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WO2013011179A2 - Vecteurs recombinants à base du virus ankara modifié (mva) avec délétion dans le gène c6l, utilisés comme vaccins contre le vih/sida et d'autres maladies - Google Patents

Vecteurs recombinants à base du virus ankara modifié (mva) avec délétion dans le gène c6l, utilisés comme vaccins contre le vih/sida et d'autres maladies Download PDF

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WO2013011179A2
WO2013011179A2 PCT/ES2012/070521 ES2012070521W WO2013011179A2 WO 2013011179 A2 WO2013011179 A2 WO 2013011179A2 ES 2012070521 W ES2012070521 W ES 2012070521W WO 2013011179 A2 WO2013011179 A2 WO 2013011179A2
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mva
hiv
cells
viral vector
recombinant mva
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WO2013011179A3 (fr
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Juan Francisco GARCÍA ARRIAZA
Carmen Elena Gómez Rodríguez
Mariano ESTEBAN RODRÍGUEZ
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Consejo Superior De Investigaciones Científicas (Csic)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention falls within the fields of molecular biology and biotechnology. Specifically it refers to recombinant viruses based on the modified Ankara virus (MVA) that express the gp120 and Gag-Pol-Nef antigens of the human immunodeficiency virus (HIV-1) of subtype B (MVA-B), on which The C6L vaccinia gene has been deleted, and they have been designed to be used as vaccines against HIV / AIDS and other diseases.
  • VMA modified Ankara virus
  • HAV-1 human immunodeficiency virus
  • MVP-B subtype B
  • MVA a highly attenuated strain of vaccinia
  • SHIV human immunodeficiency virus
  • an MVA recombinant that expresses HIV-1 antigens of subtype B, gp120 (SEQ ID No. 15 of PCT / ES2006 / 0701 14) has been constructed in our laboratory as a monomeric protein and Gag-Pol-Nef polyprotein ( SEQ ID No. 16 of PCT / ES2006 / 0701 14) (virus called MVA-B) (Patent: PCT / ES2006 / 0701 14, publication date: 1/2/2007.
  • MVA-B poxviral vectors that may increase the magnitude, amplitude, polyfunctionality and durability of immune responses against HIV-1 antigens are necessary and desirable. This is particularly relevant when a single immunogen is desirable for mass vaccination purposes to simplify immunization protocols and reduce manufacturing costs.
  • Poxviral vectors express numerous genes encoding immunomodulatory proteins that interfere with the host's anti-viral response (Alcam ⁇ , 2003. Nat. Rev. Immunol. 3: 36-50). Therefore, deletion in the MVA-B poxviral vector of vaccinia genes that are known or suggest that they may have an immunomodulatory function is a general strategy that can increase the immunogenicity of the vector against HIV-1 antigens.
  • C6L vaccinia gene which is present in the genome of MVA vaccinia strains (gene named MVA 019L, SEQ ID No: 1), Western Reserve (WR) (gene called VACV-WR_022, SEQ ID No: 3), and Copenhagen (gene named C6L, SEQ ID No: 5), but absent in the New York Vaccinia Virus (NYVAC) lineage.
  • WR Western Reserve
  • C6L SEQ ID No: 5
  • NYVAC New York Vaccinia Virus
  • C6L encodes a 157 amino acid protein with a predicted molecular weight of 18.2 kDa.
  • TLR Toll-like receptor
  • PloS Pathog. 4 e22; Graham et al., 2008. PloS Pathog. 4: e1000128; Harte et al., 2003. J Exp. Med. 197: 343-351; Kalverda et al., 2009. J. Mol. Biol. 385: 843-853; Oda et al., 2009. Structure 17: 1528-1537; Schroder et al., 2008 EMBO J. 27: 2147-2157; Stack et al., 2005. J. Exp. Med. 201: 1007-1018). Protein 06 is present, although at low levels, in mature intracellular vaccinia virions (IMV) (Chung et al., 2006. J. Virol.
  • IMV intracellular vaccinia virions
  • MVA-B AC6L a new vaccine candidate has been generated against HIV-1, called MVA-B AC6L, which contains a deletion in the MVA-B vector of the C6L vaccinia gene and which we demonstrate acts by inducing the production of type 1 interferon and activating in vivo T cell production of memory, which was not expected, but which represents an attractive alternative to increase the immunogenicity of MVA-based vaccine candidates.
  • the present invention represents a new vaccine candidate against HIV-1, called MVA-B AC6L, which contains a deletion in the MVA-B vector of the C6L vaccinia gene.
  • MVA-B AC6L replicates in cell cultures at the same level as the parental MVA-B virus, indicating that C6 is not essential for viral replication. Additionally, MVA-B AC6L induces innate immune responses by increasing IFN- ⁇ expression and IFN- ⁇ / ⁇ -induced genes (IFIT1 and IFIT2) in human THP-1 cells and monocyte-derived dendritic cells (moDCs), suggesting that C6 inhibits the IFN- ⁇ signaling pathway by blocking some unknown component involved in the induction of IFN- ⁇ .
