispersed within a water-in-oil emulsion vehicle were shown to be more effective than simple w/o emulsions of uncomplexed CD4 immunogens at competitively inhibiting B4 monoclonal antibody. Moreover these same systems were shown to be the most effective at neutralizing infectious HIV-1 virus. [00244] In the guinea pig model, the quantity and longevity of the immune responses obtained indicated that immunostimulatory complex derived from IgE/CpGl and CD4/CpG2 combinations were preferred. It was experimentally determined that compositions derived from the alternative pairings of IgE/CpG2 and C04/CpGl were adjuvanting, although not to the same extent. [00245] The possibility for fewer and lower (immunoqen/adjuvant) dose regimens is indicated by these results. In cases where reactogenicity is a problem the combination system opens up the possibility of administering a smaller volume of emulsion with similar or higher levels of immunogenicity. [00246] Specifically, this Example strongly indicates the potential of these new combination based formulations for development as efficacious dosage forms. Example 8; The Immunogenicity of IgE and CD4 Pep tide Inununogens Formulated as Immunos timula tory complex or as Insitu Gelling Polymer Solutions or in Combinations [00247] This Example illustrates the immunogenicity of IgE and CD4 peptide immunogens formulated as immunostimulatory complex with CpGl or CpG2 oligonucleotides or as in-situ gelling polymers and biocompatible solvents or as immunostimulatory complex suspended in in-situ gelling polymers and biocompatible solvents in guinea pigs, which were immunized intramuscularly. Lyophilized peptide immunogens or immunostimulatory complex derived from peptide immunogens and CpG oligonucleotides were prepared as described in Example 3. The in-situ gelling polymers were prepared as described in Example 6a/6b. [00248] To examine the immunogenicity of IgE and CD4 peptide immunogens formulated as in-situ gelling polymers (Resomer RG 504H) or as immunostimulatory complexes with CpGl or CpG2 oligonucleotides suspended in in-situ gelling polymers (Resomer® RG 504H) formed in accordance with the present invention, groups of three, 6 to 8 week old female guinea pigs (Covance Research Products Inc, Denver, PA) were immunized intramuscularly (I.M.) with the following amounts of immunogen on week 0: 300 ng of lyophilized IgE peptides/CpGl immunostimulatory complex (4:1 charge ratio) reconstituted and suspended in a final volume of 200 ^L PBS (pH 7.4), prepared as described in Table 4, or 300 ug of CD4 peptides/CpG2 immunostimulatory complex (2:1 charge ratio) reconstituted and suspended in a final volume of 200 uX PBS (pH 7.4), prepared as described in Table 4, or 300 ng of IgE peptides reconstituted and suspended in 200 uX of Resomer® RG 504H (20% by wt) dissolved in dimethyl sulfoxide (DMSO) or 300 \ig of CD4 peptides reconstituted and suspended in 200 uX of Resomer® RG 504H (20% by wt) dissolved in dimethyl sulfoxide (DMSO) or 300 Hg of lyophilized IgE peptides/CpGl immunostimulatory complex (4:1 charge ratio), prepared as described in Table 4, reconstituted and suspended in 200 jiL of Resomer® RG 504H (20% by wt) dissolved in dimethyl sulfoxide (DMSO) or 300 jig of lyophilized CD4 peptides/CpG2 immunostimulatory complex (2:1 charge ratio), prepared as described in Table 4, reconstituted and suspended in 200 jiL of Resomer® RG 504H (20% by wt) dissolved in dimethyl sulfoxide (DMSO). [00249] The guinea pigs showed no gross pathologies or behavioral changes after receiving immunostimulatory complex, in-situ gelling polymers in DMSO containing peptide immunogens or in-situ gelling polymers in DMSO containing immunostimulatory complex. Sera were obtained on weeks +3, + 6, +9, +12 and were evaluated for the presence of anti-IgE antibodies in the case of IgE immunogens or anti-CD4 antibodies in the case of CD4 immunogens, by immunogenspecific ELISAs following the procedures described in Example 7. Immunogenicity Results [00250] The serum antibody titers following single-dose immunization of IgE immunogens are shown in Figure 11 and for CD4 immunogens are shown in Figure 12. Control experiments demonstrated that unadjuvanted peptide was non-immunogenic o-r weakly immunogenic in either case. In both studies, the results of immunizations indicated that immunostimulatory complex alone were moderately adjuvanting with the titers peaking by week 9. Conversely, uncomplexed immunogens suspended in in-situ gelling polymers were also weakly adjuvanted with peak responses observed at week 12. [00251] For both IgE and CD4 immunogens, the derived immunostimulatory complexes suspended as an in-situ gelling polymer elicited the highest immune responses. These responses were seen to peak around week 9 and were sustainable through week 12. The quantity and duration of the immune responses obtained were not found with either the immunostimulatory complex alone or with uncomplexed immunogens administered in a composition including in-situ gelling polymer alone. [00252] It is expected that small molecular weight immunogens such as peptides can easily diffuse through polymer implants and gels resulting in large guantities of burst release upon injection. The physical factors that control gellation can be adjusted to retard this process; however, the mass of peptide so released is essentially unadjuvanted and subject to the standard degradation processes that are normally experienced in vivo. In addition, the small amounts of material remaining encapsulated may not be expected to be sufficient for an efficient boost once the polymer degrades, necessitating much larger doses of peptide. The residual DMSO trapped within the matrix also presents stability issues, wherein sensitive amino acids contained in the peptides could be oxidized. Furthermore it has been well established that water can penetrate these materials at varying rates depending on various factors such as gel micromorphology, polymer hydrophobicity and crystallinity45' 46. Water penetrating the matrix will promote bulk hydrolysis, the prime degradation • mechanism operating on PLG/PLGA copolymers in vivo. This process is known to be accompanied by dramatic local pH changes, which can essentially reduce the pH to 2 or 367. The free uncomplexed solubilized peptides may not be stable to such an environment, and this further limits the potential for these systems. Acid buffering agents may be employed to help to offset these problems, but cannot be considered ideal. Encapsulating a suspension of peptide immunogens in the form of an immunostimulatory complex imparts a number of stability and adjuvantation advantages for this system. Once the injection of polymer gel is made, the small amounts of complex not effectively encapsulated in the gel (presumably surface located near the gelling front) can serve to initiate or prime the immune response more effectively than uncomplexed peptide immunogen alone. The CpG oligonucleotide remains in close contact with peptide immunogen and in the form of a complex particulate may further protect and stabilize the peptide immunogen from enzymatic digestion in vivo or from chemical instabilities which may be due to the DMSO solvent contained within the matrix. [00253] Furthermore, the peptide immunogens remaining entrapped in the matrix in a particulate form would be expected to be better protected against the acidification process than free uncomplexed peptides. Immunogens presented in this form can be expected to provide a more efficient boost of immunogen to the immune system eliciting stronger and longer lasting immune responses than otherwise possible in a single—dose controlled release formulation. [00254] In control experiments, it was determined that solutions of uncomplexed peptide immunogens dissolved in polymer compositions of RG 504H (20% by wt) in DMSO gelled rapidly when placed in contact with solutions of PBS. Separating the solution from gel phase for these samples and analyzing the solutions by ultraviolet spectroscopy (at X = 280 nm) revealed that sizeable amounts of uncomplexed peptide (c.a. 50-70%) were co-extracted with the DMSO. [00255] The combination of the immunostimulatory complex and uncomplexed peptide immunogens suspended in in-situ gelling polymers have been found to synergistically enhance the overall titers for both the IgE and CD4 immunogens in these controlled release preparations. [00256] A separate study examining the effect of CpG oligonucleotide dose on immune responses was not conducted in this study. It would be expected that further improvement in the absolute titers may be obtained by employing immunostimulatory complex prepared near electrical neutrality or with an excess of negative charge supplied by either CpG oligonucleotide or alternatively an additional compatible excipient. In these compositions the majority of the peptide immunogen is bound as an immunostimulatory complex and the gellation process upon injection does not result in major loses of unadjuvanted peptide by virtue of co-extraction in the biocompatible solvent. [00257] Thus, it can be concluded that immunostimulatory complex can both stabilize peptide immunogens as immunostimulatory complex and that these compositions when combined with an in-situ gelling polymer can effectively adjuvant the immune responses in vivo. This is particularly important for these polymer systems which are intended for single-dose use. The delivery of immunogens as immunostimulatory complex suspended within an in-situ gelling polymer vehicle provides the most efficient presentation of immunogen to the immune system. The responses obtained for the combined system are significantly greater than the sum of the immune responses obtained for each system independently and are sustainable, unlike in-situ gels prepared by simplereconstitution of uncomplexed peptide immunogens from polymers in biocompatible solvents. [00258] In the guinea pig model, the quantity and longevity of the responses obtained indicated that immunostimulatory complexes derived from IgE/CpGl and CD4/CpG2 combinations were preferred. It was experimentally determined that compositions derived from the alternative pairings of IgE/CpG2 and CD4/CpGl were adjuvanting, although not to the same extent. [00259] The possibility of a single-dose regimen is indicated by these results. Specifically, this Example strongly indicates the potential of these new instantly reconstituted combination-based formulations for development as efficacious controlled release dosage form. Example 9; Preparation of Combination Immunostimulatory Complex and Mineral Salt Suspensions [00260] This Example illustrates the process of preparing a mineral salt suspension from cationic peptides derived from LHRH peptide immunogens (SEQ ID NOS: 7-9 in a 1:1:1 molar ratio in solution) or an immunostimulatory complex derived from LHRH immunogens, and CpGl oligonucleotide in various proportions. A flow diagram illustrating the process of preparing a mixed suspension as described herein is shown in Figure 16. [00261] All glassware, stir bars and pipette tips were autoclaved for 40 minutes at 121°C prior to use. All reagents were weighed, dispensed, transferred or added to reaction vessels in a laminar flow hood to prevent contamination. [0001] To a 5.0 mL glass vial equipped with a stir bar, was added 100 fig (250 uL, 0.4 mg/mL) or 1,600 pig (534 \iL, 3.0 mg/mL) of LHRH peptide immunogens dissolved in an aqueous solution. Immunostimulatory complexes with either of two charge ratios (i.e. 4:1 or 1.5:1) were prepared from LHRH immunogens and CpGl oligonucleotides in distilled deionized water. [00263] Specifically, the preparation of a 4:1 complex from 100 jig or 1,600 ug of LHRH immunogens required 7.3 \iq or 116.8 fig of CpGl oligonucleotide (2.0 mg/mL}, whereas the preparation of a 1.5:1 complex from 1,600 fig of LHRH immunogens required 350.4 fig of CpGl oligonucleotide (2.0 mg/mL), respectively. s [00264] Table 9 shows the calculations employed to determine the relative amount of CpGl oligonucleotide required for complexation with LHRH peptide immunogens for a fixed final dosage of 25 ng/0.5 mL or 400 fig/0.5 mL with respect to the specified charge ratios. / [00265] To a second 5.0 mL glass vial, was added 1.0 mL of Alhydrogel (3.2 mg Aluminum (Al)/mL) mineral salt suspension in distilled deionized water. The Alhydrogel® stock suspension employed was first pH adjusted to pH 7.1-7.4 by the addition of 0.1 N NaOH. The pH measurements were made through use of pH indicator strips with a resolution +/- 0.3 pH units. [00266] The mineral salt suspension was added to the vial containing immunostimulatory complex and residual unbound immunogens and equilibrated with stirring over 30 minutes. [0001] To the mixture of Alhydrogel® and immunostimulatory complex was added. 90 \iL of 20% NaCl for tonicity, 5.0 jiL of 2- phenoxy-ethanol (2-PE) preservative (in select cases) and additional distilled deionized water to ensure the final volume of the formulation equaled 2.0 mL. [00268] The final concentration of immunogens once formulated as a suspension of immunostimulatory complex combined with mineral salt as described above was 25 \|ig/0.5 mL or 400 fig/0.5 inL respectively. The final concentration of Alhydrogel® prepared was dependent on target species. Formulations intended for the rodents were prepared at 0.4 mg Al/0.5 mL ' whereas formulations intended for non-human primates were prepared at 0.8 mg Al/0.5 mL. In all cases the finished formulations were adjusted for tonicity (0.9% NaCl) and those employed for the non-human primates contained a preservative (0.25% v/v, 2-PE). Example 10: The Immunogenicity of LHRH Pep tide Immunogens Formulated as a Suspension of Immunostimulatory Complex with v Mineral Salt in Rodents [00269] This Example illustrates the immunogenicity of LHRH peptide immunogens formulated as immunostimulatory complexes with CpGl oligonucleotides in combination with mineral salts, which were immunized intramuscularly, in male rats. The mineral salt suspensions were prepared as described in Example 9. [0001] Groups of four, 6 to 8 week old Sprague-Dawley male rats were immunized intramuscularly (I.M.) on week 0 4 and 8 with following compositions: 25 [iq of LHRH peptides was suspended in a volume of 500 uL distilled deionized water; 25 ug of LHRH peptides was suspended in a volume of 250 uL distilled deionized water and 250 \iL of Alhydrogel® {1.6 mg Al/mL) was added; 25 p.g of LHRH peptides/CpGl immunostimulatory complex t4:l ch'arge ratio) prepared as described in Table 9 was suspended in a volume of 500 (J.L distilled deionized water; 25 fig of LHRH peptides/CpGl immunostimulatory complex (4:1 charge ratio) prepared as described in Table 9 was suspended in a volume of 250 fiL distilled deionized water and 250 ^L of Alhydrogel® (1.6 mg Al/mL) was added. To each formulation was added 45 fiL of a 20% saline solution and 455.0 uX of distilled deionized water. The final volume of each formulation was 1.0 mL. [00271] The rats showed no gross pathologies or behavioral changes after receiving mineral salts containing peptide immunogens or mineral salts containing the immunostimulatory complex. Sera were obtained on weeks +0, +4, +6, +8 and +12 and were evaluated for the presence of anti-LHRH antibodies by immunogen-specific ELISA and for serum testosterone by RIA immunoassay. Measurement of anti-LHRH antibodies [00272] Antibody activities were determined by ELISA using 96 well microtiter plates coated with the LHRH peptide54 as immunosorbent. [00273] Aliquots (100 uL) of the peptide immunogen solution at a concentration of 5 ug/mL were incubated for 1 hour at 37°C. Wells were subsequently blocked with a 3% gelatin/PBS solution for 1 hour at 37°C. The plates were then dried and used for the assay. Aliquots (100 uL) of the test immune sera, starting with a 1:100 dilution in a sample dilution buffer and 10-fold serial dilutions thereafter, were added to the peptide coated plates. The plates were incubated for 1-1.5 hours at 37°C. The plates were washed six times with 0.05% TWEEN 20 in PBS. 100 uL of horseradish peroxidase-labeled goat anti-rat IgG (Cappel) for assays performed on rat sera, 100 pL of goat anti-swine horseradish peroxidase-labeled IgG (Pierce) for assays performed on swine sera or 100 uL of horseradish peroxidase-labeled goat anti-human IgG (Anogen) for assays performed on baboon sera was added at the appropriate dilutions in conjugate dilution buffer (PBS containing 0.5M NaCl and normal goat serum). The plates were incubated for 1 hour at 37°C and washed as described above. Aliquots (100 uL) of orthophenylenediamine (OPO) substrate at 0.04% by weight (Pierce) and 0.12% H202 in sodium citrate buffer, pH 5.0, was added for 15 minutes. Reactions were stopped by addition of 50 pL 2N H2S04 and the A492 determined for each well. [00274] ELISA titers were calculated based on linear regression analysis of the adsorbances, with cutoff A4g2, set at 0.5. This cutoff was rigorous, as the values for diluted normal control samples run with each assay were less than 0.15. Measurement of Serum Testosterone [00275] Immunogens were evaluated for efficacy by RIA for serum testosterone values. Serum testosterone levels were measured using a RIA kit from Diagnostic Products (Los Angeles, Calif.) according to manufacturer's instructions. The lower detection limit for testosterone ranged from 0.01 to 0.03 nMol/L. Each sample was analyzed in duplicate. [00276] Serum samples were scored as being at castration level when the testosterone level was below limits of detection and as "near castration" at Immunogenicity Results [00277] The serum antibody titers following immunization of LHRH immunogens are shown in Figure 13a. The corresponding serum testosterone levels are shown in Figure 13b. [00278] The antibody titers determined from sera obtained from male rats in this study proved that neither the composition of LHRH peptides in buffer nor LHRH peptides in combination with the Alhydrogel® mineral salts were immunogenic. Conversely, both compositions derived from the LHRH/CpGl immunostimulatory complex alone or in combination with the Alhydrogel® mineral salt were shown to be immunogenic as depicted in Figure 13a. [00279] The antibody responses of these later two groups proved similar over the first 8 weeks. By week 12, the antibody titers found for the combined system of LHRH immunostimulatory complex and Alhydrogel® mineral salt were only marginally better (i.e. ~ 0.5 log units) than those determined for the LHRH immunostimulatory complex alone. [00280] The corresponding serum testosterone levels determined for the compositions of LHRH peptides in buffer and LHRH peptides in combination with the Alhydrogel® mineral salts similarly demonstrated that neither the LHRH peptide nor LHRH peptide with mineral salt formulations were capable of achieving immunocastration in male rats. [00281] Conversely, both the immunostimulatory complex derived from LHRH peptides/CpGl alone and the combination of LHRH peptides/OpGl oligonucleotide with Alhydrogel® mineral salt effectively imnmnocastrated all rats in each group as shown in Figure 13b. [00282] Complete immunocastration for each rat in the composition derived from the LHRH immunostimulatory complex combined with the Alhydrogel® mineral salt was achieved by week 6 (2 weeks after the first boost). A similar level of immunocastration for the composition derived from the LHRH immunostimulatory complex alone was not achieved until week 10 (2 weeks after the second boost). [00283] In this Example we have demonstrated a novel vaccination regimen to obtain effective immunocastration (as measured by serum testosterone) with either a composition prepared from LHRH immunostimulatory complex (in a 3-dose immunization strategy) or a LHRH immunostimulatory complex in combination with a mineral salt (in a two-dose immunization strategy). [00284] The Alhydrogel® mineral salt administered alone with LHRH peptide immunogens proved to be an ineffective adjuvant. However, once formulated with the LHRH immunogens in the form of an immunostimulatory complex the combination yielded synergistically far superior results. The mineral salt itself may be acting in two modes. The first may be to provide a ' depot localizing the immunostimulatory complex at the site of injection and secondly by recruiting specialized cells of the immune system which can facilitate the presentation of immunogen to the immune system. [00285] Concurrent experiments were conducted with alternative mineral salt, Adju-phos® (derived from Aluminum phosphate) and full immunocastration in all rats was achieved by week 8 solely for the system derived from the LHRH immunostimulatory complex in combination with the Adju-phos® mineral salt. Example 11: The Inmmnogenicity of LHRH Peptide Inununogens Formulated as a Suspension of Inmunostimulatory Complex with Mineral Salt in Baboons [00286] This Example illustrates the immunogenicity of LHRH peptide immunogens formulated as immunostimulatory complexes with CpGl oligonucleotides in various ratios in combination with mineral salts, which were immunized intramuscularly, in male baboons. The mineral salt suspensions were prepared as described in Example 9. [0001] Groups of two, 2 year old male baboons were immunized intramuscularly (I.M.) on weeks 0, 4 and 8 with the following compositions: 400 fig of LHRH peptides/CpGl immunostimulatory complex (4:1 or 1.5:1 charge ratio) prepaced as described in Table 9 suspended in a volume of 500 fiL distilled deionized water; 400 jxg of LHRH peptides/CpGl immunostimulatory complex (4:1 or 1.5:1 charge ratio) prepared as described in Table 9 suspended in a volume of 250 fiL distilled deionized water and 250 fiL of Alhydrogel® (3.2 mg Al/mL) was added. To each formulation was added 45 fiL of a 20% saline solution, 2.5 uL of 2-phenoxy-ethanol and 452.5 nL of distilled deionized water. The final volume of each formulation was 1.0 mL. [00288] The baboons showed no gross pathologies or behavioral changes after receiving immunostimulatory complex at either ratio or in combination with the Alhydrogel® mineral salt. Sera were obtained on weeks +0, +4, +6, +8, +12 and +14 (for the 1.5:1 complexes only) and were evaluated for the presence of anti-LHRH antibodies by immunogen-specific ELISA and for serum testosterone by RIA immunoassay. Immunogenicity Results [00289] The serum antibody titers following immunization of LHRH immunogens are shown in Figure 14a. The corresponding serum testosterone levels for each baboon are shown in Figure 14b and 14c, respectively. [00290] The antibody titers determined from sera obtained from male baboons immunized by LHRH immunostimulatory complexes prepared in either a 4:1 or 1:5:1 charge ratio or a combination of LHRH peptide immunostimulatory complexes and Alhydrogel® mineral salt prepared in either a 4:1 or 1:5:1 charge ratio indicated that all compositions were immunogenic as shown in Figure 14a. [00291] Marginally higher antibody titers were found for both systems prepared at the 4:1 charge ratio relative to the antibody titers obtained for the 1.5:1 system irrespective of whether the Alhydrogel® mineral salt was present or not. [00292] The corresponding serum testosterone levels determined from sera obtained from male baboons immunized with LHRH immunostimulatory complexes prepared at the 4:1 charge ratio without Alhydrogel® mineral salt proved totally ineffective for both baboons on trial as shown in Figure 14b. [00293] The system formulated from the LHRH immunostimulatory complexes prepared at the 1.5:1 charge ratio without Alhydrogel® mineral salt was shown to more effectively downregulate the serum testosterone response for one of the baboons in the trial, however full immunocastration was not achieved as shown in Figure 14c. [00294] For the composition prepared from a LHRH peptide immunostimulatory complex (4:1 charge ratio) in combination with Alhydrogel® full immunocastration was obtained in one baboon by week 10 and was demonstrated to be sustainable through week 14 as shown in Figure 14b. Near castration levels of serum testosterone were obtained in the other baboon in this group, although this response proved transient as shown in Figure 14b. [00295] For the composition prepared from a LHRH immunostimulatory complex (1.5:1 charge ratio) in combination with Alhydrogel* both baboons in the study were successfully immunocastrated (one baboon achieved this by week 8 and the other by week 10) and this effect proved sustainable through week 14 as shown in Figure 14c. [0001] For formulations derived from LHRH immunostimulatory complexes prepared at either a 4:1 or 1.5:1 charge ratio (LHRH peptide concentration =400 ng), the actual quantity of LHRH peptide immunogens bound in the form of an immunostimulatory complex varies significantly. Table 8 shows the proportion of LHRH immunogen administered in this form as a function of varying charge ratios. Approximately 16% (~ €3 f.ig) of the LHRH immunogens in solution are bound in the form of an immunostimulatory complex when prepared at a 4:1 charge ratio, whereas approximately 86% (~ 344 jag) are bound when prepared at the 1.5:1 ratio. [00297J The presentation of the majority of the LHRH iiranunogens in the form of an immunostimulatory complex as in the 1.5:1 ratio system provided longer lasting and earlier immunocastration responses in baboons. We have experimentally determined that the Alhydrogel® mineral salt adsorbs an additional 10% of the free unbound LHRH immunogens in solution upon addition. Thus it is likely that the mechanism by which the mineral salt is improving the efficacy of the vaccine when combined with the immunostimulatory complex is probably linked to indirect immunomodulatory effects. [00298] Concurrent experiments were conducted with an alternative mineral salt, Adju-phos® (derived from Aluminum phosphate) and full immunocastration was demonstrated in one baboon by week 10 whereas the other baboon on trial demonstrated near castration levels of serum testosterone by this time point. [00299] In this Example we have demonstrated that a LHRH immunostimulatory complex in combination with a mineral salt is an effective vaccine to achieve immunocastration (as measured by serum testosterone) in a non-human primate. [00300] Furthermore we have shown that the formulation kinetics of the immunocastration response is a function- of the initial charge ratio of LHRH immunogens to CpGl oligonucleotide and selection of mineral salt. [00301] Specifically, this Example indicates the potential of these new combination based formulations for the development of safe vaccines useful for hormone ablationin animals or humans. [00302] Moreover, this Example strongly indicates the potential of these new combination based formulations for the development of safe vaccines suitable for the treatment of androgen-sensitive prostate cancer in humans. Example 12: Preparation of Water-in-Oil Emulsions from ISA Montanide® 50v, Immunostimulatory Complex Derived from LHRH in the Presence of a IL-lfi Derived Peptide Fragment [0001] To a 10 mL vessel, was added 1,000 jxg of LHRH peptide immunogens (SEQ ID NOS: 7-9 in a 1:1:1 molar ratio in solution), dissolved in an appropriate aqueous buffer (2,500 uL, 0.4 mg/mL) and CpGl oligonucleotide to prepare an immunostimulatory complex (16:1 charge ratio) or 1,000 ug of peptide immunogens dissolved in an appropriate aqueous buffer (2,500 fiL, 0.4 mg/mL) plus a peptide derived from IL-16 (SEQ ID NO: 14, CVQGEESNDKIPC-C02H.HC1 (where C* indicates cyclization between two cysteines) in solution; or 1,000 jig of peptide immunogens dissolved in an appropriate aqueous buffer (2,500 nL, 0.4 mg/mL) was added to a mixture of CpGl and IL-1B peptide to prepare a combination of LHRH immunostimulatory complex (16:1 charge ratio) and IL-lfi peptide. Table 9 shows the calculations employed to determine the relative amount of CpGl oligonucleotide required for complexation with LHRH peptide immunogens for a fixed final dosage of 100 fig/1.0 mL with respect to the specified charge ratio. [00304] Peptide-based fragments derived from IL-lfi are known to possess adjuvanting properties.69 The IL-lfi peptide employed herein is relatively small (FW = 1421.5) and negatively charged when dissolved in standard physiological buffers. Employing the calculations described in Example 2, one nMol of IL-lIi peptide contributes 2 nMols of negative charge. As such, this molecule would not be predicted to physically interact with the LHRH immunogens and no evidence of complexation in the form of a precipitate was found upon mixing. [00305] Specifically, to prepare an immunostimulatory complex from LHRH peptide immunogens at a 16:1 charge ratio of LHRH:CpGl, 18.3 ^g CpGl oligonucleotide (9.2 ^L, 2.0 ug/mL) were used. To prepare a solution of LHRH peptide immunogens and IL-lfJ, 10 ng IL-lfi(5.0 uL, 2.0 (ig/mL) were added in an appropriate aqueous buffer. To prepare an immunostimulatory complex from LHRH peptide immunogens at a 16:1 charge ratio of LHRH:CpGl plus IL-1B, 18.3 jag CpGl oligonucleotide (9.2 (aL, 2.0 ng/mL) and 10 fig IL-115 peptide (5.0 \|iL, 2.0 ng/mL) were mixed. [00306] To each of the vessels additional aqueous solvent diluent was added so that the final volume of the aqueous . phase was fixed at 5.0 mL for preparation of ISA Montanide 50v w/o-emulsions respectively. [00307] For the preparation of the placebo group, 5.0 mL of normal saline was employed for the aqueous phase. [00308] For the LHRH peptide immunogens, normal saline was found to be suitable for cbmplexation. The calculated IP for each peptide is greater than 9.0 (Table 1), far greater than the pH of the aqueous solvent selected. [00309] This example demonstrates the advantages of stabilizing immunogens in solution in the form of an immunostimulatory complex. The complexation process has been shown to be compatible in the presence of additional immunomodulators. [00310] The diluted aqueous suspensions of LHRH immunogens or placebo solution were then slowly added to a dry 25 mL reaction vessel charged with ISA Montanide® 50v (5.0 mL). The additions were made while homogenizing (High Shear Laboratory Mixer, Sealed Unit, Silverson) the mixture at low speeds (2,000^3,000 rpm) to generate a coarse emulsion. This processing speed was maintained until the aqueous sample had been completely added and was -continued a full 2 minutes to ensure uniform pre-mixing of the aqueous and oil phases. The homogenization speed was then ramped up (5,000-8,000 rpm) and maintained for from 5 to 10 minutes further resulting in the formation of a homogeneous white finely dispersed w/oemulsion. [00311] The final concentration of immunogens once formulated ' as water-in-oil emulsions as described above was 100 jj.g/mL. Example 13: The Immunogenicity and Growth Promoting Effects of LHRH Peptide Immunogens Formulated as an Immunostimulatory Complex or with IL-lfi Peptide or as a Combination of an Immunostiiaulatory Complex with IL-lfi Peptide in a W/O Emulsion [00312] This Example illustrates the immunogenicity of LHRH peptide immunogens formulated; as immunostimulatory complexes with CpGl oligonucleotides in combination with a w/o emulsion or with an immunomodulator (IL-lIi peptide) in combination with a w/o emulsions or as immunostimulatory complexes combined with an additional immunomodulator (IL-lli peptide) in a w/oemulsion, which were immunized intramuscularly, in boars. The w/o-emulsions were prepared by homogenization as described in Example 4a and Example 12. [0001] Groups of five, 8 week old boars (30 Kg each) were immunized intramuscularly (I.M.) on week 0 and 8 with following compositions: 100 ng of LHRH peptides/CpGl immunostimulatory complex (16:1 charge ratio) prepared as described in Table 9 suspended in a final volume of 500 p.L Nad and dispersed with ISA Montanide® 50v (500 ^L) ; LHRH peptides/IL-lft peptide (1 ng) was suspended in a final volume of 500 \iL N'aCl and dispersed with ISA Montanide® 50v (500 uL) ; or LHRH peptides/(CpGl + IL-lft peptide (1 jig) ) immunostimulatory complex (16:1 charge ratio) prepared as described in Table 9 was suspended in a final volume of 500 jiL NaCl dispersed with ISA Montanide® 50v (500 uX) . A placebo control group of 500 uL NaCl dispersed with ISA Montanide® 50v (500 uL) was prepared and one unimmunized control group included in the study was surgically castrated. [00314] The boars showed no gross pathologies or behavioral changes after receiving placebo w/o emulsions, w/o emulsions containing peptide immunogens in combination with IL-115 peptide or w/o emulsions containing the immunostimulatory complex or immunostimulatory complex in combinations with IL- 1/i peptide. Sera were obtained on weeks +0, +8, +10, +12 and +14 and were evaluated for the presence of anti-LHRH antibodies by immunogen-specific ELISAs, for serum testosterone by RIA immunoassay and for weight gain. Immunogenicity Results [00315] The serum antibody titers following immunization of LHRH immunogens are shown in Figure 15a. The corresponding serum testosterone levels are shown in Figure 15b. The effects of inununocastration following immunization of LHRH immunogens versus surgical castration on the weight gain over the trial period are shown in Figure 15c. Control experiments demonstrated that unadjuvanted peptide was non-immunogenic or weakly immunogenic in all cases. [00316] The antibody titers determined from sera obtained from male boars immunized by LHRH immunostimulatory complexes, LHRH peptides adjuvanted by a cytokine, or by an LHRH immunostimulatory complex co-formulated with a cytokine administered as w/o emulsions indicated all three compositions •were immunogenic. The titers obtained at all timepoints over the trial period proved comparable. The Placebo and surgically castrated negative control groups returned no titer as shown in Figure 15a. [00317] The corresponding serum testosterone levels determined from sera obtained from boars immunized by LHRH immunostimulatory complex in a w/o emulsion or LHRH peptides adjuvanted by the IL-1B peptide in a w/o emulsion or by a combination of LHRH immunostimulatory complex and the IL-lfipeptide administered as w/o emulsion indicated that male boars in these groups were effectively immune-castrated by week 12. [00318] Notably, the compositions derived from the LHRH immunostimulatory complex in a w/o emulsion and the LHRH peptides adjuvanted by the IL-lft peptide in a w/o emulsion failed to maintain this level of serum testosterone. By week 14, serum testosterone levels from both groups were shown to be rebounding and neither group would be described as immunocastrated. [00319] The LHRH vaccine formulated as a combination of immunostimulatory complex and IL-lfi peptide as a w/o emulsion proved more effective maintaining the same level of immunocastration with no indication of early rebound as shown in Figure 15b. [00320] The Placebo negative control group returned widely fluctuating serum testosterone values at the various timepoints evaluated in agreement with the expected variations in monthly hormone levels. The surgically castrated positive control groups returned negative serum testosterone levels at all timepoints over the trial period as shown in Figure 15b. [00321] The serum testosterone level in boars prior to slaughter has been directly correlated with the quality of the meat produced. High levels of testosterone give rise to products of unappealing taste (boar taint) and thus controlling this property is an important market consideration. The standard method for ensuring negligible testosterone has been surgical castration. A viable vaccine approach must be proven effective over the interval of time when boars would normally be brought to market. The point at which the boars have reached a weight between 110-130 KGs. [00322] The growth promotion of boars immunized at 8 weeks of age in this study was tracked over 14 weeks. The boars immunocastrated by the LHRH immunostimulatory complex in a w/o, emulsion or by LHRH peptides adjuvanted solely by the IL-1I5 peptide in a w/o emulsion posted weight gains, that paralleled the surgically castrated group. These reached on average 110 KGs by week 14 as shown in Figure 15c. [00323] Unexpectedly, the group immunized by the LHRH vaccine formulated as a combination of the LHRH immunostimulatory complex and IL-l(i peptide in a w/o emulsion proved considerably more effective. All boars within this group responded rapidly reporting the highest average weight gains at all timepoints evaluated in comparison to the other groups on trial. Boars from this group attained an average weight slightly below 120 KGs by week 12 and reached 130 KGs by week 14 as shown in Figure 15c. [00324] The disparity between this group and the others on trial may be due to a synergistic combination of effects obtained when the immunostimulatory complex of LHRH/CpGl is presented to the immune system in combination with the IL-lft peptide and free uncomplexed LHRH peptide in a w/o emulsion. [00325] The combination formulation proved effective with very small amounts of LHRH/CpGl immunostimulatory complex present in suspension. In this Example a LHRH:CpGl charge ratio of 16:1 provided significant responses in all swine. [0032-5] The Placebo control group proved poorest at promoting growth over the trial period. The average weight gain for this group was significantly reduced relative to all others as shown in Figure 15c. [00327] Marginal improvements in weight gain as a consequence of immunocastration relative to placebo controls have been observed in the past70, however in this Example we have demonstrated a novel method to obtain effective immunocastration for the removal of boar taint (as measured-by serum testosterone) and obtained superior growth promotion (as measured by weight gain) over the trial period with respect to the standard method of surgical castration. [00328] Furthermore, boars immunocastrated by the LHRH vaccines formulated as a combination of an immunostimulatory complex and IL-lIi peptide in a w/o emulsion could go to market 2 weeks earlier than male boars immunized by either alternative formulation, or the present method of preference, surgical castration. [00329] Specifically, the monetary savings in terms of avoiding losses due to surgical trauma, reduced feed and housing costs and improved turnover in terms of time to market would be significant, demonstrating the advantages of this improved method of immunocastration and growth promotion. Example 14: Preparation of Water-in-Oil Emulsions from ISA Montanide® 50v and Immunostimulatory Complexes Derived from FMD [00330] To a 20 mL vessel, was added 4,000 \iq of FMD peptide immunogens (SEQ ID NOS: 12-13 in solution), dissolved in an appropriate aqueous buffer (2,000 uL, 2.0 mg/mL) or was added 4,000 \iq of peptide immunogens dissolved in an appropriate aqueous buffer (2,000 nL, 2.0 mg/mL) and CpGl oligonucleotide to prepare an immunostimulatory complex (A:1 charge ratio). f Table 9 shows the calculations employed to determine the relative amount of CpGl oligonucleotide required for complexation with the FMD peptide immunogens for a fixed final dosage of 200 ug/1.0 mL with respect to the specified charge ratio. [00331] Specifically, to prepare an irrununostimulatory complex from FMD peptide immunogens at a 4:1 charge ratio of FMD:CpGl, 125 ug CpGl oligonucleotide (62.5 \iL, 2.0 fig/mL) were used. [00332] To each of the vessels additional aqueous solvent diluent was added so that the final volume of the aqueous phase was fixed at 10.0 mL for preparation of ISA Montanide® 50v w/o-emulsions respectively. [00333] For the preparation of the placebo group, 10.0 mL of normal saline was employed for the aqueous phase. [00334] For the library of FMD peptide immunogens normal saline was found to be suitable for complexation. The average calculated IP for each peptide derived from a positional analog in the library is greater than 9.0 (Table 1), far greater than the pH of the aqueous solvent selected. [00335] The diluted aqueous suspensions of FMD immunogens or placebo solution were then slowly added to a dry 25 mL reaction vessel charged with ISA Montanide® 50v (10.0 mL). The additions were made while homogenizing (High Shear Laboratory Mixer, Sealed Unit, Silverson) the mixture at low speeds (2,000-3,000 rpm) to generate a coarse emulsion. This processing speed was maintained until the aqueous sample had been completely added and was continued a full 2 minutes to ensure uniform pre-mixing of the aqueous and oil phases. The homogenization speed was then ramped up (5,000-8,000 rpm) and maintained for from 5 to 10 minutes further resulting in the formation of a homogeneous white finely dispersed w/oemulsion. [00336] The final concentration of immunogens once formulated as water-in-oil emulsions as described above was 200 Example 15: The Immunogenicity and Protection of FMD Peptide Immunoqens Formulated as an Immunostimulatory Complex in a W/O Emulsion [00337] This Example illustrates the immunogenicity of FMD peptide immunogens formulated as a w/o emulsion or as FMD immunostimulatory complexes in combination with a w/o emulsion, which were immunized intramuscularly in cattle. The w/o-emulsions were prepared by homogenization as described in Example 4a and Example 14. [00338] Groups of three, adult cattle were immunized intramuscularly (I.M.) on week 0 and 3 with the following compositions: 400 fig of FMD peptides suspended in a final volume of 1,000 uL NaCl and dispersed with ISA Montanide® 50v (1,000 p.L) ; or FMD peptides/CpGl inununostimulatory complex (4:1 charge ratio) prepared as described in Table 9 suspended in a final volume of 1,000 jiL NaCl and dispersed with ISA Montanide® 50v (1,000 jiL) . A placebo control group of 1,000 uX NaCl emulsified with ISA Montanide® 50v (1,000 (iL) was prepared. [00339] The cattle showed no gross pathologies or behavioral changes after receiving placebo w/o emulsions or w/o emulsions containing FMD peptide immunogens or FMD immunostimulatory complexes. Sera obtained on week +5 were evaluated for the presence of FMD neutralizing antibodies and on week +6 the cattle were challenged with live virus to determine the level of protection in a trial lasting 14 days. Measurement of anti-EMD Antibodies - Neutralization Assay [00340] The quantitative neutralization assay (NA) for FMD antibody was performed with BHK-21 cells in flat-bottomed tissue-culture grade microtiter plates. The test is an equal volume test in 50 \|iL amounts. Starting from a 1:4 dilution, sera were diluted in a two-fold dilution series in duplicate. Serum samples to be tested were mixed with an equal volume of the FMDV OManisa serotype (200 TCIDSO/0.05 mL) and incubated for one hour at 37°C. A cell suspension at 106 cells/mL was prepared in- a medium containing 10% bovine serum. A volume' of 50 nL of cell suspension was added to each well. Plates were sealed and incubated at 37°C for 2-3 days. [00341] Microscopic examination was feasible after 48 hours. Fixation was effected with 10% formol/saline for 30 minutes. For staining the plates were immersed in 0.05% methylene blue in 10% formalin for 30 minutes. Positive wells (where virus had been neutralized and cells remained intact) were seen to contain blue-stained cells. Negative wells are empty. Titers were expressed as the final dilution of serum present in serum/virus mixture at the 50% end-point. Cattle Challenge Trial Protocol [00342] On day 35, all cattle, which received vaccine or placebo controls on day 0 and day 21 were separated by groups into separate containment rooms. On day 42, each animal was challenged intradermolingually (IDL) with a total of 104 BIOso FMDV 0 injected in two sites on the dorsal surface of the tongue. [00343] After challenge, the cattle were observed for development of clinical signs of FMD for 14 days and body temperature was recorded daily. Unprotected animals show lesions at sites other than the tongue. Control animals must develop a generalized infection as shown by lesions on at least three feet for the virus challenge to be considered valid.71 Immunogenicity Results [00344] The neutralizing antibody (N.A.) titers following immunization of FMD immunogens and the corresponding protection results are shown in Table 10. [00345] The N.A. titers determined from sera obtained from cattle (week +5) immunized by FMD peptide immunogens or by FMD immunostimulatory complexes administered as w/o emulsions indicated that both compositions were immunogenic. The titers obtained at the week 5 timepoint were highly variable, but in all cases were shown to be greater than 16. The minimum requirement for proof of potency for a foot-and-mouth disease vaccine as established by the Office International des Epizooties (DIE) is a N.A. titer of 16.71 Each steer in the Placebo negative control group returned negligible N.A. titers, all of which were less than 16 as shown in Table 10. [00346] Conclusive proof for protection must be obtained by challenge of the immunized groups with live virus of specified titer. Live virus was administered intradermolingually (IDL) with a total of 10* BID5o FMDV 0 injected in two sites on the dorsal surface of the tongue. [00347] The challenge protocol was initiated on week 6, one week after the N.A. titers were established for all groups and the results in Table 10 prove that the formulation derived from FMD peptides/CpGl immunostimulatory complexes in combination with unbound FMD peptides administered as a w/o emulsion was superior (3/3 protected) to the formulation derived from FMD peptides administered as a w/o emulsion alone (1/3 protected). [00348] The placebo control group confirmed that the live virus employed for the challenge was sufficiently virulent as all three cattle in this group were infected and showing signs of disease within 14 days after challenge (0/3 protected) as shown in Table 10. [00349] Both of the groups formulated with FMD immunogens or an FMD immunostimulatory complex in a w/o emulsion obtained significant N.A. antibody titers by week +5. [00350] Surprisingly, the challenge study demonstrated that the formulation of superior efficacy was derived from the formulation comprising the FMD immunostimulatory complex in the w/o emulsion. It is likely that this composition concurrently improves the N.A. responses and is more effective at upregulating a specific kind of immune response important for combating viral infections. [00351] Specifically, the immunostimulatory complex derived from FMD and CpGl presented in the form of a w/o emulsion may effectively augment the Thi arm of the immune response (e.g. IFN-gamma). This role for CpG oligonucleotides has been well established in other models and IFN-gamma itself has been shown to be an effective immunomodulator achieving immune protection against influenza virus.72 [00352] The control of foot-and-mouth disease, once an outbreak has been identified is often managed by culling infected and neighboring herds. The economic loss of important commodities like cattle, pork and sheep is often significant. The existing vaccine strategies employ killed virus, which are in some cases locally produced and of inferior quality. The manufacturing from live virus has various issues relating to production associated with it and the products themselves represent a potential safety risk. A strategy based on immunogens derived from synthetic peptides represents an improved method for effective immunization, and should be considered low risk. [00353] Specifically, this Example demonstrates that a synthetic peptide-based vaccine strategy can safely and effectively protect cattle against foot-and-mouth disease. [00354] Furthermore, this Example further demonstrates the utility of this vaccine approach for the general protection of important domestic livestock susceptible to foot-and-mouth disease. Table 1 Synthetic Peptide Immunogens And Oligonucleotides (Table Removed) IP = Theoretical lonization Potential, (ref. 68) FW = Formula Weight/I'm = Primer to target Tm (by %GQ Table 2 Molar Charge Calculations Synthetic Peptide Immunogens And Oligonucleotides (Table Removed) 1) Net charge: Calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H), a -1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for every other amino acid within the sequence. The charges are summed for each peptide and expressed as the net average charge. 2) Average Total Molar Equivalents Of+'ve Charge From Peptides: Estimates for the average charge of the combined peptide mixture are calculated by summing the overall molar charge contribution for each component within the mixture and taking the mean. 3) Average Total Peptide Formula Weight (FW): Estimates for the average Total peptide FW are calculated by summing the overall molar mass contribution for each component within the mixture and taking the mean. 4) The FMD vaccine is derived from a library of peptides. The molar contribution of each component is assumed equivalent and the average total molar equivalents of +Ve charge is similarly calculated. 5) For the FMD vaccine the average total peptide formula weight is obtained by summing the lowest and highest FW found for the various FMD peptides in the library and taking the mean. Molar Equivalents Of -'ve charge From CpG Oligonucleotides: Each phosphorothioate group contributes -1 charge. CpG1 (32 base oligomer) -1 nmol CpG1 = 31.0 nmol -Ve charge CpG2 (24 base oligomer + phosphorothioate). -1 nmol CpG2 = 24.0 nmol -Ve charge Table 3 Calculated Molar Charge Ratios Used To Prepare Complexes LHRH Peptide Immunogens And CpG1 Oligonucleotides (Table Removed) Total +'ve molar charge LHRH peptides = 4.3 nmol +'ve charge/nmol LHRH peptide (Table 2) In 100 ug LHRH peptides there are 100 u.g /4S43.9 jig/ fimol» 22.0 nmol of LHRH immunogen Total +'ve molar charge = 22.0 nmol x 4.3 nmols +'ve charge/nmol LHRH peptides = 94.6 nmols +'ve charge 2) Sample calculation: Final 8:1 molar charge ratio of LHRH immunogen to CpGl oligonucleotide. 100 ng of LHRH contributes 94.6 nmols of+'ve charge. To establish an 8:1 molar charge ratio of LHRH to CpGl in solution the required amount of LHRH that must be neutralized by CpGl is calculated as 94.6 nmols +'ve charge/8 = 11.8 nmols +'ve charge. Thus 11.8 nmols +'ve charge contributed by LHRH must be neutralized by 11.8 nmols of-'ve charge contributed by CpGl. 1 nmol of CpGl = 31 nmol -'ve charge (Table 2). Thus the total nmols of CpGl required to contribute 11.8 nmols of-'ve charge = 11.8/31 = 0.38 nmols CpGl. Table 4 Molar Charge Ratio Calculations IgE, CD4 Peptide Immunogens And CpG Oligonucleotides (Table Removed) 1) Total calculated +'ve molar charge for 100 ug or 300 ug of IgE peptides: Avg. FW = 5132.1 g/ mol (Table 2); Total +'ve molar charge IgE peptides = 7.7 nmol +'ve charge/nmol IgE peptides (Table 2) In 100 g IgE peptides there are 100 g /5132.1 g/ mol = 19.5 nmol of IgE immunogen Total +'ve molar charge = 19.5 nmol x 7.7 nmols +'ve charge/nmol IgE peptides = 150.1 nmols +'ve charge In 300 g IgE peptides there are 300 g/5132.1 g/ mol -58.5 nmol of IgE immunogen Total +'ve molar charge = 58.5 nmol x 7.7 nmols +'ve charge/nmol IgE peptides = 450.5 nmols +'ve charge 2) Total calculated +'ve molar charge for 100 ug or 300 ug of CD4 peptides: Avg. FW = 5280.6 g/ mol (Table 2); Total +'ve molar charge.CD4 peptides = 4.3 nmol +'ve charge/nmol CD4 peptides (Table 2) In 100 g CD4 peptides there are 100 g/5280.6 g/mol = 18.9 nmol of CD4 immunogen Total +'ve molar charge = 18.9 nmol x 4.3 nmols +'ve charge/nmol CD4 peptides = 81.3 nmols +'ve charge In 300 g CD4 peptides there are 300 g/5280.6 g/mol = 56.8 nmol of CD4 immunogen Total +'ve molar charge = 56.8 nmol x 4.3 nmols +'ve charge/nmol CD4 peptides = 244.2 nmols +'ve charge 3) n nmol of-'ve charge contributed by CpGl or CpG2: Total calculated -'ve molar charge for CpGl/CpG2 Oligonucleotides 1 nmol of CpGl =31 nmol -'ve charge, FW = 10,295 g/ mol (Table 1) 1 nmol of CpG2 = 24 nmol -'ve charge, FW = 7,400 g/ mol (Table 1) Table 5 Comparison Of Physical Properties Of PLG/PLGA Copolymers Dissolved In DMSO As A Function Of Weight Percent In Solution (Table Removed) Table 6 B4-HRP Inhibition Assay Of Sera Obtained From CD4 Peptides, CD4/CpG2 Iramunostimulatory Complexes Or Combinations In W/O Emulsions (Table Removed) • N.D. - samples were not assayed The value of 49.1% at week 11 for unadjuvanted CD4 peptide is unexpected and likely due to experimental error. * - Water-in-oil emulsion prepared with ISA 720 via homogenization techniques ** - Water-in-oil emulsion prepared with ISA 720 via extrusion techniques Table 7 Neutralization Of HIV-1 Strain VL135 By Immune Sera To CD4 Peptides CD4/CpG2 Immunostimulatory Complexes Or Combinations In W/O Emulsions (MT-2 microplaque assay) (Table Removed) * - Reciprocal liter prior to the addition of an equal volume of virus containing 1-1.3 logs - Water-in-oil emulsion prepared with ISA 720 by homogenization techniques * - Water-in-oil emulsion prepared with ISA 720 by extrusion techniques Table 8 Complexation Efficiency As A Function of LHRH PeptiderCpGl Molar Charge Ratio By RP-HPLC (Mass of LHRH peptide cocktail fixed @ 400 g) (Table Removed) 1) Percentage of bound peptides are calculated by comparing the RP-HPLC peak areas relative to the control LHRH composition assayed in the absence of the CpGl oligonucleotide. 2) Percentages for the overall amount of LHRH/CpG 1 complexed peptide are calculated by summing the total RP-HPLC peak areas obtained and comparing that with total summed RP-HPLC peak areas obtained from the control composition examined in the absence of CpGl oligonucleotide. Table 9. Molar Charge Ratio Calculations LHRH And FMD Peptide Immunogens And CpG Oligonucleotides (Table Removed) 1) Total calculated +'ve molar charge for 25 g, 100 g or 400 g of LHRH peptides: Avg. FW = 4543.9 g/ mol (Table 2); Total +'ve molar charge LHRH peptides = 4.3 nmol +'ve charge/nmol LHRH peptides (Table 2) In 25 g LHRH peptides there are 25 g /4543.P g/ mol = 5.5 nmol of LHRH immunogen Total +'ve molar charge = 5.5 nmol x 4.3 nmols +'ve charge/nmol LHRH peptides = 23.7 nmols +'ve charge In 100 g LHRH peptides there are 100 g /4543.9 g/ mol = 22.0 nmol of LHRH immunogen Total +'ve molar charge = 22.0 nmol x 4.3 nmols +'ve charge/nmol LHRH peptides = 94.6 nmols +'ve charge In 400 g LHRH peptides there are 400 g /4543.9 g/ m ol = 88.0 nmol of LHRH immunogen Total +'ve molar charge = 88.0 nmol x 4.3 nmols +'ve charge/nmol LHRH peptides = 378.4 nmols +'ve charge 2) Total calculated +'ve molar charge for 400 g of FMD peptides: Avg. FW = 6805 g/ mol (Table 2); Total +'ve molar charge LHRH peptides ~ 3.5 nmol +'ve charge/nmol FMD peptides (Table 2) In 400 g FMD peptides there are 400 g/6805 g/ mol = 58.8 nmol of FMD immunogen Total +'ve molar charge = 58.8 nmol x 3.5 nmols +'ve charge/nmol FMD peptides = 205.8 nmols +'ve charge 3) # nmol of-'vc charge contributed by CpGl: Total calculated -'vc molar charge for CpGl Oligonucleotides 1 nmol of CpGl =31 nmol -'ve charge, FW = 10,295 g/ mol (Table 1) Table 10. ' Neutralization Antibody Titers And Protection Data Obtained In Cattle Immunized With FMD Vaccines. (Table Removed) Challenge intradermolingually (IDL) with a total of 104 BIDso FMDV 0 injected in two sites on the dorsal surface of the tongue. We Claim: 1. A stabilized immunostimulatory complex comprising a cationic synthetic peptide immunogen comprising a target B cell or a CTL epitope, and a T helper cell epitope and anionic CpG oligonucleotide wherein the cationic synthetic peptide immunogen has a net positive charge at a pH in the range of 5.0 to 8.0 calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H), a-1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for all other amino acids in the peptide immunogen and wherein the anionic CpG oligonucleotide has a net negative charge at a pH in the range of 5.0-8. 0 and is a single-stranded DNA comprising 8 to 64 nucleotide bases with a repeat of a cytosine-guanidine motif and the number of repeats of the CpG motif is in the range of 1 to 10 wherein the CpG oligonucleotide is modifed with a phosphorothiorate or a phosphodiester, and said anionic CpG oligonucleotide is preferably selected from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO : 2, a 24 base length oligomer plus an phosphorothioate group designated as n and a combination thereof. 2. The immunostimulatory complex as claimed in claim 1, wherein the cationic peptide immunogen is a synthetic peptide. 3. The immunostimulatory complex as claimed in claim 1, wherein the net positive charge of the cationic synthetic peptide immunogen is at least +2. 4. The immunostimulatory complex as claimed in claim 3, wherein the average net positive charge of the mixture of synthetic peptide immunogents is at least +2. 5. The immunostimulatory complex as claimed in claim 1, wherein the net negative charge of the anionic oligonucleotide is at least -2. 6. The immunostimulatory complex as claimed in claim 1, wherein the CpG oligonucleotide is a single-stranded DNA molecule with 18-48 nucleotide bases and the number of repeats of CpG motif therein in the range of 3 to 8. 7. The immunostimulatory complex as claimed in claim 1, wherein the CpG oligonucleotide has the formula: 5'X'CGX2 3'wherein C and G are unmethylated and X1 is selected from the group consisting of A (adenine), G (guanine) and T (thymine); and X2 is C (cytosine) or T (thymine). 8. The immunostimulatory complex as claimed in claim 1, wherein the CpG oligonucleotide has the formula: 5'(X3)2CG(X4)23' wherein C and G are unmethylated and X3 is selected from the group consisting of A, T and G; and X4 is C or T. 9. The immunostimulatory complex as claimed in claim 1, wherein the CpG oligonucleotide is modified with a phosphorothioate. 10. The immunostimulatory complex of claim 1, wherein CpG oligonucleotide is 5'TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpG 1) SEQ ID NO : 1. 11. The immunostimulatory complex of claim 1, wherein CpG oligonucleotide is 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO: 2, a 24 base length oligomer plus an phosphorothioate group designated as n. 12. The immunostimulatory complex as claimed in claim 1, wherein the cationic synthetic peptide immunogen is a derived from a group consisting of HIV CD4, LHRH, IgE and FMD. 13. The immunostimulatory complex as claimed in claim 12, wherein the synthetic peptide derived from HIV CD4 is selected from the group consisting of SEQ ID NO: 4,5, 6 and a mixture thereof. 14. The immunostimulatory complex of claim 12, wherein the cationic peptide immunogen is a synthetic peptide derived from LHRH. 15. The immunostimulatory complex as claimed in claim 14 wherein the synthetic peptide derived from LHRH is selected from the group consisting of SEQ ID NO: 7,8, 9 and a mixture thereof. 16. The immunostimulatory complex as claimed in claim 12, wherein the synthetic peptide derived from IgE is selected from the group consisting of SEQ ID NO: 10, 11 and a mixture thereof. 17. The immunostimulatory complex as claimed in claim 12, wherein the synthetic peptide derived from FMD is selected from the group consisting of SEQ ID NO: 12, 13 and a mixture thereof. 18. A process for preparing a stabilized immunostimulatory complex as claimed in claim 1 comprising the steps of: (a) dissolving or dispersing the cationic synthetic peptide immunogen in an aqueous phase selected from the group consisting of distilled deionized water, saline, PBS or a mixture thereof such that the pH of the aqueous phase is lower than the ionization point of the peptide immunogen; (b) dissolving the anionic CpG oligonucleotide in an aqueous phase selected from the group consisting of distilled deionized water, saline, PBS or a mixture thereof; (c) adding the CpG oligonucleotide in the aqueous phase dropwise to the solution or dispersion of the cationic peptide immunogen in an amount to form a stabilized immunostimulatory complex of the peptide immunogen and the CpG oligonucleotide in a charge ratio of the cationic immunogen synthetic peptide to the CpG oligonucleotide in the range of 16: 1 to 1: 1; optionally comprising the step of (d) removing the aqueous phase of the suspension of the stabilized immunostimulatory complex obtained in step (c). 19. The process as claimed in claim 18, wherein the aqueous phase is removed by lyophilization, or spray-drying. 20. The process as claimed in claim 18, wherein the immunostimulatory complex has an average particle size in the range of 1 to 50 uM. 21. The process as claimed in claim 18 wherein the amount of the immunogen peptide and the CpG oligonucleotide added is in a charge ratio in the range of 16: 1,4: 1, 2: 1, 1.5 : 1 or I: 1 of the cationic immunogen peptide to the CpG nucleotide. 22. A process for preparing the immunostimulatory complex as claimed in claim 1 in the form of a suspension comprising the steps of: (a) preparing an immunostimulatory complex of claim 1, or preparing a solution of peptide immunogen selected from the group consisting of SEQ ID NOs: 3-13, in an aqueous phase selected from the group consisting of distilled deionized water, saline and phosphate buffered saline; (b) preparing a suspension of a mineral salt selected from the group consisting of aluminum hydroxide, aluminum phosphate, and calcium phosphate, in an aqueous phase selected from the group consisting of distilled deionized water, saline and phosphate buffered saline; (c) adding the immunostimulatory complex in the aqueous phase into an aqueous phase containing the mineral salt suspension; (d) mixing the immunostimulatory complex with the mineral salt suspension to form a mixed suspension, or whenever a solution of peptide immunogens is prepared in step (a), step (c) comprises adding the peptide solution to the suspension of the mineral salt with mixing; and step (d) comprises adding a CpG nucleotide selected from the group consisting of SEQ ID NO : 1 and SEQ ID NO : 2 with mixing to form a mixed suspension of an immunostimulatory complex and a mineral salt. 23. The process as claimed in claim 22, wherein the mineral salt is selected from the group consisting of Alhydrogel, Adjuphos® and mixtures thereof. 24. The process as claimed in claim 22, wherein the aqueous phase optionally comprises a surfactant, a tonifier, a preservative or combinations thereof. 25. The process as claimed in claim 24 wherein the aqueous phase comprises a tonifier selected from the group consisting of a PBS or saline and mixtures thereof. 26. The process as claimed in claim 24 comprising adding to the aqueous phase a preservative selected from the group consisting of 2-phenoxy-ethanol and derivatives thereof. 27. The process as claimed in claim 22 optionally comprising adding to the aqueous phase an adjuvant selected from the group consisting of MPL, MDP, DDA, Avridine, BAY-1005, DC-Choi, Murapalmitine, PCPP, a saponin, a Cholera Toxin, a heat labile Enterotoxin from E. Coli and a cytokine selected from the group consisting of IL-1R, IL-2, IL-12, IFN-y and a derivative thereof. 28. An immunostimulatory complex as claimed in any preceding claim whenever used in a pharmaceutical composition.

