Attenuated recombinant vesicular stomatitis virus vaccine vectors comprising modified matrix proteins

Abstract

The present invention relates to vesicular stomatitis virus (VSV) matrix (M) protein mutants. One mutant M protein includes a glycine changed to a glutamic acid at position 21, a leucine changed to a phenylalanine at position 111 and a methionine changed to an arginine at position 51. Another M protein mutant includes a glycine changed to a glutamic acid at position 22 and a methionine changed to an arginine at positions 48 and 51. Yet another VSV M protein mutant includes a glycine changed to a glutamic acid at position 22, a leucine changed to a phenylalanine at position 110 and a methionine changed to an arginine at positions 48 and 51. The present invention is directed also to recombinant VSVs (rVSV) having these M mutants and to vaccines based on the rVSV having the M mutants of the present invention. These new rVSVs having the mutant M were significantly attenuated and lost virulence, including neurovirulence, and are capable of inducing an immune responses against an antigen of interest. In addition, a rVSV serotype Indiana having the first described M mutant is capable of efficient replication at 31 C., and of poor replication or incapable of replication at about 37 C. or higher.

Claims

1. A prime boost combination vaccine, wherein the prime boost combination vaccine comprises: (a) an effective amount of a vaccine comprising an attenuated recombinant vesicular stomatitis virus (rVSV) of one serotype having a first modified M protein comprising the amino acid sequence of SEQ ID NO:4; and (b) an effective amount of a vaccine comprising a rVSV of another serotype having a second modified M protein comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

2. The prime boost combination vaccine of claim 1, wherein (a) is a priming vaccine and (b) is a booster vaccine.

3. The prime boost combination vaccine of claim 1, wherein (b) is a priming vaccine and (a) is a booster vaccine.

4. The prime boost combination vaccine of claim 1, wherein the two attenuated rVSVs are chimeric rVSVs that express a protein of a foreign pathogen, and wherein the two chimeric rVSVs are capable of inducing an immune response to the protein.

5. The prime boost combination vaccine of claim 4, wherein the foreign pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.

6. The prime boost combination vaccine of claim 4, wherein the foreign pathogen is a lentivirus.

7. The prime boost combination vaccine of claim 6, wherein the lentivirus is a human immunodeficiency virus (HIV) and the protein is a HIV protein.

8. The prime boost combination vaccine of claim 7, wherein the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene for expressing the HIV protein is inserted in between the G gene and the L gene.

9. The prime boost combination vaccine of claim 8, wherein the HIV gene is selected from the group of HIV genes consisting of env, gag and pol.

10. The prime boost combination vaccine of claim 4, wherein the foreign pathogen is hepatitis C virus (HCV) and the protein is a HCV protein.

11. The prime boost combination vaccine of claim 10, wherein the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene for expressing the HCV protein is inserted in between the G gene and the L gene.

12. The prime boost combination vaccine of claim 10, wherein the HCV protein is a structural or a non-structural HCV protein.

13. The prime boost combination vaccine of claim 4, wherein each one of the two vaccines comprise a mixture of the attenuated chimeric rVSVs, and wherein two of the attenuated chimeric rVSVs in the mixture have a different protein of the foreign pathogen.

14. The prime boost combination vaccine of claim 1, wherein each one of the two vaccines is capable of inducing humoral, cellular and mucosal immune responses.

15. The prime boost combination vaccine of claim 1, wherein the serotype of vaccine (a) is Indiana and the serotype of vaccine (b) is New Jersey.

16. The prime boost combination vaccine of claim 1, wherein each one of vaccine (a) and vaccine (b) further comprises an adjuvant.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments will now be described, by way of example only, with reference to the drawings, in which:

(2) FIG. 1 illustrates gene organization of the vesicular stomatitis virus (VSV), Indiana (Ind) serotype with mutations in the M gene.

(3) FIG. 2 illustrates gene organization of vesicular stomatitis virus (VSV), New Jersey (NJ) with mutations in the M gene.

(4) FIG. 3 illustrates a reverse genetics system for the recovery of VSV from cDNA.

(5) FIGS. 4A to 4D illustrate the burst size effect of mutation L111F in the M protein of rVSV.sub.Ind at permissive temperature [FIG. 4A and FIG. 4C] and at semi-permissive temperature [FIG. 4B and FIG. 4D], in baby hamster kidney (BHK) cells infected with recombinant VSVs [FIG. 4A and FIG. 4B] and in human neuroblastoma cells (SH-SY5Y) infected with the rVSV.

(6) FIG. 5 is a graph representing the burst size effect of mutations M48R+M51R, G22E, G22E/M48R+M51R, G22E/L110F, G22E/L110F/M48R+M51R in the M protein of rVSV.sub.NJ at 31 C. and 37 C. in BHK21 cells.

(7) FIG. 6A is a Western blot analysis illustrating the level of rVSV protein expression of rVSV.sub.Ind 1. WT, 2. M51R, 3. G21E 4. G21E/M51R 5. G21E/L111F and 6. G21E/L111F/M51R.

(8) FIG. 6B is a Western blot analysis illustrating the level of rVSV protein expression of rVSV.sub.NJ 1. WT, 2. M48R+M51R, 3. G22E 4. G22E/M48R+M51R 5. G22E/L110F and 6. G22E/L110F/M48R+M51R.

(9) FIGS. 7A to 7N are photographs of BHK21 cells infected with rVSV.sub.Ind having a wild type M gene (FIGS. 7B and 7I) and with rVSV.sub.Ind having different mutations in the M gene (FIGS. 7A, 7D, 7E, 7F, 7G, 7H, 7K, 7L, 7M and 7N) and photographs of uninfected BHK21 cells (FIGS. 7C and 7J). Cells were incubated at 31 C. (panels A to G) or 37 C. (FIGS. 7H to 7N).

(10) FIGS. 8A to 8L are photographs of SH-SY5Y cells infected with rVSV.sub.Ind having different mutations in the M gene (FIGS. 8C, 8D, 8E, 8F, 8I, 8J, 8K and 8L) or wild type M gene (FIGS. 8A and 8G) and photographs of uninfected SH-SY5S cells (FIGS. 8B and 8H). Cells were incubated at 31 C. (FIGS. 8A to 8F) or 37 C. (FIGS. 8G to 8L).

(11) FIGS. 9A to 9N are photographs of BHK21 cells infected with rVSV.sub.NJ having different mutations in the M gene (FIGS. 9A, 9D, 9E, 9F, 9G, 9H, 9K, 9L, 9M and 9N) or wild type M gene (FIGS. 9B and 9I), and photographs of uninfected BHK21 cells (FIGS. 9C and 9J). Cells were incubated at 31 C. (FIGS. 9A to 9G) or 37 C. (FIGS. 9H to 9N).

(12) FIGS. 10A to 10N are photographs of SH-SY5Y cells infected with rVSV.sub.NJ having different mutations in the M gene (FIGS. 10A, 10D, 10E, 10F, 10G, 10H, 10K, 10L, 10M and 10N) or having wild type M gene (FIGS. 10B and 10I), and photographs of uninfected SH-SY5Y cells (FIG. 10C and FIG. 10J). Cells were incubated at 31 C. (FIGS. 10A to 10G) or 37 C. (FIGS. 10H to 10N).

(13) FIGS. 11A to 11 D are graphs illustrating neurovirulence studies in Swiss Webster mouse by intralateral ventricular injection: FIG. 11A UV-irradiated rVSV.sub.Ind G21E/L111F/M51R, FIG. 11B rVSV.sub.Ind WT, FIG. 11C rVSV.sub.Ind M51R and FIG. 11D rVSV.sub.Ind G21E/L111F/M51R.

(14) FIGS. 12A to 12D are graphs illustrating neurovirulence studies in Swiss Webster mouse by intralateral ventricular injection: FIG. 12A rVSV.sub.NJ WT, FIG. 12B rVSV.sub.NJ M48R+M51R, FIG. 12C rVSV.sub.NJ G22E/M48R+M51R and FIG. 12D rVSV.sub.NJ G22E/L110F/M48R+M51R.

(15) FIGS. 13A to 13C are graphs illustrating the generation of rVSVs expressing HIV-1 Gag protein as a gene of interest. FIG. 13A (SEQ ID NOS: 11-15) illustrates the cloning of HIV-1 Gag-En genes into the cDNA clone of rVSV. FIG. 13B is a Western blot analysis of BHK cells infected with rVSVs expressing Gag-En and having different mutations to the M gene, and incubated at 31 C. FIG. 13C is a Western blot analysis of BHK cells infected with rVSVs expressing Gag-En and having different mutations to the M gene, and incubated at 37 C.

(16) FIG. 14 depicts a vaccination regime and a table of vaccination groups.

(17) FIG. 15 depicts a graph of the frequency of peptide specific CD8+ IFN gamma+ T cells among the different vaccination groups illustrated in the insert stimulated with dimethyl sulfoxide (DMSO).

(18) FIG. 16 is a graph illustrating the frequency of VSV N peptide specific CD8+ IFN gamma+ T cells among the different vaccination groups illustrated in the insert stimulated with VSV N.

(19) FIG. 17 is a graph illustrating the frequency of HIV-1 Gag peptide specific CD8+ IFN gamma+ T cells among the different vaccination groups illustrated in the insert stimulated with HIV-1 Gag.

(20) FIG. 18 is a graph illustrating HIV-1 Gag specific antibody responses among the different vaccination groups illustrated in the insert.

(21) FIG. 19 is a graph illustrating the frequency of VSV N peptide specific CD8+ IFN gamma+ T cells among different vaccination groups vaccinated with various doses of rVSV of Table 1.

(22) FIG. 20 is a graph illustrating the frequency of HIV-1 Gag peptide specific CD8+ IFN gamma+ T cells among different vaccination groups vaccinated with various doses of rVSV of Table 1.

(23) FIG. 21 is a graph illustrating HIV-1 Gag specific antibody responses among different vaccination groups vaccinated with various doses of rVSV of Table 1.

(24) FIG. 22 (SEQ ID NOS: 16-26) illustrates the cloning of HIV-1 gag gene linked to nucleotides encoding human B cell and T cell epitopes of gp120 and gp41 into the cDNA clone of rVSV.sub.Ind G21E/L111F/M51R and rVSV.sub.NJ G22E/M48R+M51R.

(25) FIG. 23 (SEQ ID NOS: 11-15 and 27-31, respectively) illustrates the cloning of HIV-1 gag gene linked to nucleotides encoding human B cell and T cell epitopes of gp41, nef, gp120, RT, Tat and Rev into the cDNA clone of rVSV.sub.Ind G21E/L111F/M51R and rVSV.sub.NJ G22E/M48R+M51R. It also illustrates the cloning of HIV-1 pol and env genes into the cDNA clone of rVSV.sub.Ind G21E/L111F/M51R and rVSV.sub.NJ G22E/M48R+M51R.

