Patent Grant
9943593
This application is a national stage application under 35 U.S.C. 371 of International Application No. PCT/CA2014/050614, filed Jun. 26, 2014, which in turn claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Ser. No. 61/839,798, filed Jun. 26, 2013, the contents of each of which are hereby incorporated by reference into the present disclosure.
A paper copy of the Sequence Listing and a Sequence Listing in computer readable form in .txt format titled “118494_133SequenceListing.txt”, which was submitted online on Dec. 23, 2015, and is 24 KB in size are hereby incorporated by reference. Applicants assert that the paper copy of the Sequence Listing is identical to the Sequence Listing in computer readable form.
The present invention relates, in general, to biotechnology and immunology. In particular, the present invention relates to modified matrix (M) proteins of vesicular stomatitis virus (VSV), to attenuated recombinant VSVs expressing the modified M proteins and to VSV based prime-boost vaccines that induce long-lasting humoral, cell-mediated and mucosal immune responses against foreign antigens expressed by the recombinant VSV carrying foreign genes.
Vesicular stomatitis virus (VSV) is a negative stranded RNA virus which infects most mammalian cells and expresses viral proteins up to 60% of total proteins in infected cells [Kim, G. N., and C. Y. Kang. Virology 357:41, 2007]. In nature VSV infects pigs, cattle, and horses, and causes vesicular disease around the mouth and foot. Although human infection by VSV has been reported, VSV does not cause any serious symptoms in humans [Fields, B. N., and K. Hawkins. N Engl J Med 277:989, 1967; Johnson, K. M. et al. Am J Trop Med Hyg 15:244, 1966].
VSV encodes five proteins, nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), surface glycoprotein (G), and RNA dependent RNA polymerase (L). The N, P, and L proteins of VSV are required for synthesis of positive sense and negative sense genomic RNAs and mRNA, which are necessary for the synthesis of VSV proteins.
Blocking the host cellular protein synthesis by VSV matrix (M) protein induces cell death. Changing a methionine residue at position 51 of the M protein to arginine (M51R) in the vesicular stomatitis virus Indiana serotype (VSVInd), and changing methionines at position 48 (Met48) and 51 (Met51) to Arg in the vesicular stomatitis virus New Jersey serotype (VSVNJ) M gene could negate this function of VSV M protein (Kim, G. and Kang, C., Virology, 357:41-53, 2007). While the VSVs with these Met to Arg mutations in the M protein have significantly reduced cytopathic effects, they still replicate and produce progeny viruses in the cell and in the infected animal. One of the temperature sensitive (ts) M gene mutants of VSVInd Orsay strain, tsO23 has shown limited replication in a mouse glial cell line at 37° C. and 39° C. (Rabinowitz, S. et al. Infection and Immunity 33:120-125, 1981). It has been demonstrated that the temperature sensitivity of tsO23 was the result of the improper or lack of the initiation of the viral assembly with the characteristic bullet shaped structure at 39° C. (Flood, E. et al. Virology 278:520-533, 2000; Lyles, D. et al. Virology, 217:76-87, 1996). In addition, the tsO23 lost its neurovirulence in mice even after direct inoculation into the brain (Rabinowitz, S. et al. Infection and Immunity, 33:120-125, 1981).
There are three amino acid differences between wild type M of VSV Orsay strain and M of tsO23, which are glycin (G) to glutamic acid (E) at 21st amino acid (G21E), leucine (L) to phenylalanine (F) at 111th amino acid (L111F), and histidine (H) to tyrosine (Y) at 227th amino acids (H227Y) (Morita, K. et al. J. Virol. 61:256-263, 1987). The single amino acid reversion at F111 to L in revertants implicated that the F111 appears to play a major role in the temperature sensitivity of the tsO23. However, the other two mutations may also have some role because the single reversion from the F to L at 111th position did not recover the virus completely to its wild type phenotype (Morita, K. et al. J. Virol. 61:256-263, 1987; Li, Y. et al. J. Virol. 62:3729-3737, 1988).
Given the single amino acid reversions at F111 to L in revertants, it may be advantageous to create mutations that are both less susceptible to reversion and that play a role in the temperature sensitivity of the tsO23.
