KR20060002872A - Immunogenic composition and methods - Google Patents

Abstract

A method of inducing an antigen-specific immune response in a mammalian subject includes the steps of administering to the subject an effective amount of a first composition comprising a DNA plasmid comprising a DNA sequence encoding an antigen under the control of regulatory sequences directing expression thereof in a mammalian or vertebrate cell. The method also includes administering to the subject an effective amount of a second composition comprising a recombinant vesicular stomatitis virus (rVSV) comprising a nucleic acid sequence encoding the antigen under the control of regulatory sequences directing expression thereof in the mammalian or vertebrate cell. The rVSV is in one embodiment replication competent. Kits for use in immunizations and therapeutic treatments of disease include the components and instructions for practice of this method.

Description

Immunogenic composition and methods

The present invention was partially supported by funds of the US Federal Government (National Institutes of Health, registration numbers NIH NO1-AI 05397 and NIH NO1-AI 25458). Thus, the federal government may have certain rights in the present invention.

Background of the Invention

In order to enhance the efficacy of immunogenic compositions, various compositions and methods have been reported using protein compositions, plasmid-based compositions and recombinant viral constructs as immunogenic compositions. Previous studies have demonstrated that plasmid-based immunogenic compositions initially stimulate the systemic immune system with secondary systemic immunization with conventional antigens, such as proteins or recombinant viruses, in systemic application. Xiang et al., 1997 Springer Semin . Immunopathol., 19: 257-268; Schneider, J. et al, 1998 Nature Med., 4: 397; and Sedeguh, M. et al., 1998 Proc. Natl. Acad. Sci., USA, 95: 7648; Rogers, W. O. et al, 2001 Infec. & Immun., 69 (9): 5565-72; Eo, S. K., et al, 2001 J. Immunol., 166 (9): 5473-9; Ramshaw I. A. and Ramsay, A. J., 2000 Immunol. Today, 21 (4): 163-5].

Commonly used DNA prime / live vector boost therapies include boosted vaccinia virus. Examples in recent literature include the above immunization against human immunodeficiency virus (HIV) [Hanke T. et al , 2002 Vaccine. 20 (15): 1995-8; Amara RR et al , 2002 Vaccine. 20 (15): 1949-55; Wee EG et al , 2002 J. Gen. Virol., 83 (Pt 1): 75-80; Amara RR et al, 2001 Science. 292 (5514): 69-74. Modifications that express antigens such as herpes simplex virus-2 glycoprotein D, Leishmania infantum P36 / LACK antigen, Plasmodium falciparum TRAP antigen, HIV / SIV antigen, mouse tuberculosis antigen and influenza antigen Prime-boost immunization with vaccinia virus vectors and DNA has been reported to induce specific antigen and cytokine responses. See Meseda CA et al ., 2002 J. Infect. Dis., 186 (8): 1065-73; Amara RR et al, 2002 J. Virol., 76 (15): 7625-31; Gonzalo RM et al , 2002 Vaccine, 20 (7-8): 1226-31; Schneider J. et al , 2001 Vaccine, 19 (32): 4595-602; Hel Z. et al , 2001 J. Immunol., 167 (12): 7180-91; McShane H. et al, 2001 Infec. & Immun., 69 (2): 681-6 and Degano P. et al 1999 Vaccine , 18 (7-8): 623-32 influenza and malaria models].

Plasmid Prime-Adenovirus Boost Genetic Immunization Therapy has recently been reported to induce alpha-fetoprotein-specific tumor immunity and protect swine from classical swine fever. Meng WS 2001 Cancer Res ., 61 (24): 8782-6; Hammond, J M et al , 2001 Vet. Microbio., 80 (2): 101-19; And US Pat. No. 6,210,663.

Other DNA plasmid prime-virus boost therapies have also been reported (Matano T. et al, 2001 J. Virol., 75 (23): 11891-6 (DNA Prime / Sendai Virus Vector Boost). DNA priming and recombinant poxvirus boosting have been reported (Kent, SJ et al, 1998 J. Virol., 72: 10180-8; Robinson, HL et al, 1999 Nat. Med., 5: 526-34; and Tartaglia, J. et al, 1998 AIDS Res.Human Retrovirus., 14: S291-8].

Numerous DNA prime / viral boost therapies have been evaluated, but recently described immunization therapies have some drawbacks. For example, some of these well known therapies cause disease symptoms in a subject; Other therapies can result in in vivo recombination. Other viruses are difficult to manufacture and / or have limited ability to accept foreign genes. Another virus has other safety problems than the disadvantages caused by significant existing vector immunity in humans.

It is capable of producing enhanced levels of cellular and humoral immune responses to the antigen of interest and is consistent with safety requirements, ease of manufacture and the ability to overcome the mammalian host's natural immune response to the vector during booster immunization. Useful immunization therapies are required in the art.

Summary of the Invention

The present invention provides a mammalian subject with a prime / boost therapy that shows a surprising synergistic stimulation of a cellular immune response to an antigen compared to the results obtained with DNA plasmid components or recombinant viral components when administered separately. Novel methods, compositions, and kits are provided for inducing an immune response against a pathogenic antigen or other antigen.

In one aspect, the invention provides a novel method of inducing an antigen-specific immune response in a mammalian subject. The method comprises administering to a subject an effective amount of a first composition comprising a DNA plasmid comprising a DNA sequence expressing an antigen under control of a regulatory sequence directing its expression in a mammalian cell by a DNA plasmid . The method comprises a second composition comprising rVSV comprising a nucleic acid sequence encoding said antigen under control of a regulatory sequence directing its expression in mammalian cells by recombinant vesicular stomatitis virus (rVSV). Administering an effective amount of to the subject. In one embodiment, the recombinant VSV is an attenuated, replication competent virus. In other embodiments, the recombinant VSV is a non-replicating virus. Administration of the first and second compositions can be in any order. In addition, the present invention intends to administer one of the compositions several times and then to the other composition several times. In one embodiment, cytokines are preferably co-administered.

In another aspect, the invention provides an immunogenic composition for inducing an antigen-specific immune response against an antigen in a mammalian subject. The immunogenic composition comprises a first composition comprising a DNA plasmid comprising a DNA sequence expressing an antigen under control of a regulatory sequence directing its expression by a DNA plasmid. The composition also includes one or more replication-competent recombinant vesicular stomatitis virus (VSV) comprising a nucleic acid sequence encoding the same antigen under control of a regulatory sequence directing its expression by recombinant vesicular stomatitis virus (VSV).

In another aspect, the invention provides a kit for use in a therapeutic or prophylactic method of inducing an increased level of antigen-specific immune response in a mammalian subject. The kit includes, in particular, at least one first composition comprising a DNA plasmid comprising a DNA sequence expressing an antigen under the control of a regulatory sequence directing its expression in a mammal; At least one second composition comprising a replication-competent recombinant bullous stomatitis virus (rVSV) comprising a nucleic acid sequence encoding said antigen under the control of a regulatory sequence directing its expression in said mammal; And instructions regarding the implementation of the aforementioned method.

In another aspect, the present invention provides the use of an immunogenic composition or a component thereof, in the manufacture of a medicament for inducing an animal's immune response against an antigen used in the immunogenic composition described above.

Other aspects and aspects of the invention are described in the detailed description below.

1A is a schematic of exemplary plasmid DNA encoding monkey immunodeficiency virus (SIV) gag p37 protein. This scheme shows the human cytomegalovirus (HCMV) promoter / enhancer, bovine growth hormone polyadenylation site (BGH polyA), replication origin sequence (ori) and kanamycin resistance ( kan ^R ) in which the plasmid drives expression of the gag protein. ) Contains marker genes.

FIG. 1B is a schematic of exemplary bicystronic plasmid DNA encoding two subunits p35 and p40 of Resus interleukin 12. FIG. The p35 subunit is under the control of the HCMV promoter and has an SV40 poly A site. The p40 subunit is under the control of the monkey cytomegalovirus (SCMV) promoter and has a BGH poly A site and is transversely reversed. The plasmid also contains the ori sequence and the kan ^R gene.

FIG. 2 is a bar graph showing VSV N-specific gamma interferon (IFN-ν) ELISpot response in unfractionated peripheral blood monocytes (PBMC) from animals immunized with prime / boost therapy of the present invention. The rightmost black bar represents the protocol of immunization by boosting the DNA plasmid encoding the SIV gag protein and the VSV vector encoding the influenza hemagglutinin (flu HA) protein. The light gray bars represent the protocol of the present invention comprising a priming DNA gag plasmid immunization followed by VSV boost expressing HIV gag and env proteins. White bars represent protocols comprising priming immunization with empty DNA plasmids or control DNA plasmids (con DNA) followed by immunization with VSV expressing HIV gag and env proteins. The rightmost black bar represents a protocol comprising priming immunization with DNA plasmids followed by immunization with VSV expressing flu HA protein. Each group shows results from five animals.

FIG. 3 is a bar graph showing HIVenv 6101-specific gamma interferon (IFN-ν) ELISpot response in unfractionated PBMCs from animals immunized with prime / boost therapy of the present invention. The leftmost light gray bar represents the protocol of immunization by boosting the DNA plasmid expressing SIV gag protein and VSV expressing flu HA protein. Hatched bars represent the protocol of the present invention comprising a priming DNA gag plasmid immunization followed by a VSV boost that expresses HIV gag and env proteins. Checkered bars represent protocols that include priming immunization with co-control DNA followed by immunization with VSV expressing HIV gag and env proteins. Dotted bars represent protocols comprising priming immunization with control DNA plasmids followed by immunization with VSV boost expressing flu HA protein. * Indicates when the statistically significant difference occurred at p = 0.0001.

4 is a graph showing serum anti-SIV gag p27 antibody titers by enzyme-linked immunosorbent assay (ELISA) for animals immunized with prime / boost therapy of the present invention. Plasmid DNA was administered at days 0, 4 and 8, and VSV (serum type Indiana G) and VSV (serum type Candipura G) boost were administered at 15 and 23 weeks, respectively. The protocol for immunization by boosting the DNA plasmid encoding the SIV gag protein and the VSV vector expressing the flu HA protein is indicated by (◆). The protocol of the present invention comprising a priming DNA gag plasmid immunization followed by VSV boost expressing HIV gag and env proteins is indicated by (■). Protocols comprising priming immunization with co-control DNA followed by immunization with VSV expressing HIV gag and env proteins are indicated by (▲). Protocols comprising priming immunization with a control DNA plasmid followed by immunization with VSV expressing flu HA protein are indicated by (•). Each group shows results from five animals. The statistically significant difference between groups was p = 0.0073 (*); p = 0.5941 (#) or p = 0.0027 (yen).

FIG. 5 is a graph showing SIV gag-specific spot forming cells per million cells assessed by ELISpot assay for animals immunized with prime / boost therapy of the present invention. Plasmid DNA was administered at days 0, 4 and 8, and VSV (serum type Indian G) and VSV (serum type Candipura G) boost were administered at 15 and 23 weeks, respectively. The protocol of immunization by boosting the DNA plasmid encoding the SIV gag protein and the vector encoding the flu HA protein is indicated by (◆). The protocol of the present invention comprising a priming DNA gag plasmid immunization followed by VSV boost expressing HIV gag and env proteins is indicated by (■). Priming immunization with co-control DNA followed by immunization with VSV expressing HIV gag and env proteins is indicated by (▲). (●) represents a protocol comprising priming immunization with control DNA plasmids followed by immunization with VSV expressing flu HA protein. Each group shows results from five animals. Statistical significant differences between groups are indicated in parentheses for p = 0.0001 and p = 0.0002.

FIG. 6 shows a reduced CD4 T-cell loss after several days of challenge, and the elevated immune response induced by the prime / boost combination indicated by the same symbol as FIG. 5 enhances protection from AIDS. It is a graph showing the sieving.

FIG. 7 shows the increased immune response induced by the prime / boost combination, indicated by the same symbol as in FIG. 5, as measured by the reduction of circulating virus (viral copy / ml) in plasma after several days of challenge. Graph showing enhancement.

The present invention is directed to inducing antigen-specific immune responses in mammalian or vertebrate subjects by combining certain components of the immunogenic compositions described in the prior art and optimizing the components to yield surprising synergistic results. Provide a new method. In general, the method comprises administering to the subject an effective amount of a composition comprising a DNA plasmid encoding an antigen under control of a regulatory sequence directing expression in mammalian or vertebrate cells by the DNA plasmid. The method also includes administering to the subject an effective amount of a composition comprising a replication-competent recombinant bullous stomatitis virus (rVSV). Such rVSVs comprise a nucleic acid sequence encoding an antigen under the control of regulatory sequences that direct its expression in mammalian or vertebrate cells by the rVSV.