Full Text

Stabilized Synthetic Immunogen Delivery Systems
FIELD OF THE INVENTION
[0001] The present invention relates to a stabilized
immunostimulatory complex and a method for preparing the
stabilized immunostimulatory complex. More specifically, the
present invention provides stabilized synthetic
immunostimulatory complexes that are useful in vaccine
delivery systems with improved immune responses in vivo. These
immunostimulatory complexes are also useful for preparing
vaccine formulations designed to function as a depot for
controlled release of the immunostimulatory complex. The
immunostimulatory complex may also be incorporated in
formulations designed to target specific cell types to
synergistically improve the quality of the immune responses
elicited.
BACKGROUND OF THE INVENTION
[0002] Vaccines have been successfully employed for many years
in prophylactic compositions for the prevention of infectious
disease and more recently in therapeutic compositions for the
treatment of cancers and non-infectious diseases.
[0003] Traditionally vaccines have been derived from
attenuated or killed viral or bacterial pathogens and have
proven to be very effective against diseases such as polio
virus and BordeteJIa pertussis. In spite of these successes,
there are growing concerns over the safety of such vaccines.
This has led to the development of subunit vaccines derived
from components of these pathogens or fully synthetic peptide
immunogens.
[0004] Examples of subunit vaccines include Tetanus toxoid and
hepatitis B surface antigen. These antigens are often poorly
immunogenic and require adjuvants to improve the immune
responses obtained. Well-characterized biologically active
compounds such as synthetic peptides are preferred substrates
for inducing biological responses, for safety and regulatory
purposes. However, these immunogens are not optimal, and
induce partial or negligible protective responses in animal'
models. The synthetic peptides require both stabilization and
adjuvantation for the induction of an effective immune
response in vivo.
[0005] Various methods have been employed to protect synthetic
peptide immunogens against degradation in vitro and in vivo,
mediated by various processes including chemical and physical
pathways.1 (The superscript numbers refers to publications,
which more fully describe the state of the art to which this
invention pertains. The disclosures of these references are
hereby incorporated by reference. The citation of each
reference is found at the end of this section).
[0006] Various methods have been employed to improve peptide
solubility or protect a peptide against degradation in vivo.2
These generally include simple procedures like modifying the
salt concentration and/or the pH of the solution. Peptides
have also been chemically modified by conjugation with water
soluble compounds like polyethylene glycol (PEG) or
polyethylene oxide (PEG) both to improve their aqueous
solubility and circulation time in vivo.3 It has been
documented that adjuvants derived from PEG or PEO can down
regulate the immune system.4 Thus, PEG or PEO modified
peptides would not be expected to function effectively as
adjuvants. The, addition of multiple lysines to add charge to
a peptide can improve its aqueous solubility but does not
generally result in improved immunogenicity.
[0007] The objective of these various strategies is to improve
circulation time in vivo or minimize or eliminate
immunogenicity problems associated with the physical
conditions (e.g. salt, pH, temperature, buffer type) and/or
chemical incompatibilities when peptides are employed in a
vaccine formulation.
[0008] Polyether block copolymers, comprising polycationic
polymers, were disclosed by Kabanov et al., U.S. Patent No.
5,656,6115 for stabilizing polynucleotides or oligonucleotides.
The polyether block copolymer-polynucleotide complexes are
employed to facilitate the transport of the polynucleotide
across a cell membrane for improved biological activity.
However, these polynucleotide-polyether block copolymers are
not immunogenic and are not suitable as vaccines.
[0009] Allcock et al. US patent No. 5,562,9096 describes an
immunoadjuvant derived from phosphazene polyelectrolytes. The
immunoadjuvant was admixed directly with an antigen in
solution and may be prepared as microparticles by spray drying
a solution of the polymer and the antigen or by a process
described by Cohen in US patent No. 5,149,543.7 Although,
increased adjuvanticity was shown for these systems, there are
difficulties in preparing the microparticular compositions due
to the cumbersome mechanical processes employed, which would
be difficult to scale up for commercial production.
Furthermore, the stability of the polymer-antigen complex so
formed is highly dependent on salt concentration and pH
conditions.
[0010] A different approach is described in Moss et al.
W091/040528, wherein a solid vaccine composition is prepared
from an antigen, which may be a peptide, a saponin and a
polycationic adjuvant such as OEAE-dextran. Vaccines
formulated from this combination provided improved longevity,
making such combinations suitable for use as implants.
However, the antigen must first be chemically conjugated to a
carrier molecule and exhaustively purified. The purified
antigen-carrier was then combined with a saponin and a
polycationic adjuvant to provide a solid composition. This
process provides no control over the physical properties, such
as particle size, of the product.
[0011] Numerous adjuvants and/or depot-based parenteral,
mucosal or transdermal delivery systems destined for use with
human or veterinary vaccines have been developed to enhance
the immune response. These include the use of mineral salts,
water-in-oil (w/o)-emulsions, liposomes, polymeric
microparticles, nanoparticles and gels/hydrogels.9 A large
number of clinical trials employing various (w/o)-emulsion
compositions have been conducted.
[0012] In spite of this vast body of clinical research,
typical parenteral formulations, administered subcutaneously
or intramuscularly, are prepared with adjuvants derived from
aluminum salts, such as aluminum phosphate or aluminum
hydroxide. Alum salts are suitable and effective for many
vaccines based on attenuated pathogens, killed pathogens and
subunit antigens derived from biological agents. However, the
aluminum-based adjuvants are often totally ineffective for
synthetic peptide-based immunogens because of the large dose
of peptide required and the need of much stronger
adjuvantation. The combination of a large dose of immunogen
with a weakly adjuvanting alum in a vaccine composition is not
ideal as it can lead to immunogen tolerance and
reactogenicity, i.e., undesired side reactions, such as
swelling and redness at the site of injection.
[0013] Freund's complete adjuvant (FCA), a suspension of heatkilled
Mi tuberculosis mycobacteria in mineral o-il containing
a surfactant, has been recognized as one of the most powerful
adjuvants. However, severe adverse reactions, ranging from
minor irritation to lesions and sterile abscesses at the site
of injection have been documented. Due to these adverse
reactions, FCA has been banned from human and veterinary
applications.
[0014] Thus, there is a clear need to develop adjuvants which
are safe without the toxicological and/or reactogenic problems
associated with alum or FCA and can effectively enhance
immunogenicity and prolong the effectiveness of peptide
immunogens to avoid the problem of tolerance associated with
alum. It is also most desirable to develop compositions and
methods, which can both, stabilize a peptide immunogen and
adjuvant the immune responses in a single composition.
[0015] Jones et al.10 have disclosed two specific CpG
oligonucleotides that may be co-administered with a peptidebased
malaria vaccine in Aotus monkeys to enhance immune
responses. In the Jones study, the 4-onization point (IP) of
the peptide used is 5.96. This corresponds to the pH at which
the peptide will have a theoretical zero charge.11 By virtue
of its araino acid composition, the peptide used would be
effectively uncharged at physiological pH in the aqueous
solvent selected. Thus, no complexation can take place with
the two CpG oligomers. The resultant mixture when formulated
in a w/o-emulsion is expected to be transiently adjuvanted.
To achieve a useful level of immunogenicity, multiple
injections and a large quantity of adjuvant would be required.
Further, the long-term stability of such a composition is
questionable. In fact, Jones et al. disclosed that it was
necessary to employ a large dose of CpG oligonucleotide, 500
p.g per injection. Furthermore, the methods, employed to
prepare the w/o-emulsions, cannot be easily scaled up for
commercial .applications. It is to be noted that Jones et al.
taught that different CpG oligomers are useful for different
mammalian species. For example, a CpG oligomer, CpG ODN 1826
is mitogenic for mice and a lower primate, but not for
chimpanzees or humans and the effect is not predictable.
[0016] Krieg et al., U.S. patent No. 6,194,388 Bl12 describes
unmethylated CpG oligonucleotides particularly useful for
therapeutic applications based on their ability to stimulate
immune responses when mixed with an antigen. Krieg et al., US
6,207,646 Bl13 further describes the use of unmethylated CpG
oligonucleotides to redirect a Th2 response to a Thl response'.
In both, the effectiveness of the CpG oligomers were shown by
B-cell stimulation wherein B-cells were cultured with
phosphorothioate modified CpG oligomers. There is no
disclosure or suggestion on how the CpG oligomers can be used
to provide a stabilized immunostimulatory complex or a
vaccine.
[0017] Another area of intense interest and research has been
focused on methods to formulate synthetic immunogens for
alternate delivery routes, such as mucosally, transdermally,
or orally. Mucosal immunity is mediated by the induction of
secretory immunoglobulin (slgA) found in external secretions
(e.g., intestinal, bronchial or nasal washings). It is
believed that transdermal or mucosal delivery of vaccines
would be effective against a majority of pathogenic organisms,
which gain entry via mucosal surfaces. For example, an orally
administered cholera vaccine has been shown to be far superior
to the parenterally administered analog.14
[0018] Friede et al., W099/5254915 teaches that vaccine
compositions intended for mucosal use can be derived from a
combination of an antigen with a polyoxyethylene ether or
polyoxyethylene ester as the primary adjuvant. It was
suggested that the target antigen might be a synthetic
peptide. Friede et ?.l. also suggests the addition of CpG
oligonucleotides into the vaccine composition to provide
improved responses. They showed that a combination of a
polyoxyethylene ether or polyoxyethylene ester with a CpG
oligonucleotide could improve mucosal responses when co-
administered with an antigen. However, the results showed a
lack of any adjuvanticity from simple mixtures of CpG
oligonucleotides with antigen described.
[0019] Transdermally administered vaccines represent an area
of recent interest. Ideally, devices, i.e., patches or
needle-free jet injectors can be employed to target the
intradermal Langerhan cells, i.e., dendritic cells. These
specialized cells are responsible for the effective processing
and presentation of an immunogen and can be used to directly
induce systemic humoral and cellular responses. In some
cases, intramuscular immunization was achieved by transdermal
methods.16 For example, a recent paper described a diptheria
vaccine administered as a patch. Systemic antibodies to
diptheria toxoid were found for a variety of compositions when
co-administered with adjuvants.17
[0020] Although the prior art has illustrated the potential of
various vaccine formulations, there are a number of practical
limitations for the development of synthetic peptide-based
vaccine formulations for mucosal or transdermal delivery.
These include:
1) immunogen degradation by mucosal fluids or
secretions and/or proteolytic enzymes at the mucosal
surface or within the intradermis;
2) negligible adsorption across the mucosal epithelium
or through the intradermal layers; and
3) dilution of the immunogen to a concentration below
that required to induce a suitable level of immune
responses.
[0021] Few strategies exist which both stabilize and adjuvant
a synthetic peptide-based immunogen in a single vaccine
composition. Such a composition would be essential for the
development of highly efficacious parenteral, mucosal or
transdermal peptide-based vaccines.
[0022] It is also desirable to prolong the duration of
immunogenic responses in order to reduce the number of
administrations required. This would result in improved
compliance and reduce the overall cost for vaccination.
[0023] Various methods may be employed to adjuvant synthetic
peptide-based immunogens, but normally a carrier or depot
system is required for effective long-term immunogenic
responses. Notable examples include adsorbing the immunogen
onto a mineral salt or gel. For example, encapsulating a
pepti.de immunogen within a polymeric matrix (monolithic
matrix) or gel, or layering a polymeric material around a
peptide immunogen (core-shell) may be an effective strategy.
Or, an immunogen may be incorporated in a liposome or
vesicular type of formulation, with the immunogen either
embedded in the lipid matrix or physically entrapped in the
internal aqueous phase. Another strategy may employ a
mineral-based, vegetable-based or animal-based oil, with an
aqueous solution of the immunogen in various proportions, to
prepare a water-in-oil (w/o)-emulsion or a water-in-oil-inwater
(w/o/w)-double emulsion18.
[0024] Diverse particle sizes, morphologies, surface
hydrophobicity and residual surface charge are possible
formulation dependent variables for consideration. Control of
these parameters is known to be important for the phagocytosis
of micron-sized particulates via parenteral administration19' 20
and for the uptake of particulates at specialized M-cells of
the Peyers Patches within the intestinal tract21' 22 for oral
delivery. Similarly, these parameters have been shown to be
important for access to the nasal-associated lymphoid tissue
of the nasalpharyngeal tract, a target of intranasal
delivery.23' 2"
[0025] Krone et al., U.S. Patent No. 5,700,45925 describes the
use of polyelectrolyte complexes in microparticulate form
derived from polyacids and polybases, in which the complexing
agent is a polymer. Various uses for these complexes are
described and include vaccine compositions comprising antigens
or antigenic peptides. Some of the compositions are
controlled release formulations employing potentially
biodegradable materials. In one of the examples, a method of
incorporating an antigen in polyelectrolyte complex
microparticles is described. However, the mechanical process
described for preparing microparticles by grinding the mixture
of 100 uM size particles to about 1-4 uM, is cumbersome. This
would not be easily scaled up for commercial production.
[0026] Eldridge et al.26 developed polymeric biodegradable
microspheres manufactured from poly-D,L-lactide-co-glycolide
copolymers for the controlled release of an antigen in vivo.
The polymers disclosed to be useful for encapsulating an
antigen into microparticles include poly-D,L-lactide,
polyglycolide, polycaprolactone, polyanhydrides,
polyorthoesters and poly(a-hydroxybutyric acid).
[0027] Although the controlled release of an antigen was
achieved in the prior art, difficulties were encountered when
microparticles were manufactured by methods described. The
methods described are difficult to scale-up. Moreover, the
exposure of biological materials to organic solvents and
mechanical processing can lead to denaturation and low to
modest encapsulation efficiencies. Furthermore, hydrophilic
antigens are inefficiently encapsulated in the processes
described.
[0028] Henry, et al., U.S. Pat. Nos. 5,126,141 and 5,135,75127'
28 described aqueous, thermally reversible gel compositions
formed from a polyoxyalkylene polymer and an ionic
polysaccharide for application to injured areas of the body to
prevent adhesion. Rosenberg, et al., WO93/0128629 described
the use of the same type of polyoxyalkylene polymers for the
local delivery of antisense oligonucleotides to surgically
exposed surface of blood vessels for treatment of restenosis.
Neither Henry et al. nor Rosenberg et al. taught or suggest
the use of a gel composition as a vaccine.
[0029] Dunn et al., U.S. Patent Nos. 4,938,763 and 5,702,71630/
31 describe polymeric compositions useful for the controlled
release of biologically active materials. A biocompatible
solvent was used to prepare solutions or suspensions of
antigen for direct parenteral injection, whereupon in-situ
gelling results in implant formation. Utility for a variety
of antigens including small synthetic peptide-based immunogens
was claimed. However, Dunn et al., US 5,702,71631, stated that
the controlled release compositions require up to 15% by
weight of a gel rate-retarding agent. The retarding agents
were added to modulate the gelling rate and were needed for
higher entrapment efficiencies for antigens, which are easily
extracted in vivo. As the solvent extraction is governed
largely by diffusion, this presents more of a problem for
small synthetic immunogens than for larger sub-unit or
protein-based antigens.
[0030] Neither US 4,938,76330 nor US 5,702,71631 taught nor
suggested synthetic peptide-based immunogen stabilized as an
immunostimulatory complex suspended within a biocompatible
solvent. Furthermore, neither US 4,938,76330 nor US 5,702,71631
taught nor suggested compositions which are self-adjuvanting
and can upregulate immune responses in both the priming and
boosting phases.
[0031] It is an object of this invention to develop stable
immunostimulatory complexes from synthetic peptide immunogens
and stabilizing molecules, which possess self-adjuvanting
properties in vivo. It is a further object of the present
invention to provide a simple method to stabilize a synthetic
peptide immunogen in vitro and in vivo.
[0032] It is a still further object of the present invention
to provide sustained or controlled release delivery vehicles
compatible with these stabilized synthetic peptide-based
immunostimulatory complexes.
[0033] It is a still further object of the invention to
develop formulations using a combination of stabilized
synthetic peptide-based immunostimulatory complexes and
uncomplexed immunogens in a controlled release delivery system
to achieve a synergistic enhancement of the immune response
including protective responses.
References Cited
1. Manning MC, et al. Pharmaceutical Research, 1989, 6:903-
918.
2. Monfardini C, et al., Bioconjugate Chem., 1998, 9:418-
450.
3. Roberts MJ, et al., J Pharm Sci, 1998, 87:1440-1445.
4. Hilbert AK, et al., Vaccine, 1999, 17:1065-1073.
5. Kabanov AV, et al., U.S. 5,656,611, 1997.
6. Allcock HR, et al., U.S. Patent No. 5,562,909, 1996.
7. Cohen S, et al., U.S. Patent No. 5,149,543, 1992.
8. Moss BA, et al., WO 91/04052, 1991.
9. Cox JC, et al. Vaccine, 1997, 15:248-256.
10. Jones TR, et al., Vaccine, 1999, 17:3065-3071.
11. Bjellqvist B, et al., Electrophoresis, 1993, 14:1023-
1031.
12. Kreig AM, et al., U.S. Patent No. 6,194,388 Bl, 2001.
13. Kreig AM, et al., US Patent No. 6,207,646 Bl, 2001.
14. Suharyono, et al. Lancet, 1992, 340:689-694.
15. Freide M, Hermand P., WO 99/52549, 1999.
16. Aguiar JC, et al. Vaccine, 2002, 20:275-280.
17. Scharton-Kersten T, et al. Infect Immun, 2000, 68:5306-
5313.
18. Powell MF, et al., Pharmaceutical Biotechnology, Vol. 6,
Plenum Press, New York, 1995.
19. Ikada Y, et al., J Bioactive Compat Polym, 1986, 1:32-46.
20. Kreuter J, et al. Vaccine, 1986, 4:125-129.
21. Jepson MA, et al. J Drug Targeting, 1993, 1:245-249.
22. Moldoveanu Z, et al., J' Infect Pis, 1993, 167:84-90.
23. Matsuo K, et al. Vaccine, 2000, 18:1344-1350.
24. Higaki M, et al., Vaccine, 1998, 16:741-745.
25. Krone V, et al., U.S. Patent No. 5,700,459, 1997.
26. Eldridge JH, et al., Mol Immunol, 1991, 28:287-297.
27. Henry RL, U.S. Patent No. 5,126,141,1992.
28. Henry RL, U.S. Patent No. 5,135,751, 1992.
29. Rosenberg RD, et al., WP 93/01286, 1993.
30. Dunn RL, et al., U.S. Patent No. 4,938,763, 1990.
31. Dunn RL, et al., U.S. Patent No. 5,702,716, 1997.
32. Papisov IM, et al., Advances in Polymer Science, 1988,
90, 1988,139-177.
33. Chu RS, et al., J Exp Med, 1997, 186:1623-1631.
34. Akasaka T, 'et al., Bioconjugate Chem., 2001, 12:776-785.
35. Ballico M, et al., Bioconjug Chem, 2001, 12:719-725.
36. Klinman DM, et al., Vaccine,1999, 17:19-25.
37. Krieg AM, et al., Nature 1995, 374:546-549.
38. Klinman DM, et al., Infect Immun, 1999, 67:5658-5663.
39. Nagel KM, et al., Pharmacotherapy, 1993, 13:177-188.
40. Weeratna RD, et al., Vaccine, 2000, 18:1755-1762.
41. McCluskie MJ, et al., Vaccine, 2000, 18:231-237.
42. LiCalsi C, et al., Vaccine, 1999, 17:1796-1803.
43. Romera SA, et al., Vaccine, 2001, 19:132-141.
44. Wright JC, et al., J Controlled Release, 2001, 75:1-10.
45. Graham PD, et al., J Controlled Release, 1999, 58:233-
245.
46. DesNoyer JR, et al., J Controlled Release, 2001, 70:285-
294.
47. Aguado MT, et al., Imrounobiol, 1992, 184:113-125.
48. Visscher GE, et al., J Biomed Mater Res, 1985, 19:349-
365.
49. Forbes RT, et al., J Pharm Sci, 1998, 87:13161321.
50. Overcashier DE, et al., J Pharm Sci, 1999, 88:688-695.
51. Wang CY, et al., WO 99/67293, 1999.
52. Wang CY, U.S. Patent No. 5,763,160, 1998.
53. Wang CY, U.S. Patent No. 6,090,388, 2000.
54. Ladd AE, et al., U.S. Patent No. 5,759,551, 1998.
55. Wang CY, U.S. 6,025,468, 2000.
56. Wang CY, USSN 09/865,294, 2001.
57. Wang CY, et al., U.S. Patent No. 6,107,021, 2000.
58. Wang CY, USSN 09/747802, 2001.
59. Wang CY, WO 99/66957, 1999.
60. Wang CY, WO 99/66950, 1999.
61. Mascotti DP, et al., Proc Nat head Sci, USA, 1990, ,
87:3142-3146.
62. Kabanov AV, et al., Bioconjuq Chem, 1995, 6:7-20.
63. MacDonald RC, et al., Biochim Biophys Acta, 1991,
1061:297-303.
64. Shen F, et al., Vaccine, 1999, 17:3039-3049.
65. Wang CY, et al., Proc. Nat. Acad. Sci., USA, 1999,
96:10367-10372.
66. Hanson CV, et al., J. Clin Microbiol, 1990, 28:2030-2034.
67. Park TG, et al., J Controlled Release, 1995, 33:211-222.
68. www.expasy.ch/tools/pi_tool.html
69. Hakim, I, et al. J Immunol, 1996, 157:5503-5511.
70. Zeng, XY, et al., Theriogenology, 2002, 58:1315-1326.
71. Office International des epizootics (OIE). Foot-in-mouth
disease. Manual of standards for diagnostic tests and
vaccines. Paris(France): OIE, 1996, p. 47-56 [chapter
2.1.1]
72. Proietti, E, et al., J. of Immunol, 2002, 375-383.
SUMMARY OF THE INVENTION
[0034] The present invention is directed to a stabilized
immunostimulatory complex comprising a cationic peptide and
anionic molecule or oligonucleotide or polynucleotide and a
method for stabilizing a cationic peptide by complexation with
an anionic molecule or oligonucleotide or polynucleotide via
electrostatic association. The stabilized immunostimulatory
complex may be incorporated into a pharmaceutical composition
as an immunogen delivery system.
[0035] A "cationic peptide" as described herein refers to a
peptide, which is positively charged at a pH in the range of
5.0 to 8.0. The net charge on the peptide or peptide
cocktails is calculated by assigning a +1 charge for each
lysine (K), arginine (R) or histidine (H), a -1 charge for
each aspartic acid (D) or glutamic acid (E) and a charge of 0
for the other amino acid within the sequence. The charge
contributions from the N-terminal amine (+1) and C-terminal
carboxylate (-1) end groups of each peptide effectively cancel
each other when unsubstituted. The charges are summed for
each peptide and expressed as the net average charge. A
suitable peptide immunogen has a net average positive charge
of +1. Preferably, the peptide inununogen has a net positive
charge in the range that is larger than +2.
[0036] The peptide immunogens comprise B-cell epitopes and Th
epitopes. The Th epitopes may be intrinsic to the peptide or
added thereto as described in the prior art. Suitable peptide
immunogens are described herein below.
[0037] An "anionic molecule" as described herein refers to
molecules, which are negatively charged at a pH in the range
of 5.0-8.0. The net negative charge on the oligomer or
polymer is calculated by assigning a -1 charge for each
phosphodiester or phosphorothioate group in the oligomer. A
suitable anionic oligonucleotide is a single-stranded DNA
molecule with 8 to 64 nucleotide bases, with the number of
repeats of the CpG motif in the range of 1 to 10. Preferably,
the CpG immunostimulatory single-stranded DNA molecules
contain 18-48 nucleotide bases, with the number of repeats of
CpG motif in the range of 3 to 8. ,
[0038] More preferably the anionic oligonucleotide is
represented by the formula: 5' X1CGX2 3' wherein C and G are
unmethylated; and X1 is selected from the group consisting of A
(adenine), G or T (thymine). Or, the anionic oligonucleotide is
represented by the formula: 5' (X3) 2CG (X4) 2 3' wherein C and G
are unraethylated; and X3 is selected from the group consisting
of A, T or G,. and X4 is C or T.
[0039] Most preferably, the CpG oligonucleotide is selected
from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT
TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer,
and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO:
2, a 24 base length oligomer plus a phosphorothioate group
(designated as n at the 5' end).
[0040] The resulting immunostimulatory complex is in the form
of particles with a size typically in the range from 1-50
microns and is a function of many factors including the
relative charge stoichiometry and molecular weight of the
interacting species.32 The particulated immunostimulatory
complex has the added advantage of providing adjuvantation and
upregulation of specific immune responses in vivo.
Additionally, the stabilized immunostimulatory complex is
suitable for preparing vaccine formulations by various
processes including water-in-oil emulsions, mineral salt
suspensions and polymeric gels.
[0041] The term "stabilize" as used herein may be accomplished
by the use of any material, which protects the synthetic
peptide immunogen against degradation in vitro or in vivo.
This may be accomplished by virtue of chemical modification
and/or physical association. A stabilizer may augment the
physiological properties of a synthetic peptide immunogen, an
oligosaccharide-modified glycopeptide or a lipidated peptide
to provide a more efficacious formulation.
[0042] The term "adjuvant" as described herein refers to any
material, which can enhance or upregulate the immune responses
elicited by an immunogen in humans or animals. The adjuvant
itself may or may not induce an immunogenic response.
[0043] The stabilizer may also preferably function in a
vaccine as an adjuvant, effectively upregulating the immune
responses. The stabilizer may act as an adjuvant by actively
facilitating the presentation of the immunogen to professional
processing cells of the immune system, such as macrophages and
dendritic cells. In the present invention, the stabilized
immunostimulatory complex ideally remains as an integral unit
in solution when administered.
[0044] The stabilized immunostimulatory complex may also be
formulated for controlled release and remains as a complex in
a concentrated form in a "depot" near the site of
administration. These formulations synergistically combine
the benefits of a stabilized adjuvanted immunogen coupled with
a sustained local release of immunogen to immune effector
cells. In some compositions the role of the adjuvant itself
may also involve attracting cells of the immune system to the
vicinity of the immunogen depot and stimulate such cells to
elicit an immune response.
[0045] In a second aspect of this invention, there is provided
a method for preparing a vaccine composition containing an
immunostimulatory complex. In a preferred embodiment the
immunostimulatory complex has the added advantage of being a
stabilized synthetic peptide-based immunogen in vitro and at
the same time is self-adjuvanting with upregulation of
specific immune responses in vivo.
[0046] In a third aspect of this invention, there is provided
a method for preparing a vaccine composition from the
immunostimulatory complex. The immunostimulatory complex or a
mixture of the immunostimulatory complex with the uncomplexed
immunogen may be formulated as a suspension in solution, a
water-in-oil emulsion, a suspension in combination with a
mineral salt suspension or a reconstituted suspension in a
biocompatible solution. The inununostimulatory complex alone
or in a mixture with uncomplexed immunogens may also be coformulated
in a biocompatible solvent in a polymeric gel.
[0047] This invention is further directed to the production of
useful immunogen delivery systems for administration by
various routes, including parenteral, oral, intranasal,
rectal, buccal, vaginal and transdermal routes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] The present invention is further understood with
reference to the drawings.
[0049] Figure 1 is a schematic showing the complexation
process of cationic peptide immunogens and CpG
oligonucleotides.
[0050] Figure 2 shows the typical size distribution for the
stabilized immunostimulatory complexes prepared from LHRH
peptide immunogens and CpGl oligonucleotides in various
proportions as determined by laser diffraction measurements.
[0051] Figure 3 is a schematic of the process for preparing a
water-in-oil (w/o) emulsion employing homogenization or
extrusion techniques.
[0052] Figure 4 shows a typical photomicrograph for a w/o
emulsion prepared via homogenization from ISA Montanide® 51
and LHRH:CpGl immunostimulatory complexes, wherein LHRH:CpGl
is 4:1 at a fixed final total peptide concentration of 100
ug/mL.
[0053] Figure 5 shows a typical ph6tomicrograph for a w/o
emulsion prepared via extrusion from ISA Montanide® 72-0 and
LHRH:CpGl immunostimulatory complexes, wherein LHRH:CpGl is
4:1, at a fixed final LHRH peptide concentration of 200 fig/mL.
[0054] Figure 6 is a schematic detailing the in-situ polymer
gel process employing reconstitution.
[0055] Figure 7 shows the serum IgG responses in guinea pigs
immunized intramuscularly (I.M.) in accordance with the
immunization protocols described in Example 7.
[0056] Figure 8 shows the serum IgG responses in guinea pigs
immunized intramuscularly (I.M.) in accordance with the
immunization protocols described in Example 7. No sera were
obtained for the animals immunized with the immunostimulatory
complex derived from CD4 peptides and CpG2 on week 17. This
is indicated by an asterisk in Figure 8.
[0057] Figure 9 shows the serum IgG responses in guinea pigs
immunized intramuscularly (I.M.) in accordance with the
immunization protocols described in Example 7.
[0058] Figure 10 shows the serum IgG responses in guinea pigs
immunized intramuscularly (I.M.) in accordance with the
immunization protocols described in Example 7. No sera were
obtained for the animals immunized with the immunostimulatory
complex derived from CD4 peptides and CpG2 on week 17. This
is indicated by an asterisk in Figure 10.
[0059] Figure 11 shows the serum IgG responses in guinea pigs
immunized intramuscularly (I.M.) in accordance with the
immunization protocols described in Example 8.
[0060] Figure 12 shows the serum IgG responses in guinea pigs
immunized intramuscularly (I.M.) in accordance with the
immunization protocols described in Example 8.
[0061] Figure 13a shows the serum IgG responses and 13b shows
the total serum testosterone in male rats immunized
intramuscularly (I.M.) in accordance with immunization
protocols as described in Example 10.
[0062] Figure 14a shows the serum IgG responses and 14b and
14c shows the total serum testosterone in male baboons
immunized intramuscularly (I.M.) in accordance with
immunization protocols as described in Example 11.
[0063] Figure 15a shows the serum IgG response, 15b shows the
total serum testosterone and 15c shows the average weight gain
per group in boars immunized intramuscularly (I.M.) over the
trial period in accordance with immunization protocols as
described in Example 13.
[0064] Figure 16 is a schematic detailing the process of
preparing a mixed suspension of immunostimulatory complex and
a mineral salt.
DETAILED DESCRIPTION OF THE INVENTION
[0065] In accordance with a first aspect of the invention, a
cationic peptide immunogen is complexed with an anionic
single-stranded DNA to form a stable immunostimulatory
complex.
[0066] The cationic peptide immunogen is a peptide with a net
positive charge at a pH in a range of 5.0 to 8.0. The net
charge on the peptide or peptide cocktails is calculated by
assigning a +1 charge for each lysine (K), arginine (R) or
histidine (H), a -1 charge for each aspartic acid (D) or
glutamic acid (E) and a charge of 0 for the other amino acids
in the sequence. The charge contributions from the N-terminal
amine (+1) and C-terminal carboxylate (-1) end groups of each
peptide effectively cancel each other when unsubstituted. The
charges are summed for each peptide and expressed as the net
average charge. Preferably, the net average charge of the
peptide immunogen is at least +2.
[0067] The cationic peptide immunogen may intrinsically have a
net positive charge as calculated above based on its amino
acid sequence. It may be made to have a positive charge by
the addition of a lysine, an arginine or a histidine or a
mixture of these amino acids to the N-terminal or C-terminal
of the peptide immunogen. Other synthetic moieties, such as
polyethyleneimine or polyamines, which provide a positive
charge to the peptide immunogen in aqueous solution, may also
be added.
[0068] The cationic peptide immunogen comprises a Th epitope
and a target B-cell epitope. The Th epitope may be intrinsic
to the peptide "or may be synthetically added to a peptide,
which functions as a target B-cell epitope. Suitable peptide
immunogens include peptides that elicit protective or
therapeutic immune responses and are derived from pathogens or
proteins known to cause diseases. These include: human or
animal IgE peptides for the immunotherapy of allergies, e.g.,
the IgE peptide immunogens described in WO 99/6729351; HIV
peptides for protective immunity and immunotherapy for HIV
infection described in US 5,763* 160s2; CD4 peptides for
protective immunity from HIV and immunotherapy of HIV
infection and immune disorders described in US 6,090,388s3;
Luteinizing Hormone Releasing Hormone (LHRH) peptides for
immunotherapy of androgen and estrogen-dependent tumors,
contraception and inimunocastration, and removal of boar taint
described in US 5,749,551s" and US 6,025,468SS; (3-amyloid
peptides for prevention and immunotherapy of Alzheimer's
Disease described in USSN 09/865,294s6; foot-and-mouth disease
virus peptides for protective immunity against foot-and-mouth
disease described in US 6,107,021s7; peptides from bacterial
pili for protective immunity from urinary tract infection
described in USSN 09/747802s8; -Plasmodium peptides for
protective immunity from malaria described in WO 99/669S759;
and somatostatin peptides for growth promotion in livestock
described in WO 99/66950.60 The specific peptide immunogens
named herein are examples for purposes of illustration only
and are not to be construed as limiting the scope of the
invention in any manner.
[0069] The "anionic single stranded DNA" is a polynucleotide
or oligonucleotide that is negatively charged at a pH in the
range of 5.0-8.0. The net negative charge on the
polynucleotide or oligonucleotide is calculated by assigning a
-1 charge for each phosphodiester or phosphorothioate group in
the oligomer. A suitable anionic oligonucleotide is a singlestranded
DNA molecule with 8 to 64 nucleotide bases, with a
repeated CpG motif and the number of repeats of the CpG motif
is in the range of 1 to 10. Preferably, the CpG
immunostimulatory single-stranded DNA molecules contain 18-48
nucleotide bases, with the number of repeats of CpG motif in
the range of 3 to 8.
[0070] Preferably, the anionic oligonucleotide is represented
by the formula: 5' XrCGX2 3' wherein C and G are unmethylated;
and X1 is selected from the group consisting of A (adenine), G
(guanine) and T (thymine); and X2 is C (cytosine) or T
(thymine). Or, the anionic oligonucleotide is represented by
the formula: 5' (X3)2CG(X4)2 3' wherein C and G are
unmethylated; and X3 is selected from the group consisting of
A, T or G.. and X4 is C or T. The CpG oligonucleotide may be
modified at the 5' end with a phosphorothiorate or a thiolacetamido
glycopolymer.34
[0071] Most preferably, the CpG oligonucleotide is selected
from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT
TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer,
and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO:
2, a 24 base length .oligomer plus an ph6sphorothioate bridging
group {designated as n at the 5' end).
[0001] Moreover, it is known that the DNA sequences derived
from unmethylated cytosine-guanine (CpG) dinucleotides
activate lymphocytes and can enhance a subject's immune
responses, including IL-6, TNF-a, IL-12, IFN-yresponses33.
These molecules represent preferred complementary substrates
that can both stabilize the synthetic cationic peptide
immunogens and provide a novel immunogen delivery system,
based on these findings. The stabilizing immunostimulatory^
complexes of the present invention also provide selfadjuvantation
of the immune responses in vivo without
significant dilution at the site of injection.
[0073] The formation of discrete immunostimulatory complexes
derived from cationic peptide immunogens is principally a
function of charge neutralization. It is expected that stable
complexes may be formed from CpG-containing immunostimulatory
DNA molecules derived from both natural or synthetically
modified nucleotide sequences. Furthermore, improvements in
the stability of an immunostimulatory complex may be realized
by increasing the cationic charge residing on the peptide
immunogen. These include extending the peptides with
additional lysine, arginine or histidine or other synthetic
moieties, which provides a positive charge to the modified
peptide in an aqueous solution as described above.
[0074] It is expected that non-CpG containing
immunostimulatory sequences (ISS) will be identified and
contemplated that these single-stranded DNA substrates would
prove to be useful materials to form immunostimulatory
complexes from when combined with synthetic cationic peptide
immunogens in appropriate aqueous solvents.
[0075] Modified CpG motifs are also envisioned, wherein a
defined anionic single-stranded DNA has been chemically
conjugated to another biologically functional molecule, such
as lectins, sugars or lipids for enhancing cell-specific
uptake and targeting, or polymers, copolymers and graft
copolymers such as PEG for improved circulation in vivo. The
chemically conjugated DNA.may be polyanionic and may
subsequently be complexed with a cationic peptide immunogen to
provide a modified immunostlmulatory complex with potentially
novel physical or biological properties.34' 3S
[0076] It is contemplated that block and graft co-polymers
derived from polyanionic oligomers and polyethylene glycol '
represent another class of anionic molecules, which may also
provide improved stability and improved adjuvanticity.
[0077] In another aspect of this invention an
inununostimulatory complex may be prepared from a modified CpG
oligonucleotide, wherein an additional phosphorothioate or
another bridging group has been added at the 5' end of the
oligomer for improved complexation.
[0078] Preferably, the inununostimulatory complexes have an
average aggregate particle size distribution in the range of
about 1 to 50 pM. More preferably, the immunostimulatory
complexes have an average aggregate particle size distribution
in the range of about 1 to 30 pM. Most preferably, the
immunostimulatory complexes have an average aggregate particle
size distribution in the range of about 1 to 15 uM.
[0079] There is evidence that the number of CpG motif repeats
influences the degree of intrinsic adjuvanticity and immune
stimulation, with a minimum number of CpG repeats being
required. Moreover, there is strong evidence that the
selection of flanking nucleotide bases adjacent to the CpG is
very important, as this appears to directly impact the
adjuvanticity in a species-specific manner.10' 36 For example,
it was demonstrated by Kreig et aJ.13 that enhanced
immunostimulatory activity of human cells occurred when
oligonucleotides containing CpG motifs are represented by the
formula X1X2CGX3X4 where C and G were unmethylated, and X*X2
were selected from the groups GpT, GpG, GpA and ApA and/or X3X4
were selected from the groups TpT, CpT and GpT.
[0080] Although CpG oligonucleotides can function as B-cell
mitogens37 and are useful adjuvants, it has been shown that the
immune responses generally peak 2 weeks after administration
for an antigen mixed with CpG oligonucleotides in a soluble
form. This necessitates multiple repeat injections to
maintain high antibody titers to ensure protection.38 Thus, a
method for effectively delivering constructs with these
oligonucleotides in a controlled release formulation is
strongly desired.
[0081] With respect to stability, the phosphodiester bonds in
the CpG backbone are sensitive to degradation by nucleases in
vivo39. Thus to improve the duration of the immune response,
the phosphate groups may be modified to phosphorothioate
groups.
[0082] The immunostimulatory complex of the present invention
may be formulated for delivery by numerous pathways including
parenteral, mucosal and transdermal. The immunostimulatory
complex of the present invention is particularly desirable for
vaccine formulations in that the CpG oligonucleotides in the
complex are useful adjuvants for upregulating both parenteral
and mucosal responses in vivo.40' 41
[0083] The results of our experiments show that the aggregate
particle size of the immunostimulatory complex varies based on
the ratio of the peptide immunogen to the CpG oli-gonucleotide.
The intrinsic stability of the immunostimulatory complex and
the ability to control the size of the composition increases
the potential for phagocytosis by a parenteral route.19
Mucosal immunization by targeting specific cells, such as Mcells
located on Peyer's Patches via oral route21 or the nasalassociated
lymphoid tissue (NALT) via intranasal route23 is
similarly facilitated by the use of the stabilized immunogen
of the present invention.
[0084] The immunostimulatory complex of the present invention
is prepared by a controlled self-assembling process wherein
the anionic CpG oligonucleotide in aqueous solution is added
to an aqueous solution of the cationic peptide immunogen.
Suitable aqueous solutions for the preparation of an
immunostimulatory complex is selected from the group
consisting of distilled deionized water (DOW), normal saline
(NS) or phosphate buffered saline (PBS). Distilled deionized
water and normal saline typically exhibit a pH of about 5.5,
whereas in PBS, the pH is controlled in a range of 7.2-7.4.
The complexation process is a function of the charge ratios,
the molecular weight of the interacting electrolytes, pH and
ionic strength of the medium.32
[0085] Multiply charged anionic molecules, such as the short
CpG oligomers possess a net negative charge when the pH is in
the range 5.5-7.4 in aqueous solutions. The net charge on the
peptide immunogen is dependent on the amino acid composition
of the peptide and may be affected by the pH of the aqueous
solution. Thus, the aqueous medium is selected to ensure a
net positive charge for efficient complexation. An
examination of the ionization point (IP) or point of zero
charge for the individual peptides can guide the selection
process. In general, the IP is determined by the motion of
the molecule through a pH-gradient in an isoelectric focusing
experiment.11 To ensure a peptide is positively charged, the
pH of the selected aqueous medium should be less than the
isoelectric point for the peptide in question.
[0086] To prepare an immunostimulatory complex the following
steps are followed. Firstly, the average molar positive
charge contribution is determined for a desired peptide
immunogen or for a cocktail of peptide immunogens based on the
molar ratios of peptides mixed together and the charge
contribution from each peptide component in the final vaccine
composition. Secondly, the molar negative charge contribution
is determined for the complexing oligonucleotide based on the
molar ratio of oligomer and the charge contribution from this
component in the final vaccine composition. Thirdly, the
amount of peptide immunogen, based on total average molar
positive charge, is dependent on the amount of oligonucleotide
employed for complexation and the total molar negative charge
thereof. This relationship is used to define the relative
amounts of peptide immunogens and oligonucleotides to be
combined in an aqueous solvent to form an immunostimulatory
complex. An excess of the cationic immunogen peptide may be