Currently, research groups have developed replication competent, assembly defective VSV having a G glycoprotein (G) gene deleted (ΔG) or both G and M genes deleted (ΔMG) as safer vaccine vectors (Kahn, J. et al. J. Virol. 75:11079-11087, 2001; Schwartz, J. et al., Virology 366:166-73, 2007). However, having the G gene deleted or M and G gene deleted, the VSV vector requires the supply of G or both M and G proteins in trans for the production of the assembly-defective VSV. In order to reduce the cost of producing vaccines, it is necessary to generate a system, which can produce the viral vaccine vectors that can replicate in high titre. Therefore, what is needed is a VSV vector system, which may be a full-length VSV vector, which has lost its virulence (avirulent) and still can replicate to a high titer in vitro at 31° C. cannot assemble properly at non-permissible temperatures, induce good immune responses against the gene of interest that it expresses, and having less chance of reverting back to a wild type phenotype.
Further and other objects of the invention will be realized from the following Summary of the Invention, the Discussion of the Invention and the embodiments and Examples thereof.
The inventors have generated new matrix (M) protein mutants of vesicular stomatitis virus (VSV), including VSV Indiana serotype (VSVInd) and VSV New Jersey serotype (VSVNJ). These new VSVs having the mutant M proteins are essentially non-cytolytic, significantly attenuated and avirulent, including neurovirulence (safer). Furthermore, these new VSVs having the mutant M proteins are capable of inducing immune responses against an antigen or epitope of interest. The immune response may be humoral, cellular and mucosal immune responses. In addition, the VSVInd having the M mutant can assemble and be released from the infected cells normally at 31° C., but they can not assemble properly at 37° C. or higher and the release of the viruses from infected cells is reduced significantly relative to wild type VSVInd. In addition, the M protein mutants of the present invention may be less susceptible to reversion relative to known M protein mutants of the prior art.
As such, in one embodiment, the present application relates to a modified matrix (M) protein of a vesicular stomatitis virus (VSV). In one embodiment, the modified M protein of the present invention includes an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3 including at least the following substitutions: G21E/L111A/M51R; and (ii) SEQ ID NO: 8 including at least the following substitutions: G22E/M48R/M51R. In one aspect of the present invention, SEQ ID NO:8 includes at least the following substitutions: G22E/L110A/M48R/M51R.
In one embodiment of the present invention, the modified M protein of (i) includes an amino acid sequence SEQ ID NO:4 and the modified M protein of (ii) includes the amino acid sequence SEQ ID NO:9 or SEQ ID NO:10.
In one embodiment of the present invention, the E at position 21 in (i) and the E at position 22 in (ii) are encoded by a gaa codon, and wherein the R at position 51 in (i) and (ii) and the R at position 48 in (ii) is encoded by a cga codon.
In one embodiment of the present invention, the A at position 111 of SEQ ID NO:4 and the A at position 110 of SEQ ID NO:10 is encoded by a gca codon.
In one embodiment of the present invention, the amino acid sequence of (i) is encoded by a gene comprising SEQ ID NO:2, and the amino acid sequence of (ii) is encoded by a gene comprising SEQ ID NO:6 or SEQ ID NO:7.
In another embodiment, the present invention provides for a nucleotide sequence or a gene that encodes a modified matrix (M) protein of a vesicular stomatitis virus (VSV), wherein the nucleotide sequence or gene includes a nucleotide sequence selected from SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO: 7.
In another embodiment the present invention provides for a recombinant VSV (rVSV). The rVSV of the present invention, in one embodiment, includes a nucleotide sequence or gene comprising a nucleotide sequence selected from SEQ ID NOs:2, 6 and 7.
In another embodiment the present invention provides for a recombinant vesicular stomatitis virus (rVSV). The rVSV of the present invention includes a modified matrix (M) protein according to any of the previous embodiments.
In one embodiment of the present invention, the rVSV is a recombinant vesicular stomatitis virus Indiana serotype (rVSVInd), and the modified M protein includes the amino acid sequence of SEQ ID NO: 3 including at least the following substitution: G21E/L111A/M51R.
In another embodiment of the present invention, the modified M protein of the rVSVInd includes the amino acid sequence of SEQ ID NO: 4.
In another embodiment of the present invention, the modified M protein of the rVSVInd is encoded by a gene comprising a nucleotide sequence of SEQ ID NO: 2.
In another embodiment of the present invention, the rVSVInd is capable of producing VSVInd particles at permissible temperatures and incapable of producing the particles at non-permissible temperatures.