(26) FIG. 24 illustrates a Western blot analysis of BHK21 cells infected with rVSV.sub.Ind G21E/L111F/M51R expressing Gag A, B, C, En, and RT and incubated at 31 C. and 37 C.

(27) FIG. 25 illustrates a Western blot analysis of BHK21 cells infected with rVSV.sub.NJ G22E/M48R+M51R expressing Gag A, B, C, En, and RT and incubated at 31 C. and 37 C.

(28) FIG. 26 illustrates a Western blot analysis of BHK21 cells infected with rVSV.sub.Ind G21E/L111F/M51R or rVSV.sub.NJ G22E/M48R+M51R expressing HIV-1 pol gene.

(29) FIG. 27 illustrates a Western blot analysis of BHK21 cells infected with rVSV.sub.Ind G21E/L111F/M51R or rVSV.sub.NJ G22E/M48R+M51R expressing HIV-1 gp160mss gene.

(30) FIGS. 28A to 28F are graphs illustrating VSV N and HIV-1 protein peptide specific CD8+ T cell responses among the different vaccination groups of Table 6: G1 (FIG. 28A), G2 (FIG. 28B), G3 (FIG. 28C), G4 (FIG. 28D), G5 (FIG. 28E) and G5 without Env P18 (FIG. 28F).

(31) FIG. 29 is a graph illustrating VSV N and HIV-1 protein peptide specific CD8+ T cell responses in vaccination group G6 of Table 6. The amino acid sequences of VSV N peptide and peptides from HIV-1 proteins that are used to stimulated the splenocytes are shown (SEQ ID NOS: 32-37).

(32) FIGS. 30A and 30B illustrate the humoral immune responses against HIV-1 Gag (panel A) and Gp120 (panel B) induced in vaccination groups of Table 6, which were determined by Enzyme Linked Immunosorbant Assay (ELISA).

(33) FIG. 31 depicts the HCV structural protein genes cloned into rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) and rVSV.sub.NJ(GLM).

(34) FIGS. 32A to 32D illustrate Western blot analyses of the expression of HCV Core (panel A), E1 (panel B), E2 (panel C), and NS4B (panel D) proteins from the rVSV.sub.Ind(GLM), rVSV.sub.NJ(GM), rVSV.sub.NJ(GLM).

(35) FIG. 33 depicts the HCV non-structural protein genes cloned into rVSV.sub.Ind(GLM), rVSV.sub.NJ(GM), and rVSV.sub.NJ(GLM).

(36) FIGS. 34A to 34C are Western blot analyses of the expression of HCV NS3 at 37 C. and 31 C. (FIG. 34A), NS5A at 37 C. (FIG. 34B), and NS5B at 37 C. and 31 C. (FIG. 34C) from the rVSV.sub.Ind(GLM).

(37) FIGS. 35A and 35B demonstrate the expression of HCV NS3, NS4B, NS5A, and NS5B from rVSV.sub.NJ(GM) at 37 C. (FIG. 35A) and the expression of HCV NS5AB at 37 C. (FIG. 35B). The protein expression was detected by Western blot analysis.

DETAILED DESCRIPTION OF THE INVENTION

(38) Overview

(39) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, unless indicated otherwise, except within the claims, the use of or includes and and vice versa. Non-limiting terms are not to be construed as limiting unless expressly stated or the context clearly indicates otherwise (for example including, having and comprising typically indicate including without limitation). Singular forms including in the claims such as a, an and the include the plural reference unless expressly stated otherwise. Consisting essentially of means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. Consisting of means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

(40) All numerical designations, e.g., dimensions and weight, including ranges, are approximations that typically may be varied (+) or () by increments of 0.1, 1.0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term about.

(41) The term administering includes any method of delivery of a compound of the present invention, including a pharmaceutical composition, vaccine or therapeutic agent, into a subject's system or to a particular region in or on a subject. The phrases systemic administration, administered systemically, peripheral administration and administered peripherally as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. Parenteral administration and administered parenterally means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

(42) The term amino acid is known in the art. In general the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For the amino acids relevant to the present invention the designations are: M: methionine, R: arginine, G: glycine, E: glutamic acid, L: leucine, F: phenylalanine. In certain embodiments, the amino acids used in the application of this invention are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.

(43) The term antibody as used herein is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), including polyclonal, monoclonal, recombinant and humanized antibodies and fragments thereof which specifically recognize and are able to bind an epitope of a protein. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Nonlimiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab)2, Fab, Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker. The scFvs may be covalently or non-covalently linked to form antibodies having two or more binding sites.

(44) As used herein, the term epitopes refers to sites or fragments of a polypeptide or protein having antigenic or immunogenic activity in an animal, preferably in a mammal. An epitope having immunogenic activity is a site or fragment of a polypeptide or protein that elicits an immune response in an animal. An epitope having antigenic activity is a site or fragment of a polypeptide or protein to which an antibody immunospecifically binds as determined by any method well-known to one of skill in the art, for example by immunoassays.

(45) As used herein, the term fragment in the context of a proteinaceous agent refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a peptide, polypeptide or protein. In one embodiment, a fragment of a full-length protein retains activity of the full-length protein, e.g., immunogenic activity. In another embodiment, the fragment of the full-length protein does not retain the activity of the full-length protein, e.g., non-immunogenic activity.

(46) As used herein, the term fragment in the context of a nucleic acid refers to a nucleic acid comprising an nucleic acid sequence of at least 2 contiguous nucleotides, at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, at least 15 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 35 contiguous nucleotides, at least 40 contiguous nucleotides, at least 50 contiguous nucleotides, at least 60 contiguous nucleotides, at least 70 contiguous nucleotides, at least contiguous 80 nucleotides, at least 90 contiguous nucleotides, at least 100 contiguous nucleotides, at least 125 contiguous nucleotides, at least 150 contiguous nucleotides, at least 175 contiguous nucleotides, at least 200 contiguous nucleotides, at least 250 contiguous nucleotides, at least 300 contiguous nucleotides, at least 350 contiguous nucleotides, or at least 380 contiguous nucleotides of the nucleic acid sequence encoding a peptide, polypeptide or protein. In a preferred embodiment, a fragment of a nucleic acid encodes a peptide or polypeptide that retains activity of the full-length protein, e.g., immunogenic activity. In another embodiment, the fragment of the full-length protein does not retain the activity of the full-length protein, e.g., non-immunogenic activity.

(47) The term essentially noncytolytic as used herein means that the recombinant vesicular stomatitis virus (rVSV) does not significantly damage or kill the cells it infects.

(48) The term HIV is known to one skilled in the art to refer to Human Immunodeficiency Virus. There are two types of HIV: HIV-1 and HIV-2. There are many different strains of HIV-1. The strains of HIV-1 can be classified into three groups: the major group M, the outlier group 0 and the new group N. These three groups may represent three separate introductions of simian immunodeficiency virus into humans. Within the M-group there are at least ten subtypes or clades: e.g., clade A, B, C, D, E, F, G, H, I, J, and K. A clade is a group of organisms, such as a species, whose members share homologous features derived from a common ancestor. Any reference to HIV-1 in this application includes all of these strains.

(49) The term non-infectious means of reduced to non-existent ability to infect.

(50) As used herein, the terms subject or patient are used interchangeably. As used herein, the terms subject and subjects refers to either a human or non-human animal.

(51) The term pharmaceutical delivery device refers to any device that may be used to administer a therapeutic agent or agents to a subject. Non-limiting examples of pharmaceutical delivery devices include hypodermic syringes, multichamber syringes, stents, catheters, transcutaneous patches, microneedles, microabraders, and implantable controlled release devices. In one embodiment, the term pharmaceutical delivery device refers to a dual-chambered syringe capable of mixing two compounds prior to injection.

(52) The phrase pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

(53) The phrase pharmaceutically-acceptable carrier as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

(54) The terms polynucleotide, and nucleic acid are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term recombinant polynucleotide means a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin, which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement. An oligonucleotide refers to a single stranded polynucleotide having less than about 100 nucleotides, less than about, e.g., 75, 50, 25, or 10 nucleotides.

(55) The terms polypeptide, peptide and protein (if single chain) are used interchangeably herein to refer to polymers of amino acids. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

(56) In certain embodiments, polypeptides of the invention may be synthesized chemically, ribosomally in a cell free system, or ribosomally within a cell. Chemical synthesis of polypeptides of the invention may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules (see e.g., U.S. Pat. Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; M. Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol. 264, p 90; R. E. Offord, Chemical Approaches to Protein Engineering, in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl. Acad. Sci. USA (1994) 91: 12544-12548; M. Schnlzer, et al., Science (1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).

(57) VSV is used to refer to vesicular stomatitis virus.

(58) rVSV is used to refer to a recombinant vesicular stomatitis virus.

(59) The term Indiana, and IND are used to refer to the VSV serotype Indiana (VSV.sub.Ind).

(60) The term New Jersey, and NJ are used to refer to the VSV serotype New Jersey (VSV.sub.NJ).

(61) MWT M(WT) are used to refer to a wild type M protein. The nucleotide sequence of wild type M gene of the VSV.sub.Ind may comprise the nucleotide sequence represented by SEQ ID NO: 1. The amino acid sequence of wild type M protein of the VSV.sub.Ind may comprise the amino acid sequence represented by SEQ ID NO: 3. The nucleotide sequence of wild type M gene of the VSV.sub.NJ may comprise the nucleotide sequence represented by SEQ ID NO: 5. The amino acid sequence of wild type M protein of the VSV.sub.NJ may comprise the amino acid sequence represented by SEQ ID NO: 8. M51R is used to refer to an M(WT) in the VSV.sub.Ind having a methionine changed to an arginine at position 51. G21E is used to refer to an M(WT) in VSV.sub.Ind having a glycine changed to a glutamic acid at position 21. L111F is used to refer to an M(WT) in VSV.sub.Ind having a leucine changed to a phenylalanine at position 111. G22E is used to refer to an M(WT) in VSV.sub.NJ having a glycine (G) changed to glutamic acid (E) at position 22. L110F is used to refer to an M(WT) in VSV.sub.NJ having a leucine (L) changed to a phenylalanine (F) at position 110. M48R+M51R is used to refer to an M(WT) in VSV.sub.NJ having a methionine (M) changed to an arginine (R) at positions 48 and 51. rVSV.sub.Ind M(G21E/L111F/M51R) or rVSV.sub.Ind (GLM) are used to refer to a rVSV.sub.Ind having an M(WT) having a glycine changed to a glutamic acid at position 21, a leucine changed to a phenylalanine at position 111 and a methionine changed to an arginine at position 51. rVSV.sub.NJ M(G22E/M48R+M51R) or rVSV.sub.NJ (GM) are used to refer to rVSV.sub.NJ having an M(WT) having a glycine changed to a glutamic acid at position 22 and a methionine changed to an arginine at positions 48 and 51, and rVSV.sub.NJ M(G22E/L110F/M48R+M51R) or rVSV.sub.NJ (GLM) are used to refer to rVSV.sub.NJ having an M(WT) having a glycine changed to a glutamic acid at position 22, a leucine changed to a phenylalanine at position 110 and a methionine changed to an arginine at positions 48 and 51.

