Title of Invention

NUCLEIC ACID ADJUVANTS

Abstract

The present invention relates to an adjuvant composition comprising: (i) a first nucleic acid sequence; (ii) a second nucleic acid sequence; and (iii) core carriers, wherein: (a) said first nucleic acid sequence encodes a 5' truncated bacterial ADP-ribosylating exotoxin A subunit, the truncation meaning that the subunit does not having an amino terminal bacterial signal peptide; (b) said second nucleic acid sequence encodes a 5' truncated bacterial ADP-ribosylating exotoxin B subunit, the truncation meaning that the subunit does not having an amino terminal bacterial signal peptide; (c) said first and second nucleic acid sequences are coated onto the same or different core carriers and are operably linked to, or are each operably linked to, a promoter active in mammalian cells; and (d) each of said truncated subunit coding regions encodes the subunit operably linked to a leader sequence for secretion from a mammalian cell.

Full Text

wo 03/004055 PCT/USOl/43151 NUCLEIC ACID ADJUVANTS Technical Field The invention relates to the fields of molecular biology and immunology, 5 and generally relates to nucleic acid immunization techniques. More specincaliy, the invention relates to polynucleotides encoding an adjuvant, and to immunization strategies employing sucholynucleocides. Background ] 0 Techniques for the injection of DNA and mRNA into mammalian tissue for the purposes of immunization against an expression product have been described in the art. The techniques, termed "nucleic acid immunization" herein, have been shown to elicit both humoral and cell-mediated immune responses. For example, sera from mice immunized with a DNA construct encoding the 15 envelope glycoprotein, gpl60, were shown to react with recombinant gpl60 in immunoassays, and lymphocytes from the injected mice were shown to proliferate in response to recombinant gpI20. Wang et al, (1993) Proc. Nad, Acad. Sci USA 90:4156-4160. Similarly, mice immumzed with a human growth hormone (hGH) gene demonstrated an antibody-based immune response. Tang et 20 al. (1992) Nature 356:152-154. Intramuscular injection of DNA encoding influenza nucleoprotein driven by a mammalian promoter has been shown to elicit a CDS+ CTL response that can protect mice against subsequent lethal challenge with virus. Ulmer et al. (1993) Science 259:1745-1749. Immunohistochemical studies of the injection site revealed that the DNA was 25 taken up by myeloblasts, and cvtoplasmic production of viral protein could be demonsurated for at least 6 months. Summary of the Invention It is a primarv object of the invention to provide a polynucleotide adjuvant 30 composition containing furst and second nucleic acid sequences, wherein the first nucleic acid secuence is a trucated A subunit coding region obtained or derived 1 from a bacterial ADP-ribosylating exotoxin, and the second nucleic acid sequence is a truncated B subunit coding region obtained or derived from a bacterial ADP- ribosylating exotoxin. Each of the truncated subunit coding regions has a 5' deletion and encodes a subunit peptide not having an amino terminal bacterial 5 signal peptide. The first and second nucleic acid sequences maybe present in the same or in different nucleic acid constructs. The troncated subunit cod'lng regions may be obtained or derived from the same bacterial ADP-ribosylating exotoxin and. in cer.ain preferred embodiments, the bacterial .ADP-ribosylating exotoxin is a 10 cholera toxin (CT) or an E, coli heat labile enterotoxin (LT). In addition, at least W one of the truncated subunit coding regions maybe genetically modified to detoxify the subunit peptide encoded thereby, for example wherem the tnmcated A submiit coding region has been genetically modified to disrupt or inacrivate -ADP-ribosyl transferase activity in the subunit peptide expression product. 15 It is also a primary object of the invention to provide a polynucleoride adjuvant composition contaimng first and second nucleic acid sequences, wherein the first nucleic acid sequence is a modified A subunit coding region obtained or derived from a bacterial .ADP-ribosylating exotoxin, and the second nucleic acid sequence is a B subunit coding region obtained or derived from a bacterial AD?- 20 ribosylating exotoxin. The modified A subunit coding region and said B subunit coding region each encode a mature subunit peptide, and the modified A subunit coding region has been genetically modified so as to delete a C-terminaJ KDEL or RDEL motif in the subunit peptide encoded thereby. As above, the first and second nucleic acid sequences may be present in 25 the same or in different nucleic acid constructs. The truncated subunit coding regions may be obtained or derived from the same bacterial .ADP-ribosylaring exoioxin and, in certain preferred embodiments, the bac:erial .ADP-ribosylating exotoxin is a cholera toxin (CT) or an E. ccAi heat labile eriterotoxin ('LT). In addition, at least one of the truncated subunit coding regions may be genetically modified to detoxify the subunit peptide encoded therby for example wherem the truncated A subunit coding region has been genetically modified :o disrupt or y inactivate ADP-ribosyl transferase activity in the subnnit peptide expression product. In certain aspects of the invention, the above compositions can be provided in particulate form, for example wherein the compositions are 5 particulates suitable for deliver/ from a particle delivery device. In this regard, the present compositions may be coated onto the same or a different core carrier particle and thus suitable for delivery using a particle-mediated transfection technique. Preferred core carrier particies will have an average diameter of about 0.1 to 10 µm, and may comprise a metal such as gold. Accordingly, it is a still 10 further object of the invention to provide a particle delivery device loaded with 4 (e.g., containing) a particulate composition as defined herein. It is also a primary object of the invention.to provide for the use of a composition containing a first and second nucleic acid sequence, where each sequence includes a coding region for a subunit from a bacterial ADP- 15 ribosylating exotoxin in the manufacmre of a medicament for enhancing an immime response in a vertebrate subject against an antigen of interest in the said subject. The antigen of interest and the composition are administered to the subject such that the toxin subunits encoded by the first and second nucleic acid sequences are expressed in an amount sufncient to elicit an enhanced immime 20 response against the antigen. The first nucleic acid sequence contains a truncated A subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin, and the second nucleic acid sequence contains a turncated B subunit ^"^ coding region obtained or derived from a bacterial ADP-ribosylating exotoxin, however with the proviso that each of the truncated subunit coding regions has a 25 5' deletion and encodes a subunit peptide not having an amino terminal bacterial signal peptide. It is a related primary object of the invention to provide a method for enhancing an immume resnonse asainst an antigen of interest in a subiect. The method generally entails: (a) administering the antigen of interest to the subject; 30 (b) providing an adjuvant composition compnsing first and second nucleic acid sequences, wherein the first nucleic acid sequence is a truncated A subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin, and the second nucleic acid sequence is a truncated B subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin; and (c) administering the adjuvant composition to the subject, whereby upon introduction to tlie subject, 5 the first and second nucleic acid sequences are expressed to provide subunit peptides in an amount sufficient to elicit an enhanced immune response agahist the antigen of interest. The subunit coding regions are trancated in that each coding region has a 5' deletion and encodes a subunit ::eoiide not having an amino terminal bacterial signal peptide. 10 It is yet a farther primary object of the invention to provide for the use of a composition comprising a first and second nucleic acid sequence, where each sequence includes a coding region for a subunit fi-om a bacterial ADP-ribosylating exotoxin in the m.anufacture of a medicament for enhancing an immime response in a vensbrate subject against an antigen of interest in the said 15 subject. The antigen of interest and the composition are administered to the subject such that the toxin subunits encoded by the first and second nucleic acid sequences are expressed in an amcimt sufrlcient to ehcit an erJianced irmnune 20 25 second nucleic acid sequence is a B subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin; and (c) administering the adjuvant composition to the subject, whereby upon introduction to the subject, the first and second nucleic acid sequences are expressed to provide subunit peptides in an 5 amount sufficient to elicit an enhanced immune response against the antigen of interest. The subunit coding regions are modified in that each encodes a mature subunit peptide, but the A subunit coding region has been genetically modified so as :o delete a C-:erminal IGDEL or RDEL motif in the subunit peptide encoded thereby. 10 In the uses and methods of the invention, administering the adjuvant comnositions entails transfecting cells of the subject with a polynucleotide adjuvant composirion according to the present invention. Expression cassettes and/or vectors containing the nucleic acid molecules of the present invention can be used to transfect the cells, and transfection is carried out under conditions that 15 pennit expression of the subunit peptides within the subject. The method may farther entail one or more steps of administering at least one secondary composition to the subject. The transfection procedure carried out during the immunization can be conducted either in vivo, or ex vivo (e.g., to obtain transfected cells which are 20 subsequently introduced into the subject prior to carrying out the secondary immunizadon step). When in vivo transfection is used, the nucleic acid molecules can be administered to the subject by way of intramuscular or intradermal injection of plasmid DNA or, preferably, administered to the subject using a particle-mediated delivery technique. Vaccine compositions (containing 25 the antigen of interest) can be provided in the form of any suitable vaccine composition, for example, in the form of a peptide subunit composition, in the form, of a nucleic acid vaccine composition, or in the form of a whole or split virus influenza vaccine composition. In certain methods, the antigen of interest and the adjuvant composition 30 are administered to the same site in the subject. For example, the adjuvant composition and the antigen of interest can be administred concurrently (e.g., 5 provided in a single vaccine composition). In certain preferred embodiments, the adjuvant and, optionally the antigen of interest, is administered in particulate form, for example wherein the adjuvant composition has been coated onto a core carrier particle and delivered to the subject using a particle-mediated deliver/ 5 technique. In these methods, administration of the polyr.uclectide adjuvant compositions of the present invention preferably results in an augmented cellular immune response against the co-administered antigen of interest. Such an enhanced immune response may be generally characterized by increased titers of 10 inrerferon-producing OD4 and/or CDS* T lymphocytes, increased antigen- specific cytotoxic T lymphocyte (CTL) activity, and a T helper 1-like immmune response (Thl) against the antigen of interest (characterized by increased antigen-specific antibody titers of the subclasses typically associated with cellular (hCMV) immediate early promoter and associated intron A sequence, and the coding sequence for the signal peptide of human tissue plasminogen activator co allow for secretion from mammalian cells of the tnmcated CTA expression product. The figure further contains the complete nucleic acid sequence (SEQ ID 5 NO: 1) for the pPJV2002 plasmid. Figure 5 is a restriction map and functional map of plasmid pPJV2005 that contains a truncated coding sequence for an LT subunit B (LTB) peptide, v/herein the plasmid further contains the hCMV immediate early promoter and associated inrron A sequence, and the coding sequence for the signal peptide of 5 human tissue plasminogen activator to allow for secretion from mammalian cells Formulation #2 containing the EmpVec (pWRG7054) and the pCLA-EnvT plasmid ("gpl20"); Formulation #3 containing the EmpVec (pWRG7054) combined with the pPJV2002 and pPW2003 adjuvant vectors ("CTA/B"); Formulation #4 containing :he pCLVEnvT plasmid ("gpl20") combined with the Definitioas 10 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood bv one of ordinary skill in the an to which the invention pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. 