In another embodiment of the present invention, the rVSV is a recombinant vesicular stomatitis virus New Jersey serotype (rVSVNJ), and the modified M protein includes the amino acid sequence of SEQ ID NO: 8 including at least the following substitutions: G22E/M48R/M51R. In one aspect of this embodiment, SEQ ID NO:8 includes at least the following substitutions: G22E/L110A/M48R/M51R.
In one embodiment, the modified M protein of the rVSVNJ is encoded by a gene including a nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO:7.
In one embodiment, the modified M protein of the rVSVNJ includes the amino acid sequence SEQ ID NO: 9 or SEQ ID NO:10.
In another embodiment of the present invention, the rVSV is a chimeric rVSV that expresses a protein of a foreign pathogen.
In one embodiment of the present invention, the pathogen is a viral, fungal, bacterial or parasitic pathogen.
In another embodiment, the rVSV of the present invention is essentially non-cytolytic and avirulent.
In one embodiment, the present invention provides for a vaccine. The vaccine, in one embodiment, includes an effective amount of a modified matrix (M) protein, the modified M protein being encoded by a nucleotide sequence comprising a sequence selected from: SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7.
In another embodiment, the present invention provides for a vaccine. The vaccine, in one embodiment, includes an effective amount of one or more attenuated recombinant vesicular stomatitis virus (rVSV), the one or more attenuated rVSVs including a modified matrix (M) protein, the modified M protein comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3 including at least the following substitutions: G21E/L111A/M51R, and (ii) SEQ ID NO: 8 including at least the following substitutions: G22E/M48R/M51R. In one aspect, SEQ ID NO:8 further includes the substitution L110A.
In one embodiment of the vaccine of the present invention, the rVSV is a recombinant vesicular stomatitis virus Indiana serotype (rVSVInd), and the modified M protein includes the amino acid sequence of SEQ ID NO: 3 including at least the following substitution: G21E/L111A/M51R.
In another embodiment of the vaccine of the present invention, the rVSV is a recombinant vesicular stomatitis virus Indiana serotype (rVSVInd), and the modified M protein includes the amino acid sequence of SEQ ID NO: 4.
In another embodiment of the vaccine of the present invention, the modified M protein is encoded by a gene comprising a nucleotide sequence of SEQ ID NO: 2.
In another embodiment of the vaccine of the present invention, the rVSVInd is capable of producing rVSVInd particles at permissible temperatures and incapable of producing the particles at non-permissible temperatures.
In another embodiment of the vaccine of the present invention, the rVSVInd is a full length rVSVInd.
In another embodiment of the vaccine of the present invention, the rVSV is a recombinant vesicular stomatitis virus New Jersey serotype (rVSVNJ), and the modified M protein comprises the amino acid sequence of SEQ ID NO: 8 including at least the following substitutions: G22E/M48R/M51R. In one aspect of this embodiment, SEQ ID NO:8 further includes the substitution L110A.
In one embodiment of the vaccine of the present invention, the rVSV is a recombinant vesicular stomatitis virus New Jersey serotype (rVSVNJ), and wherein the modified M protein comprises the amino acid sequence SEQ ID NO: 9 or SEQ ID NO:10.
In one embodiment of the vaccine of the present invention, the modified M protein is encoded by a gene having a nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO:7.
In another embodiment of the vaccine of the present invention, the rVSVNJ is a full length rVSVNJ.
In another embodiment of the vaccine of the present invention, 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.
In another embodiment of the vaccine of the present invention, 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.
In another embodiment of the vaccine of the present invention, the pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.
In another embodiment of the vaccine of the present invention, the pathogen is a lentivirus.
In another embodiment of the vaccine of the present invention, the lentivirus is a HIV and the protein of the foreign pathogen is a HIV protein.
In another embodiment of the vaccine of the present invention, the pathogen is HCV and the protein of the foreign pathogen is a HCV protein.
In another embodiment of the vaccine of the present invention, the E at position 21 in (i) and the E at position 22 are encoded by a gaa codon, and the R at position 51 and the R at position 48 is encoded by a cga codon.
In another embodiment of the vaccine of the present invention, the A at position 111 and the A at position 110 is encoded by a gca codon.
In another embodiment of the vaccine of the present invention, vaccine is capable of inducing a humoral, cellular and mucosal immune response.
In another embodiment of the vaccine of the present invention, the vaccine further includes an adjuvant.