(62) Overview

(63) The inventors generated novel M proteins and novel attenuated rVSVs capable of producing the novel M proteins. The novel proteins of the present invention may include: M(G21E/L111F/M51R), M(G22E/M48R+M51R), and M(G22E/L110F/M48R+M51R). The novel attenuated rVSVs of the present invention may be used as protein expression and vaccine vectors and in methods for preventing or treating infections. The rVSV of the present invention may be applied to make vaccines for the infectious diseases of human and other animals to induce cellular and humoral immune responses.

(64) Isolated Proteins and M Proteins

(65) In one embodiment, the present invention relates to isolated proteins and to nucleotide sequences that encode the isolated proteins. As such, in one embodiment the present invention relates to an isolated peptide comprising an amino acid sequence selected from amino acid sequences listed as SEQ ID NOs: 4, 9 and 10. In another embodiment, the present invention relates to isolated nucleotide sequences comprising a nucleotide sequence selected from the polynucleotides listed as SEQ ID NOs: 2, 6 and 7.

(66) In one embodiment, the present invention relates to novel VSV M proteins having at least one of the following substitutions: M(G21E/L111F/M51R), M(G22E/M48R+M51R), and M(G22E/L110F/M48R+M51R). In one aspect the present invention relates to a VSV M protein comprising an amino acid sequence selected from the amino acid sequences listed as SEQ ID NOs: 4, 9 and 10. In another embodiment the present invention relates to nucleotide sequences which encode for the novel VSV M proteins of the present invention. The nucleotide sequences may be selected from the group of sequences listed as SEQ ID NOs: 2, 6 and 7.

(67) Methods of Preventing or Treating an Infection

(68) Provided are methods of inducing an immune response, preventing or treating infections. In one embodiment, the methods may include administering to a subject: (a) an effective amount of a vaccine comprising an attenuated rVSV of one serotype having (i) a first modified M protein, the first modified M protein comprising the amino acid sequence of SEQ ID NO: 3 including at least the following substitutions: G21E/L111F/M51R, and (ii) an epitope of the pathogen; and (b) an effective amount of another vaccine comprising an attenuated rVSV of another serotype having: (i) a second modified M protein, the second modified M protein comprising the amino acid sequence of SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R, and (ii) the epitope of the pathogen. In embodiments of the present invention, the methods may include administering to a subject (a) an effective amount of rVSV.sub.Ind M(G21E/L111F/M51R), and (b) an effective amount of either a rVSV.sub.NJ M(G22E/M48R+M51R) or rVSV.sub.NJ M(G22E/L110F/M48R+M51R) in a prime-boost immunization modality.

(69) The term effective amount as used herein means an amount effective and at dosages and for periods of time necessary to achieve the desired result.

(70) In certain embodiments, (a) is administered to the subject before (b) is administered to the subject.

(71) In certain embodiments, (b) is administered to the subject more than one time over the course of treating or preventing.

(72) In certain embodiments, (a) is administered to the subject in need thereof and (b) is administered to the subject in need thereof at about weeks three, eight and sixteen post-administration of (a).

(73) In certain embodiments, (b) is administered to the subject before (a) is administered to the subject.

(74) In certain embodiments, (a) is administered to the subject more than one time over the course of treating or preventing.

(75) In certain embodiments, (b) is administered to the subject in need thereof and (a) is administered to the subject in need thereof at about weeks three, eight and sixteen post-administration of (b).

(76) Recombinant Virus

(77) In certain embodiments, present invention relates to a recombinant vesicular stomatitis virus (rVSV) which may be a full length VSV, essentially non-cytolytic, avirulent, capable of inducing an immune response in a subject, capable of reproducing virus particles to a high tire at permissive temperatures, reproducing virus particles to a low titre at semi-permissive temperatures and which may be incapable of producing virus at non-permissive temperatures, and that can express an epitope of a foreign pathogen. The rVSV of the present invention may be capable of inducing humoral, cellular and mucosal immune responses.

(78) In one embodiment, the present invention relates to rVSV.sub.Ind and rVSV.sub.NJ. The rVSV.sub.Ind may be a full length, essentially noncytolytic rVSV.sub.Ind M(G21E/L111F/M51R) capable of producing virus particles at a permissible temperature of about 31 C., and which may be incapable of or poorly capable of producing virus particles at a semi-permissive temperatures of about 37 C. and incapable of producing virus particles at non-permissive temperatures above 37 C., for example 39 C. In certain embodiments, the rVSV.sub.Ind may include a M(G21E/L111F/M51R). In certain embodiments, the rVSV.sub.Ind may include an M gene comprising a nucleotide sequence SEQ ID NO: 2.

(79) In certain embodiments, the rVSV is a full-length, essentially noncytolytic rVSV.sub.NJ M (G22E/M48R+M51R) or M(G22/L110F/M48R+M51R). In certain embodiments, the rVSV is an essentially noncytolytic rVSV.sub.NJ including an M gene, wherein the nucleotide sequence of the M gene is selected from SEQ ID NO: 6 and SEQ ID NO: 7.

(80) The rVSVs of the present invention can be prepared using techniques known in the art. In one embodiment, the rVSVs may be introduced in a host cell under conditions suitable for the replication and expression of the rVSV in the host. Accordingly, the present invention also provides a cell having a rVSV.sub.Ind wherein the amino acid sequence of the virus' M protein is modified to provide an essentially non-cytotoxic which also allows the rVSV.sub.Ind to effectively replicate at permissible temperature but may not replicate at non-permissible temperature.

(81) As such, the present invention relates also to a cell having one or more of the recombinant VSVs of the present invention.

(82) Vaccines or Immunogenic Compositions of the Invention

(83) The present invention further features vaccines or immunogenic compositions comprising one or more of the rVSVs of the present invention. In one embodiment, the present invention features vaccines or immunogenic compositions comprising an rVSV.sub.Ind and vaccines or immunogenic compositions comprising an rVSV.sub.NJ, as described above.

(84) In one embodiment, the vaccines may include rVSVs expressing an epitope of a pathogen. In another embodiment, the vaccines may include a mixture or cocktail of rVSVs expressing different epitopes of a pathogen (see Table 6, vaccination groups 5 and 6).

(85) The vaccine or immunogenic compositions of the invention are suitable for administration to subjects in a biologically compatible form in vivo. The expression biologically compatible form suitable for administration in vivo as used herein means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances maybe administered to any animal or subject, preferably humans. The vaccines of the present invention may be provided as a lyophilized preparation. The vaccines of the present invention may also be provided as a solution that can be frozen for transportation. Additionally, the vaccines may contain suitable preservatives such as glycerol or may be formulated without preservatives. If appropriate (i.e. no damage to the VSV in the vaccine), the vaccines may also contain suitable diluents, adjuvants and/or carriers.

(86) The dose of the vaccine may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances.

(87) Kits

(88) The present invention provides kits, for example for preventing or treating an infection. For example, a kit may comprise one or more pharmaceutical compositions or vaccines as described above and optionally instructions for their use. In still other embodiments, the invention provides kits comprising one or more pharmaceutical compositions or vaccines and one or more devices for accomplishing administration of such compositions.

(89) Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. In other embodiments involving kits, this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use. Such kits may have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications.

(90) Advantages and Unique Features of the rVSVs of the Present Invention

(91) Novel and Unusual Features of the Invention:

(92) Normal assembly and release of rVSV.sub.Ind M(G21E/L111F/M51) at permissive temperature (about 31 C.) made it possible to amplify the new mutant rVSVs at the permissible temperature to a high titre to make viral stock. The assembly defectiveness of rVSV.sub.Ind M(G21E/L111F/M51) at non-permissive temperature (about 37 C. (around body temperature) increased the safety of the using the rVSV in human and other animals by significantly reducing the number of progeny infectious viruses at the non-permissive temperature. The addition of these mutations to the pre-existing M51R mutation in the M protein of rVSV.sub.Ind further attenuated the virulence of VSV.sub.Ind.

(93) The three mutations, M(G22E/M48R+M51R) or four mutations, M(G22E/L110F/M48R+M51R) in the M protein of rVSV.sub.NJ did not make the virus temperature sensitive for the assembly of the virus. However, the addition of G22E mutation or G22E/L110F to the M48R+51R mutations made the VSV.sub.NJ more attenuated and became significantly less pathogenic.

(94) The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

EXAMPLES

(95) The present invention is further illustrated by the following examples which should not be construed as limiting in any way.

Example 1Introduction of Mutations into the M Genes of rVSVInd and rVSVNJ and Recovery of Recombinant VSV by Reverse Genetics

(96) Mutations were introduced into the M gene of VSV.sub.Ind (FIG. 1) and VSV.sub.NJ (FIG. 2). Nucleotide sequences encoding the amino acids at each position were mutated by the mega-primer PCR method. Each mutation is expressed as a substitution of an amino acid at a specific position (e.g., M51 in M51R) with another amino acid (e.g., R in M51R). In order to attenuate further the virulence of VSV, the inventors combined mutations (G21E and L11F) in the tsO23 with methionine to arginine mutations (M51R) in the M gene, which reduced inhibitory activity of M protein on the cellular protein synthesis, in addition, reduced the assembly of the VSV particles at non-permissive (39 C.) and semi-permissive (37 C.) temperatures. The nucleotide sequences and amino acid changes from wild type to mutants in the M genes of rVSV.sub.Ind and rVSV.sub.NJ are shown in Tables 2, 3, 4 and 5. The changed nucleotide codons are underlined and changed nucleotide sequences and amino acid sequences are bold-faced.

