Title of Invention	"VACCINE FOR THE PROPHYLACTIC OR THERAPEUTIC IMMUNIZATION AGAINST HIV AND PROCESS FOR ITS PREPARATION"
Abstract	The present invention discloses a vaccine composition for human use comprising HIV Tat or HIV Nef or HIV Nef-Tat in combination with HIV gpl20 proteins or polynucleotides encoding them together with an adjuvant comprising QS21 and 3D-MPL, wherein the vaccine in use reduces the HIV viral load in HIV infected humans and results in a maintenance of CD4+ levels over those levels found in the absence of vaccination with HIV Tat, Nef or Nef-Tat and HIV gpl20 and a process for its preparation.

Title of Invention

"VACCINE FOR THE PROPHYLACTIC OR THERAPEUTIC IMMUNIZATION AGAINST HIV AND PROCESS FOR ITS PREPARATION"

Abstract

The present invention discloses a vaccine composition for human use comprising HIV Tat or HIV Nef or HIV Nef-Tat in combination with HIV gpl20 proteins or polynucleotides encoding them together with an adjuvant comprising QS21 and 3D-MPL, wherein the vaccine in use reduces the HIV viral load in HIV infected humans and results in a maintenance of CD4+ levels over those levels found in the absence of vaccination with HIV Tat, Nef or Nef-Tat and HIV gpl20 and a process for its preparation.