In one embodiment, the present invention provides for a prime boost combination vaccine. The prime boost combination vaccine, according to one embodiment, includes: (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 comprising the amino acid sequence of SEQ ID NO:10.
In one embodiment of the prime boost combination vaccine, SEQ ID NO:4 is encoded by a gene comprising SEQ ID NO:2.
In another embodiment of the prime boost combination vaccine, SEQ ID NO:9 is encoded by a gene comprising SEQ ID NO:6 and SEQ ID NO:10 is encoded by a gene comprising SEQ ID NO:7.
In another embodiment of the prime boost combination vaccine, (a) is a priming vaccine and (b) is a booster vaccine.
In another embodiment of the prime boost combination vaccine, (b) is a priming vaccine and (a) is a booster vaccine.
In another embodiment of the prime boost combination vaccine of the present invention, 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.
In another embodiment of the prime boost combination vaccine of the present invention, the pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.
In another embodiment of the prime boost combination vaccine of the present invention, the pathogen is a lentivirus.
In another embodiment of the prime boost combination vaccine of the present invention, the lentivirus is a HIV and the protein is a HIV protein.
In another embodiment of the prime boost combination vaccine of the present invention, 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.
In another embodiment of the prime boost combination vaccine of the present invention, wherein the HIV gene is selected from the group of HIV genes consisting of env, gag and pol.
In another embodiment of the prime boost combination vaccine of the present invention, the pathogen is HCV and the epitope is a HCV protein.
In another embodiment of the prime boost combination vaccine of the present invention, 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 a gene for expressing the HCV protein is inserted in between the G gene and the L gene.
In another embodiment of the prime boost combination vaccine of the present invention, the HCV protein is a structural or a non-structural HCV protein.
In another embodiment of the prime boost combination vaccine of the present invention, each one of the two vaccines includes a mixture of the attenuated chimeric rVSVs, and at least two of the attenuated chimeric rVSVs in the mixture have a different protein of the pathogen.
In another embodiment of the prime boost combination vaccine of the present invention, each one of the two vaccines is capable of inducing humoral, cellular and mucosal immune responses.
In another embodiment of the prime boost combination vaccine of the present invention, the serotype of the rVSV of vaccine (a) is Indiana and the serotype of the rVSV of vaccine (b) is New Jersey.
In another embodiment of the prime boost combination vaccine of the present invention, each one of vaccine (a) and vaccine (b) further comprises an adjuvant.
According to another embodiment, the present invention provides for a kit. The kit, in one embodiment, includes: (a) at least one dose of an effective amount of a vaccine comprising a recombinant vesicular stomatitis virus Indiana serotype (rVSVInd) 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 a recombinant vesicular stomatitis virus New Jersey serotype (rVSVNJ) having a modified M protein comprising the amino acid sequence of SEQ ID NO:9 or the amino acid sequence of SEQ ID NO:10.
In one embodiment of the kit of the present invention (a) and (b) are formulated in a pharmaceutically acceptable carrier.
In another embodiment of the kit of the present invention SEQ ID NO:4 is encoded by a gene comprising SEQ ID NO:2.
In another embodiment of the kit of the present invention SEQ ID NO:9 is encoded by a gene comprising SEQ ID NO:6 and SEQ ID NO:10 is encoded by a gene comprising SEQ ID NO:7.
In another embodiment, the present invention provides for 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. In one embodiment the isolated peptide is provided in purified form.
In another embodiment, the present invention provides for an isolated nucleotide sequences comprising a nucleotide sequence selected from the group SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7. In one embodiment the isolated nucleotide sequences is provided in purified form.
In one embodiment, the present invention provides for a vaccine of the present invention for use to induce an immune response in a subject.
In one embodiment, the present invention relates to the prime boost combination vaccine of the present invention for use to induce an immune response in a subject.
In another embodiment, the present invention relates to a method of inducing an immune response in a subject. The method, according to one embodiment, includes administering to the subject: (a) an effective amount of a vaccine comprising an attenuated recombinant vesicular stomatitis virus (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/L111A/M51R; and (b) an effective amount of another vaccine comprising 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 one aspect of this embodiment, SEQ ID NO:8 further includes the substitution L110A.
In one embodiment of the method of inducing an immune response in a subject of the present invention (a) is administered to the subject before (b) is administered to the subject.
In another embodiment of the method of inducing an immune response in a subject of the present invention (b) is administered to the subject more than one time over the course of inducing.