The first of these two immunogenic compositions to be administered in sequence is referred to as the priming composition. The second of these two immunogenic compositions to be administered in sequence is referred to as a boosting composition. Thus, the DNA composition can be administered as a priming composition and the VSV vector composition can be administered as a boosting composition and vice versa. In one embodiment, the priming composition is administered to the subject at least once or several times prior to administering the boosting composition. Thereafter, the boosting composition is subsequently administered to the subject at least once or several times. In addition, the present invention intends to administer one of the compositions several times and then to the other composition several times. The method further intends to administer an effective amount of cytokines as one step of the method.

It has been surprisingly found that the practice of the present invention induces a CD8 + T cell response to the antigen, which is greater than that achieved by administering DNA plasmid or recombinant VSV alone in mammalian subjects. In fact, as indicated in the examples below, the immune response is greater than that expected with the additional combination of two immunogenic compositions. Thus, the immune response induced by this novel method is a dramatic synergistic increase in cellular and / or antigenic responses to antigens. For example, the maximum antigen specific cellular response to HIV-1 gag is at least three times higher than the response to administration of a single DNA plasmid immunogenic composition or nearly nine times the response to administration of a single rVSV immunogenic composition alone. See Table 1 and Figures 4 and 5 below.

Although mammalian subjects are intended to be primates, preferably humans, the present invention is not limited to the identification of mammalian subjects. The components of the method are described in detail below with reference to the documents cited herein by reference to provide details known to those skilled in the art.

A. DNA Plasmid Immunogenic Compositions

The immunogenic composition of the present invention comprises a DNA plasmid comprising a DNA sequence encoding a selected antigen against which an immune response is desired. In this plasmid, the selected antigen is under the control of regulatory sequences that direct its expression in mammalian or vertebrate cells. The components of the plasmid itself are conventional ones.

Non-viral plasmid vectors useful in the present invention contain isolated and purified DNA sequences comprising DNA sequences encoding selected immunogenic antigens. DNA molecules may be derived from viral or non-viral, eg, bacterial species designed to encode exogenous or heterologous nucleic acid sequences. Such plasmids or vectors may comprise sequences from viruses or phages. Various non-viral vectors are known in the art and include, but are not limited to, plasmids, bacterial vectors, bacteriophage vectors, "nape" DNA and DNA condensed with cationic lipids or polymers.

Examples of bacterial vectors are especially Bacillus knife meth gereng (bacille Calmette Guerin (BCG)) , Salmonella (Salmonella), Shigella (Shigella), the. Coli include (E. coli) and Listeria monocytogenes (Listeria). Suitable plasmid vectors include, for example, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pK37, pKC101, pAC105, pVA51, pKH47, pUB110, pMB9, pBR325, Col E1, pSC101, pSC101 pBR313, pML21, RSF2124, pCR1, RP4, pBAD18 and pBR328.

Examples of suitable inducible Escherichia coli expression vectors include pTrc [Amann et al. , 1988 Gene, 69: 301-315], arabinose expression vectors (eg pBAD18, Guzman et al , 1995 J. Bacteriol ., 177 : 4121-4130) and pETIId (Studier et al. , 1990 Methods in Enzymology, 185: 60-89. Target gene expression from the pTrc vector is dependent on host RNA polymerase transcription from the hybrid trp-lac fusion promoter. Target gene expression from the pETIId vector is dependent on transcription from the T7 gn10-lac fusion promoter mediated by co-expressed viral RNA polymerase T7 gn l. These viral polymerases are supplied by either host strain BL21 (DE3) or HMS I 74 (DE3) from resident probes carrying the T7 gn1 gene under the transcriptional control of the lacUV5 promoter. The pBAD system relies on an inducible arabinose promoter regulated by the araC gene. The promoter is induced in the presence of arabinose.

Promoters, and other regulatory sequences that drive antigen expression in a desired mammalian or vertebrate host, can be similarly selected from a wide variety of promoters known to be useful for this purpose. These various promoters are described below. In embodiments of the immunogenic DNA plasmid compositions described below, useful promoters are human cytomegalovirus (HCMV) promoters / enhancers (see, eg, US Pat. Nos. 5,168,062 and 5,385,839, incorporated herein by reference). And SCMV promoter enhancer.

Additional regulatory sequences for inclusion in the nucleic acid sequences, molecules or vectors of the invention include, without limitation, enhancer sequences, polyadenylation sequences, splice donor sequences and splice acceptor sequences, the beginning and the end of the polypeptide to be transcribed, respectively. Transcription initiation site and transcription termination site, ribosomal binding site for translation in transcribed fluid, epitope tag, nuclear internationalization sequence, IRES element, Goldberg-Hogness "TATA" element, restriction enzyme cleavage Sites, selectable markers, and the like. Enhancer sequences include, for example, 72bp tandem repeats of SV40 DNA or long terminal repeats of retroviruses or LTRs, and enhancer sequences are used to increase transcriptional efficiency.

Such other components useful in DNA plasmids, including replication origins, polyadenylation sequences (e.g. BGH polyA, SV40 polyA), drug resistance markers (e.g. kanamycin resistance), and the like, are described in the Examples and are specifically described below. It may also be selected from well known sequences, including the sequences to which reference is made.

The selection of promoters and other conventional vector elements is common and many such sequences are available and can be used to design plasmids useful in the present invention. See Sambrook et al . , Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Laboratory, New York, (1989) and references cited, for example, on pages 3.18-3.26 and 16.17-16.27, and Ausubel et al ., Current Protocols in Molecular Biology , John Wiley & Sons, New York (1989). All components of the plasmids useful in the present invention can be readily selected from materials known in the art by those skilled in the art and are available from the pharmaceutical industry. The choice of plasmid components and regulatory sequences is not to be considered as limiting in the present invention.

Examples of suitable DNA plasmid constructs for use in immunogenic compositions are described in detail in particular in the following patent publications, the contents of which are incorporated herein by reference: International Patent Publication No. WO98 / 17799, WO99 / 43839 and WO98 / 17799; And US Pat. No. 5,593,972; 5,817,637; 5,817,637; 5,830,876; 5,830,876; And especially 5,891,505.

Likewise, the selected antigen can be the antigen identified in the discussion below. In one embodiment of the immunogenic compositions herein, the selected antigen is an HIV-1 antigen, such as an antigen expressed by gag, pol, env, nef, vpr, vpu, vif and tat . Preferably, the antigen sequence and other components of the DNA plasmid are optimized by, for example, codon selection appropriate to the intended host and removal of any inhibitory sequences, and also discussed below in relation to antigen preparations.

Thus, such immunogenic compositions may comprise one plasmid encoding one selected antigen for expression in a host. According to the method, the plasmid composition also comprises one DNA plasmid comprising a DNA sequence encoding at least one copy of the same selected antigen. Alternatively, the composition may contain one plasmid that expresses a plurality of selected antigens. Each antigen may be under the control of an individual regulatory element or component. Alternatively, each antigen may be under the control of the same regulatory element. In another embodiment, the DNA plasmid composition may contain multiple plasmids, wherein each DNA plasmid encodes the same or different antigens.

In a further embodiment, the DNA plasmid immunogenic composition further comprises a nucleotide sequence encoding a desired cytokine, lymphokine or other genetic adjuvant, either as a separate DNA plasmid component or as part of an antigen containing DNA plasmid. It may contain. Hosts of such suitable adjuvants for which nucleic acid sequences are available are identified below. In an embodiment exemplified herein, the preferred cytokine for administration with the DNA plasmid composition of the invention is interleukin-12.

The DNA plasmid composition is preferably administered in a pharmaceutically acceptable diluent, excipient or carrier as discussed below. Although the composition may be administered by any selected route of administration, in one embodiment, a preferred method of administration is co-administration of bupivacaine as an accelerator to a composition comprising a plasmid.

In one aspect of the methods of the invention wherein the DNA composition is a priming composition, the method comprises administering one or more DNA plasmids comprising a sequence encoding an antigen for which an immune response is desired to be induced before rVSV. In another embodiment, the DNA priming composition further consists of a second plasmid encoding the selected cytokine. In another embodiment, the DNA priming composition, as illustrated below, comprises three plasmids, one plasmid expressing a first antigen, a second plasmid expressing a different second antigen, and a third plasmid expressing a selected cytokine adjuvant. Include. As described in detail in the Examples, one aspect of the DNA priming composition comprises three optimized plasmids: (1) a plasmid encoding an RNA optimized SIV gag p37 gene (see FIG. 1A); (2) a plasmid encoding two Resus IL-12 subunits p35 and p40 under separate control of the two promoters (see FIG. 1B) and (3) a plasmid encoding the HIV-1 gp160 env gene.

When used as a priming composition, such DNA plasmid compositions are administered one or preferably one or more times prior to the boosting rVSV composition. When used as a boosting composition, such DNA plasmid compositions are administered one or preferably one or more times after administration of the priming rVSV composition.

B. rVSV Immunogenic Compositions

Another immunogenic composition useful in the methods and compositions of the present invention is a replicative live attenuated bullous stomatitis virus (VSV) delivery vehicle. Such recombinant VSVs include nucleic acid sequences encoding selected antigens under the control of regulatory sequences that direct expression in mammalian or vertebrate cells. In the method of the invention, the antigen used in the DNA plasmid composition is the same antigen as used in the rVSV immunogenic composition.

VSV is a bovine virus that is a member of the taxonomy named Mononegavirales, which is a non-segmented, negative (-)-chain RNA genome that encodes four internal structural proteins and one external transmembrane protein. Include. In the 3 'to 5' direction, the gene encodes proteins named nucleocapsid (N), phosphoprotein (P), matrix protein (M), transmembrane glycoprotein (G) and polymerase (L). . Living VSV can be isolated and "rescue" using techniques known in the art (see US Pat. No. 6,044,886; No. 6,168,943; And 5,789,299; International Patent Publication No. WO 99/02657; Conzelmann, 1998, Ann. Rev. Genet ., 32 : 123-162; Roberts and Rose, 1998, Virol., 247 : 1-6; Lawson et al , 1995 Proc. Natl. Acad. Sci., USA 92 : 4477-4481. Further, for example, the genomic sequence of VSV (Indiana) is registered as accession number NC001560 in the NCBI database. Other sequences of VSV, including the VSV (candipura) sequence, are available in the database; See, for example, Accession Nos. Ay382603, Af128868, V01208, V01207, V01206, M16608, M14715, M14720 and J04350. VSV strains such as New Jersey and Indiana are also available from depository institutions such as the American Type Culture Collection (Rockville, Maryland) (eg, Accession Nos. VR-1238 and Sub. VR-1239). Other sequences are described or mentioned in documents cited throughout this specification. All documents cited herein are incorporated by reference.

The VSV genome has been shown to accept one or more foreign genes and extend to at least 3 kb. The genomes of these viruses are very stable and do not recombine and rarely undergo significant mutations. In addition, since their replication takes place in the cytoplasm and their genome consists of RNA, they can be inserted into the genome of infected host cells. In addition, these negative-chain RNA viruses have relatively simple transcriptional control sequences that can be easily manipulated for efficient foreign gene insertion. Finally, the expression level of the foreign gene can be regulated by changing the position of the foreign gene relative to the viral transcriptional promoter. The 3 'to 5' gradient of gene expression reflects the reduced likelihood of successfully passing each intergenic gene termination / gene initiation signal that is encountered as the transcriptional viral polymerase progresses along the genome template. Thus, foreign genes located near the 3 ′ terminal transcription initiation promoter are abundantly expressed, while foreign genes inserted at more distal genomic positions are much less expressed.

VSV replicates at high titers in many different cell types, and viral proteins are highly abundantly expressed. This means that not only can VSV act as a powerful functional foreign gene delivery vehicle, but it can also scale the appropriate rVSV vector to the level of manufacture in cell lines approved for human biopharmaceutical production.

rVSV has the incredible ability to transfer foreign genes encoding important protective immunogens from viral pathogens to a wide variety of different cell types and subsequently lead to abundant expression of immunogenicly-configured immunogenic proteins. Haglund, K., et al , 2000 Virol., 268 : 112-21; Kahn, JS et al , 1999 Virol., 254 : 81-91; Roberts, A. et al , 1999 J. Virol., 73 : 3723-32; Rose, NF et al , 2000 J. Virol., 74 : 10903-10; and Schlereth, B. et al. 2000 J. Virol., 74 : 4652-7. This expressed immunogen simultaneously induces both highly protective viral neutralizing antibody responses as well as protective cytotoxic T lymphocytes (CTL) (Roberts, A. et al , 1998 J. Virol., 72 : 4704-11).