(97) Wild type and mutant recombinant vesicular stomatitis viruses (rVSV) were recovered from the cDNA plasmids by reverse genetics (FIG. 3). The VSV reverse genetics employs the BHK21 cells expressing DNA dependant RNA polymerase of bacteriophage T7 (T7) and a plasmid which encodes full length genomic RNA of VSV (pVSV) and 3 plasmids expressing nucleocapsid protein (pN), phosphoprotein (pP), and VSV polymerase L protein (pL). The transcription of the full length genomic RNA and the messenger RNAs for N, P, and L proteins are under the control of T7 RNA polymerase. Internal ribosome entry site (IRES) at the upstream of each VSV N, P, and L gene enhances the translation of proteins. The plasmids are transfected into BHK-T7 cells with Lipofectamine 2000 in concentrations of 10 g of pN, 10 g of pP, 5 g of pL, and 15 g of pVSV. The culture media from the transfected cells were harvested when the cells showed about 50-70% of CPE.

Example 2the Mutation, L111F in the M Gene Significantly Reduces the Burst Sizes of rVSVInd at Semi-Permissive Temperature, 37 C.

(98) The recovered viruses were purified 3 times by plaque picking and were amplified for a larger volume of stock viruses by infecting BHK21 cells with an MOI of 0.1 at 31 C. The inventors infected BHK21 cells and human neuroblastoma cells, SH-SY5Y with an MOI of 3 of rVSVs. The infected cells were incubated at permissive temperature (31 C.) and semi-permissive temperature (37 C., body temperature) to determine the temperature sensitivity of the new M mutants in the assembly of virus particles. Culture media from the infected cells were collected every 2 hours until 10 hours after the infection, and the number of infectious viral particles in the culture media was determined by plaque assay with Vero E6 cells. The cells infected with the mutant viruses for the plaque assay were incubated at 31 C. Wild type and all mutants replicated equally well and produced similar titre of infectious viruses all along the period of 10 hrs of infection (FIG. 4A and FIG. 4C). However, the mutants of rVSV.sub.Ind, rVSV.sub.Ind-G21E/L111F and rVSV.sub.Ind-G21E/L111F/M51R replicated in significantly lower titre than wild type or M51R mutant of rVSV.sub.Ind at 37 C. The differences in producing infectious particles between the wild type and the new M mutant, rVSV.sub.Ind-G21E/L111F/M51R were as large as four logs (FIG. 4B and FIG. 4D). The error bars in FIG. 4 represent standard error of the mean. The P values for viral titers of rVSV.sub.Ind-G21E/L111F/M51R at 4 hrs, 6 hrs, 8 hrs, and 10 hrs postinfection obtained by comparing them to titers of rVSV.sub.Ind-WT are p<0.0001, p=0.0055, p=0.0040, and p=0.0015 in FIG. 4B. The P values for viral titers of rVSV.sub.Ind-G21E/L111F at 4 hrs, 6 hrs, 8 hrs, and 10 hrs postinfection obtained by comparing them to titers of rVSV.sub.Ind-WT are p<0.0001, p=0.0055, v0.0041, and p=0.0016 in FIG. 4B. The P values in FIG. 4D were <0.005. P values were computed by using two-sided independent t tests.

Example 3Mutations in the M Gene of rVSVNJ, G22E and L110F do not Reduce the Burst Size of the rVSVNJ at 37 C.

(99) BHK21 cells were infected with an MOI of 3 of rVSV.sub.NJ, wild type and M gene mutants, incubated at both 37 C. (semi-permissive temperature) and 31 C. (permissive temperature) and the culture media was harvested at 10 hrs post infection. The viral titer of each virus in the culture media was determined by plaque assay using Vero E6 cells. The average viral titre from the duplicate samples is shown in the table. Wild type and all M mutants of rVSV.sub.NJ replicated equally well at both temperatures 31 C. and 37 C. (FIG. 5), indicating the introduction of G22E and L110F mutations into the M gene of rVSV.sub.NJ did not affect the assembly and release of the virus at 37 C. The error bars in FIG. 5 represent standard error of the mean.

Example 4Assembly Defectiveness at Semi-Permissive Temperature by L111F Mutation in the M Gene of rVSVInd does not Affect the Expression of VSV Proteins

(100) BHK21 cells were infected with an MOI of 6 of rVSV.sub.Ind and rVSV.sub.NJ, wild type and M gene mutants. The infected cells were incubated at both 37 C. and 31 C. for 6 hrs. The infected cells were lysed at 6 hrs post-infection, 5 g of total protein was loaded to the SDS-PAGE gel, and rVSV proteins were detected by Western blot analysis using rabbit antiserum against VSV.sub.Ind and VSV.sub.NJ (1:5000 dilution). The result demonstrate that in spite of the mutations that reduced the burst size of the rVSV.sub.Ind at 37 C. (L111F in G21E/L111F and G21E/L111F/M51R), the level of VSV protein expression is comparable to the wild type rVSV.sub.Ind (FIG. 6A). Wild type and all M mutants of rVSV.sub.NJ expressed their proteins in a similar level at both 31 C. and 37 C. (FIG. 6B).

Example 5Combined Mutations, G21E/L111F/M51R in the M Gene of rVSVInd Reduced Cytopathogenicity Significantly in BHK21 Cells and Human Neuroblastoma Cells, SH-SY5Y

(101) In order to examine the effects of the mutations, L111F and M51R of M gene of rVSV.sub.Ind on the cytopathogenicity, the inventors infected BHK21 cells (FIG. 7) and human neuroblastoma cells (FIG. 8) with an MOI of 0.1 of rVSV.sub.Ind. The typical cytopathic effects by the VSV are rounding-up of infected cells and cell lysis. At 20 hrs after infection, the cytopathic effect caused by the rVSV.sub.Ind-G21E/L111F/M51R was compared with those by the other rVSV.sub.Ind. In 20 hrs of infection with the rVSV.sub.Ind-G21E/L111F/M51R mutant showed the most reduced cytopathic effects, none or small number of round-up cells at 37 C. and the combination of the mutations L111F and M51R (FIG. 7N and FIG. 8L) further attenuated the cytopathogenicity of the virus than the single mutation of each in both BHK21 and SH-SY5Y cells.

Example 6Reduced Cytopathic Effects in the BHK21 Cell by the rVSVNJ with the M Gene Mutations, G22E/M48R+M51R and G22E/L110F/M48R+M51R

(102) In order to examine the effects of the mutations, G22E, L110F and M48R+M51R of M gene of rVSV.sub.NJ on the cytopathogenicity, the inventors infected BHK21 cells and human neuroblastoma cells with an MOI of 0.1 of rVSV.sub.NJ. At 20 hrs after infection, the inventors compared the cytopathic effects (cell round-up and lysis) caused by the rVSV.sub.NJ wild type and other M mutants. In 20 hrs of infection with the G22E/M48R+M51R mutant and G22E/L110F/M48R+M51R mutant showed the least cytopathic effects at 37 C. and the combination of the mutations, G22E and M48R+M51R further attenuated the cytopathogenicity of the rVSV.sub.NJ in both BHK21 (FIGS. 9L and 9N) and SH-SY5Y cells (FIGS. 10L and 10N).

Example 7rVSVInd with the Mutations of G21E/L111F/M51R and rVSVNJ with the Mutations of G22E/M48R+M51R and G22E/L110F/1V148R+M51R in the M Protein was Attenuated to a Degree that Mice Injected with the Virus into the Brain Showed No Neurological Signs or any Symptoms Such as Weight Loss

(103) VSV does not show the neurotropism in host animal during the natural infection through the skin abrasion or sandfly or mosquito bites. Nevertheless, when wild type VSV is injected directly into the nose or brain in mice or monkeys, the animals demonstrate neurological symptoms. In order to examine the neurovirulence of the new M mutants of VSVs, the inventors injected Swiss Webster mice with 110.sup.6 PFU of mutant VSV and 110.sup.3 PFU of wild type VSV into the intralateral ventricle of the brain. The inventors purchased 5-week-old Swiss Webster mice with intralateral ventricular implant from Charles River laboratory. Three mice/group were injected with viruses after one week of arrival to the animal facility. After the viral injection, mice were observed for neurological signs and were weighed every two days for 4 weeks. The mice injected with 110.sup.3 PFU of wild type rVSV.sub.Ind died within four days after injection (FIG. 11B). Two mice injected with 110.sup.6 PFU of rVSV.sub.Ind-M51R (FIG. 11C) lost about 20% of body weights 6 days after injection and the body weight bounced back to normal weight at about 14 days after injection. One mice injected with the rVSV.sub.Ind-M51R did not lose the body weight through the experiment. All three mice injected with rVSV.sub.Ind-G21E/L111F/M51R showed no sign of illness and did not lose their body weight for 4 weeks until the mice were sacrificed (FIG. 11D) indicating the combination of mutations, G21E/L111F and M51R significantly attenuated the rVSV.sub.Ind and lost its neurovirulence in mice. One mice injected with rVSV.sub.NJ-WT showed paralysis in both hind legs and it was sacrificed on day 6 after injection (FIG. 12A). Two other mice injected with rVSV.sub.NJ-WT and mice injected with rVSV.sub.NJ-M48R+M51R, rVSV.sub.NJ-G22E/M48R+M51R, or rVSV.sub.NJ-G22E/L110F/M48R+M51R showed no sign of illness and weight loss during 4 weeks after injection (FIGS. 12B, 12C, and 12D). The error bars in FIGS. 11 and 12 represent standard error of the mean.

Example 8Generation of rVSVs Expressing HIV-1 Gag Protein as a Gene of Interest; HIV-1 Gag Proteins are Expressed Equally Well at Both 31 C. and 37 C.

(104) The newly generated M mutants, G21E/L111F/M51R mutant of rVSV.sub.Ind and G22E/M48R+M51R and G22E/L110F/M48R+M51R mutants of rVSV.sub.NJ demonstrated the reduced cytopathogenicity and comparable protein expressions to the wild type rVSV at 37 C. In order to be used as a vaccine vector, the new M mutants of rVSV should induce good immune responses in vivo, both humoral and cellular immune responses. When it is expressed alone in cells, HIV-1 gag proteins produce virus like particles and the virus like particles are secreted from the cells. Therefore, the gag protein was suitable protein to express from the new M mutants of rVSV to examine both cellular and humoral immune response. In addition, the CD8+ cytotoxic T cell epitope, H-2K.sup.d-restricted peptide (NH.sub.2-AMQMLKETI-COOH) (SEQ ID NO: 33) in the HIV-1 Gag is well studied in the Balb/c mouse. The inventors inserted the full-length HIV-1 gag gene linked to conserved human CD8+ T cell epitopes from gp41, gp120, and nef protein of HIV-1 (Gag-En). The Gag-En gene was inserted into the junction of G gene and L gene in the full-length cDNA clones of wild type (WT) and G21E/L111F/M51R (GLM) of rVSV.sub.Ind and wild type, G22E/M48R+M51R (GM), and G22E/L110F/M48R+M51R(GLM) of rVSV.sub.NJ. The rVSVs were recovered from the cDNA clones by reverse genetics as described in FIG. 13A and the expression of Gag-En from the rVSVs was examined by Western blot analysis using monoclonal antibody against HIV-1 p24. For the Western blot analysis, BHK21 cells were infected with MOI of 6 of rVSVs and incubated at 31 C. and 37 C., and cell lysates were prepared at 6 hrs post-infection. Total protein of 5 g was loaded onto the SDS-PAGE for the Western blot analysis. rVSV.sub.Ind(GLM)-Gag-En, rVSV.sub.NJ(GM)-Gag-En, and rVSV.sub.NJ(GLM)-Gag-En expressed Gag-En protein (64 kDa) well at semi-permissive temperature (37 C., FIG. 13C) and the expression level was similar to that at permissive temperature (31 C., FIG. 13B).