In another embodiment of the method of inducing an immune response in a subject of the present invention (a) is administered to the subject and (b) is administered to the subject at about weeks three, eight and sixteen post-administration of (a).
In another embodiment of the method of inducing an immune response in a subject of the present invention (b) is administered to the subject before (a) is administered to the subject.
In another embodiment of the method of inducing an immune response in a subject of the present invention (a) is administered to the subject more than one time over the course of inducing.
In another embodiment of the method of inducing an immune response in a subject of the present invention (b) is administered to the subject and (a) is administered to the subject at about weeks three, eight and sixteen post-administration of (b).
In another embodiment of the method of inducing an immune response in a subject of the present invention 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.
In another embodiment of the method of inducing an immune response in a subject of the present invention the pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.
In another embodiment of the method of inducing an immune response in a subject of the present invention the pathogen is a lentivirus.
In another embodiment of the method of inducing an immune response in a subject of the present invention the lentivirus is a human immunodeficiency virus (HIV) and the protein is a HIV protein.
In another embodiment of the method of inducing an immune response in a subject of the present invention 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 a gene for expressing the HIV protein is a HIV gene inserted in between the G gene and the L gene.
In another embodiment of the method of inducing an immune response in a subject of the present invention the HIV gene is selected from the group of HIV genes consisting of gag, env and pol.
In another embodiment of the method of inducing an immune response in a subject of the present invention the pathogen is hepatitis C virus (HCV) and the protein is a HCV protein.
In another embodiment of the method of inducing an immune response in a subject of the present invention 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 of the rVSV.
In another embodiment of the method of inducing an immune response in a subject of the present invention the two vaccines comprise a mixture of attenuated chimeric rVSVs, and at least two of the attenuated chimeric rVSVs in the mixture express a different protein of the pathogen.
In another embodiment of the method of inducing an immune response in a subject of the present invention each one of the two vaccines (a) and (b) induces humoral, cellular and mucosal immune responses.
In another embodiment of the method of inducing an immune response in a subject of the present invention the one serotype of vaccine (a) is Indiana and the other serotype of vaccine (b) is New Jersey.
In another embodiment of the method of inducing an immune response in a subject of the present invention each of vaccine (a) and vaccine (b) further includes an adjuvant.
Embodiments will now be described, by way of example only, with reference to the drawings, in which:
Overview
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. All publications cited and the priority document are incorporated herein by reference.
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”.
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.
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.
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.
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.
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.
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., immuogenic 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.
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.
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 O 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.
The term “non-infectious” means of reduced to non-existent ability to infect.
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.
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.
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.
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.
The terms “polynucleotide”, “nucleic acid sequence” 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.
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.
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).
“VSV” is used to refer to vesicular stomatitis virus.
“rVSV” is used to refer to a recombinant vesicular stomatitis virus.
The term “Indiana”, and “IND” are used to refer to the VSV serotype Indiana (VSVInd).
The term “New Jersey”, and “NJ” are used to refer to the VSV serotype New Jersey (VSVNJ).
“MWT” “M(WT)” are used to refer to a wild type M protein. The nucleotide sequence of wild type M gene of the VSVInd may comprise the nucleotide sequence represented by SEQ ID NO: 1. The amino acid sequence of wild type M protein of the VSVInd may comprise the amino acid sequence represented by SEQ ID NO: 3. The nucleotide sequence of wild type M gene of the VSVNJ may comprise the nucleotide 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 VSVInd having a methionine changed to an arginine at position 51. “G21E” is used to refer to an M(WT) in VSVInd having a glycine changed to a glutamic acid at position 21. “L111A” is used to refer to an M(WT) in VSVInd having a leucine (L) changed to alanine (A) 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. “L110A” is used to refer to an M(WT) in VSVNJ having a leucine (L) changed to alanine (A) at position 110. “M48R+M51R” or “M48R/M51R” is used to refer to an M(WT) in VSVNJ having a methionine (M) changed to an arginine (R) at positions 48 and 51. “rVSVInd M(G21E/L111A/M51R)” or “rVSVInd (GLM)-new” are used to refer to a rVSVInd having an M(WT) having a glycine changed to a glutamic acid at position 21, a leucine changed to alanine at position 111 and a methionine changed to an arginine at position 51. “rVSVNJ M(G22E/M48R/M51R)” “rVSVNJ M(G22E/M48R+M51R)” or “rVSVNJ (GM)” are used to refer to rVSVNJ 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. “rVSVNJ(GM)-new” refers to rVSVNJ(GM) with the codons of the present invention. “rVSVNJ M(G22E/L110F/M48R/M51R)” or “rVSVNJ M(G22E/L110F/M48R+M51R)” or “rVSVNJ (GLM)” are used to refer to rVSVNJ 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. “rVSVNJ M (G22E/L110A/M48R/M51R)”, “rVSVNJ M (G22E/L110A/M48R+M51R)” or “rVSVNJ (GLM)-new” is used to refer to rVSVNJ having an M(WT) having a glycine changed to a glutamic acid at position 22, a leucine changed to alanine at position 110 and a methionine changed to an arginine at positions 48 and 51.