employed to provide a mixture of the immunostimulatory complex
and an excess of the uncomplexed peptide. Or, an excess of
the oligonucleotide may also be employed to provide an excess
of the oligonucleotide. The relative amounts of the peptide
immunogen and the oligonucleotide selected are based on the
vaccine formulation desired.
[0087] Finally, the calculated amount of anionic
oligonucleotide in a compatible aqueous solvent is added with
mixing to the calculated amounts of cationic peptide
immunogens similarly dissolved in a compatible aqueous
solvent. The amount in nmol of the cationic peptide immunogen
used is generally in a range to provide 8 positive charges to
0.5 positive charge to the amount in nmol of the anionic
oligonucleotide to provide one negative charge. This is
referred to as the charge ratio. Where the charge ratio is
8:1, there is a large excess of the peptide immunogen. Where
the charge ratio is 1:2, there is a moderate excess of the
anionic oligonucleotide. The complex forms spontaneously in
•the form of a suspension in solution. Estimation of the
residual amounts of peptide immunogens or oligonucleotides can
be made by separating the complex from the solution and
assaying the supernatant solutions by ultraviolet
spectrophotometry (UV) or by reverse phase high performance
chromatography (RP-HPLC).
[0088] The immunostimulatory complex as prepared as a
suspension may be used as a vaccine composition. If the
immunostimulatory complex is to be injected parenterally, t'he
aqueous solvents are selected such that the final vaccine
composition is isotonic and suitable for such a purpose. In
cases where the complex is first formed in distilled deionized
water, aqueous buffers of suitable salt concentration are
added to ensure the final vaccine composition is isotonic.
[0089] The immunostimulatory complex prepared as a suspension
or solution may be lyophilized. The lyophilized composition
may then be reconstituted and incorporated into different
vaccine formulations in accordance with the desired mode of
delivery. The immunostimulatory complex of the present
invention may also be formulated as a water-in-oil emulsion,
in combination with a mineral salt suspension or a
biocompatible polymeric gel.
[0090] In accordance with a further aspect of the invention,
the invention describes a process for isolating the stabilized
immunostimulatory complexes as stable particles via
lyophilization. Reconstitution of the stabilized
immunostimulatory complex as a suspension in aqueous solvents
or biocompatible solvents shows essentially no changes in the
particle size distribution or in vivo potency. This
represents an important advantage over formulations requiring
refrigeration to maintain efficacy, such as Alum-based vaccine
compositions. This feature extends the potential utility of
these systems to include direct reconstitution prior to
immunization and alternative modes of delivery which require
stable solid state dosage forms, such as a dry powder aerosol
or nebulization for pulmonary or intranasal delivery.42
[0091] In accordance with a further aspect of the present
invention, the invention provides various processes for
preparing stable water-in-oil emulsions43 comprising the
stabilized immunostimulatory complex of the present invention.
In such an emulsion, preferably the aqueous phase comprises
the immunostimulatory complex or a mixture of the
immunostimulatory complex with the uncomplexed peptide
immunogen; and the continuous oil phase comprises a synthetic,
mineral, animal or vegetable oil. Additionally, the oil phase
may also comprise an immunostimulatory emulsifier, a
biocompatible or a metabolizable component.
[0092] In particular, the oils useful for preparing the waterin-
oil emulsions of the present invention include, but are not
limited to, synthetic oils (e.g. isopropyl myristate),
vegetable oils (e.g. peanut oil), mineral oils (e.g. Drakeol™
or Marcol™), metabolizable animal oils (e.g. squalene or
squalane), and a mixture thereof. The mineral oils are
preferred. The oil-based emulsifiers useful for stabilizing
the emulsions include, but are not limited to, the family of
mannide oleates and derivatives thereof.
[0093] The relative amount of emulsifier required is a
function of the hydrophile-lipophile balance (HLB) and the
intrinsic stability of the water-in-oil emulsion produced
under specified conditions. Methods for selecting oils and
combinations of emulsifiers are well known to those skilled in
the art.
[0001] The w/o-emulsion may comprise between 10 v/v% and 80
v/v% water in the internal aqueous phase. For most purposes,
the optimal water concentration is in the range of 30 v/v% and
50 v/v%. The internal aqueous phase is characteristically
comprised of very fine droplets with sizes typically in the
range of 1-10 jiM, preferably 1-5 uM. The preparations are
stable when maintained at room temperature or refrigerated.
[0095] Other stabilizing agents may also be used to prepare
the emulsion. These include surfactants, colloidal particles,
proteins, and other polymerizing and stabilizing agents knoton
to those skilled in the art.
[0001] The w/o-emulsion may further comprise at least one oilsoluble
lipophilic adjuvant such as 3-0-desacyl-4'-
monophosphoryl lipid A (MPL), W-acetyl-muramyl-L-alanyl-Disoglutamine
(MDP), Dimethyldioctadecylammonium bromide (DDA),
N, W-dioctadecyl-W, N' -bis(2-hydroxyethyl) propanediamine
(Avridine), N- (2-Deoxy-2-l-leucylamino-p-D-glucopyranosyl) -Noctadecyl-
dodecanoylamide hydroacetate (BAY-1005), 3p -[N-
(N, W-dimethylaminoethane)-carbamoyl] cholesterol (DC-Choi),
NAc-Mur-L-Thr-D-isoGln-sn-glycerol dipalmitoyl (Murapalmitine)
and mixtures or derivatives thereof. The w/o-emulsion may
also comprise in the dispersed phase at least one watersoluble
adjuvant, e.g., poly(di(carboxylatophenoxy)]
phosphazene(PCPP), Quillaja saponin (QS-21), Cholera Holotoxin
(CT) or Cholera Toxin B subunit(CTB), heat labile Enterotoxin
from E. Coli (LT) or heat labile Enterotoxin B subunit from E.
Coli (LTB) and cytokines such as Interleukin-1 p (IL-lfi),
Interleukin-2 (IL-2), Interleukin-12 (IL-12), interferon-y
(IFN-y)and mixtures and derivatives thereof. The water-soluble
adjuvant may be synthetic or natural. The presence of a water
soluble adjuvant with film forming properties, such as an
oligomer or polymer, can additionally serve to stabilize the
emulsion. The w/o-emulsion can facilitate presentation of the
immunogens to the immune system to provide a more efficacious
vaccine.
[0097] A water-in-oil emulsion comprising an immunostimulatory
complex, or a mixture thereof with uncomplexed immunogen may
be prepared as follows. Firstly, an immunostimulatory complex
is prepared from a peptide immunogen and an oligonucleotide in
a ratio to ensure the formation of the immunostimulatory
complex alone or in a mixture with excess residual peptide
immunogen in an aqueous solution. Secondly, the aqueous
solution is mixed with an oil containing emulsifiers and
homogenized to provide a water-in-oil emulsion wherein the
aqueous phase is dispersed in a continuous oil phase. The
water-in-oil emulsion as such is suitable for parenteral
injection.
[0098] Emulsification of the aqueous and oil phases can be
accomplished by homogenization or by transfer between two
syringes or by extruding the components through a membrane
filter of a controlled pore size. The low-energy serai-manual
methods are rapid. However, because there is considerably
less shear than other processes, the emulsion produced is not
as fine as that produced using high shear mechanical systems.
Examples of high-shear systems include rotostators,
microfluidizers, and sonifiers. Other devices similar to
these high-shear systems that are well known for
emulsification may also be employed.
[0099] In accordance with a further aspect of the present
invention, the invention provides various processes for
preparing physiologically acceptable suspensions of mineral
salts comprising the stabilized immunostimulatory complex of
the present invention. In such a mixed system, the aqueous
phase comprises a combination suspension of mineral salt and
immunostimulatory complex, which may additionally contain
residual, unbound peptide immunogens in solution.
[00100] In particular, the mineral salts useful for preparing
the full aqueous-based suspensions of the present invention
include, but are not limited to, Aluminum hydroxide (e.g.,
Alhydrogel®, Rehydragel HPA®, Rehydragel LV®} , Aluminum
phosphate (e.g., Adju-phos® or Rehyraphos®) or calcium
phosphate (e.g., Calphos®), and mixtures thereof.
[00101] Methods for selecting mineral salts and determining the
preferred concentration of mineral salt to employ or
combinations thereof are well known to those skilled in the
art.
[00102] Other stabilizing agents may also be used to prepare
the mineral salt suspension. These include surfactants, antioxidants,
physiological acceptable buffers, tonifiers,
preservatives and other agents known to those skilled in the
art.
[0001] The mineral salt suspension may further comprise at
least one additional adjuvant (e.g. MPL, MDP, DDA, N,NAvridine,
BAY-1005, DC-Choi, Murapalmitine, PCPP, QS-21, CT or
CTB, LT or LTB and cytokines such as IL-lli, IL-2, IL-12, IFNyand
mixtures and derivatives thereof). The mineral salt can
facilitate presentation of the immunogens to the immune system
in the form of a depot or attract specific cells of the immune
system by a process known as chemotaxis.
[00104] A mineral salt suspension comprising an
immunostimulatory complex, or a mixture of immunostimulatory
complex in combination with residual uncomplexed immunogens
may be prepared as follows. Firstly an immunostimulatory
complex is prepared from a peptide immunogen and an
oligonucleotide in a charge ratio to ensure the complete
complexation of all peptide immunogens and the oligonucleotide
in solution. Alternatively, an immunostimulatory complex is
prepared from a peptide immunogen and an oligonucleotide in a
charge ratio to ensure partial complexation of the peptide
immunogens and oligonucleotide components in solution;
Secondly, the aqueous suspension is combined with a mineral
salt suspension with mixing to provide a full aqueous
suspension of all components. The suspension combination as
such is suitable for parenteral injection.
[00105] In a complementary method, a mineral salt suspension
comprising an immunostimulatory complex, or a mixture thereof
with uncomplexed peptide immunogens may be prepared as
follows. Firstly, peptide immunogen is mixed with a mineral
salt suspension. Depending on the physical properties of the
mineral salt, peptide immunogens and aqueous buffer various
proportions of immunogen may be absorbed directly by the
mineral salt at this stage; Secondly, to this suspension is
added an oligonucleotide with stirring. Partial or full
complexation of the residual unbound peptide immunogens in
solution results. The suspension combination as such is
suitable for parenteral injection. In Figure 16, both methods
of preparation are shown.
[00106] In accordance with another aspect of this invention,
there is provided a process for preparing an in-situ gelling
biodegradable polymer in which a stabilized immunostimulatory
complex or a mixture of a stabilized immunostimulatory complex
and uncomplexed immunogen is dispersed. The immunostimulatory
complex may be dispersed either in solution or as a suspension
within a biocompatible solvent. The biocompatible solvent may
further comprise a soluble adjuvant that is synthetic or
natural. The solution or suspension in the biodegradable
gelling polymer is designed for the delivery of the immunogen
to a host. The in-situ gelling polymer is biodegradable and
is a copolymer of poly-D,L-lactide-co-glycolide {PLG) and
poly-D,L-lactic acid-co-glycolic acid weight in the range of about 2,000 to about 100,000 daltons
and an inherent viscosity of about 0.2 to 1.0 dl/g. The
formula of the in-site gelling polymer is:
(Figure Removed)
wherein Rl is OH or alkoxy having 1 to 5 carbons and R2 is H;
x:y is the ratio of each monomer unit of the copolymer with
x+y=l.
In the case of PLG, Rl is alkoxy and the monomer units are
lactide and glycolide and in the case of PLGA, Rl is OH and
the monomer units are lactic acid and glycolic acid.
[00107] The stabilized inununostimulatory complex or a mixture
thereof with the uncomplexed immunogen with the in~situ
gelling polymer may be prepared as a single phase or as a
suspension in a biocompatible solvent.
[00108] The biocompatible solvent useful in the present
invention is selected from the group consisting of dimethyl
sulfoxide (DMSO), N-methyl pyrrolidine (NMP), triacetin and
glycerin. DMSO is preferred. DMSO has a high capacity for
solubilizing a large quantity in weight percent of the
polymer. It has been widely used as a solvent for in-situ
gelling of polymers. DMSO may be also be used to prepare a
suspension of the stabilized immunostimulatory complex of the
present invention.
[00109] Importantly, it has been demonstrated in animal models
that there is a high tolerance for DMSO when used in small
amounts.1'1 Thus, toxicity concerns are minimal when
compositions comprising DMSO are administered via a parenteral
route.
[00113] In accordance with the present invention, a controlled
or delayed release vaccine preparation in stable form is
provided together with a method of making such a vaccine
preparation. The gel matrix of the controlled or delayed
release vaccine composition comprise a biodegradable polymer
selected from the group consisting of poly-D, L-lactide-co-'
glycolide (PLG) and poly-D, L-lactic acid-co-glycolic acid
(PLGA), polycaprolactones, polyanhydrides, polyorthoesters and
poly(a-hydroxybutyric acid), a biocompatible solvent and a
stabilized immunostimulatory complex.
[00114] The polymeric gel may further comprise at least one
additional adjuvant, e.g., MPL, MDP, DDA, A7, N- Avridine, BAY-
1005, DC-Choi, Murapalmitine, PCPP, QS-21, CT or CTB, LT or
LTB or a cytokine such as IL-IJi, IL-2, IL-12, IFN-yand
mixtures and a derivative thereof.
[00115] Advantages of the controlled released composition of
the present invention include:
(a) a. fully biodegradable and biocompatible gel
formulation;
(b) a sustained release of the immunogen for
presentation to the immune effector cells resulting
in improved immunogenicity;
(c) a high loading of the gel with a desired immunogen
in a stable composition; and
(d) a flexible mode of delivery including a suspension
of a stabilized immunostimulatory complex that is
self-adjuvanting.
[00116] The molecular weight and crystallinity of the polymer
directly impacts the entrapment efficiency in vivo. The
polymeric gelling material is miscible in polar aprotic
solvents such as DMSO. However, upon intramuscular or
subcutaneous injection, DMSO is extracted into the surrounding
body tissues with water reversibly penetrating the polymer
rich solution. This process serves as the primary mechanism
controlling in-situ gel formation. The rate at which this
process proceeds directly affects the initial burst of rele'ase
of the immunogen during the time interval when the
biocompatible solvent is actively extracted into body tissue
and exchanged with physiological solutions and before the
entrapment of the immunogen by the formation of the gel.45 It
is known that controlling the crystallization process is an
important key mechanism by which the retention -of immunogens
within the gel can be improved.46 This is intimately connected
to the internal morphology of the gel formed which limits the
diffusional pathways by which immunogens may be released.
[00117] The entrapped or retained immunostimulatory complexes
are subsequently released from the gel in limiting amounts in
a sustained fashion with a larger boost released when the bulk
of the matrix forming polymer is eroded. This varies
depending on many of the same conditions that influence
gellation, such as molecular weight, degree of crystallinity,
constitution, hydrophobicity, and the presence of additives.
[00118] The potential for an adverse toxicological response to
the solvent DMSO has been addressed in a recent study44 wherein
a device containing a peptide hormone suspended in DMSO was
surgically implanted subcutaneously in a dog and in a human.
The volume of DMSO employed in the study was 150uL. The
implant was designed to release the peptide over the course of
1 year and surgically removed at the end of the study. In
neither the dog or the human were any adverse tissue reactions
observed. The controlled release of the peptide/DMSO mixture
from the implant into physiological tissues is useful as a
model for evaluating potential toxicity concerns for a fully
biodegradable in-situ gelling polymeric composition. It is
contemplated that the amount of DMSO useful in the present
invention is essentially the same as that used in the study.
[00119] It is expected that there is an initial extraction of
the immunostimulatory complex in DMSO into body tissue before
the solubility limit of the polymer is exceeded.
Solidification takes place, retarding the release of the
stabilized immunostimulatory complex. Subsequently, the
stabilized immunostimulatory complex is released along with
DMSO by diffusion-controlled pathways or retained in the gel
with a release rate that is a function of the polymer
properties. It is apparent that the diffusivity of these
molecules within the gel is governed by several factors, such
as the internal gel morphology and porosity, the degree of
penetration by water into the gel and the hydrolysis of the
bulk of the polymer.45
[00120] in the case of the stabilized immunostimulatory complex
of the present invention, which is dispersed as a suspension
throughout the gel, the initial extraction is largely that: of
DMSO with a small quantity of the immunostimulatory complex
located near the gelling front. Thus, the small amount of the
immunostimulatory complex, not effectively entrapped during
the initial gelling phase, would be responsible for the
initial priming of the immune response in vivo. Thereafter,
it is expected that DMSO would continue to be released by
diffusion, with the bulk of the immunostimulatory complex
remaining entrapped in the gel matrix until the bulk of the
polymer has sufficiently been hydrolyzed to result in a full
release of the encapsulated immunostimulatory complex.
[00121] Specifically-, polymeric gels formulated from higher
molecular weight and more crystalline polymers derived from ochydroxy
acids degrade over a longer period of time than lower
molecular weight amorphous analogs. This phenomenon, is known
to be related to the accessibility of water to the
hydrolytically unstable ester bonds.46
[00122] It has been established that random amorphous
copolymers composed of 50% D,L-lactide and 50% glycolide with
a molecular weight in the range of from 8,000-50,000 daltons,
exhibit the highest degradation rates. 50% by weight or less
of the polymer remains after approximately 6-8 weeks, when
immersed in a PBS buffer.47
[00123] It is desirable to modify the parameters controlling
the rate of gellation, the kinetics of immunogen release and
the internal gel morphology. Specifically, use of various
pore forming agents, plasticizers, and stabilizers such as
surfactants, sugars, proteins, polymers and other excipients
known to those skilled in the art are useful for this purpose.
[00124] The preparations are stable when refrigerated or at
room temperature. The vaccine composition of the present
invention employing DMSO will freeze when refrigerated since
the freezing point of DMSO is about 18°C. It has been found
that thawing does not cause a change in the efficacy of the
vaccine in vivo.
[00125] In accordance with the present invention, the in-situ
gelling biodegradable polymer and the stabilized
immunostimulatory complex may be formulated separately. The
in-situ gelling polymer may be solubilized in a biocompatible
solvent in one vial, and the stabilized immunostimulatory
complex in dry form in a separate vial. The immunostimulatory
complex in dry form may be prepared by spray-drying, or
preferably, lyophilization.48' 49 The dried immunostimulatory
complexes can then be reconstituted in a biocompatible
solvent, and dispensed by syringe either as a solution or as a
suspension into the biodegradable polymer solution. The
mixture is then used immediately or soon thereafter for
immunization.
[00126] Employing separate vials has an added advantage of
minimizing any potential stability problems. These include
polymer degradation in the presence of the peptide immunogeri,
which may have reactive functional side chains, such as, free'
amine groups or carboxylate groups,50 or the oxidation of
selective amino acids, such as cysteine and tryptophan in the
peptide immunogen in the presence of DMSO.1
[00127] To prepare a reconstitutable in-situ gelling polymer
composition containing an immunostimulatory complex, an
immunostimulatory complex is prepared as described above.
Then, this aqueous solution is lyophilized. to form a dried
composition. The dried composition is then reconstituted as a
suspension in a biocompatible solvent containing a calculated
weight percentage of a biodegradable gelling polymer. The
final vaccine composition represents an in-situ gelling
polymer and is suitable for parenteral injection.
[00128] The compositions of the present invention are
contemplated to be useful as vaccines or for therapeutic
purposes. Other biological materials which may be modified to
possess cationic charge at pH's generally ranging from 4.0-8.0
for efficient complexation with CpG oligonucleotides, may
include proteins, protein mimetics, bacteria, bacterial
lysates, viruses, virus-infected cell lysates, antigens,
antibodies, pharmacological agents, antibiotics,
carbohydrates, lipids, cytokines, chemokines, lipidated amino
acids, glycolipids, haptens and combinations and mixtures
thereof.
[00129] These compositions can be administered parenterally via
subcutaneous or intramuscular injection. When administered,
parenterally, the immune response may be a cell-mediated
response or a local or serum antibody response to provide
neutralizing antibodies. For compositions administered
mucosally the immune responses would additionally include a
local secretory antibody response.
[00130] It will be readily apparent to those skilled in the art
that immunostimulatory complexes may also be admixed in oil- in-water encapsulation of stabilized immunostimulatory complexes within
water-in-oil-in-water (w/o/w) double emulsions, biodegradable
polymeric microparticles, lipid vesicles or liposome
structures. Most of these delivery systems are attractive for
the development of sustained release formulations.
[00131] Preferably, the peptide immunostimulatory complex of
the present invention may be employed in w/o-based emulsions
employing SEPPIC's oil-based adjuvants, in combination with
mineral salts such as Aluminum hydroxide, Aluminum phosphate
and Calcium phosphate or in a single-dose controlled release
in-situ gelling formulation based on biodegradable poly-D,Llactide-
co-glycolide (PLG) or poly-D,L-lactic acid-co-glycolic
acid (PLGA) copolymers.
[00132] Most preferably, the vaccine compositions of the
present invention comprise a stabilized immunostimulatory
complex of the peptide immunogen with a CpG oligonucleotide
admixed with the uncomplexed peptide immunogen.
[00133] It is clearly apparent to one skilled in the art, that
the various embodiments of the present invention have many
applications in the fields of medicine and in particular
vaccination, diagnosis and treatment of infections with
pathogens including bacteria and viruses. Further uses of the
present invention are described below.
Vaccine Preparation
[00134] Immunogenic compositions, suitable to be used as
vaccines, may be prepared from an immunostimulatory complex of
the present invention, as a w/o emulsion, as a suspension in
combination with a mineral salt suspension or as an in-situ
gelling polymer or a combination of these systems as disclosed
herein. The immunogenic composition containing the
immunostimulatory complex is useful for eliciting an immune
response by the host to which it has been administered. The
immune response includes the production of antibodies by the
host.
[00135] The immunogenic composition may be prepared as
injectables, as liquid solutions or suspensions, as
lyophilized or spray-dried powders or emulsions. The
composition comprising the immunostimulatory complex may be
mixed with physiologically acceptable buffers or excipients,
such as, water, saline, dextrose, glycerol, ethanol and
combinations thereof. The vaccine may further contain
additional substances such as wetting or emulsifying agents,
pH buffering agents, or adjuvants to further enhance the
effectiveness thereof. The vaccine may further contain
additional biocompatible substances, specifically in
conjunction with the in-situ gelling polymers such as dimethyl
sulfoxide (DMSO), N-methyl pyrrolidine (NMP), triacetin,
glycerin, and poly vinyl pyrrolidone (PVP).
[00136] The vaccine of the present invention may be
administered parenterally, e.g., by injection subcutaneously,
intramuscularly or transdermally. The vaccines of the present
invention may be administered mucosally via oral, intranasal,
rectal, vaginal or ocular routes.
[00137] The vaccines are administered in a manner compatible
with the formulation, and in such amount as to. be
therapeutically effective, protective and immunogenic. The
quantity to be administered depends on the subject or species
to be treated, including, for example, the capacity of the
subject's or species' immune system to synthesize antibodies,
and if needed, to produce cell-mediated immune responses.
[00138] Precise amounts of emulsifying oils, mineral salts or
gelling polymers and material having biological activity
required to be administered for effect depend on the judgement
of the practitioner or veterinarian. However, suitable dosage
ranges are readily determinable by one skilled in the art and
may be of the order of micrograms to milligrams. Suitable
regimes for initial administration and booster doses are also
variable, but may include an initial administration followed
by subsequent administrations. The dosage of the vaccine may
also depend on the route of administration and thus vary from
one host or species to another.
EXAMPLES
[00139] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific Examples. These Examples
are described solely for purposes of illustration and are not
intended to limit the scope of the invention. Changes in the
form and substitution of equivalents are contemplated as
circumstances may suggest it as necessary to achieve a
particular goal. Although specific terms have been employed
herein, such terms are intended in a descriptive sense and not
for purposes of limitations.
[00140] Methods of chemistry, organic chemistry, polymer
chemistry, protein biochemistry and immunology used but not
explicitly described in this disclosure and these Examples are
amply reported in the scientific literature and are well
within the ability, of those skilled in the art.
Preparation of Immunostimulatory complex
[00141] In general, an immunostimulatory complex of a synthetic
peptide immunogen and a CpG oligonucleotide in aqueous
solutions is prepared by the dropwise addition of a stock
peptide solution in an appropriate aqueous solvent into a vial
containing a gently stirred stock solution of CpG
oligonucleotide dissolved in an appropriate aqueous solvent.
The reverse mode of addition is equally effective. Compatible
aqueous solvents include, but are not limited to, distilled '
deionized water, normal saline (NS =0.9% NaCl) or phosphate
buffered saline (PBS = 10 mM Phosphate buffer, 0.9% NaCl) or
mixtures thereof. The complexation process is largely
unaffected by physiologic buffers, providing flexibility when
selecting a compatible solvent system for both the synthetic
peptide inununogen and the CpG oligonucleotide.
[00142] The complex forms immediately and can be identified
visually by the observation of a fine precipitate suspended in
solution. The quantity of suspension so formed is a function
of the relative amounts of the CpG oligonucleotide to the
cationic peptide in solution. The precipitation process is
controlled by the electrostatic neutralization of oppositely
charged molecules. In a thermodynamically favourable process,
the highly charged polyanionic single-stranded DMA binds with
the positively charged cationic peptide immunogen.61
[00143] The CpG oligonucleotide is selected from a group
consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3'
(CpGl) SEQ ID NO: 1, a 32 base length oligomer, and 5'nTC GTC
GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO: 2, a 24 base
length oligomer plus an phosphorothioate group (designated as
n at the 5' end). The CpG oligonucleotides were synthesized
by Oligo's Etc. (Wilsonville, Oregon), and are obtained in a
lyophilized dry state. These materials were reconstituted in
the appropriate aqueous solvent prior to use. CpGl possesses
a CpG motif sequestered within a sequence of 8 nucleotide
bases and may provide stronger adjuvantation in vivo and
improved stability by binding cationic peptides with higher
affinities, than shorter oligonucleotides. A phosphorothioate
modified group at the 5' end of CpG2 increases the molar
negative charge density and potentially promotes improved
binding.
[00144] The peptide immunogens were synthesized and the
appropriate aqueous buffer used to ensure that the peptide is
cationic in solution. This is an important consideration in
vaccines where complexation of the peptide immune-gen to the
CpG oligonucleotide is desired. The ionization point or IP
for each peptide immunogen and the pH of the medium is used to
guide the selection of the appropriate buffer. The pH for an
aqueous mixture of a stock peptide solution dissolved in
distilled deionized water or normal saline (NS) was
approximately 5.5, whereas in phosphate buffered saline (PBS)
the pH of the stock peptide solutions was significantly higher
at approximately 7.2. Careful selection of aqueous solvent
systems is made to ensure full protonation for peptides
derived from amino acids with weakly basic side chains,
notably Histidine.
[00145] Table 1 list the physical properties of the synthetic
peptide immunogens and CpG oligonucleotides used to form
immunostimulatory complexes. Three exemplary peptide
immunogen targets are depicted in Table 1. A cocktail of two
or three peptide immunogens or in one case a combinatorial
library of peptides containing analogs of the peptide have
been employed to prepare each vaccine. Each peptide immunogen
comprises two segments, a B-cell target epitope and a T-helper
epitope. The Th epitope is included to improve the
immunogenicity of the peptide immu-riogen.
[00146] The B-cell and T-help epitopes were selected after
screening libraries of peptides in the appropriate animal
models. Detailed information regarding the identification and
composition of these constructs can be found by referring to
US patent Nos. 6,090,38853, US patent No. 5,759,551s' and
WO99/6729351 and US 6, 107, 021. 5V SEQ ID NOS: 7-9 in Table 1
comprise the LHRH immunogen peptides and are useful in a
vaccine for prostate cancer immunotherapy, designed for
hormone ablation treatment. SEQ ID NOS: 10-11 are useful In
an anti-IgE immunotherapeutic vaccine for the treatment of
allergy. SEQ ID NOS: 4-6 are useful in an anti-CD4
immunotherapeutic vaccine for the treatment of HIV infection.
SEQ ID NOS: 12-13 comprise a combinatorial library of FMD
peptides and are useful in an anti-FMD vaccine for protective
immunity against foot-and-mouth disease.
[00147] The immunostimulatory complex of the present invention
may be prepared with various ratios of cationic peptides to
CpG oligonucleotides to provide different physical properties,
such as the size of the microparticulate complexes. Table 2
shows the calculated average molar positive charge and average
formula weight for the peptide immunogen in the mixture.
Table 2 also provides the calculated average molar negative
charge contribution from CpGl (S.EQ ID NO: 1) and CpG2 NO: 2), respectively.
Example 1: Preparation of Tmmunostimulatory complex of LHRH
Immunogens and CpGl Oligonucleotides
[00148] This Example illustrates the preparation of
immunostimulatory complex from LHRH peptide immunogens and
CpGl oligonucleotides in various proportions. A flow diagram
of the process of complex formation as described herein is
shown in Figure 1.
[00149] All glassware, stir bars and pipette tips were
autoclaved for 40 minutes at 121°C prior to use. -All reagents
were weighed, dispensed, transferred or added to reaction
vessels in a laminar flow hood to prevent contamination.
[0001] A LHRH peptide immunogen stock solution was prepared by
mixing a 1:1:1 molar ratio of peptides of SEQ ID NOS: 7-9 at a
concentration of 3 mg/mL in distilled deionized water. 33 jaL
of the stock solution (100 jig of the peptide immunogens) was
added to each of a series of 2 mL vials equipped with micro
stir bars. To this solution was added 0.5 mL of distilled
deionized water as a diluent. A stock solution of 2.0 ng/|iL
of CpGl oligonucleotide was prepared in distilled deionized
water. Various amount of the CpGl oligonucleotide stock
solution was added to each vial to form the immunostimulatory
complex. The amount of CpGl oligonucleotide added to each
vial was determined by calculation to provide a charge ratio
of LHRH:CpGl ranging from 8:1, with a large excess of LHRH, to
1:2 with an excess of CpGl. The respective amounts of CpGl
used to prepare these compositions are shown in Table 3. It
is to be noted that the ratio of LHRH:CpGl are represented as
molar charge ratios and is based on the calculations shown in
Table 3.
[00151] The additions were made at room temperature with
continuous stirring and equilibrated for 30 min. In all
cases, an immediate clouding of the reaction mixture was
observed upon addition of the CpG oligonucleotide stock
solution. After complete addition of the CpGl
oligonucleotide, a fine white particulate suspension was
observed. The particles gradually settled and could be easily
resuspended with gentle shaking.
[0001] The solid microparticulate complexes can be essentially
removed after settling and allows the separated supernatant
solutions to be analyzed'by ultraviolet spectroscopy for
residual uncomplexed peptide immunogens (at X = 280 nm) or for
residual CpGl oligonucleotide (at A. = 260 nm) . For the
immunostimulatory complexes prepared using an excess of LHRH,
wherein LHRH:CpGl = 8:1, 4:1 or 2:1, excess amounts of peptide
were detected.
[00153] The result obtained by ultraviolet spectroscopy is an
estimate and may be ±20% of the number obtained for the
following reasons. The peptide chromophores have sizeably
smaller extinction coefficients as compared to CpG
oligonucleotides and the wavelength maxima used to detect the
peptides and CpGl are fairly close to each other. Thus, the
estimates for free residual peptide may possibly be
exaggerated. Further, a small amount of nanoparticles of
peptide CpG complex may be present in the supernatant. The
interpretation of these results is further complicated by the
observation that increasing the excess amount of the peptide
immunogen relative to CpG generally results in complex
aggregates with smaller average particle sizes.
[0001] It is observable from Figure 2 that the ranking of
LHRH/CpGl complexes with respect to average aggregate particle
size distribution is in the order LHRH:CpGl 2:1 > 4:1 > 8:1.
It is expected that the efficiency of the complexation process
would vary based on the physical properties of the peptide
immunogens and CpG oligonucleotides selected and the relative
ratio of each in the vaccine composition. For the LHRH:CpGl
system, the residual levels of uncomplexed peptide as
determined by UV spectroscopy range from 60-90% (LHRH:CpGl =
8:1), 40-80% (LHRH:CpGl = 4:1} and 25-65% LHRH:CpGl = 2:1)
over the background, respectively. For the LHRH:CpGl complex
prepared at a 1:1 charge ratio there was very little
detectable concentration of residual peptide immunogen, ~3%,
or residual CpG oligonucleotide, r2%.' The large increase in
the aggregate size of this complex coupled with the
essentially complete complexation of immunogen is consistent
with expected polyelectrolyte behavior at neutral charge. For
the immunostimulatory complex LHRH:CpGl = 1:2 charge ratio, an
excess of CpGl, 48% residual level of CpGl was found at X =
260 nm. This amount of residual CpGl approximates the
quantity of CpGl expected if the first equivalent of CpGl was
fully complexed with the peptide immunogen in solution.
[00155] The results of the UV method demonstrate that
immunostimulatory complex compositions prepared with a high
excess of peptide to oligonucleotide (e.g. LHRHrCpGl =8:1
charge ratio) results in a significant amount of peptide free
in solution. Similarly, immunostimulatory complexes prepared
from a moderate excess of oligonucleotide to peptide (e.g.
LHRHrCpGl = 1:2 charge ratio) result in compositions with
excess free oligonucleotide. The presence of excess
oligonucleotide can serve to stabilize smaller aggregates as
shown in Figure 2.
[00156] This example demonstrates that there may be no
practical advantages to preparing immunostimulatory complex
with a high excess of LHRH, LHRH:CpGl =8:1 charge ratio,
wherein a significant amount of peptide remains free in
solution. Similarly, there is no practical advantage for
immunostimulatory complex prepared with a moderate excess of
CpGl, LHRHrCpGl = 1:2 charge ratio, wherein it is reasonable
to assume that after complete complexation at the point of
electrical neutrality, excess oligonucleotide can serve only
to stabilize the aggregates as shown in Figure 2. This result
does reveal that compatible anionic molecules and/or polymers
may be sequentially added to a preformed 1:1 electrically
neutral complex in order to reduce the effective particle size
of the composition. This presents a novel strategy for
complete immunostimulatory complexation coupled with particle
size control.
[00157] It is an object of this invention to effectively bind
the peptide immunogens in solution for certain applications to
maximize the stability of the vaccine in vivo. Thus
immunostimulatory complex prepared with charge ratios of
peptide immunogens to CpG oligonucleotides ranging from 4:1 to
1:1 respectively are preferred. It is another object of this
invention to maximize the adjuvanticity of the
immunostimulatory complex in vivo by using smaller more
discrete particles {~10 microns or less) for presentation to
the immune system.
[00158] It has been found that the presence of residual free
and uncomplexed peptide is more desirable for more complex
vaccine formulations such as water-in-oil emulsions or
absorption on to mineral salts. In these formulations,
adjuvantation of the immune responses may result from
immunogens bound as immunostimulatory complex and also from
uncomplexed immunogens dispersed within the w/o emulsion or
adsorbed on the mineral salt directly. Thus, the
immunostimulatory complexes prepared with charge ratios of
peptide immunogens to CpG oligonucleotides ranging from 8:1 to
2:1 are found to be useful for these applications.
[00159] More preferably, a combination of maximal peptide
complexation for stability and small particle size for
improved adjuvanticity is found for immunostimulatory
complexes prepared with charge ratios of peptide immunogens to
CpG oligonucleotides ranging from 4:1 to 2:1.
[00160] The most preferred immunostimulatory complex are those
prepared to possess physical properties, which make them
suitable for alternative delivery modalities. Specifically,
average particle sizes on the order of 10 microns or less are
desirable in particular for rectal, vaginal, oral and nasal
'delivery.
Example 2: Quantisation of Peptide Immunogen and
Oligonucleotide Complexation Efficiency by RP-HPLC
[0001] This Example.illustrates a preferable method for
determining the complexation efficiency with respect to the
peptide immunogen and oligonucleotide vaccine components
employing reverse phase high performance liquid chromatography
(RP-HPLC). This technique allows the quantitation of each
residual uncomplexed individual peptide in a peptide cocktail
mixture to be separated and identified (at X = 226 nm) and can
be used to verify full complexation of the CpG oligonucleotide
(at A. = 260 nm) . The solid microparticulate complexes can be
separated from the supernatant solution by centrifuging
followed by filtration. Two separate RP-HPLC programs are run
on supernatant samples to identify and quantitate residual
LHRH peptide immunogens and CpGl oligonucleotides in solution.
[00162] The peptide(s) were resolved by high performance liquid
chromatography (HPLC) using the Vydac 4.6 x 250 mm C-18
column, Cat. number 218TP54, with a gradient from 95% of
solution A (0.05% TFA in HPLC grade water) and 5% solution B
(0.05% TFA in HPLC grade acetonitrile) to 24% of solution A
and 76% solution B in 40 minutes at the flow rate of 1
mL/minute. The UV wavelength absorbance was monitored at 226
nm. Peptide identity was determined by retention time, using
standard peptides.
[00163] The oligonucleotide(s) were resolved by high
performance liquid chromatography (HPLC) using the PerSeptive
Biosystem 4.6 x 100 mm Oligo R3 column, Cat. number R330-050,
with a gradient from 95% of solution A ( 0.1 M TEAA in HPLC
grade water, pH=8) and 5% solution B (HPLC grade acetonitrile)
to 24% of solution A and 76% solution B in 40 minutes at the
flow rate of I mL/minute. The UV wavelength absorbance was
monitored at 260 nm. Peptide identity was determined by
retention time, using standard oligonucleotide.
[00164] Immunostimulatory complexes of LHRH and CpGl ranging
from 8:1 to 1:1 were prepared for this study as described in
Example 1 and Example 11. The respective amounts of CpGl used
to prepare these compositions are shown in Table 3 and Table
9. It is to be noted that the ratio of LHRH:CpGl are
represented as molar charge ratios and is based on the
calculations shown in Table 3 and Table 9.
[00165] For the immunostimulatory complexes prepared using an
excess of LHRH, wherein LHRH:CpGl = 8:1, 4:1 or 2:1, nonequivalent
amounts of residual peptide were detected by RPHPLC
in the supernatant solutions indicating that the
complexation process is selective. This technique enables a
ranking of LHRH peptide immunogens with preferences for CpGl
oligonucleotide in solution based on binding affinity. In all
cases no residual uncomplexed CpGl oligonucleotide could be
detected by RP-HPLC, indicating full complexation of this
component.
[00166] For the immunostimulatory complex prepared using an
equivalent amount of LHRH to CpGl oligonucleotide based on
charge ratio (LHRH:CpGl = 1:1), essentially no peptide and no
CpGl was detected by RP-HPLC indicating full complexation of
all components.
[00167] The complete set of results for these analyses is
depicted in Table 8. It is clear that the binding of LHRH
peptide immunogens with CpGl oligonucleotide may be ranked as
p607E > p667 > p500. The three peptides have near identical
ionization points and all three are positively charged as
calculated in Table 1. The preference of CpGl oligonucleotide
for p607E (+4 charge) over p667 (-1-5 charge) both of which are
preferred to p500 (+4 charge) is likely related to the
molecular weight and the distribution of charges within these
peptides.
Example 3: Preparation of Dried Immunostimulatory Complex
[00168] This Example illustrates the procedure used to prepaxe
an immunostimulatory complex in a dry state.
[00169] Suspensions of LHRH/CpGl complexes, prepared as
described in Example 1, in 0.5-1.0 mL in aqueous solvent,
distilled deionized water, normal saline or phosphate buffered
saline, were placed in a dry ice/acetone bath and frozen for
15 minutes. The frozen samples were then placed on a freezedryer
(Vertis 25LEZ) and the water removed by sublimation at
200 millitorr over three days. This procedure provided a near
transparent glassy finished product in the vial. The
appearance of the residual solid recovered depends on the
aqueous solvent used and can range from a near transparent
glass to a white fluffy solid.
[00170] Reconstitution of the dried materials in- the same
volume of aqueous solvent regenerated a suspension of discrete
particles. The particle size distributions, determined as
described in Example 2, showed essentially no change.
[00171] This demonstrated that the drying and resuspension
process does not effect the physical properties of the
prepared immunostimulatory complexes. Thus, a vaccine
composition comprising the immunostimulatory complexes of the
present invention may be provided in the form of a suspension,
a solid or a dried powder.
Example 4a: Preparation of Water—in-Oil Emulsions Using High
Shear Homogenization
[00172] This Example illustrates the process of preparing a
water-in-oil (w/o)-emulsion from cationic peptides derived
from LHRH peptide immunogens (SEQ ID NOS: 7-9 in a 1:1:1 molar
ratio in solution), IgE peptide immunogens (SEQ ID NO: 10-11
in a 2:1 molar ratio in solution) CD4 peptide immunogens (SEQ
ID No: 4-6 in a 2:1:1 molar ratio in solution) or