Full Text	"VACCINE FOR THE PROPHYLACTIC OR THERAPEUTIC IMMUNIZATION AGAINST HIV AND PROCESS FOR ITS PREPARATION" The present invention relates to novel uses of HIV proteins in medicine and vaccine compositions containing such HIV proteins. In particular, the invention relates to the use of HIV Tat and HIV gpl20 proteins in combination. Furthermore, the invention relates to the use of HIV Nef and HIV gp!20 proteins in combination. HIV-1 is the primary cause of the acquired immune deficiency syndrome (AIDS) which is regarded as one of the world's major health problems. Although extensive research throughout the world has been conducted to produce a vaccine, such efforts thus far have not been successful. The HIV envelope glycoprotein gpl20 is the viral protein that is used for attachment to the host cell. This attachment is mediated by the binding to two surface molecules of helper T cells and macrophages, known as CD4 and one of the two chemokine receptors CCR-4 or CXCR-5. The gpl20 protein is first expressed as a larger precursor molecule (gpl60), which is then cleaved post-translationally to yield gpl20 and gp41. The gpl20 protein is retained on the surface of the virion by linkage to the gp41 molecule, which is inserted into the viral membrane. The gpl20 protein is the principal target of neutralizing antibodies, but unfortunately the most immunogenic regions of the proteins (V3 loop) are also the most variable parts of the protein. Therefore, the use of gp120 (or its precursor gp 160) as a vaccine antigen to elicit neutralizing antibodies is thought to be of limited use for a broadly protective vaccine. The gp!20 protein does also contain epitopes that are recognized by cytotoxic T lymphocytes (CTL). These effector cells are able to eliminate virus-infected cells, and therefore constitute a second major antiviral immune mechanism. In contrast to the target regions of neutralizing antibodies some CTL epitopes appear to be relatively conserved among different HIV strains. For this reason gpl20 and gp!60 are considered to be useful antigenic components in vaccines that aim at eliciting cell-mediated immune responses (particularly CTL). 1 Non-envelope proteins of HIV-1 have been described and include for example internal structural proteins such as the products of the gag and pol genes and, other non-structural proteins such as Rev, Nef, Vif and Tat (Greene et al., New England J. Med, 324, 5, 308 et seq (1991) and Bryant et al. (Ed. Pizzo), Pediatr. Infect. Dis. J., ll,5,390etseq(1992). HIV Tat and Nef proteins are early proteins, that is, they are expressed early in infection and in the absence of structural protein. In a conference presentation (C. David Pauza, Immunization with Tat toxoid attenuates SHIV89.6PD infection in rhesus macaques, 12th Cent Gardes meeting, Mames-La-Coquette, 26.10.1999), experiments were described in which rhesus macaques were immunised with Tat toxoid alone or in combination with an envelope glycoprotein gpl60 vaccine combination (one dose recombinant vaccinia virus and one dose recombinant protein). However, the results observed showed that the presence of the envelope glycoprotein gave no advantage over experiments performed with Tat alone. However, we have found that a Tat- and/or Nef-containing imrnunogen (especially a Nef-Tat fusion protein) acts synergistically with gpl20 in protecting rhesus monkeys from a pathogenic challenge with chimeric human-simian immunodeficiency virus (SHIV). To date the SHIV infection of rhesus macaques is considered to be the most relevant animal model for human AIDS. Therefore, we have used this preclinical model to evaluate the protective efficacy of vaccines containing a gpl20 antigen and a Nef- and Tat-containing antigen either alone or in combination. Analysis of two markers of viral infection and pathogenieity, the percentage of CD4-positive cells in the peripheral blood and the concentration of free SHIV RNA genomes in the plasma of the monkeys, indicated that the two antigens acted in synergy. Immunization with either gp 120 or NefTat + SIV Nef alone did not result in any difference compared to immunization with an adjuvant alone. In contrast, the administration of the combination of gpl20 and NefTat + SIV Nef, antigens resulted in a marked improvement of the two above-mentioned parameters in all animals of those particular experimental group. 2 Thus, according to the present invention there is provided a new use of HIV Tat and/or Nef protein together with HIV gpl20 in the manufacture of a vaccine for the prophylactic or therapeutic immunisation of humans against HIV. As described above, the NefTat protein, the SIV Nef protein and gpl20 protein together give an enhanced response over that which is observed when either NefTat 4-SIV Nef, or gpl20 are used alone. This enhanced response, or synergy can be seen in a decrease in viral load as a result of vaccination with these combined proteins. Alternatively, or additionally the enhanced response manifests itself by a maintenance of CD4+ levels over those levels found in the absence of vaccination with HIV NefTat, SIV Nef and HIV gpl20. The synergistic effect is attributed to the combination of gpl20 and Tat, or gp!20 and Nef, or gp!20 and both Nef and Tat. The addition of other HIV proteins may further enhance the synergistic effect, which was observed between gpl20 and Tat and/or Nef. These other proteins may also act synergistically with individual components of the gpl20, Tat and/or Nef-containing vaccine, not requiring the presence of the full original antigen combination. The additional proteins may be regulatory proteins of HIV such as Rev, Vif, Vpu, and Vpr. They may also be structural proteins derived from the HIV gag or pot genes. The HIV gag gene encodes a precursor protein p55, which can assemble spontaneously into immature virus-like particles (VLPs). The precursor is then proteolytically cleaved into the major structural proteins p24 (capsid) and p18 (matrix), and into several smaller proteins. Both the precursor protein p55 and its major derivatives p24 and pl8 may be considered as appropriate vaccine antigens which may further enhance the synergistic effect observed between gp120 and Tat and/or Nef. The precursor p55 and the capsid protein p24 may be used as VLPs or as monomeric proteins. The HIV Tat protein in the vaccine of the present invention may, optionally be linked to an HIV Nef protein, for example as a fusion protein. 3 The HIV Tat protein, the HIV Nef protein or the NefTat fusion protein in the present invention may have a C terrnir al Histidine tail which preferably comprises between 5-10 Histidine residues. The presence of an histidine (or 'His') tail aids purification. In a preferred embodiment the proteins are expressed with a Histidine tail comprising between 5 to 10 and preferably six Histidine residues. These are advantageous in aiding purification. Separate expression, in yeast (Saccharomyces cerevisiae), of Nef (Macreadie I.G. et al, 1993, Yeast 9 (6) 565-573) and Tat (Braddock M et ah, 1989, Cell 58 (2) 269-79) has been reported. Nef protein and the Gag proteins p55 and pi 8 are myristilated. The expression of Nef and Tat separately in a Pichia expression system (Nef-His and Tat-His constructs), and the expression of a fusion construct Nef-Tat-His have been described previously in WO99/16884. The DNA and amino acid sequences of representative Nef-His (Seq. ID. No.s 8 and 9), Tat-His (Seq. ID. No.s 10 and 1l)and of Nef-Tat-His fusion proteins (Seq. ID. No.s 12 and 13) are set forth in Figure 1. The HIV proteins of the present invention may be used in their native conformation, or more preferably, may be modified for vaccine use. These modifications may either be required for technical reasons relating to the method of purification, or they may be used to biologically inactivate one or several functional properties of the Tat or Nef protein. Thus the invention encompasses derivatives of HIV proteins which may be, for example mutated proteins. The term 'mutated' is used herein to mean a molecule which has undergone deletion, addition or substitution of one or more amino acids using well known techniques for site directed mutagenesis or any other conventional method. For example, a mutant Tat protein may be mutated so that it is biologically inactive whilst still maintaining its immunogenic epitopes. One possible mutated tat gene, constructed by D.Clements (Tulane University), (originating from BH10 molecular clone) bears mutations in the active site region (Lys4l->Ala)and in RGD motif (Arg78->Lys and Asp80->Glu) ( Virology 235: 48-64, 1997). 4 A mutated Tat is illustrated in Figure 1 (Seq. ID. No.s 22 and 23) as is a Nef-Tat Mutant-His (Seq. ID. No.s 24 and 25). The HIV Tat or Nef proteins in the vaccine of the present invention may be modified by chemical methods during the purification process to render the proteins stable and monomeric. One method to prevent oxidative aggregation of a protein such as Tat or Nef is the use of chemical modifications of the protein's thiol groups. In a first step the disulphide bridges are reduced by treatment with a reducing agent such as DTT, beta-mercaptoethanol, or gluthatione. In a second step the resulting thiols are blocked by reaction with an alkylating agent (for example, the protein can be carboxyamidated/carbamidomethylated using iodoacetamide). Such chemical modification does not modify functional properties of Tat or Nef as assessed by cell binding assays and inhibition of lymphoproliferation of human peripheral blood mononuclear cells. The HIV Tat protein and HIV gpl20 proteins can be purified by the methods outlined in the attached examples. The vaccine of the present invention will contain an immunoprotective or immunotherapeutic quantity of the Tat and/or Nef or NefTat and gpl20 antigens and may be prepared by conventional techniques. Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757. The amount of protein in the vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise 1-1000 ?g of each 5 protein, preferably 2-200 ?g, most preferably 4-40 ?g of Tat or Nef or NefTat and preferably 1-150 ?g, most preferably 2-25 ?g of gpl20. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody litres and other responses in subjects. One particular example of a vaccine dose will comprise 20 fig of NefTat and 5 or 20 fig of gpl20. Following an initial vaccination, subjects may receive a boost in about 4 weeks, and a subsequent second . booster immunisation. The proteins of the present invention are preferably adjuvanted in the vaccine formulation of the invention. Adjuvants are described in general in Vaccine Design -the Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995. Suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes. In the formulation of the invention it is preferred that the adjuvant composition induces a preferential Thl response. However it will be understood that other responses, including other humoral responses, are not excluded. An immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system. The resultant immune response may be broadly distinguished into two extreme catagories, being humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed Thl-type responses (cell-mediated response), and Th2-type immune responses (humoral response). Extreme Th 1 -type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice Thl-type responses are often characterised by the generation of 6 antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. Th2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM. It can be considered that the driving force behind the development of these two types of immune responses are cytokines, a number of identified protein messengers which serve to help the ceils of the immune system and steer the eventual immune response to either a Thl or Th2 response. Thus high levels of Thl -type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of Th2-type cytokines tend to favour the induction of humoral immune responses to the antigen. It is important to remember that the distinction of Thl and Th2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly Thl or predominantly Th2. However, it is often convenient to consider the families of cytokines in terms of that described in rnurine CD4 +ve T cell clones by Mosmann and Coffinan (Mosmann. T.R. and Coffman, R.L. (1989) TH1 and TH2 cells: different patterns oflymphokine secretion had to different functional properties. Annual Review of Immunology, 7, p 145-173). Traditionally, Thl-type responses are associated with the production of the INF-y and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of Thl-type immune responses are not produced by T-cells, such as IL-I2. In contrast, Th2- type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10 and tumour necrosis factor-p(TNF-p). It is known that certain vaccine adjuvants are particularly suited to the stimulation of either Th 1 or Th2 - type cytokine responses. Traditionally the best indicators of the Thl:Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Thl or Th2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement of the IgG 1 :IgG2a ratio of antigen specific antibody responses. ¦7 Thus, a Thl-type adjuvant is cne which stimulates isolated T-cell populations to produce high levels of Thl-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific ixnxnunoglohuhn responses associated with Thl-type isotype. Preferred Thl-type immunostimulants which may be formulated to produce adjuvants suitable for use in the present invention include and are not restricted to the following. Monophosphoryl lipid A, in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL), is a preferred Thl-type imrmmostirnulant for use in the invention. 3D-MPL is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. It can be purified and prepared by the methods taught in GB 2I22204B, which reference also discloses the preparation of diphosphoryl lipid A, and 3-O-deacylated variants thereof. Other purified and synthetic lipopoiysaccharides have been described (US 6,005,099 and EP 0 729 473 Bl; Hilgers et al., 1986, Int.Arch.AHergy.Immunol, 79(4):392-6; Hilgers etal., 1987, Immunology, 60(l):141-6; and EP 0 549 074 Bl). A preferred form of 3D-MPL is in the form of a particulate formulation having a small particle size less than 0.2jxm in diameter, and its method of manufacture is disclosed in EP 0 689 454. Saponins are also preferred Thl immunostimulants in accordance with the invention. Saponins are well known adjuvants and are taught in: Lacaille-Dubois, M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363-386). For example, Quit A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof, are described in US 5,057,540 and "Saponins as vaccine adjuvants", Kensil, C. R-, Crit Rev Ther Drug Carrier Syst, 1996, 12 (l-2):L-55; and EP 0 362 279 BL The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants, and the method of their production is disclosed in US Patent No. 5,057,540 and EP 0 362 279 Bl. Also described in these references is the use of QS7 (a non-haemolytic fraction of Quil-A) which acts as a potent adjuvant for systemic vaccines. Use of QS21 is further described in Kensil et al, (199l J. 8 Immunology vol 146,431-437). Combinations of QS21 and polysorbate or cyclodextrin are also known (WO 99/10008). Paniculate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in WO 96/33739 and WO 96/11711. Another preferred Imnnmostimulant is an irnmunostirmiiatory oiigonucleotide containing unmethylated CpG dinucleotides ("CpG"). CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et ai. J.Immunot, 1998, I60(2):870-S76; McCluskie and Davis, JJmmunol, 1998, I61(9):4463-6). Historically, it was observed that the DNA fraction of BCG could exert an anti-tumour effect. In further studies, synthetic oligonucleotides derived from BCG gene sequences were shown to be capable of inducing immunostimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a central CG motif, carried this activity. The central role of the CG motif in Jmirmnostimulation was later elucidated in a publication by Krieg, Nature 374, p546 1995. Detailed analysis has shown that the CG motif has to be in a certain sequence context, and that such sequences are common in bacterial DNA but are rare in vertebrate DNA. The immunostimuiaiory sequence is often: Purine, Purine, C, G, pyrimidine, pyrimidme; wherein the CG motif is not methylated, but other unmethylated CpG sequences are known to be irnmunostimuiatory and may be used in the present invention. In certain combinations of the six nucleotides a palindromic sequence is present. Several of these motifs, either as repeats of one motif or a combination of different motifs, can be present in the same oligonucleotide. The presence of one or more of these immunostimulatory sequences containing oligonucleotides can activate various immune subsets, including natural killer celts (which produce interferon y and have cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8), 1977). Other unmethylated CpG containing sequences not having this consensus sequence have also now been shown to be irnmunomodulatory. 9 CpG when formulated into vaccines, is generally administered in free solution together with free antigen (WO 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (WO 98/16247), or formulated with a carrier such as aluminium hydroxide ((Hepatitis surface antigen) Davis et al supra ; Brazolot-Millan et al, Proc.Natl.Acad.Scl, USA, 1998, 95(26), 15553-8). Such immunostimulants as described above may be formulated together with carriers, such as for example liposomes, oil in water emulsions, and or metallic salts, including aluminium salts (such as aluminium hydroxide). For example, 3D-MPL may be formulated with aluminium hydroxide (EP 0 689 454) or oil in water emulsions (WO 95/17210); QS21 maybe advantageously formulated with cholesterol containing liposomes (WO 96/33739), oil in water emulsions (WO 95/17210) or alum (WO 98/15287); CpG may be formulated with alum (Davis et al. supra ; Brazolot-Millan supra) or with other cationic carriers. Combinations of immunostimulants are also preferred, in particular a combination of a monophosphoryl lipid A and a saponin derivative (WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241), more particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153. Alternatively, a combination of CpG plus a saponin such as QS21 also forms a potent adjuvant for use in the present invention. Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, together with an aluminium salt. An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched in cholesterol containing liposomes (DQ) as disclosed in WO 96/33739. A particularly potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is another preferred formulation for use in the invention. 10 Another preferred formulation comprises a CpG oligonucleotide alone or together with an aluminium salt In another aspect of the invention, the vaccine may contain DNA encoding one or more of the Tat, Nef and gpI20 polypeptides, such that the polypeptide is generated in situ. The DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems such as plasmid DNA, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Holland, Crit. Rev, Therap. Drug Carrier Systems 15:143-198, 1998 and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). When the expression system is a recombinant live microorganism, such as a virus or bacterium, the gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses. Viruses and bacteria used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox, modified poxviruses e.g. Modified Virus Ankara (MVA))t alphaviruses (Sindbis virus, Semliki Forest Virus, Venezueliart Equine Encephalitis Virus), flaviviruses (yellow fever virus, Dengue virus, Japanese encephalitis virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella, Neisseria, BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention. Thus, the Nef, Tat and gpl20 componerflts of a preferred vaccine according to the invention may be provided in the form of polynucleotides encoding the desired proteins. Furthermore, immunisations according to the invention may be performed with a combination of protein and DNA-based formulations. Prime-boost immunisations are considered to be effective in inducing broad immune responses. Adjuvanted protein vaccines induce mainly antibodies and T helper immune responses, while delivery of DNA as a plasmid or a live vector induces strong cytotoxic T lymphocyte (CTL) It responses. Thus, the combination of protein and DNA vaccination will provide for a wide variety of immune respc rises: This is particularly relevant in the context of HIV, since both neutralising antibodies and CTL are thought to be important for the immune defence against HIV. In accordance with the invention a schedule for vaccination with gp!20, Nef and Tat, alone or in combination, may comprise the sequential ("prime-boost") or simultaneous administration of protein antigens and DNA encoding the above-mentioned proteins. The DNA may be delivered as plasmid DNA or in the form of a recombinant live vector, e.g. a poxvirus vector or any other suitable live vector such as those described herein. Protein antigens may be injected once or several times followed by one or more DNA administrations, or DNA may be used first for one or more administrations followed by one or more protein immunisations. A particular example of prime-boost immunisation according to the invention involves priming with DNA in the form of a recombinant live vector such as a modified poxvirus vector, for example Modified Virus Ankara (MVA) or a alphavinis, for example Venezuelian Equine Encephalitis Virus followed by boosting with a protein, preferably an adjuvanted protein. Thus the invention further provides a pharmaceutical kit comprising: a) a composition comprising one or more of gpl20, Nef and Tat proteins together with a pharmaceutically acceptable excipient; and b) a composition comprising one or more of gpl20, Nef and Tat-encoding polynucleotides together with a pharmaceutically acceptable excipient; with the proviso that at least one of (a) or (b) comprises gpl20 with Nef and/or Tat and/or Nef-Tat. Compositions a) and b) may be administered separately, in any order, or together. Preferably a) comprises all three of gpl20, Nef and Tat proteins. Preferably b) comprises all three of gpl20, Nef and Tat DNA. Most preferably the Nef and Tat are in the form of a NefTat fusion protein. In a further aspect of the present invention there is provided a method of manufacture of a vaccine formulation as herein described, wherein the method comprises admixing 12 a combination of proteins according to the invention. The protein composition may be mixed with a suitable adjuvant and, optionally, a carrier. Particularly preferred adjuvant and/or carrier combinations for use in the formulations according to the invention are as follows: i) 3D-MPL + QS21 in DQ ii) Alum + 3D-MPL iii) Alum + QS21 in DQ + 3D-MPL iv) Alum + CpG v) 3D-MPL + QS21 in DQ + oil in water emulsion vi) CpG The invention is illustrated in the accompanying examples and Figures: 13 EXAMPLES General The Nef gene from the Bru/Lai isolate (Cell 40: 9-17, 1985) was selected for the constructs of these experiments since this gene is among those that are most closely related to the consensus Nef. The starting material for the Bra/Lai Nef gene was a U70bp DNA fragment cloned on the mammalian expression vector pcDNA3 (pcDNA3/Nef). The Tat gene originates from the BH10 molecular clone. This gene was received as an HTLV III cDNA clone named pCVl and described in Science, 229, p69-73, 1985. The expression of the Nef and Tat genes could be in Pichia or any other host. Example 1. EXPRESSION OF HIV-1 nef AND tat SEQUENCES IN PICHIA PASTORIS. Nef protein, Tat protein and the fusion Nef-Tat were expressed in the methylotrophic yeast Pichia pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. To express these HI\M genes a modified version of the integrative vector PHIL-D2 (INVITROGEN) was used. This vector was modified in such a way that expression of heterologous protein starts immediately after the native ATG codon of the AOX1 gene and will produce recombinant protein with a tail of one glycine and six histidines residues . This PHIL-D2-MOD vector was constructed by cloning an oligonucleotide linker between the adjacent AsuII and EcoRI sites of PHIL-D2 vector (see Figure 2). In addition to the His tail, this linker carries Ncol, Spel and Xbal restriction sites between which nef tat and nef tat fusion were inserted. 14 1.1 CONSTRUCTION OF THE INTEGRATIVE VECTORS pRIT14597 (encoding Nef-His protein), pRlT14598 (encoding Tat-Hls protein) and pRIT14599 (encoding fusion Nef-Tat-His). The nefgme was amplified by PCR from the pc0NA3/Nef plasniid with primers 01 and 02. Ncol PRIMER 01 (Seq ID NO 1): SWTCGTCCATG.GGT.GGCAAG.TGG.T 3' Spel PRIMER 02 (Seq ID NO 2): 5' CGGCTACTAGTGCAGTTCTTGAA 3* The PCR fragment obtained and the iategrative PHIL-D2-MOD vector were both restricted by Ncol and Spel, purified on agarose gel and iigated to create the integrative plasmid pRJT14597 (see Figure 2). The tat gene was amplified by PCR from a derivative of the pCVl plasmid with primers 05 and 04: Spel PRIMER 04 (Seq ID NO 4): 5' CGGCTACTAGTTTCCTTCGGGCCT 3' Ncol PRIMER 05 (Seq ID NO 5): 5'ATCGTCCATGGAGCCAGTAGATC 3' An Ncol restriction site was introduced at the 5' end of the PCR fragment while a Spel site was introduced at the 3' end with primer 04. The PCR fragment obtained 15 and the PHIL-D2-MOD vecto- were both restricted by Ncol and Spel, purified on agarose gel and Iigated to create the initegrative plasmid pRIT14598. To construct pRIT14599, a 910bp DNA fragment corresponding to the nef-tat-His coding sequence was Iigated between the EcoRI blunted(T4 polymerase) and Ncol sites of the PHIL-D2-M0D vector. The nef-tat-His coding fragment was obtained by Xbal blunted(T4 polymerase) and Ncol digestions of pRIT14596. 1.2 TRANSFORMATION OF PICHIA PASTORIS STRAIN GS115(his4). To obtain Pichia pastoris strains expressing Nef-His, Tat-His and the fusion Nef-Tat-His, strain GS115 was transformed with linear NotI fragments carrying the respective expression cassettes plus the HIS4 gene to complement his4 in the host genome.Transformation of GS115 with Notl-linear fragments favors recombination at the AOXI locus. Multicopy integrant clones were selected by quantitative dot blot analysis and the type of integration, insertion (Mut+phenotype) or transplacement (Mutsphenotype), was determined. From each transformation, one transformant showing a high production level for the recombinant protein was selected : Strain Y1738 (Mut+ phenotype) producing the recombinant Nef-His protein, a myristylated 215 amino acids protein which is composed of: °Myristic acid °A methionine, created by the use of Ncol cloning site of PHIL-D2-MOD vector °205 a.a. of Nef protein(starting at a.a.2 and extending to a.a.206) °A threonine and a serine created by the cloning procedure (cloning at Spel site of PHIL-D2-M0D vector. °One glycine and six histidines. 16 Strain Y1739 (Mut phenotype) producing the Tat-His protein, a 95 amino acid protein which is composed of: °A methionine created by the use of Ncol cloning site °85 a.a. of the Tat protein(starting at a.a.2 and extending to a.a.86) °A threonine and a serine introduced by cloning procedure °One glycine and six histidines Strain Yl737(Muts phenotype) producing the recombinant Nef-Tat-His fusion protein, a myristylated 302 amino acids protein which is composed of: °Myristic acid °A methionine, created by the use of Ncol cloning site °205a.a. of Nef protein(starting at a.a.2 and extending to a.a.206) °A threonine and a serine created by the cloning procedure °85a.a. of the Tat protein(starting at a.a.2 and extending to a.a.86) °A threonine and a serine introduced by the cloning procedure °One glycine and six histidines Example 2. EXPRESSION OF HIV-1 Tat-MUTANT IN PICHIA PASTORIS A mutant recombinant Tat protein has also been expressed. The mutant Tat protein must be biologically inactive while maintaining its immunogenic epitopes. A double mutant tat gene, constructed by D.Clements (Tulane University) was selected for these constructs. This tat gene (originates from BH10 molecular clone) bears mutations in the active site region (Lys41-Ala)and in RGD motif (Arg78-Lys and Asp80-»Glu) (Virology 235: 48-64, 1997). 17 The mutant tat gene was received as a cDNA fragment subcloned between the EcoRl and Hindlll sites within a CMV expression plasmid (pCMVLys41/KGE) 2.1 CONSTRUCTION OF THE INTEGRATIVE VECTORS pRIT14912(encoding Tat mutant-His protein) and pRIT14913(encoding fusion Nef-Tat mutant-His). The tat mutant gene was amplified by PCR from the pCMVLys41/KGE plasmid with primers 05 and 04 (see section 1.1 construction of pRIT14598) An Ncol restriction site was introduced at the 5* end of the PCR fragment while a Spel site was introduced at the 3* end with primer 04. The PCR fragment obtained and the PHIL-D2-MOD vector were both restricted by Ncol and Spel, purified on agarose gel and ligated to create the integrative plasmid pRIT14912 To construct pRIT14913, the tat mutant gene was amplified by PCR from the pCMVLys41/KGE plasmid with primers 03 and 04. Spel PRIMER 03 (Seq ID NO 3): 5* ATCGTACTAGT.GAG.CCA.GTA.GAT.C 3* Spel PRIMER 04 (Seq ID NO 4): 5' CGGCTACTAGTTTCCTTCGGGCCT 3' The PCR fragment obtained and the plasmid pRIT14597 (expressing Nef-His protein) were both digested by Spel restriction enzyme, purified on agarose gel and ligated to create the integrative plasmid pRIT14913 2.2 TRANSFORMATION OF PICHIA PASTORIS STRAIN GS115. 