15 In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. The term 'adjuvant" intends any material or composition capable of specifically or non-specifically altering, enhancing, directing, redirecting, potentiating or initiating an antigen-specific immune response. Thus, 20 coadministration of an adjuvant with an antigen may result in a lower dose or fewer doses of antigen being necessary to achieve a desired immune response in the subject to which the antigen is administered, or coadmiristration may result in f ... a qualitatively and'or quantitatively different immune response in the subject. 25 30 ^■f^^ll^ An "adjuvant composition" intends any phannaceutical composition containing an adjuvant Adjuvant compositions can be delivered in the methods of the invention while in any suitable phannaceutical fonn, for example, as a liquid, powder, cream, lotion, emulsion, gel or the like. However, preferred 5 adjuvant compositions will be in particulate form. It is intended, although not always explicitly stated, that molecules havmg similar biological activity as witd-type or purified peptide or chemical adjuvants. For purposes of the present invention, a 'humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T-lymphocytes and/'cr ocher white blood ceils. The term 'nucleic acid immunization" is used herein to refer to the 5 iniroducrion of a nucleic acid molecule encoding one or more selected antigens into a host cell for the in vivo expression of the antigen or antigens. The term also encompasses introduction of a nucleic acid molecule encoding one or more selected adjuvants into a host cell for the in vnvo expression of the adjuvanta or adjuvants. The nucleic acid molecule can be introduced directly into the a central processing unit and used for bioinfonnatics applications such as functional genomics and homology searching. A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). 5 Typically. 'Vector constructy' "expression vector," and "gene transfer vector." mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. A 'plasmid' is a vector in the form of an extrachromosomal genetic element. 10 A nucleic acid sequence which "encodes" a selected adjuvant and/or effecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the 5 nucleic acid sequence and the promoter sequence can still be considered 'operabiy linked" to the coding sequence. 'Recombinant" is used herein to describe a nucleic acid molecule (polynucleotide) of genomic, cDNA, semisyathetic, or syntheric or.gin whihc by virtue of its origin or manipulation is not associated with all or a pordon of the 10 polynucleotide with which it is associated in nature and/or is linked to a polynucleotide other than that to which it is linked in nature. Two nucieic acid sequences which are contained within a single recombinanr nucleic acid molecule are 'heterologous" relative to each other when they are not normally associated with each other in nature. whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in 5 Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Davhoff. Atlas of Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with singie-stranded-specific nucleasels), and size determination of the digested fragmens. Two DNA, or two 5 polypeptide sequences are 'substantially homologous" to each other when the w^^- By "core carrier" is meant a carrier on which a guest nucleic acid (e.g., DNA, RSA) is coated in order to impart a denned particle size as well as a sufficiently high density to achieve the momentum required for cell membrane penetration, such that the guest molecule can be delivered using panicle-mediated that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodimenis of the invention only, and is not intended to be limiting. 5 The present invention provides novel compositions containing nucleic acid sequences, wherein a first sequence in the composition is a coding sequence lor an A subunit obtained or derived from an ADP-ribosylating baccerial toxin, and a second sequence in the composition is a coding sequence for a B subunit obtained or derived from an .ADP-ribosyiating bacterial toxin. The fust and 10 second sequences are useful in immunization methods wherein they are delivered extremely similar molecules, and are at least about 70-80% homologous at the amino acid level. The CT and LT toxins are hexamers, composed of a single molecule of an A subunit surrounded by a doughnut-shaped ring composed of 5 molecules of the 5 3 subunit. The A subunit possesses an ADP-ribosylase activity resulting in G protein modifications that lead to cAMP and protein Idnase A upregulation following internlization of the A subunit in a manmrialian cell. The A subunit can be nicked by exogenous proteases yielding the Al and A2 fragments linlced via a single disulfide bridge. The A subunit of CT contains a C-terminal KDEL triggering activity, respectively. Regarding the B subunit of LT in particular, binding to the GMl receptor on B cells results in polyclonal activation, occurs in the absence of signiiicant proliferation, and involves the upregulation c: a number of important molecules such as MHC class II, B7, CD40, ICAM-1 and 5 IL2-Ra. For T cells, addition of CT or LT holotoxins or recombinant 3 subunits is a coding region for a modified, mature A subunit peptide obtained or derived from an ADP-ribosylating exotoxin, and (b) the second nucleic acid sequence is a coding region for a mature B subunit peptide obtained or derived from an ADP- ribosylating exotoxin. The first nucleic acid sequence contains a coding region 5 that provides for a "modified" A subunit peptide in that the said coding region has been genetically altered so as to delete a four amino acid residue C-termnai KDEL or RDZL motif normally found in the encoded subumil peotide. In still a further embodiment, a composition is provided which includes aspects of the invention, one or both of the toxin subunit peptide coding regions can be further genetically modified to detoxify rhe subunit peptide(s) encoded thereby. For example, genetically altered toxin mutants which have disrupted A\DP-ribcsylat:ng acnviry, tripsin cleavage site mutations, or disrapted birding 5 activity are known in the art. See, e.g., Burnetts at al. (1994) 'Recombinant n"iicrobial ADP-ribosylating toxins of Bordetella peniisis. Vibrio ckolerae. ana enterotoxigenic Escherichia coli: structure, function and toxoid vaccine prime the replication reaction. The product of the first round of replication is itself a template for subsequent replication, thus repeated successive cycles of replication result in geometric amplification of the DNA fragment delimited by the primer pair used, 5 Once the relevant sequences for the ADP-ribosylating exotoxin subunits of mterest have been obtained, they can be linked together to provide one or mors conuguous nucleic acid molecules using standard cloning or molecular biology techniques. More particularly, after sequence information for the selected toxin subunit combination has been obtained, the coding sequences can be combined w vectors which include control sequences operably linked to the inserted sequence or sequences, thus providing expression cassettes that allow for expression of the toxin subunit peptides in vivo in a targeted subject species. exotoxin subunit peptide coding sequences of interest, the expression cassettes can be provided in a suitable transfection vector such as a DNA plasmid vector or a viral vector for subsequent administration to a subject.. In this regard, the polynuclectide compositions of the invention can be used as standalone adjuvant lower or fewer doses of the antigen are required to generate an eScient immune response. As used herein, the term 'cc-administered' such as when an AD?- nbosylating exotoxin subunit encoding adjuvant composition according :c the 5 present invention is "co-administered' with an antigen of Interest (e.g., a vaccine w Vh^^g^' ccmmon allergens can also be obtained from molds or fung] such as Altemaria, Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor and thermophilic actinomycetes; penicillin and tetracycline are common ancibiotic Vjl^^^ associated with colorectal cancer, gplOO or MARTI antigens associated with melanoma, and the PSA antigen associated with prostate cancer. The p53 gene sequence is know-n (see e.g., Harris et al. -(1986) MoL Call Biol. 6:4650-4656) ' and is deposited with GenBank under Accession No. Ml4694. Thus, the 5 adjuvant composirions of the present invention can be used to carry out immunotherapeutic methods for treating cervical, breast, colorectal, prostate, lung cancers and melanomas. / isolate DNA. Polyiucleodde sequences can also be produced synthetically, rather than cloned. w 1^ which have an integrated defective provirus (the "helper") that expresses all of the genes of the virus but cannot package its own genome due to a deletion of the packaging signal, known as the psi sequence. Thus, the cell line produces empty- viral shells. Producer lines can be derived from the packaging lines which, in 5 addition to the helper, contain a viral vector which includes sequences required in A-\V expression vectors are constructed using kno'vvn techniques to at least provide as operatively linked components in the direction of transcription, 20 control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region. The control elements are selected to be functional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded (5' and 3') with functional AAVITR sequences. Suitable AAV constructs can be designed using techniques well Conventional Pharmaceutical Preparations Formulation of a preparation comprising the polynucleotide molecules of 5 the present invention, with or without addition of an antigen composition, can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the