In addition to the efficacy of rVSV, these fleshed viral gene transfer vehicles are safe because wild-type VSV causes little or no disease symptoms or pathology in healthy people despite significant viral replication. Tesh, RB et al , 1969 Am. J. Epidemiol ., 90 : 255-61. In addition, few people are infected with VSV and already have immunity to VSV. If further attenuated, such rVSV compositions are suitable for use in immunocompromised or otherwise less healthy human subjects. An important advantage of using VSV vectors in this method is the large number of serotypes of VSV due to changes or modifications of the viral attachment protein G of VSV. Thus, different serotypes of VSV vectors bearing the same heterologous antigen can be used for repeated administration to avoid any interfering neutralizing antibody response generated against VSV G proteins by the host's immune system.

Recombinant VSV can be designed to retain selected antigens and regulatory sequences inserted at any position of VSV under the control of a viral transcriptional promoter using techniques already described in the art. In one embodiment, the heterologous gene encoding the selected antigen is inserted between the G and L coding regions of VSV. In other embodiments, the heterologous gene can be fused to a G protein site. In another embodiment, the heterologous gene is fused to a site at or adjacent to any one of the other VSV genes.

In another embodiment, the gene is 'shuffling' to different sites in the genome. In particular, the N gene is shuffled to different sites in the genome. Cloning to produce shuffled recombinant cDNA sequences involves modifications of the original VSV plasmid backbone (pVSV-XN1). The three variants of this original vector comprise plasmids in the following sequence, outside the normal gene sequence (3′- NPMGL- 5 ′): i) 3′- PNMGL- 5 ′; ii) 3'- PMNGL- 5 'and iii) 3'- PMGNL- 5'. The cloning strategy used to generate such plasmids is described in Ball, LA et al . 1999 J. Virol ., 73: 4705-12. The technique takes advantage of the fact that the gene-terminal / gene initiation signals found between each coding sequence are nearly identical and that rearranged genes can be constructed without introducing any nucleotide substitutions. Alternatively, a small number of effective point mutations can be introduced into the non-coding sequence to create customary restriction sites that promote genome rearrangement.

In another embodiment of these vectors, the carboxy-terminal coding sequence of the 29 amino acid cytoplasmic domain of the G gene is truncated by deleting amino acids from the 5 'C terminus of the G gene. Alternatively, the entire G gene is deleted. In one embodiment, the entire cytoplasmic domain of the G gene is removed. In another embodiment, at least 28 amino acids in the cytoplasmic domain are removed. In another embodiment, about 20 amino acids in the cytoplasmic domain are deleted. In yet further embodiments, up to about 10 amino acids in the cytoplasmic domain are deleted.

Reportedly, both the shuffled genomic strategy and the G protein modification strategy cause partial growth defects. Flanagan, EB, et al 2001 J. Virol. 75 : 6107-14; Schnell, M. J. et al . 1998 EMBO J., 17 : 1289-96. Such VSV modifications are expected to induce more attenuated phenotypes or even non-replicating VSVs for use in various aspects of the present invention. Johnson, JE et al , 1998 Virol., 251 : 244- 52.37; Johnson, JE, et al., 1997 J. Virol., 71 : 5060-8.

Similarly, the selected antigen can be the antigen identified in the discussion below. In one embodiment of the immunogenic compositions herein, the antigen of choice is the HIV-1 gag and / or env ( gp160) gene of clade B virus isolate. In another embodiment, the antigen is an HIV-1 pol, nef, vpr, vpu, vif or tat gene. Preferably, the antigen sequence is optimized by codon selection and / or removal of any inhibitory sequences appropriate for the intended host, also discussed below in connection with antigen preparation.

To solve the problem of any potential vector replication efficiency reduction with sequential administration, successful booster immunization is possible using a vector set of similar design, each carrying a G gene from a different VSV serotype. The primary amino acid sequences of G proteins from VSV Indiana, New Jersey and Candipura are sufficiently different so that existing immunity to one does not interfere with infection and replication of the other. Thus, neutralizing antibody responses generated by rVSV (Indiana) should not interfere with the replication of rVSV (New Jersey) or rVSV (Candipura). Vector sets that can enable successful sequential immunization can be prepared by replacing G genes from VSV Indiana with VSV candipura or branched homologs from VSV New Jersey to form three immunologically different vectors.

Thus, such rVSV immunogenic compositions may comprise one rVSV encoding a single antigen selected for expression in a host. According to the method, an rVSV immunogenic composition comprises one rVSV comprising a nucleic acid sequence encoding one or more copies of the same selected antigen. Alternatively, the composition may contain one rVSV that expresses a plurality of selected antigens. Each antigen may be under the control of an individual regulatory element or component. Alternatively, each antigen may be under the control of the same regulatory element. In another embodiment, the rVSV composition may contain multiple rVSVs, each of which encodes the same or different antigen.

In another embodiment, the rVSV immunogenic composition may further contain or be administered with cytokines, lymphokines or genetic adjuvants. Cytokines may be administered as proteins or in plasmids as described above or encoded by inserting cytokine coding sequences into recombinant VSV. For example, cytokine coding sequences can be inserted at any location in the VSV genome and expressed from viral transcriptional promoters. Hosts of such suitable adjuvants for which nucleic acid sequences are available are identified below. In an embodiment exemplified herein, the preferred cytokine for administration with the rVSV composition of the invention is interleukin-12.

The rVSV composition is preferably administered in a pharmaceutically acceptable diluent, excipient or carrier as discussed below. Although the composition may be administered by any selected route of administration, in one embodiment the preferred method of administration is intranasal administration.

In one aspect of the methods of the invention wherein the rVSV composition is a boosting composition, the method comprises administering one or more rVSV immunogenic compositions after administering the DNA immunogenic priming composition. The rVSV immunogenic composition expresses the same antigen as expressed by the priming composition. In one embodiment, the rVSV composition comprises an additional recombinant virus that encodes a selected cytokine. In another embodiment, the rVSV comprises a cytokine, eg, IL-12, present in the same rVSV that expresses the antigen. In another embodiment, multiple rVSV compositions are later administered as boosters. In one embodiment, two or more rVSV compositions are administered following the priming composition. In another embodiment, three or more rVSV compositions are administered following the priming composition.

Preferably, as discussed above, each subsequent rVSV composition has a different serotype but the same antigen coding sequence. The different serotypes are selected from known natural serotypes and any synthetic serotypes provided by manipulation of VSV G proteins. Among the known methods of altering the G protein of rVSV are described in WO99 / 32648 and Rose, N. F. et al. 2000 J. Virol., 74: 10903-10.

In other embodiments, each rVSV has a different antigen coding sequence but the same VSV G protein. In another embodiment, each rVSV has a different antigen coding sequence and a different VSV G protein. As described in detail in the Examples, one embodiment of a series of rVSV boosting compositions comprises two optimized rVSVs containing the HIV-1 gp160 env gene, one rVSV is Indiana serotype and the other is candipura serotype to be.

According to the invention, such rVSV immunogenic compositions may be administered as a boosting composition after administration of a priming DNA immunogenic composition presenting the same antigen to the host. In any embodiment of the method of the invention, the second and any additional rVSVs are administered as a booster following the first rVSV administration. In one embodiment the additional rVSV booster is of the same serotype with the same antigen. Alternatively, additional rVSV boosters with different serotypes are administered sequentially before administering the boosting DNA immunogenic composition. Administration of three or more boosters has been shown to be useful. When used as a boosting composition, the rVSV immunogenic composition is administered continuously after the priming DNA composition. When used as a priming composition, the rVSV composition is administered once or preferably several times. In one embodiment the additional rVSV booster is of the same serotype with the same antigen. Alternatively, additional rVSV boosters with different serotypes are administered sequentially before administering the boosting DNA immunogenic composition.

Examples of suitable rVSV constructs capable of expressing HIV-1 protein in vivo are described in detail in the following publications as examples of: Rose et al , 2000 Virol., 268 : 112-121; Rose et al , 2000 J. Virol., 74 : 10903-10910; Rose et al 2001 Cell, 106 : 539-549; Rose et al , 2002 J. Virol., 76 : 2730-2738; Rose et al , 2002 J. Virol., 76 : 7506-7517; Which is incorporated herein by reference]. Additional rVSV vectors can be further attenuated by progressive truncation of VSV G protein or by shuffling VSV structural genes as described above. Non-replicating VSVs can also be used in accordance with the present invention. It is expected that rVSVs that exhibit a desirable balance of attenuation and immunogenicity will be useful in the present invention.

Some exemplary rVSV constructs illustrated in the Examples below have the following recombinant genomes:

(1) 3 'N- P- M- G-HIV env -L-5'

(2) 3 'N- P- M- G-HIV gag -L-5'

(3) 3 'N- P- M- G-SIV gag -L-5'

In one embodiment, the methods and immunogenic compositions of the invention use one of the rVSV constructs described above. In another embodiment, the methods and immunogenic compositions of the invention use both rVSV-HIV env and rVSV-HIV gag . The latter immunogenic composition, along with the humoral immune response against the intrinsically constructed gp160, simultaneously induces CTLs (for Env and Gag) that can kill HIV-1 virus infected cells. HIV-1 gp160 expressed in this manner binds to CD4 / coceptor and undergoes conformational changes associated with receptor binding. Appropriate gp160 / receptor interactions expose potential viral neutralizing determinants present on gp160 that are required for induction of antibody-mediated protection from infection. LaCasse, RA et al, 1999 Science. 283 : 357-62.

C. Antigens for Use in Immunogenic Compositions of the Invention

Antigenic or immunogenic compositions useful in the methods and compositions of the present invention enhance immune responses in vertebrate hosts against selected antigens. The antigen of choice may be a protein, polypeptide, fragment or fusion thereof derived from a pathogenic virus, bacteria, fungus or parasite when expressed by plasmid DNA or VSV. Alternatively, the antigen of choice may be a protein, polypeptide, peptide, fragment or fusion thereof derived from a cancer cell or tumor cell. In other embodiments, the antigen of choice may be a protein, polypeptide, peptide, fragment or fusion thereof derived from an allergen to interfere with the production of IgE to modulate an allergic response to the allergen. In another embodiment, the selected antigen is a protein, polypeptide, fragment or fusion thereof derived from a molecule or a portion thereof that refers to a molecule (an automolecule) produced in an amount or in a position undesired by the host, eg For example, they may be from amyloid precursor proteins for preventing or treating diseases characterized by amyloid deposition in vertebrate hosts. In one aspect of the invention, the antigen of choice can be a protein, polypeptide, peptide, fragment or fusion thereof derived from HIV-1.

The present invention also provides a method for preparing an immune response of a vertebrate host from (1) pathogenic viruses, bacteria, fungi or parasites, and (2) cancer cells or tumor cells for inducing a therapeutic or prophylactic anticancer effect in a vertebrate host. Cancer antigens or tumor-associated antigens, (3) allergens to modulate allergic reactions to allergens by interfering with IgE production, or (4) to reduce undesirable effects, in an undesirable manner by the host. A method of increasing the ability of an immunogenic composition to contain an antigen selected from a molecule or portion thereof produced in or at a position.

In another embodiment, preferred viral immunogenic compositions using the prime / boost therapy of the invention include, without limitation, human immunodeficiency virus, monkey immunodeficiency virus, respiratory syncytial virus, parainfluenza virus type 1 to 3, influenza virus , Herpes simplex virus, human cytomegalovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human papillomavirus, poliovirus, rotavirus, calcivirus, measles virus, mumps virus, rubella virus, Adenovirus, Rabies virus, Dog distemper virus, Linderpes virus, Human metapneumovirus, Avian pneumovirus (formerly turkey non-bronchialitis virus), Hendra virus, Nipa virus, Coronavirus, Parvovirus, Infectious nonbronchiolitis virus Composition for the prevention and / or treatment of diseases caused by feline leukemia virus, feline infectious peritonitis virus, avian infectious cyst disease virus, Newcastle disease virus, Marek's disease virus, porcine respiratory and genital syndrome virus, equine arteritis virus and various encephalitis viruses It includes.