Overview
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/L111A/M51R), and M(G22E/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.
Isolated Proteins and M Proteins
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 and 9. 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 and 6. The isolated peptides and nucleotide sequences may be provided in purified form. The nucleotides and amino acid sequences of the present invention may be artificially made or synthesized by known methods in the art.
In one embodiment, the present invention relates to novel VSV M proteins having at least one of the following substitutions: M(G21E/L111A/M51R), M(G22E/M48R/M51R) or M(G22E/L110A/M48R/M51R).
The E at positions 21 or 22 as the case may be, is encoded by the codon gaa and R at positions 48 and 51, as the case may be, is encoded by codon cga. The A at position 111 may preferably be encoded by the codon gca.
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.
Methods of Preventing or Treating an Infection
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 the following substitutions: G21E/L111A/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 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 rVSVInd M(G21E/L111A/M51R), and (b) an effective amount of rVSVNJ M(G22E/M48R/M51R) or M(G22E/L110A/M48R/M51R) in a prime-boost immunization modality. The E at positions 21 or 22 as the case may be, is encoded by the codon gaa and R at positions 48 and 51, as the case may be, is encoded by codon cga. The A at position 111 or 110 may preferably be encoded by the codon gca.
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.
In certain embodiments, (a) is administered to the subject before (b) is administered to the subject.
In certain embodiments, (b) is administered to the subject more than one time over the course of treating or preventing.
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).
In certain embodiments, (b) is administered to the subject before (a) is administered to the subject.
In certain embodiments, (a) is administered to the subject more than one time over the course of treating or preventing.
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).
Recombinant Virus
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.
In one embodiment, the present invention relates to rVSVInd and rVSVNJ. The rVSVInd may be a full length, essentially noncytolytic rVSVInd M(G21E/L111A/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 rVSVInd may include a M(G21E/L111A/M51R). In certain embodiments, the rVSVInd may include an M gene comprising a nucleotide sequence SEQ ID NO: 2.
In certain embodiments, the rVSV is a full-length, essentially noncytolytic rVSVNJ M (G22E/M48R/M51R) or M(G22/L110A/M48R/M51R). In certain embodiments, the rVSV is an essentially noncytolytic rVSVNJ including an M gene, wherein the nucleotide sequence of the M gene is selected from SEQ ID NO: 6 and SEQ ID NO: 7.
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 rVSVInd wherein the amino acid sequence of the virus' M protein is modified to provide an essentially non-cytotoxic which also allows the rVSVInd to effectively replicate at permissible temperature but may not replicate at non-permissible temperature.
As such, the present invention relates also to a cell having one or more of the recombinant VSVs of the present invention.
Vaccines or Immunogenic Compositions of the Invention
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 rVSVInd and vaccines or immunogenic compositions comprising an rVSVNJ, as described above.
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).
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.
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.
Kits
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.
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.
Advantages and Unique Features of the rVSVs of the Present Invention
Novel and Unusual Features of the Invention:
Normal assembly and release of rVSVInd M(G21E/L111A/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 rVSVInd M(G21E/L111A/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 rVSVInd further attenuated the virulence of VSVInd.
Prior to the present invention it was unknown that including the substitution L111A would result in rVSV having normal assembly at permissive temperatures and assembly defectiveness at non-permissive temperatures. The advantage of having a L111A substitution instead of L111F is that the former has less chance of reverting back to a wild type form.
The E at positions 21, which may be encoded by the codon gaa and R at positions 48 and 51, as the case may be, being encoded by codon cga decreases the chances of the mutants reverting back to wild type.