ordinarily' skilled artisan. For example, compositions containing one or more suitable vectors can be combined with one or more pharmaceutically acceptable excipients or vehicles to 10 provide a hquid preparation. auxiliary substances is available in REMINGTON'S PKL\RMACEUTIC.AI SCIENCES (IVIack Pub. Co., N.J. 1991), incorporated herein by reference. Certain facilitators of nucleic acid uptake and'or expression ('transfection w polylysine, polyarginine, polyomithine, spermine, spermidine, as well as conj agates of these molecules. The formulated compositions will thus typically include one of more poiynucleotide molecule (e.g., plasraid vector) containing the coding sequences dextran-mediated transfection, calcium phosphate precipitation, electroporation, and direct microinjection of into nuclei). However, deliver/will most typically be via conventional needle aiid svrmge for the liquid compositions and for liquid 1)^ prepared and suitably purified, the above-described plasmid vector constructs can be coated onto carrier particles {e.g., core carriers) using a variety' of techniques known in the art. Carrier particles are selected from materials which have a suitable density in the range of panicle sizes typically used for intracellular 5 delivery from an appropriate particle delivery' device. The optimum carrier W|^^^ 0 15 Following their formation, core carrier particles coated with the nucleic acid preparations of the present invention, alone or in combination with e.g., antigen preparations, are delivered to a subject using particle-mediated deliver}' techniques. The coated particles are administered to the subject to be treated in a maimer compatible with the dosage formulation, and in an amount that will be effective to bring about the desired adjuvant effect'immune response. The amount of the composition to be delivered which, in the case of nucleic acid w acid, rartaric acid, glycine, high molecular weight pclyeihyiene glycols i7EGs), -0 and combination thereof. A thorough discussion of pharmaceuticaily acceptable sxcipients, carriers, stabilizers and other auxiliary substances is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991), irxorporated herein by referepxe. packaged prior to use, can comprise a hermetically sealed container enclosing a suitable amount of the particles comprising a suitable nucleic acid construct and/or a selected antigen (e.g., to provide a multicomponent vaccine composition). The particulate compositions can be packaged as a sterile 5 formulation, and the hermetically sealed container can thus be desiened to preserve sterility of the formulation until use in the methods of :he invention. If desired, the containers can be adapted for direct use in a panicle delivery device. Such containers can take the form of capsules, foil pouches, sachets, cassertes, and the like. Appropriate panicle delivery devices (e.g., needleless s>Tinces) are 10 described herein. w surface, and the densicy and kinematic viscosity of the targeted skin tissue. In this regard, optimal particle densities for use in needleless injection generally range ber.veen about 0.1 and 25 g./cm3. preferably between about 0.9 and 1.5 g.'cm3 and h w . r/ Enhancing Immune Responses 5 In another embodiment of the invention, a method for enhancing an immune response against a co-administered antigen of interest is provided. In essence, the method entails (a) administering an antigen of interest to a subject. additionai or arciliary" components to the subjec:. For rxample, a secondary 30 vaccine ccmposinon can be administered, wherein the secondary' composition can comprise a nucleic acid vaccine, or the secondan' vaccine composition can 1"* Experimental Below are examples of specific embodiments for canning out the present 20 invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in anyway. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.). but some experimental error and deviation should, of course, be allowed for. 4S In order tc facilitate insertion into an expression vector, Primers 1 and 2 contain extra sequences at their 5' ends (outside the region of homology to the CTA coding sequence) which include recognition sites for Xhel and BamHI, 20 respectively. Primers 1 and 2 were designed to lead to PCR generation of a fragment of the A subunit coding sequence starting a: nucleotide position 16- and ending at nucleotide position S86 (GenBank Accession PD30O53). This region Primer 4: 5'— CCT GGA TCC TTA ATT TGC CAT ACT AAT TGC —3' (SEQ ID NO: 10). In order to facilitate insertion-into an expression vector, Primers 3 and 4 5 contain extra sequences at their 5' ends (outside the region of homology to the CTB coding sequence) which include recognition sites for Nhel and BamKL respectively. Primers 3 and 4 were designed lo lead to PCR generation of a fraement of the B subunit coding sequence starring at nucieotide position 946 and ending at nucleotide position 1257 (GenBank Accession #D30053). This region i G encompasses the entire coding sequence for the mature subunit B peptide but k does not include the sequence encoding the bacterial signal peptide found at the ('minus KDEL). respectively. The parental cloning vector pWRG7054 contains the human cvtomegalovirus immediate early promoter with the associated intrcn A sequence. In addition, the coding sequence for the signal peptide of human tissue plasminogen acdvator is included in pWRG7054 to allow for the secretion ■k^^ respectively. Primers 6 and 7 were designed to lead to PCR generation of a fragment of the A snbunit coding sequence starting at nucleotide position 145 and More panicularly, the sequence from the RNA segment #7 (rha: encodes 30 the M2 protein) of influenza virus strain A'Xagoshima 10/95 (H3N2) was used as a model to design PCR primers to facilitate cloning of the manure M2 coding 53 sequence from AySydney/5/97 (H3N2). The A/Kagoshima sequence was used for primer design since the sequence of RNA segment 7 of A'Sydney has not yet been determined. The high degree of conservation among M2 sequences was after which. 0.5µl of reverse transcriptase from the kit was added. The reaction was incubated at 37°C for 15 minutes to complete the reverse transcription step. The PCR reaction step was completed by addition of the following 4,0 \xg of DNA per mg of gold, and a single batch contained 26 mg of gold. DNA was precipitated onto the gold particles by addition of a 1/10 volume of 10% CaCl2 during continuous agitation of the tube on a rotary/mixer. DNA-gold complexes were washed three times with absolute ethanol, and then injected into Formulation #5: pM2-FL DNA vector and pPJV2002 coprecipitated onto single batch of gold (2 |ig pM2-FL DNA per mg gold, and 1 µg of pPJV2002 DNA per mg gold), 0.5 mg gold per cartridge; and V washed three times, and a streptavidin-horse radish peroxidase conjugate (Southern Biotechnology) diluted to 1 :8000 in PBS/0.1 % Tween-20 was added and the plate incubated for 1 hour at room temperature. Following three J o As' can be seen, al! experimental groups immunized with a formulanon containing cne or more of the CT-encading adjuvant vectors (Fcnnulations #2-6') Example 4 Enhancement of .Antigen-Specific Cellular Responses to a DNA Vaccine Encoding the Hepatitis B Surface Antigen (HBsAg) Using Adjuvant Plasmid Vectors Encoding the CT-A and CT-B Subunit Peptides. A Hepatitis B surface antigen (HBsAg) vector plasmid was construacted as follows. To generate the HbsAg coding region, the p.AM6 constract (obtained from the Amxerican Type Culture Collection "ATCC'} with pWRG712S (1 jig DRV0.5 mg gold per delivery). Mice (4 per expenmental group) were immunized and boosted at 4 weeks, then sacrificed at 2 10% fetal bovine serum (FBS) for 1-2 hr at RT. The medium was removed and spleen cells dispensed into the wells with a total of 1x106 cells per well. For wells in which less than 1xlO6 cells from immunized animals was added cells from naive animals were used, to bring the total to 1x106 Cells were incubated 5 overnight in a tissue culture incubator in the presence of the peptide as described above. The plates were then washed 2 time witli PBS and 1 time with distilled The cellular immune responses measured in the two groupS OF mace indicate a significant erhancement of cellular responses by the CT adjuvant 20 vectors. More particularly, the results of the CDS-specific IFN-v ELISA assay are depicted below in Table 3. 63 20. adjuvanted formulation is indicative of a greater amount of IFY-ý secreted by these cells in response to antigen. Cel]s from naive mice did not yield a iL measurable OD value in the IFN-ý ELISA. The results of the CD8-specific IFN'-ý EIJSPOT assay are depicted below in Table 4. ■^ ■:: ^'.) 64 The.above data demonstrate that the novel adjuvant compositions ox the present invention have a potent ability to enhance the cellular immune responses 20 to a coadministered antigen, in this case a HBsAg expressed from a DNA vaccine. 65 Example 5 Enhancement of Humoral and Cellular Immune Responses to HTV-1 gpl20 Antigen Using Simultaneous Delivers of a gpl20 .Antigen Vector and CT.VCTB Adjuvant Vectors. 5 A plasmid vector endoding HTV-l gpl20 was constructed as follows. The vector was constructed starting with a Bluescript (Stxatagene, La Jolla, CA) plasmid backbone, the human cytomegalovirus (hCMTV) immediate early product with the truncation point lying 12S amino acid residues downstream of the gp 120/gp41 processing site. A second plasmid vector encoding HIV-l rev. termed "pC-rev"' herein. pWRG7054 DNA per mg gold, and 1 fig each of pPJV2002 and pPJV2003 DNA per mg gold), 0.5 mg gold per cartridge; V Serum antibody responses to the HIV gp 120 antigen were tested using an ELISA assay on specimens collected at week 5 and week 6.5 (post-prime and post-boost, respectively). For the ELISA, Costar hih binding ELAplates were stimulated in medium alone (negative control), "or in medium with 1 µg/ml of a HIV gpl20 peptide having the following sequence: RIQRGPGRAPVITGK (SEQ ID NO: 21). Following a 24 hour incubation at 37°C in 5% CO2, the plates were km anrigen in an animal model. In addition, rhe enhanced IFX-y production seen iu these studies indicates that use of the CT adjuvant vector combinations of the present invention provides a robust Thl-like immune response m :he in-iunized animals. 