OA19257A - Vesicular stomatitis virus for prime boost vaccines. - Google Patents

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.

Description

[0121] Unless defined otherwise, ail technical and scientifîc terms used herein hâve the same meaning as commonly understood b y one of ordinary skill in the art to which this invention belongs. Also, unless indicated otherwise, except within the daims, the use of “or” includes “and” and vice versa. Non-limitîng 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 daims such as “a”, “an” and “the” include the plural référencé unless expressly stated otherwise. “Consisting essentially of’ means any recited éléments are necessarily included, éléments that would materially affect the basic and novel characteristics of the listed éléments are exduded, and other éléments may optionally be included. “Consisting of ’ means that ail éléments other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

[0122] AU numerical désignations, e.g., dimensions and weight, including ranges, are approximations that typically may be varied ( + ) or ( - ) by incréments of 0.1, 1.0, or 10,0, as appropriate. AU numerical désignations may be understood as preceded by the term “about”.

[0123] The term “administering” includes any method of delivery of a compound of the présent invention, including a pharmaceutical composition, vaccine or therapcutic agent, into a subject's System or to a partïcular région in or on a subject. The phrases “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripheraily” as used herein mean the administration of a compound, drug or other material other than directly into the centra] nervous System, such that it entera the patient’s system and, thus, is subject to metabolistn and other like processes, for example, subcutaneous administration. “Parentéral administration” and “administered parenterally” means modes of administration other than entera] and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, txanstracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoîd, intraspinal and intrastemal injection and infusion.

[0124] 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 Biochemica] Nomenclature (see Biochemistry (1972) 11:1726-1732). For the amino acids relevant to the présent invention the désignations are; M: méthionine, 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, thréonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.

[0125] The tenu “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 recogruze and are able to bïnd 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 molécule that are capable of selectively reactîng with a certain protein. Nonlimiting examples of such proteolytic and/or recombinant fragments include Fab, F^b^, 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.

[0126] As used herein, the term “epitopes” refers to sites or fragments of a polypeptide or protein having antigenic or immunogenic activity in an animal, prefcrably 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 immunospecifîcally binds as determined by any method well-known to one of skill in the art, for example by immunoassays.

[0127] 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 fiill-length protein retains activity of the full19257 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.

[0128] As used herein, the term “fragment” in the context of a nucleic acid refers to a nucleic acid comprising an nucleic acid sequence of ai least 2 contiguous

nucléotides,	at	least 5 contiguous nucléotides, at	least	10 contiguous
nucléotides,	at	least 15 contiguous nucléotides, at	least	20 contiguous
nucléotides,	at	least 25 contiguous nucléotides, at	least	30 contiguous
nucléotides,	at	least 35 contiguous nucléotides, at	least	40 contiguous
nucléotides,	at	least 50 contiguous nucléotides, at	least	60 contiguous
nucléotides,	at	least 70 contiguous nucléotides, at	least	contiguous 80
nucléotides,	at	least 90 contiguous nucléotides, at	least	100 contiguous
nucléotides,	at	least 125 contiguous nucléotides, at	least	150 contiguous
nucléotides,	at	least 175 contiguous nucléotides, at	least	200 contiguous
nucléotides,	at	least 250 contiguous nucléotides, at	least	300 contiguous

nucléotides, at least 350 contiguous nucléotides, or at least 380 contiguous nucléotides 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 rctains activity of the full-length protein, e.g., immuogenic activity. In another embodiment, the fragment of the full-length protein does not retain the activity of the full-length protein, e.g., nonimmunogenic activity.

[0129] 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.

[0130] 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 outlief” group O and the “new” group

N. These three groups may represcnt 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 ail of these strains.

[0131] The term “non-infectious” means of reduced to non-existent ability to infect.

[0132] 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 nonhurnan animal.

[0133] 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 hypodennic syringes, multichamber syringes, stents, cathéters, 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.

[0134] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, matériels, 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 animais without excessive toxicity, irritation, allergie response, or other problem or complication, commensurate with a reasonàble benefit/risk ratio.

[0135] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable matériel, composition or vebicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in cairying 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 ingrédients 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 com starch and potato starch;

(3) cellulose, and its dérivatives, 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 supposîtory waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (II) polyols, such as ¹⁰ glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnésium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonie saline; (18) Ringefs solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other ¹⁵ non-toxic compatible substances employed in pharmaceutical formulations.

[0136] The ternis “polynucleotide”, and “nuclcic acid are used interchangeably to refer to a polymeric form of nucléotides of any length, either deoxyribonucleotides or ribonuclcotides, or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding régions of a gene or gene fragment, locî (locus) defined from linkage analysis, exons, ²⁰ 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 modifîed nucléotides, such as methylated nucléotides and nucléotide analogs. If présent, modifications to the nucléotide structure may be imparted before or after assembly of the polymer. The sequence of nucléotides may be interrupted by non-nucleotide components. A polynucleotide may be further modifîed after polymerization, such as by conjugation with a labeling component The term “recombinant” polynucleotide means a polynucleotide of genomic, cDNA, seini-synthctic, or synthetic origin, which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement. An “oligonucleotide” refera to a single strandcd polynucleotide having less than about 100 nucléotides, less than about, e.g., 75, 50, 25, or 10 nucléotides.

[0137] The teims polypeptide”, “peptide” and “protein” (if single Chain) are used înterchangeably herein to refer to polymers of amino acids. The polymer may be linear or branched, it may comprise modîfied amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modifîed; for example, disulfide bond formation, glycosylation, lipidation, acétylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0138] 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 stepwîse 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 intennediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the fiill 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 molécules (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. Wlodawcr, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al., Biochemisfry (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- OSbrd, “Chemical

Approaches to Protein Engineering”, .in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plénum 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. KChang, 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).

[0139] “VS V” is used to refer to vesicular stomatitis virus.

[0140] rVSV” is used to refer to a recombinant vesicular stomatitis virus.

[0141] The teim “Indiana”, and “IND are used to refer to the VSV serotype Indiana (VSV_Ind).

[0142] The term “New Jersey”, and “NJ” are used to refer to the VSV serotype New Jersey (VSV_NJ).

[0143] “MWT” “M(WT)” are used to refer to a wild type M protein. The nucléotide sequence of wild type M gene of the VSV_[nd may comprise the nucléotide sequence represented by SEQ ID NO: 1. The amino acid sequence of wild type M protein of the VSV_tod may comprise the amino acid sequence represented by SEQ ID NO: 3. The nucléotide sequence of wild type M gene of the VSV_W may comprise the nucléotide sequence represented by SEQ ID NO: 5. The amino acid sequence of wild type M protein of the VSVnj may comprise the amino acid sequence represented by SEQ ID NO: 8. “M51R” is used to refer to an M(WT) in the VSVm having a méthionine changed to an arginine at position 51. “G21E is used to refer to an M(WT) in VSV_ind having a glycine changed to a glutamic acid at position 21. “El 11F” is used to refer to an M(WT) in VSVfod having a leucine changed to a phenylalanine at position 111. “G22E” is used to refer to an M(WT) in VSVnj having a glycine (G) changed to glutamic acid (E) at position 22. “L110F” is used to refer to an M(WT) in VSVnj having a leucine (L) changed to a phenylalanine (F) at position 110. “M48R+M51 R” is used to refer to an M(WT) in VSVhj having a méthionine (M) changed to an arginine (R) at positions 48 and 51. rVSVw M(G21E/L111F/M51R)” or ‘ïVSVkd (GLM)” are used to refer to a rVSVtd having an M(WT) having a glycine changed to a glutamic acid at position 21, a leucine changed to a 5 phenylalanine at position 111 and a méthionine changed to an arginine at position 51. “rVSV_Ni M(G22E/M48R+M51R)” or “rVSV_w (GM)” are used to refer to rVSVjqj having an M(WT) having a glycine changed to a glutamic acid at position 22 and a méthionine changed to an arginine at positions 48 and 51, and “rVSV_w M(G22E/L110F/M48R+M51R)” or *WSV_w (GLM)” are used to ¹⁰ refer to rVSV_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 méthionine changed to an arginine at positions 48 and 51.

[0144] OverView

[0145] The inventors generated novel M proteins and novel attenuated rVSVs capable 1 A of producing the novel M proteins. The novel proteins of the présent invention may include: M(G21E/L111F/M51R), M(G22E/M48R+M51R), and

M(G22E/L110F/M48R+M51R). The novel attenuated rVSVs of the présent invention may be used as protein expression and vaccine vectors and in methods for preventing or treaüng infections. The rVSV of the présent invention may be 20 applied to make vaccines for the infectious diseases of human and other animais to induce cellular and humoral immune responses.