  • IFIT1 and IFIT2 IFN- ⁇ / ⁇ -induced genes
  • MVA-B AC6L significantly increases the magnitude and polyfunctionality of the immune response of CD4 + and CD8 + memory T cells specific against HIV-1, which is mainly mediated by effector phenotype CD8 + T cells, in both immunization groups.
  • the response of specific CD4 + memory T cells against HIV-1, induced by MVA-B and MVA-B AC6L was preferentially specific against Env.
  • MVA-B induces specific CD8 + memory T cell immune responses against Env and Gag
  • MVA-B AC6L preferentially induces specific CD8 + memory T cell immune responses against Gag-Pol-Nef (GPN ).
  • MVA-B AC6L increases antibody levels against Env.
  • MVA-B AC6L represents a new vaccine candidate against HIV-1 that, not being obvious, has an immunological benefit by increasing IFN- ⁇ -dependent responses in human cells and increasing the humoral response and the magnitude and quality of the specific immune responses of T-cell memory against HIV-1 antigens.
  • the results obtained would allow to extend the scope of protection to improve the immunogenicity of new recombinant vectors based on MVA, which express other heterologous antigens (examples , malaria, leishmania, hepatitis C virus and prostate cancer) by deleting the C6L gene, in order to be used as vaccines against these diseases.
  • MVA-B AC6L The construction of MVA-B AC6L described in the present invention and the tests in which both its in vitro behavior, as well as the innate immune response induced in human cells, and its immunogenic capacity in mice against HIV-1 antigens are evaluated they are described in more detail with the help of the figures and the examples that appear hereinafter.
  • the present invention refers to a viral vector based on a recombinant MVA virus, characterized in that the nucleotide sequence encoding said vector comprises:
  • the mutation in the sequence SEQ ID No: 1 is a partial or total deletion. In an even more preferred embodiment, the mutation in the sequence SEQ ID No: 1 is a total deletion.
  • the nucleotide sequence encoding said vector comprises at least one nucleotide sequence encoding a heterologous antigen selected from the Next group: HIV antigen, malaria antigen, leishmaniasis antigen, hepatitis C virus antigen and prostate cancer antigen.
  • the nucleotide sequence encoding said vector comprises the nucleotide sequences encoding the following HIV antigens: gp120 antigens (SEQ ID No. 15 of PCT / ES2006 / 0701 14) and Gag-Pol-Nef (SEQ ID No. 16 of PCT / ES2006 / 0701 14) of HIV of subtype B, or of any other subtype, under the control of the early / late viral synthetic promoter inserted into the TK viral locus.
  • the present invention also refers to the method of manufacturing the viral vector based on a recombinant MVA virus defined above, characterized by comprising the following steps:
  • step b) purifying the viral vector based on a recombinant MVA virus defined above, obtained in step b).
  • the present invention also refers to the use of the viral vector based on a recombinant MVA virus defined above, as an immunogen to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer.
  • said vector is used as an immunogen to prevent or treat HIV disease.
  • the viral vector based on a recombinant MVA virus defined above, as part of an immunization protocol to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer, in which at least one dose of vaccination is administered to the individual.
  • said vector is administered to the individual a single dose of vaccination to:
  • said vector is used as part of an immunization protocol in which the individual is administered at least one dose of vaccination, to prevent or treat HIV disease.
  • said vector is used as part of an immunization protocol in which the individual is administered at least one vaccination dose or several vaccination doses of a combination of heterologous vectors (proteins, DNA, VLPs, attenuated viral vectors) to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer.
  • heterologous vectors proteins, DNA, VLPs, attenuated viral vectors
  • the present invention also refers to the use of the immunogenic composition or vaccine defined above, to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer.
  • said immunogenic composition or vaccine is for preventing or treating HIV.
  • the present invention refers to the following terms:
  • MVA recombinant virus or “MVA” refers interchangeably to: a highly attenuated strain of vaccinia, called Ankara modified virus (MVA), obtained after 576 serial passes in chicken embryonic cell cultures (CEF).
  • MVA Ankara modified virus
  • MVA-B recombinant virus or "MVA-B parental virus” or "MVA-B poxviral vector” refers interchangeably to: poxvirus based on the modified Ankara virus (MVA) expressing the gp120 and Gag antigens -Pol- Nef of human immunodeficiency virus (HIV-1) of subtype B (MVA-B).
  • MVA modified Ankara virus
  • HAV-1 human immunodeficiency virus
  • parental is used when comparing MVA-B with MVA-B AC6L, since the deletion of C6L is made on the genome of MVA-B.
  • viral vector or "recombinant viral vector” refers to: a modified virus that acts as a vehicle for introducing exogenous genetic material into a cell.
