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Definitions

• the present invention relates to novel vaccine vectors derived from Leporipoxviruses and in particular from the myxomatosis virus.
• the genus Leporipoxvirus belongs to the family of Poxviridae. This genus, whose type species is the myxomatosis virus (VM), includes poxviruses specific to Leporidae. Myxomatosis virus, also known as Myxoma virus, is the causative agent of myxomatosis, a major infectious disease in European rabbits, and endemic in Europe. Poxviridae have an intracytoplasmic replicative cycle in which different viral forms are successively observed: IMV (mature intracellular virions), EVI (enveloped intracellular virions) and EEV (enveloped extracellular virions).
• IMV mature intracellular virions
• EVI enveloped intracellular virions
• EEV enveloped extracellular virions
• IMVs are formed in the cytoplasm of infected cells, and have a single membrane. They represent the vast majority of viral progeny and are released during lysis of the host cell. A small part of IMV acquires an additional double envelope derived from the Golgi apparatus or endosomes, constituting the IEV viral forms. IEVs migrate to the cell surface and fuse with the cell membrane. They lose their outermost envelope and release EEV into the extracellular environment.
• Recombinant poxviruses have been shown to be effective as vaccination vectors by their ability to elicit an immune response against foreign antigens. Their exclusively cytoplasmic replicative cycle avoids possible complications resulting from the integration of viral DNA into the genome of the infected cell. They are able to integrate large fragments of foreign DNA (more than 25 kbp) by homologous recombination. They are also able to correctly express the proteins of interest and to induce a good immune response. Leporipoxviruses also have the advantage of having a very narrow host spectrum in vivo which gives them a significant safety of use.
• transgenes are inserted in intergenic zones, or in zones containing genes that are not essential for viral replication.
• the majority of the insertion sites that have been identified are located near the genomic endings, where non-essential genes are located in the viral cycle (MACKETT et al., J Virol, 49, 857-864, 1984, PERKUS et al. al., Virology, 180, 406-410, 1991).
• PCT Application WO 02072852 describes the use of recombinant Leporipoxviruses for obtaining vaccine vectors that can be used in animals other than Leporidae, especially in birds, felines, canines, pigs, sheep, cattle, horses, as well as in humans. It is indicated that in these vectors, the transgene expressing the vaccine antigen of interest may be inserted at the level of the gene coding for thymidine kinase, or at one of the genes coding for the virulence factors MI1L, SERP- 1, -2 and -3 or MGF.
• poxviruses With regard to poxviruses, the only vectors of this type described to date have been obtained from recombinant vaccinia virus in which the vaccine antigens of interest are fused to the B5R envelope protein of EEV virions (KATZ and MOSS, AIDS Res Hum Retroviruses, 13, 1497-1500, 1997, BARCHICHAT and KATZ, Virus Res, 90, 243-251, 2002, KWAK et al., Virology, 322, 337-348, 2004).
• KATZ and MOSS AIDS Res Hum Retroviruses, 13, 1497-1500, 1997, BARCHICHAT and KATZ, Virus Res, 90, 243-251, 2002, KWAK et al., Virology, 322, 337-348, 2004.
• the inventors have now identified two Leporipoxvirus envelope proteins to which an antigen of interest can be fused, without this fusion altering the functions of these proteins in viral morphogenesis.
• M071L and M022L proteins called M071L and M022L in the myxomatosis virus (CAMERON et al., Virology, 264, 298-31, 1999).
• M071L GenBank: NPJD51785, GI: 9633707
• M071L is a 324 aa protein encoded by a late gene; it has a molecular weight of about 36.8 kDa; it is integrated into the IMV membrane of which it constitutes the immunodominant antigenic component.
• gpO71L NP_051960, GI: 9633880
• H3L protein which is involved in the maturation of virus particles (DA Fonseca et al., J. Virol., 74, 7518-7528, 2000).
• M071L has 90% identity and 94% sequence similarity with gpO71L and 35% identity and 61% similarity with H3L.
• sequence of the M071L protein of the myxomatosis virus (identical to the GenBank sequence NP__051785) is represented in the attached sequence listing under the number SEQ ID NO: 1; the sequence of the M022L protein of the myxomatosis virus (identical to the GenBank NP_051736 sequence) is represented in the attached sequence listing under the number SEQ ID NO: 2.