18 Pichia pastoris strains expressing Tat mutant-His protein and the fusion Nef-Tat mutant-His were obtained, by applying integration and recombinant strain selection strategies previously described in section 1.2 . Two recombinant strains producing Tat mutant-His protein ,a 95 amino-acids protein, were selected: YI775 (Mut+ phenotype) and Y1776(Muts phenotype). One recombinant strain expressing Nef-Tat mutant-His fusion protein, a 302 amino-acids protein was selected: Yl 774(Mut+ phenotype). Example 3: FERMENTATION OF PICHIA PASTORIS PRODUCING RECOMBINANT TAT-HIS. A typical process is described in the table hereafter. Fermentation includes a growth phase (feeding with a glycerol-based medium according to an appropriate curve) leading to a high cell density culture and an induction phase (feeding with a methanol and a salts/micro-elements solution). During fermentation the growth is followed by taking samples and measuring their absorbance at 620 run. During the induction phase methanol was added via a pump and its concentration monitored by Gas chromatography (on culture samples) and by on-line gas analysis with a Mass spectrometer. After fermentation the cells were recovered by centrifugation at 5020g during 30' at 2-8°C and the cell paste stored at -20°C. For further work cell paste was thawed, resuspended at an OD (at 620 nm) of 150 in a buffer (Na2HPO4 pH7 50 mM, PMSF 5%, Isopropanol 4 mM) and disrupted by 4 passages in a DynoMill (room 0.6L, 3000 rpm, 6L/H, beads diameter of 0.40-0.70 mm). For evaluation of the expression samples were removed during the induction, disrupted and analyzed by SDS-Page or Western blot. On Coomassie blue stained SDS-gels the recombinant Tat-his was clearly identified as an intense band presenting a maximal intensity after around 72-96H induction. 19 20 Media used for fermentation: 2 Example 4: PURIFICATION OF Nef-Tat-His FUSION PROTEIN (PICHIA PASTORIS) The purification scheme has been developed from I46g of recombinant Pichia pastoris cells (wet weight) or 2L Dyno-mill homogenate OD 55. The chromatographic steps are performed at room temperature. Between steps , Nef»Tat positive fractions are kept overnight in the cold room (+4°C) ; for longer time, samples are frozen at -20°C. 23 24 25 26 ¦^ Level Of purity estimated by SDS-PAGE is shown in Figure 6 (Daiichi Silver Staining. Coomassie blue G250. Western blotting): After dialysis and sterile filtration steps: > 95% -> Recovery (evaluated by a colorimetric protein assay: DOC TCA BCA) 2,8 mg of oxidized Nef-Tat-his protein are purified from 73 g of recombinani Pichia pastoris cells (wet weight) or 1 L of Dyno-mill homogenate OD 50. Example 6: PURIFICATION OF REDUCED TAT-HIS PROTEIN (PICHIA PASTORIS) The purification scheme has been developed from 160 g of recombinant Pichia pastoris cells (wet weight) or 2L Dyno-mill homogenate OD 66. The chromatographic steps are performed at room temperature. Between steps, Tat positive fractions are kept overnight in the cold room (+4°C) ; for longer time, samples are frozen at -20°C. 27 28 "^ Level of purity estimated by SDS-PAGE as shown in Figure 7(Daiichi Silver Staining. Coomassie blue G250. Western blotting): After dialysis and sterile filtration steps: > 95% "^ Recovery (evaluated by a colorimetric protein assay: DOC TCA BCA) 48 mg of reduced Tat-his protein are purified from 160 g of recornbinant Pichia pastoris cells (wet weight) or 2 L of Dyno-mill homogenate OD 66. 29 The purification scheme has been developed from 74 g of recombinant Pichia pastoris cells (wet weight) or 1L Dyno-mill homogenate OD60. The chromatographic steps are performed at room temperature. Between steps, Tat positive fractions are kept overnight in the cold room (+4°C) ; for longer time, samples are frozen at -20°C. "^ Level of purity estimated by SPS-FAGE as shown in Figure g fDaiichi Silver Staining, Coomassie blue G250. Western blotting): After dialysis and sterile filtration steps: > 95% "^ Recovery (evaluated by a colorimetric protein assay; DOC TCA BCA) 31 19 mg of oxidized Ta;-his protein are purified from 74 g of reeombinant Pichia pastoris cells (wet weight) or 1 L of Dyno-mill homogenate OD 60. Example S: PURIFICATION OF SIV REDUCED NEF-HIS PROTEIN (PICHIA PASTORIS) 32 The purification scheme has been developed from 340 g of reeombinant Pichia pastoris cells (wet weight) or 4 L Dyno-mill homogenate OD 100. The chromatographic steps are performed at room temperature. Between steps , Nef positive fractions are kept overnight in the cold room (+4°C) ; for longer time, samples are frozen at -20°C. •> Level of purity estimated by SDS-PAGE as shown in Figure 9 (Daiichi Silver Staining. Coomassie blue G250. Western blotting"): After dialysis and sterile filtration steps: > 95% "^ Recovery (evaluated by a colorimetric protein assay: DOC TCA BCA) 33 20 mg of SIV reduced Nef-his protein are purified irorn 340 g of recombinant Pichia pastoris cells (wet weight) or 4 L of Dyno-miH homogenate OD 100. Example 9: PURIFICATION OF HIV REDUCED NEF-HIS PROTEIN (PICHIA PASTORIS) 34 The purification scheme has been developed from 160 g of recombinant Pichia pastoris cells (wet weight) or 3 L Dyno-miH homogenate OD 50. The chromatographic steps are performed at: room temperature. Between steps , Nef positive fractions are kept overnight in the cold room (+4°C); for longer time, samples are frozen at ~20°C. ¦> Level of purity estimated by SDS-PAGE as shown in Figure 10 (Daiichi Silver Staining. Coomassie blue G250, Western blotting): After dialysis and sterile filtration steps: > 95% -^ Recovery (evaluated by a colorimetric protein assay: DOC TCA BCA) 35 20 mg of HIV reduced Nef-his protein are purified from 160 g of recombinant Pichia pistons cells (wet weight) or 3 L of Dyno-mill homogenate OD 50. Example 10: EXPRESSION OF nef SEQUENCE IN PICHIA PASTORIS In order to evaluate Nef and Tat antigens in the pathogenic SHIV challenge model, we have expressed the Nef protein of simian immunodeficiency virus (SIV) of macaques, SIVmac239 ( Aids Research and Human Retroviruses, 6:1221-1231,1990). In the Nef coding region , SIV mac 239 has an in-frame stop codon after 92aa predicting a truncated product of only 10kD. The remainder of the Nef reading frame is open and would be predicted to encode a protein of 263aa (30kD) in its fully open form. Our starting material for SIVmac239 nefgene was a DNA fragment corresponding to the complete coding sequence, cloned on the LX5N plasmid (received from Dr R.C. Desrosiers, Southborough,MA,USA). This SIV nef gene is mutated at the premature stop codon (nucleotide G at position 9353 replaces the original T nucleotide) in order to express the full-length SIVmac239 Nef protein. To express this SIV nef gene in Pichia pastoris, the PHIL-D2-MOD Vector (previously used for the expression of HIV-1 nef and tat sequences) was used. The recombinant protein is expressed under the control of the inducible alcohol oxidase (AOX1) promoter and the c-terminus of the protein is elongated by a Histidine affinity tail that will facilitate the purification. 10.1 CONSTRUCTION OF THE INTEGRATIVE VECTOR PRIT 14908 To construct pRIT 14908 , the SIV nef gene was amplified by PCR from the pLX5N/SIV-NEF plasmid with primers SNEF1 and SNEF2. 36 PRIMER SNEF1: 5' ATCGTCCATG.GGTGGAGCTATTTT 3' Ncol PRIMER SNEF2; 5' CGGCTACTAGTGCGAGTTTCCTT 3+ Spel The SIV nef DNA region amplified starts at nucleotide 9077 and terminates at nucleotide 9865 ( Aids Research and Human Retroviruses, 6:1221-1231,1990). An Ncol restriction site (with carries the ATG codon of the nef gene) was introduced at the 5' end of the PCR fragment while a Spel site was introduced at the 3* end. The PCR fragment obtained and the integrative PHIL-D2-MOD vector were both restricted by Ncol and Spel. Since one Ncol restriction site is present on the SIV nef amplified sequence (at position 9286), two fragments of respectively ±200bp and ± 600bp were obtained, purified on agarose gel and ligated to PHIL-D2-MOD vector. The resulting recomblnant plasmid received, after verification of the nef amplified region by automated sequencing, the pRIT 14908 denomination. 10.2 TRANSFORMATION OF PICH1A PASTORIS STRAIN GS115(his4). To obtain Pichia oastoris strain expressing SIV nef His, strain GS115 was transformed with a linear Noil fragment carrying only the expression cassette and the fflS4gene(Fig.Il). This linear NotI DNA fragment ,with homologies at both ends with A0X1 resident P.pastoris gene, favors recombination at the A0X1 locus. Multicopy integrant clones were selected by quantitative dot blot analysis . One transformant showing the best production level for the recombinant protein was selected and received the Y1772 denomination. Strain Y1772 produces the recombinant SIV Nef-His protein, a 272 amino acids protein which would be composed of: °Myristic acid °A methionine, created by the use of Ncol cloning site of PHIL-D2-MOD vector . 37 °262 amino acids (aa) of Nef protein (starting at aa 2 and extending to aa 263, see Figure 12) °A threonine and a serine created by the cloning procedure (cloning at Spel site of PHIL-D2-M0D vector (Fig. 11). °One glycine and six histidines. Nucleic and Protein sequences are shown on figure 12. 10.3 CHARACTERIZATION OF THE EXPRESSED PRODUCT OF STRAIN Y1772. Expression level After 16 hours induction in medium containing 1% methanol as carbon source, abundance of the recombinant Nef-His protein, was estimated at 10% of total protein (Fig. 13 , lanes 3-4). Solubility Induced cultures of recombinant strain Y1772 producing the Nef-His protein were centriruged. Cell pellets were resuspended in breaking buffer, disrupted with 0.5mm glass beads and the cell extracts were centrifiiged. The proteins contained in the insoluble pellet (P) and in the soluble supernatant (S) were compared on a Coomassie Blue stained SDS-PAGE10%. As shown in figure 13, the majority of the recombinant protein from strain Y1772 (lanes 3-4) is associated with the insoluble fraction. Strain Y1772 which presents a satisfactory recombinant protein expression level is used for the production and purification of SIV Nef-His protein. Example 11: EXPRESSION OF GP120 IN CHO 38 A stable CHO-K1 cell line which produces a recombinant gPI20 glycoprotein has been established. Recombinant gP120 glycoprotein is a recombinant truncated form of the gP120 envelope protein of HIV-1 isolate W61D. The protein is excreted into the cell culture medium, from which it is subsequently purified. Construction of gp!20 transfection plasmid pRJT13968 The envelope DNA coding sequence (including the 5'exon of tat and rev) of HIV-1 isolate W61D was obtained (Dr. Tersmette, CCB, Amsterdam) as a genomic gpl60 envelope containing plasmid W61D (Nco-Xhol). The plasmid was designated pRIT13965. In order to construct a gpI20 expression cassette a stop codon had to be inserted at the ammo acid glu 515 codon of the gp!60 encoding sequence in pRIT13965 using a primer oligonucleotide sequence (DIR 131) and PCR technology. Primer DIR 131 contains three stop codons (in all open reading frames) and a Sail restriction site. The complete gpl20 envelope sequence was then reconstituted from the N-terminal BamHl-Dral fragment (170 bp) of a gpl60 plasmid subclone pW61d env (pRIT13966) derived from pRIT13965, and the Dral-Sall fragment (510 bp) generated by PCR from pRIT13965. Both fragments were gel purified and ligated together into the E.coli plasmid pUC18, cut first by Sail (klenow treated), and then by BamHl. This resulted in plasmid pRITl3967. The gene sequence of the Xmal-Sall fragment (1580 bp) containing the gp!20 coding cassette was sequenced and found to be identical to the predicted sequence. Plasmid RIT13967 was ligated into the CHO GS-expression vector pEE 14 (Celitech Ltd., UK) by cutting first with Bell (klenow treated) and then by Xmal. The resulting plasmid was designated pRIT13968. Preparation of Master Cell Bank The gpl20-construct (pRIT13968) was transfected into CHO cells by the classical CaPO4-precipitation/grycero! shock procedure. Two days later the CHOK1 cells were subjected to selective growth medium (GMEM +¦ methionine sulfoximine (MSX) 25 pM + Glutamate + asparagine 10% Foetal calf serum ). Three chosen 39 transfectant clones were farther amplified in 175m2 flasks and few cell vials were stored at -80°C. C-env 23,9 was selected for further expansion. A small prebank of cells was prepared and 20 ampoules were frozen. For preparation of the prebank and the MCB, cells were grown in GMEM culture medium, supplemented with 7.5 % fetal calf serum and containing 50 uM MSX. These cell cultures were tested for sterility and mycoplasma and proved to be negative. The Master Cell Bank CHOKI env 23,9 (at passage 12) was prepared using cells derived from the premaster cell bank. Briefly, two ampoules of the premaster seed were seeded in medium supplemented with 7.5% dialysed foetal bovine serum. The cells were distributed in four culture flasks and cultured at 37°C. After cell attachment the culture medium was changed with fresh medium supplemented with 50 uM MSX. At confluence, cells were collected by trypsination and subcultured with a 1/8 split ratio in T-flasks - roller bottle - cell factory units. Cells were collected from cell factory units by trypsination and centrifugation. The cell pellet was resuspended in culture medium supplemented with DMSO as cryogenic preservative. Ampoules were prelabelled, autoclaved and heat-sealed (250 vials). They were checked for leaks and stored overnight at -70DC before storage in liquid nitrogen. Cell Culture And Production Of Crude Harvest Two vials from a master cell bank are thawed rapidly. Cells are pooled and inoculated in two T-flasks at 37° ± 1°C with an appropriate culture medium supplemented with 7.5 % dialysed foetal bovine (FBS) serum. When reaching confluence (passage 13), ceils are collected by trypsinisation, pooled and expanded in 10 T-flasks as above. Confluent cells (passage 14) are trypsinised and expanded serially in 2 cell factory units (each 6000 cm2; passage 15), then in 10 cell factories (passage 16). The growth culture medium is supplemented with 7.5 % dialysed foetal bovine (FBS) serum and 1% MSX. When cells reach confluence, the growth culture medium is discarded and replaced by "production medium" containing only 1 % dialysed foetal bovine serum and no MSX. Supernatant is collected every two 40 days (48 hrs-interval) for up to 32 days. The harvested culture fluids are clarified immediately through a 1.2-0.22 $im filter unit and kept at -20°C before purification. Example 12: PURIFICATION OF HIV GP 120 (W610 CHO) FROM CELL CULTURE FLUID AH purification steps are performed in a cold room at 2-8°C. pH of buffers are adjusted at this temperature and are filtered on 0.2 urn filter. They are tested for pyrogen content (LAL assay). Optical density at 280 nm, pH and conductivity of column eluates are continuously monitored. (0 Clarified Culture Fluid The harvested clarified cell culture fluid (CCF) is filter-sterilized and Tris buffer, pH 8.0 is added to 30 mM final concentration. CCF is stored frozen at -20°C until purification. (K) Hvdrophobic Interaction Chromatographv After thawing, ammonium sulphate is added to the clarified culture fluid up to 1 M. The solution is passed overnight on a TSK/TOYOPEARL-BUTYL 650 M (TOSOHAAS) column, equilibrated in 30 mM Tris buffer- pH 8.0 - 1 M ammonium sulphate. Under these conditions, the antigen binds to the gel matrix. The column is washed with a decreasing stepwise ammonium sulphate gradient. The antigen is eluted at 30 mM Tris buffer- pH 8.0 - 0.25 M ammonium sulphate. (iii) Anion-exchange Chromatographv After reducing the conductivity of the solution between 5 and 6 mS/cm, the gP120 pool of fractions is loaded onto a Q-sepharose Fast Flow (Pharmacia) column, equilibrated in Tris-saline buffer - pH 8.0. The column is operated on a negative mode, i.e. gP120 does not bind to the gel, while most of the impurities are retained. (iv) Concentration and diafiltration by ultrafiltration In order to increase the protein concentration, the gP120 pool is loaded on a FILTRON membrane "Omega Screen Channel", with a 50 kDa cut-off. At the end of the concentration, the buffer is exchanged by diafiltration with 5 mM phosphate 41 buffer containing CaCl2 0.3 mM, pH 7.0. If further processing is not performed immediately, the gP120 pool is stored frozen at -20°C. After thawing the solution is filtered onto a 0.2 uM membrane in order to remove insoluble materiel. (v) Chromatographv on hydroxvapatite The gP120 UF pool is loaded onto a macro-Prep Ceramic Hydroxyapatite, type II (Biorad) column equilibrated in 5 mM phosphate buffer + CaCl2 0.3 mM, pH 7.0. The column is washed with the same buffer. The antigen passes through the column and impurities bind to the column. (vi) Cation exchange chromatographv The gP120 pool is loaded on a CM/TOYOPEARL-650 S (TOSOHAAS) column equilibrated in acetate buffer 20 mM, pH 5.0. The column is washed with the same buffer, then acetate 20 mM, pH 5.0 and NaCl 10 mM. The antigen is then eluted by the same buffer containing 80 mM NaCl. (vii) Ultrafiltration In order to augment the virus clearance capacity of the purification process, an additional ultrafiltration step is carried out. The gP120 pool is subjected to ultrafiltration onto a FILTRON membrane "Omega Screen Channel", cut-off 150 kDa. This pore-size membrane does not retain the antigen. After the process, the diluted antigen is concentrated on the same type of membrane (Filtron) but with a cut-off of 50 kDa. (viii) Size exclusion Gel Chromatographv The gP120 pool is applied to a SUPERDEX 200 (PHARMACIA) column in order to exchange the buffer and to eliminate residual contaminants. The column is eluted with phosphate buffer saline (PBS). (ix) Sterile filtration and storage Fractions are sterilized by filtration on a 0.2 uM PVDF membrane (Millipore). After sterile filtration, the purified bulk is stored frozen at -20°C up to formulation. The purification scheme is summarized by the flow sheet below. 42 => Level of purity of the purified bulk estimated by SDS-PAGE analysis (Silver staining / Coomassie Blue / Western Blotting) is > 95%. => Production yield is around 2.5 mg /L CCF (according to Lowry assay) -Global purification yield is around 25% (according to Elisa assay) 43 => Purified material is stable 1 week at 37°C (according to WB analysis) Example 13: VACCINE PREPARATION A vaccine prepared in accordance with the invention comprises the expression products of one or more DNA recombinants encoding an antigen. Furthermore, the formulations comprise a mixture of 3 de -O-acylated monophosphoryl lipid A 3D-MPL and QS21 in an oil/water emulsion or an oligonucleotide containing unmethylated CpG dinucleotide motifs and aluminium hydroxide as carrier. 3D-MPL: is a chemically detoxified form of the lipopolysaccharide (LPS) of the Gram-negative bacteria Salmonella minnesota. Experiments performed at Smith Kline Beecham Biologicals have shown that 3D-MPL combined with various vehicles strongly enhances both the humoral immunity and a TH1 type of cellular immunity. QS21: is a saponin purified from a crude extract of the bark of the Quillaja Saponaria Molina tree, which has a strong adjuvant activity: it induces both antigen-specific lymphoproliferation and CTLs to several antigens. Experiments performed at Smith Kline Beecham Biologicals have demonstrated a clear synergistic effect of combinations of 3D-MPL and QS21 in the induction of both humoral and THI type cellular immune responses. The oil/water emulsion is composed of 2 oils (a tocopherol and squalene), and of PBS containing Tween 80 as emulsifier. The emulsion comprises 5% squalene, 5% 44 tocopherol, 2% Tween 80 and has an average particle size of 180 nm (see WO 95/17210). Experiments performed at Smith Kline Beecham Biologicals have proven that the adjunction of this OAV emulsion to 3D-MPL/QS21 further increases their immunostimulant properties. Preparation of the oil/water emulsion (2 fold concentrate) Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100ml two fold concentrate emulsion 5g of DL alpha tocopherol and 5ml of squalene are vortexed to mix thoroughly. 90ml of PBS/Tween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe and finally microfluidised by using an Ml 10S Microfluidics machine. The resulting oil droplets have a size of approximately 180 nm. Preparation of oil in water formulation. Antigens (100 ?g gpl20, 20 ?g NefTat, and 20 ug SIV Nef, alone or in combination) were diluted in 10 fold concentrated PBS pH 6.8 and H2O before consecutive addition of the oil in water emulsion, 3D-MPL (50?g), QS21 (50?g) and 1 ?g/ml thiomersal as preservative at 5 min interval. The emulsion volume is equal to 50% of the total volume (250ul for a dose of 500?l). All incubations were carried out at room temperature with agitation. CpG oligonucleotide (CpG) is a synthetic unmethylated oligonucleotide containing one or several CpG sequence motifs. CpG is a very potent inducer of TH1 type immunity compared to the oil in water formulation that induces mainly a mixed THI/TH2 response. CpG induces lower level of antibodies than the oil in water formulation and a good cell mediated immune response. CpG is expected to induce lower local reactogenicity. 45 Preparation of CpG oligonucleotide solution: CpG dry powder is dissolved in H2O to give a solution of 5 mg/ml CpG. Preparation of CpG formulation. The 3 antigens were dialyzed against NaCl 150 mM to eliminate the phosphate ions that inhibit the adsorption of gpl20 on aluminium hydroxide. The antigens diluted in H2O (100 ?g gpl20, 20 ?g NefTat and 20 ?g SIV Nef) were incubated with the CpG solution (500 ?g CpG) for 30 min before adsorption on A1(OH)3 to favor a potential interaction between the His tail of NefTat and Nef antigens and the oligonucleotide (stronger immunostimulatory effect of CpG described when bound to the antigen compared to free CpG). Then were consecutively added at 5 min interval A1(OH)3 (500 ?g), 10 fold concentrated NaCl and 1 ?g/ml thiomersal as preservative. All incubations were carried out at room temperature with agitation. Example 14: IMMUNIZATION AND SHIV CHALLENGE EXPERIMENT IN RHESUS MONKEYS. First Study Groups of 4 rhesus monkeys were immunized intramuscularly at 0, 1 and 3 months with the following vaccine compositions: Group 1: Adjuvant 2 + gpl20 Group 2: Adjuvant 2 + gpl20 + NefTat + SIV Nef Group 3; Adjuvant 2 + NefTat + SIV Nef Group 4 Adjuvant 6 +gpl20 + NefTat + SIV Nef Group 5 Adjuvant 2 + NefTat + SIV Nef Group 6 Adjuvant 2 46 Adjuvant 2 comprises squalene/tocopherol/Tween 80/3D-MPL/QS2I and Adjuvant 6 comprises alum and CpG. Tat* represents mutated Tat, in which Lys4I-»Ala and in RGD motif Arg78->Lys and Asp80->Glu (Virology 235: 48-64, 1997). One month after the last immunization all animals were challenged with a pathogenic SHIV (strain 89.6p). From the week of challenge (wkl6) blood samples were taken periodically at the indicated time points to determine the % of CD4-positive cells among peripheral blood mononuclear cells by FACS analysis (Figure 14) and the concentration of RNA viral genomes in the plasma by bDNA assay (Figure 15). Results All animals become infected after challenge with SHIV89.6P. CD4-positive cells decline after challenge in all animals of groups 1,3,5 and 6 except one animal in each of groups 1 and 6 (control group). All animals in group 2 exhibit a slight decrease in CD4-positive cells and recover to baseline levels over time. A similartrend is observed in group 4 animals (Figure 14). Virus load data are almost the inverse of CD4 data. Virus load declines below the level of detection in ¾ group 2 animals (and in the one control animal that maintains its CD4-positive cells), and the fourth animal shows only marginal virus load. Most of the other animals maintain a high or intermediate virus load (Figure 15). Surprisingly, anti-Tat and anti-Nef antibody titres measured by ELISA were 2 to 3-fold higher in Group 3 (with mutated Tat) than in Group 5 (the equivalent Group with non-mutated Tat) throughout the course of the study. At week 68 (56 weeks post challenge) all animals from the groups that had received the full antigen combination (groups 2 and 4) were still alive, while most of the animals in the other groupshad to be euthanized due to AIDS-like symptoms. The surviving animals per group were: 47 Group I: 2/4 Group 2: 4/4 Group 3: 0/4 Group 4 4/4 Group 5 0/4 Group 6 1/4 Conclusions The combination of gp 120 and NefTat (in the presence of SIV Nef) prevents the loss of CD4-positive cells, reduces the virus load in animals infected with pathogenic SHIVg9.6P, and delays or prevents the development of AIDS-Iike disease symptoms, while gpl20 or NefTat/SIV Nef alone do not protect from the pathologic consequences of the SHIV challenge. The adjuvant 2 which is an oil in water emulsion comprising squalene, tocopherol and Tween 80, together with 3D-MPL and QS2I seems to have a stronger effect on the study endpoints than the alum / CpG adjuvant. Second study A second rhesus monkey SHIV challenge study was conducted to confirm the efficacy of the candidate vaccine gpl20/NefTat + adjuvant and to compare different Tat-based antigens. The study was conducted by a different laboratory. The design of the study was as follows. Groups of 6 rhesus monkeys were immunized at 0,4 and 12 weeks with injections i.m. and challenged at week 16 with a standard dose of pathogenic SHIVg9.6P. Group I is the repeat of Group 2 in the first study. 48 Group 1: Adjuvant 2 + gp!20 + NefTat + SIV Nef Group 2: Adjuvant 2 + gpI20 + Tat (oxidised) Group 3: Adjuvant 2 + gp 120 + Tat (reduced) Group 4 Adjuvant 2 animals This is a confirmation of the result obtained in the first SHIV study. Due to the lack of susceptibility of the study animals, the virus load parameter could not be used to demonstrate a vaccine effect. Taken together, the combination of gpl20 and Tat and Nef HIV antigens provides protection against the pathologic consequences of HIV infection, as evidenced in a SHIV model. The Tat alone antigens in combination with gp!20 also provide some protection from the decline of CD4-positive cells. The effect is less pronounced than with the gpl20/NefTat/SIV Nef antigen combination, but it demonstrates that gpl20 and Tat are able to mediate some protective efficacy against SHIV-induced disease manifestations. The second SHIV challenge study was performed with rhesus monkeys from a source completely unrelated to the source of animals from the first study. Both parameters, % of CD4-positive cells and plasma virus load, suggest that the animals in the second study were less susceptible to SHIV-induced disease, and that there was considerably greater variability among the animals. Nonetheless, a beneficial effect on the maintenance of CD4-positive cells of the gpl20/NefTat/SIV Nef vaccine was seen with the experimental vaccine containing gpl20/NefTat and SIV Nef. This indicates that the vaccine effect was not only repeated in a separate study, but furthermore demonstrated in an unrelated monkey population. 50 WE CLAIM : 1. A process for the preparation of a vaccine formulation for use in the prophylactic or therapeutic immunisation of humans against HIV, comprising the steps of admixing a) an HIV Tat protein or polynucleotide as defined by SEQ ID Nos 10 or 11, or a derivative sequence which is with or without a C-teminal His-tail, or mutated, or chemicaily modified; or b) an HIV Nef protein or polynucleotide as defined by SEQ ID Nos 8 or 9, or a derivative sequence which is with or without a C-teminal His-tail. or mutated, or chemically modified; or c) an HIV Tat protein or polynucleotide linked to an HIV Nef protein or polynucleotide (Nef-Tat) as defined by SEQ ID Nos 12 or 13, or a derivative sequence which is with or without a C-teminal His-tail, or mutated, or chemically modified; and an HIV gpl20 protein or polynucleotide; wherein the Tat, Nef of Nef-Tat act in synergy with gpl20 in the treatment or prevention of HIV wherein the vaccine in use reduces the HIV viral load in HIV infected humans and results in a maintenance of CD4+ levels over those levels found in the absence of vaccination with HIV Tat, Nef or Nef-Tat and HIV gpl20. 2. The process, as claimed in claim 1, wherein the vaccine optionally comprises an antigen selected from the group consisting of: gag, rev, vif, vpr, vpu. 3. The process, as claimed in claim 1 or claim 2S wherein the Tat protein is a mutated protein. 4. The process, as claimed in any one of claims 1 - 3, wherein the Tat, Nef or Nef-Tat protein is reduced. 5. The process, as claimed in any one of claims 1 - 4, wherein the Tat, Nef or Nef-Tat protein is carbamidomethylated. 6. The process, as claimed in any one of claims 1 - 4, wherein the Tat, Nef or Nef-Tat protein is oxidised. 51 7. The process, as claimed in any one of claims 1 - 6, wherein an optional adjuvant is used in the admixture. 8. The process, as claimed in claim 7, wherein the adjuvant is a TH1 inducing adjuvant. 9. The process, as claimed in claim 7 or claim 8, wherein the adjuvant comprises monophosphoryl lipid A or a derivative thereof such as 3-de-O-acylated monophsphoryl lipid A. 10. The process, as claimed in any one of claims 7-9, wherein an optional saponin adjuvant is used in the admixture. 11. The process, as claimed in any one of claims 7-10, wherein an optional oil in water emulsion is used in the admixture. 12. The process, as claimed in claim 7 or claim 8, wherein the adjuvant comprises CpG motif- containing oligonucleotides. 13. The process, as claimed in claim 12, wherein an aluminium salt is used as a second adjuvant. 14. A vaccine composition for human use comprising HIV Tat or HIV Nef or HIV Nef-Tat in combination with HIV gpl20 proteins or polynucleotides encoding them together with an adjuvant comprising QS21 and 3D-MPL, wherein the vaccine in use reduces the viral load in HIV infected humans and results in a maintenance of CD4+ levels over those found in the absence of vaccination with HIV Tat, Nef, or Nef-Tat and HIV gpl20. The present invention discloses a vaccine composition for human use comprising HIV Tat or HIV Nef or HIV Nef-Tat in combination with HIV gpl20 proteins or polynucleotides encoding them together with an adjuvant comprising QS21 and 3D-MPL, wherein the vaccine in use reduces the HIV viral load in HIV infected humans and results in a maintenance of CD4+ levels over those levels found in the absence of vaccination with HIV Tat, Nef or Nef-Tat and HIV gpl20 and a process for its preparation.