  • MVA-B AC6L vector refers to: an isolated MVA virus, characterized in that it expresses the gp120 and Gag-Pol-Nef antigens of the human immunodeficiency virus (HIV-1) of subtype B, and has a deletion in the polynucleotide sequence encoding an amino acid sequence homologous to that of the C6L gene (SEQ ID No: 1), and is presented for use as a pharmaceutical composition or medicament.
  • the deletion of the C6L gene in MVA-B includes positions 19068 to 19541 of the MVA genome.
  • C6L vaccinia gene refers to: a vaccinia gene, whose function was unknown prior to the presentation of this invention.
  • the C6L gene is present in the genome of MVA vaccinia strains (gene named MVA 019L, positions 19068 to 19541 of the MVA genome, SEQ ID No: 1), Western Reserve (WR) (gene named VACV-WR_022, positions 16401 to 16856 of WR, SEQ ID No: 3), and Copenhagen (gene named C6L, positions 19484 to 19939, SEQ ID No: 5), but absent in the New York Vaccinia Virus (NYVAC) lineage.
  • MVA 019L positions 19068 to 19541 of the MVA genome, SEQ ID No: 1
  • WR Western Reserve
  • VACV-WR_022 genes 16401 to 16856 of WR
  • Copenhagen gene named C6L, positions 19484 to 19939, SEQ ID No: 5
  • Plasmid or "transfer vector” refers to a circular fragment of extrachromosomal double stranded circular or linear DNA, which is found inside almost all bacteria, and they act and replicate independently to bacterial chromosomal DNA and can be transferred from one bacterium to another. They are used as vectors in genetic manipulation.
  • virus refers to: a microscopic infectious entity that can only multiply within the cells of other organisms. More specifically, it refers to viruses belonging to the Poxviridae family, which is a family of interrelated DNA viruses called poxviruses, infectious for vertebrate and invertebrate animals,
  • composition refers to: those substances that are present in a given sample and in certain quantities.
  • heterologous antigen refers to: a molecule (usually a protein or a polysaccharide), which triggers the formation of antibodies and can cause an immune response. It preferably refers to those antigens of a viral species other than vaccinia that are inserted into a viral vaccinia vector.
  • HIV antigens refers to: human immunodeficiency virus (HIV) antigens, which are expressed from the recombinant MVA viruses that contain them. It includes any HIV antigen encoded from the HIV genome.
  • HIV human immunodeficiency virus
  • malaria antigens refers to: malaria antigens, which are expressed from the recombinant MVA viruses that contain them. It includes any malaria antigen encoded from the genome of the pathogen of the genus Plasmodium, which generates the disease, the term “leishmaniasis antigens” refers to: leishmania antigens, which are expressed from the recombinant MVA viruses that They contain them. It includes any leishmania antigen encoded from the genome of the pathogen of the genus Leishmania, which generates the disease.
  • hepatitis C virus antigens refers to: hepatitis C virus antigens, which are expressed from the recombinant MVA viruses that contain them. Include any virus antigen of hepatitis C encoded from the hepatitis C virus genome.
  • prostate cancer antigens refers to: prostate cancer antigens (including PSCA and STEAP antigens), which are expressed as From the recombinant MVA viruses that contain them
  • the term “disease vaccine” refers to: an immunogenic or antigenic preparation or composition used to establish the immune system's response to a disease. They are prepared or combinations of immunogens or antigens that once inside the body cause the immune system response, through the production of antibodies, and generate immunological memory producing permanent or transient immunity.
  • vaccine against prostate cancer refers to: an antigen preparation used to establish the immune system's response to prostate cancer.
  • mutation refers to: an alteration or change in the genetic information of a living being and that, therefore, will produce a change of characteristics, which occurs suddenly and spontaneously, and that can be transmitted or inherited To the offspring.
  • the genetic unit capable of mutating is the gene that is the unit of hereditary information that is part of the DNA.
  • deletion refers to: a special type of mutation that involves the loss of a DNA fragment, which can range from the loss of a single nucleotide (point deletion) to the loss of large regions
  • mutation partial refers to: the loss of a DNA fragment of a gene, which does not cause the total loss thereof
  • total deletion refers to: the loss of a DNA fragment of a gene, which causes the total loss of it.
  • immunogen refers to: those antigens that elicit an immune response.
  • immuno protocol refers to: method used for the administration of an immunogenic agent or a vaccine to an organism to generate an immune response.
  • the term: "induce an immune response of CD4 + and CD8 + T cells from specific antigen memory” refers to: the ability of the vaccine administered in the immunization protocol to stimulate the host's immune response by generating CD4 + and CD8 + T cells that are capable of specifically recognizing the administered antigen, the term:” inducing the expression of IFN- ⁇ in innate immune cells “refers to: the ability of the administered vaccine to stimulate the production of IFN- ⁇ by innate immune cells, such as macrophages and dendritic cells.
  • Increase IFN- ⁇ -dependent responses in human cells refers to: the ability of the administered vaccine to further stimulate those immune responses that occur as a result of the production of IFN- ⁇ by cells Innate human immune systems, such as macrophages and dendritic cells.