In another aspect, preferred bacterial immunogenic compositions using the prime / boost regimen of the present invention, without limitation, Haemophilus influenzae (Haemophilus influenzae) (type determination is possible (typable) and type determination is not possible (nontypable) both), Haemophilus cotton Taunus ( Haemophilus somnus ), Moraxella catarrhalis , Streptococcus pneumoniae , Streptococcus pyogenes , Streptococcus agalactiae , Streptococcus agalactiae , Streptococcus agalactiae Streptococcus faecalis), Helicobacter pylori (Helicobacter pylori), Nathan Serie menin giti display (Neisseria meningitidis), O (Neisseria gonorrhoeae), Chlamydia trachomatis (Chlamydia trachomatis), Chlamydia pneumoniae (Chlamydia pneumoniae) on Nathan Serie Kono, chlamydia Citadines City (Chlamydia psittaci), Bordetella Pere System (Bordetella pertussis), Alor EO caucus Ortiz de-Tees (Alloiococcus otiditis), Salmonella typhimurium (Salmonella typhi), Salmonella typhimurium (Salmonella typhimurium), can Salmonella cholera device (Salmonella choleraesuis), Escherichia coli (Escherichia coli) , Shigella Vibrio cholerae (Shigella, Vibrio cholerae), Corynebacterium diphtheria in (Corynebacterium diphtheriae), Mycobacterium-to-Vere Cool tuberculosis (Mycobacterium tuberculosis), Mycobacterium Oh Away-Mycobacterium (Mycobacterium avium - Mycobacterium) intracellular complex, Proteus Mira Billy's (Proteus mirabilis), Proteus vulgaris (Proteus vulgaris), Streptococcus aureus (Staphylococcus aureus), Streptococcus epi more misses (Staphylococcus epidermidis), Clostridium tetani (Clostridium tetani ), leptospira interrogating elegans (leptospira interrogans), Abu borelri D'Perry (Borrelia burgdorferi), Paz Chateau Pasteurella H. Molina urticae (Pasteurella haemolytica), Pasteurella Pas Chateau far the Cauchy (Pasteurella multocida), evil Martino Bacillus player right pneumoniae (Actinobacillus pleuropneumoniae) And compositions relating to the prevention and / or treatment of diseases caused by Mycoplasma gallisepticum .

In another aspect, a preferred immunogenic composition for using the prime / boost regimen of the present invention, fungal pathogens are, Aspergillus unrestricted switch (Aspergillis), Blas Sat My process (Blastomyces), Candida (Candida), koksi video Compositions relating to the prevention and / or treatment of diseases caused by Coccidiodes , Cryptococcus and Histoplasma .

In another embodiment, preferred immunogenic compositions for parasites using the prime / boost therapy of the invention include, without limitation, Leishmania major , Ascaris , Trichuris , Giardia , juhyeol fluke (Schistosoma), Cryptosporidium (Cryptosporidium), trichomonas (trichomonas), Toxoplasma gon diimide (Toxoplasma gondii) and New-necked seutiseu karini including the composition of the prophylaxis and / or treatment of induced disease by (Pneumocystis carinii) do.

In another embodiment, preferred immunogenic compositions for inducing a therapeutic or prophylactic anticancer effect in a vertebrate host using prime / boost therapy of the invention include, without limitation, prostate specific antigens, cancer embryo antigens, MUC- 1, Her2, CA-125 and MAGE-3, including cancer antigens or compositions with tumor-associated antigens.

The nucleotide and protein sequences of the known antigens listed above are readily available publicly through deatabases such as NCBI, or from other sources such as American type culture collections and universities.

Preferred immunogenic compositions for modulating responses to allergens in vertebrate hosts using the prime / boost therapy of the present invention include compositions containing allergens or fragments thereof. Examples of such allergens are described in US Pat. No. 5,830,877 and WO 99/51259, which is incorporated herein by reference. Such allergens include, without limitation, pollen, insect poison, animal dandruff, fungal spores and drugs such as penicillin. These immunogenic compositions interfere with the production of IGE antibodies known to cause allergen responses.

Preferred immunogenic compositions for modulating responses to autologous molecules in vertebrates using the prime / boost therapy of the present invention include compositions containing autologous molecules or fragments thereof. Examples of such automolecules include β-chain insulin involved in diabetes, G17 molecules involved in gastroesophageal reflux disease and antigens that downregulate autoimmune responses in diseases such as multiple sclerosis, lupus and rheumatoid arthritis. Examples also include β-amyloid peptides (also referred to as Aβ peptides), which are internal 39-43 amino acid fragments of amyloid precursor protein (APP) produced by the processing of APP by β and γ secretase enzymes. The Aβ1-42 peptide has the sequence of SEQ ID NO: 1 as follows: Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala.

In the selection and use of antigenic sequences for the design of the DNA plasmids and rVSV constructs of the invention, alteration of codon usage and / or removal of inhibitory sequences of selected antigen coding gene sequences as well as the DNA plasmid into which these sequences are inserted It is also desirable to. Removal of inhibitory sequences is described in US Pat. No. 5,965,726; 5,972,596; 5,972,596; No. 6,174,666; No. 6,291,664; And 6,414,132; And the techniques discussed in International Patent Publication No. WO01 / 46408, which is incorporated herein by reference. Briefly, the technique involves mutating the identified inhibitor / labile sequences in the selected gene, preferably using multiple point mutations.

In one particular embodiment illustrated below, the immunogenic plasmids and rVSV compositions of the present invention preferably comprise one or more sequences optimized to encode HIV-antigens such as gag, pol and nef antigens, or immunogenic fragments or fusions thereof. use. The gag and env genes of chimeric monkey-human immunodeficiency virus (SHIV) (89.6P) are useful for making rVSV, so protection from infection and alleviation of viral load and disease are demonstrated in the Rhesus macaque model. Can be. The following example demonstrates the use of SHIV analogues, namely SIV gag and HIV 89.6P env .

D. Promoters Useful in Immunogenic DNA Plasmids or rVSV Constructs

Suitable promoters for use in any of the components of the present invention can be readily selected from constitutive promoters, inducible promoters, tissue-specific promoters and the like. Examples of constitutive promoters that are nonspecific in activity and used in nucleic acids encoding antigens of the invention include, without limitation, retroviral Laus sarcoma virus (RSV) promoter, retroviral LTR promoter (optionally used with RSV enhancer), Cytomegalovirus (CMV) promoter (optionally used with CMV enhancer) (Boshart et al , 1985 Cell , 41 : 521-530), SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phospho Glycerol kinase (PGK) promoter and EF1α promoter (Invitrogen). Inducible promoters regulated by externally supplied compounds include, but are not limited to, arabinose promoter, zinc-induced sheep metallothionine (MT) promoter, dexamethasone (Dex) -induced mouse mammary tumor virus (MMTV) Promoter, T7 polymerase promoter system (WO 98/10088); Thalpihormone insect promoters [No et al , 1996 Proc. Natl. Acad. Sci. USA , 93 : 3346-3351], tetracycline - inhibitory system [Gossen et al , 1992 Proc. Natl. Acad. Sci. USA , 89 : 5547-5551], tetracycline-induced systems (Gossen et al , 1995 Science , 268: 1766-1769, also Harvey et al , 1998 Curr. Opin. Chem. Biol. , 2: 512-518], RU486-inducible system (Wang et al , 1997 Nat. Biotech. , 15: 239-243 and Wang et al , 1997 Gene Ther. , 4: 432-441] and rapamycin-induced systems (Magari et al , 1997 J. Clin. Invest. , 100 : 2865-2872].

Another kind of inducible promoter that may be useful in this category is a promoter that is regulated by certain physiological conditions, eg, temperature or acute phase, or only in replicating cells. Useful tissue-specific promoters include synthetic muscle promoters that are more active than skeletal muscle β-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, and natural promoters. Li et al ., 1999 Nat. Biotech ., 17 : 241-245. Examples of tissue-specific promoters are particularly described in liver [albumin, Miyatake et al . 1997 J. Virol ., 71 : 5124-32; Hepatitis B virus core promoter, Sandig et al ., 1996 Gene Ther ., 3 : 1002-9; Alpha-fetoprotein (AFP), Arbuthnot et al ., 1996 Hum. Gene Ther ., 7 : 1503-14], bone [osteocalcin, Stein et al ., 1997 Mol. Biol. Rep ., 24 : 185-96; bone sialoprotein, Chen et al ., 1996 J. Bone Miner. Res ., 11 : 654-64, lymphocytes [CD2, Hansal et al ., 1988 J. Immunol ., 161 : 1063-8; Immunoglobulin heavy chains; T cell receptor α chain], neurons [neuron-specific enolase (NSE) promoter, Andersen et al . 1993 Cell. Mol. Neurobiol ., 13 : 503-15; Neurofibrillary light chain gene, Piccioli et al ., 1991 Proc. Natl. Acad. Sci. USA , 88 : 5611-5; Promoters for neuron-specific ngf gene, Piccioli et al ., 1995 Neuron , 15 : 373-84 are known. For further listings useful in this category, see International Patent Publication No. WO00 / 55335.

E. Carriers, Excipients, Adjuvant and Formulations Useful in Immunogenic Compositions of the Invention

Immunogenic compositions useful in the present invention further comprise immunologically acceptable diluents or pharmaceutically acceptable carriers such as sterile water or sterile isotonic saline regardless of whether they are DNA plasmid compositions or rVSV compositions. Include. Immunogenic compositions may also be mixed with such diluents or carriers in conventional manner. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, and the like. Suitable carriers are apparent to those skilled in the art and depend largely on the route of administration.

Other additional ingredients that may be present in the immunogenic compositions of the invention are adjuvants, preservatives, surfactants and chemical stabilizers, suspending agents or dispersing agents. Typically, stabilizers, adjuvants and preservatives are optimized to determine the best formulation for efficacy in the subject human or animal.

1. Supplements

Adjuvants are substances that enhance the immune response when administered in combination with an immunogen or antigen. Interleukin 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (US Pat. No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and mutant forms thereof) ), Interferon-α, β and γ, granulocyte-macrophage colony stimulating factor (US Pat. No. 5,078,996 and ATCC Accession No. 39900), macrophage colony stimulating factor, granulocyte colony stimulating factor, GSF and tumor necrosis factor α and Numerous cytokines or lymphokines, including but not limited to β, have immunomodulatory activity and can therefore be used as adjuvants. Other adjuvants useful in the present invention include chemokines, including but not limited to MCP-1, MIP-1α, MIP-1β and RANTES. Selective molecules such as L-selectin, P-selectin and E-selectin may also be useful as adjuvants. Still other useful adjuvants include, but are not limited to members of mucin-like molecules, such as integrin sequences such as CD34, GlyCAM-1 and MadCAM-1, LFA-1, VLA-1, Mac-1 and p150.95. ; Members of immunoglobulin superfamily such as PECAM, ICAM (eg ICAM-1, ICAM-2 and ICAM-3), CD2 and LFA-3; Costimulatory molecules such as CD40 and CD40L; Growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epithelial growth factor, B7.2, PDGF, BL-1 and vascular endothelial growth factor; Receptor molecules comprising Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2 and DR6 Included. Another adjuvant molecule includes caspase (ICE) (see, eg, International Patent Publications WO98 / 17799 and WO99 / 43839, which are incorporated herein by reference).

Suitable adjuvants for enhancing the immune response include, without limitation, MPL ^™ (3-O-deacylated monophosphoryl lipid A; described in US Pat. No. 4,912,094, which is incorporated herein by reference; manufactured by Coryxa ), Hamilton, MT). Synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, also available from Hamilton, MT and described in US Pat. No. 6,113,918, which is incorporated herein by reference. Suitable for use as an adjuvant. One such AGP is 2-[(R) -3-tetradecanoyloxytetradecarbonylamino] ethyl 2-deoxy-4-O-phosphono-3-, also known as 529 (formerly known as RC529). O-[(R) -3-tetradecaneoxytetradecanoyl] -2-[(R) -3-tetradecanoyloxytetradecanoyl-amino] -bD-glucopyranoside. The 529 adjuvant is formulated in an aqueous form or as a stable emulsion.