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"VACCINE FOR THE PROPHYLACTIC OR THERAPEUTIC IMMUNIZATION AGAINST HIV AND PROCESS FOR ITS PREPARATION"
The present invention relates to novel uses of HIV proteins in medicine and vaccine compositions containing such HIV proteins. In particular, the invention relates to the use of HIV Tat and HIV gpl20 proteins in combination. Furthermore, the invention relates to the use of HIV Nef and HIV gp!20 proteins in combination.
HIV-1 is the primary cause of the acquired immune deficiency syndrome (AIDS) which is regarded as one of the world's major health problems. Although extensive research throughout the world has been conducted to produce a vaccine, such efforts thus far have not been successful.
The HIV envelope glycoprotein gpl20 is the viral protein that is used for attachment to the host cell. This attachment is mediated by the binding to two surface molecules of helper T cells and macrophages, known as CD4 and one of the two chemokine receptors CCR-4 or CXCR-5. The gpl20 protein is first expressed as a larger precursor molecule (gpl60), which is then cleaved post-translationally to yield gpl20 and gp41. The gpl20 protein is retained on the surface of the virion by linkage to the gp41 molecule, which is inserted into the viral membrane.
The gpl20 protein is the principal target of neutralizing antibodies, but unfortunately the most immunogenic regions of the proteins (V3 loop) are also the most variable parts of the protein. Therefore, the use of gp120 (or its precursor gp 160) as a vaccine antigen to elicit neutralizing antibodies is thought to be of limited use for a broadly protective vaccine. The gp!20 protein does also contain epitopes that are recognized by cytotoxic T lymphocytes (CTL). These effector cells are able to eliminate virus-infected cells, and therefore constitute a second major antiviral immune mechanism. In contrast to the target regions of neutralizing antibodies some CTL epitopes appear to be relatively conserved among different HIV strains. For this reason gpl20 and gp!60 are considered to be useful antigenic components in vaccines that aim at eliciting cell-mediated immune responses (particularly CTL).