  • the term: "increase the magnitude and quality of memory immune responses of specific T cells against antigens” refers to: the ability of the administered vaccine to further stimulate the number and proportion of memory T cells that are capable of specifically recognizing the administered antigen.
  • quality refers to the ability of memory T cells to be polyfunctional, that is, to secrete different cytokines at the same time, such as for example IFN- ⁇ , IL-2, or TNFa.
  • EM and TEMRA phenotypes refers to: two subpopulations of memory T cells, which are defined as a function of the expression of different surface markers such as CD44 and CD62L, and which are called EM ("Effector memory” or effector memory T cells. CD44 + / CD62L “ )) and TEMRA (" Effector memory terminally differentiated “or terminally differentiated effector memory T cells. CD44 " / CD62L " ).
  • the term "homology”, as used herein, refers to the similarity between two structures, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences. Two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure. If Particular of the invention MVA-B AC6L, although the deletion of the C6L vaccinia gene has been performed on MVA-B, is sufficient to allow a person skilled in the art to obtain new recombinant vectors based on MVA, which express other heterologous antigens (malaria , leishmania, hepatitis C virus and prostate cancer) by deleting the C6L gene, in order to be used as vaccines against these diseases.
  • heterologous antigens malaria , leishmania, hepatitis C virus and prostate cancer
  • the sequences of the Gag-Pol-Nef subtype B-type HIV (from isolate IIIB) and gp120 (from isolate BX08) sequences are indicated under control of the early / late viral synthetic promoter (sE / L) inserted into the TK viral locus (J2R gene) (adapted from Gómez et al., 2007. Vaccine 25: 2863-2885).
  • the molecular weight marker (1 Kb) with the corresponding sizes (in base pairs) is indicated on the left.
  • the Mock column represents uninfected cells.
  • C Expression of HIV-1 Bxo8gp120 and INBGPN proteins in infected DF-1 cells (2 PFU / cell) with MVA-B and MVA-B AC6L, at 24 hours post-infection.
  • D MVA-B and MVA-B AC6L viral growth kinetics in infected DF-1 cells (0.01 PFU / cell) at different times and titrated by a plaque immunostaining assay with anti-WR antibodies. The average of 3 independent experiments is represented.
  • DF-1 cells were infected with 5 PFU / MVA-B and MVA-B AC6L (A) or WR and MVA (B) cells in the presence or absence of AraC (B).
  • Cell extracts were collected at 24 hours post-infection (A) or at the indicated times (B) and analyzed by SDS-PAGE.
  • C6 vaccinia protein was detected by Western blotting using a polyclonal rabbit serum against C6.
  • C DF-1 cells were infected with WR, MVA, MVA-B or MVA-B AC6L for 18 hours. The location of C6 was analyzed by immunofluorescence and the cells were stained with DAPI (stained cell nucleus), purified rabbit polyclonal antibody anti-C6 and anti-14K.
  • MVA-B AC6L induces the production of IFN- ⁇ and IFN type I-induced genes in macrophages and dendritic cells.
  • Human macrophages THP-1 (A) and moDCs (B, C, D) were infected with MVA, MVA-B and MVA-B AC6L (5 PFU / cell in A, and 0.2, 0.02 or 0.002 PFU / cell in B, C and D).
  • the RNA was extracted and the levels of IFN- ⁇ messenger RNA, genes induced by type I IFN (IFIT1 and IFIT2), Chemokines and HPRT were analyzed by RT-PCR.
  • the results are expressed as the ratio between the levels of messenger RNA of the gene versus the levels of messenger RNA of HPRT. UA: arbitrary units.
  • the data represent the mean ⁇ standard deviation of the samples in duplicate.
  • C, D human moDCs were infected with 0.2, 0.02 and 0.002 PFU / cell of MVA, MVA-B and MVA-B AC6L. 6 hours later, cell-free supernatants were collected to quantify IFN- ⁇ concentration by ELISA (C) and IFN type I concentration using the HL1 16 cell line, which expresses luciferase under the control of a promoter induced by IFN type I (D).
  • the results are expressed by absorbance values at 450 nm (C), and in luciferase units (D). Data represent the mean ⁇ standard deviation of duplicates and are representative of 2 independent experiments.
  • the TLR signaling path is composed of several TLRs located on the cell surface or in intracellular compartments.
  • the activation of these TLRs leads to the recruitment of several adapter proteins and kinases and the subsequent activation of transcription factors (IRFs, NF- ⁇ and ATF2 / c-Jun) that are translocated to the nucleus and activate the transcription of type I IFN and cytokines proinflammatory
  • VACV encodes several immunomodulatory proteins that interfere with the TLR signaling pathway (indicated by rectangles): A46 inhibits TLR adapter molecules such as MyD88, TRIF, MAL and TRAM; A52 inhibits molecules such as IRAK-2 and TRAF6; K7 interacts with DDX3 and prevents the induction of I FN via TBKI / lkk- ⁇ -; and B14 inhibits ⁇ phosphorylation and therefore prevents the activation of N F-KB.