Example 9Vaccination Regimen with rVSV in Mice

(105) Six Balb/c mice per group were vaccinated with the prime-boost regimen illustrated in FIG. 14. Mice were grouped according to the vaccine vector type (wild type vs. mutant) and regimen, e. g., priming and boosting with the same serotype of rVSV or alternating the two serotypes for priming and boost. The mice were prime-vaccinated intramuscularly with 510.sup.6 PFU of rVSVs at age of 6 weeks. Three weeks after the priming, mice were boost-vaccinated with the same dose of rVSV as the prime vaccination. A week after the booster injection, splenocytes and sera were collected to detect the HIV-1 Gag specific CD8+ T cell immune responses and humoral immune responses.

Example 10Priming with rVSVInd(GLM)-HIV-1 Gag-En and Booster with rVSVNJ(GLM)-HIV-1 Gag-En Induces Strongest CD8+ T Cell Immune Responses Against HIV-1 Gag

(106) The T cells stimulated by the interaction with MHC I molecule on the antigen presenting cells loaded with the peptide enhances the secretion of interferon- (IFN-), which indicates the antigen specific T cell immune responses. The splenocytes were double stained with FITC-anti-CD8 and APC-anti-IFN- for CD8+ T cells with the increased intracellular INF-. In order to examine the HIV-1 Gag protein specific CD8+ T cell immune response, splenocytes were isolated and 110.sup.6 cells were stimulated with H-2K.sup.d-restricted HIV-1 Gag peptide, NH.sub.2-AMQMLKETI-COOH (SEQ ID NO: 33), the cells were stained with FITC rat anti-mouse CD8 for the CD8 molecules and stained with APC rat anti-mouse IFN- for intracellular IFN-. The secretion of IFN- was blocked with Brefeldin A before staining them. VSV specific CD8+ T cell immune responses were examined with the use of nucleocapsid specific peptide, IN275: NH2-MPYLIDFGL-COOH (SEQ ID NO: 32). Peptide specific CD8+-IFN-+ T cells were counted with FACSCalibur, a flowcytometer. The splenocytes treated with DMSO (solvent for the peptide, FIG. 15) did not stimulate the CD8+ T cells indicating that treating the splenocytes with the VSV N peptides and HIV-1 Gag peptides stimulated specifically CD8+ T cells in FIG. 16 and FIG. 17. Prime and boost immunization by alternating two serotypes of rVSV(WT) or rVSV(GLM) mutants induced stronger CD8+ T cell immune responses against VSV as well as HIV-1 Gag proteins than using a single serotype of rVSV for the prime and boost vaccinations as seen in groups 1, 2, 5, 6, 9, and 10. (FIG. 16 and FIG. 17). With the vaccination with the new M mutants or rVSV, CD8+ T cell responses against HIV-1 Gag protein was induced better when mice were vaccinated with rVSV.sub.Ind(GLM)-Gag for priming and rVSV.sub.NJ(GLM)-Gag for boosting than vice versa (FIG. 17, group 6 vs. group 10). P value on the group 6 in FIG. 17 was computed by using two-sided independent sample t test, and the result was compared to that for the group 10. The error bars in FIGS. 16 and 17 represent standard error of the mean.

Example 11HIV-1 Gag Specific Humoral Immune Responses

(107) Generation of HIV-1 Gag specific antibody was examined with the serum collected at a week after the boost immunization. The Gag specific antibody titer was determined by the indirect enzyme-linked immunosorbent assay (ELISA). For the ELISA, 96 well ELISA plate was coated with recombinant p55 at a concentration of 125 ng/well. The mouse serum was diluted 1:100. The antibody bound to the antigen, p55 was detected with secondary antibody, sheep anti-mouse IgG-HRP. The enzymatic activity of HRP was detected by adding substrates, a mixture of hydrogen peroxide and tetramethylbenzidine. The OD of each sample was read at the wavelength of 450 with the microplate reader. The humoral immune responses against HIV-1 Gag (generation of antibody against HIV-1 Gag) was induced well in mice vaccinated with the new M mutants, and the best humoral immune responses against HIV-1 Gag were induced when two serotypes of rVSV(WT) or rVSV(GLM) were alternated for prime and booster injection (FIG. 18, groups 5, 6, 9, and 10). As for the utilization of the new M mutants for inducing humoral immune responses against foreign proteins, priming with rVSV.sub.Ind(GLM)-Gag and booster with rVSV.sub.NJ(GLM)-Gag worked better than vice versa (FIG. 18, group 6 vs. group 10). The error bars in FIG. 18 represent standard error of the mean.

Example 12Increasing Doses of rVSVInd(GLM), rVSVNJ(GM), and rVSVNJ(GLM) for Vaccination Induced Stronger Immune Responses in Mice

(108) As illustrated in FIG. 17 and FIG. 18, stronger HIV-1 Gag specific CD8+ T cell immune responses and humoral immune responses were induced when mice were prime vaccinated with rVSV.sub.Ind(GLM)-Gag and booster vaccinated with rVSV.sub.NJ(GLM)-Gag. However the immune responses were not as strong as those induced with rVSV.sub.Ind(WT)-Gag and rVSV.sub.NJ(WT)-Gag. Therefore, the inventors wanted to examine whether they could increase the immune responses, if they increase the doses of the rVSV.sub.Ind(GLM)-Gag and rVSV.sub.NJ(GLM)-Gag. Six (6) week old Balb/c mice, 6 mice/group, were vaccinated with rVSV.sub.Ind(GLM)-Gag for priming and rVSV.sub.NJ(GLM)-Gag or rVSV.sub.NJ(GM)-Gag for booster. Because the rVSV.sub.NJ(GM) was as attenuated as the rVSV.sub.NJ(GLM) in vitro and in vivo, the inventors included rVSV.sub.NJ(GM)-Gag as a booster virus, and compared the immune responses to that induced by rVSV.sub.NJ(GLM)-Gag. Mice groups were vaccinated with various doses of rVSV.sub.Ind(GLM)-Gag, rVSV.sub.NJ(GLM)-Gag, and rVSV.sub.NJ(GLM)-Gag; 510.sup.6 PFU/mouse, 510.sup.7 PFU/mouse, 510.sup.8 PFU/mouse, and 510.sup.9 PFU/mouse (Table 1).

(109) Mice were vaccinated according to the schedule as seen in FIG. 14 and were sacrificed a week after booster vaccination for splenocytes and serum. The splenocytes were double stained with FITC-anti-CD8 and APC-anti-IFN- for CD8+ T cells with the increased intracellular INF-. In order to examine the HIV-1 Gag protein specific CD8+ T cell immune response, splenocytes were isolated and 110.sup.6 cells were stimulated with H-2Kd-restricted HIV-1 Gag peptide, NH2-AMQMLKETI-COOH (SEQ ID NO: 33), the cells were stained with FITC rat anti-mouse CD8 for the CD8 molecules and stained with APC rat anti-mouse IFN- for intracellular IFN-. The secretion of IFN- was blocked with Brefeldin A before staining them. VSV specific CD8+ T cell immune responses were examined with the use of nucleocapsid specific peptide, IN275: NH2-MPYLIDFGL-COOH (SEQ ID NO: 32). Peptide specific CD8+-IFN-+ T cells were counted with FACSCalibur, a flowcytometer.

(110) CD8+ T cell immune responses against VSV N protein were very similar in all vaccination groups with different doses of rVSV.sub.Ind(GLM)-Gag, rVSV.sub.NJ(GLM)-Gag, or rVSV.sub.NJ(GM)-Gag (FIG. 19). The HIV-1 Gag specific CD8+ T cell immune responses after prime vaccination with rVSV.sub.Ind(GLM)-Gag and booster with rVSV.sub.NJ(GLM)-Gag looked increased a little bit when the vaccine doses were increased, but differences were not statistically significant (FIG. 20, groups 2, 3, 4, 5). However, when mice were prime vaccinated with rVSV.sub.Ind(GLM)-Gag and booster vaccinated with rVSV.sub.NJ(GM)-Gag, the frequency of the HIV-1 Gag specific CD8+ T cells were increased significantly with the increasing dose of 510.sup.8 PFU. The HIV-1 Gag specific CD8+ T cell response was strongest in a group vaccinated with 510.sup.9 PFU/mouse (FIG. 20. groups 6, 7, 8, 9). P values on the groups 8 and 9 in FIG. 20 were computed by using two-sided independent sample t test, the result of group 8 was compared to that for the group 7, and the result of group 9 was compared to that for the group 8. The error bars in FIGS. 19 and 20 represent standard error of the mean.

(111) Generation of antibody against HIV-1 Gag protein was increased with the increasing doses of rVSV.sub.Ind(GLM)-Gag for priming and booster with rVSV.sub.NJ(GLM)-Gag or rVSV.sub.NJ(GM)-Gag (FIG. 21). Booster vaccination with rVSV.sub.NJ(GLM)-Gag or with rVSV.sub.NJ(GM)-Gag generated similar level of Gag specific antibodies in various doses of the virus. The error bars in FIG. 21 represents standard error of the mean.