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Non-envelope proteins of HIV-1 have been described and include for example internal structural proteins such as the products of the gag and pol genes and, other non-structural proteins such as Rev, Nef, Vif and Tat (Greene et al., New England J. Med, 324, 5, 308 et seq (1991) and Bryant et al. (Ed. Pizzo), Pediatr. Infect. Dis. J., ll,5,390etseq(1992).
HIV Tat and Nef proteins are early proteins, that is, they are expressed early in infection and in the absence of structural protein.
In a conference presentation (C. David Pauza, Immunization with Tat toxoid attenuates SHIV89.6PD infection in rhesus macaques, 12th Cent Gardes meeting, Mames-La-Coquette, 26.10.1999), experiments were described in which rhesus macaques were immunised with Tat toxoid alone or in combination with an envelope glycoprotein gpl60 vaccine combination (one dose recombinant vaccinia virus and one dose recombinant protein). However, the results observed showed that the presence of the envelope glycoprotein gave no advantage over experiments performed with Tat alone.
However, we have found that a Tat- and/or Nef-containing imrnunogen (especially a Nef-Tat fusion protein) acts synergistically with gpl20 in protecting rhesus monkeys from a pathogenic challenge with chimeric human-simian immunodeficiency virus (SHIV). To date the SHIV infection of rhesus macaques is considered to be the most relevant animal model for human AIDS. Therefore, we have used this preclinical model to evaluate the protective efficacy of vaccines containing a gpl20 antigen and a Nef- and Tat-containing antigen either alone or in combination. Analysis of two markers of viral infection and pathogenieity, the percentage of CD4-positive cells in the peripheral blood and the concentration of free SHIV RNA genomes in the plasma of the monkeys, indicated that the two antigens acted in synergy. Immunization with either gp 120 or NefTat + SIV Nef alone did not result in any difference compared to immunization with an adjuvant alone. In contrast, the administration of the combination of gpl20 and NefTat + SIV Nef, antigens resulted in a marked improvement of the two above-mentioned parameters in all animals of those particular experimental group.
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Thus, according to the present invention there is provided a new use of HIV Tat and/or Nef protein together with HIV gpl20 in the manufacture of a vaccine for the prophylactic or therapeutic immunisation of humans against HIV.
As described above, the NefTat protein, the SIV Nef protein and gpl20 protein together give an enhanced response over that which is observed when either NefTat 4-SIV Nef, or gpl20 are used alone. This enhanced response, or synergy can be seen in a decrease in viral load as a result of vaccination with these combined proteins. Alternatively, or additionally the enhanced response manifests itself by a maintenance of CD4+ levels over those levels found in the absence of vaccination with HIV NefTat, SIV Nef and HIV gpl20. The synergistic effect is attributed to the combination of gpl20 and Tat, or gp!20 and Nef, or gp!20 and both Nef and Tat.
The addition of other HIV proteins may further enhance the synergistic effect, which was observed between gpl20 and Tat and/or Nef. These other proteins may also act synergistically with individual components of the gpl20, Tat and/or Nef-containing vaccine, not requiring the presence of the full original antigen combination. The additional proteins may be regulatory proteins of HIV such as Rev, Vif, Vpu, and Vpr. They may also be structural proteins derived from the HIV gag or pot genes.
The HIV gag gene encodes a precursor protein p55, which can assemble spontaneously into immature virus-like particles (VLPs). The precursor is then proteolytically cleaved into the major structural proteins p24 (capsid) and p18 (matrix), and into several smaller proteins. Both the precursor protein p55 and its major derivatives p24 and pl8 may be considered as appropriate vaccine antigens which may further enhance the synergistic effect observed between gp120 and Tat and/or Nef. The precursor p55 and the capsid protein p24 may be used as VLPs or as monomeric proteins.
The HIV Tat protein in the vaccine of the present invention may, optionally be linked to an HIV Nef protein, for example as a fusion protein.
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The HIV Tat protein, the HIV Nef protein or the NefTat fusion protein in the present invention may have a C terrnir al Histidine tail which preferably comprises between 5-10 Histidine residues. The presence of an histidine (or 'His') tail aids purification.
In a preferred embodiment the proteins are expressed with a Histidine tail comprising between 5 to 10 and preferably six Histidine residues. These are advantageous in aiding purification. Separate expression, in yeast (Saccharomyces cerevisiae), of Nef (Macreadie I.G. et al, 1993, Yeast 9 (6) 565-573) and Tat (Braddock M et ah, 1989, Cell 58 (2) 269-79) has been reported. Nef protein and the Gag proteins p55 and pi 8 are myristilated. The expression of Nef and Tat separately in a Pichia expression system (Nef-His and Tat-His constructs), and the expression of a fusion construct Nef-Tat-His have been described previously in WO99/16884.
The DNA and amino acid sequences of representative Nef-His (Seq. ID. No.s 8 and 9), Tat-His (Seq. ID. No.s 10 and 1l)and of Nef-Tat-His fusion proteins (Seq. ID. No.s 12 and 13) are set forth in Figure 1.
The HIV proteins of the present invention may be used in their native conformation, or more preferably, may be modified for vaccine use. These modifications may either be required for technical reasons relating to the method of purification, or they may be used to biologically inactivate one or several functional properties of the Tat or Nef protein. Thus the invention encompasses derivatives of HIV proteins which may be, for example mutated proteins. The term 'mutated' is used herein to mean a molecule which has undergone deletion, addition or substitution of one or more amino acids using well known techniques for site directed mutagenesis or any other conventional method.
For example, a mutant Tat protein may be mutated so that it is biologically inactive whilst still maintaining its immunogenic epitopes. One possible mutated tat gene, constructed by D.Clements (Tulane University), (originating from BH10 molecular clone) bears mutations in the active site region (Lys4l->Ala)and in RGD motif (Arg78->Lys and Asp80->Glu) ( Virology 235: 48-64, 1997).
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A mutated Tat is illustrated in Figure 1 (Seq. ID. No.s 22 and 23) as is a Nef-Tat Mutant-His (Seq. ID. No.s 24 and 25).
The HIV Tat or Nef proteins in the vaccine of the present invention may be modified by chemical methods during the purification process to render the proteins stable and monomeric. One method to prevent oxidative aggregation of a protein such as Tat or Nef is the use of chemical modifications of the protein's thiol groups. In a first step the disulphide bridges are reduced by treatment with a reducing agent such as DTT, beta-mercaptoethanol, or gluthatione. In a second step the resulting thiols are blocked by reaction with an alkylating agent (for example, the protein can be carboxyamidated/carbamidomethylated using iodoacetamide). Such chemical modification does not modify functional properties of Tat or Nef as assessed by cell binding assays and inhibition of lymphoproliferation of human peripheral blood mononuclear cells.
The HIV Tat protein and HIV gpl20 proteins can be purified by the methods outlined in the attached examples.
The vaccine of the present invention will contain an immunoprotective or immunotherapeutic quantity of the Tat and/or Nef or NefTat and gpl20 antigens and may be prepared by conventional techniques.
Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.
The amount of protein in the vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise 1-1000 ?g of each
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protein, preferably 2-200 ?g, most preferably 4-40 ?g of Tat or Nef or NefTat and preferably 1-150 ?g, most preferably 2-25 ?g of gpl20. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody litres and other responses in subjects. One particular example of a vaccine dose will comprise 20 fig of NefTat and 5 or 20 fig of gpl20. Following an initial vaccination, subjects may receive a boost in about 4 weeks, and a subsequent second . booster immunisation.
The proteins of the present invention are preferably adjuvanted in the vaccine formulation of the invention. Adjuvants are described in general in Vaccine Design -the Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995.
Suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.
In the formulation of the invention it is preferred that the adjuvant composition induces a preferential Thl response. However it will be understood that other responses, including other humoral responses, are not excluded.
An immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system. The resultant immune response may be broadly distinguished into two extreme catagories, being humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed Thl-type responses (cell-mediated response), and Th2-type immune responses (humoral response).
Extreme Th 1 -type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice Thl-type responses are often characterised by the generation of
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antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. Th2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
It can be considered that the driving force behind the development of these two types of immune responses are cytokines, a number of identified protein messengers which serve to help the ceils of the immune system and steer the eventual immune response to either a Thl or Th2 response. Thus high levels of Thl -type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of Th2-type cytokines tend to favour the induction of humoral immune responses to the antigen.
It is important to remember that the distinction of Thl and Th2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly Thl or predominantly Th2. However, it is often convenient to consider the families of cytokines in terms of that described in rnurine CD4 +ve T cell clones by Mosmann and Coffinan (Mosmann. T.R. and Coffman, R.L. (1989) TH1 and TH2 cells: different patterns oflymphokine secretion had to different functional properties. Annual Review of Immunology, 7, p 145-173). Traditionally, Thl-type responses are associated with the production of the INF-y and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of Thl-type immune responses are not produced by T-cells, such as IL-I2. In contrast, Th2- type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10 and tumour necrosis factor-p(TNF-p).
It is known that certain vaccine adjuvants are particularly suited to the stimulation of either Th 1 or Th2 - type cytokine responses. Traditionally the best indicators of the Thl:Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Thl or Th2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement of the IgG 1 :IgG2a ratio of antigen specific antibody responses.
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Thus, a Thl-type adjuvant is cne which stimulates isolated T-cell populations to produce high levels of Thl-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific ixnxnunoglohuhn responses associated with Thl-type isotype.
Preferred Thl-type immunostimulants which may be formulated to produce adjuvants suitable for use in the present invention include and are not restricted to the following.
Monophosphoryl lipid A, in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL), is a preferred Thl-type imrmmostirnulant for use in the invention. 3D-MPL is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. It can be purified and prepared by the methods taught in GB 2I22204B, which reference also discloses the preparation of diphosphoryl lipid A, and 3-O-deacylated variants thereof. Other purified and synthetic lipopoiysaccharides have been described (US 6,005,099 and EP 0 729 473 Bl; Hilgers et al., 1986, Int.Arch.AHergy.Immunol, 79(4):392-6; Hilgers etal., 1987, Immunology, 60(l):141-6; and EP 0 549 074 Bl). A preferred form of 3D-MPL is in the form of a particulate formulation having a small particle size less than 0.2jxm in diameter, and its method of manufacture is disclosed in EP 0 689 454.
Saponins are also preferred Thl immunostimulants in accordance with the invention. Saponins are well known adjuvants and are taught in: Lacaille-Dubois, M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363-386). For example, Quit A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof, are described in US 5,057,540 and "Saponins as vaccine adjuvants", Kensil, C. R-, Crit Rev Ther Drug Carrier Syst, 1996, 12 (l-2):L-55; and EP 0 362 279 BL The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants, and the method of their production is disclosed in US Patent No. 5,057,540 and EP 0 362 279 Bl. Also described in these references is the use of QS7 (a non-haemolytic fraction of Quil-A) which acts as a potent adjuvant for systemic vaccines. Use of QS21 is further described in Kensil et al, (199l J.
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Immunology vol 146,431-437). Combinations of QS21 and polysorbate or cyclodextrin are also known (WO 99/10008). Paniculate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in WO 96/33739 and WO 96/11711.
Another preferred Imnnmostimulant is an irnmunostirmiiatory oiigonucleotide containing unmethylated CpG dinucleotides ("CpG"). CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et ai. J.Immunot, 1998, I60(2):870-S76; McCluskie and Davis, JJmmunol, 1998, I61(9):4463-6). Historically, it was observed that the DNA fraction of BCG could exert an anti-tumour effect. In further studies, synthetic oligonucleotides derived from BCG gene sequences were shown to be capable of inducing immunostimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a central CG motif, carried this activity. The central role of the CG motif in Jmirmnostimulation was later elucidated in a publication by Krieg, Nature 374, p546 1995. Detailed analysis has shown that the CG motif has to be in a certain sequence context, and that such sequences are common in bacterial DNA but are rare in vertebrate DNA. The immunostimuiaiory sequence is often: Purine, Purine, C, G, pyrimidine, pyrimidme; wherein the CG motif is not methylated, but other unmethylated CpG sequences are known to be irnmunostimuiatory and may be used in the present invention.
In certain combinations of the six nucleotides a palindromic sequence is present. Several of these motifs, either as repeats of one motif or a combination of different motifs, can be present in the same oligonucleotide. The presence of one or more of these immunostimulatory sequences containing oligonucleotides can activate various immune subsets, including natural killer celts (which produce interferon y and have cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8), 1977). Other unmethylated CpG containing sequences not having this consensus sequence have also now been shown to be irnmunomodulatory.
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CpG when formulated into vaccines, is generally administered in free solution together with free antigen (WO 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (WO 98/16247), or formulated with a carrier such as aluminium hydroxide ((Hepatitis surface antigen) Davis et al supra ; Brazolot-Millan et al, Proc.Natl.Acad.Scl, USA, 1998, 95(26), 15553-8).
Such immunostimulants as described above may be formulated together with carriers, such as for example liposomes, oil in water emulsions, and or metallic salts, including aluminium salts (such as aluminium hydroxide). For example, 3D-MPL may be formulated with aluminium hydroxide (EP 0 689 454) or oil in water emulsions (WO 95/17210); QS21 maybe advantageously formulated with cholesterol containing liposomes (WO 96/33739), oil in water emulsions (WO 95/17210) or alum (WO 98/15287); CpG may be formulated with alum (Davis et al. supra ; Brazolot-Millan supra) or with other cationic carriers.
Combinations of immunostimulants are also preferred, in particular a combination of a monophosphoryl lipid A and a saponin derivative (WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241), more particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153. Alternatively, a combination of CpG plus a saponin such as QS21 also forms a potent adjuvant for use in the present invention.
Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, together with an aluminium salt. An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched in cholesterol containing liposomes (DQ) as disclosed in WO 96/33739.
A particularly potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is another preferred formulation for use in the invention.
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Another preferred formulation comprises a CpG oligonucleotide alone or together with an aluminium salt
In another aspect of the invention, the vaccine may contain DNA encoding one or more of the Tat, Nef and gpI20 polypeptides, such that the polypeptide is generated in situ. The DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems such as plasmid DNA, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Holland, Crit. Rev, Therap. Drug Carrier Systems 15:143-198, 1998 and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). When the expression system is a recombinant live microorganism, such as a virus or bacterium, the gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses. Viruses and bacteria used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox, modified poxviruses e.g. Modified Virus Ankara (MVA))t alphaviruses (Sindbis virus, Semliki Forest Virus, Venezueliart Equine Encephalitis Virus), flaviviruses (yellow fever virus, Dengue virus, Japanese encephalitis virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella, Neisseria, BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention.
Thus, the Nef, Tat and gpl20 componerflts of a preferred vaccine according to the invention may be provided in the form of polynucleotides encoding the desired proteins.
Furthermore, immunisations according to the invention may be performed with a combination of protein and DNA-based formulations. Prime-boost immunisations are considered to be effective in inducing broad immune responses. Adjuvanted protein vaccines induce mainly antibodies and T helper immune responses, while delivery of DNA as a plasmid or a live vector induces strong cytotoxic T lymphocyte (CTL)
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responses. Thus, the combination of protein and DNA vaccination will provide for a wide variety of immune respc rises: This is particularly relevant in the context of HIV, since both neutralising antibodies and CTL are thought to be important for the immune defence against HIV.
In accordance with the invention a schedule for vaccination with gp!20, Nef and Tat, alone or in combination, may comprise the sequential ("prime-boost") or simultaneous administration of protein antigens and DNA encoding the above-mentioned proteins. The DNA may be delivered as plasmid DNA or in the form of a recombinant live vector, e.g. a poxvirus vector or any other suitable live vector such as those described herein. Protein antigens may be injected once or several times followed by one or more DNA administrations, or DNA may be used first for one or more administrations followed by one or more protein immunisations.
A particular example of prime-boost immunisation according to the invention involves priming with DNA in the form of a recombinant live vector such as a modified poxvirus vector, for example Modified Virus Ankara (MVA) or a alphavinis, for example Venezuelian Equine Encephalitis Virus followed by boosting with a protein, preferably an adjuvanted protein.
Thus the invention further provides a pharmaceutical kit comprising:
a) a composition comprising one or more of gpl20, Nef and Tat proteins
together with a pharmaceutically acceptable excipient; and
b) a composition comprising one or more of gpl20, Nef and Tat-encoding
polynucleotides together with a pharmaceutically acceptable excipient;
with the proviso that at least one of (a) or (b) comprises gpl20 with Nef and/or Tat and/or Nef-Tat.
Compositions a) and b) may be administered separately, in any order, or together. Preferably a) comprises all three of gpl20, Nef and Tat proteins. Preferably b) comprises all three of gpl20, Nef and Tat DNA. Most preferably the Nef and Tat are in the form of a NefTat fusion protein.
In a further aspect of the present invention there is provided a method of manufacture of a vaccine formulation as herein described, wherein the method comprises admixing
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a combination of proteins according to the invention. The protein composition may be mixed with a suitable adjuvant and, optionally, a carrier.
Particularly preferred adjuvant and/or carrier combinations for use in the formulations
according to the invention are as follows:
i) 3D-MPL + QS21 in DQ
ii) Alum + 3D-MPL
iii) Alum + QS21 in DQ + 3D-MPL
iv) Alum + CpG
v) 3D-MPL + QS21 in DQ + oil in water emulsion
vi) CpG
The invention is illustrated in the accompanying examples and Figures:
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EXAMPLES
General
The Nef gene from the Bru/Lai isolate (Cell 40: 9-17, 1985) was selected for the constructs of these experiments since this gene is among those that are most closely related to the consensus Nef.
The starting material for the Bra/Lai Nef gene was a U70bp DNA fragment cloned on the mammalian expression vector pcDNA3 (pcDNA3/Nef).
The Tat gene originates from the BH10 molecular clone. This gene was received as an HTLV III cDNA clone named pCVl and described in Science, 229, p69-73, 1985.
The expression of the Nef and Tat genes could be in Pichia or any other host.
Example 1. EXPRESSION OF HIV-1 nef AND tat SEQUENCES IN PICHIA PASTORIS.
Nef protein, Tat protein and the fusion Nef-Tat were expressed in the methylotrophic yeast Pichia pastoris under the control of the inducible alcohol oxidase (AOX1) promoter.
To express these HI\M genes a modified version of the integrative vector PHIL-D2 (INVITROGEN) was used. This vector was modified in such a way that expression of heterologous protein starts immediately after the native ATG codon of the AOX1 gene and will produce recombinant protein with a tail of one glycine and six histidines residues . This PHIL-D2-MOD vector was constructed by cloning an oligonucleotide linker between the adjacent AsuII and EcoRI sites of PHIL-D2 vector (see Figure 2). In addition to the His tail, this linker carries Ncol, Spel and Xbal restriction sites between which nef tat and nef tat fusion were inserted.
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1.1 CONSTRUCTION OF THE INTEGRATIVE VECTORS pRIT14597 (encoding Nef-His protein), pRlT14598 (encoding Tat-Hls protein) and pRIT14599 (encoding fusion Nef-Tat-His).
The nefgme was amplified by PCR from the pc0NA3/Nef plasniid with primers 01 and 02.
Ncol PRIMER 01 (Seq ID NO 1): SWTCGTCCATG.GGT.GGCAAG.TGG.T 3'
Spel PRIMER 02 (Seq ID NO 2): 5' CGGCTACTAGTGCAGTTCTTGAA 3*
The PCR fragment obtained and the iategrative PHIL-D2-MOD vector were both restricted by Ncol and Spel, purified on agarose gel and iigated to create the integrative plasmid pRJT14597 (see Figure 2).
The tat gene was amplified by PCR from a derivative of the pCVl plasmid with primers 05 and 04:
Spel PRIMER 04 (Seq ID NO 4): 5' CGGCTACTAGTTTCCTTCGGGCCT 3'
Ncol PRIMER 05 (Seq ID NO 5): 5'ATCGTCCATGGAGCCAGTAGATC 3'
An Ncol restriction site was introduced at the 5' end of the PCR fragment while a Spel site was introduced at the 3' end with primer 04. The PCR fragment obtained
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and the PHIL-D2-MOD vecto- were both restricted by Ncol and Spel, purified on agarose gel and Iigated to create the initegrative plasmid pRIT14598.
To construct pRIT14599, a 910bp DNA fragment corresponding to the nef-tat-His coding sequence was Iigated between the EcoRI blunted(T4 polymerase) and Ncol sites of the PHIL-D2-M0D vector. The nef-tat-His coding fragment was obtained by Xbal blunted(T4 polymerase) and Ncol digestions of pRIT14596.
1.2 TRANSFORMATION OF PICHIA PASTORIS STRAIN GS115(his4).
To obtain Pichia pastoris strains expressing Nef-His, Tat-His and the fusion Nef-Tat-His, strain GS115 was transformed with linear NotI fragments carrying the respective expression cassettes plus the HIS4 gene to complement his4 in the host genome.Transformation of GS115 with Notl-linear fragments favors recombination at the AOXI locus.
Multicopy integrant clones were selected by quantitative dot blot analysis and the type of integration, insertion (Mut+phenotype) or transplacement (Mutsphenotype), was determined.
From each transformation, one transformant showing a high production level for the recombinant protein was selected :
Strain Y1738 (Mut+ phenotype) producing the recombinant Nef-His protein, a myristylated 215 amino acids protein which is composed of:
°Myristic acid
°A methionine, created by the use of Ncol cloning site of PHIL-D2-MOD vector
°205 a.a. of Nef protein(starting at a.a.2 and extending to a.a.206)
°A threonine and a serine created by the cloning procedure (cloning at Spel
site of PHIL-D2-M0D vector. °One glycine and six histidines.
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Strain Y1739 (Mut phenotype) producing the Tat-His protein, a 95 amino acid protein which is composed of:
°A methionine created by the use of Ncol cloning site
°85 a.a. of the Tat protein(starting at a.a.2 and extending to a.a.86)
°A threonine and a serine introduced by cloning procedure °One glycine and six histidines
Strain Yl737(Muts phenotype) producing the recombinant Nef-Tat-His fusion protein, a myristylated 302 amino acids protein which is composed of:
°Myristic acid
°A methionine, created by the use of Ncol cloning site
°205a.a. of Nef protein(starting at a.a.2 and extending to a.a.206)
°A threonine and a serine created by the cloning procedure
°85a.a. of the Tat protein(starting at a.a.2 and extending to a.a.86)
°A threonine and a serine introduced by the cloning procedure
°One glycine and six histidines
Example 2. EXPRESSION OF HIV-1 Tat-MUTANT IN PICHIA PASTORIS
A mutant recombinant Tat protein has also been expressed. The mutant Tat protein must be biologically inactive while maintaining its immunogenic epitopes.
A double mutant tat gene, constructed by D.Clements (Tulane University) was selected for these constructs.
This tat gene (originates from BH10 molecular clone) bears mutations in the active site region (Lys41-*Ala)and in RGD motif (Arg78-*Lys and Asp80-»Glu) (Virology 235: 48-64, 1997).
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The mutant tat gene was received as a cDNA fragment subcloned between the EcoRl and Hindlll sites within a CMV expression plasmid (pCMVLys41/KGE)
2.1 CONSTRUCTION OF THE INTEGRATIVE VECTORS
pRIT14912(encoding Tat mutant-His protein) and pRIT14913(encoding fusion
Nef-Tat mutant-His).
The tat mutant gene was amplified by PCR from the pCMVLys41/KGE plasmid with primers 05 and 04 (see section 1.1 construction of pRIT14598)
An Ncol restriction site was introduced at the 5* end of the PCR fragment while a Spel site was introduced at the 3* end with primer 04. The PCR fragment obtained and the PHIL-D2-MOD vector were both restricted by Ncol and Spel, purified on agarose gel and ligated to create the integrative plasmid pRIT14912
To construct pRIT14913, the tat mutant gene was amplified by PCR from the pCMVLys41/KGE plasmid with primers 03 and 04.
Spel PRIMER 03 (Seq ID NO 3): 5* ATCGTACTAGT.GAG.CCA.GTA.GAT.C 3*
Spel PRIMER 04 (Seq ID NO 4): 5' CGGCTACTAGTTTCCTTCGGGCCT 3'
The PCR fragment obtained and the plasmid pRIT14597 (expressing Nef-His protein) were both digested by Spel restriction enzyme, purified on agarose gel and ligated to create the integrative plasmid pRIT14913
2.2 TRANSFORMATION OF PICHIA PASTORIS STRAIN GS115.
18