  • RLRs retinoic acid-inducible gene I (RIG-l) -like receptors”
  • VACV vaccinia virus
  • VACV encodes immunomodulatory proteins that interfere with the RLR signaling path (indicated by rectangles): K7 interacts with DDX3 and prevents the induction of IFN via TBKI / lkk- ⁇ -; and C6 inhibits the phosphorylation of IRF3 although its mechanism is still unknown. Figure 6.
  • MVA-B AC6L activates the IRF3 signaling path.
  • THP-1 cells differentiated with PMA were infected at a multiplicity of infection of 5 PFU / cell with MVA-B, MVA-B AC6L, MVA or with medium (Mock). At different post-infection times the cells were washed with cold PBS and lysed for 10 minutes at 4 ° C with "Cell Lysis Buffer" (Cell Signaling). The reaction mixtures were centrifuged for 10 min at 13,000 rpm. The protein concentration of the supernatants was determined using the "bicinchoninic acid protein assay" (Pierce Biotechnology). 30 g of total protein was loaded into 10% polyacrylamide gels (w / v) and transferred to nitrocellulose membranes.
  • A IFN- ⁇ secreting splenocytes specific to the HIV-1 Gag-B peptide were quantified by an ELISPOT assay. Data represent the mean ⁇ standard deviation of triplicates. ** represents p ⁇ 0.005.
  • BD Phenotypic analysis by flow cytometry of CD4 + T cells and specific CD8 + against HIV-1 Env, Gag and GPN antigens.
  • CD44 and CD62L were used to identify the sub-populations of memory called "central memory” (CM: CD44 + / CD62L + ), "effector memory” (MS: CD44 + / CD62L “ ) and” terminally differentiated effector memory “(TEMRA: CD447CD62L “ ).
  • CM central memory
  • MS effector memory
  • TEMRA CD447CD62L
  • the production of IFN- ⁇ and IL-2 was analyzed by intracellular marking (ICS).
  • B A representative flow cytometry test is shown. Sub-populations of memory T cells are drawn by density drawings. The dots represent IFN-y and IL-2 producing T cells.
  • C The percentage of specific CD4 + and CD8 + memory T cells against HIV-1 Env, Gag and GPN antigens is represented.
  • the frequencies were calculated representing the number of memory T cells producing IFN-y and / or IL-2 versus the total number of splenocytes CD4 + and CD8 + .
  • the values of the unstimulated controls were subtracted in all cases. ** represents p ⁇ 0.005.
  • the circles represent the proportion of CM, MS and TEMRA within the specific CD4 + and CD8 + memory T cells against HIV-1 Env, Gag and GPN antigens.
  • AD The data are derived from an experiment, representative of 2 experiments performed.
  • the polyfunctionality of the specific CD4 + (left part) and CD8 + memory T cells (right side) against HIV-1 Env + Gag + GPN antigens is defined based on IFN-y and / or IL-2 production. All possible combinations of responses are shown on the X axis.
  • the percentages of IFN-y and / or IL-2 producing memory T cells among the total CD4 + and CD8 + T cells are shown on the Y axis.
  • Each part of the circle corresponds to the proportion of CD4 + or CD8 + T cells producing IFN- ⁇ , IL-2 or IFN-y + IL-2 within the total of specific CD4 + or CD8 + memory T cells versus HIV-1 antigens.
  • the size of Each circle represents the magnitude of the specific induced memory immune response against HIV-1 antigens.
  • Anti-gp120 antibody titers were determined by ELISA.
  • the titles represent the last dilution of the serum that gave a signal 3 times higher than the signals obtained with the serum of naive mice.
  • the dotted line represents the ELISA detection limit.
  • the horizontal bar represents the average value and the values obtained by each mouse are represented by circles. * represents p ⁇ 0.05.
  • Gómez CE Najera JL, Jiménez V, Bieler K, Wild J, et al. (2007) Generation and immunogenicity of novel HIV / AIDS vaccine candidates targeting HIV-1 Env / Gag-Pol-Nef antigens of clade C. Vaccine 25: 1969-1992.
  • Gómez CE Najera JL, Krupa M, Esteban M (2008) The poxvirus vectors MVA and NYVAC as gene delivery systems for vaccination against infectious diseases and cancer. Curr Gene Ther 8: 97-120.
  • Poxvirus K7 protein adopts a Bcl-2 fold: biochemical mapping of its interactions with human DEAD box RNA helicase DDX3. J Mol Biol 385: 843-853.
  • Example 1 Generation of the invention MVA-B AC6L.
  • the generated recombinant virus, presented in this invention, has been referred to as MVA-B AC6L.
  • a schematic diagram of the MVA-B AC6L deletion mutant is depicted in Figure 1 A.