Example 13Generation of rVSVInd(GLM) and rVSVNJ(GM) Expressing HIV-1 Proteins; Gag, Pol, Env, and RT

(112) For HIV-1 vaccines utilizing the rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) as vaccine vectors, the inventors included HIV genes encoding most of the large polyproteins, which are cleaved into functional proteins-Gag and Env and proteins with enzymatic activities (pol gene products). In addition, HIV-1 gag gene was linked to nucleotides encoding peptide epitopes for T cells and B cells in humans. The inventors have generated rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) carrying cassettes encoding HIV-1 Gag linked to B-cell epitopes from HIV Env protein derived from multiple viral clades (FIG. 22). The inventors have generated the rVSV.sub.Ind(GLM)-Gag-En and rVSV.sub.NJ(GM)-Gag-En, which expresses gag gene and T cell peptide epitopes from Nef, Gp120, and Gp41 (FIG. 23). The inventors generated rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) with HIV-1 Gag-RT, which encodes gag protein with peptide epitopes from RT, Tat, and Rev proteins (FIG. 23). In addition, the inventors generated rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) with pol gene and env gene, respectively (FIG. 23). Previously, the inventors have demonstrated that replacement of signal peptide of HIV-1 envelope protein with the honeybee melittin signal peptide dramatically increased the rates of Gp120 expression, glycosylation, and secretion (Yan Li et al., PNAS, 93: 9606-9611, 1996). The increased expression and secretion of Gp120 are expected to enhance the induction of the T cell and B cell immune responses against HIV-1 gp120. Therefore, the inventors generated rVSVs with HIV-1 env gene with melittin signal sequence (gp160mss). The recombinant VSVs with the HIV-1 proteins were plaque purified and were amplified for the stock viruses. The expression of HIV-1 proteins at 31 C. and 37 C. from the mutant VSV was examined by Western blot analysis. The Gag proteins tagged with T cell and B cell epitopes from various HIV-1 proteins were expressed well from rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) at both 31 C. and 37 C. (FIG. 24 and FIG. 25). The cloned HIV-1 pol gene in the rVSV genome encodes a polyprotein, which is cleaved into protease (PR, p11), reverse transcriptase (RT, p66/p51), and integrase (IN, p31). The inventors detected two subunits of RT products, p66 and p51. The p51 is generated from the p66 by proteolytic cleavage of a fragment of p15 at the carboxy terminus. The RT products p66 and p51 were expressed equally well from both rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) and at both 31 C. and 37 C. (FIG. 26). The env gene encodes glycoprotein Gp160, which is cleaved into Gp120 of receptor binding protein and gp41 of fusion inducing cytoplasmic and transmembrane protein. Gp160 is cleaved by the cellular trans-golgi resident protease furin. The Gp160 expressed in BHK21 cells from the rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) was not cleaved to Gp120 and Gp41 or the cleavage was not very efficient, although the level of expression was good at both 31 C. and 37 C. (FIG. 27). Our previous data on the expression of the Gp160 in BHK21 cells and cleavage into Gp120 and Gp41 demonstrated that the processing was not very efficient (Kunyu Wu et al., Journal of General Virol., 90:1135-1140, 2009).

Example 14Immunization Studies in Mice with the rVSVInd(GLM) and rVSVNJ(GM) with HIV-1 Proteins, Gag, Gp160 and RT

(113) The preliminary prime-boost immunization studies with new M mutant rVSVs, rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) revealed that priming mice with the rVSV.sub.Ind(GLM) and boosting with rVSV.sub.NJ(GM) induced better CD8+ T cell immune response (FIG. 17) and humoral immune responses (FIG. 18) against the foreign gene than the other way around. With the rVSV.sub.Ind(GLM)-Gag priming and rVSV.sub.NJ(GM)-Gag boosting, the dosage of 510.sup.8 pfu of each virus induced better Gag specific CD8+ T cell responses than lower dosages, although the B cell responses against Gag was similar to that induced with the lower dosages (FIG. 20 and FIG. 21). The inventors chose 510.sup.8 pfu as a dosage for immunization studies with rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) expressing HIV-1 Gag, Gp160, and RT.

(114) The inventors prime immunized Balb/c mice with rVSV.sub.Ind(GLM) expressing HIV-1 proteins and boost immunized with rVSV.sub.NJ(GM) expressing HIV-1 proteins in three weeks after the priming as described in the table 6. The inventors examined how the immune responses against the individual HIV-1 proteins are induced with individual injection or with the mixed injection of three different viruses expressing separate proteins. One week after the boost immunization, the inventors sacrificed the immunized mice for their splenocytes and sera. The 110.sup.6 splenocytes were stimulated with H-2K.sup.d-restricted HIV-1 protein specific peptides, which are Gag, Env P18, RT354, RT464, and RT472. The peptide sequences are shown in the FIG. 29. The stimulated splenocytes were double stained for the cell surface CD8 molecules and intracellular IFN- of T cells with FITC rat anti-mouse CD8 and APC rat anti-mouse IFN-. The regimen, prime-boost immunization with rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) induced peptide specific CD8+ T cell immune responses with different degree depending on the peptides the inventors used for the stimulation. CD8+ T cells against Env peptide stimulated CD8+ T cells the most up to 19%-23% of CD8+ T cells on average (FIG. 28C, G3 and FIG. 28E, G5). About 4.5% or 3% of CD8+ T cells were responsive to the Gag protein in mice immunized with rVSV expressing Gag alone (FIG. 28B, G2) or with mixed three rVSVs expressing Gag A, Gag B, and Gag C linked with various B and T cell epitopes of HIV-1 Gp120 and Gp41 (FIG. 29, G6). RT specific CD8+ T cells were generated in about 2% of CD8+ T cells in mice injected with rVSV with pol gene (FIG. 28D, G4). CD8+ T cell responses against individual HIV-1 protein, Gag and RT were stronger when mice were injected with single rVSV expressing individual protein (FIG. 28B, G2 and FIG. 28D, G4) than when injected with mixed rVSVs expressing single protein (FIG. 28E, G5). Env specific CD8+ T cell responses were strongest and were similar in both single injection and mixed injections. The CD8+ T cells responses against Env peptide epitope are immunodominant in Balb/c mice. Currently, the inventors are uncertain why mixed injections with three different rVSVs expressing Gag, Env, and RT induced comparatively lower CD8+ T cell responses against VSV N, gag, and RT than with the single virus injection. It is possible that the competition of each virus for the antigen presenting cells in the mixed virus injection lowered the CD8+ T cells against Gag and RT. Because rVSV is common vector for HIV-1 proteins in mice either injected with single rVSV or mixed rVSVs, VSV N supposed to induce similar or better CD8+ T cell responses in the mixed injection. However, the CD8+ T cell responses against VSV N in mice are lower even with the single injections when the Env is expressed (FIG. 28C, G3) as in the mixed injection (FIG. 28E, G5). Therefore, it may be the result of immune dominance of Env protein in Balb/c mice, which affects negatively to the presentation of other proteins to the CD8+ T cells when Env is expressed together. For the combined immunizations in mice, all three rVSVs were injected at the single site, which may have resulted in the competition of different proteins for the antigen presentation in the same antigen presentation cells. The inventors will test the immunization with the mixed injections at more than one injection sites to induce CD8+ T cell responses.

(115) HIV-1 Gag proteins expressed in cells can form virus like particles (VLP) and the VLP can be released from the cells. The released VLPs can induce humoral immune responses against Gag proteins. HIV-1 env gene encodes glycosylated surface proteins, which are processed through ER-golgi network to be properly folded and cleaved into transmembrane subunit Gp41 and surface unit Gp120. The Gp41 and Gp120 are associated by non-covalent bond and mature to form a trimer of Gp41 and Gp120 heterodimer. The matured trimer of Gp41 and Gp120 are exported to the cell surface. The Gp120 tends to fall off from the cell surface because of the weak bondage between Gp41 and Gp120. Therefore, antibody against Gp120 can be induced to the cell surface Gp120 or fallen off Gp120. Antibody titer against HIV-1 Gag protein and Gp120 was determined by ELISA. For the Gag antibody the microplate was coated with 125 ng/well of recombinant p55 (Pierce Biotechnology, RP-4921) and 50 l of mice sera was tested with the dilution of 1:100, 1:200, and 1:400. Gag antibody was produced in mice injected with rVSV Gag-En alone (FIG. 30A, G2) and with mixed viruses rVSV-Gag A+rVSV Gag B+rVSV Gag C (FIG. 30A, G6). For the Gp120 ELISA, 96 well microplate was coated with 250 ng/well of HIV-1 SF162 Gp140 trimer from clade B (NIH AIDS reagent program, cat#12026). Mice sera were diluted in 1:100, 1:200, and 1:400 and 50 l of the diluted serum was added to the microplate for the ELISA. Antibody specific to Gp120 was generated in mice injected with rVSV-HIV-1 Gp160 alone and in mice injected with the mixed viruses expressing each protein of Gag, Gp160, and RT (FIG. 30B, G3 and G5). The titer of Gp120 antibody was slightly lower, although not statistically significant, in mice injected with mixed viruses than in mice injected with single virus expressing Gp160.

(116) The newly generated M mutants of rVSVs, rVSV.sub.Ind(GLM) and rVSV.sub.NJ(GM) induced CD8+ T cell and humoral immune responses against various HIV-1 proteins expressed from the vector after injecting mice with an individual virus expressing single virus or with mixed viruses expressing various HIV-1 proteins.

Example 15Generation of rVSVInd(GLM), rVSVNJ(GM), and rVSVNJ(GLM) with Hepatitis C Virus Structural Proteins

(117) Generally, the humoral immune response is the first line of defence mediated by adaptive immune responses against any pathogens. Although humoral immune responses against HCV and its role in the prevention of HCV infection is not well studied compared to the HCV specific cellular immune responses, it is worthy to include vaccines which can induce HCV specific antibodies. It has been demonstrated that nucleocapsid protein core and surface glycoproteins E1 and E2 form virus-like particles (VLP) which can be released from the cells (Blanchard, E. et al., J. Virol. 76:4073-4079, 2002). In addition, it has been demonstrated that HCV protein p7, a viroporin forming an ion channel in the ER membrane, takes part in releasing the HCV particles from the infected cell (Steinmann, E. et al., PLoS Pathogens 3:962-972, 2007). Another HCV transmembrane protein NS4B forms a membranous web structure, which mainly consists of the ER membrane. The membranous web formed by NS4B is a microstructure for the production of progeny HCV. It is not well known what functions NS4B has in the replication of HCV, but it is appealing to include NS4B into vaccine candidates simply because of its nature forming a membranous structure of ER in which other HCV proteins, especially Core, E1, E2, p7 are localized. Therefore, including Core, E1, E2, P7, and NS4B together into the vaccine may induce humoral and cellular immune responses.

