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  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20111—Lyssavirus, e.g. rabies virus
  • C12N2760/20141—Use of virus, viral particle or viral elements as a vector
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  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20111—Lyssavirus, e.g. rabies virus
  • C12N2760/20161—Methods of inactivation or attenuation
  • C12N2760/20162—Methods of inactivation or attenuation by genetic engineering

Definitions

• VACCINES The present invention relates to the use of novirhabdovirus for the production of vaccines.
• Novirhabdoviruses are negative RNA viruses of the Rhabdoviridae family.
• Novirhabdovirus includes various pathogenic species of aquatic animals, among which there are in particular two pathogenic species of fish, and mainly salmonids: the infectious hematopoietic necrosis virus (VNHI) and the viral haemorrhagic septicemia virus (VSHV).
• VNHI infectious hematopoietic necrosis virus
• VSHV viral haemorrhagic septicemia virus
• novirhabdoviruses The genome structure of novirhabdoviruses is similar to that of mammalian rhabdoviruses, but differs from the latter by the presence of an additional gene, coding for a non-structural protein, called NV protein (for “non-virion”). , whose function remains unknown at present.
• NV protein for “non-virion”.
• the genome of novirhabdoviruses comprises six genes, the organization of which can be schematized as follows:
• N represents the gene coding for the nucleoprotein associated with the viral RNA
• P represents the gene coding for the phosphoprotein associated with the viral polymerase
• M represents the gene coding for the matrix protein
• G represents the gene coding for the glycoprotein envelope G
• NV represents the gene coding for the protein
• NV RNA dependent RNA polymerase
• VNHI and VSHV cause significant damage in fish farms, particularly in fry.
• Vaccines capable of conferring protective immunity against these viruses have been proposed. They are based either on the use of killed or inactivated viruses, or on the use of glycoprotein G, which can induce the synthesis of neutralizing antibodies capable of conferring protective immunity. It has thus been proposed to use vaccines based on recombinant glycoprotein G, or naked DNA vaccines carrying the gene coding for glycoprotein G. However, these vaccines must be administered by injection, and it is very difficult in practice to use this method of administration on thousands of young fry.
• the present invention more particularly relates to the use of novirhabdoviruses in which the gene for the NV protein is inactivated for obtaining medicaments.
• novirhabdovirus in which the NV gene is inactivated means any novirhabdovirus carrying a mutation which results in the absence of production of the NV protein, or the production of a non-functional NV protein, without abolishing the production or function of the other viral proteins.
• the gene of the NV protein can for example be inactivated by the deletion of at least a part of said gene or, advantageously of all of it, or possibly by nonsense mutation, shift of the reading frame, etc. It is generally preferred to carry out inactivation by deletion of all or a significant portion of the gene, which makes it possible to avoid any risk of reversion to the wild virus.
• the subject of the present invention is any recombinant novirhabdovirus whose gene of the NV protein is inactivated, and which contains in its genome at least one heterologous gene coding for a protein of therapeutic interest.
• said protein of therapeutic interest is chosen from vaccine antigens of organisms, and in particular of viruses, pathogenic for fish, and in particular from capsid or envelope glycoproteins fish pathogens.
• vaccine antigens of organisms and in particular of viruses, pathogenic for fish, and in particular from capsid or envelope glycoproteins fish pathogens.
• glycoprotein E2 and the glycoprotein El of the salmonid sleeping sickness virus VILLOING et al., J. Virol. 74, 173-183, 2000, and Dis. Aquat. Org ., 40, 19-27, 2000
• the glycoprotein G of a rhabdovirus in particular the glycoprotein G of a novirhabdovirus of a species other than that from which the recombinant novirhabdovirus according to the invention is derived.
• said heterologous gene is inserted at the location of the inactivated gene of the NV protein.
• a recombinant novirhabdovirus in accordance with the invention is chosen from:
• Recombinant novirhabdoviruses in accordance with the invention can be obtained by methods known per se, in particular by the method described in US Pat. No. 6,033,886 for the preparation of rhabdoviruses, which consists in co-transfecting a host cell expressing an RNA polymerase with the complementary DNA (cDNA) of the viral genome, and DNA molecules coding for the viral proteins N, P, and L.
• cDNA complementary DNA
• the cDNA of the genome of a recombinant novirhabdovirus according to the invention is obtained from the cDNA of the corresponding wild virus, by introduction of one or more mutations inactivating the NV gene, and insertion of the heterologous gene of interest. These modifications can be made by conventional techniques of genetic engineering.
• the present invention also encompasses the cDNA of the genome of a recombinant novirhabdovirus according to the invention, as well as any recombinant vector comprising said cDNA.
• a subject of the present invention is also medicaments comprising a novirhabdovirus in which the NV gene is inactivated, and in particular vaccines comprising a recombinant novirhabdovirus in accordance with the invention.
• the vaccines according to the invention can be used in fish, and in particular salmonids, such as farmed trout and salmon.
• recombinant novirhabdoviruses in accordance with the invention which simultaneously express the glycoprotein G of the original novirhabdovirus, and at least one vaccine antigen from another pathogenic organism, makes it possible to obtain multivalent vaccines, capable of imparting a protection against at least two pathogens.