Pichia pastoris strains expressing Tat mutant-His protein and the fusion Nef-Tat mutant-His were obtained, by applying integration and recombinant strain selection strategies previously described in section 1.2 .
Two recombinant strains producing Tat mutant-His protein ,a 95 amino-acids protein, were selected: YI775 (Mut+ phenotype) and Y1776(Muts phenotype).
One recombinant strain expressing Nef-Tat mutant-His fusion protein, a 302 amino-acids protein was selected: Yl 774(Mut+ phenotype).
Example 3: FERMENTATION OF PICHIA PASTORIS PRODUCING RECOMBINANT TAT-HIS.
A typical process is described in the table hereafter.
Fermentation includes a growth phase (feeding with a glycerol-based medium according to an appropriate curve) leading to a high cell density culture and an induction phase (feeding with a methanol and a salts/micro-elements solution). During fermentation the growth is followed by taking samples and measuring their absorbance at 620 run. During the induction phase methanol was added via a pump and its concentration monitored by Gas chromatography (on culture samples) and by on-line gas analysis with a Mass spectrometer. After fermentation the cells were recovered by centrifugation at 5020g during 30' at 2-8°C and the cell paste stored at -20°C. For further work cell paste was thawed, resuspended at an OD (at 620 nm) of 150 in a buffer (Na2HPO4 pH7 50 mM, PMSF 5%, Isopropanol 4 mM) and disrupted by 4 passages in a DynoMill (room 0.6L, 3000 rpm, 6L/H, beads diameter of 0.40-0.70 mm).
For evaluation of the expression samples were removed during the induction, disrupted and analyzed by SDS-Page or Western blot. On Coomassie blue stained SDS-gels the recombinant Tat-his was clearly identified as an intense band presenting a maximal intensity after around 72-96H induction.
19