Other auxiliaries include mineral oil and water emulsions, aluminum salts (e.g. aluminum hydroxide, aluminum phosphate), ampigen, Avridine, L121 / squalene, D-lactide-polylactide / Glucosides, pluronic polyols, muramyl dipeptides, killed bordetella, saponins described in US Pat. No. 5,057,540, which is incorporated herein by reference, such as Stimulon QS-21 (Antigenics, Framingham , MA.)) And particles produced therefrom, for example, ISCOMS (immunostimulatory complexes), mycobacterium tuberculosis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligos containing CpG motifs Nucleotides; see US Pat. No. 6,207,646; Which is incorporated herein by reference], whooping cough toxin (PT) or E. E. coli heat-labile toxins (LT), in particular LT-K63, LT-R72, PT-K9 / G129 (WO 93/13302 and WO 92/19265, which are incorporated herein by reference) Is included.

In addition, cholera toxins, including those described in WO 00/18434, wherein glutamic acid at amino acid position 29 is replaced by another amino acid (other than aspartic acid), preferably histidine, and Mutants thereof are useful as adjuvants. Similar CT toxins or mutants are disclosed in WO 02/098368, wherein isoleucine at amino acid position 16 is replaced with another amino acid alone or with substitution of serine at amino acid position 68, or amino acid 72 Valine at position is replaced with another amino acid). Other CT toxins are disclosed in WO 02/098369, wherein arginine at amino acid position 25 is replaced with another amino acid, and / or amino acid is inserted at amino acid position 49, and two amino acids are amino acid 35 And inserted at position 36).

In one embodiment exemplified below, preferably the adjuvant is IL-12 expressed from a plasmid. See US Pat. No. 5,457,038, which is incorporated herein by reference; 5,648,467; 5,648,467; 5,723,127 and 6,168,923. Such IL-12 expressing plasmids are incorporated into the immunogenic DNA plasmid-containing priming compositions of the examples. However, the plasmid is administered to a mammalian or vertebrate host alone with the rVSV composition (or expressed by rVSV) or between the priming composition and the boosting composition. In a preferred embodiment, the cytokine is administered as a nucleic acid composition comprising the DNA sequence encoding the cytokine under the control of regulatory sequences directing expression in mammalian cells. In another useful embodiment, the cytokine expression plasmid is administered with the DNA composition. In another embodiment, the cytokine is administered between the prime and the boosting composition. In another step, the cytokine is administered in conjunction with the boosting step. In another embodiment, the cytokine is administered with both the priming composition and the boosting composition.

If the priming composition is a DNA plasmid composition, as in the examples below, the plasmid composition may comprise a DNA sequence encoding a cytokine under the control of regulatory sequences directing expression in mammalian cells. In some embodiments, the cytokine-encoding sequence is on the same DNA plasmid as the antigen coding sequence. In other embodiments, the cytokine coding sequence is on a DNA plasmid different from the DNA plasmid encoding the antigen.

2. Accelerators or Co-Formulations

In addition to the carriers as described above, immunogenic compositions consisting of polynucleotide molecules preferably comprise any polynucleotide promoter or "co-agent", eg, local anesthetics, peptides, cationic lipids, liposomes or lipid particles. Lipids, polyvalent cations such as polylysine, branched three-dimensional polycations such as dendrimers, carbohydrates, cationic amphiphiles, detergents, benzyl ammonium surfactants or other compounds that promote polynucleotide delivery to cells. Such promoters include local anesthetic bupivacaine or tetracaine (see US Pat. No. 5,593,972; 5,817,637; 5,817,637; 5,380,876; 5,380,876; 5,981,505 and 6,383,512 and International Patent Publication No. WO98 / 17799, which is incorporated herein by reference. Other non-exclusive examples of such promoters or co-formulations useful in the present invention include US Pat. No. 5,703,055; No. 739,118; , 837,533; International Patent Publication No. WO96 / 10038, published April 4, 1996; And International Patent Publication No. WO94 / 16737, published August 8, 1994, each of which is incorporated herein by reference.

Most preferably, local anesthetics are present in an amount to form one or more complexes with the nucleic acid molecule. When a local anesthetic is mixed with a nucleic acid molecule or plasmid of the present invention, it encapsulates DNA and forms various small complexes or particles that are homogeneous. Thus, in one embodiment of the immunogenic compositions of the invention, the complex is formed by mixing a local anesthetic with at least one plasmid of the invention. The single complex resulting from this mixture may contain various combinations of different plasmids. Alternatively, in another embodiment of the composition of the present invention, the local anesthetic may be mixed separately with each plasmid. Then, when all plasmids are administered as a single cyclic administration, the individual mixtures are combined as a single composition to ensure that the desired ratio of plasmids are present in a single immunogenic composition. Alternatively, local anesthetics and respective plasmids can be mixed separately and administered separately to obtain the desired ratio.

Where the term “complex” or “one or more complexes” or “complexes” is subsequently used to define this aspect of an immunogenic composition, the term is understood to include one or more complexes. Each complex contains a mixture of plasmids or a mixture of complexes formed separately. Each complex may contain only one type of plasmid or complex, or a mixture of plasmids or complexes, wherein each complex contains polycistronic DNA. Preferably, the composite is about 50 to about 150 nm in diameter. If the promoter used is a local anesthetic, preferably bupivacaine, an amount of from about 0.1% to about 1.0% by weight based on the total weight of the polynucleotide composition is preferred. See International Patent Publication No. WO99 / 21591, which is incorporated herein by reference and teaches incorporation of benzylammonium surfactants, preferably administered in an amount of about 0.001 to 0.03% by weight as a co-agent. According to the invention, the amount of local anesthetic is present in a ratio of 0.01 to 2.5% w / v local anesthetic to 1 to 10 μg / ml nucleic acid relative to the nucleic acid molecule. Another such range is 0.05-1.25% w / v local anesthetic versus 100 μg / ml-1 mg / ml nucleic acid.

3. Other Additives to Immunogenic Compositions

Other additives may be included in the immunogenic compositions of the invention, including preservatives, stabilizing components, surfactants, and the like.

Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol and parachlorophenol.

Suitable stabilizing components that can be used include, for example, casamino acid, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate and milk powder.

Suitable surfactant materials include, but are not limited to Freund's complete adjuvant, quinone analogues, hexadecylamine, octadecylamine, octadecylamino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide, methoxyhexadecylglycerol and fluro Nick polyols; Polyamines such as pyran, dextransulfate, poly IC, carbopol; Peptides such as muramyl peptides and dipeptides, dimethylglycine, tuftsin; Oil emulsions; And mineral gels such as aluminum phosphate and the like and immunostimulatory complexes (ISCOMS). The plasmid and rVSV can also be incorporated into liposomes for use as immunogenic compositions. The immunogenic composition may contain other additives suitable for the selected mode of administration of the composition. Compositions of the present invention may also include lyophilized polynucleotides that can be used with other pharmaceutically acceptable excipients for developing powders, solutions or suspensions. Remington: The Science and Practice of Pharmacy, Vol. 2, 19 ^th edition (1995), for example Chapter 95 Aerosols; And International Patent Publication No. WO99 / 45966, the teachings of which are incorporated herein by reference.

Such immunogenic compositions may contain additives suitable for administration via any conventional route of administration. In some preferred embodiments, the immunogenic compositions of the invention are prepared for human subject administration, for example in the form of solutions, powders, aerosols, tablets, capsules, enteric coated tablets or capsules or suppositories. Thus, immunogenic compositions also include, but are not limited to, suppositories, solvents, emulsions, pastes, and implanted sustained release or biodegradable formulations in oily or aqueous vehicles. In one embodiment of the formulation for parenteral administration, the active ingredient is provided in anhydrous (ie, powder or granule) form for reconstitution of the reconstituted composition into a suitable vehicle (eg, sterile pyrogen-free water) prior to parenteral administration. do. Other useful, parenteral administrable formulations include formulations comprising the active ingredient in microcrystalline form, as a liposome preparation, or as the active ingredient in a biodegradable polymer system. Sustained release or implantable compositions may include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, poorly soluble polymers or poorly soluble salts.

The immunogenic compositions of the invention are not limited by the choice of conventional physiologically acceptable excipients, adjuvants or other ingredients useful in pharmaceutical formulations of the above types. The preparation of these pharmaceutically acceptable compositions from the aforementioned ingredients, with appropriate pH isotonicity, stability and other conventional characteristics, is within the skill of the art.

F. Dosage and Route for the Immunogenic Compositions of the Invention

In general, the selection of an appropriate “effective amount” or dosage for the components of an immunogenic composition of the invention also determines the type of antigen in the immunogenic composition (s) used, as well as most particularly the overall health of the immunized subject. And physical condition of the subject, including age and weight. The method and route of administration and the presence of additional components in the immunogenic composition can also affect the dosage and amount of the plasmid and rVSV composition. Selection and up or down adjustment of such effective amounts are within the skill of the art. The amount of plasmid and rVSV required to induce an immune response, preferably a protective response or cause an exogenous effect in the patient without significant side effects depends on these factors. Suitable dosages are readily determined by one skilled in the art.

The antigenic or immunogenic compositions of the invention are intranasal, oral, vaginal, rectal, parenteral, intradermal, transdermal (WO 98/20734, which is incorporated herein by reference), intramuscular, peritoneal Administration to humans or non-human vertebrates is via a variety of routes, including intra, subcutaneous, intravenous and intraarterial administration. The appropriate route is selected by the attending physician according to the nature of the immunogenic composition used and the assessment of the age, weight, sex and general health of the patient and the antigens and similar factors present in the immunogenic composition.

In the examples provided below, the immunogenic DNA composition is administered intramuscularly (i.m.). In one embodiment, it is preferred to administer the rVSV composition intranasally rather than intramuscularly. However, the dosage and mode of administration are not limited in the present invention.

Likewise, the order of administration of the immunogenic compositions and the time period between individual administrations can be selected by the attending physician and the person skilled in the art according to the exact mode and physical characteristics of the host for the application of the method. Such optimization is expected to be within the skill of the art.

G. Kit Ingredients

In another aspect, the present invention provides a pharmaceutical kit for immediate implementation of an immunogenic, prophylactic or therapeutic regimen for the treatment of a disease or condition noted above, wherein an immune response to the antigen is desired. The kit is designed for use in a method of inducing a high level of antigen-specific immune response in a mammal or vertebrate subject. The kit contains one or more immunogenic compositions comprising a DNA plasmid comprising a DNA sequence expressing an antigen under control of a regulatory sequence directing expression in mammalian or vertebrate cells. Preferably, multiple prepackaged doses of the DNA immunogenic composition are provided in a multi-dose kit. The kit comprises one or more immunogenic compositions comprising a replication competent recombinant bullous stomatitis virus (rVSV) comprising a nucleic acid sequence encoding the same antigen under control of a regulatory sequence directing expression in a mammalian or vertebrate subject. It contains. Preferably, multiple prepackaged doses of the rVSV immunogenic composition are provided in a multi-dose kit.

If the above immunogenic composition also does not contain a DNA plasmid and / or rVSV expressing a cytokine such as IL-12, the kit may optionally contain multiple prepackaged doses of individual cytokine compositions or multiple dose cytokine compositions. It also contains. Such cytokine compositions are generally nucleic acid compositions that comprise a DNA sequence encoding a selected cytokine under the control of regulatory sequences directing expression in a mammal or vertebrate subject.

The kit also contains instructions regarding the use of the immunogenic composition in the prime / boost method described herein. The kit may include instructions for carrying out a particular assay, various carriers, excipients, diluents, adjuvants, and the like, as well as devices for administering the composition, such as syringes, spray devices, and the like. Other ingredients may include disposable gloves, decontamination instructions, swabs or containers, in particular compositions.

In order to better understand the present invention, the following examples are presented. The examples are for illustrative purposes only and are not intended to limit the scope of the invention. All publications, publications, and patents cited in the following examples are incorporated herein by reference.

As demonstrated in the examples below, the prime / boost protocol of the present invention induces a surprising synergistic effect on antigen-specific cellular and humoral immune responses in immunized subjects. Indeed, the synergistic nature of the response to the composition of the present invention when these reactions induced by the prime / boost protocol of the present invention are compared with the results of administration of only a large number of priming compositions or a plurality of boosting compositions Is fairly obvious. Combined preferred antigen presentation, followed by administration of the DNA plasmid composition followed by rVSV boost administration, results in an increase in antigen-specific T cells in the immunized subject, which increase significantly exceeds any additional response. Likewise, the increase in humoral response to the desired antigen is unexpectedly high when both DNA plasmids and rVSV immunogenic compositions are used in the immunization protocol. See, for example, Table 1 below and FIGS. 4 and 5.