71 Example 6 Enhancement of .Antibody Responses to Hepatitis B Core and Surface Antigens Using Simultaneous Delivery of a Vector Encoding HBcAs and HBsAe with CTA/CTB Adjuvant Vectors. polyA sequence (bGHpA) were each obtained from the JW4303 vector construct (gift of Dr. Harriet Robinson, University of Massachusetts) and inserted into a plasmid backbone. The resultant construct was cut with 'Nhel filled with polvmerase and then cut with BamHI to generate a vector fragment Formulation #1: Control, the HBcAg/IIBsAg vector (pWRG7193) alone, 2 µg DNA per mg gold, 0.5 mg gold per cartridge; and 2(: control group (Formulation =1) and adjuvant test group (Formulation;=21 were 2S5 and 662 mIUVml, respectively, demonstrating the ability of the present adjuvant plasmids (pPJV2006 and pPJV2(303) to augment immune responses :o an antigen er.coded by a separate vector. Example 7 Enhancement of Cellular Thl-Like Immune Response to a DNA Vaccine Usine Plasmid Vectors Encoding CT or LT Subunits, 5 The pJvI2-FL DNA vaccine vector encoding the M2 protein of influenza A virus was employed to test the adjuvant effects of the pPJV2002. pPJV2003, ppJV'2004, pPJV2005, pPJV'2006 and pPA'2007 adjuvant vectors in the context cf particle-mediated DNA vaccination. Particle-mediated DNA vaccination was performed by precipitating the M2 DNA vaccine vector, either v/ith our without \f0 Formulation #2: pM2-FL DNA vector combined with the CTA and CTB (pPJV2002 and pPJV2003) DNA vectors prior to precipitation onto a single batch. of gold, 2.1 [ig total DNA per mg gold (1.0 µg of each DNA adjuvant vecicr per mg gold, 0.1 µg pM2-FL), 0.5 mg gold per cartridge; of gold, 2.1 µg total DNA per mg gold (1.0 µg of each DNA adjuvant vector per mg gold, 0.1 |ig pM2-FL), 0.5 mg gold per cartridge; PowderJect® XR-1 particle delivery device (Powder: ect Vaccines Inc., Madison. \VI) at a helium pressure of 400 p.s.i,. Serum samples were collected two weeks following the second or booster immunization. Individual serum samples were assayed for M2-3peciiic antibody 5 responses for both IgGl and IgG2a subclasses usmg the ELISA assay of Example 3 above for the determina::on of total IgG titer, except that a secondary .antibody conjugate specific for eidger IgGl or IgG2a was employed. The goat anti-mouse As can be seen with reference to Table 5, addition of either of the A or B subunits, or the various combinations of A and B subunits to the M2 DNA vaccine formulation resulted in sienificant reductions in the IgGl-to-l2:G2a ratio v/ith 1.5 mM MgCL (Promega Corp., Madison, WT); 0.400 [iM of each primer; 200 [iM or each cLNTP (USB Inc., Cleveland, OH); 2,5 µg Taq polymerass (Promega Corp., Madison, WD; i.O ng template DNA; water to 100 µl; and a and EcoR1 to generate a core antigen insertion fragment; the modified pWRGVUS plasmid was cut with Pstl and Mfel to generate a vector fragment, and the core antigen insertion fragment was ligated into the vector fragment, resulting in the dual surface/core antigen plasmid construct. Fonnulution #3: ("CT w/o TPA") 1 µg dual surface/core antigen DNA plasmid vector, 0.5 jig CTA w/o TPA DNA plasmid vector, 0.5 [ig CTB w/'o TPA DNA plasmid vector; Formulation #12: ("LTB") 1µg dual surface/core antigen DNA plasmid vector, 1 µg pJV2O05 (LTB) DNA plasmid vector; and w Finally, the clearlv observable difference in adjuvant effect between the secreted (Formulations #2 and =8) and the non-secreted ('Formulaticns -3 and -9) heins establish that the observed adjuvant effects are not due :o CvG monts within the adjuvant vectors since the signai-ccntaining and non signai-ccntaining 30 vectors do not have any difference in bacterial DNA (CpG) content yet exhibit S5 significant differences in their ability to augment surface antigen-specific IFN-7 responses. In a second study, the above-described DNA vaccine formulations were adminisiered to eight groups of mice using the PowderJect® XR-1 particle 5 delivery device (PowderJect Vaccines Inc., Madison, UT). Each experimental group contained 5 animals, and each ammal received two immunizations with the combinations of the present adjuvant plasmid vectors to provide vaccine compositions. After immunization, the immunized animals were challenged with HSV-2 virus. and the protective effect of the various vaccine compositions was determined. 1 from the HSV-2 genome, for example the genomic region spanning from approximately nucleotide 114589 to 134980 of the HSV-2 genome, or anEcoRI fragment that spans nucleotides 110931 to 139697 of the HSV-2 genome. The sequence of the HSV-2 genome is available form published sources, for example 5 the sequence deposited with GenBank un der Accession Number NC_001798. In the study, the above-described DNA vaccine fcrmulatains were administered to five different groups of mice using the PowderJect XR-1 15 panicle delivery device (PowderJect Vaccines Inc., Madison, WT). Each experimental group contained 12 animals, and each animal received two immunizations (single shot applied to the abdomen) with the respective formulation with a four vveelc restmg period beUveen inmiunizaiions. A sixth group of mice was established as a negative (naivej control, and did not receive 20 any vaccinations. 4 mice from each group were sacked 2 weeks arier the second immunization and used for IFN-y ELISPOT assays (data not shown). Two v/eeks post second immunization, all remaining mice (8/group) were challenged with 1X10° FPU of HSV-2 virus, strain MS, via intra-nasal insullation. The survival graph depicting the results of the challenge study is 25 depicted in Figure 14. As can be seen, 100% of the naive animals succumbed within 4 days post challenge. The naive animals are depicted on the graph by the curve. In addition, 100% of the animals receiving the ICP27 antigen plasn:d vector alone (Formulation #1) died within 7 days post challenge. The animals receiving Formulation #1 are depicted on the graph by the curve. In marked 30 contrast, the 25% (2/S) of the anim.als receiving the ICP27 piasnrid adjuvanicd with the low dose CT (Formulation 3) were protected tcm the viral challenge, 89 and 38% (3/8) of the animals receiving the ICP27 adjuvanted with the high dose CT (Formulation #2) were protected from the viral challenge. The animals receiving Formulation #3 are depicted on the graph by the (■) curve. The animals receiving Formulation #2 are depicted on the graph by the (♦) cruve. 5 Finally, both the low dose LT-adjuvanted (Formulation =5) and the high dose LT- adjuvanted (Formulation #4) ICP27 vaccine provided complete (100%) protection in the immunized animals. The animals receiving Formulation #5 are depicted on the graph by the curve. The animals receving Formulation =4 are depicted on the graph by the (O) cur\'e. 10 Accordingly, novel polynucleotide adjuvant molecules, compositions comprising those adjuvant molecules, and conventional and nucleic acid 15 immunization techniques have been described. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spi wo 03/004055 PCT/USOl/43151 NUCLEIC ACID ADJUVANTS Technical Field The invention relates to the fields of molecular biology and immunology, 5 and generally relates to nucleic acid immunization techniques. More specincaliy, the invention relates to polynucleotides encoding an adjuvant, and to immunization strategies employing sucholynucleocides. Background ] 0 Techniques for the injection of DNA and mRNA into mammalian tissue for the purposes of immunization against an expression product have been described in the art. The techniques, termed "nucleic acid immunization" herein, have been shown to elicit both humoral and cell-mediated immune responses. For example, sera from mice immunized with a DNA construct encoding the 15 envelope glycoprotein, gpl60, were shown to react with recombinant gpl60 in immunoassays, and lymphocytes from the injected mice were shown to proliferate in response to recombinant gpI20. Wang et al, (1993) Proc. Nad, Acad. Sci USA 90:4156-4160. Similarly, mice immumzed with a human growth hormone (hGH) gene demonstrated an antibody-based immune response. Tang et 20 al. (1992) Nature 356:152-154. Intramuscular injection of DNA encoding influenza nucleoprotein driven by a mammalian promoter has been shown to elicit a CDS+ CTL response that can protect mice against subsequent lethal challenge with virus. Ulmer et al. (1993) Science 259:1745-1749. Immunohistochemical studies of the injection site revealed that the DNA was 25 taken up by myeloblasts, and cvtoplasmic production of viral protein could be demonsurated for at least 6 months. Summary of the Invention It is a primarv object of the invention to provide a polynucleotide adjuvant 30 composition containing furst and second nucleic acid sequences, wherein the first nucleic acid secuence is a trucated A subunit coding region obtained or derived 1 from a bacterial ADP-ribosylating exotoxin, and the second nucleic acid sequence is a truncated B subunit coding region obtained or derived from a bacterial ADP- ribosylating exotoxin. Each of the truncated subunit coding regions has a 5' deletion and encodes a subunit peptide not having an amino terminal bacterial 5 signal peptide. The first and second nucleic acid sequences maybe present in the same or in different nucleic acid constructs. The troncated subunit cod'lng regions may be obtained or derived from the same bacterial ADP-ribosylating exotoxin and. in cer.ain preferred embodiments, the bacterial .ADP-ribosylating exotoxin is a 10 cholera toxin (CT) or an E, coli heat labile enterotoxin (LT). In addition, at least W one of the truncated subunit coding regions maybe genetically modified to detoxify the subunit peptide encoded thereby, for example wherem the tnmcated A submiit coding region has been genetically modified to disrupt or inacrivate -ADP-ribosyl transferase activity in the subunit peptide expression product. 15 It is also a primary object of the invention to provide a polynucleoride adjuvant composition contaimng first and second nucleic acid sequences, wherein the first nucleic acid sequence is a modified A subunit coding region obtained or derived from a bacterial .ADP-ribosylating exotoxin, and the second nucleic acid sequence is a B subunit coding region obtained or derived from a bacterial AD?- 20 ribosylating exotoxin. The modified A subunit coding region and said B subunit coding region each encode a mature subunit peptide, and the modified A subunit coding region has been genetically modified so as to delete a C-terminaJ KDEL or RDEL motif in the subunit peptide encoded thereby. As above, the first and second nucleic acid sequences may be present in 25 the same or in different nucleic acid constructs. The truncated subunit coding regions may be obtained or derived from the same bacterial .ADP-ribosylaring exoioxin and, in certain preferred embodiments, the bac:erial .ADP-ribosylating exotoxin is a cholera toxin (CT) or an E. ccAi heat labile eriterotoxin ('LT). In addition, at least one of the truncated subunit coding regions may be genetically modified to detoxify the subunit peptide encoded therby for example wherem the truncated A subunit coding region has been genetically modified :o disrupt or y inactivate ADP-ribosyl transferase activity in the subnnit peptide expression product. In certain aspects of the invention, the above compositions can be provided in particulate form, for example wherein the compositions are 5 particulates suitable for deliver/ from a particle delivery device. In this regard, the present compositions may be coated onto the same or a different core carrier particle and thus suitable for delivery using a particle-mediated transfection technique. Preferred core carrier particies will have an average diameter of about 0.1 to 10 µm, and may comprise a metal such as gold. Accordingly, it is a still 10 further object of the invention to provide a particle delivery device loaded with 4 (e.g., containing) a particulate composition as defined herein. It is also a primary object of the invention.to provide for the use of a composition containing a first and second nucleic acid sequence, where each sequence includes a coding region for a subunit from a bacterial ADP- 15 ribosylating exotoxin in the manufacmre of a medicament for enhancing an immime response in a vertebrate subject against an antigen of interest in the said subject. The antigen of interest and the composition are administered to the subject such that the toxin subunits encoded by the first and second nucleic acid sequences are expressed in an amount sufncient to elicit an enhanced immime 20 response against the antigen. The first nucleic acid sequence contains a truncated A subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin, and the second nucleic acid sequence contains a turncated B subunit ^"^ coding region obtained or derived from a bacterial ADP-ribosylating exotoxin, however with the proviso that each of the truncated subunit coding regions has a 25 5' deletion and encodes a subunit peptide not having an amino terminal bacterial signal peptide. It is a related primary object of the invention to provide a method for enhancing an immume resnonse asainst an antigen of interest in a subiect. The method generally entails: (a) administering the antigen of interest to the subject; 30 (b) providing an adjuvant composition compnsing first and second nucleic acid sequences, wherein the first nucleic acid sequence is a truncated A subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin, and the second nucleic acid sequence is a truncated B subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin; and (c) administering the adjuvant composition to the subject, whereby upon introduction to tlie subject, 5 the first and second nucleic acid sequences are expressed to provide subunit peptides in an amount sufficient to elicit an enhanced immune response agahist the antigen of interest. The subunit coding regions are trancated in that each coding region has a 5' deletion and encodes a subunit ::eoiide not having an amino terminal bacterial signal peptide. 