immunostimulatory complex derived from LHRH, IgE or CD4
immunogens and CpGl or CpG2 oligonucleotides in various
proportions using homogenization techniques. A flow diagram'
illustrating the process of emulsion formation via
homogenization as described herein is shown in Figure 3.
[00173] All glassware, stir bars and pipette tips were
autoclaved for 40 minutes at 121°C prior to use. All reagents
were weighed, dispensed, transferred or added to reaction
vessels in a laminar flow hood to prevent contamination.
[00174] The w/o-emulsions were optimized for stability with
respect to the volume ratio of the aqueous to oil phases
required. For compositions employing Montanide® ISA 720 oils
(SEPPIC, Inc) the ratio of water to oil was 30:70 by volume.
For compositions employing ISA-Montanide® 51 or ISA Montanide®
50v oils (SEPPIC, Inc.) the ratio of water to oil was 50:50 by
volume.
Example 4b: Preparation of Water-in-Oil Emulsions from ISA
Montanide® 720 and Immunostimulatory complex
[0001] To a 10 mL vessel, was added 3,333 ug of peptide
immunogens dissolved in an appropriate aqueous buffer (1,111
uL, 3 mg/mL) or an immunostimulatory complex prepared from
3,333 \iq of peptide immunogens dissolved in an appropriate
aqueous buffer (1,111 ^L, 3 mg/mL) and either CpGl or CpG2
oligonucleotides. Table 3 and Table 4, shows the calculations
for determining relative amounts of each reagent employed,
[00176] Specifically, to prepare an immunostimulatory complex
from LHRH peptide immunogens at a 4:1 charge ratio of
LHRH:CpGl, 244 ng CpGl oligonucleotide (122 uX, 2.0
were used.
[00177] Specifically, to prepare an immunostimulatory complex
of IgE peptide immunogens at a 4:1 charge ratio of IgE:CpGl,
387 jag of CpGl oligonucleotide (193.5 ^.L, 2.0 |ig/^L) were
used. To form a 1:1 neutral complex of IgE:CpGl, 1,548 |ig of
CpGl oligonucleotide (774.0 uX, 2.0 ng/uL) were used.
[00178] Specifically, to prepare an immunostimulatory complex
of CD4 peptide immunogens at a 2:1 charge ratio of CD4:CpG2,
402 ug of CpG2 oligonucleotide (201 ^L, 2.0 ug/u.L) were used.
To form a 1:2 charge ratio of CD4:CpG2, 1608 fig of CpG2
oligonucleotide (804 ^iL, 2.0 ng/^L) were used.
[00179J To each of the vessels additional diluent aqueous
solvent was added so that the final volume of the aqueous
phase was fixed at 3.0 mL for preparation of ISA Montanide®
720 w/o-emulsions respectively.
[00180] For LHRH or IgE peptides normal saline or PBS was found
to be suitable for complexation. The calculated IP for each
peptide immunogen is greater than 9.0 (Table 1), far greater
than the pH of the aqueous solvent selected.
[00181] In the case of CD4 peptides the choice of aqueous
solvent proved important. Upon dilution with either normal
saline or PBS a solid precipitate was observed to quickly form
in solution. This instability would preclude use of this
immunogen combination by parenteral routes. An examination of
the peptide immunogens revealed that peptide sequence ID No: 6
(Table 1) has a calculated ionization point of 6.91. In PBS
(pH ~ 7.2), this peptide would tend to aggregate and be
expected to exhibit instability. A solution to this problem
was found by first preparing the immunostimulatory complex in
distilled deionized water followed by dilution with saline or
PBS of sufficient ionic strength to ensure that the suspension
was isotonic and suitable for injection.
[00182] This example demonstrates the advantages of stabilizing
immunogens in solution in the form of an immunostimulatory
complex of LHRH, IgE or CD4 peptides.
[00183] The diluted aqueous solutions or suspensions were then
slowly added to a dry 25 mL reaction vessel charged with 7.0
mL of ISA Montanide® 720. The additions were made while
homogenizing (High Shear Laboratory Mixer, Sealed Unit,
Silverson) the mixture at low speeds (2,000-3,000 rpm) to
generate a coarse emulsion. This processing speed was
maintained until the aqueous sample had been completely added
and was continued a full 2 minutes to ensure uniform premixing
of the aqueous and oil phases. The homogenization
speed was then ramped up (5,000-8,000 rpm) and maintained for
from 5 to 10 minutes further resulting in the formation of a
homogeneous white finely dispersed w/o-emulsion.
[0001] The final concentration of immunogens once formulated
as suspensions or in water-in-oil emulsions as described above
was 200 u.g/mL.
Example 4c; Stability Evaluation for Water-in-Oil Emulsions
Prepared by Homogenization Methods.
[00185] The consistency and stability of the w/o-emulsions
prepared by homogenization was checked by a variety of
methods. To prove that the emulsion was water-in-oil (w/o)
and not oil-in-water (o/w) or water-in-oil-in-water (w/o/w), a
droplet of the composition was added to a beaker containing
distilled deionized water. A droplet of a w/o-emulsion will
float on the surface and not disperse into water. Conversely,
a droplet from an o/w emulsion will instantly disperse into
water and a droplet from a w/o/w double emulsion will disperse
both on the surface and into the bulk of the aqueous phase.
Droplets from the emulsions prepared from ISA Montanide® 720
were observed to float on the surface with minimal dispersal
and droplets from the emulsions prepared from ISA Montanide®
51 oils were observed to float on the surface with essentially
no dispersal. These results indicated that the emulsions were
w/o and further that the tendency towards dispersal was higher
for the w/o-emulsion prepared from ISA Montanide® 720. This
is related to the initial viscosity of the oils themselves and
the viscosity of the resultant emulsions. This is an
important consideration for maximizing the depot potential of
the resultant vaccine formulation.
[00186] The apparent viscosity of the finished emulsions and
oils were checked (Brookfield DV-1 + rotational viscometer) for
lot to lot consistency and for long-term stability trials.
ISA Montanide® 720 had a viscosity of -15 mPa at 25°C whereas
the w/o emulsion prepared from ISA Montanide® 720 had a
viscosity of ~45-50 mPa at 25°C. This provided a fairly fluid
product, which is desirable for facilitating the handling, and
dispensing of the vaccine with a syringe.
[00187] In contrast, ISA Montanide® 51 had a viscosity of -50
mPa at 20°C whereas the w/o-emulsions prepared from ISA
Montanide® had a viscosity of - 1,500-2,900 mPa at 25°C. The
wide variation in viscosity was found to be a function of
buffer selection. (PBS = -2,900 mPa, NS = -2,500 mPa and
distilled deionized water = -1,500 mPa) Material of this high
viscosity can present some difficulties with respect to
transferring and dispensing with a syringe. However, the
long-term stability of these compositions was improved. The
long-term stability of the emulsion was evaluated by placing 1
mL of each emulsion in a 1.5 mL eppendorf vial and
centrifuging the contents under high speed (5,000 rpm) for 10
minutes. These conditions do not simulate actual storage
conditions but can be used to predict the resistance of the
emulsion to separation. In the case of ISA Montanide® 720, 5-
10% of the volume separated out with a clear or straw yellow
oil phase observed on the surface. In the case of ISA
Montanide® 51, 0-2% of the volume separated out with a clear
or straw yellow oil phase observed on the surface. The higher
viscosity of the ISA Montanide® 51 emulsion products accounts
for the greater stability to sedimentation and resistance to
separation.
[00188] The particle size and distribution for the w/o-emulsion
was further characterized by optical microscopy (Nikon DIAPHOT
200) . A photomicrograph of each composition was obtained and
an estimate of the size range of particles was made using a
computer-generated scale. The scale itself is externally
referenced against standards of known particle sizes (MIST
traceable microparticles - Duke Scientific). For w/o
emulsions prepared from either ISA Montanide® 720 or ISA
Montanide® 51 and peptide immunogens, the particles sizes were
essentially the same (c.a. 1-2 microns) with minimal
aggregation or coalescence. For w/o-emulsions prepared from
either ISA Montanide® 720 or ISA Montanide® 51 and
immunostimulatory complex the particle sizes were slightly
larger (c.a. 1-3 microns) with minimal aggregation or
coalescence. Figure 4 is a photomicrograph obtained from a
w/o-emulsion prepared via homogenization from ISA Montanide®
51 and an immunostimulatory complex derived from 100 ng of
LHRH peptide immunogens, with LHRH:CpGl in a final charge
ratio of 4:1.
[00189] The initial average particle size was on the order of
10 microns. The process of homogenizing an aqueous suspension
of immunostimulatory complexes under high shear resulted in a
smaller average aggregate particle size. A stable w/o-
emulsion with droplets in the size in the range of 1-3 microns
was obtained.
Example 5a: General Preparation of Water-in-Oil Emulsions
Using Low Shear Extrusion
[00190] This Example illustrates the process of w/o-emulsion
formation from cationic peptides derived from LHRH, IgE or CD4
immunogens or immunostimulatory complex derived from LHRH, IgE
or CD4 immunogens and CpGl or CpG2 oligonucleotides in various
proportions using extrusion techniques. Table 3 and Table 4,
shows the general calculations for determining the relative
amounts of each reagent employed. A flow diagram illustrating
the process of emulsion formation via extrusion as described
herein is shown in Figure 3.
100191] All glassware, stir bars and pipette tips and the
entire extruder mechanism were autoclaved for 40 minutes at
121°C prior to use. All reagents were weighed, dispensed,
transferred or added to reaction vessels in a laminar flow
hood to prevent contamination.
[00192] The extrusion process involves repeatedly passing an
aqueous phase loaded in one syringe into an oil phase loaded
in a second syringe through a narrow bore tube joining the two
syringes. Emulsification occurs as the fluids are driven
through the narrow bore under pressure, typically 100 psi. By
contrast, a homogenizer system typically operates at a
pressure that is greater than 1,000 psi. The number of return
passages necessary for the above extrusion process often
exceeds 20 to 30 before a visually uniform w/o-emulsion is
generated. This manual extrusion process cannot generate
significant shear and the number of exchanges required to
efficiently produce a w/o-emulsion is highly variable. The
physical properties of the w/o-emulsions are inconsistent and
the overall stability and consequently in vivo potency are
typically highly irreproducible. In spite of these problems,
there are a number of possible applications for products
produced by this process.
[0001] TO address these shortcomings an extruder mechanism has
been developed(LiposoFast™ Basic, Avestin, Inc., Ottawa,
Canada). The device consists of two syringes (0.5 mL or 1.0
mL) fitted via luer locks with a narrow bore passage connected
to a holder with a polycarbonate membrane of a defined pore
size placed in between the two syringes. The device as
originally designed was for the preparation of liposomes of a
controlled size.63 The application of such a device with a
compatible oil-based product for the preparation of w/o
emulsions appears not to have been contemplated. The membrane
pore size can be selected (Whatman Nucleopore, 0.05 ^M - 10
fiM) . The smaller pore size allows for the extrusion of
dispersions under increased shear. The larger pore size can
be selected for formulating wherein the particulates are
larger sized. Using this device, the efficiency of
emulsification was increased. Fewer return passages were
required to provide a more uniform and stable product. The in
vivo potency of such a preparation would be predicted to be
more reproducible. However, there are still limitations due
to the small maximum volume, c.a. 1.0 mL, which can be
practically employed and the practical restrictions on the
choice for the oil component.
[00194] The process works well for the preparation of w/oemulsions
derived from ISA Montanide® 720. However, the
higher viscosities of emulsions derived from ISA Montanide® 51
results in significant backpressure, precluding the use of
this extrusion device. As such, this method can be best viewed
as a process for preparing instant w/o emulsions from oils
with apparent viscosities of less than 1,500 mPa.
[00195] In particular, where the costs associated with storage
and stability of vaccines are of concern or where patient
compliance is an issue or application as a palliative medicine
is involved, this method of delivery may be cost effective and
practical. Ideally, trained practitioners such as doctors or
pharmacists can be relied upon for preparing instant w/oformulations
on-site for general, use.
[00196] This device and protocol may be used for the
preparation of instantaneous o/w, and w/o/w, microemulsions
for which controlled shear and extrusion are required, or for
the preparation of refined products.
Example 5b: Preparation of Water-in-Oil Emulsions from ISA
Montanide® 720
[0001] To a 1.0 mL glass syringe (gas tight), was added 333 fig
of LHRH, IgE or CD4 peptide immunogens dissolved in an
appropriate aqueous buffer (111 uL, 3 mg/mL) or an
immunostimulatory complex (with a 4:1 charge ratio) prepared
from 333 fig of LHRH or IgE peptide immunogens or an
immunostimulatory complex (with a 2:1 charge ratio) prepared
from 333 fig of CD4 peptide immunogens dissolved in an
appropriate aqueous solvent (111 fiL, 3 mg/mL) and CpGl or CpG2
oligonucleotides in appropriate ratios as described in Tables
3 and Table 4, respectively.
[00198] Specifically, to prepare immunostimulatory complex from
LHRH peptide immunogens at a charge ratio of LHRH:CpGl of 4:1,
24.3 ug CpGl oligonucleotide (12.2 uL, 2.0 ug/mL) were added.
[00199] To prepare immunostimulatory complex from IgE peptide
immunogens at a charge ratio of IgE:CpGl of 4:1, 38.7 fig of
CpGl oligonucleotide (19.4 uL, 2.0 ug/uL) were added or to
form a 1:1 neutral complex of IgE:CpGl, 154.8 fig of CpGl
oligonucleotide (77.4 fiL, 2.0 ug/uL) were added.
[00200] To prepare immunostimulatory complex from CD4 peptide
immunogens at a charge ratio of CD4:CpG2 of 2:1, 40 ng of CpG2
oligonucleotide (20 uX, 2.0 jig/fiL) were added or to form a
complex at a charge ratio of CD4:CpG2 of 1:2, 160 \iq of CpG2
oligonucleotide (80 (iL, 2.0 ng/jxL) were added.
[00201] Additional diluent aqueous solvent was added so that
the final volume of the aqueous phase was 300 ui.
[00202] To a second 1 . 0 mL glass syringe (gas tight), was added
700 u.L of ISA Montanide® 720. The syringes were connected via
luer locks to an extrusion-housing unit containing a membrane
holder and support for the polycarbonate membrane filter.
Membrane filters with pore sizes 3 \iM or 5 ^iM were selected
for w/o-emulsions to be prepared with peptide immunogens in
the aqueous phase. Whereas, melnbrane filters with pore sizes
5 nM or 10 fiM were selected for the preparation of w/o
emulsions with immunostimulatory complex suspended in the
aqueous phase. The aqueous phase was then first passed
through the membrane into the oil phase, typically with great
ease. The subsequent exchanges require additional pressure
with the increased backpressure generated during the emulsion
process. After 8-12 passages the backpressure upon extrusion
had equalized and a homogeneous white emulsion was typically
obtained.
[00203] The final concentration of immunogens once formulated
as solutions or as immunostimulatory complex in water-in-oil
emulsions as described above was 200
Example 5c: Stability Evaluation for Water-in-Oil Emulsions
Prepared by Extrusion
[0001] Stability tests similar to those conducted in Example
4c, prepared by the extrusion processes confirmed that these
compositions were w/o-emulsion. Droplets placed on the
surface of distilled deionized water were observed to float
with minimal dispersal. The viscosities of the resultant
emulsions were found to range from ~35-40 mPa, a slight
reduction when compared to the high energy homogenized system
in Example 4c. The size distribution of the emulsion droplets
A
was larger than the analogous homogenized systems. About 1-3
microns for w/o emulsions prepared from peptide immunogens or
2-5 microns for w/o emulsions prepared from immunostimulatory
complex. For comparison, Figure 5 is a photomicrograph
obtained from a w/o emulsion sample prepared via extrusion
from ISA Montanide® 720 and an immunostimulatory complex
derived from 200 jag of LHRH peptide immunogens and CpGl
oligonucleotides in a final charge ratio of LHRH:CpGl of 4:1.
[00205] In general, the size of the droplets obtained by the
low energy extrusion process as shown in Figure 5 were larger
and more highly aggregated than the high energy homogenized
droplets as shown in Figure 4. Overall, these instant w/o
emulsions are sufficiently stable for immediate or same day
use.
Example 6a: Preparation of In-situ Polymeric Gels
[00206] This Example illustrates the process of in-situ gel
matrix formation and encapsulation of immunostimulatory
complex derived from IgE or CD4 immunogens and CpGl or CpG2
oligonucleotides in various proportions using direct
reconstitution techniques. A flow diagram showing the process
of preparing an in-situ gel formulation with either peptide
immunogens or itnmunostimulatory complex via reconstitution as
described herein is shown in Figure 6.
[00207] All glassware, stir bars and pipette tips were
autoclaved for 40 minutes at 121°C prior to use. All reagents
were weighed, dispensed, transferred or added to reaction
vessels in a laminar flow hood to prevent contamination.
[00208] In general, varying weight percentages of PLG or PLGA
copolymers (Boehringer Ingelheim) were dissolved in
biocompatible solvents such as anhydrous dimethyl sulfoxide
(DMSO, Aldrich). The solubilization process required vigorous
agitation for higher molecular weight polymers with continuous
stirring maintained for from 4-6 hours to ensure complete
dissolution. The polymer solution was then filtered through
an organic solvent compatible membrane of pore size 0.45
microns (Phenomenex, PTFE). To this was added a solution of
cationic peptide immunogens or more preferably a suspension of
immunostimulatory complex in an appropriate biocompatible
solvent. The peptide immunogen or peptide/CpG complex or a
mixture thereof was first lyophilized (as described in Example
3) and subsequently dissolved or resuspended in an appropriate
biocompatible solvent, such as DMSO.
[00209] The use of polar aprotic solvents, such as DMSO, may
present some problems with respect to long-term peptide
stability. DMSO is known as a powerful oxidizing agent and
peptides containing sensitive amino acids such as cysteine and
tryptophan may be chemically incompatible in these solutions.
Thus, peptide immunogens containing these residues may have
to be reconstituted on site for immediate use.
[00210] The lyophilized peptide immunogens or immunostimulatory
complex or a mixture thereof was reconstituted directly in the
vial into a solution of polymer in DMSO at the time of use,
thereby avoiding prolonged exposure of the peptide immunogens
to DMSO. The dissolution of the peptide immunogen or the
resuspension of immunostimulatory complex or a mixture of the
immunostimulatory complex with peptide immunogens was rapid,
requiring gentle shaking to ensure sample uniformity.
[00211] There are minimal stability and manufacturing issues
expected for these generic compositions. The polymer
solutions in anhydrous DMSO are not as susceptible to
hydrolytic degradation as compared with peptide immunogens
dissolved or suspended in aqueous solutions. The polymer
solution may be frozen (DMSO freezes at c.a. 18°C) with no
detectable changes in the physical properties upon thawing.
The peptide or immunostimulatory complex isolated in the
lyophilized dry state would also be expected to exhibit
increased stability in the absence of water.
[00212] The mixture of polymer and peptide immunogens or
immunostimulatory complex as such constitutes a single-phase
solution or a suspension suitable for subcutaneous or
intramuscular injection.
[00213] Of importance for either system is the viscosity of the
solution or suspension. This directly influences the ability
to syringe and inject the compositions by a subcutaneous or
intramuscular route.
[00214] The apparent viscosity of these systems is a function
of various factors including the constitution, molecular
weight, crystallinity and intrinsic viscosity of the PLG or
PLGA copolymers. These factors delimit the useful amount by
weight for each polymer that can be dissolved while
maintaining practical flow characteristics. Table 5 shows the
physical properties for selected PLG or PLGA polymers and the
corresponding amount in weight percentages that may be
dissolved in DMSO to obtain solution viscosities of practical
use. The apparent viscosity for these solutions was
determined by a Brookfield DV-1+ rotational viscometer.
[00215] 100 mPa was arbitrarily chosen as the upper desired
limit for these compositions. In most cases a solution or
suspension formulated as an in-situ gelling polymer solution
with an apparent viscosity less than 200 mPa can be uniformly
delivered through conventional syringes.
hydrolytic degradation as compared with peptide immunogens
dissolved or suspended in aqueous solutions. The polymer
solution may be frozen (DMSO freezes at c.a. 18°C) with no
detectable changes in the physical properties upon thawing.
The peptide or immunostimulatory complex isolated in the
lyophilized dry state would also be expected to exhibit
increased stability in the absence of water.
[00212] The mixture of polymer and peptide immunogens or
immunostimulatory complex as such constitutes a single-phase
solution or a suspension suitable for subcutaneous or
intramuscular injection.
[00213] Of importance for either system is the viscosity of the
solution or suspension. This directly influences the ability
to syringe and inject the compositions by a subcutaneous or
intramuscular route.
[00214] The apparent viscosity of these systems is a function
of various factors including the constitution, molecular
weight, crystallinity and intrinsic viscosity of the PLG or
PLGA copolymers. These factors delimit the useful amount by
weight for each polymer that can be dissolved while
maintaining practical flow characteristics. Table 5 shows the
physical properties for selected PLG or PLGA polymers and the
corresponding amount in weight percentages that may be
dissolved in DMSO to obtain solution viscosities of practical
use. The apparent viscosity for these solutions was
determined by a Brookfield DV-1+ rotational viscometer.
[00215] 100 mPa was arbitrarily chosen as the upper desired
limit for these compositions. In most cases a solution or
suspension formulated as an in-situ gelling polymer solution
with an apparent viscosity less than 200 mPa can be uniformly
delivered through conventional syringes.
was 200 ng/mL. The immunogen or immunostimulatory complex was
immediately reconstituted in solution or suspension with
gentle shaking to ensure content uniformity.
[00219] The apparent viscosities of the solutions prepared from
these polymers at the specified weight ratios were found to be
close to 100 mPa. (see Table 5). The concentrations for
Resomer® RG 503H, Resomer® RG 504H and Resomer® RG 756 for a
solution viscosity of around 100 mPa were critical, Resomer®
RG 503H and Resomer® RG 504H have much lower inherent
viscosities and both are derived from PLGA with amorphous
character, wherein the monomer composition was around 50% D,Llactide
and 50% glycolide. These materials would be expected
to degrade at a rate of 6-8 weeks for 50% of the polymer in
vivo. Thus, the encapsulated components would have a twothree
month release profile with gels prepared from Resomer®
RG 503H or Resomer® RG 504 H. Gels prepared from these
materials would be most suited for short-term single-dose
controlled release applications. Conversely, Resomer® RG 756
is a more crystalline polymer composed of 75% D,L-lactide to
25% glycolide residues. It may require from 4-6 months for
50% of the polymer to degrade in vivo and the release rate for
encapsulated components would consequently be more protracted.
Resomer® RG 756 would be expected to be more desirable for
long-term single-dose controlled release applications.
Example 7: The Immunogenicity of IgE and CD4 Pep tide
Immunogens Formulated as Immunostimulatory complex or as W/O
Emulsion or in Combinations
[00220] This Example illustrates the immunogenicity of IgE and
CD4 peptide inununogens formulated as immunostimulatory
complexes with CpGl or CpG2 oligonucleotides, as a w/oemulsion
or as immunostimulatory complex dispersed in a w/oemulsion
in guinea pigs, which were immunized intramuscularly.
was 200 p.g/mL. The immunogen or immunostimulatory complex was
immediately reconstituted in solution or suspension with
gentle shaking to ensure content uniformity.
[00219] The apparent viscosities of the solutions prepared from
these polymers at the specified weight ratios were found to be
close to 100 mPa. (see Table 5). The concentrations for
Resomer® RG 503H, Resomer® RG 504H and Resomer® RG 15-6 for a
solution viscosity of around 100 mPa were critical, Resomer®
RG 503H and Resomer® RG 504H have much lower inherent
viscosities and both are derived from PLGA with amorphous
character, wherein the monomer composition was around 50% D,Llactide
and 50% glycolide. These materials would be expected
to degrade at a rate of 6-8 weeks for 50% of the polymer in
vivo. Thus, the encapsulated components would have a twothree
month release profile with gels prepared from Resomer®
RG 503H or Resomer® RG 504H. Gels prepared from these
materials would be most suited for short-term single-dose
controlled release applications. Conversely, Resomer® RG 756
is a more crystalline polymer composed of 75% D,L-lactide to
25% glycolide residues. It may require from 4-6 months for
50% of the polymer to degrade in vivo and the release rate for
encapsulated components would consequently be more protracted.
Resomer® RG 756 would be expected to be more desirable for
long-term single-dose controlled release applications.
Example 7: The Immunogen i city of IgE and CD4 Pep tide
Inununogens Formulated as Immunostimulatory complex or as W/O
Emulsion or in Combinations
[00220] This Example illustrates the immunogenicity of IgE and
CD4 peptide immunogens formulated as immunostimulatory
complexes with CpGl or CpG2 oligonucleotides, as a w/oemulsion
or as immunostimulatory complex dispersed in a w/oemulsion
in guinea pigs, which were immunized intramuscularly.
The w/o-emulsion.s were prepared by homogenization as
described in Example 4a/4b or by extrusion as described in
Example 5a/5b.
[00221] Groups of three, 6 to 8 week old female guinea pigs
(Covance Research Products Inc., Denver, PA) were immunized'
intramuscularly (I.M.) on week 0, .3 and 6 with the following
compositions: 100 ng of the IgE peptides/CpGl
immunostimulatory complex (4:1 charge ratio and 1:1 charge
ratio) prepared as described in Table 4 suspended in a final
volume of 250 fiL PBS, pH 7.4; CD4 peptides/CpG2
immunostimulatory complexes (2:1 charge ratio and 1:2 charge
ratio) prepared as described in Table 4 in 75 ^L suspended in
a final volume of 250 nL PBS' PH 7-4'"' I9E peptides in 75 fiL
PBS, pH 7.4 emulsified with ISA Montanide® 720 (175 \iL) ; CD4
peptides in 75 fiL distilled deionized water emulsified with
ISA Montanide® 720 (175 fiL) ; IgE peptide/CpGl
immunostimulatory complex in 75 nL PBS, pH 7.4 prepared as
described in Table 4 (4:1 charge ratio and 1:1 neutral charge
ratio) emulsified with ISA Montanide® 720 (175 nL) , or CD4
peptides/CpG2 immunostimulatory complexes prepared as
described in Table 4 in 75 \)iL distilled deionized water (2:1
charge ratio and 1:2 charge ratio) emulsified with ISA
Montanide® 720 (175
[00222] The guinea pigs showed no gross pathologies or
behavioral changes after receiving immunostimulatory
complexes, w/o emulsions containing peptide immunogens or w/o
emulsions containing the immunostimulatory complexes. Sera
were obtained on weeks +3, + 5, +9, +11 and +17 and were
evaluated for the presence of anti-IgE antibodies in the case
of IgE immunogens or anti-CD4 antibodies in the case of CD4
immunogens, by immunogen-specif ic ELISAs.
Measurement of anti-IgE antibodies
[00223] Anti-IgE peptide titers were determined by IgE peptide
ELISA and cross-reactivities to human IgE by human IgE ELISA.
Peptide ELISAs for determination of anti-IgE peptide
reactivity were conducted in microtiter plates coated with the
target antigen site peptide without the T helper site, as
described64. For determination of anti-human IgE crossreactivity,
human IgE ELISAs were conducted in microtiter
plates coated in a likewise fashion with a human IgE myeloma
protein (American Biosystems, Inc. cat. no. A113) at 5 ug/ml.
[00224] Captured anti-peptide or anti-IgE antibodies were
detected by horseradish peroxidase-labeled anti-guinea pig IgG
goat antibody. ELISA titers, expressed as logio of reciprocal
dilution, were calculated based on linear regression analysis
of the absorbances, with cutoff A value was rigorous, as the values for diluted normal guinea
pig control samples run with each assay were less than 0.15.
Hyperimmune guinea pig anti-IgE peptide immunogen antiserum
was used as a positive control. Pre-immune sera were used as
negative controls.
Measurement of anti-CD4 Antibodies
[00225] ELISAs for binding to recombinant soluble CD4 were done
in 96 well microtiter plates coated with rsCD4 (American Bio-
Technologies) at 0.25 pg/mL, using 100 pL per well in lOmM
NaHC03 buffer, pH 9.5. Wells were blocked with 250 pL of 3%
gelatin, washed with 0.05% TWEEN 20 in phosphate-bufferedsaline
(PBS) and dried. Test wells were reacted with 100 pL of
diluted immune sera for 1 hour at 37 C. Wells were washed
with 0.05% TWEEN 20 in PBS, reacted with 100 pL of horseradish
peroxidase-labeled goat anti-mouse IgG (Pierce) diluted 1:1000
in 1% goat serum, 0.05% TWEEN® 20 in PBS, and washed. 100 pL
of orthophenylenediamine (OPD) substrate at 0.04% by weight
(Pierce) and 0.12% H202 in sodium citrate buffer, pH 5.0, was
added for 15 minutes. Reactions were stopped by addition of
100 uL 1.0 M H2SC>4 and £492 determined. Hyperimmune guinea pig
anti-CD4 peptide immunogen antiserum was used as a positive
control. Pre-immune sera were used as negative control.
Measurement of Functional Antigenicity by Competitive ELISA
[00226] In this competitive ELISA, functional antigenicity was
quantitated for CD4 immunogens by testing the evoked
antibodies for the capacity to competitively inhibit a
functional monoclonal antibody, mAb B4, whose known
specificity was for the CD4 complex on the host cell surface
that binds HIV. This anti-binding site monoclonal antibody
has been well characterized for its high affinity for the HIV
binding complex, for binding to domain 1 of soluble
recombinant CD4 (rsCD4), and for its ability to neutralize
HIV-1 primary isolates.65
[0001] The anti-binding site monoclonal antibody was purified
by protein A affinity chromatography and conjugated to
horseradish peroxidase. The mAb B4-HRP conjugate was used in
the assay as a tracer, at a concentration of 0.5 ng/ml. 96-
well microtiter plates were coated with recombinant soluble
CD4 protein, 1 ng/ml in 0.1 M sodium carbonate buffer, pH 9.5,
with overnight incubation. Reactions were done in the
microtiter wells in 100 fil total volume of PBS/goat
serum/gelatin/TWEEN® 20, with serially diluted immune serum
(guinea pig, swine, or baboon) and 30 \il of the mAb B4-HRP
working stock. Diluted serum and mAb B4-HRP were preincubated
prior to adding the mixture to the well. The
positive control for the competition ELISA was 5 p.1 of
unlabelled-anti-binding site mAb at 0.5 jag/ml in normal serum,
the negative control is normal serum. The serum/antibody
dilution mixture, 100 pi was added to a coated well and
incubated for one hour at 37°. The plates were drained and
washed and bound mAb B4-HRP was detected by reaction with a
chromogen. The chromogen was 3,3',5,5'-tetramethylbenzidine
(TMB) and TMB-bound mAb B4 conjugate was detected at h^. K
calibration curve was obtained with purified mAb B4 serially
diluted from 10 ug/ml into normal serum of the appropriate
species so as to obtain an mAb equivalent value for the
dilutions of immune sera which competitively inhibit the
binding to human recombinant soluble CD4 of the mAb B4-HRP.
Virus Neutralization Assay
[00228] The MT-2 microplaque assay was carried out as
described66 except that heat-inactivated sera were serially
diluted in 50% high glucose DMEM with 15% FBS, antibiotics, 2%
glutamine and bicarbonate buffer, and 50% pooled, defibrinated
normal human plasma. In this assay, diluted serum is
incubated with 20 pfu of HIV in microtiter wells. HIVsensitive
MT-2 cells are added and formed into monolayers by
centrifugal force under molten agarose. Residual virus
infectivity is detected by the presence of propidium iodidestained
plaques one week later. The endpoint is the serum
dilution at which there was a 50% or 90% reduction in the
plaque count.
Immunogenicity Results
[00229] The serum antibody titers following immunization of IgE
immunogens are shown in Figure 7 (homogenized systems) and
Figure 9 (extruded systems) and for CD4 immunogens are shown
in Figure 8 (homogenized systems) and Figure 10 (extruded
systems). Table 6 compares the competitive inhibition of a B4
monoclonal antibody assayed on sera obtained from the CD4
peptide study (homogenized and extruded w/o emulsion systems)
on week 9, week 11 and week 17, respectively. Table 7
compares virus neutralization activity (50% and 90% inhibition
respectively) assayed on sera obtained from the CD4 peptide
study (homogenized and extruded w/o emulsion systems) on week
9 and week 11, respectively. Control experiments demonstrated
that unadjuvanted peptide was non-immunogenic or weakly
immunogenic in all cases.
[00230] For both IgE and CD4 vaccines, the results of
immunizations indicated that immunostimulatory complexes were
adjuvanting and titers by week 9 were slightly less than or
comparable to those obtained with w/o emulsions, irrespective
of whether the emulsions were prepared by homogenized or
extruded techniques as shown in Figures 7-10.
[00231] The combination systems with inununostimulatory complex
dispersed as w/o emulsions prepared by either method
consistently provided the highest sustained immune responses.
As depicted in Figures 7-10, the antibody titers elicited
from week 11 through week 17, for both IgE and CD4 immunogens
were found to be on the order of a log unit or more higher
than antibody titers obtained with either the
inununostimulatory complex alone or the w/o emulsions with
peptide alone. The sole exception to this being the CD4 w/o
emulsions prepared by homogenization, where this separation is
not found until week 17.
[00232] This observation is further supported by data obtained
from the CD4 peptide competitive inhibition and virus
neutralization studies highlighted in Tables 6 and 7, wherein
the immune sera to the w/o emulsion combination systems with
CD4 peptide/CpG oligonucleotides (charge ratio CD4:CpG2 = 2:1)
competitively inhibited the highest level of B4 monoclonal
antibody compared to the immune sera to the simple w/o
emulsions or immunostimulatory complex with CD4 peptides
alone. Moreover, the same formulations are shown to be the
most effective at eliciting neutralizing activity against
infectious virus.
[00233] Small differences are noted between the homogenized or
extruded preparations and include the rate with which the
titers elicited to IgE or CD4 immunogens obtained by ELISA
were observed to peak. The immune responses peaked earlier
(wk 9) for the extruded w/o emulsions, although the duration
of the responses obtained are good (essentially equivalent by
week 11 and slightly reduced by week 17). The analogous
homogenized system peaked a little later (week 11) and
provided sustained responses as can be seen by the high titers
persisting at week 17.
[00234] This trend is also supported by data obtained from the
CD4 peptide/mAb B4-HRP competitive inhibition assay and the"
virus neutralization studies highlighted in Tables 6 and 7.
[00235] By week 9, assays on sera obtained from animals
immunized by the w/o emulsion system prepared from uncomplexed
CD4 peptides via homogenization (87.2%) or by extrusion
(88.6%) have been shown to competitively inhibit high levels
of B4 monoclonal antibody. These were found to be the same
order of magnitude (within experimental error) as for sera
obtained from animals immunized with the w/o emulsion
combination systems of CD4 peptide/CpG2 oligonucleotides (2:1)
prepared via homogenization (70.1%) or by extrusion (94.9%).
By week 17, assays on sera obtained from animals immunized by
the w/o emulsion system prepared from uncomplexed CD4 peptides
via homogenization (11.2%) or by extrusion (42.6%)
competitively inhibited dramatically lower levels of B4
monoclonal antibody. The degree of inhibition were no longer
generally comparable with that obtained for sera from animals
immunized with the w/o emulsion combination systems of CD4
peptide/CpG2 oligonucleotides (2:1) prepared via
homogenization (85.0%) or by extrusion (77.2%) processes.
[00236] Moreover, the same trend is shown for sera obtained
from formulations of C04 peptides when assayed for
neutralization of infectious HIV-1 virus.
[00237] By week 9, assays on sera obtained from animals
immunized by the w/o emulsion system prepared from uncomplexed
A
CD4 peptides via homogenization (73% for 50% virus
neutralization or 27% for 90% virus neutralization) or by
extrusion (31% for 50% virus neutralization or 12% for 90%
virus neutralization) neutralized essentially the same
percentages of infectious HIV-1 virus (within experimental
error). Assays on sera obtained from animals immunized with
the w/o emulsion combination systems of CD4 peptide/CpG2
oligonucleotides (2:1) prepared via homogenization (12% for
50% virus neutralization or neutralization) or by extrusion (103% for 50% virus
neutralization or 33% for 90% virus neutralization) has shown
that the homogenized system is significantly less effective at
generating neutralizing antibodies against infectious HIV-1
virus at this time point. By week 11, assays on sera obtained
from animals immunized by the w/o emulsion system prepared
from uncomplexed CD4 peptides via homogenization (57% for 50%
virus neutralization or 17% for 90% virus neutralization) or
by extrusion ( 90% virus neutralization) demonstrated that the capacity to
neutralize infectious HIV-1 virus is reduced or eliminated
(within experimental error). Assays on sera obtained from
animals immunized with the w/o emulsion combination systems of
CD4 peptide/CpG oligonucleotides (2:1) prepared via
homogenization (40% for 50% virus neutralization or 30% for
90% virus neutralization) or by extrusion (120% for 50% virus
neutralization or 39% for 90% virus neutralization) has
clearly shown that both the homogenized and extruded systems
were now effective at eliciting neutralizing antibody
responses against infectious HIV. The response for the
homogenized system was shown to be delayed relative to the
extruded composition.
[00238] One possible explanation for these trends may be that
w/o emulsions prepared via low energy extrusion processes can
interact or present complexed immunogens more efficiently at earlier time points to immune effector cells, whereas the high
energy homogenization process, provides a more uniform
emulsion and essentially acts as a more efficient depot. The
immune responses obtained by such a system would be
anticipated to be delayed and potentially more sustainable.
[00239] The combination of the immunostimulatory complex and
residual uncomplexed peptides dispersed in w/o emulsions were
found to synergistically enhance the overall titers for both
the IgE and CD4 immunogens. The method of preparation can
influence the kinetics and duration of the responses, with
both homogenization and extrusion shown to be compatible for
preparing an efficacious vaccine.
[00240] In addition, in a separate study we examined the effect
of CpG dose on immune responses. For both IgE and CD4
immunogens, immunostimulatory complexes were prepared from
varying doses of CpGl or CpG2 oligonucleotides (i.e. IgErCpGl
= 4:1 or 1:1 and CD4:CpG2 = 2:1 or 1:2) and administered in
either an aqueous buffer or dispersed in a w/o emulsion. All
compositions were found to be immunogenic, however, in both
studies the smaller relative dose of oligonucleotide {excess
of synthetic peptide immunogen to oligonucleotide) elicited
the superior immune responses in terms of absolute titer and
of duration. In the case of IgE peptides/CpGl
oligonucleotides the ranking follows the order 4:1 charge
ratio > 1:1 neutral charge ratio whereas for CD4 peptides/CpG2
oligonucleotides the ranking follows the order 2:1 charge
ratio > 1:2 charge ratio. Most often improved adjuvanticity
can be correlated with increasing doses of adjuvants. In
these Examples, we have demonstrated the unexpected advantages
of co-formulating stabilized immunostimulatory complex and
uncomplexed peptide for maximum synergy of immune responses.
[00241] Thus, it can be concluded that immunostimulatory
complex prepared from IgE or CD4 immunogens as described in
this Example can effectively adjuvant immune responses in
vivo. The responses obtained are comparable to those found
for these same immunogens dispersed in w/o emulsions, however,
it is anticipated that fewer reactogenicity issues would
result from use of the fully aqueous dispersable
immunostimulatory complex of synthetic immunogens as opposed
to dispersing synthetic immunogens as w/o emulsions.
[00242] In this Example we have demonstrated that the delivery
of IgE or CD4 immunogens as immunostimulatory complexes in
combination with uncomplexed peptides dispersed within a
water-in-oil emulsion vehicle provided the most effective
presentation of immunogen to the immune system. The immune
responses obtained for the combined system are significantly
greater than the sum of the immune responses obtained for each
system independently.
[00243] Furthermore, assays of sera obtained from animals
immunized with combinations of immunostimulatory complex of
CD4 immunogens and uncomplexed CD4 immunogens dispersed within
a water-in-oil emulsion vehicle were shown to be more
effective than simple w/o emulsions of uncomplexed CD4
immunogens at competitively inhibiting B4 monoclonal antibody.
Moreover these same systems were shown to be the most
effective at neutralizing infectious HIV-1 virus.
[00244] In the guinea pig model, the quantity and longevity of
the immune responses obtained indicated that immunostimulatory
complex derived from IgE/CpGl and CD4/CpG2 combinations were
preferred. It was experimentally determined that compositions
derived from the alternative pairings of IgE/CpG2 and C04/CpGl
were adjuvanting, although not to the same extent.
[00245] The possibility for fewer and lower
(immunoqen/adjuvant) dose regimens is indicated by these
results. In cases where reactogenicity is a problem the
combination system opens up the possibility of administering a
smaller volume of emulsion with similar or higher levels of
immunogenicity.
[00246] Specifically, this Example strongly indicates the
potential of these new combination based formulations for
development as efficacious dosage forms.
Example 8; The Immunogenicity of IgE and CD4 Pep tide
Inununogens Formulated as Immunos timula tory complex or as Insitu
Gelling Polymer Solutions or in Combinations
[00247] This Example illustrates the immunogenicity of IgE and
CD4 peptide immunogens formulated as immunostimulatory complex
with CpGl or CpG2 oligonucleotides or as in-situ gelling
polymers and biocompatible solvents or as immunostimulatory
complex suspended in in-situ gelling polymers and
biocompatible solvents in guinea pigs, which were immunized
intramuscularly. Lyophilized peptide immunogens or
immunostimulatory complex derived from peptide immunogens and
CpG oligonucleotides were prepared as described in Example 3.
The in-situ gelling polymers were prepared as described in
Example 6a/6b.
[00248] To examine the immunogenicity of IgE and CD4 peptide
immunogens formulated as in-situ gelling polymers (Resomer RG
504H) or as immunostimulatory complexes with CpGl or CpG2
oligonucleotides suspended in in-situ gelling polymers
(Resomer® RG 504H) formed in accordance with the present
invention, groups of three, 6 to 8 week old female guinea pigs
(Covance Research Products Inc, Denver, PA) were immunized
intramuscularly (I.M.) with the following amounts of immunogen
on week 0: 300 ng of lyophilized IgE peptides/CpGl
immunostimulatory complex (4:1 charge ratio) reconstituted and
suspended in a final volume of 200 ^L PBS (pH 7.4), prepared
as described in Table 4, or 300 ug of CD4 peptides/CpG2
immunostimulatory complex (2:1 charge ratio) reconstituted and
suspended in a final volume of 200 uX PBS (pH 7.4), prepared
as described in Table 4, or 300 ng of IgE peptides
reconstituted and suspended in 200 uX of Resomer® RG 504H (20%
by wt) dissolved in dimethyl sulfoxide (DMSO) or 300 \ig of CD4
peptides reconstituted and suspended in 200 uX of Resomer® RG