[0146] Isolated Proteins and MProteins

[0147] In one embodiment, the présent invention relates to isolated proteins and to nucléotide sequences that encode the isolated proteins. As such, in one ₂₅ embodiment the présent invention relates to an isolated peptide comprising an amino aejd sequence selected from amino acid sequences listed as SEQ ID NOs; 4, 9 and 10. In another embodiment, the présent invention relates to isolated nucléotide sequences comprising a nucléotide sequence selected from the polynucleotides listed as SEQ ID NOs: 2,6 and 7.

[0148] In one embodiment, the présent invention relates to novel VSV M proteins having at least one of the following substitutions: M(G2IEZL111F/M51R), M(G22E/M48R+M51R), and M(G22E/L110F/M48R+M51R). In one aspect the présent 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 présent invention relates to nucléotide sequences which encode for the novel VSV M proteins of the présent invention. The nucléotide sequences may be selected from the group of sequences listed as SEQ ID NOs: 2,6 and 7.

[0149] Methods ofPreventing or Treating an Infection

[0150] 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 fîrst 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 présent invention, the methods may include administering to a subject (a) an effective amount of rVSV^ M(G21E/L111F/M51R), and (b) an effective amount of either a rVSV_NJ M(G22E/M48R+M51R) or rVSV_NJ M(G22E/L110F/M48R+M51R) in a prime-boost immunization modality.

[0151] The tenu “effective amount” as used herein means an amount effective and at dosages and for periods of time necessary to achieve the desired resuit.

[0152] In certain embodiments, (a) is administered to the subject before (b) is administered to the subject.

[0153] In certain embodiments, (b) is administered to the subject more than one dîne over the course of treating or preventing.

[0154] 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).

[0155] In certain embodiments, (b) is administered to the subject before (a) is administered to the subject.

[0156] In certain embodiments, (a) is administered to the subject more than one time over the course of treating or preventing.

[0157] 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).

[0158] Recombinant Virus

[0159] In certain embodiments, présent invention relates to a recombinant vesicular stomatitis virus (rVSV) which may be a full length VSV, essentially noncytolytic, avirulent, capable of inducing an immune response in a subject, capable of reproducing virus particles to a high tire at permissive températures, reproducing virus particles to a low titre at semi-permissive températures and which may be incapable of producing virus at non-permissive températures, and that can express an epitope of a foreign pathogen. The rVSV of the présent invention may be capable of inducing humoral, cellular and mucosal immune responses.

[0160] In one embodiment, the présent invention relates to rVSVfaj and tVSVnj. The rVSVina may be a iull length, essentially noncytolytic rYSY^ M(G21E/L111F/M51R) capable of producing virus particles at a permissible température of about 31°C, and which may be incapable of or poorly capable of producing virus particles at a semi-permissive températures of about 37°C and incapable of producing virus particles at non-permissive températures above 37°C, for example 39^ÛC. In certain embodiments, the rVSV_tad may include a M(G21E7L111F/M51R). In certain embodiments, the rVSV_M may include an M gene comprising a nucléotide sequence SEQ ID NO: 2.

[0161] In certain embodiments, the rVSV is a full-length, essentially noncytolytic rVSVw M (G22E/M48R+M51R) or M(G22/L110F/M48R+M51R). In certain embodiments, the rVSV is an essentially noncytolytic rVSVNj including an M gene, wherein the nucléotide sequence of the M gene is selectcd from SEQ ID NO: 6 and SEQ ID NO: 7.

[0162] The rVSVs of the présent 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 réplication and expression of the rVSV in the host. Accordingly, the présent invention also provides a cell having a rVSV_bd wherein the amino acid sequence of the virus’ M protein is modified to provide an essentially non-cytotoxic which also allows the rVSV_bd to effectively replicate at pennissible température but may not replicate at non-permissible température.

[0163] As such, the présent invention relates also to a cell having one or more of the recombinant VSVs of the présent invention.

[0164] Vaccines or Immunogenic Compositions of the Invention

[0165] The présent invention further features vaccines or immunogenic compositions comprising one or more of the rVSVs of the présent invention. In one embodiment, the présent invention features vaccines or immunogenic compositions comprising an rVSVhd and vaccines or immunogenic compositions comprising an rVSVNj, as described above.

[0166] 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 af rVSVs expressing different epitopes of a pathogen (ses Table 6, vaccination groups 5 and 6).

[0167] 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 présent invention may be provided as a lyophilized préparation. The vaccines of the présent invention may also be provided as a solution that can be frozen for transportation, Additionally, the vaccines may contain suitable preservatives such as glycerol ormaybe formulated withoutpreservatives. Ifappropriate (i.e. no damage to the VSV in the vaccine), the vaccines may also contain suitable diluents, adjuvants and/or carriers.

[0168] The dose of the vaccine may vary according to factors such as the disease State, âge, sex, and weight of the individual, and the àbility of antibody to elicit a desired response in the individual. Dosage régime may be adjusted to provide the optimum therapeutic response. The dose of the vaccine may also be varied to provide optimum preveutative dose response depending upon the circumstances.

[0169] Kits

[0170] The présent 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.

[0171] Kit components may be packaged for either manual or partïally or wholly automated practice of the foregoing methods. In other embodiments involving kits, this invention contemplâtes a kit including compositions of the présent invention, and optionally instructions for theîr use. Such kits may hâve a variety of uses, including, for example, imagjng, diagnosis, therapy, and other applications.

[0172] Advantages and Unique Features of the rVSVs of theprésent invention

[0173] Novel and Unusual features of the invention:

[0174] Normal assembly and release of rVSV_!ntl M(G21E/L111F/M51) at permissive température (about 31°C) made it possible to amplify the new mutant rVSVs at the permissible température to a high titre to make viral stock. The assembly defectiveness of rVSVjad M(G21E/L111F/M51) at non-pennissive température (about 37°C (around body température) increased the safety of the using the rVSV in human and other animais by significantly reducing the number of progeny infectious viruses at the non-permissive température, The addition of these mutations to the pre-existing M51R mutation in tire M protein of rVSV_tacl further attenuated the virulence of VSV^.

[0175] The three mutations, M(G22E/M48R+M51R) or four mutations, M(G22E/L110F/M48R+M51R) in the M protein of rVSV^j did not make the virus température sensitive for the assembly of the virus. However, the addition of G22E mutation or G22E/L110F to the M48R+51R mutations made the VSVnj more attenuated and becamc significantly less pathogenic.

[0176] The above disclosure generally describes the présent invention. A more complété understanding can be obtained by reference to the following spécifie Examples. These Examples are described solcly for puiposes of illustration and are not intended to lirait the scope of the invention. Changes in form and substitution of équivalents are contemplated as circumstances may suggest or render expédient. Although spécifie tenns bave beeu employed herein, such ternis are intended in a descriptive sense and not for purposes of limitation.

[0177] EXAMPLES

[0178] The présent invention is further illustrated by the following examples which 5 should not be construed as limiting in any way.

[0179] Example 1 - Introduction of Mutations into the M genes ef rVSVud and rVSVwj and Recovery of Recombinant VSV by Reverse Genetics

[0180] Mutations were introduced into the M gene of VSV^ (FIG. 1) and VSV_NJ (FIG.

2). Nucléotide sequeuces 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 spécifie 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 LI1F) in the tsO23 with méthionine 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 semipermissive (37 °C) températures. The nucléotide sequeuces and amino acid changes from wild type to mutants in the M genes of rVSV_M and rVSV_NJ are shown in Tables 2, 3, 4 and 5. The changed nucléotide codons are underlined ²⁰ and changed nucléotide sequences and amino acid séquences are bold-faced.

[0181] 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 dépendant RNA polymerase of bactériophage T7 (T7) and a plasmid which encodes ftill 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. Internai ribosome

[0182]

[0183] 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 Lîpofectamine™ 2000 in concentrations of 10 pg of pN, 10 pg of pP, μ5 g of pL, and 15 pg of pVSV. The culture media from the transfected cells were harvested when the cells showed about 50-70% of CPE.

Example 2 - The mutation, L111F in the M gene signifïcantly reduces the burst sizes of rVSV_!nd at semi-permissive température, 37°C.