• the inventors have found that when a sequence coding for an exogenous protein is fused to the sequence coding for M071L, so as to obtain a chimeric protein comprising said exogenous protein fused at the C-terminal end of M071L, this chimeric protein was expressed comparable to the native M071L protein.
• this C-terminal fusion does not interfere with the function of the M071L protein in viral morphogenesis, and in modified Myxoma virus expressing the chimeric protein, it is localized as the native M071L protein, at the level of the IMV membrane.
• the exogenous protein is presented on the surface of IMV and can also induce an immune response.
• the inventors have also found that, on the other hand, when the exogenous protein was fused to the N-terminus of M071L, it was not possible to obtain a recombinant virus expressing the fused protein, suggesting a lethal modification of the protein. M071L.
• the inventors have found that the fusion of an exogenous protein at its C-terminal end produced a non-functional chimeric protein, and resulted in the absence of formation of viral particles.
• the exogenous protein is fused to the N-terminal end of M022L, the chimeric protein obtained preserves, in modified Myxoma virus expressing this protein, the function and the localization of the native M022L protein, and allows the presentation of the exogenous protein on the surface of EEV and the induction of a humoral and cellular immune response directed against this exogenous protein.
• modified Myxoma viruses expressing the chimeric proteins described above although they have a morphology and a capacity for replication comparable to those of the wild strain from which they originate, have a considerably reduced pathogenicity. .
• the subject of the present invention is therefore a modified Leporipoxvirus characterized in that it expresses a chimeric coat protein selected from: a) a chimeric coat protein comprising:
• a region A consisting of a protein having at least 85%, preferably at least 90%, and most preferably at least 95% identity, or at least 90%, preferably at least 92%, and most preferably at least 96% sequence similarity with the M071L protein defined by the sequence SEQ ID NO: 1; a region B constituted by an exogenous protein of interest, said region B being fused to the C-terminal end of said region A; b) a chimeric coat protein comprising:
• region A consisting of a protein having at least 85%, preferably at least 90%, and most preferably at least 95% identity, or at least 90%, preferably at least 92%, and most preferably at least 96% sequence similarity with the M022L protein defined by the sequence SEQ ID NO: 2;
• region B consisting of an exogenous protein of interest, said region B being fused to the N-terminus of said region A directly, or possibly via a peptide linker of a few amino acids.
• the percentages of identity and similarity referred to herein are calculated using the BLASTP program, using the default parameters, on a comparison window consisting of the complete sequence of the protein.
• the exogenous protein of interest is expressed on the surface of IMV in the case of Leporipoxviruses expressing a chimeric protein as defined in a) above, and on the surface of EEV in the case of Leporipoxviruses expressing a chimeric protein as defined in (b) above.
• a modified Leporipoxvirus expresses a chimeric protein as defined in a) above, and a chimeric protein as defined in b) above. These two chimeric proteins may contain the same protein of interest or two different proteins of interest.
• the present invention also encompasses any modified Leporipoxvirus expressing a chimeric protein comprising a Leporipoxvirus IMV membrane protein fused with an exogenous protein of interest.
• the exogenous protein of interest can be any protein against which it is desired to induce a response.
• humoral and / or cellular immune system Its size can vary from a few amino acids to a few hundred amino acids.
• It may be in particular an antigenic protein derived from a viral, bacterial or parasitic pathogen, or an antigenic fragment of said protein, or a recombinant protein combining various antigenic fragments, originating from the same antigen or different antigens.
• Modified Leporipoxviruses according to the invention can be obtained from wild Leporipoxviruses, or attenuated Leporipoxviruses. These may include myxomatosis virus or rabbit fibroid virus.
• strains of myxomatosis virus from which can be constructed modified viruses according to the invention include: strain LA ⁇ SANNE (ATCC VR-115), strain Moses (ATCC VR-II 6), the strains described in Application FR 2736358, and in particular strains LEON 162 (L162): CNCM 1-1595; HUNGARIAN (HG): CNCM 1-1593; FINISTERE (F9): CNCM 1-1596; R 801: CNCM 1-1598; TOULOUSE 1 (Tl): CNCM 1-1592, as well as the attenuated strain SG 33 (CNCM I-1594).