Previously the inventors developed attenuated rVSVInd with the mutations of G21E, M51R, and L111F in the M gene. The amino acid changes were accomplished by changing nucleotides. Nucleotide codon for Glycine 21, GGG was changed to GAA for Glutamic acid with 2 nucleotide changes. Nucleotide codon for Methionine51, ATG was changed to AGG for Arginine with 1 nucleotide change. Nucleotide codon for Leucine, TTG was changed to TTT for Phenylalanine with 1 nucleotide change. The initial nucleotide changes to mutate M51 to R and L111 to F were only one in each amino acid. However, it is very likely that, if we change more nucleotides to change amino acids, will diminish the chances of the changed amino acids reverting back to the original amino acids. Therefore, the iventors mutated AGG of M51R to CGA to have the nucleotide codon changed completely in all 3 nucleotides. TTT of Phenylalanine was mutated to GCA of Alanine to have 3 nucleotides were completely changed. The resulting mutations in the M protein of rVSVInd (GLM)-New are G21E/L111A/M51R with nucleotide changes of GAA(E)/GCA(A)/CGA(R). More nucleotides changes in a codon for a amino acid change will increase the stability of the temperature sensitive mutations in the rVSVInd(GLM). Likewise more nucleotides for M48R and M51R in the rVSVNJ(GMM) mutant. The initial changes in the nucleotide codon for the mutations G22E/M48R/M51R were GAA(E)/AGG(R)/AGG(R). These nucleotide codons were further changed to GAA(E)/CGA(R)/CGA(R) to have all three nucleotides for the Arginine changed.
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.
The present invention is further illustrated by the following examples which should not be construed as limiting in any way.
Mutations were introduced into the M gene of VSVInd (
Wild type and mutant recombinant vesicular stomatitis viruses (rVSV) were recovered from the cDNA plasmids by reverse genetics (
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 (
BHK21 cells were infected with an MOI of 3 of rVSVNJ, 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 rVSVNJ replicated equally well at both temperatures 31° C. and 37° C. (
BHK21 cells were infected with an MOI of 6 of rVSVInd and rVSVNJ, 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 μg 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 VSVNJ (1:5000 dilution). The result 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 (
In order to examine the effects of the mutations, L111F and M51R of M gene of rVSVInd on the cytopathogenicity, the inventors infected BHK21 cells (
In order to examine the effects of the mutations, G22E, L110F and M48R+M51R of M gene of rVSVNJ on the cytopathogenicity, the inventors infected BHK21 cells and human neuroblastoma cells with an MOI of 0.1 of rVSVNJ. At 20 hrs after infection, the inventors compared the cytopathic effects (cell round-up and lysis) caused by the rVSVNJ 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 rVSVNJ in both BHK21 (
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 1×106 PFU of mutant VSV and 1×103 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 1×103 PFU of wild type rVSVInd died within four days after injection (
The newly generated M mutants, G21E/L111F/M51R mutant of rVSVInd and G22E/M48R+M51R and G22E/L110F/M48R+M51R mutants of rVSVNJ 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-2Kd-restricted peptide (NH2-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 rVSVInd and wild type, G22E/M48R+M51R (GM), and G22E/L110F/M48R+M51R(GLM) of rVSVNJ. The rVSVs were recovered from the cDNA clones by reverse genetics as described in
Six Balb/c mice per group were vaccinated with the prime-boost regimen illustrated in
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-γ (IFN-γ), which indicates the antigen specific T cell immune responses. The splenocytes 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 specific CD8+ T cell immune response, splenocytes were isolated and 1×106 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-γ for intracellular IFN-γ. The secretion of IFN-γ 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-γ+ T cells were counted with FACSCalibur, a flowcytometer. The splenocytes treated with DMSO (solvent for the peptide,
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 (
As illustrated in
Mice were vaccinated according to the schedule as seen in
CD8+ T cell immune responses against VSV N protein were very similar in all vaccination groups with different doses of rVSVInd(GLM)-Gag, rVSVNJ(GLM)-Gag, or rVSVNJ(GM)-Gag (
Generation of antibody against HIV-1 Gag protein was increased with the increasing doses of rVSVInd(GLM)-Gag for priming and booster with rVSVNJ(GLM)-Gag or rVSVNJ(GM)-Gag (
For HIV-1 vaccines utilizing the rVSVInd(GLM) and rVSVNJ(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 rVSVInd(GLM) and rVSVNJ(GM) carrying cassettes encoding HIV-1 Gag linked to B-cell epitopes from HIV Env protein derived from multiple viral clades (
The preliminary prime-boost immunization studies with new M mutant rVSVs, rVSVInd(GLM) and rVSVNJ(GM) revealed that priming mice with the rVSVInd(GLM) and boosting with rVSVNJ(GM) induced better CD8+ T cell immune response (
The inventors prime immunized Balb/c mice with rVSVInd(GLM) expressing HIV-1 proteins and boost immunized with rVSVNJ(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 1×106 splenocytes were stimulated with H-2Kd-restricted HIV-1 protein specific peptides, which are Gag, Env P18, RT354, RT464, and RT472. The peptide sequences are shown in the
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 μl 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 (
The newly generated M mutants of rVSVs, rVSVInd(GLM) and rVSVNJ(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.