• a recombinant novirhabdovirus according to the invention which expresses, in addition to the glycoprotein G of the novirhabdovirus from which it is derived, the glycoprotein G of a novirhabdovirus of a different species makes it possible to confer protection with respect to the two novirhabdovirus species concerned.
• the vaccines according to the invention have the advantage of also being able to be administered by balneation, that is to say by simple addition of the water vaccine from the breeding pond where the animals to be vaccinated are kept. This method of administration is therefore much simpler to implement than the injection method.
• it makes it possible to solve the problems posed by the vaccination of young fry, which are the most sensitive to diseases induced by novirhabdoviruses, but also to the stress linked to vaccination by injection.
• the absence of multiplication in fish of the recombinant novirhabdoviruses in accordance with the invention allows their use without risk of dissemination in the environment and of contamination of wild species.
• This plasmid contains the complete cDNA of the genome of the NHI virus, cloned downstream of the promoter of the RNA polymerase from phage T7 and upstream of a ribozyme sequence of the hepatitis virus virus and of the transcription terminator of the RNA.
• phage T7 polymerase in the vector pBlueScript SK (STRATAGENE).
• a recombinant NHI virus in which the NV gene has been excised and replaced by the reporter gene GFP (Green Fluorescent Protein) was constructed as described by BIACCHESI et al. (publication cited above).
• the plasmid obtained is called pINHV- ⁇ NV-GFP
• Recombinant NHI viruses in which the NV gene has been replaced by the NV gene or the G gene from SHV have been constructed as follows:
• NV and G genes were obtained from the 07-71 strain of SHV (SCHUETZE H. et al. Virus Genes., 19 (1),
• EPC epithelio a papulosum cyprinid
• VHS has a multiplicity of infection of 1. Twenty four hours post-infection, the cells are lysed and the total RNAs are extracted (RNAgents Total RNA Isolation System kit, PROMEGA). Approximately 1 ⁇ g of RNAs are denatured 10 min at room temperature in the presence of 40 mM of hydroxy-methyl-mercury.
• the denatured RNA is then placed in a reaction mixture containing 80 mM of ⁇ -mercaptoethanol, 10 mM of dithiothreitol, 1 mM of dNTP, 40 U RNasin (GIBCO-BRL), 25 p ol of the primer 3'G VHSV or 3'NV VHSV primer, and 200 U of SUPERSCRIPT II, in buffer IX (GIBCO-BRL).
• the mixture is maintained for 1 hour at 42 ° C.
• the cDNAs obtained are amplified by PCR with the 5 'G VHSV primer or the 5'NV VHSV primer, in the presence of TAQ polymerase (GIBCO BRL).
• the PCR products thus obtained are purified and subcloned in a TA-cloning plasmid (pGEM-T).
• the G or NV VHSV DNA fragments are excised by Spel and Smal digestion.
• Either of these fragments is inserted in place of the NV gene in the Eagl-Pstl fragment of the cDNA of the genome of the NHI virus, and the .Eaçrl-Pstl fragment thus modified introduced into the plasmid pINHV as indicated above.
• the plasmids obtained are respectively designated pINHV- ⁇ NV-NVSHV and pINHV- ⁇ NV-GSHV.
• Three expression plasmids comprising respectively the genes coding for nucleoprotein N, phosphoprotein P, and RNA polymerase RNA dependent L of NHI were constructed, as described by BIACCHESI et al. (publication cited above). These constructs are respectively called pT7-N, pT7-P and pT7-L.
• the plasmid pINHV, the plasmid pINHV- ⁇ NV-NVSHV or the plasmid pINHV- ⁇ NV-GSHV, and the 3 plasmids, pT7-N, pT7-P, pT7-L, at respective doses of 1 ⁇ g, 0.5 ⁇ g, 0.2 ⁇ g, 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 phage T7 RNA polymerase (vTF7-3, FUERST et al. Proc. Natl. Acad. Sci.
• the cells are incubated for 5 hours at 37 ° C. then washed with MEM culture medium (without serum) and incubated for 7 days at 14 ° C. in MEM culture medium containing 2% fetal calf serum.
• the cells and the supernatant are frozen / thawed, and clarified by centrifugation for 10 minutes at 10,000 rpm. The supernatant is used at a 1/10 dilution to infect a carpet of EPC cells. Viruses are produced in the supernatant 3-4 days post-infection.
• the structure of the viruses obtained having the entire genome of the wild virus (rNHI), or resulting from the deletion of the NV gene and its replacement by the gene coding for GFP (rNHI- ⁇ NV-GFP), the NV gene from VSHV (rNHI- ⁇ NV-NVSHV), or the G gene of VSHV (rNHI- ⁇ NV-GSHV), is shown schematically in Figure 1.
• rNHI- ⁇ NV-GFP the NV gene from VSHV
• rNHI- ⁇ NV-NVSHV the G gene of VSHV
• Viral stocks of each of the viruses produced were constituted by successive passages in cell culture (EPC) of the supernatant collected 7 days after transfection (PO supernatant). Cells were infected at a multiplicity of infection (MOI) of 1. After 3 passages, the supernatants were collected at various' time post-infection, and titrated by limiting dilution to establish a growth curve.
• EPC cell culture
• MOI multiplicity of infection
• EXAMPLE 2 PATHOGENICITY AND VACCINE PROPERTIES OF RECOMBINANT NOVIRHABDOVIRUSES
• NHI- ⁇ NV-NVSHV virus rNHI- ⁇ NV-NVSHV virus
• NHI- ⁇ NV-GSHV virus rNHI- ⁇ NV-GSHV virus
• viruses used are the following: wtIHNV: wild NHI virus, French strain 32/87; wtVHSV: wild SHV virus, strain 07-71; wtSVCV: Spring Carpemia Viremia Virus
• rlHNV wild type NHI virus produced as described in Example 1;
• G-SHV recombinant NHI virus carrying the G gene of the SHV virus in place of the G gene of NHI;
• G-SVCV recombinant NHI virus carrying the G gene of the SVCV virus in place of the G gene of NHI;
• G-IHN-SHV recombinant IHN virus carrying the G gene of the virus
• ⁇ NV-GFP recombinant NHI virus carrying the GFP gene in place of the NHI NV gene
• NVSHV recombinant NHI virus carrying the NV gene of the SHV virus in place of the NV gene of NHI.

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