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 température (31°C) and semi-permissive température (37 °C, body température) to détermine the température 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 detennined by plaque assay with Veto E6 cells. The cells infected with the mutant viruses for the plaque assay were incubated at 31^ÛC. Wild type and ail mutants replicated equally well and produced similar titre of infectious viruses ail along the period of 10 hrs of infection (FIG. 4A and C). However, the mutants of rVSVua, rVSV]_od-G21E/LlllF and rVSV_fad-G21E/LUlFZM51R replicated in significantly lower titre than wild type or M51R mutant of rVSV_tod at 37°C. The différences in producing infectious particles between the wild type and the new M mutant, rVSV[nd-G21E/Ll 11F/M51R were as large as four logs (FIG. 4B and D). The error bars in FIG. 4 represent standard error of the mean. The P values for viral titers of rVSV_Jna-G21E/LlllF/M51R at 4hrs, 6hrs, 8hrs, and lOhrs postinfection obtained by comparing them to titers of rVSVicd-WT are pO.OOOl, p==O,OO55, p=0.0040, and p=0.0015 in FIG 4B. The P values for viral titers of rVSV_fad-G21E/LlllF at 4hrs, 6hrs, 8hrs, and lOhrs postinfection obtained by comparing them to titers of rVSVfod-WT are p<0.0001, p=0.0055, p=0.0041, 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.

[0184] Example 3 - Mutations in the M gene of rVSV_KJ, G22E and L110F do not reduce the burst swe of the rVSV« at 37 °C.

[0185] BHK21 cells were infected with an MOI of 3 of rVSV_w, wild type and M gene mutants, incubated at both 37^QC (semi-permissive température) and 31°C (permissive température) 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 ail M mutants of rVSV_NJ replicated equally well at both températures 31*C and 37°C (FIG. 5), indicating the introduction of G22E and L1I0F mutations into the M gene of rVSV_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.

[0186] Example 4 — Assembly defectiveness at semi-permissive température by L111F mutation in the M gene of rVSV_Jnd does not affect the expression of VSV proteins

[0187] BHK21 cells were infected with an MOI of 6 of rVSVind and rVSVw, 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 pg of total protein was loaded to the SDS-PAGE gel, and rVSV proteins were detected by Western blot analysis using rabbit antiserum against VSVind and VSVni (1:5000 dilution). The resuit demonstrate that in spite of the mutations that reduced the burst size of the rVSVind at 37°C (L111F in G21E/L111F and G21E/L111F/M51R), the level of VSV protein expression is comparable to the wild type rVSVind (FIG. 6A). Wild type and ail M mutants of rVSV_NJ expressed their proteins in a similar level at both 31°C and 37’C (FIG. 6B).

[0188] Example 5 - Combinée! Mutations, G21E/L111F/M51R in the M gene of rVSV_tBd reduced cytopathogenicity significantly in BHK21 cells and human neuroblastoma cells, SH-SY5Y.

[0189] In order to examine the effects of the mutations, L111F and M51R of M gene of rVSVind on the cytopathogenicity, the inventera infected BHK21 cells (FIG. 7) and human neuroblastoma cells (FIG. 8) with an MOI of 0.1 of rVSVind. The typical cytopatbic effects by the VSV are rounding-up of infected cells and cell lysis. At 20 hra after infection, the cytopathic effect caused by the rVSViœiG21E/L11IFZM51R was compared with those by the other rVSVind. Ih 20 hrs of infection with the rVSV|_nd-G21E/LlllFZM51R 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 eachinbothBHK21 and SH-SY5Y cells.

[0190] Example 6 - Reduced cytopathic effects in the BHK21 cell by the rVSV_w with the M gene mutations, G22E/M48R+M51R and G22E/L110F/M48R+M51R.

[0191] In order to examine the effects of the mutations, G22E, L110F and M48R+M51R of M gene of rVSV^ on the cytopathogenicity, the inventera infected BHK2I cells and human neuroblastoma cells with an MOI of 0.1 of rVSVfu· At 20 hrs after infection, the inventera compared the cytopathic effects (cell round-up and lysis) caused by the rVSV^j 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^j in both BHK21 (FIG. 9L and 9N) and SH-SY5Y cells (FIG. 10L and 10N).

[0192] Example 7 - rVSV_lnd with the mutations of G21E/L111F/M51R and rVSVNj with the mutations of G22E/M48R+M51R and

G22E/L110F/M48R+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.

[0193] VSV docs 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 animais demonstxate neurological symptoms. In order to examine the neurovirulence of the new M mutants of VSVs, the inventors injected Swiss Webster mice with 1X10^s PFU of mutant VSV and 1X10³ PFU of wild type VSV into the intralateral ventricle of the brain. The inventors purchased 5week-old Swiss Webster mice with intralateral ventticular implant from Charles River laboratory. Three mice/group were injected with viruses after one week of arrivai 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 1X10³ PFU of wild type rVSVInd died within four days after injection (FIG. 11B). Two mice iiyected with 1X10⁶ PFU of rVSVtad-M51R (FIG. 1 IC) lost about 20% ofbody 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 rVSVind-M51R did not lose the body weight through the experiment Ail three mice injected with rVSVind-G21E/LlllF/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/LH1F and M51R significantly attenuated the rVSV^d and lost its neurovirulence in mice, One mice injected with rVSVNj-WT showed paralysie in both hind legs and it was sacrificed on day 6 after injection (FIG. 12A). Two other mice injected with rVSV_Nj-WT and mice injected with rVSV_Nr M48R+M51R, rVSV_NJ-G22E/M48R+M5 IR, or rVSV_Nr G22E/L110F/M48R+M51R showed no sign of illness and weight loss during 4 weeks after injection (FIG. 12B, 12C, and 12D). The error bars in FIG. 11 and 12 represent standard error of the mean.

[0194] Example 8 - Génération of rVSVs expressing HIV-l Gag protein as s gene of interest; HIV-l Gag proteins are expressed equally well at both 31’C and 37‘C.

[0195] The newly generated M mutants, G21E7L1HF/M51R mutant of rVSVj^ and G22E/M48R+M51R and G22E/L110F/M48R+M51R mutants of rVSV_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-l 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<restricted peptide (NH₂-AMQMLKETI-COOH) (SEQ ID NO: 33) in the HIV-l Gag is well studied in the Balb/c mouse. The inventors inserted the fulllength HIV-l gag gene linked to conserved human CD8+ T cell epitopes from gp41, gpl20, and nef protein of HIV-l (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/LH1F/M51R (GLM) of rVSV_w and wild type, G22E/M48R+M51R (GM), and G22E/L110F/M48R+M51R(GLM) of rVSV_NJ. The rVSVs were recovered from the cDNA clones by reverse genetics as described in FIG. 3 and the expression of Gag-En from the rVSVs was examined by Western blot analysis using monoclonal antibody against HIV-l 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 lysâtes were prepared at 6 hrs post-infection. Total protein of 5 pg was loaded onto the SDS-PAGE for the Western blot analysis. rVSVind(GLM)-Gag-En, rVSVpj(GM)-Gag-En, and rVSVNj(GLM)-Gag-En expressed Gag-En protein (64 kDa) well at semipermissive température (37°C, FIG. 13C) and the expression level was similar to that at permissive température (31°C, FIG. 13B).

[0196] Exampk 9 - Vaccination Regimen with rVSV in Mice

[0197] 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 altemating the two sérotypes for priming and boost. The mice were prime-vaccmated intramuscularly with 5X10^$ PFU of rVSVs at âge 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, splénocytes and sera were collected to detect the HIV-1 Gag spécifie CD8+ T cell immune responses and humoral immune responses.

[0198] Example 10 - Priming with rVSV_lnd(GLM)-HrV-l Gag-En and Booster with rVSVNj(GLM)-HIV-l Gag-En induces strongest CD8+ T cell immune responses against HIV-1 Gag.

[0199] The T cells stimulated by the interaction with MHC I molécule on the antigen presenting cells loaded with the peptide enhances the sécrétion of înterferon-γ (IFN-γ), which indicates the antigen spécifie T cell immune responses. The splénocytes were double stained with FITC-anti-CD8 and APC-anti-IFN-γ for CD8+ T cells with the increased intracellular INF-γ. In order to examine the HIV-1 Gag protein spécifie CD8+ T cell immune response, splénocytes were isolated and 1X10^é cells were stimulated with H-2K^d-restricted HIV-1 Gag peptide, NHi-AMQMLKETI-COOH (SEQ ID NO: 33), the cells were stained with FITC rat anti-mouse CD8 for the CD8 molécules and stained with APC rat anti-mouse IFN-y for intracellular IFN-γ. The sécrétion of IFN-y was blocked j with Brefeldin A befbre staining them- VSV spécifie CD8+ T cell immune responses were examined with the use of nucleocapsid spécifie peptide, IN275: NH2-MPYLIDFGL-COOH (SEQ ID NO: 32). Peptide spécifie CD8+-IFN-y+ T cells were counted with FACSCalibur, a floweytometer. The splénocytes treatedI p with DMSO (solvent for the peptide, FIG. 15) did not stimulate the CD8+ T| cells indicating that treating the splénocytes with the VSV N peptides and HIV-I

I i ï

Gag peptides stimulated specifically CD8+ T celis in FIG. 16 and FIG. 17. Prime and boost immunization by altemating two serotypes of rVSV(WT) or rVSV(GLM) mutants induced stronger CD8+ T cell immune responses against VSV as well as HCV-1 Gag proteins than using a single serotype of rVSV for 5 the prime and boost vaccinations as seen in groups 1,2, 5,6, 9, and 10. (FIG. 16 and FJG. 17). With the vaccination with the new M mutants or rVSV, CD8+ T cell responses against HIV-1 Gag protein was induced better when mi ce were vaccinated with rVSV_tali(GLM)-Gag for priming and rVSV_NXGLM)-Gag for boosting than vice versa (FIG 17, group 6 vs. group 10). P value on the group 6 in FIG. 17 was compuied by using two-sided indépendant sample t test, and the resuit was compared to that for the group 10. The error bars in FIG. 16 and 17 represent standard error of the mean.