Example 1 Preparation of DNA Plasmids

This example describes exemplary plasmids useful in one aspect of the invention set forth in Examples 2 and 3. These plasmids are not limited in the present invention but have been optimized for use in subsequent experiments. The following DNA immunogenic compositions were designed using standard recombinant DNA techniques. Expressing HIV or SIV gag gene DNA backbone vectors include HCMV promoter, BGH poly A termination sequence, Co1E1 bacterial replication origin (ori); And kanamycin resistance gene for selection.

A. SIV gag p37 DNA plasmid

Plasmid WLV102 is a bacterial plasmid that expresses SIV gag p37 as the selected antigen for which an immune response is desired. Plasmid WLV102 (4383 bp) is an RNA optimized truncated gag gene (p37) from SIV inserted into DNA plasmid expression vector WLV001 [see; Qiu et al , 1999 J. Virol., 73 : 9145-9152. gag gene, US Pat. No. 5,965,726; 5,972,596; No. 6,174,666; 6,291,664; And 6,414,132; And RNA optimization by inactivating inhibitory sequences using the techniques described in detail in WO 01/46408, which is incorporated herein by reference, to allow the gag gene to be expressed at a high level of Rev independent.

The WLV102 plasmid backbone consists of three genetic units. The first genetic unit is a genetic element from the HCMV early early promoter / enhancer, see Boshart et al . 1985, cited above] and BGH polyadenylation signals (Goodwin EC and Rottman FM, 1982 J. Biol. Chem. 267 : 16330-16334]. The gag gene is cloned between SalI and EcoRI sites. The second component is the chimeric kanamycin resistance gene ( km ^r ) gene, adenyl 4′-nucleotidyl transferase type 1a (US Pat. No. 5,851,804). The gene is designed to confer resistance to a limited number of aminoglycosides, which allows the selection of bacteria containing plasmids. The third component is the ColE1 bacterial replication origin required for the propagation of the plasmid during bacterial fermentation. This plasmid is illustrated in Figure 1A.

B. Plasmids Encoding Cytokine-rIL-12 DNA

The Resus IL-12 WLV104 plasmid is a dual promoter construct that expresses a heterodimeric form of Resus IL-12. Plasmid WLV103 is a dual promoter expression plasmid consisting of two genes encoding human IL12 proteins p35 and p40. The plasmid has a total of 6259 nucleotides. Each cistron in WLV103 containing two interleukin-12 units p35 or p40 is under the control of an individual regulatory element. The p35 subunit is under the control of the HCMV promoter / enhancer and the SV40 polyadenylation signal (cloned between SalI and MluI sites). The p40 subunit is under the control of the SCMV promoter and has a BGH polyadenylation signal (cloned into the XhoI site).

The plasmid backbone consists of several components. The first component is the genetic element from the HCMV early promoter / enhancer and the SV 40 polyadenylation signal [see; Fitzgerald M and Shenk T., 1981 Cell, 24 : 251-260]. The second component is the SCMV promoter, see Jeang et al . 1987 J. Virol., 61 : 1559-1570] and a BGH polyadenylation signal. The third component is the chimeric kanamycin resistance gene ( km ^r ) gene, adenyl 4 ′ nucleotidyl transferase type 1a. The fourth component is the ColE1 bacterial origin of replication required for plasmid propagation in bacteria.

The resulting plasmid vectors SIV gag / HIV gag and IL-12 DNA were analyzed using restriction enzyme digestion. The plasmids were transiently transfected into Rhabdosarcoma cells grown in DMEM + 10% fetal calf serum (FCS) medium and antibiotics. The cells were then subjected to appropriate expression of viral proteins using specific monoclonal antibodies against HIV gag (ABI) and polyclonal serum from Resus (from SIV infected animals) in SIV gag in Western blot assays. Was analyzed. IL-12 was detected in the supernatant using an ELISA kit (R & D Systems) that detects p70 protein.

Plasmid vectors were grown in transformed DH10B cells grown in LB medium supplemented with kanamycin and purified using a Qiagen kit according to the manufacturer's wish. DNA was then analyzed by electrophoresis on agarose gel against known standards.

Each plasmid was formulated at a total volume of 4.0 cc at a concentration of 2.5 mg / ml in 0.25% bupivacaine for use in the following examples.

Example 2: Preparation of Recombinant VSV Vectors

A. VSV Genome cDNA Cloning

The genetic background for VSV genomic cDNA engineering is pVSV-XN1 (Schnell, MJ, et al 1996 J. Virol., 70 : 2318-23). The clone contains a modified form of the VSV Indiana strain (VSV _i ) cDNA sequence. Modifications include the addition of two unique restriction endonuclease recognition sites (XhoI and NheI) and the added VSV gene-initiation and VSV gene-termination signal copy. If foreign genes such as HIV-1 _89.6p env gp160 or SIV gag p55 or HIV-1 gag are inserted between the XhoI and NheI sites for convenience, these foreign genes are present at positions suitable for expression controlled by VSV transcriptional control signals. . In addition, the VSV cDNA sequence is flanked by cis-acting DNA sequences required to facilitate rescue of live viral copies. T7 RNA polymerase promoter directs the transcription of primary transcripts across viral cDNA. Ribozymes cleave the primary transcript after the T7 RNA polymerase terminates transcription to form the ends of the RNA genome.

First, the gag gene (HIV Clade B) was inserted between the G and L genes to modify the rVSV _i genomic clone (pVSV-XN1) to prepare plasmid prVSVi- gag . Similarly, separate rVSV _i cDNA clones containing the HIV env gene (genomic clone prVSVi-env) were prepared. Gag and env cDNA sequences were prepared for amplification of coding region sequences from plasmid templates containing HIV HXBc2 gag gene or HIV 6101 strain env gene and for insertion into pVSV-XN1. Primers used for PCR amplification contain terminal restriction enzyme cleavage sites suitable for subsequent cloning; The 5 'primer contained the XhoI site and the 3' primer contained the NheI site. The amplified coding region sequences were inserted individually into pVSV-XN1 to produce clones containing gag and clones containing env .

The env gene inserted into pVSV-XN1 was a modified form encoding the HIV Env / VSV G fusion protein. Cell surface expression of Env after infection with the rVSV-HIV Env vector has been shown to be enhanced when the cytoplasmic tail of Env is replaced by a shorter cytoplasmic tail of VSV G protein using redundant PCR procedures. Johnson, et al , 1998, cited above; Johnson et al., 1997, cited above. The vector backbone of these two plasmids was altered by changing the Indiana G gene to a gene of Candidipura or New Jersey serotype according to published techniques. G gene exchange was achieved using a unique MluI site in the M gene and an engineered XhoI site in VSV cDNA clones.

VSV G gene coding sequences from Candidipura strains or New Jersey strains were PCR amplified using 5 'primers extending across the MluI site in the M gene. 3 'PCR primers were used that contain XhoI sites as well as sequences homologous to the 3' end of the G gene, as well as additional sequences corresponding to the gene-terminal / gene-initiation signal. The original Indiana G gene was cleaved from the plasmid backbone using MluI and XhoI, and then the G gene coding sequence amplified with the primers was used to replace the original Indian G gene.

B. Rescue of rVSV from cDNA Clone

RNA transcribed from the genomic cDNA plasmid or genomic cDNA plasmid was not sufficient to initiate the viral replication cycle after being introduced into the cell. Viral RNA was not in itself an active template for translation or replication. Thus, recombinant viruses must be recovered from various VSV canine cDNA constructs. Remedies known in the art allow for the recovery of viruses from cloned DNA. Successful viral rescue indicates that VSV genomic RNA is present intracellularly with the VSV N protein surrounding the viral genomic RNA, as well as the P and L proteins that form the viral RNA-dependent RNA polymerase required for viral mRNA synthesis and genome replication. It was necessary. Production of all these viral components in cultured cells was achieved by cotransfection of the plasmid for VSV genomic cDNA and the expression plasmid encoding VSV N, P and L proteins.

All these plasmids are designed for transcription by phage polymerase T7 RNA; Thus, the transfected cells were also infected with recombinant vaccinia virus expressing phage polymerase (MVA / T7 or VTF7-3). Standard procedures used for VSV rescue are described in Lawson et al . 1995, cited above; And Schnell et al ., 1996 J. Virol., 70 : 2318-23. This process has been modified to be more efficient for rescue of highly attenuated viruses and to make the process consistent with regulatory guidelines. In brief, the method is described as follows.

Qualified Vero cells in 12.5 cm ² flasks were transfected with rVSV genomic clones and supported plasmids encoding VSV N, P and L using a calcium phosphate transfection procedure. When initiating transfection, sufficient certified MVA / T7 was added to provide multiplicity of infection of 2 plaque-forming units per cell. Three hours after initiation of transfection, cells were subjected to a three hour heat shock step to improve the rescue efficiency of some negative chain RNA viruses (Parks, CL et al, 1999 J. Virol., 73 : 3560-6). 48-72 hours after transfection, cells and culture medium were transferred to larger flasks containing established monolayers of Vero cells and then incubated for at least one day to allow amplified rescued virus. After this amplification step, the collected virus was filtered to remove the most contaminating VSV / T7 and used for clonal isolation of rVSV strains.

The nucleotide sequence of the viral genome was determined using the consensus sequencing method to analyze the RNA viral genome. Briefly, purified viral RNA was reverse transcribed to generate cDNA, and then overlapping regions of the genome were amplified by PCR using gene-specific primers. The PCR product was gel purified and then cycle-sequenced using fluorescent dye-terminator. Sequencing reaction products were purified and analyzed in an automated sequencer.

C. Specific Constructs Used in the Examples

To prepare specific recombinant VSV vectors used in the following experiments, recombinant VSV encoding HIV-1 89.6P gp160 (rVSV HIV-1envG) and SIVmac239 gag p55 (rVSV SIVgag) fused to the transmembrane region of the VSV G protein. Were mixed and used as experimental immunogenic composition. RVSV (rVSV fluHA) encoding influenza hemagglutinin (flu HA) protein was used as a control composition. Recombinant VSV vectors have already been generated using genes encoding the desired antigen inserted between VSV G and L transcription units. Prepared as described. Rose et al, 2000 J. Virol. 74: 10903-10. The following constructs are described in Rose et al, supra.

1. Plasmid Construction

Plasmids containing the candipura glycoprotein [ G (Ch)] gene (Masters, PS, 1989 Virol., 171 : 285-290) were kindly provided by Amiya Banerjee, Cleveland Clinic. To construct a VSV vector containing a G (Ch) gene in place of the Indiana glycoprotein [ G (I)] gene, the Xho I site was first prepared using the complementary primers 5'-CCCCTAGTGGGATCTCCAGTGATATTTGGAC (SEQ ID NO: 2) and 5'-GTCCAAATATCACTGGAGATCCCACTAGGGG (SEQ ID NO: 3) and using oligonucleotide-directed mutagenesis using the Stratagene QuikChange mutagenesis kit. Removed in G (Ch) gene. Mutation of CTC G AG (SEQ ID NO: 4) to CTC C AG (SEQ ID NO: 5) removed the Xho I site without affecting the amino acid sequence of G (Ch). The gene was then amplified by PCR using Vent DNA polymerase (New England Biolabs). The forward primer was 5'-GATCGATCGAATTC ACGCGT AACATGACTTCTTCAG (SEQ ID NO: 6) containing the Mlu I site (underlined) downstream of the ATG start codon for the G (Ch) protein. The reverse primer was 5'-GAACG GTCGA CGCGC CTCGAG CG TGATATCTGTT AG TTTTTTTCATA TCATGTTGTTGGGCTTGAAGATC (SEQ ID NO: 7) followed by the VSV transcription initiation and termination signal following the complement of the 3 'coding sequence of G (Ch) (underlined). Sal I and Xho I sites (in bold).