• strain LA ⁇ SANNE ATCC VR-115
• strain Moses ATCC VR-II 6
• the strains described in Application FR 2736358 and in particular strains LEON 162 (L162): CNCM 1-1595; HUNGARIAN (HG): CNCM 1-1593; FINISTERE (F9): CNCM 1-1596; R 801: CNCM 1-1598; TOULOUSE 1 (T
• strains of rabbit fibroid virus from which can be constructed modified viruses according to the invention include: the strain
• the modified Leporipoxviruses according to the invention can be produced by conventional techniques used for the production of recombinant poxviruses, which implement a homologous recombination between the genome of a poxvirus in which it is desired to insert a sequence of interest, and a transfer vector, containing the sequence that one wishes to insert, framed by sequences of the genome of the poxvirus flanking the site where the insertion must take place. Homologous recombination occurs in host cells infected with the selected poxvirus and transfected with the transfer vector.
• the present invention also relates to tools for the production of modified Leporipoxviruses in accordance with the invention, and in particular: a polynucleotide encoding a chimeric envelope protein, as defined above, an expression cassette containing said polynucleotide under the control of a suitable promoter, and a recombinant vector containing said expression cassette.
• the promoter used in the expression cassettes in accordance with the invention is that of the gene coding for the envelope protein to which the exogenous protein of interest is fused.
• These expression cassettes may also include a marker gene facilitating the selection of recombinant viruses. This may be, for example, a gene conferring a selective advantage on recombinant viruses, such as an antibiotic resistance gene, such as mycophenolic acid or geneticin, or a gene allowing the visualization of the plaques. lyse containing the recombinant virus, such as the LacZ gene, the gus gene, or a gene encoding a fluorescent protein.
• Host cells that can be used for producing the modified compounds according to the invention are those usually used for the production of Leporipoxvirus; these include normal or tumorous rabbit cells, normal or tumor monkey cells, avian, normal or tumor cells, or human tumor cells.
• the modified Leporipoxviruses in accordance with the invention can be used to obtain vaccines, not only in leporids, but also in other animal species, including birds and mammals, and in particular sheep, cattle, swine, equines, canines, felines, and primates, especially humans.
• Said vaccines can be formulated for parenteral use, for example intradermally, intramuscularly, or subcutaneously, orally, or mucosally, for example intranasal.
• the amount of virus modified per vaccine dose is chosen so as to allow a level of expression of the exogenous protein of sufficient interest to induce an immune response against this protein. It may depend in particular on the nature of said exogenous protein, the species and the age of the subject to be vaccinated, the type of immune response (cellular or humoral) that it is desired to favor. Usually, it will be between 10 3 and 10 9 pfu (plaque forming units) per vaccine dose.
• Three recombinant viruses derived from the strain strain Jardin 1 were constructed. Two produce the M022L protein fused with either GFP (Green Fluorescent Protein) or with the ectodomain of the M2 protein (M2e) (GenBank: AY340089) of the avian influenza A virus in its amino-terminal part (Tl-N-gfp- M022L and Tl-N-M2e-M022L). The third product protein M071L fused with M2e in its carboxy-terminal part (Tl-C-M2e-M071L). Material and methods :
• the myxomatous viruses of strain Jardin 1 (Tl), as well as the modified viruses are propagated on RK13 cells in medium consisting of DMEM (Dubelcco's Minimal Eagle Medium, Gibco®) supplemented with penicillin at 100 IU / ml final and streptomycin at 100 ⁇ g / ml, supplemented with 2% fetal calf serum (FCS).
• DMEM Dubelcco's Minimal Eagle Medium, Gibco®
• FCS fetal calf serum
• the selection of recombinant viruses gpt + was carried out in the selection medium MX-HAT consisting of DMEM 5% SVF supplemented with mycophenolic acid 25 ⁇ g / ml (Sigma®), with Xanthine 250 ⁇ g / ml (Sigma®), with hypoxanthine 15 ⁇ g / ml , Aminopterin 0.176 ⁇ g / mL and Thymidine 4 ⁇ g / mL.