[0200] Example 11 - HIV-1 Gag spécifie humoral immune responses

[0201] Génération of HIV-1 Gag spécifie antibody was examined with the sérum ⁵ collected at a week after the boost immunization. The Gag spécifie antibody titer was determined by the indirect enzyme-liriked immunosorbent assay (ELISA). For the ELISA, 96 well ELISA plate was coated with recombinant p55 at a concentration of 125 ng/well. The mouse sérum was diluted 1:100. The antibody bound to the antigen, p55 was detected with secondary antibody, sheep 20 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 (génération of antibody against HIV-1 Gag) was induced well in mice vaccinated with the new M 25 mutants, and the beat humoral immune responses against HIV-1 Gag were induced when two serotypes of rVSV(WT) or rVSV(GLM) were altemated 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 rVSVuiKGLMj-Gag and booster with rVSV_NJ(GLM)-Gag worked better than vice versa (Gig. 18, group 6 vs. group 10). The error bars in FIG. 18 represent standard crror of the mean.

[0202] Exemple 12 - In créas in g doses of rVSV^GLM). rVSV_Nj(GM), and rVSV^j(GLM) for vaccination induced stronger immune responses in mice

[0203] As illustrated in FIG. 17 and FIG. 18, stronger HIV-1 Gag spécifie CD8+ T cell immune responses and humoral immune responses were induced when mice were prime vaccinated with rVSViad(GLM)-Gag and booster vaccinated with rVSVNj(GLM)-Gag. However the immune responses were not as strong as those induced with rVSVt,d(WT)-Gag and rVSVxj(WT)-Gag. Thereforc, the inventors wanted. to examine whether they could increase the immune responses, if they increase the doses of the rVSVumCGLMj-Gag and rVSVNj(GLM)-Gag. Six (6) week old Balb/c mice, 6 mice/group, were vaccinated with rVSV_ind(GLM)-Gag for priming and rVSVxj(GLM)-Gag or rVSV_w(GM)-Gag for booster. Because the iVSVnj(GM) was as attenuated as the rVSV^GLM) in vitro and in vivo, the inventors included rVSVNj(GM)-Gag as a booster virus, and compared the immune responses to that induced by rVSVNj(GLM)-Gag. Mice groupa were vaccinated with various doses of rVSV_tad(GLM)-Gag, rVSV_Nj(GLM)-Gag, and rVSV_NJ(GLM)-Gag; 5X10⁶ PFU/mouse, 5X10⁷ PFU/mouse, 5X10⁸ PFU/mouse, and 5X10⁹ PFU/mouse (Table 1).

[0204] Mice were vaccinated according to the schedule as seen in FIG. 14 and were sacrifîced a week after booster vaccination for splénocytes and sérum. The splénocytes were double stained with FITC-anti-CD8 and APC-anti-IFN-γ for CD8+ T cells with the increased intracellular INF-γ. In order to examine the HIV-1 Gag protein spécifie CD8+ T cell immune response, splénocytes were isolated and 1X10⁶ cells were stimulated with H-2Kd-restricted HIV-1 Gag peptide, NH2-AMQMLKETI-C00H (SEQ ID NO: 33), the cells were stained with FITC rat anti-mouse CD8 for the CD8 molécules and stained with APC rat anti-mouse IFN-γ for intracellular IFN-γ. The sécrétion of EFN-γ was blocked with Brefeldin A before staining them. VSV spécifie CD8+ T cell immune responses were examined with the use of nucleocapsid spécifie peptide, IN275: NH2-MPYLÏDFGL-COOH (SEQ ID NO: 32), Peptide spécifie CD8+-IFN-y+ T cells were counted with FACSCalibur, a floweytometer.

[0205] CD8+ T cell immune responses against VSV N protein were very similar in ail vaccination groupe with different doses of rVSVind(GLM)-Gag, rVSV_w(GLM)-Gag, or rVSVNj(GM)-Gag (FIG. 19). The HIV-1 Gag spécifie CD8+ T cell immune responses after prime vaccination with rVSVind(GLM)Gag and booster with rVSVNj(GLM)-Gag looked increased a little bit when the vaccine doses were increased, but différences were not statistically significant (FIG. 20, groups 2, 3, 4, 5), However, when mice were prime vaccinated with rVSVhd(GLM)-Gag and booster vaccinated with rVSVwKGMJ-Gag, the frequency of the HIV-1 Gag spécifie CD8+ T cells were increased sïgnificantly with the increasing dose of 5X10⁸ PFU. The HIV-1 Gag spécifie CD8+ T cell response was strongest in a group vaccinated with 5X10⁹ 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 resuit of group 8 was compared to that for the group 7, and the resuit of group 9 was compared to that for the group 8. The errer bars in FIG. 19 and 20 represent standard errer of the mean.

[0206] Génération of antibody against HIV-1 Gag protein was increased with the increasing doses of rVSVjnd(GLM)-Gag for priming and booster with rVSV_NJ(GLM)-Gag or rVSV_NJ(GM)-Gag (FIG. 21). Booster vaccination -with rVSV_NJ(GLM)-Gag or with rVSV_w(GM)-Gag generated similar level of Gag spécifie antibodies in various doses of the virus. The error bars in FIG. 21 represents standard error of the mean.

[0207] Exemple 13 - Génération of rVSVind(GLM) and tVSVnj(GM) expressing HIV-1 proteins; Gag, Pol, Env, and RT

[0208] For HIV-ï vaccines utilizing the rVSVind(GLM) and rVSVmiGM) 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 nucléotides encoding peptide epitopes for T cells and B cells in humans. The inventors hâve generated rVSVhXGLM) and rVSVw(GM) cairying cassettes encoding HIV-1 Gag linked to B-cell epitopes from HIV Env protein derived from multiple viral clades (FIG. 22). The inventors hâve generated the rVSV_tod(GLM)-Gag-En and rVSVw(GM)-Gag-En, which expresses gag gene and T cell peptide epitopes from Nef, Gpl 20, and Gp41 (FIG. 23). The inventors generated rVSV_îsd(GLM) and rVSV_NJ(GM) with HIV1 Gag-RT, which encodes gag protein with peptide epitopes from RT, Tat, and Rev proteins (FIG. 23). In addition, the inventors generated rVSVi_nd(GLM) and rVSVNj(GM) with pol gene and env gene, respectively (FIG. 23). Previously, the inventors hâve demonstrated that replacement of signal peptide of HTV-1 envelope protein with the honeybee melittin signal peptide dramatically increased the rates of Gpl20 expression, glycosylation, and sécrétion (Yan U et al., PNAS, 93: 9606-9611, 1996). The increased expression and sécrétion of Gpl20 are expected to enhance the induction of the T cell and B cell immune responses against HIV-1 gpl20. Therefore, the inventors generated rVSVs with HTV-1 env gene with melittin signal sequence (gpl60mss). 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 ^PC 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 varions HTV-1 proteins were expressed well from rVSV_w(GLM) and rVSV_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, pli), 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 pl5 at frie carboxy terminus. The RT products p66 and p51 were expressed equally well from both rVSV_tod(GLM) and tVSV_nj(GM) and at both 31 ^DC and 37 °C (FIG 26). The env gene encodes glycoprotein Gpl60, which is cleaved into Gpl20 of receptor binding protein and gp41 of fusion inducing cytoplasmic and transmembrane protein. Gpl 60 is cleaved by the cellular trans-golgi résident protease furin. The Gpl60 expressed in BHK21 cells from the rVSVj^GLM) and rVSV^j(GM) was not cleaved to Gpl20 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 Gpl60 in BHK21 cells and cleavage into Gpl20 and Gp41 demonstrated that the processing was not very efficient (Kunyu Wu et al., Journal of General Virol., 90:1135-1140,2009).

