Recombinant novirhabdovirus usable as an antigen vector

Abstract

The invention relates to a recombinant novirhabdovirus expressing a chimeric protein comprising the sequence of an antigen of interest flanked, at the N-terminus, by a signal peptide, and at the C-terminus, by a polypeptide comprising at least one portion of the transmembrane domain of a rhabdovirus G protein. Said recombinant novirhabdovirus can be used especially for inducing an immune response to the antigen of interest.

Claims

1. A recombinant novirhabdovirus, the genome of which comprises the genes encoding the endogenous N, P, M, G and L proteins of said novirhabdovirus, and an exogenous gene encoding a chimeric protein, wherein said chimeric protein comprises the sequence of an antigenic protein of interest fused, at its N-terminal end, with a novirhabdovirus G protein signal peptide and, at its C-terminal end, with a novirhabdovirus G protein sequence fragment, said fragment comprising the transmembrane domain of said novirhabdovirus G protein or a portion thereof, said portion of the transmembrane domain comprises at least 15 consecutive C-terminal amino acids of said domain and wherein said antigenic protein of interest is not a glycoprotein.

2. The recombinant novirhabdovirus as claimed in claim 1, further comprising, the intravirion domain of a novirhabdovirus G protein, or a portion of said intravirion domain, said intravirion domain or said portion thereof being C-terminal to the transmembrane domain or the portion thereof, and said intravirion domain or the portion thereof following said transmembrane domain or the portion thereof.

3. The recombinant novirhabdovirus as claimed in claim 2, wherein said portion of the intravirion domain comprises at least three consecutive N-terminal amino acids of said domain.

4. The recombinant novirhabdovirus as claimed in claim 1, wherein said recombinant novirhabdovirus is devoid of the endogenous NV gene, and in that the gene encoding the chimeric protein is inserted as a replacement for said NV gene.

5. The recombinant novirhabdovirus as claimed in claim 1, wherein said recombinant novirhabdovirus contains the endogenous NV gene, and in that the gene encoding the chimeric protein is inserted into an additional transcription unit placed in an intergenic region of the viral genome.

6. The recombinant novirhabdovirus as claimed in claim 5, further comprising at least two additional transcription units, each of which comprising an exogenous gene encoding-said chimeric protein.

7. The recombinant novirhabdovirus as claimed in claim 1, wherein the recombinant novirhabdovirus is chosen from an infectious hematopoietic necrosis virus and a viral hemorrhagic septicaemia virus.

8. An isolated cDNA encoding the genome of a recombinant novirhabdovirus as claimed in claim 1.

9. A method of inducing an immune response in a subject, comprising administering to the subject a vaccine comprising the recombinant novirhabdovirus of claim 1, wherein the immune response is directed against the antigen of interest expressed by said novirhabdovirus.

10. The method of claim 9 wherein said vaccine is selected the group consisting of an antiviral vaccine, an antibacterial vaccine, an antifungal vaccine, an antiparasitic vaccine and an antitumor vaccine.

11. A method of producing antibodies in a subject, comprising administering to the subject the recombinant novirhabdovirus of claim 1, wherein the antibodies are directed against the antigen of interest expressed by said novirhabdovirus.

Description

EXAMPLE 1

Construction of a Recombinant Novirhabdovirus Containing a Gene Encoding a Chimeric Protein Comprising Domain III Of West Nile Virus (wnv) Glycoprotein E

(1) The constructions were carried out using the pVHSV plasmid, described by Biacchesi et al. (2010, mentioned above). This plasmid contains the complete cDNA of the genome of a VHSV (strain 23-75, GenBank FN665788), cloned downstream of the T7 phage RNA polymerase promoter and upstream of a ribozyme sequence of the hepatitis virus and of the T7 phage RNA polymerase transcription terminator, in the pBlueScript SK vector (Stratagene).

(2) The pVHSV plasmid contains a unique PsiI restriction site in the intergenic region between the N and P genes. This site is used to insert an additional transcription unit, containing a sequence encoding a fusion protein made up of the sequence of domain III of the West Nile Virus glycoprotein E (GenBank AF481864) preceded by the signal peptide of the VHSV G protein (strain 23-75, GenBank CBJ23832.1.), and followed by the 42 C-terminal amino acids of the VHSV G protein (strain 23-75).

(3) The construction of this additional transcription unit is described in detail hereinafter.

(4) The sequence encoding domain III of the West Nile Virus glycoprotein E, that encoding the signal peptide of the VHSV G protein, and that encoding the 42 C-terminal amino acids of the VHSV G protein were amplified by PCR using appropriate primers.

(5) A first PCR amplification was carried out on the West Nile Virus cDNA, using the following primers:

(6) TABLE-US-00001 SPSHVDIIIF: (SEQIDNO:1) 5-ACTAGTATGGACACCACGATCACCACTCCGCTCATTCTCATTCTGAT CACCTGCGGAGCAGCTAGCGGAACAACCTATGGCGTCTGTTCAAAGG- 3,whichcontainsanSpeIsite(ACTAGT)andan NheIsite(GCTAGC) and DIIISHVTMR: (SEQIDNO:2) 5-GGCCCCTCCCACAACCCCCATCCCAGATAACGCTCCTTTGAGGGTGG TTGTAAAGG-3.

(7) A second PCR amplification was carried out on the VHSV cDNA, using the following primers:

(8) TABLE-US-00002 DIIITMSHVF: (SEQIDNO:3) 5-CCTTTACAACCACCCTCAAAGGAGCGTTATCTGGGATGGGGGTTGTG GGAGGGGCC-3, and SHVTMR: (SEQIDNO:4) 5-TACGTATCAGACCGTCTGACTTCTAGAGAACTGC-3, whichcontainsanSnaBIsite(TACGTA).