(118) For the HCV structural protein vaccines using the new M mutant rVSVs, the inventors inserted the HCV core gene, E1E2P7 and NS4B genes together (connected by the VSV intergenic junction sequences), and CoreE1E2P7 and NS4B genes together (connected by the VSV intergenic junction sequences) to the junction of VSV G gene and L (FIG. 31). The rVSVs; rVSV.sub.Ind(GLM)-FC, rVSV.sub.Ind(GLM)-CE1E2P7/NS4B, rVSV.sub.NJ(GM)-FC, and rVSV.sub.NJ(GLM)-E1E2P7/NS4B were generated by reverse genetics. The inventors had a trouble to generate the rVSV.sub.NJ(GM) with CE1E2P7/NS4B from the plasmid DNA, therefore, the inventors removed the core and cloned E1E2P7/NS4B into the plasmid of rVSV.sub.NJ(GLM) and generated the rVSV.sub.NJ(GLM)-E1E2P7/NS4B. The expression of Core, E1, E2, and NS4B from the recombinant VSV at 31 C. and 37 C. was examined by Western blot analysis using antibodies against each protein (FIG. 32). The proteins were expressed similarly in quantity at both temperatures. Core (FIG. 32A), E1 (FIG. 32B), and E2 FIG. 32C) were expressed well from the rVSV.sub.Ind(GLM)-CE1E2P7/NS4B, but the expression of NB4B was lower than the other proteins (FIG. 32D).

Example 16Generation of rVSVInd(GLM), rVSVNJ(GM), and rVSVNJ(GLM) with HCV Non-Structural Proteins

(119) The inventors want to target most of the HCV proteins including core, E1, E2, NS3, NS4A, NS4B, NS5A and NS5B proteins in order to induce HCV specific CD8+ T cell and CD4+ T cell immune responses to multiple proteins. HCV nonstructural (NS) proteins-NS3, NS4A, NS4B, NS5A, and NS5B cover more than half of the HCV polyprotein. The NS proteins are cleaved into individual proteins by NS3 with the help of NS4A. Several studies demonstrate that patients who recover from the acute HCV infection develop strong CD4+ T cell and CD8+ T cell responses against multiple epitopes in the NS3 protein (Diepolder, H. M. J. Virol. 71:6011-6019, 1997; Lamonaca, V. et al. Hepatology 30:1088-1098, 1999; Shoukry, N. H. et al. J. Immunol. 172:483-492, 2004) indicating that including NS3 in the vaccine candidate is important to elicit successful cellular immune responses against HCV. NS3 protein, a serine protease and RNA helicase associates with NS4A and resides on the ER membrane (Sato, S. et al. J. Virol. 69:4255-4260, 1995; Failla, C. et al. J. Virol. 69:1769-1777, 1995). NS5A protein, a phosphoprotein is believed to be involved in HCV RNA synthesis together with NS5B protein, a RNA dependent RNA polymerase (Shirota, Y. et al., J. Biol. Chem., 277:11149-11155, 2002; Shimakami, et al. J. Virol. 78:2738-2748, 2004). NS5B protein is a RNA dependent RNA polymerase that synthesizes positive sense HCV genomic RNA as well as intermediate negative sense genomic RNA (Beherens, S. E. EMBO J. 15:12-22, 1996). NS5B protein is a tail-anchored protein and associates with ER membrane through its carboxyl terminal 20 amino acids (Yamashita, T. J. Biol. Chem. 273:15479-15486, 1998; Hagedorn, C. H. Curr. Top. Microbiol. Immunol. 242:225-260, 2000).

(120) The inventors cloned HCV NS genes as a gene for a single protein or a gene for a polyprotein of 2 or 3 NS proteins. The NS genes NS3, NS34AB, NS5A, NS5B, NS5AB were cloned into the junction at G gene and L gene of pVSV.sub.Ind(GLM) and pVSV.sub.NJ(GM) (FIG. 33). The inventors generated rVSV.sub.Ind(GLM)-NS3, rVSV.sub.Ind(GLM)-NS34AB, rVSV.sub.Ind(GLM)-NS5A, rVSV.sub.Ind(GLM)-NS5B, and rVSV.sub.Ind(GLM)-NS5AB, and the expression of the NS proteins at 31 C. and 37 C. were determined by Western blot analysis using antibodies against each NS protein (FIG. 34). The inventors generated rVSV.sub.NJ(GM)-NS3, rVSV.sub.NJ(GM)-NS4B, rVSV.sub.NJ(GM)-NS34AB, rVSV.sub.NJ(GM)-NS5A, rVSV.sub.NJ(GM)-NS5B, and rVSV.sub.NJ(GM)-NS5AB. The expression of each protein from the rVSV.sub.NJ vector at 37 C. was examined by Western blot analysis using antibodies against each NS protein (FIG. 35). Although the expression level of NS proteins from the rVSV.sub.Ind and rVSV.sub.NJ was different, it was good enough to use them as HCV vaccine.

(121) TABLE-US-00001 TABLE 1 Mouse Vaccination Groups and Regimen Prime Boost Vacci- Titer Titer nation (PFU)/ (PFU)/ # of Groups Viruses 50 l Viruses 50 l Mice 1 Ind (WT)-Gag 5 10.sup.6 NJ (WT)-Gag 5 10.sup.6 6 2 Ind (GLM)-Gag 5 10.sup.6 NJ (GLM)-Gag 5 10.sup.6 6 3 Ind (GLM)-Gag 5 10.sup.7 NJ (GLM)-Gag 5 10.sup.7 6 4 Ind (GLM)-Gag 5 10.sup.8 NJ (GLM)-Gag 5 10.sup.8 6 5 Ind (GLM)-Gag 5 10.sup.9 NJ (GLM)-Gag 5 10.sup.9 6 6 Ind (GLM)-Gag 5 10.sup.6 NJ (GM)-Gag 5 10.sup.6 6 7 Ind (GLM)-Gag 5 10.sup.7 NJ (GM)-Gag 5 10.sup.7 6 8 Ind (GLM)-Gag 5 10.sup.8 NJ (GM)-Gag 5 10.sup.8 6 9 Ind (GLM)-Gag 5 10.sup.9 NJ (GM)-Gag 5 10.sup.9 6 10 NJ (WT)-Gag 5 10.sup.6 Ind (WT)-Gag 5 10.sup.6 6 Total 60 Mice

(122) TABLE-US-00002 TABLE2 NeucleotideSequenceComparisonbetweenMGenesofVSVIndiana serotype,WildType(SEQIDNO:1)andaMutantG21E/L111F/M51R (SEQIDNO2) 150 SEQIDNO:1: ATGAGTTCCTTAAAGAAGATTCTCGGTCTGAAGGGGAAAGGTAAGAAATC SEQIDNO:2: ATGAGTTCCTTAAAGAAGATTCTCGGTCTGAAGGGGAAAGGTAAGAAATC 51100 SEQIDNO:1: TAAGAAATTAGGGATCGCACCACCCCCTTATGAAGAGGACACTAACATGG SEQIDNO:2: TAAGAAATTAGAAATCGCACCACCCCCTTATGAAGAGGACACTAACATGG 101150 SEQIDNO:1: AGTATGCTCCGAGCGCTCCAATTGACAAATCCTATTTTGGAGTTGACGAG SEQIDNO:2: AGTATGCTCCGAGCGCTCCAATTGACAAATCCTATTTTGGAGTTGACGAG 151200 SEQIDNO:1: ATGGACACTCATGATCCGCATCAATTAAGATATGAGAAATTCTTCTTTAC SEQIDNO:2: AGGGACACTCATGATCCGCATCAATTAAGATATGAGAAATTCTTCTTTAC 201250 SEQIDNO:1: AGTGAAAATGACGGTTAGATCTAATCGTCCGTTCAGAACATACTCAGATG SEQIDNO:2: AGTGAAAATGACGGTTAGATCTAATCGTCCGTTCAGAACATACTCAGATG 251300 SEQIDNO:1: TGGCAGCCGCTGTATCCCATTGGGATCACATGTACATCGGAATGGCAGGG SEQIDNO:2: TGGCAGCCGCTGTATCCCATTGGGATCACATGTACATCGGAATGGCAGGG 301350 SEQIDNO:1: AAACGTCCCTTCTACAAGATCTTGGCTTTTTTGGGTTCTTCTAATCTAAA SEQIDNO:2: AAACGTCCCTTCTACAAGATCTTGGCTTTTTTTGGTTCTTCTAATCTAAA 351400 SEQIDNO:1: GGCCACTCCAGCGGTATTGGCAGATCAAGGTCAACCAGAGTATCACGCTC SEQIDNO:2: GGCCACTCCAGCGGTATTGGCAGATCAAGGTCAACCAGAGTATCACGCTC 401450 SEQIDNO:1: ACTGTGAAGGCAGGGCTTATTTGCCACACAGAATGGGGAAGACCCCTCCC SEQIDNO:2: ACTGTGAAGGCAGGGCTTATTTGCCACACAGAATGGGGAAGACCCCTCCC 451500 SEQIDNO:1: ATGCTCAATGTACCAGAGCACTTCAGAAGACCATTCAATATAGGTCTTTA SEQIDNO:2: ATGCTCAATGTACCAGAGCACTTCAGAAGACCATTCAATATAGGTCTTTA 501550 SEQIDNO:1: CAAGGGAACGGTTGAGCTCACAATGACCATCTACGATGATGAGTCACTGG SEQIDNO:2: CAAGGGAACGGTTGAGCTCACAATGACCATCTACGATGATGAGTCACTGG 551600 SEQIDNO:1: AAGCAGCTCCTATGATCTGGGATCATTTCAATTCTTCCAAATTTTCTGAT SEQIDNO:2: AAGCAGCTCCTATGATCTGGGATCATTTCAATTCTTCCAAATTTTCTGAT 601650 SEQIDNO:1: TTCAGAGATAAGGCCTTAATGTTTGGCCTGATTGTCGAGAAAAAGGCATC SEQIDNO:2: TTCAGAGATAAGGCCTTAATGTTTGGCCTGATTGTCGAGAAAAAGGCATC 651700 SEQIDNO:1: TGGAGCTTGGGTCCTGGATTCTGTCAGCCACTTCAAATGA SEQIDNO:2: TGGAGCTTGGGTCCTGGATTCTGTCAGCCACTTCAAATGA

(123) TABLE-US-00003 TABLE3 AminoAcidSequenceComparisonbetweenMProteinsofVSVIndiana serotypeWildType(SEQIDNO:3)andaMutantG21E/L111F/M51R (SEQIDNO:4) 12150 SEQIDNO:3: MSSLKKILGLKGKGKKSKKLGIAPPPYEEDTNMEYAPSAPIDKSYFGVDE SEQIDNO:4: MSSLKKILGLKGKGKKSKKLEIAPPPYEEDTNMEYAPSAPIDKSYFGVDE 51100 SEQIDNO:3: MDTHDPHQLRYEKFFFTVKMTVRSNRPFRTYSDVAAAVSHWDHMYIGMAG SEQIDNO:4: RDTHDPHQLRYEKFFFTVKMTVRSNRPFRTYSDVAAAVSHWDHMYIGMAG 101111150 SEQIDNO:3: KRPFYKILAFLGSSNLKATPAVLADQGQPEYHAHCEGRAYLPHRMGKTPP SEQIDNO:4: KRPFYKILAFFGSSNLKATPAVLADQGQPEYHAHCEGRAYLPHRMGKTPP 151200 SEQIDNO:3: MLNVPEHFRRPFNIGLYKGTVELTMTIYDDESLEAAPMIWDHFNSSKFSD SEQIDNO:4: MLNVPEHFRRPFNIGLYKGTVELTMTIYDDESLEAAPMIWDHFNSSKFSD 201229250 SEQIDNO:3: FRDKALMFGLIVEKKASGAWVLDSVSHFK SEQIDNO:4: FRDKALMFGLIVEKKASGAWVLDSVSHFK