[0209] Example 14 - Immunization studies in mice with the rVSVi_nd(GLM) and rVSV_hJ(GM) with HIV-1 proteins, Gag, Gpl60 and RT

[0210] The preliminary prime-boost immunization studies with new M mutant rVSVs, rVSV]_Kd(GLM) and rVSVju(GM) rwealed that priming mice with the rVSV_fcd(GLM) and boosting with tVSVnj(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 rVSVir^GLMJ-Gag priming and rVSV_NI(GM)-Gag boosting, the dosage of 5X10⁸ pfu of each virus induced better Gag spécifie 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 inventais chose 5X10⁸ pfu as a dosage for immunization studies with rVSV_lnd(GLM) and fVSVnj(GM) expressing HJV-1 Gag, Gpl60, and RT.

[0211] The inventors prime immunized Balb/c mice with rVSVind(GLM) expressing HIV-1 proteins and boost immunized with rVSVm(GM) expressing HIV-1 proteins in three weeks after the priming as described in the table 6. Die 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 sacrifîced the immunized mice for their splénocytes and sera. The 1X10⁶ splénocytes were stimulated with H-2K^d-restricted HFV-1 protein spécifie peptides, which are Gag, Env P18, RT354, RT464, and RT472. The peptide séquences are shown in the FIG. 29. The stimulated splénocytes were double stained for the cell surface CD8 molécules and intracellular IFN-γ of T cells with FITC rat anti-mouse CD8 and APC rat anti-mouse IFN-γ. The regimen, prime-boost immunization with rVSV^GLM) and rVSV_NJ(GM) induced peptide spécifie 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. 28, G3 and 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. 28, 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 Gpl20 and Gp41 (FIG. 29, G6). RT spécifie CD8+ T cells were generated in about 2% of CD8+ ¹⁵ T cells in mice injected with rVSV with pol gene (FIG. 28, 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. 28, G2 and G4) than when injected with mixed rVSVs expressing single protein (FIG.

28, G5). Env spécifie CD8+ T cell responses were strongest and were similar in ²⁰ 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 uncertam 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 compétition of each virus for the antigen presenting cells in the mixed virus injection lowered the CD8+ T cells againt 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 ₃₀ is expressed (FIG. 28, G3) as in the mixed injection (FIG. 28, G5). Therefore, it

may be the resuit of immune dominance of Env protein in Balb/c mice, which affects negatively to the présentation of other proteins to the CD8+ T cells when Env is exprcssed togethcr. For the combined immunizations in mice, ail three rVSVs were injected at the single site, which may hâve resulted in the 5 compétition of different proteins for the antigen présentation in the saine antigen présentation cells. The inventors will test the immunization with the mixed injections at more than one injection sites to induce CD8+ T cell responses.

[0212] 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 10 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 Gpl20. The Gp41 and Gpl20 are associated by non-covalent bond and mature to form a trimer of Gp41 and Gpl20 heterodimer. Thematured trimer of Gp41 and Gpl20 ⁵ are exported to the cell surface, The Gpl20 tends to fall off from the cell surface because of the weak bondage between Gp41 and Gpl20. Thereforc, antibody against Gpl20 can be induced to the cell surface Gpl20 or fallen off Gpl20. Antibody titer against HIV-1 Gag protein and Gpl 20 was determined by ELIS A. For the Gag antibody the nncroplate was coated with 125 ng/well of recombinant p55 (Pierce Biotechnology, RP-4921) and 50 μΐ of mice sera was ²⁰ 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 Gpl20 ELISA, 96 well microplate was coated with 250 ng/well of HIV-1 SF162 Gpl40 trimer from clade B (NIH AIDS reagent program, cat# 12026).

Mice sera were diluted in 1:100, 1:200, and 1:400 and 50 μΐ of the diluted sérum was added to the microplate for the ELISA. Antibody spécifie to GpI20 was generated in mice injected with rVSV-HIV-1 Gpl60 alone and in mice injected with themixed viruses expressing each protein of Gag, Gpl60, and RT (FIG. 30B, G3 and G5). The titer of Gpl20 antibody was slightiy lowcr, although not statistically significant, in mice injected with mixed viruses than in mice injected with single virus expressing Gpl 60.

[0213] The newly generated M mutants of rVSVs, rVSV_Ind(GLM) and rVSV_NJ(GM) induced CD8+ T cell and humoral immune responses against various HIV-l proteins expressed from the vector after injecting mice with an individual virus expressing single virus or with mixed viruses expressing various HTV-1 proteins.

[0214] Example 15 - Génération of rVSVi_nd(GLM), rVSV_NJ(GM), and rVSVxj(GLM) with Hepatitis C Virus Structural Protems

[0215] 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 rôle in the prévention of HCV infection is not well studied compared to the HCV spécifie cellular immune responses, it is worthy to include vaccines which can induce HCV spécifie antibodjes. It has been demonstrated that nucleocapsid protein core and surface glycoproteins El 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 fonns a membranous web structure, which mainly consista 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 inactions NS4B has in the réplication 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, El, E2, p7 are localized. Therefore, including Core, El, E2, P7, and NS4B together into the vaccine may induce humoral and cellular immune responses.

[0216] For the HCV structural protein vaccines using the new M mutant rVSVs, the inventors inserted the HCV core gene, E1E2P7 and NS4B genes together (connecter! by the VSV intergenic junction sequences), and CoreElE2P7 and NS4B genes together (connected by the VSV intergenic junction séquences) to the junction of VSV G gene and L (FIG. 31). The rVSVs; rVSV_tod(GLM)-FC, rVSV_Ind(GLM)-CE!E2P7/NS4B, rVSV_NJ(GM)-FC, and rVSV_NJ(GLM)E1E2P7/NS4B were generated by reverse genetics. The inventors had a trouble to generate the rVSV_NJ(GM) with CE1E2P7/NS4B ftom the plasmid DNA, therefore, the inventors removed the core and cloned E1E2P7/NS4B into the plasmid of tVSVnKGLM) and generated the _rVSV_w(GLM)-ElE2P7/NS4B. The expression of Core, El, E2, and NS4B from the recombinant VSV at 31^qC and 37°C was examined by Western blot analysis using antibodics against each protein (FIG, 32). The proteins were expressed similarly in quantity at both températures. Core (FIG. 32A), El (FIG. 32B), and E2 FIG. 32C) were expressed well from the rVSVj_nd(GLM)-CElE2P7/NS4B, but the expression of NB4B was lower than the other proteins (FIG. 32D).

[0217] Exemple 16 - Génération of rVSV|_nd(GLM), rVSV_NJ(GM), and rVSVxf(GLM)with HCV Non-Structural Proteins

[0218] The inventors want to target most of the HCV proteins including core, El, E2, NS3, NS4A, NS4B, NS5A and NS5B proteins in order to induce HCV spécifie 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 wbo recover from the acute HCV infection develope strong CD4+ T cell and CD8+ T cell responses against multiple epitopes in the NS3 protein | (Diepolder, H. Μ. J. Virol. 71:6011-6019, 1997; Lamonaca, V, et al.I

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 importantI to elicit successful cellular immune responses against HCV. NS3 protein, a serine protease and RNA helicase associâtes with NS4A and résides on the ER membrane (Sato, S. et al. J. Virol. 69:4255-4260,1995; Faille, 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 dépendent

RNA polymerase (Shirota, Y. et al., J. Biol. Chem., 277:11149-11155, 2002;

Shiraakami, et al. J. Virol. 78:2738-2748, 2004). NS5B protein is a RNA dépendent RNA polymerase that synthesizes positive sense HCV genomic RNA as well as intermediate négative sense genomic RNA (Beherens, S. E. EMBO J. 15:12-22,1996). NS5B protein is a tail-anchored protein and associâtes with ER membrane through its carboxyl terminal 20 amino acids (Yamashita, T. J. Biol.

Chem. 273:15479-15486, 1998; Hagedoni, C. H. Curr. Top. Microbiol. Immunol. 242:225-260,2000).

[0219] The inventons 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, NS34AJB, NS5A, NS5B, 15

NS5AB were cloned into the junction at G gene and L gene of pVSV^GLM) and pVSVkj(GM) (FIG. 33). The inventons generated rVSV_tod(GLM)-NS3, rVSV[nd(GLM)-NS34AB, rVSV_M(GLM)-NS5A, rVSV_M(GLM)-NS5B, and rVSV^GLMJ-NSSAB, 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^GMj-NSS, rVSV_NJ(GM)NS4B, iVSVnj(GM)-NS34AB, rVSV_Nj(GM)-NS5A, rVSV_NJ(GM)-NS5B, and rVSV_w(GM)-NS5AB. The expression of each protein from the tVSVnj 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 rVSVind and rVSV_NJ was different, it was good enough to use them as HCV vaccine.