10 It is yet a farther primary object of the invention to provide for the use of a composition comprising a first and second nucleic acid sequence, where each sequence includes a coding region for a subunit fi-om a bacterial ADP-ribosylating exotoxin in the m.anufacture of a medicament for enhancing an immime response in a vensbrate subject against an antigen of interest in the said 15 subject. The antigen of interest and the composition are administered to the subject such that the toxin subunits encoded by the first and second nucleic acid sequences are expressed in an amcimt sufrlcient to ehcit an erJianced irmnune 20 25 second nucleic acid sequence is a B subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin; and (c) administering the adjuvant composition to the subject, whereby upon introduction to the subject, the first and second nucleic acid sequences are expressed to provide subunit peptides in an 5 amount sufficient to elicit an enhanced immune response against the antigen of interest. The subunit coding regions are modified in that each encodes a mature subunit peptide, but the A subunit coding region has been genetically modified so as :o delete a C-:erminal IGDEL or RDEL motif in the subunit peptide encoded thereby. 10 In the uses and methods of the invention, administering the adjuvant comnositions entails transfecting cells of the subject with a polynucleotide adjuvant composirion according to the present invention. Expression cassettes and/or vectors containing the nucleic acid molecules of the present invention can be used to transfect the cells, and transfection is carried out under conditions that 15 pennit expression of the subunit peptides within the subject. The method may farther entail one or more steps of administering at least one secondary composition to the subject. The transfection procedure carried out during the immunization can be conducted either in vivo, or ex vivo (e.g., to obtain transfected cells which are 20 subsequently introduced into the subject prior to carrying out the secondary immunizadon step). When in vivo transfection is used, the nucleic acid molecules can be administered to the subject by way of intramuscular or intradermal injection of plasmid DNA or, preferably, administered to the subject using a particle-mediated delivery technique. Vaccine compositions (containing 25 the antigen of interest) can be provided in the form of any suitable vaccine composition, for example, in the form of a peptide subunit composition, in the form, of a nucleic acid vaccine composition, or in the form of a whole or split virus influenza vaccine composition. In certain methods, the antigen of interest and the adjuvant composition 30 are administered to the same site in the subject. For example, the adjuvant composition and the antigen of interest can be administred concurrently (e.g., 5 provided in a single vaccine composition). In certain preferred embodiments, the adjuvant and, optionally the antigen of interest, is administered in particulate form, for example wherein the adjuvant composition has been coated onto a core carrier particle and delivered to the subject using a particle-mediated deliver/ 5 technique. In these methods, administration of the polyr.uclectide adjuvant compositions of the present invention preferably results in an augmented cellular immune response against the co-administered antigen of interest. Such an enhanced immune response may be generally characterized by increased titers of 10 inrerferon-producing OD4 and/or CDS* T lymphocytes, increased antigen- specific cytotoxic T lymphocyte (CTL) activity, and a T helper 1-like immmune response (Thl) against the antigen of interest (characterized by increased antigen-specific antibody titers of the subclasses typically associated with cellular (hCMV) immediate early promoter and associated intron A sequence, and the coding sequence for the signal peptide of human tissue plasminogen activator co allow for secretion from mammalian cells of the tnmcated CTA expression product. The figure further contains the complete nucleic acid sequence (SEQ ID 5 NO: 1) for the pPJV2002 plasmid. Figure 5 is a restriction map and functional map of plasmid pPJV2005 that contains a truncated coding sequence for an LT subunit B (LTB) peptide, v/herein the plasmid further contains the hCMV immediate early promoter and associated inrron A sequence, and the coding sequence for the signal peptide of 5 human tissue plasminogen activator to allow for secretion from mammalian cells Formulation #2 containing the EmpVec (pWRG7054) and the pCLA-EnvT plasmid ("gpl20"); Formulation #3 containing the EmpVec (pWRG7054) combined with the pPJV2002 and pPW2003 adjuvant vectors ("CTA/B"); Formulation #4 containing :he pCLVEnvT plasmid ("gpl20") combined with the Definitioas 10 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood bv one of ordinary skill in the an to which the invention pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. 15 In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. The term 'adjuvant" intends any material or composition capable of specifically or non-specifically altering, enhancing, directing, redirecting, potentiating or initiating an antigen-specific immune response. Thus, 20 coadministration of an adjuvant with an antigen may result in a lower dose or fewer doses of antigen being necessary to achieve a desired immune response in the subject to which the antigen is administered, or coadmiristration may result in f ... a qualitatively and'or quantitatively different immune response in the subject. 25 30 ^■f^^ll^ An "adjuvant composition" intends any phannaceutical composition containing an adjuvant Adjuvant compositions can be delivered in the methods of the invention while in any suitable phannaceutical fonn, for example, as a liquid, powder, cream, lotion, emulsion, gel or the like. However, preferred 5 adjuvant compositions will be in particulate form. It is intended, although not always explicitly stated, that molecules havmg similar biological activity as witd-type or purified peptide or chemical adjuvants. For purposes of the present invention, a 'humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T-lymphocytes and/'cr ocher white blood ceils. The term 'nucleic acid immunization" is used herein to refer to the 5 iniroducrion of a nucleic acid molecule encoding one or more selected antigens into a host cell for the in vivo expression of the antigen or antigens. The term also encompasses introduction of a nucleic acid molecule encoding one or more selected adjuvants into a host cell for the in vnvo expression of the adjuvanta or adjuvants. The nucleic acid molecule can be introduced directly into the a central processing unit and used for bioinfonnatics applications such as functional genomics and homology searching. A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). 5 Typically. 'Vector constructy' "expression vector," and "gene transfer vector." mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. A 'plasmid' is a vector in the form of an extrachromosomal genetic element. 10 A nucleic acid sequence which "encodes" a selected adjuvant and/or effecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the 5 nucleic acid sequence and the promoter sequence can still be considered 'operabiy linked" to the coding sequence. 'Recombinant" is used herein to describe a nucleic acid molecule (polynucleotide) of genomic, cDNA, semisyathetic, or syntheric or.gin whihc by virtue of its origin or manipulation is not associated with all or a pordon of the 10 polynucleotide with which it is associated in nature and/or is linked to a polynucleotide other than that to which it is linked in nature. Two nucieic acid sequences which are contained within a single recombinanr nucleic acid molecule are 'heterologous" relative to each other when they are not normally associated with each other in nature. whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in 5 Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Davhoff. Atlas of Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with singie-stranded-specific nucleasels), and size determination of the digested fragmens. Two DNA, or two 5 polypeptide sequences are 'substantially homologous" to each other when the w^^- By "core carrier" is meant a carrier on which a guest nucleic acid (e.g., DNA, RSA) is coated in order to impart a denned particle size as well as a sufficiently high density to achieve the momentum required for cell membrane penetration, such that the guest molecule can be delivered using panicle-mediated that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodimenis of the invention only, and is not intended to be limiting. 