  • the manufacturing method of MVA-B AC6L includes the following e ⁇ apas:
  • MVA-B recombinant virus which contains the nucleotide sequences that code for the HIV antigens of subtype B gp120 and Gag-Pol-Nef, with the plasmid generated in a) which directs the deletion of the C6L gene (SEQ ID No: 1), and
  • MVA-B AC6L purify the viral vector called MVA-B AC6L, based on a recombinant MVA-B virus with deletion of the C6L gene (SEQ ID No: 1), obtained in step b).
  • the plasmid or transfer vector called pGem-RG-C6L wm was constructed to be able to generate the MVA-B AC6L deletion mutant, which has a deletion in the vaccinia C6L gene (The MVA 019L gene of MVA, SEQ ID No: 1 , is equivalent to the VACV-WR_022 gene of the vaccinia Wesirin Reserve (WR) strain, SEQ ID No: 3, and to the C6L gene of the Copenhagen vaccinia strain, SEQ ID No: 5.
  • the corresponding nomenclature is used to the Copenhagen genes to refer to the MVA genes).
  • pGem-RG-C6L wm was obtained by sequential cloning of five DNA fragments that contain the dsRed2, rsGFP and flanking (left and right) recombination sequences of the C6L gene in plasmid pGem-7Zf (-) (Promega).
  • plasmid pGem-Red-GFP wm (4540) was performed pb), which contains the dsRed2 and rsGFP genes under the control of an early / late synthetic viral promoter (E / L) and which was previously described (Garc ⁇ a-Arriaza et al., 2010. PLoS One 5: e12395).
  • the dsRed2 gene under the control of an early / late synthetic viral promoter (E / L) was amplified by PCR of plasmid pG-dsRed2 using Red2-B oligonucleotides (SEQ ID No: 15. 5 ' - GAACTAGGATCCTAA CTCGAGAAA-3 ' ) (includes the restriction site Bam Hl) and Red2-N (SEQ ID No: 16.
  • 5 ' -C GTTG GTCTAGAG AG AAAA ATTG -3 ' (includes the restriction site Xbal) and GFP-E (SEQ ID No: 18.
  • 5 ' -CTATAGAATTCTCAAGCTATGC-3 ' ) (comprises the restriction site Eco Rl) (832 bp), digested with Xba I and Eco Rl and cloned into plasmid pGem-Red wm previously digested with the same restriction enzymes to generate pGem-Red-GFP wm (4540 bp).
  • the MVA-B genome was used as a template for PCR amplification of the right flank of the C6L gene (Nucleotides 18689-19079 in the MVA genome. 391 bp), using the oligonucleotides RFC6L-Aatll-F (SEQ ID No : 7. Nucleotides 18689-18714 in the MVA genome) (comprising the Aatll restriction site) and RFC6L-Xbal-R (SEQ ID No: 8.
  • Nucleotides 19054-1979 in the MVA genome (comprises the restriction site Xbal)
  • This right flank was digested with Aatll and Xbal and cloned into the plasmid pGem-Red-GFP wm previously digested with the same restriction enzymes to generate pGem-RG-RFsC6L wm (4898 bp).
  • the repeated right flank of the C6L gene (Nucleotides 18689-19079 in the MVA genome. 391 bp) was amplified by PCR from the MVA-B genome with the oligonucleotides RF ' C6L-Xmal-F (SEQ ID No: 9.
  • Nucleotides 18689-18714 in the MVA genome (comprising the Xmal restriction site) and RF ' C6L-Clal-R (SEQ ID No: 10.
  • Nucleotides 19054-1979 in the MVA genome (comprises the Clal restriction site ), digested with Xmal and Clal and inserted into the plasmid pGem-RG-RFsC6L wm digested with Xmal / Clal to generate pGem-RG-RFdC6L wm (5259 bp).
  • the left flank of the C6L gene (Nucleotides 19530-19942 in the MVA genome.
  • LFC6L-Clal-F (SEQ ID No: 1 1. Nucleotides 19530-19555 in the MVA genome) (comprising the Clal restriction site) and LFC6L-BamHI- R (SEQ ID No: 12. Nucleotides 19917-19942 in the MVA genome) (comprising the BamH I restriction site), digested with Clal and BamHI and inserted into the plasmid pGem-RG-RFdC6L wm digested with Clal / Bam Hl . The resulting plasmid pGem-RG-C6L wm (5642 bp) was confirmed by DNA sequence analysis and directs the deletion of the C6L gene from the MVA and MVA-B genome.
  • MVA-B AC6L is carried out in cell cultures by means of a recombination process between the MVA-B virus and the plasmid pGem-RG-C6L wm, which contains the right and left flanks of the C6L gene.
  • MVA-B AC6L was constructed by selection in cell cultures of viral plaques that coexpress dsRed2 / rsGFP (express proteins with red and green fluorescence respectively), using the dsRed2 and rsGFP genes as transient selection markers, as previously described. (Garc ⁇ a-Arriaza et al., 2010.
  • PLoS One 5 e12395.