(9) The two amplification products were mixed, and a third PCR amplification was carried out on the mix, using the primers:

(10) TABLE-US-00003 SPSHVF: (SEQIDNO:5) 5-ACTAGTATGGACACCACGATCACCACTCCGC-3,which containsanSpeIsite(ACTAGT), and (SEQIDNO:4) SHVTMR.

(11) The product of this third amplification (SPg-DIII-TMg), which contains the sequence encoding the signal peptide of the VHSV G protein, in reading frame with that encoding domain III of the West Nile Virus glycoprotein E, and that encoding the 42 C-terminal amino acids of the VHSV G protein, was cloned into a pJet1.2 vector (Fermentas). The SPg-DIII-TMg insert was excised from this vector by SpeI/SnaBI digestion and cloned, in place of the tdTomato gene, into the pVSHV-dTomato plasmid (described by Biacchesi et al., 2010, mentioned above) previously digested with SpeI/SnaBI, so as to obtain the final construct pSHV-SPg-DIII-TMg.

(12) Recombinant Novirhabdovirus Production:

(13) Three expression plasmids comprising respectively the genes encoding the nucleoprotein N, the phosphoprotein P, and the RNA-dependent RNA polymerase L of VHSV were constructed, as described by Biacchesi et al. (2010, publication mentioned above). These constructs are respectively called pT7-N, pT7-P and pT7-L.

(14) The pVHSV plasmid or the pVHSV-SPg-DIII-TMg plasmid, at a dose of 1 g, and the 3 pT7-N, pT7-P and pT7-L plasmids, at respective doses of 0.25 g. 0.2 g and 0.2 g, are introduced, by transfection in the presence of lipofectamine (Gibco-BRL), into EPC cells previously infected with a recombinant vaccinia virus expressing the T7 phage RNA polymerase (vTF7-3, Fuerst et al., Proc. Natl. Acad. Sci. USA, 92, 4477-4481, 1986).

(15) After transfection, the cells are incubated for 5 hours at 37 C. and then washed with MEM culture medium (serum free) and incubated for 7 days at 14 C. in MEM culture medium containing 2% of foetal calf serum. The cells and the supernatant are frozen/thawed, and clarified by centrifugation for 10 minutes at 10 000 revolutions/min. The supernatant is used at the 1/10 dilution to infect a layer of EPC cells (Epithelioma Papulosum Cyprini cells, derived from carp epithelial cells). The viruses are produced in the supernatant 3-4 days post-infection.

(16) The viruses obtained are respectively called rVHSV, in the case of the virus possessing the genome of the wild-type virus, and rVHSV-SPg-DIII-TMg in the case of the virus containing the gene encoding the fusion protein.

(17) Viral stocks of each of the viruses produced were formed by successive passages in cell culture of the supernatant taken 7 days after transfection (PO supernatant) on EPC cells. The cells are infected at a multiplicity of infection (MOI) of 1. After 3 passages, the supernatants were removed at various times post-infection, and titrated by limiting dilution in order to establish a growth curve.

(18) The growth curves established for the rVHSV and rVHSV-SPg-DIII-TMg viruses show that the rVHSV-SPg-DIII-TMg virus multiplies in cell culture as well as the rVHSV virus.

(19) The expression of domain III of the West Nile Virus glycoprotein E in the cells infected with rVHSV-SPg-DIII-TMg was verified at 2 days post-infection, by means of an indirect immunofluorescence test using an anti-DIII monoclonal antibody, on live infected cells or infected cells fixed with alcohol/acetone.

EXAMPLE 2

Exression of Domain III of the West Nile Virus Glycoprotein E at the Surface of the Novirhabdovirus

(20) EPC cells were infected as described in example 1 above, with the rVHSV virus or with the rVHSV-SPg-DIII-TMg virus.

(21) Three days after the infection, the culture supernatant was recovered, and the viruses were purified on a sucrose gradient using this supernatant.

(22) The viral proteins were separated by SDS-PAGE electrophoresis and visualized after staining with Coomassie blue or after Western blot transfer and incubation with a monoclonal antibody directed against domain III of the WNV glycoprotein E.

(23) The results are shown in FIG. 1.

(24) Legend of FIG. 1:

(25) A: Left-hand panel: SDS PAGE gel, stained with Coomassie blue, of the rVHSV and rVHSV-SPg-DIII-TMg viruses purified on a sucrose gradient before transfer for Western blot; right-hand panel: Western blot transfer with the monoclonal antibody directed against domain III of the WNV glycoprotein E; B: SDS PAGE Gels of purified virus stained with Coomassie blue. M: Molecular weight marker, 1: rVHSV 2: rVHSV-SPg-DIII-TMg.

(26) These results show that domain III of the WNV glycoprotein E is strongly expressed in the particles of the rVHSV-SPg-DIII-TMg virus.

(27) The purified rVHSV-SPg-DIII-TMg viral particles were also observed by electron microscopy after immunolabeling with colloidal gold using either an antibody directed against domain III of the WNV glycoprotein E, or an antibody directed against the VHSV glycoprotein G.

(28) The results are shown in FIG. 2. The black dots at the surface of the viral particles indicate the presence of domain III of the WNV glycoprotein E (FIG. 2A), and also of the VHSV glycoprotein G (FIG. 2B) at the surface of the rVHSV-SPg-DIII-TMg viruses.

(29) This shows that the antigen of interest is very strongly and very effectively expressed at the surface of the viral particles.