5 The present invention provides novel compositions containing nucleic acid sequences, wherein a first sequence in the composition is a coding sequence lor an A subunit obtained or derived from an ADP-ribosylating baccerial toxin, and a second sequence in the composition is a coding sequence for a B subunit obtained or derived from an .ADP-ribosyiating bacterial toxin. The fust and 10 second sequences are useful in immunization methods wherein they are delivered extremely similar molecules, and are at least about 70-80% homologous at the amino acid level. The CT and LT toxins are hexamers, composed of a single molecule of an A subunit surrounded by a doughnut-shaped ring composed of 5 molecules of the 5 3 subunit. The A subunit possesses an ADP-ribosylase activity resulting in G protein modifications that lead to cAMP and protein Idnase A upregulation following internlization of the A subunit in a manmrialian cell. The A subunit can be nicked by exogenous proteases yielding the Al and A2 fragments linlced via a single disulfide bridge. The A subunit of CT contains a C-terminal KDEL triggering activity, respectively. Regarding the B subunit of LT in particular, binding to the GMl receptor on B cells results in polyclonal activation, occurs in the absence of signiiicant proliferation, and involves the upregulation c: a number of important molecules such as MHC class II, B7, CD40, ICAM-1 and 5 IL2-Ra. For T cells, addition of CT or LT holotoxins or recombinant 3 subunits is a coding region for a modified, mature A subunit peptide obtained or derived from an ADP-ribosylating exotoxin, and (b) the second nucleic acid sequence is a coding region for a mature B subunit peptide obtained or derived from an ADP- ribosylating exotoxin. The first nucleic acid sequence contains a coding region 5 that provides for a "modified" A subunit peptide in that the said coding region has been genetically altered so as to delete a four amino acid residue C-termnai KDEL or RDZL motif normally found in the encoded subumil peotide. In still a further embodiment, a composition is provided which includes aspects of the invention, one or both of the toxin subunit peptide coding regions can be further genetically modified to detoxify rhe subunit peptide(s) encoded thereby. For example, genetically altered toxin mutants which have disrupted A\DP-ribcsylat:ng acnviry, tripsin cleavage site mutations, or disrapted birding 5 activity are known in the art. See, e.g., Burnetts at al. (1994) 'Recombinant n"iicrobial ADP-ribosylating toxins of Bordetella peniisis. Vibrio ckolerae. ana enterotoxigenic Escherichia coli: structure, function and toxoid vaccine prime the replication reaction. The product of the first round of replication is itself a template for subsequent replication, thus repeated successive cycles of replication result in geometric amplification of the DNA fragment delimited by the primer pair used, 5 Once the relevant sequences for the ADP-ribosylating exotoxin subunits of mterest have been obtained, they can be linked together to provide one or mors conuguous nucleic acid molecules using standard cloning or molecular biology techniques. More particularly, after sequence information for the selected toxin subunit combination has been obtained, the coding sequences can be combined w vectors which include control sequences operably linked to the inserted sequence or sequences, thus providing expression cassettes that allow for expression of the toxin subunit peptides in vivo in a targeted subject species. exotoxin subunit peptide coding sequences of interest, the expression cassettes can be provided in a suitable transfection vector such as a DNA plasmid vector or a viral vector for subsequent administration to a subject.. In this regard, the polynuclectide compositions of the invention can be used as standalone adjuvant lower or fewer doses of the antigen are required to generate an eScient immune response. As used herein, the term 'cc-administered' such as when an AD?- nbosylating exotoxin subunit encoding adjuvant composition according :c the 5 present invention is "co-administered' with an antigen of Interest (e.g., a vaccine w Vh^^g^' ccmmon allergens can also be obtained from molds or fung] such as Altemaria, Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor and thermophilic actinomycetes; penicillin and tetracycline are common ancibiotic Vjl^^^ associated with colorectal cancer, gplOO or MARTI antigens associated with melanoma, and the PSA antigen associated with prostate cancer. The p53 gene sequence is know-n (see e.g., Harris et al. -(1986) MoL Call Biol. 6:4650-4656) ' and is deposited with GenBank under Accession No. Ml4694. Thus, the 5 adjuvant composirions of the present invention can be used to carry out immunotherapeutic methods for treating cervical, breast, colorectal, prostate, lung cancers and melanomas. / isolate DNA. Polyiucleodde sequences can also be produced synthetically, rather than cloned. w 1^ which have an integrated defective provirus (the "helper") that expresses all of the genes of the virus but cannot package its own genome due to a deletion of the packaging signal, known as the psi sequence. Thus, the cell line produces empty- viral shells. Producer lines can be derived from the packaging lines which, in 5 addition to the helper, contain a viral vector which includes sequences required in A-\V expression vectors are constructed using kno'vvn techniques to at least provide as operatively linked components in the direction of transcription, 20 control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region. The control elements are selected to be functional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded (5' and 3') with functional AAVITR sequences. Suitable AAV constructs can be designed using techniques well Conventional Pharmaceutical Preparations Formulation of a preparation comprising the polynucleotide molecules of 5 the present invention, with or without addition of an antigen composition, can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the ordinarily' skilled artisan. For example, compositions containing one or more suitable vectors can be combined with one or more pharmaceutically acceptable excipients or vehicles to 10 provide a hquid preparation. auxiliary substances is available in REMINGTON'S PKL\RMACEUTIC.AI SCIENCES (IVIack Pub. Co., N.J. 1991), incorporated herein by reference. Certain facilitators of nucleic acid uptake and'or expression ('transfection w polylysine, polyarginine, polyomithine, spermine, spermidine, as well as conj agates of these molecules. The formulated compositions will thus typically include one of more poiynucleotide molecule (e.g., plasraid vector) containing the coding sequences dextran-mediated transfection, calcium phosphate precipitation, electroporation, and direct microinjection of into nuclei). However, deliver/will most typically be via conventional needle aiid svrmge for the liquid compositions and for liquid 1)^ prepared and suitably purified, the above-described plasmid vector constructs can be coated onto carrier particles {e.g., core carriers) using a variety' of techniques known in the art. Carrier particles are selected from materials which have a suitable density in the range of panicle sizes typically used for intracellular 5 delivery from an appropriate particle delivery' device. The optimum carrier W|^^^ 0 15 Following their formation, core carrier particles coated with the nucleic acid preparations of the present invention, alone or in combination with e.g., antigen preparations, are delivered to a subject using particle-mediated deliver}' techniques. The coated particles are administered to the subject to be treated in a maimer compatible with the dosage formulation, and in an amount that will be effective to bring about the desired adjuvant effect'immune response. The amount of the composition to be delivered which, in the case of nucleic acid w acid, rartaric acid, glycine, high molecular weight pclyeihyiene glycols i7EGs), -0 and combination thereof. A thorough discussion of pharmaceuticaily acceptable sxcipients, carriers, stabilizers and other auxiliary substances is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991), irxorporated herein by referepxe. packaged prior to use, can comprise a hermetically sealed container enclosing a suitable amount of the particles comprising a suitable nucleic acid construct and/or a selected antigen (e.g., to provide a multicomponent vaccine composition). The particulate compositions can be packaged as a sterile 5 formulation, and the hermetically sealed container can thus be desiened to preserve sterility of the formulation until use in the methods of :he invention. If desired, the containers can be adapted for direct use in a panicle delivery device. Such containers can take the form of capsules, foil pouches, sachets, cassertes, and the like. Appropriate panicle delivery devices (e.g., needleless s>Tinces) are 10 described herein. w surface, and the densicy and kinematic viscosity of the targeted skin tissue. In this regard, optimal particle densities for use in needleless injection generally range ber.veen about 0.1 and 25 g./cm3. preferably between about 0.9 and 1.5 g.'cm3 and h w . r/ Enhancing Immune Responses 5 In another embodiment of the invention, a method for enhancing an immune response against a co-administered antigen of interest is provided. In essence, the method entails (a) administering an antigen of interest to a subject. additionai or arciliary" components to the subjec:. For rxample, a secondary 30 vaccine ccmposinon can be administered, wherein the secondary' composition can comprise a nucleic acid vaccine, or the secondan' vaccine composition can 1"* Experimental Below are examples of specific embodiments for canning out the present 20 invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in anyway. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.). but some experimental error and deviation should, of course, be allowed for. 4S In order tc facilitate insertion into an expression vector, Primers 1 and 2 contain extra sequences at their 5' ends (outside the region of homology to the CTA coding sequence) which include recognition sites for Xhel and BamHI, 20 respectively. Primers 1 and 2 were designed to lead to PCR generation of a fragment of the A subunit coding sequence starting a: nucleotide position 16- and ending at nucleotide position S86 (GenBank Accession PD30O53). This region Primer 4: 5'— CCT GGA TCC TTA ATT TGC CAT ACT AAT TGC —3' (SEQ ID NO: 10). In order to facilitate insertion-into an expression vector, Primers 3 and 4 5 contain extra sequences at their 5' ends (outside the region of homology to the CTB coding sequence) which include recognition sites for Nhel and BamKL respectively. Primers 3 and 4 were designed lo lead to PCR generation of a fraement of the B subunit coding sequence starring at nucieotide position 946 and ending at nucleotide position 1257 (GenBank Accession #D30053). This region i G encompasses the entire coding sequence for the mature subunit B peptide but k does not include the sequence encoding the bacterial signal peptide found at the ('minus KDEL). respectively. The parental cloning vector pWRG7054 contains the human cvtomegalovirus immediate early promoter with the associated intrcn A sequence. In addition, the coding sequence for the signal peptide of human tissue plasminogen acdvator is included in pWRG7054 to allow for the secretion ■k^^ respectively. Primers 6 and 7 were designed to lead to PCR generation of a fragment of the A snbunit coding sequence starting at nucleotide position 145 and More panicularly, the sequence from the RNA segment #7 (rha: encodes 30 the M2 protein) of influenza virus strain A'Xagoshima 10/95 (H3N2) was used as a model to design PCR primers to facilitate cloning of the manure M2 coding 53 sequence from AySydney/5/97 (H3N2). The A/Kagoshima sequence was used for primer design since the sequence of RNA segment 7 of A'Sydney has not yet been determined. The high degree of conservation among M2 sequences was after which. 