  • 3 x 10 6 DF-1 cells were infected with MVA-B at a multiplicity of infection of 0.05 PFU / cell and then transfected 1 h later with 6 g of plasmid pGem-RG-C6L wm DNA using Lipofectamine (Invitrogen) according to the manufacturer's instructions. After 72 hours, the cells were collected, lysed by freeze-thaw cycles, sonicated and used for the selection of recombinant viruses.
  • the MVA-B AC6L deletion mutant was selected from the viral progeny obtained after 6 consecutive rounds of plaque purification in DF-1 cells and during this process the plaques were selected from those with red / green fluorescence.
  • the viruses were selected from plaques expressing both fluorescent proteins (dsRed2 and rsGFP, red and green fluorescence).
  • the viral progeny of the selected plaques only expresses a fluorescent marker (Red2 or GFP) and in the last two passes (6 passes in total) the viruses of the selected plaques do not express any fluorescent marker due to loss of the dsRed2 and rsGFP genes.
  • the MVA-B deletion mutant was obtained AC6L and the deletion of the C6L gene was confirmed by PCR amplification of the C6L locus, using oligonucleotides RFC6L-Aatll-F and LFC6L-BamHI-R (described previously) and subsequent analysis by DNA sequencing.
  • the C6L gene See SEQ ID No: 1.474 nucleotides, positions 19068-19541 in the MVA genome
  • the deletion of the C6L gene in MVA-B includes positions 19068 to 19541 of the MVA genome.
  • the MVA-B AC6L recombinant virus obtained was grown in DF-1 cells to obtain a viral preparation called P2, which was grown in chicken embryonic cells (CEF), and purified by centrifugation through two sucrose mattresses at 36% (w / v) in 10 mM Tris-HCI pH 9, as previously described (Ram ⁇ rez et al., 2000. J Virol, 74 (2): 923-933.
  • MVA-B AC6L was titled DF-1 cells by an immunostaining assay, using a rabbit polyclonal antibody against the WR vaccinia strain (National Center for Biotechnology; 1: 1000) followed by anti-rabbit-HRP (Sigma; 1: 1000), as It has been previously described (Antoine et al., 1998. Virology 244: 365-396).
  • the preparation of MVA-B AC6L is free of mycoplasmas or bacteria.
  • plasmid pGem-RG- C6L wm (or a similar one containing the left and right flanks of the C6L gene) can be used by a technology similar to that proposed here to delegate the C6L gene on any recombinant MVA virus that expresses other heterologous antigens other than the one presented here (HIV subtype B), such as antigens of malaria, leishmania, hepatitis C virus, prostate cancer, etc; in order to be able to be used as vaccines against these diseases.
  • HAV subtype B heterologous antigens of malaria
  • leishmania hepatitis C virus
  • prostate cancer etc.
  • MVA-B AC6L deletion mutant demonstrates that C6 protein is not essential for MVA replication.
  • C6L deletion alters virus replication the growth of MVA-B AC6L and MVA-B in DF-1 cells was compared. Viral kinetics studies revealed that the deletion of C6L in the MVA-B genome does not affect viral replication. Therefore, C6L is not essential for viral propagation in cell cultures ( Figure 1 D).
  • MVA-B AC6L is an attenuated virus that does not replicate in mammalian cells ( Figure 1 E).
  • the open reading frame (ORF) of C6 (MVA gene 019L, 157 aa, 18.2 kDa. See SEQ ID No: 1) was amplified by PCR using the C6L-Nhel-F oligonucleotides (SEQ ID No: 13) (comprising the Nhel) and C6L-BamHI-R restriction site (SEQ ID No: 14) (comprising the BamHI restriction site), and the MVA DNA as a template.
  • the amplified product (Nucleotides 19068-19541 in the MVA genome. 488 bp) was digested with Nhel and BamHI and cloned into plasmid pET-27b (+) (Novagen).
  • the ligation product was used to transform E.coli strain BL21, and the plasmid of a positive kanamycin resistant colony was sequenced to confirm that it contains the C6L gene sequence.
  • the plasmid generated was called pET-27b-C6L (5837 bp).
  • Plasmid pET-27b (+) provides a tail of 6 histidines at the carboxy terminal end of the C6 protein, generating a recombinant C6 protein of about 24 kDa.
  • Kanamycin-resistant colonies were grown in Luria broth medium to an optical density of 0.5 to 595 nm. IPTG was added (0.5 mM) and the culture was grown for 4 more hours.
  • the cells were centrifuged and for cell lysis the cells were resuspended in 50 mM Tris-HCI, pH 7.5, 0.3 M NaCI, 8 M Urea, and incubated with lysozyme (1 mg / ml) for 30 minutes in the presence of phenylmethylsulfonyl fluoride (1 mM).
  • the suspension was frozen-thawed twice, the cell debris was removed by centrifugation and the supernatant was incubated with Probound resin (Invitrogen). Elution was carried out with different concentrations of imidazole (100 to 500 mM) in 50 mM Tris-HCI, pH 7.5, 0.3 M NaCI.