PCR product was digested with Mlu I and Sal I and digested with Mlu I and Xho I to VSV G (I) The coding sequence was cloned ( Sal I and Xho I leave a suitable terminal for ligation) and cloned into the pVSVXN-1 vector (Schnell, et al 1996 J. Virol. 70 : 2318-2323] The plasmid derived by this method was named pVSV (GCh) XN-1, which expresses foreign genes flanked by Xho I and Nhe I sites unique between the G (Ch) and L genes. Contains site

Plasmid using the same procedure as described above Vectors containing the G (NJ) protein gene were generated from pNJG. Gallione, CJ, and JK Rose. 1983 J. Virol. 46 : 162-169. Forward primer was 5'-GATCGATCGAA TTC ACGCGT AATATGTTGTCTTATCTAATCTTTGC (SEQ ID NO: 8), and reverse primer was 5'-GGAACG GTCGA CGCGC CTCGAG CG TGATATCTGTT AG TTTTTTTCATA TTAACGGAAATGAGCCATTTCCACG (SEQ ID NO: 9). Sites indicated in bold and underlined sequences are for the candipura construct described above, with subsequent cloning steps also as described above. The final vector plasmid derived was named pVSV (GNJ) XN-1, which is the G (NJ) gene and VSV L Xho I unique between genes and It contains a foreign gene expression site flanked by the Nhe I site.

To generate a vector containing the HIV Env 89.6 G gene, VSV Genes encoding 89.6 envelope proteins with G cytoplasmic tails were cut from pVSV-89.6gp160G using Xho I and Nhe I (Johnson et al 1997, cited above) vector pVSV (GNJ) XN-1 Or cloned between Xho I and Nhe I sites in pVSV (GCh) XN-1. The gp160G gene encodes all the transmembrane and transmembrane domains of all gp120 and gp41, and four of the 89.6 Env cytoplasmic domains fused to the 26 C-terminal amino acids of the VSV G cytoplasmic domain starting with the sequence IHLC (SEQ ID NO: 11). Amino acid (NRVR) (SEQ ID NO: 10).

2. Recovery of Recombinant Virus

Recombinant VSV was recovered using established methods (Lawson et al 1995, cited above). Briefly, young hamster kidney (BHK) cells were grown to about 60% confluency in a 10-cm dish. The cells were then infected with vTF7-3, a recombinant vaccinia virus that expresses a T7 RNA polymerase at 10 infection multiplicity (MOI) . Fuerst et al 1987 Mol. Cell. Biol. 7: 2538-2544. After 1 hour, the plate of each cell was placed into , plasmid encoding 5 μg of pBS-P, 1 μg of pBS-L, and one of the three full length recombinants as described above 10 μg transfection. Transfection was carried out using cationic liposome reagents containing dimethyldioctadecyl ammonium bromide and dioleyl-phosphatidylethanolamine (Rose et al, 1991 Biotechniques 10: 520-525). The cells were then incubated at 38 ° C. for 48 hours. The cell supernatant was passed through a 0.2-μm filter to remove the majority of vaccinia virus and then applied to fresh BHK cells at 37 ° C. for 48 hours. For some recoveries additional plasmids encoding VSV G (pBS-G), 4 μg / plate, were included along with N, P and L support plasmids. Recovery of the infectious virus was confirmed by scanning the BHK cell monolayer for VSV cytopathic effect. Viral plaques were then isolated from BHK cells and virus from a single plaque was added to 10-cm diameter plates of BHK cells to propagate virus stock from individual plaques. The stock was then stored at 80 ° C. The titers obtained for VSV (GI) -89.6G, VSV (GCh) -89.6G, and VSV (GNJ) -89.6G are both 10 ⁷ to 10 ⁸ PFU after freezing and thawing, a process that reduces VSV titers by about threefold. It is in the range of / ml.

Each VSV construct was diluted with sterile DME to a final volume of 0.8 cc for use in the next experiment.

Example 3: Prime / Boost Immunization Therapy

A. Immunization Protocol

Resus macaques (5 per group) are either SIV gag p37 polyproteins (groups 1 and 2) or empty DNA plasmids (group 3) with 5 mg of isostronic DNA plasmid encoding Resus IL-12 p35 and IL-12 p40. And 4) immunized by intramuscular injection of 10 mg. All these DNA plasmids and formulations thereof are as described in detail in Example 1. The DNA immunization schedule provides a second booster immunization at weeks 4 and 8 following the initial immunization on day 0. The needles and syringes were used to inject 1cc per site into four sites of the deltoid and quadriceps muscles.

Then, at 15 weeks, the macaques were recombinant vesicular stomatitis virus (rVSV) vector (5 × 10 ⁶ pfu) and SIV gag p55 of serotype Indiana (I) of Example 2 containing the HIV-1 gp160 env gene. Intranasally with a second rVSV (I) (5 × 10 ⁶ pfu) containing genes (groups 2 and 3), or rVSV (I) (1 × 10 ⁷ pfu) containing groups of influenza hemagglutinin genes (groups 1 and 4) Inoculation (0.4 cc / nostril, using a small pipette) was immunized. Macaque again contains the rVSV (serum candipura, Ch) based vector (5 × 10 ⁶ pfu) of Example 2 containing the HIV-1 gp160 env gene and the second rVSV (Ch) containing the SIV gag p55 gene ( Intranasal inoculation (0.4 cc / nostril, small pipette) with 5 × 10 ⁶ pfu) (groups 2 and 3) or rVSV (Ch) (1 × 10 ⁷ pfu) (groups 1 and 4) containing the influenza hemagglutinin gene Boosted).

Macaques were closely monitored for induction of cellular and humoral immune responses in peripheral blood and antibody responses at various mucosal surfaces. This monitoring includes enzyme-linked immunospto assay (ELISpot) of SIV gag p55 peptide pool for gamma interferon, ELISpot and gamma interferon of HIV-1 env 6101 peptide pool for gamma interferon. Performance of ELISpot of VSV N peptide pool. ELISpot assay detected a cellular immune response in PBL.

The humoral immune response was determined by serum (Fig. 4), nasal lavage, rectal secretions and saliva for anti-SIV gag p27 IgG titer by ELISA and anti-HIV-1gp160 env titer by ELISA (data not shown). Data are not presented). For sera, the antibodies used in the ELISA were standard antibodies against the chimeric viruses SHIV89.6 and SHIV89.

B. ELISpot Assay to Detect Cytokine- Secreting Rat and Human Cells

First, to detect and quantify individual antibody-secreting B cells, a filter immune plaque assay, otherwise called enzyme linked immunospot assay (ELISpot), was developed. This technique originally provided a fast and versatile alternative to conventional plaque-forming cell assays. Recent modifications have improved the sensitivity of the ELISpot assay to the extent that cells producing as few as 100 specific protein molecules per second can be detected. This assay uses relatively high concentrations of certain proteins (eg, cytokines) in an environment immediately adjacent to protein-secreting cells. These cell products are captured and detected using high affinity antibodies.

The ELISpot assay uses two high affinity cytokine-specific antibodies, directed against different epitopes on the same cytokine molecule: a combination of two monoclonal antibodies or one monoclonal antibody and one multivalent antiserum . ELISpots generate spots based on colorimetric reactions that detect cytokines secreted by single cells. Spots originally represent the "footprint" of cytokine-producing antibodies. Spots (ie, spot forming cells or SFCs) can be quantified permanently and visually, microscopically or electronically.

ELISpot assays were performed as follows: 96 well flat ELISpot plates (Millipore, Bedford, Mass.) Were subjected to mouse anti-human γ interferon (hIFN-ν monoclonal antibodies (clone 27, BD-Pharmingen, San Diego CA) Coated overnight at a concentration of 1 μg / mL, washed 10 times with 1 × PBS supplemented with 0.25% Tween-20 and blocked for 2 hours with PBS containing 5% fetal calf serum (FBS). Crop peripheral blood lymphocytes (PBL) were isolated from freshly harvested heparinized whole blood by Ficoll-Hypaque density gradient centrifugation and 50 μg / mL Complete culture medium (5% FCS, 2) containing PHA-M (Sigma), a pool of 15 amino acid peptides overlapped by 11 amino acids spanning the entire SIV gag open reading frame (1 μM final peptide concentration) or sole medium resuspended in RPMI 1640 medium supplemented with mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin sulfate, 1 mM sodium pyruvate, 1 mM HEPES, 100 μM non-essential amino acid.

Injection cell number was 2 × 10 ⁵ PBL / well in 100 μl and analyzed in duplicate wells. Cells were incubated at 37 ° C. for 16 hours and then removed from the plates by first washing with deionized water and then 10 times with 1 × PBS containing 0.25% Tween-20. Plates were then treated with rabbit polyclonal anti-hIFN-v biotinylation detector antibody (0.2 μg / well, Biosource, Camarillo, Calif.) Diluted with 1 × PBS containing 1% BSA and at room temperature for 2 hours. Incubated for 2 hours. The plates were then washed with 1 × PBS containing 0.25% Tween-20 and 100 μl of streptavidin-alkaline phosphatase conjugate diluted 1: 500 with 1 × PBS containing 5% FBS and 0.005% Tween-20 per well. (Corporate: Southern Biotech, Birmingham AL) and incubated for an additional 2.5 hours at room temperature. Plates were washed 10 times with 1 × PBS containing 0.25% Tween-20 to remove unbound conjugate. The chromogenic substrate (100 μl / well, 1-step NBT / BCIP, Pierce, Rockford, IL) is then added for 3 to 5 minutes, washed with water, the plate is air dried and the resulting spot is inverted Counting was performed visually using a microscope.

ELISpot assays were performed on the present invention to determine the number of CD8 + T cells (CTL) and CD4 + T cells induced in response to the prime / boost immunization method of the present invention as measured by the production of gamma interferon. This assessment was performed over 25 weeks for the treated macaques (three DNA primes and two VSV boosts).

C. Results

After primary DNA immunization, SIVgag-specific IFN-νELISpot responses were readily detected in 10 SIV gag / rIL-12 DNA immunized macaques (average 254 SFC / 10 ⁶ cells). After the second SIV gag / IL-12 DNA immunization, 10 out of 10 immunized macaques showed a high level of ELISpot response (mean 1133 SFC / 10 ⁶ cells) and a third order of gag / IL-12 DNA. Administration boosted the gag-specific ELISpot response in the majority of animals (average 1506 SFC / 10 ⁶ cells). After the first rVSV HIVenv / rVSV SIVgag boost, the mean SIV gag ELISpot response was significantly higher than the response receiving only a single rVSV HIVenv / rVSV SIVgag immunization (mean 386 SFC / 10 ⁶ cells) (3772 SFC / 10 ⁶ cells). These results support the fact that the use of cytokines enhances DNA prime, thereby increasing the immunogenicity of rVSV immunization.

These results are reported in Table 1 below and in FIGS. 2, 3, 4 and 5. 2 depicts the rVSV N-specific IFN-νELISpot response in unfractionated peripheral blood monocytes (PBMCs) from these animals immunized after 25 weeks. 3 depicts HIVenv 6101-specific IFN-νELISpot responses for the same sample. * Indicates a statistically significant difference occurring at p = 0-.0001. 4 shows the results of serum antibody responses when anti-SIV gag p27 IgG titers by ELISA and anti-HIV-1gp160 env titers by ELISA were measured. The immunization protocol by boosting the DNA plasmid expressing the SIV gag protein and the VSV vector expressing the flu HA protein is represented by (◆). The protocol of the present invention comprising a priming DNA gag plasmid immunization followed by VSV boost expressing HIV gag and env proteins is indicated by (■). Priming immunization with co-control DNA followed by immunization with VSV expressing HIV gag and env proteins is indicated by (▲). Protocols comprising priming immunization with a control DNA plasmid followed by immunization with VSV expressing flu HA protein are indicated by (•). Each group shows results from five animals. The statistically significant difference between groups was p = 0.0073 (*); p = 0.5941 (#) or p = 0.0027 (yen).

5 depicts the mean SIV gag-specific IFN-γELISpot response for the same sample. The protocol of immunization by boosting the DNA plasmid encoding the SIV gag protein and the vector encoding the flu HA protein is indicated by (◆). The protocol of the present invention comprising a priming DNA gag plasmid immunization followed by VSV boost expressing HIV gag and env proteins is indicated by (■). Priming immunization with co-control DNA followed by immunization with VSV expressing HIV gag and env proteins is indicated by (▲). (●) represents a protocol comprising priming immunization with control DNA plasmids followed by immunization with VSV expressing flu HA protein. Each group shows results from five animals. Statistical significant differences between groups are indicated in parentheses for p = 0.0001 and p = 0.0002.

In Table 1 the same results are reported in tabular form.