• MX-HAT consisting of DMEM 5% SVF supplemented with mycophenolic acid 25 ⁇ g / ml (Sigma®), with Xanthine 250 ⁇ g / ml (Sigma®), with hypoxanthine 15 ⁇ g / ml , Aminopterin 0.176 ⁇ g / mL and Thymidine 4 ⁇ g / mL.
• the M022L gene was amplified by PCR using primers F-GFPM022L and R-GFP-M022L and cloned in amino-terminal fusion with GFP (Green Fluorescent Protein) in the plasmid pEGFP-F (Clontech) resulting in plasmid pGFPM022L.
• the GFP-M022L fragment was removed from the pGFP-M022L by double NheI and BamHI digestion, followed by treatment with the Klenow fragment of DNA polymerase I (Invitrogen) in order to produce ends on both sides. francs.
• the plasmid pCR2-promM022L was opened with EcoRV and then dephosphorylated with "Shrimp Alkaline Phosphatase" (Promega).
• the GFP-M022L fragment was cloned into pCR2promM022L resulting in plasmid pPROM-GFP-M022L.
• Simple underlining indicates restriction sites
• wave underlining indicates the zone of hybridization of the long primers between them.
• the sequence of the gpt selection gene under the control of the early-late mixed promoter p7 / 5 was obtained by PCR with Taq Expand High Fidelity (Roche) on the plasmid pRB-gpt with the primers F-gpt-Xho I and R -gpt-EcoR I.
• the PCR product ( ⁇ 750 bp) was inserted directly into the pGEM®-T commercial vector (Promega) to give the pGEMT-gpt vector.
• the left recombination box was obtained by PCR amplification of the 3 'sequence of the M070R gene ( ⁇ 500 bp) of the Tl strain of the myxomatous virus with the primers F-M070RNco I and R-M070R-EcoR I.
• Straight recombination was obtained by PCR amplification of the 5 'sequence of the M071L gene ( ⁇ 450 bp) of the Tl strain of myxomatous virus with the primers F-M071L-Nde I and R-M071L-Pst I.
• the sequence corresponding to the ectodomain of M2 was obtained and amplified by PCR ( ⁇ 120 bp) using two pairs of primers: a pair of long overlapping primers, T5-promEcoR I and T6-Nco I, and a pair of short primers corresponding to the 5 'end of each long primer, Pl-EcoR I and P2-Nco I.
• the PCR products digested with the appropriate restriction enzymes were sequentially inserted into the pGEMT-gpt vector digested with these same enzymes. .
• Figure 2A The restriction sites used are indicated to the left of the white arrows. On the right are indicated genes or gene fragments inserted in the previous vector thanks to the same enzymes
• the left recombination box was obtained by PCR amplification of the 3 'sequence of the M022L gene ( ⁇ 500 bp) of the Tl strain of the myxomatous virus with the primers F-M022LNco I and R-M022L-Apa I.
• Straight recombination was obtained by PCR amplification of the 5 'sequence of the M023R gene ( ⁇ 400 bp) of the Tl strain of the myxomatous virus with the primers F-M023R-Xho I and R-M023R-Pst I.
• the sequence corresponding to the ectodomain of M2 was obtained and amplified by PCR ( ⁇ 120 bp) using two pairs of primers: a pair of long overlapping primers, T5-prom-M022L and T ⁇ -Nco I, and a pair of primers 5 'end of each long primer, Pl-promM022L and P2-Nco I.
• the PCR products digested with the appropriate restriction enzymes were inserted sequential in the pGEMT-gpt vector digested with the same enzymes.
• the RK13 cells were infected 48 h after culturing with T1 virus at a MOI of 0.05. After 2 hours of adsorption at 37 ° C., the inoculum is removed and the cells rinsed 3 times with OptiMEM. The cells thus infected are then transfected with the mixture of 10 ⁇ g of plasmid DNA (each of the abovementioned transfer plasmids) and 20 ⁇ g of lipofectamine (Invitrogen) deposited on these cells for 5 hours. The cells are then rinsed 3 times and put back into DMEM medium plus PS, 2% FCS in culture at 37 ° C., 5% CO 2 until a cytopathic effect of about 80-90% is obtained. .