  • the eluted fractions were grouped, loaded into desalted columns following the manufacturer's instructions (GE-Healthcare, Freiburg, Germany), and were collected.
  • the protein was quantified using the Bradford assay, fractionated by 12% SDS-PAGE and analyzed by Western blot using an anti-His tag antibody (1: 5000) to detect the presence of vaccinia C6 protein.
  • Fractions containing C6 protein (with an estimated purity of 90%) were stored in aliquots at -20 ° C.
  • the C6 protein (1 150 g) was injected into "New Zealand White” rabbits to produce anti-C6 serum and anti-C6 rabbit polyclonal antibodies (Biomedal Laboratories, Seville).
  • Example 5 C6 is expressed early in a viral infection.
  • C6 The intracellular location of the C6 protein was examined by immunofluorescence in DF-1 cells infected with different strains of vaccinia (Figure 2C).
  • C6 was detected in the cytoplasm, presumably in viral factories, of DF-1 cells infected with WR, MVA and MVA-B, but not with MVA-B AC6L.
  • the reduced fluorescence intensity of C6 indicates the expression of low protein levels compared to the late A27 protein.
  • Example 6.- MVA-B AC6L increases IFN- ⁇ expression in human macrophages and dendritic cells.
  • MVA-B AC6L was a more potent inducer than MVA and MVA-B at low infective doses (0.002 PFU / ml, Figure 3B).
  • MVA-B AC6L stimulates the release to the medium by moDCs of higher levels of IFN- ⁇ ( Figure 3C) and IFN type I than MVA and MVA-B ( Figure 3D).
  • Example 7 MVA-B AC6L induces phosphorylation of IRF3 in THP-1 cells.
  • Example 8.- MVA-B AC6L increases the magnitude and polyfunctionality of the response of specific memory T cells against HIV-1.
  • IFN- ⁇ ELISPOT revealed that, compared to MVA-B, MVA-B AC6L increased the response of specific IFN- ⁇ secreting memory T cells against the Gag-B peptide of HIV-1 by 2.1 times (p ⁇ 0.005) (an HIV-1 peptide representative of the Gag antigen) ( Figure 7A). MVA, used as a control, did not induce any specific HIV-1 memory response.
  • the phenotype of HIV-1-specific memory T cells induced after immunization with DNA-B / MVA-B and DNA-B / MVA-B AC6L was characterized by polychromatic flow cytometry using ICS.
  • Splenic CD4 + and CD8 + T cells were co-stained for surface markers CD44 and CD62L in order to define the different memory sub-populations: naive (CD447CD62L + ), "central memory” (CM: CD44 + / CD62L + ), "effector memory” (EM: CD44 + / CD62L " ) and” effector terminal memory differentiated "(TEMRA: CD447CD62L " ).
  • IFN- ⁇ and IL-2 were also evaluated after in vitro stimulation with different "poles" of HIV-1 peptides (Env-pool, Gag-pool and GPN-pool) that cover the entire HIV sequences -1 present in the poxviral vector (Figure 7B).
  • DNA-B / MVA-B AC6L increased the polyfunctionality of specific CD4 + and CD8 + T memory cells against HIV-1, consisting of cells that produce both IFN- ⁇ and IL-2 [CD4 + T cells: 34 % in DNA-B / MVA-B AC6L vs. 16% in DNA-B / MVA-B, (p ⁇ 0.005); CD8 + T cells: 29% in DNA-B / MVA-B AC6L vs. 16% in DNA-B / MVA-B, (p ⁇ 0.005)] ( Figure 8).
  • DNA-B / MVA-B AC6L significantly increases the magnitude and polyfunctionality of the response of specific CD4 + and CD8 + T memory cells against HIV-1, with the majority of the response mediated by EM and TEMRA T cells.
  • DNA-B / MVA-B and DNA-B / MVA-B AC6L After vaccination with DNA-B / MVA-B and DNA-B / MVA-B AC6L, the responses of specific CD4 + T memory cells against HIV-1 were preferentially specific against Env.
  • DNA-B / MVA-B AC6L induced immunodominance against specific CD8 + T memory cells against GPN, while DNA-B / MVA-B preferentially induced specific CD8 + T memory cells against Env and Gag.

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Abstract

La présente invention relève du domaine de la biologie moléculaire et de la biotechnologie. Elle concerne plus spécifiquement des virus recombinants à base du virus Ankara modifié (MVA) qui expriment les antigènes gp120 et Gag-Pol-Nef du virus de l'immunodéficience humaine (VIH-1) de sous-type B (MVA-B), sur lesquels le gène de vaccine C6L a connu une délétion, et qui sont conçus pour être utilisés comme vaccins contre le VIH/SIDA et d'autres maladies.
PCT/ES2012/070521 2011-07-19 2012-07-11 Vecteurs recombinants à base du virus ankara modifié (mva) avec délétion dans le gène c6l, utilisés comme vaccins contre le vih/sida et d'autres maladies WO2013011179A2 (fr)

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