Mean SIV gag-specific IFN-gamma ELISpot response in Resus macaque after SIV gag / resus IL-12 DNA prime and rVSV-SIVgag / rVSV-HIVenv boost Group Protocol Number / Animal Count  Prime Boost Mean SIV gag-specific IFNγ ELISpot Response (# SFC / 10 ⁶ PBL) ^a 1, prime alone (n = 5) SIVgag / IL-12 DNA VSV FluHA 471 2, prime / boost (n = 5) SIVgag / IL-12 DNA VSV-SIVgag / HIVenv 2,338 3, boost alone (n = 5) Ball dna VSV-SIVgag / HIVenv 420 4, control group (n = 5) Ball dna VSV FluHA 55 ^a Mean SIV gag-specific ELISpot response is reported 25 weeks after initial DNA immunization.

The results of this assay demonstrate the surprising synergistic effect of the prime / boost therapy of the present invention compared to the results of administering only a plurality of priming compositions and only to a plurality of boosting compositions. Combined desired antigen presentation, followed by DNA plasmid followed by rVSV boost, results in an increase in antigen-specific T cells in the immunized subject, which increase significantly exceeds any additional response. Likewise, the increase in humoral response to the desired antigen is unexpectedly high when both DNA plasmids and rVSV immunogenic compositions are used in the immunization protocol. See, for example, Table 1 below and FIGS. 4 and 5.

Example 4 Macaque Model for Immunization Against HIV

Resus (Rh) macaques are immunized according to the same protocol using the prime / boost strategy of Example 3 and the plasmid and VSV vector described in Example 3. About 32 weeks after administration of the first priming composition, maca was challenged with the pathogenic SIV / HIV recombinant virus SHIV89.6P at a dose of 50% monkey infection (MID ₅₀ ) 330 [Reimann et al , 1996 J. .. Virol, 70: 3198-3206; Reimann et al 1996 J. Virol ., 70 : 6922-6928.

After about 50 to 70 days of challenge, animals are monitored for disease. Animals are monitored for induction of cell mediated responses and antibody responses immediately before and 1 and 2 weeks after each immunization. Serum collected before and after 2, 4, 6, 8, and 12 weeks of challenge was tested for neutralizing antibody responses against HIV-1 89.6, 89.6P, 6101, and other Clade B primary isolates. Try it. During the experimental immunization period, CD4 / CD8 counts are monitored immediately before challenge and every two weeks after challenge. Immediately prior to challenge and weekly after challenge, viral load in serum is measured by branched DNA analysis. Other body fluids (vaginal, rectal and nasal secretions and saliva) of animals are tested for cellular and humoral immune responses.

Cell-mediated immune responses to the desired antigens are analyzed using some of the most appropriate cell basic assays, including ⁵¹ Cr-release CTL assay, soluble MHC type I tetramer staining, ELISpot assay and intracellular cytokine assay. Mucosal antibody responses are assessed by ELISA techniques optimized for use with low membrane samples. Serum antibody response is assessed by standard ELISA techniques. In addition, sera from all immunized animals are tested for neutralizing antibody responses against HIV Env 6101 and other primary clade B isolates. FIG. 6 illustrates that the elevated immune response induced with the prime / boost combination of the present invention increased protection from AIDS, as measured by reduced CD4 T-cell loss at least 250 days post challenge.

In addition to monitoring the immune response induced with the immunogenic composition, the propagation potential of the liver viral vector is assessed by measuring the level at which it is released and the time period over which it is released. Nasal washes obtained at frequent intervals during the first three weeks after each immunization are tested for the presence of live VSV. Plasma viral loads are also examined for the presence of live virus copies. FIG. 7 demonstrates that the elevated immune response induced with this prime / boost combination reduced circulating virus in plasma at least 250 days after challenge.

The result of this experiment is that unlike control animals, antigen-specific CD8 + and CD4 + T cells are highly likely to be very high in animals immunized according to the invention. Animals immunized according to the prime / boost methodology of the present invention are expected to develop AIDS in non-immunized animals, while animals remain healthy after HIV exposure.

Comparison of this protocol with other known prime / boost methodologies provides that the methods of the present invention are provided by safety for immunized animals and by immunization alone or multiple DNA priming compositions alone or by one or multiple rVSV vector immunizations alone. It is expected that this will demonstrate an advantage in inducing higher levels of anti-HIV CTL and antibodies. Repeated prime / boost according to the present invention is synergistic and therefore prophylactically beneficial for subjects previously exposed and therapeutically advantageous for subjects already infected with HIV.

All documents cited herein are hereby incorporated by reference. Various modifications and minor changes in the methods and compositions are believed to be apparent to those skilled in the art.

<110> Wyeth <120> Immunogenic composition and methods <130> AM-101319PCT <150> US 60 / 457,876 <151> 2003-03-26 <150> US 60 / 546,733 <151> 2004-02-23 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 42 <212> PRT <213> Artificial <220> <223> beta-amyloid 1-42 peptide <400> 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile             20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala         35 40 <210> 2 <211> 31 <212> DNA <213> Artificial <220> <223> complementary primer <400> 2 cccctagtgg gatctccagt gatatttgga c 31 <210> 3 <211> 31 <212> DNA <213> Artificial <220> <223> complementary primer <400> 3 gtccaaatat cactggagat cccactaggg g 31 <210> 4 <211> 6 <212> DNA <213> Artificial <220> <223> complementary primer <400> 4 ctcgag 6 <210> 5 <211> 6 <212> DNA <213> Artificial <220> <223> complementary primer <400> 5 ctccag 6 <210> 6 <211> 36 <212> DNA <213> Artificial <220> <223> forward primer <400> 6 gatcgatcga attcacgcgt aacatgactt cttcag 36 <210> 7 <211> 70 <212> DNA <213> Artificial <220> <223> reverse primer <400> 7 gaacggtcga cgcgcctcga gcgtgatatc tgttagtttt tttcatatca tgttgttggg 60 cttgaagatc 70 <210> 8 <211> 46 <212> DNA <213> Artificial <220> <223> forward primer <400> 8 gatcgatcga attcacgcgt aatatgttgt cttatctaat ctttgc 46 <210> 9 <211> 73 <212> DNA <213> Artificial <220> <223> reverse primer <400> 9 ggaacggtcg acgcgcctcg agcgtgatat ctgttagttt ttttcatatt aacggaaatg 60 agccatttcc acg 73 <210> 10 <211> 4 <212> PRT HIV Env 89.6 G gene <400> 10 Asn Arg Val Arg One <210> 11 <211> 4 <212> PRT <213> VSV G cytoplasmic tail <400> 11 Ile His Leu Cys One

Claims (45)

(a) administering to a mammalian subject an effective amount of a first composition comprising a DNA plasmid comprising an DNA encoding an antigen under control of a regulatory sequence directing its expression by a DNA plasmid; And (b) an effective amount of a second composition comprising recombinant VSV comprising the nucleic acid sequence encoding said antigen under the control of a regulatory sequence directing its expression by recombinant vesicular stomatitis virus (VSV); A method of inducing an antigen-specific immune response in a mammalian subject, comprising administering to the subject. The method of claim 1, wherein the VSV is replication competent. The method of claim 1, wherein the VSV is non-replicable. The method of claim 1, wherein the first composition is administered to the subject one or more times prior to administering the second composition. The method of claim 4, wherein the second composition is administered to the subject one or more times. The method of claim 1, wherein the second composition is administered to the subject one or more times prior to administering the first composition. The method of claim 6, wherein the first composition is administered to the subject one or more times. The method of claim 1, further comprising administering to the subject an effective amount of cytokine in step (a). The method of claim 8, wherein the cytokine is administered as a nucleic acid composition comprising a DNA plasmid comprising a DNA sequence encoding such cytokine under the control of a regulatory sequence directing its expression by a DNA plasmid. The method of claim 9, wherein the cytokine is selected from the group consisting of IL-12, IL-15, GM-CSF and combinations thereof. The method of claim 9, wherein the cytokine coding sequence is on the same DNA plasmid as the antigen coding sequence. The method of claim 9, wherein the cytokine coding sequence is on a DNA plasmid different from the DNA plasmid encoding the antigen. The method of claim 1, further comprising administering to the mammal an effective amount of cytokine in step (b). The method of claim 13, wherein the cytokine is administered in the form of a protein. The method of claim 14, wherein the cytokine is selected from the group consisting of IL-12, IL-15, GM-CSF, and combinations thereof. The method of claim 1, wherein the antigen is a protein, polypeptide, peptide, fragment or fusion thereof, wherein the protein is derived from a member selected from the group consisting of bacteria, viruses, fungi, parasites, cancer cells, tumor cells, allergens and autologous molecules. How to be. The method of claim 16, wherein the virus is a human or monkey immunodeficiency virus. The method of claim 17, wherein the antigen is selected from the group consisting of gag, pol, env, nef, vpr, vpu, vif and tat , and immunogenic fragments or fusions thereof. The method of claim 1, wherein the first composition comprises one DNA plasmid comprising a DNA sequence encoding one or more copies of the same or different antigens. The method of claim 1, wherein the first composition comprises one or more DNA plasmids where each DNA plasmid encodes the same or different antigens. The method of claim 1, wherein the immune response comprises an increase in CD8 + T cell response to the antigen that is greater than that achieved by administering a DNA plasmid or recombinant VSV alone. The method of claim 1, wherein the immune response comprises a synergistic increase in antibody response to the antigen than is achieved by administering the first or second composition alone. The method of claim 1, wherein the mammalian subject is a primate. The method of claim 23, wherein the mammalian subject is a human. The method of claim 1, further comprising administering at least two DNA plasmids each comprising a sequence encoding a different antigen prior to administering the recombinant VSV in step (a). The method of claim 1, further comprising administering two or more recombinant VSVs in step (b). The method of claim 26, wherein each recombinant VSV has a different VSV serotype from a different VSV G protein, but having the same antigen coding sequence. The method of claim 26, wherein each recombinant VSV has a different antigen coding sequence but has the same VSV G protein. The method of claim 26, wherein each recombinant VSV has a different VSV G protein from a different antigen coding sequence. The method of claim 26, wherein the second recombinant VSV and any additional recombinant VSV are administered as a booster after the first recombinant VSV. The method of claim 30 further comprising administering three or more boosters. The method of claim 1, wherein the DNA plasmid composition is administered in a pharmaceutically acceptable diluent, excipient or carrier. 33. The method of claim 32, wherein the excipient comprises bupivacaine. The method of claim 1, wherein the rVSV composition is administered in a pharmaceutically acceptable diluent, excipient or carrier. (a) a first composition comprising a DNA plasmid comprising a DNA sequence encoding an antigen under the control of a regulatory sequence directing its expression by a DNA plasmid; And (b) at least one recombinant VSV comprising a nucleic acid sequence encoding said antigen under the control of a regulatory sequence directing its expression by recombinant bullous stomatitis virus (VSV). Immunogenic Compositions for Inducing Antigen-Specific Immune Responses. 36. The immunogenic composition of claim 35 wherein the VSV is replication competent. 36. The immunogenic composition of claim 35 wherein the VSV is non-replicating. 36. The immunogenic composition of claim 35 further comprising a cytokine composition. The immunogenic composition of claim 38, wherein the cytokine composition comprises a nucleic acid composition comprising a DNA sequence encoding a cytokine under the control of a regulatory sequence directing its expression by a DNA plasmid.  At least one first composition comprising a DNA plasmid comprising a DNA sequence encoding an antigen under control of a regulatory sequence directing its expression by a DNA plasmid; At least one second composition comprising a recombinant VSV comprising a nucleic acid sequence encoding said antigen under control of a regulatory sequence directing its expression by recombinant bullous stomatitis virus (VSV); And A kit for use in a method of inducing an antigen-specific immune response in a mammalian subject comprising instructions for carrying out the method according to claim 1. The kit of claim 40, wherein the VSV is replication competent. The kit of claim 40, wherein the VSV is non-replicable. 41. The kit of claim 40, further comprising a cytokine composition. The kit of claim 43 wherein the cytokine composition comprises a nucleic acid composition containing a DNA plasmid comprising a DNA sequence encoding a cytokine under the control of a regulatory sequence directing its expression by a DNA plasmid. In the manufacture of a medicament for inducing an immune response against an antigen in an animal, (a) a first comprising a DNA plasmid comprising the DNA sequence encoding the antigen under the control of a regulatory sequence directing its expression by a DNA plasmid Composition; And (b) the nucleic acid sequence encoding said antigen is under the control of a regulatory sequence directing its expression by recombinant bullous stomatitis virus (VSV).

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