20

Media used for fermentation:

2
Example 4: PURIFICATION OF Nef-Tat-His FUSION PROTEIN (PICHIA PASTORIS)
The purification scheme has been developed from I46g of recombinant Pichia pastoris cells (wet weight) or 2L Dyno-mill homogenate OD 55. The chromatographic steps are performed at room temperature. Between steps , Nef»Tat positive fractions are kept overnight in the cold room (+4°C) ; for longer time, samples are frozen at -20°C.

23

24

25

26

¦^ Level Of purity estimated by SDS-PAGE is shown in Figure 6 (Daiichi Silver Staining. Coomassie blue G250. Western blotting):
After dialysis and sterile filtration steps: > 95%
-> Recovery (evaluated by a colorimetric protein assay: DOC TCA BCA)
2,8 mg of oxidized Nef-Tat-his protein are purified from 73 g of recombinani Pichia pastoris cells (wet weight) or 1 L of Dyno-mill homogenate OD 50.
Example 6: PURIFICATION OF REDUCED TAT-HIS PROTEIN (PICHIA PASTORIS)
The purification scheme has been developed from 160 g of recombinant Pichia pastoris cells (wet weight) or 2L Dyno-mill homogenate OD 66. The chromatographic steps are performed at room temperature. Between steps, Tat positive fractions are kept overnight in the cold room (+4°C) ; for longer time, samples are frozen at -20°C.
27

28

"^ Level of purity estimated by SDS-PAGE as shown in Figure 7(Daiichi Silver Staining. Coomassie blue G250. Western blotting):
After dialysis and sterile filtration steps: > 95% "^ Recovery (evaluated by a colorimetric protein assay: DOC TCA BCA)
48 mg of reduced Tat-his protein are purified from 160 g of recornbinant Pichia pastoris cells (wet weight) or 2 L of Dyno-mill homogenate OD 66.
29

The purification scheme has been developed from 74 g of recombinant Pichia pastoris cells (wet weight) or 1L Dyno-mill homogenate OD60. The chromatographic steps are performed at room temperature. Between steps, Tat positive fractions are kept overnight in the cold room (+4°C) ; for longer time, samples are frozen at -20°C.

"^ Level of purity estimated by SPS-FAGE as shown in Figure g fDaiichi Silver Staining, Coomassie blue G250. Western blotting):
After dialysis and sterile filtration steps: > 95% "^ Recovery (evaluated by a colorimetric protein assay; DOC TCA BCA)
31

19 mg of oxidized Ta;-his protein are purified from 74 g of reeombinant Pichia pastoris cells (wet weight) or 1 L of Dyno-mill homogenate OD 60.
Example S: PURIFICATION OF SIV REDUCED NEF-HIS PROTEIN (PICHIA PASTORIS)
32
The purification scheme has been developed from 340 g of reeombinant Pichia pastoris cells (wet weight) or 4 L Dyno-mill homogenate OD 100. The chromatographic steps are performed at room temperature. Between steps , Nef positive fractions are kept overnight in the cold room (+4°C) ; for longer time, samples are frozen at -20°C.

•> Level of purity estimated by SDS-PAGE as shown in Figure 9 (Daiichi Silver Staining. Coomassie blue G250. Western blotting"):
After dialysis and sterile filtration steps: > 95% "^ Recovery (evaluated by a colorimetric protein assay: DOC TCA BCA)
33

20 mg of SIV reduced Nef-his protein are purified irorn 340 g of recombinant Pichia pastoris cells (wet weight) or 4 L of Dyno-miH homogenate OD 100.
Example 9: PURIFICATION OF HIV REDUCED NEF-HIS PROTEIN (PICHIA
PASTORIS)
34
The purification scheme has been developed from 160 g of recombinant Pichia pastoris cells (wet weight) or 3 L Dyno-miH homogenate OD 50. The chromatographic steps are performed at: room temperature. Between steps , Nef positive fractions are kept overnight in the cold room (+4°C); for longer time, samples are frozen at ~20°C.

¦> Level of purity estimated by SDS-PAGE as shown in Figure 10 (Daiichi Silver Staining. Coomassie blue G250, Western blotting):
After dialysis and sterile filtration steps: > 95% -^ Recovery (evaluated by a colorimetric protein assay: DOC TCA BCA)
35

20 mg of HIV reduced Nef-his protein are purified from 160 g of recombinant Pichia pistons cells (wet weight) or 3 L of Dyno-mill homogenate OD 50.
Example 10: EXPRESSION OF nef SEQUENCE IN PICHIA PASTORIS
In order to evaluate Nef and Tat antigens in the pathogenic SHIV challenge model, we have expressed the Nef protein of simian immunodeficiency virus (SIV) of macaques, SIVmac239 ( Aids Research and Human Retroviruses, 6:1221-1231,1990). In the Nef coding region , SIV mac 239 has an in-frame stop codon after 92aa predicting a truncated product of only 10kD. The remainder of the Nef reading frame is open and would be predicted to encode a protein of 263aa (30kD) in its fully open form.
Our starting material for SIVmac239 nefgene was a DNA fragment corresponding to the complete coding sequence, cloned on the LX5N plasmid (received from Dr R.C. Desrosiers, Southborough,MA,USA).
This SIV nef gene is mutated at the premature stop codon (nucleotide G at position 9353 replaces the original T nucleotide) in order to express the full-length SIVmac239 Nef protein.
To express this SIV nef gene in Pichia pastoris, the PHIL-D2-MOD Vector (previously used for the expression of HIV-1 nef and tat sequences) was used. The recombinant protein is expressed under the control of the inducible alcohol oxidase (AOX1) promoter and the c-terminus of the protein is elongated by a Histidine affinity tail that will facilitate the purification.
10.1 CONSTRUCTION OF THE INTEGRATIVE VECTOR PRIT 14908
To construct pRIT 14908 , the SIV nef gene was amplified by PCR from the pLX5N/SIV-NEF plasmid with primers SNEF1 and SNEF2.
36

PRIMER SNEF1: 5' ATCGTCCATG.GGTGGAGCTATTTT 3'
Ncol
PRIMER SNEF2; 5' CGGCTACTAGTGCGAGTTTCCTT 3+
Spel
The SIV nef DNA region amplified starts at nucleotide 9077 and terminates at nucleotide 9865 ( Aids Research and Human Retroviruses, 6:1221-1231,1990).
An Ncol restriction site (with carries the ATG codon of the nef gene) was introduced at the 5' end of the PCR fragment while a Spel site was introduced at the 3* end. The PCR fragment obtained and the integrative PHIL-D2-MOD vector were both restricted by Ncol and Spel. Since one Ncol restriction site is present on the SIV nef amplified sequence (at position 9286), two fragments of respectively ±200bp and ± 600bp were obtained, purified on agarose gel and ligated to PHIL-D2-MOD vector. The resulting recomblnant plasmid received, after verification of the nef amplified region by automated sequencing, the pRIT 14908 denomination.
10.2 TRANSFORMATION OF PICH1A PASTORIS STRAIN GS115(his4).
To obtain Pichia oastoris strain expressing SIV nef His, strain GS115 was transformed with a linear Noil fragment carrying only the expression cassette and the fflS4gene(Fig.Il).
This linear NotI DNA fragment ,with homologies at both ends with A0X1 resident P.pastoris gene, favors recombination at the A0X1 locus. Multicopy integrant clones were selected by quantitative dot blot analysis . One transformant showing the best production level for the recombinant protein was selected and received the Y1772 denomination.
Strain Y1772 produces the recombinant SIV Nef-His protein, a 272 amino acids
protein which would be composed of:
°Myristic acid
°A methionine, created by the use of Ncol cloning site of PHIL-D2-MOD vector .
37

°262 amino acids (aa) of Nef protein (starting at aa 2 and extending to aa 263, see
Figure 12)
°A threonine and a serine created by the cloning procedure (cloning at Spel site of
PHIL-D2-M0D vector (Fig. 11).
°One glycine and six histidines.
Nucleic and Protein sequences are shown on figure 12.
10.3 CHARACTERIZATION OF THE EXPRESSED PRODUCT OF STRAIN
Y1772.
Expression level
After 16 hours induction in medium containing 1% methanol as carbon source, abundance of the recombinant Nef-His protein, was estimated at 10% of total protein (Fig. 13 , lanes 3-4).
Solubility
Induced cultures of recombinant strain Y1772 producing the Nef-His protein were centriruged. Cell pellets were resuspended in breaking buffer, disrupted with 0.5mm glass beads and the cell extracts were centrifiiged. The proteins contained in the insoluble pellet (P) and in the soluble supernatant (S) were compared on a Coomassie Blue stained SDS-PAGE10%.
As shown in figure 13, the majority of the recombinant protein from strain Y1772 (lanes 3-4) is associated with the insoluble fraction.
Strain Y1772 which presents a satisfactory recombinant protein expression level is used for the production and purification of SIV Nef-His protein.
Example 11: EXPRESSION OF GP120 IN CHO
38

A stable CHO-K1 cell line which produces a recombinant gPI20 glycoprotein has been established. Recombinant gP120 glycoprotein is a recombinant truncated form of the gP120 envelope protein of HIV-1 isolate W61D. The protein is excreted into the cell culture medium, from which it is subsequently purified.
Construction of gp!20 transfection plasmid pRJT13968
The envelope DNA coding sequence (including the 5'exon of tat and rev) of HIV-1
isolate W61D was obtained (Dr. Tersmette, CCB, Amsterdam) as a genomic gpl60
envelope containing plasmid W61D (Nco-Xhol). The plasmid was designated
pRIT13965.
In order to construct a gpI20 expression cassette a stop codon had to be inserted at the ammo acid glu 515 codon of the gp!60 encoding sequence in pRIT13965 using a primer oligonucleotide sequence (DIR 131) and PCR technology. Primer DIR 131 contains three stop codons (in all open reading frames) and a Sail restriction site.
The complete gpl20 envelope sequence was then reconstituted from the N-terminal BamHl-Dral fragment (170 bp) of a gpl60 plasmid subclone pW61d env (pRIT13966) derived from pRIT13965, and the Dral-Sall fragment (510 bp) generated by PCR from pRIT13965. Both fragments were gel purified and ligated together into the E.coli plasmid pUC18, cut first by Sail (klenow treated), and then by BamHl. This resulted in plasmid pRITl3967. The gene sequence of the Xmal-Sall fragment (1580 bp) containing the gp!20 coding cassette was sequenced and found to be identical to the predicted sequence. Plasmid RIT13967 was ligated into the CHO GS-expression vector pEE 14 (Celitech Ltd., UK) by cutting first with Bell (klenow treated) and then by Xmal. The resulting plasmid was designated pRIT13968.
Preparation of Master Cell Bank
The gpl20-construct (pRIT13968) was transfected into CHO cells by the classical CaPO4-precipitation/grycero! shock procedure. Two days later the CHOK1 cells were subjected to selective growth medium (GMEM +¦ methionine sulfoximine (MSX) 25 pM + Glutamate + asparagine *10% Foetal calf serum ). Three chosen
39

transfectant clones were farther amplified in 175m2 flasks and few cell vials were stored at -80°C. C-env 23,9 was selected for further expansion.
A small prebank of cells was prepared and 20 ampoules were frozen. For preparation of the prebank and the MCB, cells were grown in GMEM culture medium, supplemented with 7.5 % fetal calf serum and containing 50 uM MSX. These cell cultures were tested for sterility and mycoplasma and proved to be
negative.
The Master Cell Bank CHOKI env 23,9 (at passage 12) was prepared using cells derived from the premaster cell bank. Briefly, two ampoules of the premaster seed were seeded in medium supplemented with 7.5% dialysed foetal bovine serum. The cells were distributed in four culture flasks and cultured at 37°C. After cell attachment the culture medium was changed with fresh medium supplemented with 50 uM MSX. At confluence, cells were collected by trypsination and subcultured with a 1/8 split ratio in T-flasks - roller bottle - cell factory units. Cells were collected from cell factory units by trypsination and centrifugation. The cell pellet was resuspended in culture medium supplemented with DMSO as cryogenic preservative. Ampoules were prelabelled, autoclaved and heat-sealed (250 vials). They were checked for leaks and stored overnight at -70DC before storage in liquid nitrogen.
Cell Culture And Production Of Crude Harvest
Two vials from a master cell bank are thawed rapidly. Cells are pooled and inoculated in two T-flasks at 37° ± 1°C with an appropriate culture medium supplemented with 7.5 % dialysed foetal bovine (FBS) serum. When reaching confluence (passage 13), ceils are collected by trypsinisation, pooled and expanded in 10 T-flasks as above. Confluent cells (passage 14) are trypsinised and expanded serially in 2 cell factory units (each 6000 cm2; passage 15), then in 10 cell factories (passage 16). The growth culture medium is supplemented with 7.5 % dialysed foetal bovine (FBS) serum and 1% MSX. When cells reach confluence, the growth culture medium is discarded and replaced by "production medium" containing only 1 % dialysed foetal bovine serum and no MSX. Supernatant is collected every two
40

days (48 hrs-interval) for up to 32 days. The harvested culture fluids are clarified immediately through a 1.2-0.22 $im filter unit and kept at -20°C before purification.
Example 12: PURIFICATION OF HIV GP 120 (W610 CHO) FROM CELL CULTURE FLUID
AH purification steps are performed in a cold room at 2-8°C. pH of buffers are adjusted at this temperature and are filtered on 0.2 urn filter. They are tested for pyrogen content (LAL assay). Optical density at 280 nm, pH and conductivity of column eluates are continuously monitored.
(0 Clarified Culture Fluid
The harvested clarified cell culture fluid (CCF) is filter-sterilized and Tris buffer, pH
8.0 is added to 30 mM final concentration. CCF is stored frozen at -20°C until
purification.
(K) Hvdrophobic Interaction Chromatographv
After thawing, ammonium sulphate is added to the clarified culture fluid up to 1 M. The solution is passed overnight on a TSK/TOYOPEARL-BUTYL 650 M (TOSOHAAS) column, equilibrated in 30 mM Tris buffer- pH 8.0 - 1 M ammonium sulphate. Under these conditions, the antigen binds to the gel matrix. The column is washed with a decreasing stepwise ammonium sulphate gradient. The antigen is eluted at 30 mM Tris buffer- pH 8.0 - 0.25 M ammonium sulphate.
(iii) Anion-exchange Chromatographv
After reducing the conductivity of the solution between 5 and 6 mS/cm, the gP120 pool of fractions is loaded onto a Q-sepharose Fast Flow (Pharmacia) column, equilibrated in Tris-saline buffer - pH 8.0. The column is operated on a negative mode, i.e. gP120 does not bind to the gel, while most of the impurities are retained.
(iv) Concentration and diafiltration by ultrafiltration In order to increase the protein concentration, the gP120 pool is loaded on a FILTRON membrane "Omega Screen Channel", with a 50 kDa cut-off. At the end of the concentration, the buffer is exchanged by diafiltration with 5 mM phosphate
41

buffer containing CaCl2 0.3 mM, pH 7.0. If further processing is not performed
immediately, the gP120 pool is stored frozen at -20°C. After thawing the solution is filtered onto a 0.2 uM membrane in order to remove insoluble materiel.
(v) Chromatographv on hydroxvapatite
The gP120 UF pool is loaded onto a macro-Prep Ceramic Hydroxyapatite, type II
(Biorad) column equilibrated in 5 mM phosphate buffer + CaCl2 0.3 mM, pH 7.0.
The column is washed with the same buffer. The antigen passes through the column and impurities bind to the column.
(vi) Cation exchange chromatographv
The gP120 pool is loaded on a CM/TOYOPEARL-650 S (TOSOHAAS) column equilibrated in acetate buffer 20 mM, pH 5.0. The column is washed with the same buffer, then acetate 20 mM, pH 5.0 and NaCl 10 mM. The antigen is then eluted by the same buffer containing 80 mM NaCl.
(vii) Ultrafiltration
In order to augment the virus clearance capacity of the purification process, an additional ultrafiltration step is carried out. The gP120 pool is subjected to ultrafiltration onto a FILTRON membrane "Omega Screen Channel", cut-off 150 kDa. This pore-size membrane does not retain the antigen. After the process, the diluted antigen is concentrated on the same type of membrane (Filtron) but with a cut-off of 50 kDa.
(viii) Size exclusion Gel Chromatographv
The gP120 pool is applied to a SUPERDEX 200 (PHARMACIA) column in order to exchange the buffer and to eliminate residual contaminants. The column is eluted with phosphate buffer saline (PBS).
(ix) Sterile filtration and storage
Fractions are sterilized by filtration on a 0.2 uM PVDF membrane (Millipore). After sterile filtration, the purified bulk is stored frozen at -20°C up to formulation. The purification scheme is summarized by the flow sheet below.
42