Table 1. Mouse Vaccination Groups and Regimen

Vaccination Groups	Prime	Boost	“ofMice
	Viruses	Titer (PFUySOpl	Viruses	Titer (PFU)/50 μΐ
1	Ind (WT)-Gag	5X10e	NJ(WT)-Gag	5X10“	â
2	Ind (GLM)-Gag	5X1Ô4	NJ (GLM)-Gag	5X10^	6
3	Ind (GLM)-Gag	5X107	NJ(GLM)-Gag	5X107	6
4	Ind (GLM)-Gag	5X10“	NJ (GLM)-Gag	5X10“	6
5	Ind (GLM)-Gag	5X10y	NJ(GLM)-Gag	5X10^	6
6	Ind (GLM)-Gag	5X106	NJ (GM) Gag	5X106	6
7	Ind (GLM)-Gag	5X10'	NJ(GM)-Gag	5X10’	6
8	Ind (GLM)-Gag	5X10“	NJ(GM)-Gag	5X10“	6
9	Ind (GLM)-Gag	5X10s	NJ(GM)-Gag	5X10ÿ	6
10	NJ (WT)-Gag	5X106	Ind (WT)-Gag	5X10“	6
Total Mice		60

Table 2. Neuckotide Sequence Comparison between M Genes of VSV Indiana serotype, Wild Type (SEQ ID NO: 1) and a Mutant G21E/L111F/M51R (SEQ ID NO 2)

50

SEQ ID NO; 1; ATGAGTTCCT TAAAGAAGAT TCTCGGTCTG AAGGGGAAAG GTAAGAAATC

SEQ ID KO: 2: ATGAGTTCCT TAAAGAAGAT TCTCGGTCTG AAGGGGAAAG GTAAGAAATC

SEQ ID NO:

SEQ ID NO:

100

1: TAAGAAATTA gggatcgcac caccccctta tgaagaggac actaacatgg

2: TAAGAAATTA gaaatcgcac caccccctta tgaagaggac actaacatgg

SEQ ID NO:

SEQ ID NO:

1:

2:

101 agtatgctcc gagcgctcca attgacaaat agtatgctcc gagcgctcca attgacaaat

150 cctattttgg agttgacgag cctattttgg agttgacgag

151 200

SEQ ID NO: 1: ATGGACACTC ATGATCCGCA TCAATTAAGA TATGAGAAAT TCTTCTTTAC

SEQ ID NO: 2: AGGGACACTC ATGATCCGCA TCAATTAAGA TATGAGAAAT TCTTCTTTAC

SEQ ID NO:

SEQ ID NO:

SEQ ID NO;

SEQ ID NO:

SEQ ID NO:

SEQ ID NO:

²⁰¹ 250

1: AGTGAAAATG ACGGTTAGAT CTAATCGTCC GTTCAGAACA TACTCAGATG

2: AGTGAAAATG ACGGTTAGAT CTAATCGTCC GTTCAGAACA TACTCAGATG

251 300

1: TGGCAGCCGC TGTATCCCAT TGGGATCACA TGTACATCGG AATGGCAGGG

2: TGGCAGCCGC TGTATCCCAT TGGGATCACA TGTACATCGG AATGGCAGGG

301

1: AAACGTCCCT TCTACAAGAT CTTGGCTTTT TTGGGTTCTT

2: AAACGTCCCT TCTACAAGAT CTTGGCTTTT TTTGGTTCTT

350

CTAATCTAAA

CTAATCTAAA

	SEQ ID SEQ ID	NO: 1: NO: 2:	351 GGCCACTCCA GCGGTATTGG CAGATCAAGG TCAACCAGAG	400 TATCACGCTC TATCACGCTC
GGCCACTCCA	GCGGTATTGG CAGATCAAGG TCAACCAGAG
10	SEQ ID	NO: 1:	401 ACTGTGAAGG	CAGGGCTTAT TTGCCACACA GAATGGGGAA	450 GACCCCTCCC
	SEQ ID	NO: 2:	ACTGTGAAGG	CAGGGCTTAT TTGCCACACA GAATGGGGAA	GACCCCTCCC
	SEQ ID	NO: 1:	451 ATGCTCAATG	taccagagca cttcagaaga ccattcaata	500 TAGGTCTTTA
	SEQ ID	NO: 2s	ATGCTCAATG	TACCAGAGCA CTTCAGAAGA CCATTCAATA	TAGGTCTTTA
	SEQ ID	NO: 1:	501 CAAGGGAACG	GTTGAGCTCA CAATGACCAT CTACGATGAT	550 GAGTCACTGG
15	SEQ ID	NO: 2:	caagggaacg	GTTGAGCTCA CAATGACCAT CTACGATGAT	GAGTCACTGG
	SEQ ID	NO: 1:	551 AAGCAGCTCC	TATGATCTGG GATCATTTCA ATTCTTCCAA	600 ATTTTCTGAT
	SEQ ID	NO: 2:	AAGCAGCTCC	TATGATCTGG GATCATTTCA ATTCTTCCAA	ATTTTCTGAT
	SEQ ID	NO: 1:	601 TTCAGAGATA	AGGCCTTAAT GTTTGGCCTG ATTGTCGAGA	650 1 AAAAGGCATC 1
	SEQ ID	NO: 2:	TTCAGAGATA	AGGCCTTAAT GTTTGGCCTG ATTGTCGAGA	AAAAGGCATC |
20	SEQ ID	NO: 1:	651 TGGAGCTTGG	GTCCTGGATT CTGTCAGCCA CTTCAAATGA	700 j 1
SEQ ID	NO: 2:	TGGAGCTTGG	GTCCTGGATT CTGTCAGCCA CTTCAAATGA	r

Table 3. Anûno Acid Sequence Compamon between M Proteins of VSV Indiana serotype Wild Type (SEQID NO: 3) and aMutant G21E/L111F/M51R (SEQ ID NO: 4)

2150

SEQ ID NO: 3: MSSLKKILGL KGKGKKSKKL GIAPPPYEED TNMEYAPSAP IDKSYFGVDE SEQ ID NO: 4: MSSLKKILGL KGKGKKSKKL EIAPPPYEED TNMEYAPSAP IDKSYFGVDE

51100

SEQ ID NO: 3: MDTHDPHQLR YEKFFFTVKM TVRSNRPFRT YSDVAAAVSH WDHMYIGMAG

SEQ ID NO: 4: RDTHDPHQLR YEKFFFTVKM TVRSNRPFRT YSDVAAAVSH WDHMYIGMAG

101 111150

SEQ ID NO: 3: KRPFYKILAF LGSSNLKATP AVLADQGQPE YHAHCEGRAY LPHRMGKTPP

SEQ ID NO: 4: KRPFYKILAF FGSSNLKATP AVLADQGQPE YHAHCEGRAY LPHRMGKTPP

SEQ ID NO:

SEQ ID NO:

151200

3: MLNVPEHFRR PFNIGLYKGT VELTMTIYDD ESLEAAHMIW DHFNSSKFSD

4: MLNVPEHFRR PFNIGLYKGT VELTMTIYDD ESLEAAPMIW DHFNSSKFSD

201229

SEQ ID NO: 3: FRDKALMFGL IVEKKASGAW VLDSVSHFK SEQ ID NO: 4: FRDKALMFGL IVEKKASGAW VLDSVSHFK

250

Table 4. Nucléotide Sequence Comparison between M Genes of VSV New Jersey serotype Wild Type (SEQ ID NO: 5) and Mutants, G22E/M48R+M51R (SEQ ID NO: 6) and G22E/L110F/M48R+M51R (SEQ Π) NO: 7)

150

SEQ ID NO: 5: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA SEQ ID NO: 6: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA Π>ΝΟ:7: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA

51100

SEQ ID NO: 5 :ATCAAAGAAA CTAGGCTTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG

SEQ Œ> NO: 6 :ATCAAAGAAA CTAGAATTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG

SEQ ID NO: 7 îATCAAAGAAA CTAGAAÏTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG

101150

SEQ ID NO: 5 lAGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG AATGGAGGAT