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.
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 (
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 develope 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).
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 pVSVInd(GLM) and pVSVNJ(GM) (
Mutations were introduced into the M gene of VSVInd and VSVNJ. 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) in the tsO23 with methionine to arginine mutations (M51R) and L111A 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 rVSVInd and rVSVNJ 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.
Wild type and mutant recombinant vesicular stomatitis viruses (rVSV) were recovered from the cDNA plasmids by reverse genetics. 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 μg of pN, 10 μg of pP, μ5 g of pL, and 15 μg of pVSV. The culture media from the transfected cells were harvested when the cells showed about 50-70% of CPE.
First, AGG of M51R in the M gene of prVSVInd(GLM) was changed to CGA by megaprimer method. The primers, Ind(GLM) M (F) and Ind (GLM) M(M51R) was used to amplify the part of M gene with the additional nucleotide changes by polymerase chain reaction. The first PCR product was used as a megaprimer together with Ind(GLM) M (R) to amplify the whole M gene with the nucleotide changes, and the second PCR product was cloned into pBluescript II KS vector (Invitrogen) at the restriction site Not I. After confirmation of the nucleotide changes in the M gene clone with the CGA at the R51, the additional nucleotide change GCA for the Alanine was introduced by megaprimer PCR method using the primers Ind(GLM) M(F) and Ind(GLM) M(L111A), and Ind(GLM) M(R). The PCR product was cloned again into the pBluescript II KS vector at the Not I site. The M gene with the nucleotide changes of GAA(E)/CGA(R)/GCA(A) was cut from the pBluescript II KS vector with restriction enzymes, Pac I and Not I and cloned into the prVSVInd(GLM), which M gene was removed by cutting with Pac I and Not I.
The nucleotides for the M48R and M51R in the M gene of rVSVNJ was changed by the megaprimer PCR method. The part of M gene with the nucleotide changes were amplified with the primers, NJ-M(F) and NJ-M(M48R+M51R), and the PCR product was used as a megaprimer with the primer NJ-G(R) to amplify the full length NJ M gene and G gene in order to use the restriction enzyme site, Sac I. The M and G PCR product was cut with Pac I and replaced the M and G gene of prVSVNJ(GMM).
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 with an MOI of 0.1 of rVSVInd(GLM) and rVSVInd(GLM)-New. 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 at 20 hrs post-infection. 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. Both rVSVInd(GLM) and rVSVInd(GLM)-New showed the same temperature sensitivity at 37° C. indicating that that the additional nucleotide changes in the M gene did not alter the temperature sensitivity of the rVSVInd(GLM). The mutation L111A shows the same temperature sensitivity as L111F indicating that the mutation L111A is not a silent mutation.
The inventors further demonstrated the stability of the new rVSV vector after 20 passages at 37 C (about body temperature) and at 31 C (the temperature for virus amplification) (data not shown). The new GLM mutant (G21E/L111A/M51R) with further nucleotide changes (codons gca for A and cga for R) did not change back to wild type sequences (reversion) after 20 passages in contrast to the old GLM mutant (G21E/L110F/M51R). The result demonstrated that the new GLM mutant is more stable and safer to use for vaccination in humans and other animals.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2014/050614 | 6/26/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/205579 | 12/31/2014 | WO | A |
| Entry |
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| Number | Date | Country | |
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| 20160144022 A1 | May 2016 | US |
| Number | Date | Country | |
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| 61839798 | Jun 2013 | US |