• plasmid DNA each of the abovementioned transfer plasmids
• lipofectamine Invitrogen
• recombination mixtures consisting of the cell lysate containing wild-type virus and recombinant virus undergo three cycles of freezing / thawing.
• ten BP100 each containing 9.10 6 RK13 cells of 48h are infected with the recombination mixture.
• the liquid medium is replaced by solid medium (MEME + PS, 1% low melting point agarose, 25 mM final hepes, 3% final NaHCO 3 , 2% FCS).
• solid medium MEME + PS, 1% low melting point agarose, 25 mM final hepes, 3% final NaHCO 3 , 2% FCS.
• the lysis plaques containing the recombinant viruses are identified after observation by fluorescence microscopy and isolated.
• the RK13 cells are pre-treated for 5h30 with the selection medium before the application of the recombination mixtures, then the inoculums are replaced by an appropriate volume of selection medium, before the application of the medium.
• solid supplied with MPA, Xanthine and HAT.
• the purification of the recombinant viruses is then carried out by several passages / isolations in limiting dilution: a mother of recombinant virus is isolated, undergoes three cycles of freezing / thawing and spread at different dilutions to isolate a daughter beach which will undergo again this same protocol.
• the viruses thus obtained are then amplified and titrated. Results:
• the Tl-N-gfp-M022L, Tl-N-M2e-M022L and Tl-C-M2e-MO71L viruses form lysis plaques in RK13 cells comparable to those formed by the wild-type T1 virus. indicates that these fusions do not alter viability or viral proliferation in vitro.
• the observation of lysis plaques obtained after infection with Tl-N-gfp-M022L virus under a fluorescence microscope shows, on the other hand, that the fusion protein is expressed in all ranges.
• the recombinant viruses obtained in Example 1 were tested in vivo (rabbits, mice and sheep), on the one hand to explore the pathogenicity for the target species of the VM (rabbits) and on the other hand to verify the implementation in place of an immune response against transgenic products in the target species and non-target species of the VM.
• mice of the BaIbC type were infected intraperitoneally with 5.10 pfu of Tl-N-M2e-M022L or Tl-C-M2e-M071L virus at the rate of two injections at 21-day intervals .
• a batch of two uninfected mice served as a control.
• the mice were monitored (presence of lesions, general status assessment) regularly up to 42 days post-inoculation, then sacrificed and bled to extract the sera.
• the presence of anti-GFP antibodies in the sera of rabbits inoculated with the virus Tl-NgfpM022L was tested by immunofluorescence.
• the RK13 cells were transfected with 2 ⁇ g of plasmid pEGFP-F (clontech) using 3 ⁇ l of Fugen ⁇ (Roche). After 48 hours of culturing at 37 ° C., 5% CO2, the cells are fixed with 4% paraformaldheddy in PBS. A detection of GFP by immunofluorescence is then performed. The cells are permeabilized with 0.1% Triton X100 in PBS. After rinsing, 300 ⁇ l of the differently diluted sera (1/100, 1/300) in PBS Tween are deposited on the cells.
• the presence of anti-M2e antibodies in rabbit and mouse sera inoculated with Tl-N-M2e-M022L or Tl-C-M2e-M071L viruses was tested by immunofluorescence.
• the RK13 cells were infected with avian H7N1 influenza virus (MOI of 0.3) or with influenza virus of the human H1N1 type (MOI of 0.1) and then fixed at 8 h.
• the detection of the M2 protein follows the protocol mentioned above, the sera of rabbits and mice being diluted respectively to 1 / 200th and 1 / 100th.
• the anti-mouse IgG and anti-IgG secondary antibodies of FITC-coupled rabbits were used at the 1: 200 dilution.
• the cellular response against the product of the fused transgene was explored in both sheep inoculated with Tl-N-gfp-M022L virus.
• the principle is to measure by lymphoproliferation flow cytometry after restimulation by gfp PBMCs isolated and cultivated in the presence of a fluorochrome (CFSE).
• CFSE fluorochrome
• the blood is collected on EDTA tube, then diluted (1: 2) with PBS.
• the mixture is loaded onto a density gradient (FicollPaque plus, Amersham) and then centrifuged at 800g for 20 minutes.