504H (20% by wt) dissolved in dimethyl sulfoxide (DMSO) or 300
Hg of lyophilized IgE peptides/CpGl immunostimulatory complex
(4:1 charge ratio), prepared as described in Table 4,
reconstituted and suspended in 200 jiL of Resomer® RG 504H (20%
by wt) dissolved in dimethyl sulfoxide (DMSO) or 300 jig of
lyophilized CD4 peptides/CpG2 immunostimulatory complex (2:1
charge ratio), prepared as described in Table 4, reconstituted
and suspended in 200 jiL of Resomer® RG 504H (20% by wt)
dissolved in dimethyl sulfoxide (DMSO).
[00249] The guinea pigs showed no gross pathologies or
behavioral changes after receiving immunostimulatory complex,
in-situ gelling polymers in DMSO containing peptide immunogens
or in-situ gelling polymers in DMSO containing
immunostimulatory complex. Sera were obtained on weeks +3,
+ 6, +9, +12 and were evaluated for the presence of anti-IgE
antibodies in the case of IgE immunogens or anti-CD4
antibodies in the case of CD4 immunogens, by immunogenspecific
ELISAs following the procedures described in Example
7.
Immunogenicity Results
[00250] The serum antibody titers following single-dose
immunization of IgE immunogens are shown in Figure 11 and for
CD4 immunogens are shown in Figure 12. Control experiments
demonstrated that unadjuvanted peptide was non-immunogenic o-r
weakly immunogenic in either case. In both studies, the
results of immunizations indicated that immunostimulatory
complex alone were moderately adjuvanting with the titers
peaking by week 9. Conversely, uncomplexed immunogens
suspended in in-situ gelling polymers were also weakly
adjuvanted with peak responses observed at week 12.
[00251] For both IgE and CD4 immunogens, the derived
immunostimulatory complexes suspended as an in-situ gelling
polymer elicited the highest immune responses. These
responses were seen to peak around week 9 and were sustainable
through week 12. The quantity and duration of the immune
responses obtained were not found with either the
immunostimulatory complex alone or with uncomplexed immunogens
administered in a composition including in-situ gelling
polymer alone.
[00252] It is expected that small molecular weight immunogens
such as peptides can easily diffuse through polymer implants
and gels resulting in large guantities of burst release upon
injection. The physical factors that control gellation can be
adjusted to retard this process; however, the mass of peptide
so released is essentially unadjuvanted and subject to the
standard degradation processes that are normally experienced
in vivo. In addition, the small amounts of material remaining
encapsulated may not be expected to be sufficient for an
efficient boost once the polymer degrades, necessitating much
larger doses of peptide. The residual DMSO trapped within the
matrix also presents stability issues, wherein sensitive amino
acids contained in the peptides could be oxidized.
Furthermore it has been well established that water can
penetrate these materials at varying rates depending on
various factors such as gel micromorphology, polymer
hydrophobicity and crystallinity45' 46. Water penetrating the
matrix will promote bulk hydrolysis, the prime degradation •
mechanism operating on PLG/PLGA copolymers in vivo. This
process is known to be accompanied by dramatic local pH
changes, which can essentially reduce the pH to 2 or 367. The
free uncomplexed solubilized peptides may not be stable to
such an environment, and this further limits the potential for
these systems. Acid buffering agents may be employed to help
to offset these problems, but cannot be considered ideal.
Encapsulating a suspension of peptide immunogens in the form
of an immunostimulatory complex imparts a number of stability
and adjuvantation advantages for this system. Once the
injection of polymer gel is made, the small amounts of complex
not effectively encapsulated in the gel (presumably surface
located near the gelling front) can serve to initiate or prime
the immune response more effectively than uncomplexed peptide
immunogen alone. The CpG oligonucleotide remains in close
contact with peptide immunogen and in the form of a complex
particulate may further protect and stabilize the peptide
immunogen from enzymatic digestion in vivo or from chemical
instabilities which may be due to the DMSO solvent contained
within the matrix.
[00253] Furthermore, the peptide immunogens remaining entrapped
in the matrix in a particulate form would be expected to be
better protected against the acidification process than free
uncomplexed peptides. Immunogens presented in this form can
be expected to provide a more efficient boost of immunogen to
the immune system eliciting stronger and longer lasting immune
responses than otherwise possible in a single—dose controlled
release formulation.
[00254] In control experiments, it was determined that
solutions of uncomplexed peptide immunogens dissolved in
polymer compositions of RG 504H (20% by wt) in DMSO gelled
rapidly when placed in contact with solutions of PBS.
Separating the solution from gel phase for these samples and
analyzing the solutions by ultraviolet spectroscopy (at X =
280 nm) revealed that sizeable amounts of uncomplexed peptide
(c.a. 50-70%) were co-extracted with the DMSO.
[00255] The combination of the immunostimulatory complex and
uncomplexed peptide immunogens suspended in in-situ gelling
polymers have been found to synergistically enhance the
overall titers for both the IgE and CD4 immunogens in these
controlled release preparations.
[00256] A separate study examining the effect of CpG
oligonucleotide dose on immune responses was not conducted in
this study. It would be expected that further improvement in
the absolute titers may be obtained by employing
immunostimulatory complex prepared near electrical neutrality
or with an excess of negative charge supplied by either CpG
oligonucleotide or alternatively an additional compatible
excipient. In these compositions the majority of the peptide
immunogen is bound as an immunostimulatory complex and the
gellation process upon injection does not result in major
loses of unadjuvanted peptide by virtue of co-extraction in
the biocompatible solvent.
[00257] Thus, it can be concluded that immunostimulatory
complex can both stabilize peptide immunogens as
immunostimulatory complex and that these compositions when
combined with an in-situ gelling polymer can effectively
adjuvant the immune responses in vivo. This is particularly
important for these polymer systems which are intended for
single-dose use. The delivery of immunogens as
immunostimulatory complex suspended within an in-situ gelling
polymer vehicle provides the most efficient presentation of
immunogen to the immune system. The responses obtained for
the combined system are significantly greater than the sum of
the immune responses obtained for each system independently
and are sustainable, unlike in-situ gels prepared by simplereconstitution
of uncomplexed peptide immunogens from polymers
in biocompatible solvents.
[00258] In the guinea pig model, the quantity and longevity of
the responses obtained indicated that immunostimulatory
complexes derived from IgE/CpGl and CD4/CpG2 combinations were
preferred. It was experimentally determined that compositions
derived from the alternative pairings of IgE/CpG2 and CD4/CpGl
were adjuvanting, although not to the same extent.
[00259] The possibility of a single-dose regimen is indicated
by these results. Specifically, this Example strongly
indicates the potential of these new instantly reconstituted
combination-based formulations for development as efficacious
controlled release dosage form.
Example 9; Preparation of Combination Immunostimulatory
Complex and Mineral Salt Suspensions
[00260] This Example illustrates the process of preparing a
mineral salt suspension from cationic peptides derived from
LHRH peptide immunogens (SEQ ID NOS: 7-9 in a 1:1:1 molar
ratio in solution) or an immunostimulatory complex derived
from LHRH immunogens, and CpGl oligonucleotide in various
proportions. A flow diagram illustrating the process of
preparing a mixed suspension as described herein is shown in
Figure 16.
[00261] All glassware, stir bars and pipette tips were
autoclaved for 40 minutes at 121°C prior to use. All reagents
were weighed, dispensed, transferred or added to reaction
vessels in a laminar flow hood to prevent contamination.
[0001] To a 5.0 mL glass vial equipped with a stir bar, was
added 100 fig (250 uL, 0.4 mg/mL) or 1,600 pig (534 \iL, 3.0
mg/mL) of LHRH peptide immunogens dissolved in an aqueous
solution. Immunostimulatory complexes with either of two
charge ratios (i.e. 4:1 or 1.5:1) were prepared from LHRH
immunogens and CpGl oligonucleotides in distilled deionized
water.
[00263] Specifically, the preparation of a 4:1 complex from 100
jig or 1,600 ug of LHRH immunogens required 7.3 \iq or 116.8 fig
of CpGl oligonucleotide (2.0 mg/mL}, whereas the preparation
of a 1.5:1 complex from 1,600 fig of LHRH immunogens required
350.4 fig of CpGl oligonucleotide (2.0 mg/mL), respectively.
s
[00264] Table 9 shows the calculations employed to determine
the relative amount of CpGl oligonucleotide required for
complexation with LHRH peptide immunogens for a fixed final
dosage of 25 ng/0.5 mL or 400 fig/0.5 mL with respect to the
specified charge ratios.
/
[00265] To a second 5.0 mL glass vial, was added 1.0 mL of
Alhydrogel* (3.2 mg Aluminum (Al)/mL) mineral salt suspension
in distilled deionized water. The Alhydrogel® stock
suspension employed was first pH adjusted to pH 7.1-7.4 by the
addition of 0.1 N NaOH. The pH measurements were made through
use of pH indicator strips with a resolution +/- 0.3 pH units.
[00266] The mineral salt suspension was added to the vial
containing immunostimulatory complex and residual unbound
immunogens and equilibrated with stirring over 30 minutes.
[0001] To the mixture of Alhydrogel® and immunostimulatory
complex was added. 90 \iL of 20% NaCl for tonicity, 5.0 jiL of 2-
phenoxy-ethanol (2-PE) preservative (in select cases) and
additional distilled deionized water to ensure the final
volume of the formulation equaled 2.0 mL.
[00268] The final concentration of immunogens once formulated
as a suspension of immunostimulatory complex combined with
mineral salt as described above was 25 |ig/0.5 mL or 400 fig/0.5
inL respectively. The final concentration of Alhydrogel®
prepared was dependent on target species. Formulations
intended for the rodents were prepared at 0.4 mg Al/0.5 mL '
whereas formulations intended for non-human primates were
prepared at 0.8 mg Al/0.5 mL. In all cases the finished
formulations were adjusted for tonicity (0.9% NaCl) and those
employed for the non-human primates contained a preservative
(0.25% v/v, 2-PE).
Example 10: The Immunogenicity of LHRH Pep tide Immunogens
Formulated as a Suspension of Immunostimulatory Complex with
v
Mineral Salt in Rodents
[00269] This Example illustrates the immunogenicity of LHRH
peptide immunogens formulated as immunostimulatory complexes
with CpGl oligonucleotides in combination with mineral salts,
which were immunized intramuscularly, in male rats. The
mineral salt suspensions were prepared as described in Example
9.
[0001] Groups of four, 6 to 8 week old Sprague-Dawley male
rats were immunized intramuscularly (I.M.) on week 0 4 and 8
with following compositions: 25 [iq of LHRH peptides was
suspended in a volume of 500 uL distilled deionized water; 25
ug of LHRH peptides was suspended in a volume of 250 uL
distilled deionized water and 250 \iL of Alhydrogel® {1.6 mg
Al/mL) was added; 25 p.g of LHRH peptides/CpGl
immunostimulatory complex t4:l ch'arge ratio) prepared as
described in Table 9 was suspended in a volume of 500 (J.L
distilled deionized water; 25 fig of LHRH peptides/CpGl
immunostimulatory complex (4:1 charge ratio) prepared as
described in Table 9 was suspended in a volume of 250 fiL
distilled deionized water and 250 ^L of Alhydrogel® (1.6 mg
Al/mL) was added. To each formulation was added 45 fiL of a
20% saline solution and 455.0 uX of distilled deionized water.
The final volume of each formulation was 1.0 mL.
[00271] The rats showed no gross pathologies or behavioral
changes after receiving mineral salts containing peptide
immunogens or mineral salts containing the immunostimulatory
complex. Sera were obtained on weeks +0, +4, +6, +8 and +12
and were evaluated for the presence of anti-LHRH antibodies by
immunogen-specific ELISA and for serum testosterone by RIA
immunoassay.
Measurement of anti-LHRH antibodies
[00272] Antibody activities were determined by ELISA using 96
well microtiter plates coated with the LHRH peptide54 as
immunosorbent.
[00273] Aliquots (100 uL) of the peptide immunogen solution at
a concentration of 5 ug/mL were incubated for 1 hour at 37°C.
Wells were subsequently blocked with a 3% gelatin/PBS solution
for 1 hour at 37°C. The plates were then dried and used for
the assay. Aliquots (100 uL) of the test immune sera, starting
with a 1:100 dilution in a sample dilution buffer and 10-fold
serial dilutions thereafter, were added to the peptide coated
plates. The plates were incubated for 1-1.5 hours at 37°C.
The plates were washed six times with 0.05% TWEEN 20 in PBS.
100 uL of horseradish peroxidase-labeled goat anti-rat IgG
(Cappel) for assays performed on rat sera, 100 pL of goat
anti-swine horseradish peroxidase-labeled IgG (Pierce) for
assays performed on swine sera or 100 uL of horseradish
peroxidase-labeled goat anti-human IgG (Anogen) for assays
performed on baboon sera was added at the appropriate
dilutions in conjugate dilution buffer (PBS containing 0.5M
NaCl and normal goat serum). The plates were incubated for 1
hour at 37°C and washed as described above. Aliquots (100 uL)
of orthophenylenediamine (OPO) substrate at 0.04% by weight
(Pierce) and 0.12% H202 in sodium citrate buffer, pH 5.0, was
added for 15 minutes. Reactions were stopped by addition of
50 pL 2N H2S04 and the A492 determined for each well.
[00274] ELISA titers were calculated based on linear regression
analysis of the adsorbances, with cutoff A4g2, set at 0.5.
This cutoff was rigorous, as the values for diluted normal
control samples run with each assay were less than 0.15.
Measurement of Serum Testosterone
[00275] Immunogens were evaluated for efficacy by RIA for serum
testosterone values. Serum testosterone levels were measured
using a RIA kit from Diagnostic Products (Los Angeles, Calif.)
according to manufacturer's instructions. The lower detection
limit for testosterone ranged from 0.01 to 0.03 nMol/L. Each
sample was analyzed in duplicate.
[00276] Serum samples were scored as being at castration level
when the testosterone level was below limits of detection and
as "near castration" at Immunogenicity Results
[00277] The serum antibody titers following immunization of
LHRH immunogens are shown in Figure 13a. The corresponding
serum testosterone levels are shown in Figure 13b.
[00278] The antibody titers determined from sera obtained from
male rats in this study proved that neither the composition of
LHRH peptides in buffer nor LHRH peptides in combination with
the Alhydrogel® mineral salts were immunogenic. Conversely,
both compositions derived from the LHRH/CpGl immunostimulatory
complex alone or in combination with the Alhydrogel® mineral
salt were shown to be immunogenic as depicted in Figure 13a.
[00279] The antibody responses of these later two groups proved
similar over the first 8 weeks. By week 12, the antibody
titers found for the combined system of LHRH immunostimulatory
complex and Alhydrogel® mineral salt were only marginally
better (i.e. ~ 0.5 log units) than those determined for the
LHRH immunostimulatory complex alone.
[00280] The corresponding serum testosterone levels determined
for the compositions of LHRH peptides in buffer and LHRH
peptides in combination with the Alhydrogel® mineral salts
similarly demonstrated that neither the LHRH peptide nor LHRH
peptide with mineral salt formulations were capable of
achieving immunocastration in male rats.
[00281] Conversely, both the immunostimulatory complex derived
from LHRH peptides/CpGl alone and the combination of LHRH
peptides/OpGl oligonucleotide with Alhydrogel® mineral salt
effectively imnmnocastrated all rats in each group as shown in
Figure 13b.
[00282] Complete immunocastration for each rat in the
composition derived from the LHRH immunostimulatory complex
combined with the Alhydrogel® mineral salt was achieved by
week 6 (2 weeks after the first boost). A similar level of
immunocastration for the composition derived from the LHRH
immunostimulatory complex alone was not achieved until week 10
(2 weeks after the second boost).
[00283] In this Example we have demonstrated a novel
vaccination regimen to obtain effective immunocastration (as
measured by serum testosterone) with either a composition
prepared from LHRH immunostimulatory complex (in a 3-dose
immunization strategy) or a LHRH immunostimulatory complex in
combination with a mineral salt (in a two-dose immunization
strategy).
[00284] The Alhydrogel® mineral salt administered alone with
LHRH peptide immunogens proved to be an ineffective adjuvant.
However, once formulated with the LHRH immunogens in the form
of an immunostimulatory complex the combination yielded
synergistically far superior results. The mineral salt itself
may be acting in two modes. The first may be to provide a '
depot localizing the immunostimulatory complex at the site of
injection and secondly by recruiting specialized cells of the
immune system which can facilitate the presentation of
immunogen to the immune system.
[00285] Concurrent experiments were conducted with alternative
mineral salt, Adju-phos® (derived from Aluminum phosphate) and
full immunocastration in all rats was achieved by week 8
solely for the system derived from the LHRH immunostimulatory
complex in combination with the Adju-phos® mineral salt.
Example 11: The Inmmnogenicity of LHRH Peptide Inununogens
Formulated as a Suspension of Inmunostimulatory Complex with
Mineral Salt in Baboons
[00286] This Example illustrates the immunogenicity of LHRH
peptide immunogens formulated as immunostimulatory complexes
with CpGl oligonucleotides in various ratios in combination
with mineral salts, which were immunized intramuscularly, in
male baboons. The mineral salt suspensions were prepared as
described in Example 9.
[0001] Groups of two, 2 year old male baboons were immunized
intramuscularly (I.M.) on weeks 0, 4 and 8 with the following
compositions: 400 fig of LHRH peptides/CpGl immunostimulatory
complex (4:1 or 1.5:1 charge ratio) prepaced as described in
Table 9 suspended in a volume of 500 fiL distilled deionized
water; 400 jxg of LHRH peptides/CpGl immunostimulatory complex
(4:1 or 1.5:1 charge ratio) prepared as described in Table 9
suspended in a volume of 250 fiL distilled deionized water and
250 fiL of Alhydrogel® (3.2 mg Al/mL) was added. To each
formulation was added 45 fiL of a 20% saline solution, 2.5 uL
of 2-phenoxy-ethanol and 452.5 nL of distilled deionized
water. The final volume of each formulation was 1.0 mL.
[00288] The baboons showed no gross pathologies or behavioral
changes after receiving immunostimulatory complex at either
ratio or in combination with the Alhydrogel® mineral salt.
Sera were obtained on weeks +0, +4, +6, +8, +12 and +14 (for
the 1.5:1 complexes only) and were evaluated for the presence
of anti-LHRH antibodies by immunogen-specific ELISA and for
serum testosterone by RIA immunoassay.
Immunogenicity Results
[00289] The serum antibody titers following immunization of
LHRH immunogens are shown in Figure 14a. The corresponding
serum testosterone levels for each baboon are shown in Figure
14b and 14c, respectively.
[00290] The antibody titers determined from sera obtained from
male baboons immunized by LHRH immunostimulatory complexes
prepared in either a 4:1 or 1:5:1 charge ratio or a
combination of LHRH peptide immunostimulatory complexes and
Alhydrogel® mineral salt prepared in either a 4:1 or 1:5:1
charge ratio indicated that all compositions were immunogenic
as shown in Figure 14a.
[00291] Marginally higher antibody titers were found for both
systems prepared at the 4:1 charge ratio relative to the
antibody titers obtained for the 1.5:1 system irrespective of
whether the Alhydrogel® mineral salt was present or not.
[00292] The corresponding serum testosterone levels determined
from sera obtained from male baboons immunized with LHRH
immunostimulatory complexes prepared at the 4:1 charge ratio
without Alhydrogel® mineral salt proved totally ineffective
for both baboons on trial as shown in Figure 14b.
[00293] The system formulated from the LHRH immunostimulatory
complexes prepared at the 1.5:1 charge ratio without
Alhydrogel® mineral salt was shown to more effectively
downregulate the serum testosterone response for one of the
baboons in the trial, however full immunocastration was not
achieved as shown in Figure 14c.
[00294] For the composition prepared from a LHRH peptide
immunostimulatory complex (4:1 charge ratio) in combination
with Alhydrogel® full immunocastration was obtained in one
baboon by week 10 and was demonstrated to be sustainable
through week 14 as shown in Figure 14b. Near castration
levels of serum testosterone were obtained in the other baboon
in this group, although this response proved transient as
shown in Figure 14b.
[00295] For the composition prepared from a LHRH
immunostimulatory complex (1.5:1 charge ratio) in combination
with Alhydrogel* both baboons in the study were successfully
immunocastrated (one baboon achieved this by week 8 and the
other by week 10) and this effect proved sustainable through
week 14 as shown in Figure 14c.
[0001] For formulations derived from LHRH immunostimulatory
complexes prepared at either a 4:1 or 1.5:1 charge ratio (LHRH
peptide concentration =400 ng), the actual quantity of LHRH
peptide immunogens bound in the form of an immunostimulatory
complex varies significantly. Table 8 shows the proportion of
LHRH immunogen administered in this form as a function of
varying charge ratios. Approximately 16% (~ €3 f.ig) of the LHRH
immunogens in solution are bound in the form of an
immunostimulatory complex when prepared at a 4:1 charge ratio,
whereas approximately 86% (~ 344 jag) are bound when prepared at
the 1.5:1 ratio.
[00297J The presentation of the majority of the LHRH iiranunogens
in the form of an immunostimulatory complex as in the 1.5:1
ratio system provided longer lasting and earlier
immunocastration responses in baboons. We have experimentally
determined that the Alhydrogel® mineral salt adsorbs an
additional 10% of the free unbound LHRH immunogens in solution
upon addition. Thus it is likely that the mechanism by which
the mineral salt is improving the efficacy of the vaccine when
combined with the immunostimulatory complex is probably linked
to indirect immunomodulatory effects.
[00298] Concurrent experiments were conducted with an
alternative mineral salt, Adju-phos® (derived from Aluminum
phosphate) and full immunocastration was demonstrated in one
baboon by week 10 whereas the other baboon on trial
demonstrated near castration levels of serum testosterone by
this time point.
[00299] In this Example we have demonstrated that a LHRH
immunostimulatory complex in combination with a mineral salt
is an effective vaccine to achieve immunocastration (as
measured by serum testosterone) in a non-human primate.
[00300] Furthermore we have shown that the formulation kinetics
of the immunocastration response is a function- of the initial
charge ratio of LHRH immunogens to CpGl oligonucleotide and
selection of mineral salt.
[00301] Specifically, this Example indicates the potential of
these new combination based formulations for the development
of safe vaccines useful for hormone ablationin animals or
humans.
[00302] Moreover, this Example strongly indicates the potential
of these new combination based formulations for the
development of safe vaccines suitable for the treatment of
androgen-sensitive prostate cancer in humans.
Example 12: Preparation of Water-in-Oil Emulsions from ISA
Montanide® 50v, Immunostimulatory Complex Derived from LHRH in
the Presence of a IL-lfi Derived Peptide Fragment
[0001] To a 10 mL vessel, was added 1,000 jxg of LHRH peptide
immunogens (SEQ ID NOS: 7-9 in a 1:1:1 molar ratio in
solution), dissolved in an appropriate aqueous buffer (2,500
uL, 0.4 mg/mL) and CpGl oligonucleotide to prepare an
immunostimulatory complex (16:1 charge ratio) or 1,000 ug of
peptide immunogens dissolved in an appropriate aqueous buffer
(2,500 fiL, 0.4 mg/mL) plus a peptide derived from IL-16 (SEQ
ID NO: 14, C*VQGEESNDKIPC*-C02H.HC1 (where C* indicates
cyclization between two cysteines) in solution; or 1,000 jig of
peptide immunogens dissolved in an appropriate aqueous buffer
(2,500 nL, 0.4 mg/mL) was added to a mixture of CpGl and IL-1B
peptide to prepare a combination of LHRH immunostimulatory
complex (16:1 charge ratio) and IL-lfi peptide. Table 9 shows
the calculations employed to determine the relative amount of
CpGl oligonucleotide required for complexation with LHRH
peptide immunogens for a fixed final dosage of 100 fig/1.0 mL
with respect to the specified charge ratio.
[00304] Peptide-based fragments derived from IL-lfi are known to
possess adjuvanting properties.69 The IL-lfi peptide employed
herein is relatively small (FW = 1421.5) and negatively
charged when dissolved in standard physiological buffers.
Employing the calculations described in Example 2, one nMol of
IL-lIi peptide contributes 2 nMols of negative charge. As
such, this molecule would not be predicted to physically
interact with the LHRH immunogens and no evidence of
complexation in the form of a precipitate was found upon
mixing.
[00305] Specifically, to prepare an immunostimulatory complex
from LHRH peptide immunogens at a 16:1 charge ratio of
LHRH:CpGl, 18.3 ^g CpGl oligonucleotide (9.2 ^L, 2.0 ug/mL)
were used. To prepare a solution of LHRH peptide immunogens
and IL-lfJ, 10 ng IL-lfi(5.0 uL, 2.0 (ig/mL) were added in an
appropriate aqueous buffer. To prepare an immunostimulatory
complex from LHRH peptide immunogens at a 16:1 charge ratio of
LHRH:CpGl plus IL-1B, 18.3 jag CpGl oligonucleotide (9.2 (aL,
2.0 ng/mL) and 10 fig IL-115 peptide (5.0 |iL, 2.0 ng/mL) were
mixed.
[00306] To each of the vessels additional aqueous solvent
diluent was added so that the final volume of the aqueous .
phase was fixed at 5.0 mL for preparation of ISA Montanide
50v w/o-emulsions respectively.
[00307] For the preparation of the placebo group, 5.0 mL of
normal saline was employed for the aqueous phase.
[00308] For the LHRH peptide immunogens, normal saline was
found to be suitable for cbmplexation. The calculated IP for
each peptide is greater than 9.0 (Table 1), far greater than
the pH of the aqueous solvent selected.
[00309] This example demonstrates the advantages of stabilizing
immunogens in solution in the form of an immunostimulatory
complex. The complexation process has been shown to be
compatible in the presence of additional immunomodulators.
[00310] The diluted aqueous suspensions of LHRH immunogens or
placebo solution were then slowly added to a dry 25 mL
reaction vessel charged with ISA Montanide® 50v (5.0 mL). The
additions were made while homogenizing (High Shear Laboratory
Mixer, Sealed Unit, Silverson) the mixture at low speeds
(2,000^3,000 rpm) to generate a coarse emulsion. This
processing speed was maintained until the aqueous sample had
been completely added and was -continued a full 2 minutes to
ensure uniform pre-mixing of the aqueous and oil phases. The
homogenization speed was then ramped up (5,000-8,000 rpm) and
maintained for from 5 to 10 minutes further resulting in the
formation of a homogeneous white finely dispersed w/oemulsion.
[00311] The final concentration of immunogens once formulated '
as water-in-oil emulsions as described above was 100 jj.g/mL.
Example 13: The Immunogenicity and Growth Promoting Effects
of LHRH Peptide Immunogens Formulated as an Immunostimulatory
Complex or with IL-lfi Peptide or as a Combination of an
Immunostiiaulatory Complex with IL-lfi Peptide in a W/O Emulsion
[00312] This Example illustrates the immunogenicity of LHRH
peptide immunogens formulated; as immunostimulatory complexes
with CpGl oligonucleotides in combination with a w/o emulsion
or with an immunomodulator (IL-lIi peptide) in combination with
a w/o emulsions or as immunostimulatory complexes combined
with an additional immunomodulator (IL-lli peptide) in a w/oemulsion,
which were immunized intramuscularly, in boars. The
w/o-emulsions were prepared by homogenization as described in
Example 4a and Example 12.
[0001] Groups of five, 8 week old boars (30 Kg each) were
immunized intramuscularly (I.M.) on week 0 and 8 with
following compositions: 100 ng of LHRH peptides/CpGl
immunostimulatory complex (16:1 charge ratio) prepared as
described in Table 9 suspended in a final volume of 500 p.L
Nad and dispersed with ISA Montanide® 50v (500 ^L) ; LHRH
peptides/IL-lft peptide (1 ng) was suspended in a final volume
of 500 \iL N'aCl and dispersed with ISA Montanide® 50v (500 uL) ;
or LHRH peptides/(CpGl + IL-lft peptide (1 jig) )
immunostimulatory complex (16:1 charge ratio) prepared as
described in Table 9 was suspended in a final volume of 500 jiL
NaCl dispersed with ISA Montanide® 50v (500 uX) . A placebo
control group of 500 uL NaCl dispersed with ISA Montanide® 50v
(500 uL) was prepared and one unimmunized control group
included in the study was surgically castrated.
[00314] The boars showed no gross pathologies or behavioral
changes after receiving placebo w/o emulsions, w/o emulsions
containing peptide immunogens in combination with IL-115
peptide or w/o emulsions containing the immunostimulatory
complex or immunostimulatory complex in combinations with IL-
1/i peptide. Sera were obtained on weeks +0, +8, +10, +12 and
+14 and were evaluated for the presence of anti-LHRH
antibodies by immunogen-specific ELISAs, for serum
testosterone by RIA immunoassay and for weight gain.
Immunogenicity Results
[00315] The serum antibody titers following immunization of
LHRH immunogens are shown in Figure 15a. The corresponding
serum testosterone levels are shown in Figure 15b. The
effects of inununocastration following immunization of LHRH
immunogens versus surgical castration on the weight gain over
the trial period are shown in Figure 15c. Control experiments
demonstrated that unadjuvanted peptide was non-immunogenic or
weakly immunogenic in all cases.
[00316] The antibody titers determined from sera obtained from
male boars immunized by LHRH immunostimulatory complexes, LHRH
peptides adjuvanted by a cytokine, or by an LHRH
immunostimulatory complex co-formulated with a cytokine
administered as w/o emulsions indicated all three compositions
•were immunogenic. The titers obtained at all timepoints over
the trial period proved comparable. The Placebo and
surgically castrated negative control groups returned no titer
as shown in Figure 15a.
[00317] The corresponding serum testosterone levels determined
from sera obtained from boars immunized by LHRH
immunostimulatory complex in a w/o emulsion or LHRH peptides
adjuvanted by the IL-1B peptide in a w/o emulsion or by a
combination of LHRH immunostimulatory complex and the IL-lfipeptide
administered as w/o emulsion indicated that male boars
in these groups were effectively immune-castrated by week 12.
[00318] Notably, the compositions derived from the LHRH
immunostimulatory complex in a w/o emulsion and the LHRH
peptides adjuvanted by the IL-lft peptide in a w/o emulsion
failed to maintain this level of serum testosterone. By week
14, serum testosterone levels from both groups were shown to
be rebounding and neither group would be described as
immunocastrated.
[00319] The LHRH vaccine formulated as a combination of
immunostimulatory complex and IL-lfi peptide as a w/o emulsion
proved more effective maintaining the same level of
immunocastration with no indication of early rebound as shown
in Figure 15b.
[00320] The Placebo negative control group returned widely
fluctuating serum testosterone values at the various
timepoints evaluated in agreement with the expected variations
in monthly hormone levels. The surgically castrated positive
control groups returned negative serum testosterone levels at
all timepoints over the trial period as shown in Figure 15b.
[00321] The serum testosterone level in boars prior to
slaughter has been directly correlated with the quality of the
meat produced. High levels of testosterone give rise to
products of unappealing taste (boar taint) and thus
controlling this property is an important market
consideration. The standard method for ensuring negligible
testosterone has been surgical castration. A viable vaccine
approach must be proven effective over the interval of time
when boars would normally be brought to market. The point at
which the boars have reached a weight between 110-130 KGs.
[00322] The growth promotion of boars immunized at 8 weeks of
age in this study was tracked over 14 weeks. The boars
immunocastrated by the LHRH immunostimulatory complex in a w/o,
emulsion or by LHRH peptides adjuvanted solely by the IL-1I5
peptide in a w/o emulsion posted weight gains, that paralleled
the surgically castrated group. These reached on average 110
KGs by week 14 as shown in Figure 15c.
[00323] Unexpectedly, the group immunized by the LHRH vaccine
formulated as a combination of the LHRH immunostimulatory
complex and IL-l(i peptide in a w/o emulsion proved
considerably more effective. All boars within this group
responded rapidly reporting the highest average weight gains
at all timepoints evaluated in comparison to the other groups
on trial. Boars from this group attained an average weight
slightly below 120 KGs by week 12 and reached 130 KGs by week
14 as shown in Figure 15c.
[00324] The disparity between this group and the others on
trial may be due to a synergistic combination of effects
obtained when the immunostimulatory complex of LHRH/CpGl is
presented to the immune system in combination with the IL-lft
peptide and free uncomplexed LHRH peptide in a w/o emulsion.
[00325] The combination formulation proved effective with very
small amounts of LHRH/CpGl immunostimulatory complex present
in suspension. In this Example a LHRH:CpGl charge ratio of
16:1 provided significant responses in all swine.
[0032-5] The Placebo control group proved poorest at promoting
growth over the trial period. The average weight gain for
this group was significantly reduced relative to all others as
shown in Figure 15c.
[00327] Marginal improvements in weight gain as a consequence
of immunocastration relative to placebo controls have been
observed in the past70, however in this Example we have
demonstrated a novel method to obtain effective
immunocastration for the removal of boar taint (as measured-by
serum testosterone) and obtained superior growth promotion (as
measured by weight gain) over the trial period with respect to
the standard method of surgical castration.
[00328] Furthermore, boars immunocastrated by the LHRH vaccines
formulated as a combination of an immunostimulatory complex
and IL-lIi peptide in a w/o emulsion could go to market 2 weeks
earlier than male boars immunized by either alternative
formulation, or the present method of preference, surgical
castration.
[00329] Specifically, the monetary savings in terms of avoiding
losses due to surgical trauma, reduced feed and housing costs
and improved turnover in terms of time to market would be
significant, demonstrating the advantages of this improved
method of immunocastration and growth promotion.
Example 14: Preparation of Water-in-Oil Emulsions from ISA
Montanide® 50v and Immunostimulatory Complexes Derived from
FMD
[00330] To a 20 mL vessel, was added 4,000 \iq of FMD peptide
immunogens (SEQ ID NOS: 12-13 in solution), dissolved in an
appropriate aqueous buffer (2,000 uL, 2.0 mg/mL) or was added
4,000 \iq of peptide immunogens dissolved in an appropriate
aqueous buffer (2,000 nL, 2.0 mg/mL) and CpGl oligonucleotide
to prepare an immunostimulatory complex (A:1 charge ratio).
f
Table 9 shows the calculations employed to determine the
relative amount of CpGl oligonucleotide required for
complexation with the FMD peptide immunogens for a fixed final
dosage of 200 ug/1.0 mL with respect to the specified charge
ratio.
[00331] Specifically, to prepare an irrununostimulatory complex
from FMD peptide immunogens at a 4:1 charge ratio of FMD:CpGl,
125 ug CpGl oligonucleotide (62.5 \iL, 2.0 fig/mL) were used.
[00332] To each of the vessels additional aqueous solvent
diluent was added so that the final volume of the aqueous
phase was fixed at 10.0 mL for preparation of ISA Montanide®
50v w/o-emulsions respectively.
[00333] For the preparation of the placebo group, 10.0 mL of
normal saline was employed for the aqueous phase.
[00334] For the library of FMD peptide immunogens normal saline
was found to be suitable for complexation. The average
calculated IP for each peptide derived from a positional
analog in the library is greater than 9.0 (Table 1), far
greater than the pH of the aqueous solvent selected.
[00335] The diluted aqueous suspensions of FMD immunogens or
placebo solution were then slowly added to a dry 25 mL
reaction vessel charged with ISA Montanide® 50v (10.0 mL).
The additions were made while homogenizing (High Shear
Laboratory Mixer, Sealed Unit, Silverson) the mixture at low
speeds (2,000-3,000 rpm) to generate a coarse emulsion. This
processing speed was maintained until the aqueous sample had
been completely added and was continued a full 2 minutes to
ensure uniform pre-mixing of the aqueous and oil phases. The
homogenization speed was then ramped up (5,000-8,000 rpm) and
maintained for from 5 to 10 minutes further resulting in the
formation of a homogeneous white finely dispersed w/oemulsion.
[00336] The final concentration of immunogens once formulated
as water-in-oil emulsions as described above was 200
Example 15: The Immunogenicity and Protection of FMD Peptide
Immunoqens Formulated as an Immunostimulatory Complex in a W/O
Emulsion
[00337] This Example illustrates the immunogenicity of FMD
peptide immunogens formulated as a w/o emulsion or as FMD
immunostimulatory complexes in combination with a w/o
emulsion, which were immunized intramuscularly in cattle. The
w/o-emulsions were prepared by homogenization as described in
Example 4a and Example 14.
[00338] Groups of three, adult cattle were immunized
intramuscularly (I.M.) on week 0 and 3 with the following
compositions: 400 fig of FMD peptides suspended in a final
volume of 1,000 uL NaCl and dispersed with ISA Montanide® 50v
(1,000 p.L) ; or FMD peptides/CpGl inununostimulatory complex
(4:1 charge ratio) prepared as described in Table 9 suspended
in a final volume of 1,000 jiL NaCl and dispersed with ISA
Montanide® 50v (1,000 jiL) . A placebo control group of 1,000
uX NaCl emulsified with ISA Montanide® 50v (1,000 (iL) was
prepared.
[00339] The cattle showed no gross pathologies or behavioral
changes after receiving placebo w/o emulsions or w/o emulsions
containing FMD peptide immunogens or FMD immunostimulatory
complexes. Sera obtained on week +5 were evaluated for the
presence of FMD neutralizing antibodies and on week +6 the
cattle were challenged with live virus to determine the level
of protection in a trial lasting 14 days.
Measurement of anti-EMD Antibodies - Neutralization Assay
[00340] The quantitative neutralization assay (NA) for FMD
antibody was performed with BHK-21 cells in flat-bottomed
tissue-culture grade microtiter plates. The test is an equal
volume test in 50 |iL amounts. Starting from a 1:4 dilution,
sera were diluted in a two-fold dilution series in duplicate.
Serum samples to be tested were mixed with an equal volume of
the FMDV OManisa serotype (200 TCIDSO/0.05 mL) and incubated for
one hour at 37°C. A cell suspension at 106 cells/mL was
prepared in- a medium containing 10% bovine serum. A volume' of
50 nL of cell suspension was added to each well. Plates were
sealed and incubated at 37°C for 2-3 days.
[00341] Microscopic examination was feasible after 48 hours.
Fixation was effected with 10% formol/saline for 30 minutes.
For staining the plates were immersed in 0.05% methylene blue
in 10% formalin for 30 minutes. Positive wells (where virus
had been neutralized and cells remained intact) were seen to
contain blue-stained cells. Negative wells are empty. Titers
were expressed as the final dilution of serum present in
serum/virus mixture at the 50% end-point.
Cattle Challenge Trial Protocol
[00342] On day 35, all cattle, which received vaccine or
placebo controls on day 0 and day 21 were separated by groups
into separate containment rooms. On day 42, each animal was
challenged intradermolingually (IDL) with a total of 104 BIOso
FMDV 0 injected in two sites on the dorsal surface of the
tongue.
[00343] After challenge, the cattle were observed for
development of clinical signs of FMD for 14 days and body
temperature was recorded daily. Unprotected animals show
lesions at sites other than the tongue. Control animals must
develop a generalized infection as shown by lesions on at
least three feet for the virus challenge to be considered
valid.71
Immunogenicity Results
[00344] The neutralizing antibody (N.A.) titers following
immunization of FMD immunogens and the corresponding
protection results are shown in Table 10.
[00345] The N.A. titers determined from sera obtained from
cattle (week +5) immunized by FMD peptide immunogens or by FMD
immunostimulatory complexes administered as w/o emulsions
indicated that both compositions were immunogenic. The titers
obtained at the week 5 timepoint were highly variable, but in
all cases were shown to be greater than 16. The minimum
requirement for proof of potency for a foot-and-mouth disease
vaccine as established by the Office International des
Epizooties (DIE) is a N.A. titer of 16.71 Each steer in the
Placebo negative control group returned negligible N.A.
titers, all of which were less than 16 as shown in Table 10.
[00346] Conclusive proof for protection must be obtained by
challenge of the immunized groups with live virus of specified
titer. Live virus was administered intradermolingually (IDL)
with a total of 10* BID5o FMDV 0 injected in two sites on the
dorsal surface of the tongue.
[00347] The challenge protocol was initiated on week 6, one
week after the N.A. titers were established for all groups and
the results in Table 10 prove that the formulation derived
from FMD peptides/CpGl immunostimulatory complexes in
combination with unbound FMD peptides administered as a w/o
emulsion was superior (3/3 protected) to the formulation
derived from FMD peptides administered as a w/o emulsion alone
(1/3 protected).
[00348] The placebo control group confirmed that the live virus
employed for the challenge was sufficiently virulent as all
three cattle in this group were infected and showing signs of
disease within 14 days after challenge (0/3 protected) as
shown in Table 10.
[00349] Both of the groups formulated with FMD immunogens or an
FMD immunostimulatory complex in a w/o emulsion obtained
significant N.A. antibody titers by week +5.
[00350] Surprisingly, the challenge study demonstrated that the
formulation of superior efficacy was derived from the
formulation comprising the FMD immunostimulatory complex in
the w/o emulsion. It is likely that this composition
concurrently improves the N.A. responses and is more effective
at upregulating a specific kind of immune response important
for combating viral infections.
[00351] Specifically, the immunostimulatory complex derived
from FMD and CpGl presented in the form of a w/o emulsion may
effectively augment the Thi arm of the immune response (e.g.
IFN-gamma). This role for CpG oligonucleotides has been well
established in other models and IFN-gamma itself has been
shown to be an effective immunomodulator achieving immune
protection against influenza virus.72
[00352] The control of foot-and-mouth disease, once an outbreak
has been identified is often managed by culling infected and
neighboring herds. The economic loss of important commodities
like cattle, pork and sheep is often significant. The
existing vaccine strategies employ killed virus, which are in
some cases locally produced and of inferior quality. The
manufacturing from live virus has various issues relating to
production associated with it and the products themselves
represent a potential safety risk. A strategy based on
immunogens derived from synthetic peptides represents an
improved method for effective immunization, and should be
considered low risk.
[00353] Specifically, this Example demonstrates that a
synthetic peptide-based vaccine strategy can safely and
effectively protect cattle against foot-and-mouth disease.
[00354] Furthermore, this Example further demonstrates the
utility of this vaccine approach for the general protection of
important domestic livestock susceptible to foot-and-mouth
disease.
Table 1
Synthetic Peptide Immunogens And Oligonucleotides
(Table Removed)
IP = Theoretical lonization Potential, (ref. 68)
FW = Formula Weight/I'm = Primer to target Tm (by %GQ
Table 2
Molar Charge Calculations
Synthetic Peptide Immunogens And Oligonucleotides
(Table Removed)