=> Level of purity of the purified bulk estimated by SDS-PAGE analysis (Silver staining / Coomassie Blue / Western Blotting) is > 95%.
=> Production yield is around 2.5 mg /L CCF (according to Lowry assay) -Global purification yield is around 25% (according to Elisa assay)
43
=> Purified material is stable 1 week at 37°C (according to WB analysis)

Example 13: VACCINE PREPARATION
A vaccine prepared in accordance with the invention comprises the expression products of one or more DNA recombinants encoding an antigen. Furthermore, the formulations comprise a mixture of 3 de -O-acylated monophosphoryl lipid A 3D-MPL and QS21 in an oil/water emulsion or an oligonucleotide containing unmethylated CpG dinucleotide motifs and aluminium hydroxide as carrier.
3D-MPL: is a chemically detoxified form of the lipopolysaccharide (LPS) of the Gram-negative bacteria Salmonella minnesota.
Experiments performed at Smith Kline Beecham Biologicals have shown that 3D-MPL combined with various vehicles strongly enhances both the humoral immunity and a TH1 type of cellular immunity.
QS21: is a saponin purified from a crude extract of the bark of the Quillaja Saponaria Molina tree, which has a strong adjuvant activity: it induces both antigen-specific lymphoproliferation and CTLs to several antigens.
Experiments performed at Smith Kline Beecham Biologicals have demonstrated a clear synergistic effect of combinations of 3D-MPL and QS21 in the induction of both humoral and THI type cellular immune responses.
The oil/water emulsion is composed of 2 oils (a tocopherol and squalene), and of PBS containing Tween 80 as emulsifier. The emulsion comprises 5% squalene, 5%
44

tocopherol, 2% Tween 80 and has an average particle size of 180 nm (see WO 95/17210).
Experiments performed at Smith Kline Beecham Biologicals have proven that the adjunction of this OAV emulsion to 3D-MPL/QS21 further increases their immunostimulant properties.
Preparation of the oil/water emulsion (2 fold concentrate)
Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100ml two fold concentrate emulsion 5g of DL alpha tocopherol and 5ml of squalene are vortexed to mix thoroughly. 90ml of PBS/Tween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe and finally microfluidised by using an Ml 10S Microfluidics machine. The resulting oil droplets have a size of approximately 180 nm.
Preparation of oil in water formulation.
Antigens (100 ?g gpl20, 20 ?g NefTat, and 20 ug SIV Nef, alone or in combination) were diluted in 10 fold concentrated PBS pH 6.8 and H2O before consecutive addition of the oil in water emulsion, 3D-MPL (50?g), QS21 (50?g) and 1 ?g/ml thiomersal as preservative at 5 min interval. The emulsion volume is equal to 50% of the total volume (250ul for a dose of 500?l).
All incubations were carried out at room temperature with agitation.
CpG oligonucleotide (CpG) is a synthetic unmethylated oligonucleotide containing one or several CpG sequence motifs. CpG is a very potent inducer of TH1 type immunity compared to the oil in water formulation that induces mainly a mixed THI/TH2 response. CpG induces lower level of antibodies than the oil in water formulation and a good cell mediated immune response. CpG is expected to induce lower local reactogenicity.
45

Preparation of CpG oligonucleotide solution: CpG dry powder is dissolved in H2O to give a solution of 5 mg/ml CpG.
Preparation of CpG formulation.
The 3 antigens were dialyzed against NaCl 150 mM to eliminate the phosphate ions that inhibit the adsorption of gpl20 on aluminium hydroxide.
The antigens diluted in H2O (100 ?g gpl20, 20 ?g NefTat and 20 ?g SIV Nef) were incubated with the CpG solution (500 ?g CpG) for 30 min before adsorption on A1(OH)3 to favor a potential interaction between the His tail of NefTat and Nef antigens and the oligonucleotide (stronger immunostimulatory effect of CpG described when bound to the antigen compared to free CpG). Then were consecutively added at 5 min interval A1(OH)3 (500 ?g), 10 fold concentrated NaCl and 1 ?g/ml thiomersal as preservative.
All incubations were carried out at room temperature with agitation.
Example 14: IMMUNIZATION AND SHIV CHALLENGE EXPERIMENT IN RHESUS MONKEYS.
First Study
Groups of 4 rhesus monkeys were immunized intramuscularly at 0, 1 and 3 months with the following vaccine compositions:
Group 1: Adjuvant 2 + gpl20
Group 2: Adjuvant 2 + gpl20 + NefTat + SIV Nef
Group 3; Adjuvant 2 + NefTat* + SIV Nef
Group 4 Adjuvant 6 +gpl20 + NefTat + SIV Nef
Group 5 Adjuvant 2 + NefTat + SIV Nef
Group 6 Adjuvant 2
46

Adjuvant 2 comprises squalene/tocopherol/Tween 80/3D-MPL/QS2I and Adjuvant 6 comprises alum and CpG.
Tat* represents mutated Tat, in which Lys4I-»Ala and in RGD motif Arg78->Lys and Asp80->Glu (Virology 235: 48-64, 1997).
One month after the last immunization all animals were challenged with a pathogenic SHIV (strain 89.6p). From the week of challenge (wkl6) blood samples were taken periodically at the indicated time points to determine the % of CD4-positive cells among peripheral blood mononuclear cells by FACS analysis (Figure 14) and the concentration of RNA viral genomes in the plasma by bDNA assay (Figure 15).
Results
All animals become infected after challenge with SHIV89.6P.
CD4-positive cells decline after challenge in all animals of groups 1,3,5 and 6 except one animal in each of groups 1 and 6 (control group). All animals in group 2 exhibit a slight decrease in CD4-positive cells and recover to baseline levels over time. A similartrend is observed in group 4 animals (Figure 14).
Virus load data are almost the inverse of CD4 data. Virus load declines below the level of detection in ¾ group 2 animals (and in the one control animal that maintains its CD4-positive cells), and the fourth animal shows only marginal virus load. Most of the other animals maintain a high or intermediate virus load (Figure 15).
Surprisingly, anti-Tat and anti-Nef antibody titres measured by ELISA were 2 to 3-fold higher in Group 3 (with mutated Tat) than in Group 5 (the equivalent Group with non-mutated Tat) throughout the course of the study.
At week 68 (56 weeks post challenge) all animals from the groups that had received the full antigen combination (groups 2 and 4) were still alive, while most of the animals in the other groupshad to be euthanized due to AIDS-like symptoms. The surviving animals per group were:
47

Group I: 2/4
Group 2: 4/4
Group 3: 0/4
Group 4 4/4
Group 5 0/4
Group 6 1/4
Conclusions
The combination of gp 120 and NefTat (in the presence of SIV Nef) prevents the loss of CD4-positive cells, reduces the virus load in animals infected with pathogenic SHIVg9.6P, and delays or prevents the development of AIDS-Iike disease symptoms, while gpl20 or NefTat/SIV Nef alone do not protect from the pathologic consequences of the SHIV challenge.
The adjuvant 2 which is an oil in water emulsion comprising squalene, tocopherol and Tween 80, together with 3D-MPL and QS2I seems to have a stronger effect on the study endpoints than the alum / CpG adjuvant.
Second study
A second rhesus monkey SHIV challenge study was conducted to confirm the efficacy of the candidate vaccine gpl20/NefTat + adjuvant and to compare different Tat-based antigens. The study was conducted by a different laboratory.
The design of the study was as follows.
Groups of 6 rhesus monkeys were immunized at 0,4 and 12 weeks with injections i.m. and challenged at week 16 with a standard dose of pathogenic SHIVg9.6P.
Group I is the repeat of Group 2 in the first study.
48

Group 1: Adjuvant 2 + gp!20 + NefTat + SIV Nef
Group 2: Adjuvant 2 + gpI20 + Tat (oxidised)
Group 3: Adjuvant 2 + gp 120 + Tat (reduced)
Group 4 Adjuvant 2

animals This is a confirmation of the result obtained in the first SHIV study. Due to the lack of susceptibility of the study animals, the virus load parameter could not be used to demonstrate a vaccine effect. Taken together, the combination of gpl20 and Tat and Nef HIV antigens provides protection against the pathologic consequences of HIV infection, as evidenced in a SHIV model.
The Tat alone antigens in combination with gp!20 also provide some protection from the decline of CD4-positive cells. The effect is less pronounced than with the gpl20/NefTat/SIV Nef antigen combination, but it demonstrates that gpl20 and Tat are able to mediate some protective efficacy against SHIV-induced disease manifestations.
The second SHIV challenge study was performed with rhesus monkeys from a source completely unrelated to the source of animals from the first study. Both parameters, % of CD4-positive cells and plasma virus load, suggest that the animals in the second study were less susceptible to SHIV-induced disease, and that there was considerably greater variability among the animals. Nonetheless, a beneficial effect on the maintenance of CD4-positive cells of the gpl20/NefTat/SIV Nef vaccine was seen with the experimental vaccine containing gpl20/NefTat and SIV Nef. This indicates that the vaccine effect was not only repeated in a separate study, but furthermore demonstrated in an unrelated monkey population.
50

WE CLAIM :
1. A process for the preparation of a vaccine formulation for use in the prophylactic or therapeutic immunisation of humans against HIV, comprising the steps of admixing
a) an HIV Tat protein or polynucleotide as defined by SEQ ID Nos 10 or 11, or a
derivative sequence which is with or without a C-teminal His-tail, or mutated, or
chemicaily modified; or
b) an HIV Nef protein or polynucleotide as defined by SEQ ID Nos 8 or 9, or a derivative
sequence which is with or without a C-teminal His-tail. or mutated, or chemically
modified; or
c) an HIV Tat protein or polynucleotide linked to an HIV Nef protein or polynucleotide
(Nef-Tat) as defined by SEQ ID Nos 12 or 13, or a derivative sequence which is with
or without a C-teminal His-tail, or mutated, or chemically modified;
and an HIV gpl20 protein or polynucleotide;
wherein the Tat, Nef of Nef-Tat act in synergy with gpl20 in the treatment or prevention of HIV wherein the vaccine in use reduces the HIV viral load in HIV infected humans and results in a maintenance of CD4+ levels over those levels found in the absence of vaccination with HIV Tat, Nef or Nef-Tat and HIV gpl20.
2. The process, as claimed in claim 1, wherein the vaccine optionally comprises an antigen
selected from the group consisting of: gag, rev, vif, vpr, vpu.
3. The process, as claimed in claim 1 or claim 2S wherein the Tat protein is a mutated protein.
4. The process, as claimed in any one of claims 1 - 3, wherein the Tat, Nef or Nef-Tat protein is
reduced.
5. The process, as claimed in any one of claims 1 - 4, wherein the Tat, Nef or Nef-Tat protein is
carbamidomethylated.
6. The process, as claimed in any one of claims 1 - 4, wherein the Tat, Nef or Nef-Tat protein is
oxidised.
51

7. The process, as claimed in any one of claims 1 - 6, wherein an optional adjuvant is used in
the admixture.
8. The process, as claimed in claim 7, wherein the adjuvant is a TH1 inducing adjuvant.
9. The process, as claimed in claim 7 or claim 8, wherein the adjuvant comprises
monophosphoryl lipid A or a derivative thereof such as 3-de-O-acylated monophsphoryl lipid A.
10. The process, as claimed in any one of claims 7-9, wherein an optional saponin adjuvant is
used in the admixture.
11. The process, as claimed in any one of claims 7-10, wherein an optional oil in water
emulsion is used in the admixture.
12. The process, as claimed in claim 7 or claim 8, wherein the adjuvant comprises CpG motif-
containing oligonucleotides.
13. The process, as claimed in claim 12, wherein an aluminium salt is used as a second adjuvant.
14. A vaccine composition for human use comprising HIV Tat or HIV Nef or HIV Nef-Tat in
combination with HIV gpl20 proteins or polynucleotides encoding them together with an adjuvant
comprising QS21 and 3D-MPL, wherein the vaccine in use reduces the viral load in HIV infected
humans and results in a maintenance of CD4+ levels over those found in the absence of vaccination
with HIV Tat, Nef, or Nef-Tat and HIV gpl20.
The present invention discloses a vaccine composition for human use comprising HIV Tat or HIV Nef or HIV Nef-Tat in combination with HIV gpl20 proteins or polynucleotides encoding them together with an adjuvant comprising QS21 and 3D-MPL, wherein the vaccine in use reduces the HIV viral load in HIV infected humans and results in a maintenance of CD4+ levels over those levels found in the absence of vaccination with HIV Tat, Nef or Nef-Tat and HIV gpl20
and a process for its preparation.

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Patent Number

209777

Indian Patent Application Number

IN/PCT/2002/00930/KOL

PG Journal Number

36/2007

Publication Date

07-Sep-2007

Grant Date

06-Sep-2007

Date of Filing

17-Jul-2002

Name of Patentee

SMITHKLINE BEECHAM BIOLOGICALS S.A.

Applicant Address

RUE DE L'INSTITUT 89, B-1330 RIXENSART,BELGIUM

Inventors:

#	Inventor's Name	Inventor's Address
1	VOSS GERALD	SMITHKLINE BEECHAM BIOLOGICALS S.A.,RUE DEL'INSTITUT 89, B-1330 RIXENSART,BELGIUM

PCT International Classification Number

A61K 39/21

PCT International Application Number

PCT/EP01/00944

PCT International Filing date

2001-01-29

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	PCT/EP00/05998	2000-06-28	U.K.
2	0013806.5	2000-06-06	U.K.
3	0002200.4	2000-01-31	U.K.
4	0009336.9	2000-04-14	U.K.