• the PBMCs peripheral blood mononuclear cells
• the PBMCs are cultured at 37 ° C. under a 5% CO 2 atmosphere.
• the RPMIc culture medium consists of RPMI 1640 Glutamax, 25 mM Hepes (Gibco-BRL), to which is added 10% of fetal calf serum (FCS), 1% of sodium pyruvate (Gibco-BRL), 1% non-essential amino acids (Gibco-BRL), 1% ⁇ -mercaptoethanol (Gibco-BRL),
• the PBMCs are labeled with CFSE (Molecular Probes).
• the cells are taken up in PBS at a concentration of 10 7 cells / ml.
• a 2X solution (2.5 ⁇ M) of CFSE in PBS is added volume by volume to the cells. Everything is incubated 10 minutes away from light.
• SVF is added volume to volume to neutralize the marking.
• the mixture is incubated for 3 minutes.
• the tube is then supplemented with RPMIc and centrifuged at 300 g for 10 minutes at 4 ° C. The washing is repeated in a new tube.
• the cell concentration is adjusted to 6.10 6 cells / ml with RPMIc.
• the cells are cultured in P24 plates at a rate of 3.10 6 cells per well under 1M.
• rGFP (Clonetech) protein (10 to 0.1 ⁇ g)
• the cells are thus cultured at 37 ° C., under a 5% CO 2 atmosphere. After 6 days of incubation, the cells are harvested, washed in FACS buffer (PBS, 2.5 mM EDTA, 0.5% BSA) and labeled with anti-CD2 antibodies coupled to A647 and anti-CD4 coupled to phycoerythrin (Serotec). . Viability is determined by the addition of propidium iodide at 1 ⁇ g / ml just prior to acquisition (BD Biosciences Pharmingen). The acquisition is performed with a FACScalibur (Becton Dickinson). The analysis is performed with the Flowjo software. Results:
• the recombinant MVs are non-pathogenic:
• the sera of rabbits infected with Tl-N-gfp-M022L virus were taken at 0, 10, 20 and 30 days post-infection.
• the presence of anti-GFP antibodies was tested in immunofluorescence on cells transfected with the plasmid pEGFP-F allowing transient expression of the GFP protein in eukaryotic cells.
• the GFP emitting in the green the detection was carried out thanks to a red fluorochrome, the TRITC. It is thus well check the presence of anti-GFP antibodies by colocalization of natural GFP fluorescence and immunostaining ⁇ 1.
• the results obtained with the serum of a rabbit taken at day 30 post-infection with the Tl-N-gfp-M022L virus are shown in Figure 3.
• the left panels (A, C and E) show the location of the GFP .
• the panels on the right (B, D, and F) show the labeling obtained with the aid of various sera: polyclonal serum anti-myxomatous virus (B), serum rabbit 40451 taken at J30 postinfection diluted 1/100 ( D), and this same serum diluted to 1/300 (F).
• FIG. 4A Serums of rabbits infected with OJ (Tl-N-M2e-M022L in 1 and Tl-C-M2e-M071L in 2) and in J30 (Tl-N-M2e-M022L in 3 and Tl-C-M2e ⁇ M071L in 4)
• mice were immunized with either of the M2e-expressing viruses. The sera were taken 42 days after inoculation and tested by immunofluorescence.
• FIG. 4B serum of uninfected mice (1) and infected with Tl-N-M2e-M022L virus (2) or Tl-C-M2e-M071L virus (3)
• Panel A control ewe: in 1, cells restimulated with culture medium; in 2, cells restimulated with 3 ⁇ g of GFP
• Panel B ewes inoculated with Tl-Ngfp-M022L: in 1, 2 and 3, restimulated cells with respectively 10, 3 and 1 ⁇ g of GFP; at 4, cells restimulated with VM, and in 5 cells restimulated with culture medium.
• the abscissa axis measures the intensity of the green fluorescence (CFSE) and the ordinate axis the intensity of the red fluorescence (CD4 labeling).
• CFSE green fluorescence
• CD4 labeling the intensity of the red fluorescence
• Leporipoxviruses thus constitute non-replicative vaccine vectors, particularly interesting for use in species other than leporids.

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