(124) TABLE-US-00004 TABLE4 NucleotideSequenceComparisonbetweenMGenesofVSVNewJersey serotypeWildType(SEQIDNO:5)andMutants,G22E/M48R+ M51R (SEQIDNO:6)andG22E/L110F/M48R+ M51R(SEQIDNO:7) 150 SEQIDNO:5: ATGAGTTCCTTCAAAAAGATTCTGGGATTTTCTTCAAAAAGTCACAAGAA SEQIDNO:6: ATGAGTTCCTTCAAAAAGATTCTGGGATTTTCTTCAAAAAGTCACAAGAA IDNO:7: ATGAGTTCCTTCAAAAAGATTCTGGGATTTTCTTCAAAAAGTCACAAGAA 51100 SEQIDNO:5: ATCAAAGAAACTAGGCTTGCCACCTCCTTATGAGGAATCAAGTCCTATGG SEQIDNO:6: ATCAAAGAAACTAGAATTGCCACCTCCTTATGAGGAATCAAGTCCTATGG SEQIDNO:7: ATCAAAGAAACTAGAATTGCCACCTCCTTATGAGGAATCAAGTCCTATGG 101150 SEQIDNO:5: AGATTCAACCATCTGCCCCATTATCAAATGACTTCTTCGGAATGGAGGAT SEQIDNO:6: AGATTCAACCATCTGCCCCATTATCAAATGACTTCTTCGGAAGGGAGGAT SEQIDNO:7: AGATTCAACCATCTGCCCCATTATCAAATGACTTCTTCGGAAGGGAGGAT 151200 SEQIDNO:5: ATGGATTTATATGATAAGGACTCCTTGAGATATGAGAAGTTCCGCTTTAT SEQIDNO:6: AGGGATTTATATGATAAGGACTCCTTGAGATATGAGAAGTTCCGCTTTAT SEQIDNO:7: AGGGATTTATATGATAAGGACTCCTTGAGATATGAGAAGTTCCGCTTTAT 201250 SEQIDNO:5: GTTGAAGATGACTGTTAGAGCTAACAAGCCCTTCAGATCGTATGATGATG SEQIDNO:6: GTTGAAGATGACTGTTAGAGCTAACAAGCCCTTCAGATCGTATGATGATG SEQIDNO:7: GTTGAAGATGACTGTTAGAGCTAACAAGCCCTTCAGATCGTATGATGATG 251300 SEQIDNO:5: TCACCGCAGCGGTATCACAATGGGATAATTCATACATTGGAATGGTTGGA SEQIDNO:6: TCACCGCAGCGGTATCACAATGGGATAATTCATACATTGGAATGGTTGGA SEQIDNO:7: TCACCGCAGCGGTATCACAATGGGATAATTCATACATTGGAATGGTTGGA 301350 SEQIDNO:5: AAGCGTCCTTTCTACAAGATAATTGCTCTGATTGGCTCCAGTCATCTGCA SEQIDNO:6: AAGCGTCCTTTCTACAAGATAATTGCTCTGATTGGCTCCAGTCATCTGCA SEQIDNO:7: AAGCGTCCTTTCTACAAGATAATTGCTTTTATTGGCTCCAGTCATCTGCA 351400 SEQIDNO:5: AGCAACTCCAGCTGTGTTGGCAGACTTAAATCAACCAGAGTATTATGCCA SEQIDNO:6: AGCAACTCCAGCTGTGTTGGCAGACTTAAATCAACCAGAGTATTATGCCA SEQIDNO:7: AGCAACTCCAGCTGTGTTGGCAGACTTAAATCAACCAGAGTATTATGCCA 401450 SEQIDNO:5: CACTAACAGGTCGTTGTTTTCTTCCTCACCGACTCGGATTGATCCCACCG SEQIDNO:6: CACTAACAGGTCGTTGTTTTCTTCCTCACCGACTCGGATTGATCCCACCG SEQIDNO:7: CACTAACAGGTCGTTGTTTTCTTCCTCACCGACTCGGATTGATCCCACCG 451500 SEQIDNO:5: ATGTTTAATGTGTCCGAAACTTTCAGAAAACCATTCAATATTGGGATATA SEQIDNO:6: ATGTTTAATGTGTCCGAAACTTTCAGAAAACCATTCAATATTGGGATATA SEQIDNO:7: ATGTTTAATGTGTCCGAAACTTTCAGAAAACCATTCAATATTGGGATATA 501550 SEQIDNO:5: CAAAGGGACTCTCGACTTCACCTTTACAGTTTCAGATGATGAGTCTAATG SEQIDNO:6: CAAAGGGACTCTCGACTTCACCTTTACAGTTTCAGATGATGAGTCTAATG SEQIDNO:7: CAAAGGGACTCTCGACTTCACCTTTACAGTTTCAGATGATGAGTCTAATG 551600 SEQIDNO:5: AAAAAGTCCCTCATGTTTGGGAATACATGAACCCAAAATATCAATCTCAG SEQIDNO:6: AAAAAGTCCCTCATGTTTGGGAATACATGAACCCAAAATATCAATCTCAG SEQIDNO:7 AAAAAGTCCCTCATGTTTGGGAATACATGAACCCAAAATATCAATCTCAG 601650 SEQIDNO:5: ATCCAAAAAGAAGGGCTTAAATTCGGATTGATTTTAAGCAAGAAAGCAAC SEQIDNO:6: ATCCAAAAAGAAGGGCTTAAATTCGGATTGATTTTAAGCAAGAAAGCAAC SEQIDNO:7: ATCCAAAAAGAAGGGCTTAAATTCGGATTGATTTTAAGCAAGAAAGCAAC 651700 SEQIDNO:5: GGGAACTTGGGTGTTAGACCAATTGAGTCCGTTTAA SEQIDNO:6: GGGAACTTGGGTGTTAGACCAATTGAGTCCGTTTAA SEQIDNO:7: GGGAACTTGGGTGTTAGACCAATTGAGTCCGTTTAA

(125) TABLE-US-00005 TABLE5 AminoAcidSequenceComparisonbetweenMProteinsofVSVNewJersey serotypeWildType(SEQIDNO:8)andMutants,G22E/M48R+ M51R (SEQIDNO:9)andG22E/L110F/M48R+ M51R(SEQIDNO:10) 1224850 SEQIDNO:8: MSSFKKILGFSSKSHKKSKKLGLPPPYEESSPMEIQPSAPLSNDFFGMED SEQIDNO:9: MSSFKKILGFSSKSHKKSKKLELPPPYEESSPMEIQPSAPLSNDFFGRED SEQIDNO:10: MSSFKKILGFSSKSHKKSKKLELPPPYEESSPMEIQPSAPLSNDFFGRED 51100 SEQIDNO:8: MDLYDKDSLRYEKFRFMLKMTVRANKPFRSYDDVTAAVSQWDNSYIGMVG SEQIDNO:9: RDLYDKDSLRYEKFRFMLKMTVRANKPFRSYDDVTAAVSQWDNSYIGMVG SEQIDNO:10: RDLYDKDSLRYEKFRFMLKMTVRANKPFRSYDDVTAAVSQWDNSYIGMVG 101110150 SEQIDNO:8: KRPFYKIIALIGSSHLQATPAVLADLNQPEYYATLTGRCFLPHRLGLIPP SEQIDNO:9: KRPFYKIIALIGSSHLQATPAVLADLNQPEYYATLTGRCFLPHRLGLIPP SEQIDNO:10: KRPFYKIIAFIGSSHLQATPAVLADLNQPEYYATLTGRCFLPHRLGLIPP 151200 SEQIDNO:8: MFNVSETFRKPFNIGIYKGTLDFTFTVSDDESNEKVPHVWEYMNPKYQSQ SEQIDNO:9: MFNVSETFRKPFNIGIYKGTLDFTFTVSDDESNEKVPHVWEYMNPKYQSQ SEQIDNO:10: MFNVSETFRKPFNIGIYKGTLDFTFTVSDDESNEKVPHVWEYMNPKYQSQ 201250 SEQIDNO:8: IQKEGLKFGLILSKKATGTWVLDQLSPFK SEQIDNO:9: IQKEGLKFGLILSKKATGTWVLDQLSPFK SEQIDNO:10: IQKEGLKFGLILSKKATGTWVLDQLSPFK

(126) TABLE-US-00006 TABLE 6 Vaccination studies in mice for broad range CD8+ T cell responses and humoral immune responses against HIV-1 proteins Vacci- nation rVSV w/HIV-1 Proteins Groups Prime Boost G 1 rVSV.sub.Ind(GLM) rVSV.sub.NJ(GM) G 2 rVSV.sub.Ind(GLM)-HIV-1 Gag-En rVSV.sub.NJ(GM)-HIV-1 Gag-En G 3 rVSV.sub.Ind(GLM)-HIV-1 rVSV.sub.NJ(GM)-HIV-1 Gp160mss Gp160mss G 4 rVSV.sub.Ind(GLM)-HIV-1 Pol rVSV.sub.NJ(GM)-HIV-1 Pol G 5 rVSV.sub.Ind(GLM)-HIV-1 rVSV.sub.NJ(GM)-HIV-1 Gag-En + rVSV.sub.Ind(GLM)- Gag-En + rVSV.sub.NJ(GM)- Gp160mss + rVSV.sub.Ind(GLM)- Gp160mss + rVSV.sub.NJ(GM)- HIV-1 Pol HIV1 Pol G 6 rVSV.sub.Ind(GLM)-HIV-1 Gag-A + rVSV.sub.NJ(GM)-HIV-1 Gag-A + rVSV.sub.Ind(GLM)-HIV-1 Gag-B + rVSV.sub.NJ(GM)-HIV-1 Gag-B + rVSV.sub.Ind(GLM)-HIV-1 Gag-C rVSV.sub.NJ(GM)-HIV-1 Gag-C