0.5µl of reverse transcriptase from the kit was added. The reaction was incubated at 37°C for 15 minutes to complete the reverse transcription step. The PCR reaction step was completed by addition of the following 4,0 \xg of DNA per mg of gold, and a single batch contained 26 mg of gold. DNA was precipitated onto the gold particles by addition of a 1/10 volume of 10% CaCl2 during continuous agitation of the tube on a rotary/mixer. DNA-gold complexes were washed three times with absolute ethanol, and then injected into Formulation #5: pM2-FL DNA vector and pPJV2002 coprecipitated onto single batch of gold (2 |ig pM2-FL DNA per mg gold, and 1 µg of pPJV2002 DNA per mg gold), 0.5 mg gold per cartridge; and V washed three times, and a streptavidin-horse radish peroxidase conjugate (Southern Biotechnology) diluted to 1 :8000 in PBS/0.1 % Tween-20 was added and the plate incubated for 1 hour at room temperature. Following three J o As' can be seen, al! experimental groups immunized with a formulanon containing cne or more of the CT-encading adjuvant vectors (Fcnnulations #2-6') Example 4 Enhancement of .Antigen-Specific Cellular Responses to a DNA Vaccine Encoding the Hepatitis B Surface Antigen (HBsAg) Using Adjuvant Plasmid Vectors Encoding the CT-A and CT-B Subunit Peptides. A Hepatitis B surface antigen (HBsAg) vector plasmid was construacted as follows. To generate the HbsAg coding region, the p.AM6 constract (obtained from the Amxerican Type Culture Collection "ATCC'} with pWRG712S (1 jig DRV0.5 mg gold per delivery). Mice (4 per expenmental group) were immunized and boosted at 4 weeks, then sacrificed at 2 10% fetal bovine serum (FBS) for 1-2 hr at RT. The medium was removed and spleen cells dispensed into the wells with a total of 1x106 cells per well. For wells in which less than 1xlO6 cells from immunized animals was added cells from naive animals were used, to bring the total to 1x106 Cells were incubated 5 overnight in a tissue culture incubator in the presence of the peptide as described above. The plates were then washed 2 time witli PBS and 1 time with distilled The cellular immune responses measured in the two groupS OF mace indicate a significant erhancement of cellular responses by the CT adjuvant 20 vectors. More particularly, the results of the CDS-specific IFN-v ELISA assay are depicted below in Table 3. 63 20. adjuvanted formulation is indicative of a greater amount of IFY-ý secreted by these cells in response to antigen. Cel]s from naive mice did not yield a iL measurable OD value in the IFN-ý ELISA. The results of the CD8-specific IFN'-ý EIJSPOT assay are depicted below in Table 4. ■^ ■:: ^'.) 64 The.above data demonstrate that the novel adjuvant compositions ox the present invention have a potent ability to enhance the cellular immune responses 20 to a coadministered antigen, in this case a HBsAg expressed from a DNA vaccine. 65 Example 5 Enhancement of Humoral and Cellular Immune Responses to HTV-1 gpl20 Antigen Using Simultaneous Delivers of a gpl20 .Antigen Vector and CT.VCTB Adjuvant Vectors. 5 A plasmid vector endoding HTV-l gpl20 was constructed as follows. The vector was constructed starting with a Bluescript (Stxatagene, La Jolla, CA) plasmid backbone, the human cytomegalovirus (hCMTV) immediate early product with the truncation point lying 12S amino acid residues downstream of the gp 120/gp41 processing site. A second plasmid vector encoding HIV-l rev. termed "pC-rev"' herein. pWRG7054 DNA per mg gold, and 1 fig each of pPJV2002 and pPJV2003 DNA per mg gold), 0.5 mg gold per cartridge; V Serum antibody responses to the HIV gp 120 antigen were tested using an ELISA assay on specimens collected at week 5 and week 6.5 (post-prime and post-boost, respectively). For the ELISA, Costar hih binding ELAplates were stimulated in medium alone (negative control), "or in medium with 1 µg/ml of a HIV gpl20 peptide having the following sequence: RIQRGPGRAPVITGK (SEQ ID NO: 21). Following a 24 hour incubation at 37°C in 5% CO2, the plates were km anrigen in an animal model. In addition, rhe enhanced IFX-y production seen iu these studies indicates that use of the CT adjuvant vector combinations of the present invention provides a robust Thl-like immune response m :he in-iunized animals. 71 Example 6 Enhancement of .Antibody Responses to Hepatitis B Core and Surface Antigens Using Simultaneous Delivery of a Vector Encoding HBcAs and HBsAe with CTA/CTB Adjuvant Vectors. polyA sequence (bGHpA) were each obtained from the JW4303 vector construct (gift of Dr. Harriet Robinson, University of Massachusetts) and inserted into a plasmid backbone. The resultant construct was cut with 'Nhel filled with polvmerase and then cut with BamHI to generate a vector fragment Formulation #1: Control, the HBcAg/IIBsAg vector (pWRG7193) alone, 2 µg DNA per mg gold, 0.5 mg gold per cartridge; and 2(: control group (Formulation =1) and adjuvant test group (Formulation;=21 were 2S5 and 662 mIUVml, respectively, demonstrating the ability of the present adjuvant plasmids (pPJV2006 and pPJV2(303) to augment immune responses :o an antigen er.coded by a separate vector. Example 7 Enhancement of Cellular Thl-Like Immune Response to a DNA Vaccine Usine Plasmid Vectors Encoding CT or LT Subunits, 5 The pJvI2-FL DNA vaccine vector encoding the M2 protein of influenza A virus was employed to test the adjuvant effects of the pPJV2002. pPJV2003, ppJV'2004, pPJV2005, pPJV'2006 and pPA'2007 adjuvant vectors in the context cf particle-mediated DNA vaccination. Particle-mediated DNA vaccination was performed by precipitating the M2 DNA vaccine vector, either v/ith our without \f0 Formulation #2: pM2-FL DNA vector combined with the CTA and CTB (pPJV2002 and pPJV2003) DNA vectors prior to precipitation onto a single batch. of gold, 2.1 [ig total DNA per mg gold (1.0 µg of each DNA adjuvant vecicr per mg gold, 0.1 µg pM2-FL), 0.5 mg gold per cartridge; of gold, 2.1 µg total DNA per mg gold (1.0 µg of each DNA adjuvant vector per mg gold, 0.1 |ig pM2-FL), 0.5 mg gold per cartridge; PowderJect® XR-1 particle delivery device (Powder: ect Vaccines Inc., Madison. \VI) at a helium pressure of 400 p.s.i,. Serum samples were collected two weeks following the second or booster immunization. Individual serum samples were assayed for M2-3peciiic antibody 5 responses for both IgGl and IgG2a subclasses usmg the ELISA assay of Example 3 above for the determina::on of total IgG titer, except that a secondary .antibody conjugate specific for eidger IgGl or IgG2a was employed. The goat anti-mouse As can be seen with reference to Table 5, addition of either of the A or B subunits, or the various combinations of A and B subunits to the M2 DNA vaccine formulation resulted in sienificant reductions in the IgGl-to-l2:G2a ratio v/ith 1.5 mM MgCL (Promega Corp., Madison, WT); 0.400 [iM of each primer; 200 [iM or each cLNTP (USB Inc., Cleveland, OH); 2,5 µg Taq polymerass (Promega Corp., Madison, WD; i.O ng template DNA; water to 100 µl; and a and EcoR1 to generate a core antigen insertion fragment; the modified pWRGVUS plasmid was cut with Pstl and Mfel to generate a vector fragment, and the core antigen insertion fragment was ligated into the vector fragment, resulting in the dual surface/core antigen plasmid construct. Fonnulution #3: ("CT w/o TPA") 1 µg dual surface/core antigen DNA plasmid vector, 0.5 jig CTA w/o TPA DNA plasmid vector, 0.5 [ig CTB w/'o TPA DNA plasmid vector; Formulation #12: ("LTB") 1µg dual surface/core antigen DNA plasmid vector, 1 µg pJV2O05 (LTB) DNA plasmid vector; and w Finally, the clearlv observable difference in adjuvant effect between the secreted (Formulations #2 and =8) and the non-secreted ('Formulaticns -3 and -9) heins establish that the observed adjuvant effects are not due :o CvG monts within the adjuvant vectors since the signai-ccntaining and non signai-ccntaining 30 vectors do not have any difference in bacterial DNA (CpG) content yet exhibit S5 significant differences in their ability to augment surface antigen-specific IFN-7 responses. In a second study, the above-described DNA vaccine formulations were adminisiered to eight groups of mice using the PowderJect® XR-1 particle 5 delivery device (PowderJect Vaccines Inc., Madison, UT). Each experimental group contained 5 animals, and each ammal received two immunizations with the combinations of the present adjuvant plasmid vectors to provide vaccine compositions. After immunization, the immunized animals were challenged with HSV-2 virus. and the protective effect of the various vaccine compositions was determined. 1 from the HSV-2 genome, for example the genomic region spanning from approximately nucleotide 114589 to 134980 of the HSV-2 genome, or anEcoRI fragment that spans nucleotides 110931 to 139697 of the HSV-2 genome. The sequence of the HSV-2 genome is available form published sources, for example 5 the sequence deposited with GenBank un der Accession Number NC_001798. In the study, the above-described DNA vaccine fcrmulatains were administered to five different groups of mice using the PowderJect XR-1 15 panicle delivery device (PowderJect Vaccines Inc., Madison, WT). Each experimental group contained 12 animals, and each animal received two immunizations (single shot applied to the abdomen) with the respective formulation with a four vveelc restmg period beUveen inmiunizaiions. A sixth group of mice was established as a negative (naivej control, and did not receive 20 any vaccinations. 4 mice from each group were sacked 2 weeks arier the second immunization and used for IFN-y ELISPOT assays (data not shown). Two v/eeks post second immunization, all remaining mice (8/group) were challenged with 1X10° FPU of HSV-2 virus, strain MS, via intra-nasal insullation. The survival graph depicting the results of the challenge study is 25 depicted in Figure 14. As can be seen, 100% of the naive animals succumbed within 4 days post challenge. The naive animals are depicted on the graph by the curve. In addition, 100% of the animals receiving the ICP27 antigen plasn:d vector alone (Formulation #1) died within 7 days post challenge. The animals receiving Formulation #1 are depicted on the graph by the curve. In marked 30 contrast, the 25% (2/S) of the anim.als receiving the ICP27 piasnrid adjuvanicd with the low dose CT (Formulation 3) were protected tcm the viral challenge, 89 and 38% (3/8) of the animals receiving the ICP27 adjuvanted with the high dose CT (Formulation #2) were protected from the viral challenge. The animals receiving Formulation #3 are depicted on the graph by the (■) curve. The animals receiving Formulation #2 are depicted on the graph by the (♦) cruve. 5 Finally, both the low dose LT-adjuvanted (Formulation =5) and the high dose LT- adjuvanted (Formulation #4) ICP27 vaccine provided complete (100%) protection in the immunized animals. The animals receiving Formulation #5 are depicted on the graph by the curve. The animals receving Formulation =4 are depicted on the graph by the (O) cur\'e. 10 Accordingly, novel polynucleotide adjuvant molecules, compositions comprising those adjuvant molecules, and conventional and nucleic acid 15 immunization techniques have been described. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims. rit and the scope of the invention as defined by the appended claims. Claims What iS claimed is: 5 1.. A composition comprising first and second nucleic acid sequences, wherein said first nucleic acid sequence is a trucated A subunit coding region obtained or derived from a bacterial .ADP-ribosylating exotoxin. and said second nucleic acid sequence; is a truncated B subunic coding region obtained or derived from a bacterial .ADP-ribosylaiing exotoxin, with the proviso 10 that each of said truncated subunit coding regions has a 5' deletion and encodes a subunit peptide not having an amino terminal bacterial signal peptide. 