1) Net charge: Calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H),
a -1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for every other amino
acid within the sequence. The charges are summed for each peptide and expressed as the net
average charge.
2) Average Total Molar Equivalents Of+'ve Charge From Peptides: Estimates for the average
charge of the combined peptide mixture are calculated by summing the overall molar charge
contribution for each component within the mixture and taking the mean.
3) Average Total Peptide Formula Weight (FW): Estimates for the average Total peptide FW are
calculated by summing the overall molar mass contribution for each component within the mixture and
taking the mean.
4) The FMD vaccine is derived from a library of peptides. The molar contribution of each component
is assumed equivalent and the average total molar equivalents of +Ve charge is similarly calculated.
5) For the FMD vaccine the average total peptide formula weight is obtained by summing the lowest
and highest FW found for the various FMD peptides in the library and taking the mean.
Molar Equivalents Of -'ve charge From CpG Oligonucleotides: Each phosphorothioate group
contributes -1 charge.
CpG1 (32 base oligomer) -1 nmol CpG1 = 31.0 nmol -Ve charge
CpG2 (24 base oligomer + phosphorothioate). -1 nmol CpG2 = 24.0 nmol -Ve charge
Table 3
Calculated Molar Charge Ratios Used To Prepare Complexes
LHRH Peptide Immunogens And CpG1 Oligonucleotides
(Table Removed)