SEQ ID NO: 6 :AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG AAGGGAGGAT

SEQ IDNO: 7 :AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG AAGGGAGGAT

151200

SEQ ID NO: 5 :ATGGATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT

SEQIDNO: 6

:AGGGATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT

SEQIDNO;? xAGGGATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT

SEQID NO: 5 SEQIDNO: 6 SEQIDNO:?	201 250 :GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG :GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG :GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG
SEQ ID NO: 5 SEQIDNO: 6 SEQIDNO: 7	251 300 ;TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA :TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA :TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA
SEQ Œ> NO: 5 SEQIDNO: 6 SEQIDNO:?	301 350 :AAGCGTCCTT TCTACAAGAT AATTGCTCTG ATTGGCTCCA GTCATCTGCA :AAGCGTCCTT TCTACAAGAT AATTGCTCTG ATTGGCTCCA GTCATCTGCA îAAGCGTCCTT TCTACAAGAT AATTGCTTTT ATTGGCTCCA GTCATCTGCA
SEQIDNO: 5 SEQIDNO: 6 SEQIDNO: 7	351 400 îAGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA :AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA ;AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA
SEQ ID NO: 5 SEQIDNO: 6 SEQIDNO: 7	401 450 :CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG :CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG :CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG
SEQIDNO: 5 SEQIDNO: 6 SEQIDNO:?	451 500 :ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA :ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA sATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA
SEQIDNO: 5 SEQIDNO: 6 SEQIDNO:?	501 550 :CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG îCAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG :CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG
SEQIDNO; 5 SEQIDNO: 6 SEQIDNO:?	551 600 :AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG :AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG :AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG
SEQIDNO: 5	601 650 :ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAAC

SEQIDNO: 6 :ATCCAAAAAG SEQIDNO: 7 :ATCCAAAAAG	AAGGGCTTAA ATTCGGATTG AAGGGCTTÂA ATTCGGATTG	ATTTTAAGCA AGAAAGCAAC ATTTTAAGCA AGAAAGCAAC
651 SEQIDNO: 5 zGGGAACTTGG	GTGTTAGACC AATTGAGTCC	700 GTTTAA
SEQIDNO: 6 :GGGAACTTGG	GTGTTAGACC AATTGAGTCC	GTTTAA
SEQIDNO: 7 :GGGAACTTGG	GTGTTAGACC AATTGAGTCC	GTTTAA

Table 5. Amino Acid Sequence Comparison between M Proteins of VSV New Jersey serotype Wild Type (SEQ Π) NO: 8)and Mutants, G22E/M48R+M51R (SEQ ID NO: 9) and G22E/L110F/M48R+M51R (SEQ ID NO: 10)

SEQIDNO:8 :MSSFKKILGF SSKSHKKSKK

SEQ ID NO:9 :MSSFKKILGF SSKSHKKSKK

SEQIDNO: 10: MSSFKKILGF SSKSHKKSKK

48 50

LGLPPPYEES SPMEIQPSAP LSNDFFGMED LELPPPYEES SPMEIQPSAP LSNDFFGRED LELPPPYEES SPMEIQPSAP LSNDFFGRED

100

SEQ ID NO: 8 :MDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG SEQ ID NO: 9 :RDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG SEQIDNO: 10: RDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG

101 110

SEQIDNO: 8 :KRPFYKIIAI> IGSSHLQATP

SEQIDNO.· 9 :KRPFYKIIAI« IGSSHLQATP

SEQIDNO: I0:KRPFYKIIAF IGSSHLQATP

150 AVLADLNQPE YYATLTGRCF LPHRLGLIPP AVLADLNQPE YYATLTGRCF LPHRLGLIPP AVLADLNQPE YYATLTGRCF LPHRLGLIPP

151

SEQIDNO: 8 -.MFNVSETFRK PFNIGIYKGT

SEQIDNO:9 :MFNVSETFRK PFNIGIYKGT

SEQIDNO: 10: MFNVSETFRK PFNIGIYKGT

200

LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ LDFTFTVSDD esnekvphvw eymnpkyqsq LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ

201

SEQIDNO: 8 :IQKEGLKFGL ILSKKATGTW VLDQLSPFK

SEQIDNO: 9 :IQKEGLKFGL ILSKKATGTW VLDQLSPFK

SEQIDNO: 10:IQKEGLKFGL ILSKKATGTW VLDQLSPFK

250

Table 6. Vaccination studies in mice for broad range CD8+ T cell responses and humoral immune responses against HIV-1 proteins

Vaccination Groups	rVSV w/HIV-1 Proteins
	Prime	Boost
G1	rVSVind(GLM)	rVSVw(GM)
G2	rVSV^GLMJ-HIV-l Gag-En	rVSVNJ(GM)-HIV-l Gag-En
G3	rVSVM(GLM)-HIV-l Gpl60mss	rVSVNJ(GM)-HlV-l GplâOmss
G4	rVSVM(GLM)-HIV-l Pol	iVSVnKGWO-HIV-I Pol
G5	rVSVmdCGLMJ-HIV-l Gag-En + rVSVinXGLMi-GpieOmss + rVSVtad(GLM)-HrV-l Pol	rVSVNKGM)-HIV-l Gag-En + rVSVN)(GM)-GplâOmss + rVSVNJ(GM)-HIVl Pol
G6	rVSVM(GLM)-HIV-l Gag-A+ rVSVtad(GLM)-HIV-l Gag-B+ rVSV^GLNfl-HrV-l Gag-C	rVSVw(GM)-HIV-l Gag-A+ rVSVNJ(GM)-HIV-l Gag-B+ rVSVNi(GM)-HIV-l Gag-C

Claims (15)

1. l. A modifîed matrix (M) protein of a vesicular stomatitis virus (VSV), characterized in that the modified M protein comprises an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3 including at least the following substitutions: G21E/L111F/M51R; and (ii) SEQ ID NO: 8 including at least the following
2. 2. The modified M protein of daim 1, characterized in that SEQ ID NO:8 further includes the following substitution: L110F.
3. 3 5. The vaccine of any one of daims 18 to 34, characterized in that said vaccine is capable of inducing a humoral, cellular and mucosal immune response,

   36, The vaccine of any one of daims 18 to 3 5, characterized in that said vaccine further indudes an adjuvant.

   37. Use of an of attenuated rVSV of one serotype having 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/LU1F/M51R; in the manufacture of a fist vaccine and an attenuated rVSV of another serotype having 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 in the manufacture of a second vaccine, said vaccines being for inducing an inmmune response in a subject by administration of effective amounts of said vaccines to said subject.

   38. The use of claim 37, characterized in that the second M protein further ïncludes the following substitution: L110F.

   39. The use of claim 37, characterized in that the first vaccine is for administration to the subject before the second vaccine.

   40. The use of claim 39, characterized in that the first vaccine is for administration to the subject more than one time over the course of inducing.

   41. The use of claim 37, characterized in that the first vaccine is for administration to the subject and the second vaccine is for administration to the subject at about weeks three, eight and sixteen post- administration of the first vaccine.

   42. The use of claim 37, characterized in that ±e second vaccine is for administration to the subject before the first vaccine.

   43. The use of claim 42, characterized in that the first vaccine is for administration to the subject more than one time over the course of inducing.

   44. The use of claim 42, characterized in that the second vaccine is for administration to the subject and the first vaccine is administration to the subject at about weeks three, eight and sixteen post- administration of the second vaccine.

   45. The use of claim 37, characterized in that the two rVSVs are chimeric rVSVs that express a protein of a foreign pathogen, and wherein the two rVSVs are capable of inducing an immune response to the protein.

   46. The use of claim 45, characterized in that the pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.

   47. The use of claim 45, characterized in that the pathogen is a lentivirus.

   48. The use according to claim 37, characterized in that the lentivirus is a HIV and the protein is a HIV protein.

   49. The use of claim 48, characterized in that the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dépendent RNA polymerase (L) gene, and wherein a gene for expressing the HIV protein is a HIV gene inserted in between the G gene and the L gene,

   50. The use of claim 49, characterized in that the HIV gene is selected from the group of HIV genes consisting of gag, env and pol.

   51. The use of claim 45, characterized in that the pathogen is HCV and the protein is a HCV protein.

   52. The use of claim 51 , characterized in that the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dépendent RNA polymerase (L) gene, and wherein a gene for expressing the HCV protein is inserted in between the G gene and the L gene of the rVSV.

   5

   53. The use of claim 45, characterized in that the two vaccines comprise a mixture of attenuated chimeric rVSVs, wherein at least two of the attenuated chimeric rVSVs m the mixture express a different protein of the pathogen.

   54. The use of any one of daims 37 to 53, characterized in that each one of the two vaccines (a) and (b) induccs humoral, cellular and mucosal immune responses.

   10

   55. The use of any one of daims 37 to 54, characterized in that the one serotype of vaccine (a) is Indiana and the other serotype of vaccine (b) is New Jersey.

   56. The use of any one of daims 37 to 54, characterized in that each of the first vaccine and the second vaccine (b) further comprises an adjuvant.

   57. A prime boost combination vaccine, characterized in that the prime boost combination vaccine comprises: (a) an effective amount of a vaccine comprising an attenuated 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.

   58. The prime boost combination vaccine of claim 57, characterized in that the second M protein further includes the following substitution: Ll I0F.

   59. The prime boost combination vaccine of claim 57, characterized in that (a) is a priming vaccine and (b) is a booster vaccine.