2.. The composition of claim 1, wherein said first and second nucleic acid sequences are present in a single nucleic acid construe:. 15 3. The composition of claim 2, wherein said nucleic acid construct is a plasmid vector 4. The composition of claim 2, wherein the first and second nucleic 20 acid sequences are operably linked to a transcriptional control element. 5. The composition of claim 4, vvherein said transcriptional conrrol element is a heterologous promoter. 25 6. The composition of claim I wherein said first and second nucleic acid sequences are present in separate nucleic acid constructs. 7. The ccmposition of claim 6, vvherein said separate nucleic acid constructs are piasmid vectors. 91 8. The composition of claim 1, wherein the tmncated subunit coding regions are obtained or derived from the same bacterial .ADP-ribosylating exotoxin. 5 9. The composition of claim 8, wherein said bacterial ADP- ribosylaring exotoxin is a cholera toxin (CT). 10. The composition of claim S, wherein said bacterial ADP- ribosyating exotoxin is an E. coli heat labile enterotoxin (LT). 10 11. The composition of claim 1, wherein at least one of the truncated subunit coding regions has been genetically modified to detoxify the subunit peptide encoded thereby. 15 12. The composition of claim 11, wherein :he truncated A subunit coding region has been genetically modified to disrjp: or macrivate .ADP-ribosyi transferase activity in the subunit peptide encoded thereby. 13. The composition of claim 1, whersin the truncated A subunit 20 cocing reeion has been further senetically modified so as to delete a C-terminal KDEL or RDEL motif in the subunit peptide encoded thereby. 14. The composition of claim 1 further comprising an antigen of interest. 15. The composirion of claim 14, wherein said antigen is fron: a bacterial. \'iral or parasitic pathogen. 15. The composition of claim 1, further compriding a third nucleic 92 17. The composirion of claim 16, vvherein said antigen is fron a bacterial, vixal or parasitic pathogen. w wo 03/004055 PCT/USOl/43151 27. The composition of claim 25, wherein the core carrier particle comprises a metal. 94 35. The composition of claim 33, wherein the first and second nucleic acid sequences are operably linked to a transcnptional control element. 44. The composition of claim 32, wherein the modified A subunit coding region and the B subunit coding region have each been truncated by a 5' deietion whereby each of said truncated subunit coding regions encodes a subunit peptide not having an amino terminal bacterial signal peptide. 5 45. The composition of claim 32 furher ccmprising an antigen of interest. 46. The composition of claim 45, wherein said antigen is from a 10 bacterial, viral or parasitic pathogen. 47. The composition of claim 32 further comprising a third nucleic acid sequence that encodes an antigen of interest. If 4S. The composition of claim 47. wherein said antigen is from a bacteriaL viral or parasitic pathogen. 49. The composition of claim 47. wherein said third nucleic acid sequence is present in a nucleic acid construct that does not contain said first or 20 said second nucleic acid sequence. 50. The composition of claim 49, wherein the nucleic acid construe: W containing the third nucleic acid sequence is a plasmid vector. 25 51. The composition of claim 47, wherein said third nucleic acid sequence is present in a nucleic acid construct that also contains at least one of said first or said second nucleic acid sequence. 52. The composition of claim 51. wherein the nucleic acid construct 36 containing the third nucluic acid sequence is a pasmid vector. 96 WO 03/004055 PCT/USOl/431^1 53. The composition of claim 32, wherein said composition is in a particulate form. 54. The composition of claim 53, wherein said paniculate ccmposiiion 5 is suitable for transdermal deliver/ via a particle delivery device. 55. The composition of claim 52 further comprising a pharmaceutically accepable vehicle or excinienc. 110 56. A com.position according to claim 55. wherein the first and second nucleic acid sequences are coated onto a core carrier panicle. 57. The composition of claim 56, wherein the core carrier panicle has an average diameter of about 0.1 to about 10µm. 15 58. The composition of claim 56, wherein the core canier pvirticle comprises a metal. 59. The composition of claim 5S, wherein the metal is gold. 20 60. A panicle delivery device, wherein said device is loaded with a paniculate vaccine composition as defined in claim. 54. 61. The composition of claim 32 further comprising a transfecrion 25 facilitating agent. 62. The composition of claim 61, wherein the n-ansfection facihtating agent is a liposome. 3} 63. Use of a composition com.pnsmg a first and second nucleic acid sequence, each said sequence including a coding region for a suburjt from a 97 NVO 03/004055 PCT/TSOl/43151 bacterial ADP-ribosylating exotoxin in the manufacture of a medicament for enhancing an immune response in a vertebrate subject against an antigen of interest in the said subject by administering the antigen of interest and the said composition to the subject whereby the toxin subunits encoded by the nrsi and 5 second nucleic,acid sequences are expressed in an amount sufficient to elicit an enhanced immune response against the antigen, wherein said nrst nucleic acid sequence comains a truncated A subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin, and said second nucleic acid sequence contains a truncated B subunit coding region obtained or derived from a bactenal 10 .ADP-ribosylating exotoxin, with the proviso that each of said truncated subunix coding regions has a 5' deletion and encodes a subunit peptide not having an amino terminal bacterial signal peptide. 64. Use according to claim 63, wherein the antigen of interest and the 15 composition are administered to the same site in the subject. 65. Use according to claim 63, wherein the antigen of interest and the composition are administered concurrently. 20 66. Use according to claim 65, wherein the antigen of interest and the composition are combined to provide a single vaccine composition. 67. Use according to claim 63, wherein the antigen of interest is from a bacterial, viral or parasitic pathogen. 25 6S. Use according to claim 67, wherein a third nucleic acid sequence is admimsiered :o the subject and the third sequence encodes said antigen of intrest. ?'.' 69. Use according to claim 63, wherein :he first and second nucleic acid sequences are administered to the subject in paniculate form. 98 wo 03/004055 PCT/US01/4315I 70. Use according to claim 69, wherein the composirion containing the first and second nucleic acid sequences are coated onto a core carrier panicle and administered to the subject using a particle-mediated delivery technique. 5 71. Use accordine to claim 63, v/herein the subiect is human. 72. Use of a composition comprising a first and second nucleic acid sequence, each said sequence including a coding region for a subunit from a . bacterial.ADP- ribosylaning exotoxin in the manufacture of a medicaracn: for 10 enhancing an immune response in a vertebrate subject against an antigen of interest in the said subject by administering the antigen of interest and the said ■ composition to the subject whereby the toxin subunits encoded by the first and second nucleic acid sequences are expressed in an amount sufficient to elicit an enhanced immune response against the antigen, wherein said first nucleic acid 15 sequence contains a modified A subunit coding region obtained or derived from a bacterial ADP-nbosylating exotoxin, and said second nucleic acid sequence contains a B subunit coding region obtained or derived from a bacterial .VDP-ribosylating exotoxin, with the proviso that said modifed A subunit coding region and said B subunit coding region each encode a mature subunit peptide, 20 and with the further proviso that the modified A subunit coding region has been genetically modined so as to delete a C-terminal KDEL or RDEL motif in the subunit peptide encoded thereby. 73. Use according to claim 72, wherein the antigen of interest and the 25 composition are administered to the same site in the subject. 74. Use according to claim 72. wherein the antigen of interest and ihe composition are administered concurrently. jvJ 75. Use according to claim 74, wherem the antigen of interss: and the composition are com.bmed to provide a single vaccine compcsition. 99 76. Use according to claim 72, wherein the antigen of interest is from a bacterial, viral or parasitic pathogen. 77. Use according to claim 76, wherein a third nucleic acid sequence 5 is administered lo the subject and the third sequence encodes said antigen of interest. 78. Use according to claim 72, wherein the first and second nucleic acid sequences are administered to the subject m particulate form. 10 79. Use according to claim 7S, wherein the composition comprising the first and second nucleic acid sequences are coated onto a core carrier panicle and administered to the subject using a particle-mediated delivery technique. 15 SO. Use according to claim. 72, wherein the subject is human. SI. A method for enhancing an hnmune response against an antigen of interest in a subject, the method comprising: (a) administering the antigen of interest to the subject; 20 (b) providing an adjuvant composition comprising first and second nucleic acid sequences, wherein said first nucleic acid sequence is a truncated A subunit coding region obtained or derived from a bacterial .ADP-ribosylating exotoxin and said second nucleic acid sequence is a truncated B subunit coding resion obtained or derived from a bacterial -ADP-ribosvlatins exotoxin, with the 25 proviso that each of said truncated subunit coding regions has a 5' deletion and encodes a subunit peptide not having an amino tenninal bacterial signal peptide; znc (c) administering said adjuvant composition to the subject, whereby upon introduction to the subject, the first and second nucleic acid sequences are 30 expressed to provide subunit peptides in an amount sufficient to elicit said enhnced immune response against the antigen of interest. 100 S2. A method for enhancing an immune resDonse asainst an anti2en of interest m a subject, the method comprising: (a) adminisering the antigen of interest to the subject; (b) providing an adjuvant composition comprismg first and second 5 nucleic acid sequences, wherein said first nucleic acid sequence is a modified A subunit coding reg:on obtained or derived from a bacterial .AOP-ribosyiatirg exotoxin, and said second nucleic acid sequence is a 3 subumt coding region obtained or derived from a bacteria! ADP-ribosvlatine exotoxin, with the proviso that said nocified A subunit coding region and said 3 subunit coding region each 10 encode a mature subumt peptide, and with the further proviso that the mocitied A subunit coding region has been genetically modified so as to delete a C-terminal KDEL or RDEL motif in the subumt peptide encoded thereby; and (c) administering said adjuvant composition to the subject, whereby upon introduction to the subject, the first and second nucleic acid sequences are 15 expressed.to provide subunit peptides in an amount sufficient to ehcit said enhanccd immmunc response against the antigen of inicrcst. 10 i 83. A composition substantially as herein described with reference to the accompanying drawings.

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