Total +'ve molar charge LHRH peptides = 4.3 nmol +'ve charge/nmol LHRH peptide (Table 2)
In 100 ug LHRH peptides there are 100 u.g /4S43.9 jig/ fimol» 22.0 nmol of LHRH immunogen
Total +'ve molar charge = 22.0 nmol x 4.3 nmols +'ve charge/nmol LHRH peptides = 94.6 nmols +'ve charge
2) Sample calculation:
Final 8:1 molar charge ratio of LHRH immunogen to CpGl oligonucleotide.
100 ng of LHRH contributes 94.6 nmols of+'ve charge.
To establish an 8:1 molar charge ratio of LHRH to CpGl in solution the required amount of LHRH that must be neutralized by CpGl is
calculated as 94.6 nmols +'ve charge/8 = 11.8 nmols +'ve charge.
Thus 11.8 nmols +'ve charge contributed by LHRH must be neutralized by 11.8 nmols of-'ve charge contributed by CpGl.
1 nmol of CpGl = 31 nmol -'ve charge (Table 2).
Thus the total nmols of CpGl required to contribute 11.8 nmols of-'ve charge = 11.8/31 = 0.38 nmols CpGl.
Table 4
Molar Charge Ratio Calculations
IgE, CD4 Peptide Immunogens And CpG Oligonucleotides
(Table Removed)