   60. The prime boost combination vaccine of claim 57, characterized in that (b) is a priming vaccine and (a) is a booster vaccine.

   61. The prime boost combination vaccine of claim 57, characterized in that the two attenuated rVSVs are chimeric rVSVs that express a protein of a foreign pathogen, and wherein the two chimeric rVSVs aie capable of inducing an immune response to the protein.

   62. The prime boost combination vaccine of claim 61, characterized in that the pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.

   63. The prime boost combination vaccine of claim 61, characterized in that the pathogen is a lentivirus.

   5

   64. The prime boost combination vaccine of claim 63, characterized in that the lentivirus is a HIV and the protein is a HIV protein.

   65. The prime boost combination vaccine of claim 64, characterized in that the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dépendent RNA polymerase (L) gene, and wherein a gene for expressing the HIV protein is inserted inbetween the G gene and the L gene,

   66. The prime boost combination vaccine of claim 65, characterized in that the HIV gene is selected from the group of HIV genes consisting of env, gag and pot.

   67. The prime boost combination vaccine of claim 61, characterized in that the pathogen is HCV and the epitope is a HCV protein.

   15

   68. The prime boost combination vaccine of claim 67, characterized in that the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dépendent RNA polymerase (L) gene, and wherein a gene for expressing the HCV protein is inserted in between the G gene and the L gene.

   69. The prime boost combination vaccine of claim 67, characterized in that the HCV ²⁰ protein is a structural or a non-structural HCV protein.

   70. The prime boost combination vaccine of any one of claims 61 to 69, characterized in that each one of the two vaccines comprise a mixture of the attenuated chimeric rVSVs, and wherein at least two of the attenuated chimeric rVSVs in the mixture hâve a different protein of the pathogen.

   25

   71. The prime boost combination vaccine of any one of claims 57 to 70, characterized in that each one of the two vaccines is capable of inducîng humoral, cellular and mucosal immune responses.

   72. The prime boost combination vaccine of any one of daims 57 to 71, characterized in that the serotype of vaccine (a) is Indiana and the serotype of vaccine (b) is New Jersey.

   73. The prime boost combination vaccine of any one of daims 57 to 72, characterized in 5 that each one of vaccine (a) and vaccine (b) fiirther comprises an adjuvant.

   74. A kit comprising: (a) at least one dose of an effective amount of a vaccine comprising a rVSV^ having a modified M protein comprising the amino acid sequence of SEQ ID NO:4, and (b) at least one dose of an effective amount of a vaccine comprising rVSV_w having a modifîed M protein comprising the amino acid

   3. The modified M protein of claim 1, characterized in that said modified M protein comprises an amino acid sequence selected from the group consisting of: SEQ ID
4. 4. A recombinant VSV (rVSV), characterized in that said rVSV comprises a modifîed matrix (M) protein, the modified M protein comprising an amino acid sequence selected from the group consisting of: (i) SEQ JD NO: 3 including at least the following substitutions: G21E/L111F/M51R; and (ii) SEQ ID NO; 8 including at least

   15 the following substitutions: G22E/M48R+M5 IR.
5. 5. The rVSV of claim 4, characterized in that the rVSV is a chimeric rVSV that expresses a protein of a foreign pathogen, and wherein said chimeric rVSV is capable of inducing an immune response to said protein.

   5 substitutions: G22EZM48R+M51R.
6. 6. The rVSV of claim 5, characterized in that said pathogen is a viral, fiingal, bacterial 2o ^cr parasitic pathogen.
7. 7. The rVSV of claim 4, characterized in that the rVSV is essentially non-cytolytic and avirulent.
8. 8. The rVSV of claim 4, characterized in that said rVSV is a recombinant vesicular stomatitis virus Indiana serotype (rVSVfaa), and wherein the modified M protein ²⁵ comprises the amino acid sequence of SEQ ID NO: 3 including at least the following substitution: G21E/L111F/M51R.
9. 9. The rVSV of claim 8, characterized in that the modified M protein comprises the amino acid sequence of SEQ ID NO: 4.
10. 10 sequence of SEQ IDNO:9 or SEQ IDNO:IO.

    75. The kit of claim 62 characterized in that (a) and (b) are formulated in a pharmaceutically acceptable carrier.

    76, An isolated peptide comprising an amino acid sequence selected from the group of amino acid sequences listed as SEQ ID NOs: 4,9 and 10.

    10 comprises the amino acid sequence of SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51 R.

    10. The rVSV of claim 8, characterized in that the modified M protein is encoded by a gene comprising a nucléotide sequence of SEQ ID NO: 2.

    5

    10 NO:4, SEQ ID NO:9 and SEQ ID NO: 10.
11. 11. The rVSV according to any one of daims 8-10, characterized in that the rVSVbd is capable of producing VSV_M particles at permissible températures and incapable of producing the particles at non-permissible températures.
12. 12. The rVSV of claim 4, characterized in that said rVSV is a recombinant vesicular stomatitis virus New Jersey serotype (rVSVm), and wherein the modified M protein
13. 13. The rVSV ofclaim 12, characterized in that the modified M protein is encoded by a gene having a nucléotide sequence of SEQ ID NO: 6.
14. 14. The rVSV of claim 12, characterized in that the modified M protein comprises the

    15 amino acid sequence SEQ ID NO: 9.

    15. The rVSV of claim 12, characterized in that the modified M protein fiirther includes the following substitution: L110F.

    16. The rVSV of claim 15, characterized in that the modified M protein is encoded by a gene having a nucléotide sequence of SEQ ID NO: 7.

    17. The rVSV of claim 15, characterized in that the modified M protein comprises the amino acid sequence SEQ ID NO: 10.

    18. A vaccine, characterized in that the vaccine comprises an effective amount of one or more attenuated rVSVs, the one or more attenuated rVSVs including a modified matrix (M) protein, the modified M protein comprising an amino acid sequence

    25 selected from the group consisting of: (i) SEQ ID NO: 3 including at least the following substitutions: G21E/L111F/M51R, and (ii) SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R.

    19. The vaccine of daim 18, characterized in that said rVSV is a recombinant vesicular stomatitis virus Indiana serotype (rVSV_fcd), and wherein the modified M protein comprises the amino acid sequence of SEQ ID NO: 3 including at least the following substitution: G21E/L111F/M51R.

    5

    20. The vaccine of claim 19, characterized in that the modified M protein comprises the amino acid sequence of SEQ ID NO: 4.

    21. The vaccine of claim 19, characterized in that the modified M protein is encoded by a gene comprising a nucléotide sequence of SEQ ID NO: 2.

    22. The vaccine of any one of daims 19 to 20, characterized in that the rVSVind is ^{capable of} P^roducmg VSν_ω particles at permissïble températures and incapable of producing the particles at non-pennissible températures.

    23. The vaccine of claim 18, characterized in that said rVSV is a recombinant vesicular stomatitis virus New Jersey serotype (rVSV_NJ), and wherein the modified M protein comprises the amino acid sequence of SEQ ID NO: 8 including at least the following 15 substitutions: G22E/M48R+M51R.

    24. The vaccine of daim 23, characterized in that the modified M protein is encoded by a gene having a nucléotide sequence of SEQ ED NO: 6.

    25. The vaccine of claim 23, characterized in that the modified M protein comprises the amino acid sequence SEQ ID NO: 9.

    2o

    26. The vaccine of claim 23, characterized in that the modified M protein further includes the following substitution: L11 OF.

    27. The vaccine of daim 26, characterized in that the modified M protein is encoded by a gene having a nucléotide sequence of SEQ ID NO: 7.

    28. The vaccine of daim 26, characterized in that the modified M protein comprises the 25 amino acid sequence SEQ ID NO: 10.

    29. The vaccine of any one of daims 18 to 28, characterized in that the rVSV is a chimeric rVSV that expresses a protein of a foreign pathogen, and wherein said chimcric rVSV is capable of inducing an immune response to said protein.

    30. The vaccine of claim 29, characterized in that the vaccine comprises a mixture of attenuated chimeric rVSVs, wherein at least two chimeric rVSVs in the mixture express a different protein of the foreign pathogen.

    31. The vaccine of claim 29, characterized in that the pathogen is a viral, a fungal, a ⁵ bacterial or a parasitic pathogen,

    32. The vaccine of claim 29, characterized in that the pathogen is a lentivirus.

    33. The vaccine according to claim 33, characterized in that the lentivirus is a HIV and the epitope is a HIV protein.

    34. The vaccine of claim 29, characterized in that the pathogen is HCV and the epitope ¹⁰ is a HCV protein.
15. ₁₅ 77. An isolated nucléotide sequences for encoding the isolated peptide ofdaim 76.