1) Total calculated +'ve molar charge for 100 ug or 300 ug of IgE peptides:
Avg. FW = 5132.1 g/ mol (Table 2); Total +'ve molar charge IgE peptides = 7.7 nmol +'ve charge/nmol IgE peptides (Table 2)
In 100 g IgE peptides there are 100 g /5132.1 g/ mol = 19.5 nmol of IgE immunogen
Total +'ve molar charge = 19.5 nmol x 7.7 nmols +'ve charge/nmol IgE peptides = 150.1 nmols +'ve charge
In 300 g IgE peptides there are 300 g/5132.1 g/ mol -58.5 nmol of IgE immunogen
Total +'ve molar charge = 58.5 nmol x 7.7 nmols +'ve charge/nmol IgE peptides = 450.5 nmols +'ve charge
2) Total calculated +'ve molar charge for 100 ug or 300 ug of CD4 peptides:
Avg. FW = 5280.6 g/ mol (Table 2); Total +'ve molar charge.CD4 peptides = 4.3 nmol +'ve charge/nmol CD4 peptides (Table 2)
In 100 g CD4 peptides there are 100 g/5280.6 g/mol = 18.9 nmol of CD4 immunogen
Total +'ve molar charge = 18.9 nmol x 4.3 nmols +'ve charge/nmol CD4 peptides = 81.3 nmols +'ve charge
In 300 g CD4 peptides there are 300 g/5280.6 g/mol = 56.8 nmol of CD4 immunogen
Total +'ve molar charge = 56.8 nmol x 4.3 nmols +'ve charge/nmol CD4 peptides = 244.2 nmols +'ve charge
3) n nmol of-'ve charge contributed by CpGl or CpG2:
Total calculated -'ve molar charge for CpGl/CpG2 Oligonucleotides
1 nmol of CpGl =31 nmol -'ve charge, FW = 10,295 g/ mol (Table 1)
1 nmol of CpG2 = 24 nmol -'ve charge, FW = 7,400 g/ mol (Table 1)
Table 5
Comparison Of Physical Properties Of PLG/PLGA Copolymers Dissolved
In DMSO As A Function Of Weight Percent In Solution
(Table Removed)

Table 6
B4-HRP Inhibition Assay Of Sera Obtained From CD4 Peptides, CD4/CpG2
Iramunostimulatory Complexes Or Combinations In W/O Emulsions
(Table Removed)

• N.D. - samples were not assayed
The value of 49.1% at week 11 for unadjuvanted CD4 peptide is unexpected and likely due to experimental error.
*** - Water-in-oil emulsion prepared with ISA 720 via homogenization techniques
**** - Water-in-oil emulsion prepared with ISA 720 via extrusion techniques
Table 7
Neutralization Of HIV-1 Strain VL135 By Immune Sera To CD4 Peptides
CD4/CpG2 Immunostimulatory Complexes Or Combinations In W/O Emulsions
(MT-2 microplaque assay)
(Table Removed)

* - Reciprocal liter prior to the addition of an equal volume of virus containing 1-1.3 logs
** - Water-in-oil emulsion prepared with ISA 720 by homogenization techniques
*** - Water-in-oil emulsion prepared with ISA 720 by extrusion techniques
Table 8
Complexation Efficiency As A Function of LHRH PeptiderCpGl
Molar Charge Ratio By RP-HPLC
(Mass of LHRH peptide cocktail fixed @ 400 g)
(Table Removed)

1) Percentage of bound peptides are calculated by comparing the RP-HPLC peak areas relative to the control LHRH
composition assayed in the absence of the CpGl oligonucleotide.
2) Percentages for the overall amount of LHRH/CpG 1 complexed peptide are calculated by summing the total RP-HPLC
peak areas obtained and comparing that with total summed RP-HPLC peak areas obtained from the control composition
examined in the absence of CpGl oligonucleotide.
Table 9.
Molar Charge Ratio Calculations
LHRH And FMD Peptide Immunogens And CpG Oligonucleotides
(Table Removed)
1) Total calculated +'ve molar charge for 25 g, 100 g or 400 g of LHRH peptides:
Avg. FW = 4543.9 g/ mol (Table 2); Total +'ve molar charge LHRH peptides = 4.3 nmol +'ve charge/nmol LHRH peptides (Table 2)
In 25 g LHRH peptides there are 25 g /4543.P g/ mol = 5.5 nmol of LHRH immunogen
Total +'ve molar charge = 5.5 nmol x 4.3 nmols +'ve charge/nmol LHRH peptides = 23.7 nmols +'ve charge
In 100 g LHRH peptides there are 100 g /4543.9 g/ mol = 22.0 nmol of LHRH immunogen
Total +'ve molar charge = 22.0 nmol x 4.3 nmols +'ve charge/nmol LHRH peptides = 94.6 nmols +'ve charge
In 400 g LHRH peptides there are 400 g /4543.9 g/ m ol = 88.0 nmol of LHRH immunogen
Total +'ve molar charge = 88.0 nmol x 4.3 nmols +'ve charge/nmol LHRH peptides = 378.4 nmols +'ve charge
2) Total calculated +'ve molar charge for 400 g of FMD peptides:
Avg. FW = 6805 g/ mol (Table 2); Total +'ve molar charge LHRH peptides ~ 3.5 nmol +'ve charge/nmol FMD peptides (Table 2)
In 400 g FMD peptides there are 400 g/6805 g/ mol = 58.8 nmol of FMD immunogen
Total +'ve molar charge = 58.8 nmol x 3.5 nmols +'ve charge/nmol FMD peptides = 205.8 nmols +'ve charge
3) # nmol of-'vc charge contributed by CpGl:
Total calculated -'vc molar charge for CpGl Oligonucleotides
1 nmol of CpGl =31 nmol -'ve charge, FW = 10,295 g/ mol (Table 1)
Table 10. '
Neutralization Antibody Titers And Protection Data Obtained
In Cattle Immunized With FMD Vaccines.
(Table Removed)

Challenge intradermolingually (IDL) with a total of 104 BIDso FMDV 0 injected in two sites on the dorsal surface of the tongue.

We Claim:
1. A stabilized immunostimulatory complex comprising a cationic synthetic peptide immunogen comprising a target B cell or a CTL epitope, and a T helper cell epitope and anionic CpG oligonucleotide wherein the cationic synthetic peptide immunogen has a net positive charge at a pH in the range of 5.0 to 8.0 calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H), a-1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for all other amino acids in the peptide immunogen and wherein the anionic CpG oligonucleotide has a net negative charge at a pH in the range of 5.0-8. 0 and is a single-stranded DNA comprising 8 to 64 nucleotide bases with a repeat of a cytosine-guanidine motif and the number of repeats of the CpG motif is in the range of 1 to 10 wherein the CpG oligonucleotide is modifed with a phosphorothiorate or a phosphodiester, and said anionic CpG oligonucleotide is preferably selected from a group consisting of 5' TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpGl) SEQ ID NO: 1, a 32 base length oligomer and 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO : 2, a 24 base length oligomer plus an phosphorothioate group designated as n and a combination thereof.
2. The immunostimulatory complex as claimed in claim 1, wherein the cationic peptide immunogen is a synthetic peptide.
3. The immunostimulatory complex as claimed in claim 1, wherein the net positive charge of the cationic synthetic peptide immunogen is at least +2.
4. The immunostimulatory complex as claimed in claim 3, wherein the average net positive charge of the mixture of synthetic peptide immunogents is at least +2.
5. The immunostimulatory complex as claimed in claim 1, wherein the net negative charge of the anionic oligonucleotide is at least -2.
6. The immunostimulatory complex as claimed in claim 1, wherein the CpG oligonucleotide is a single-stranded DNA molecule with 18-48 nucleotide bases and the number of repeats of CpG motif therein in the range of 3 to 8.

7. The immunostimulatory complex as claimed in claim 1, wherein the CpG oligonucleotide has the formula: 5'X'CGX2 3'wherein C and G are unmethylated and X1 is selected from the group consisting of A (adenine), G (guanine) and T (thymine); and X2 is C (cytosine) or T (thymine).
8. The immunostimulatory complex as claimed in claim 1, wherein the CpG oligonucleotide has the formula: 5'(X3)2CG(X4)23' wherein C and G are unmethylated and X3 is selected from the group consisting of A, T and G; and X4 is C or T.
9. The immunostimulatory complex as claimed in claim 1, wherein the CpG oligonucleotide is modified with a phosphorothioate.
10. The immunostimulatory complex of claim 1, wherein CpG oligonucleotide is 5'TCG TCG TTT TGT CGT TTT GTC GTT TTG TCG TT 3' (CpG 1) SEQ ID NO : 1.
11. The immunostimulatory complex of claim 1, wherein CpG oligonucleotide is 5'nTC GTC GTT TTG TCG TTT TGT CGT T 3' (CpG2) SEQ ID NO: 2, a 24 base length oligomer plus an phosphorothioate group designated as n.
12. The immunostimulatory complex as claimed in claim 1, wherein the cationic synthetic peptide immunogen is a derived from a group consisting of HIV CD4, LHRH, IgE and FMD.
13. The immunostimulatory complex as claimed in claim 12, wherein the synthetic peptide derived from HIV CD4 is selected from the group consisting of SEQ ID NO: 4,5, 6 and a mixture thereof.
14. The immunostimulatory complex of claim 12, wherein the cationic peptide immunogen is a synthetic peptide derived from LHRH.

15. The immunostimulatory complex as claimed in claim 14 wherein the synthetic peptide derived from LHRH is selected from the group consisting of SEQ ID NO: 7,8, 9 and a mixture thereof.
16. The immunostimulatory complex as claimed in claim 12, wherein the synthetic peptide derived from IgE is selected from the group consisting of SEQ ID NO: 10, 11 and a mixture thereof.
17. The immunostimulatory complex as claimed in claim 12, wherein the synthetic peptide derived from FMD is selected from the group consisting of SEQ ID NO: 12, 13 and a mixture thereof.
18. A process for preparing a stabilized immunostimulatory complex as claimed in claim 1 comprising the steps of:
(a) dissolving or dispersing the cationic synthetic peptide immunogen in an
aqueous phase selected from the group consisting of distilled deionized water,
saline, PBS or a mixture thereof such that the pH of the aqueous phase is lower
than the ionization point of the peptide immunogen;
(b) dissolving the anionic CpG oligonucleotide in an aqueous phase
selected from the group consisting of distilled deionized water, saline, PBS or a
mixture thereof;
(c) adding the CpG oligonucleotide in the aqueous phase dropwise to the
solution or dispersion of the cationic peptide immunogen in an amount to form a
stabilized immunostimulatory complex of the peptide immunogen and the CpG
oligonucleotide in a charge ratio of the cationic immunogen synthetic peptide to
the CpG oligonucleotide in the range of 16: 1 to 1: 1; optionally comprising the
step of
(d) removing the aqueous phase of the suspension of the stabilized
immunostimulatory complex obtained in step (c).
19. The process as claimed in claim 18, wherein the aqueous phase is removed by
lyophilization, or spray-drying.

20. The process as claimed in claim 18, wherein the immunostimulatory complex has an average particle size in the range of 1 to 50 uM.
21. The process as claimed in claim 18 wherein the amount of the immunogen peptide and the CpG oligonucleotide added is in a charge ratio in the range of 16: 1,4: 1, 2: 1, 1.5 : 1 or I: 1 of the cationic immunogen peptide to the CpG nucleotide.
22. A process for preparing the immunostimulatory complex as claimed in claim 1 in the form of a suspension comprising the steps of:

(a) preparing an immunostimulatory complex of claim 1, or preparing a solution of peptide immunogen selected from the group consisting of SEQ ID NOs: 3-13, in an aqueous phase selected from the group consisting of distilled deionized water, saline and phosphate buffered saline;
(b) preparing a suspension of a mineral salt selected from the group consisting of aluminum hydroxide, aluminum phosphate, and calcium phosphate, in an aqueous phase selected from the group consisting of distilled deionized water, saline and phosphate buffered saline;
(c) adding the immunostimulatory complex in the aqueous phase into an aqueous phase containing the mineral salt suspension;
(d) mixing the immunostimulatory complex with the mineral salt
suspension to form a mixed suspension,
or whenever a solution of peptide immunogens is prepared in step (a),
step (c) comprises adding the peptide solution to the suspension of the
mineral salt with mixing; and
step (d) comprises adding a CpG nucleotide selected from the group
consisting of SEQ ID NO : 1 and SEQ ID NO : 2 with mixing to form a mixed
suspension of an immunostimulatory complex and a mineral salt.
23. The process as claimed in claim 22, wherein the mineral salt is selected from the group consisting of Alhydrogel, Adjuphos® and mixtures thereof.
24. The process as claimed in claim 22, wherein the aqueous phase optionally comprises a surfactant, a tonifier, a preservative or combinations thereof.

25. The process as claimed in claim 24 wherein the aqueous phase comprises a tonifier selected from the group consisting of a PBS or saline and mixtures thereof.
26. The process as claimed in claim 24 comprising adding to the aqueous phase a preservative selected from the group consisting of 2-phenoxy-ethanol and derivatives thereof.
27. The process as claimed in claim 22 optionally comprising adding to the aqueous phase an adjuvant selected from the group consisting of MPL, MDP, DDA, Avridine, BAY-1005, DC-Choi, Murapalmitine, PCPP, a saponin, a Cholera Toxin, a heat labile Enterotoxin from E. Coli and a cytokine selected from the group consisting of IL-1R, IL-2, IL-12, IFN-y and a derivative thereof.
28. An immunostimulatory complex as claimed in any preceding claim whenever used in a pharmaceutical composition.

Documents:

1935-DELNP-2004-Abstract-(01-05-2009).pdf

1935-DELNP-2004-Abstract-(07-07-2009).pdf

1935-delnp-2004-abstract.pdf

1935-DELNP-2004-Claims-(01-05-2009).pdf

1935-DELNP-2004-Claims-(07-07-2009).pdf

1935-delnp-2004-claims.pdf

1935-DELNP-2004-Correspondence-Others (10-11-2009).pdf

1935-DELNP-2004-Correspondence-Others-(01-05-2009).pdf

1935-DELNP-2004-Correspondence-Others-(07-07-2009).pdf

1935-DELNP-2004-Correspondence-Others-(19-05-2009).pdf

1935-delnp-2004-correspondence-others.pdf

1935-delnp-2004-description (complete).pdf

1935-delnp-2004-drawings.pdf

1935-DELNP-2004-Form-1-(01-05-2009).pdf

1935-delnp-2004-form-1.pdf

1935-delnp-2004-form-18.pdf

1935-DELNP-2004-Form-2-(01-05-2009).pdf

1935-delnp-2004-form-2.pdf

1935-DELNP-2004-Form-26-(19-05-2009).pdf

1935-delnp-2004-form-3.pdf

1935-delnp-2004-form-5.pdf

1935-delnp-2004-pct-101.pdf

1935-delnp-2004-pct-401.pdf

« Previous Patent

Next Patent »

Patent Number

238664

Indian Patent Application Number

1935/DELNP/2004

PG Journal Number

9/2010

Publication Date

26-Feb-2010

Grant Date

16-Feb-2010

Date of Filing

06-Jul-2004

Name of Patentee

UNITED BIOMEDICAL INC.

Applicant Address

25 DAVIDS DRIVE, HAUPPAUGE, NY 11788 (USA).

Inventors:

#	Inventor's Name	Inventor's Address
1	SOKOLL, KENNETH, K.	6 STRATTON LANE, STONY BROOK, NY 11790 (USA).

PCT International Classification Number

A61K

PCT International Application Number

PCT/US03/04711

PCT International Filing date

2003-02-14

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10/076,674	2002-02-14	U.S.A.
2	10/355,161	2003-01-31	U.S.A.