WO2014162031A1 - Vecteurs recombinés dérivés du virus ankara modifié (mva) utilisés comme vaccins préventifs et thérapeutiques contre l'hépatite c - Google Patents

Vecteurs recombinés dérivés du virus ankara modifié (mva) utilisés comme vaccins préventifs et thérapeutiques contre l'hépatite c Download PDF

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WO2014162031A1
WO2014162031A1 PCT/ES2014/070246 ES2014070246W WO2014162031A1 WO 2014162031 A1 WO2014162031 A1 WO 2014162031A1 ES 2014070246 W ES2014070246 W ES 2014070246W WO 2014162031 A1 WO2014162031 A1 WO 2014162031A1
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hcv
mva
cells
virus
cell
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PCT/ES2014/070246
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Spanish (es)
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Mariano Esteban Rodriguez
Carmen Elena Gómez Rodríguez
Beatriz PERDIGUERO DE LA TORRE
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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
    • A61K39/29—Hepatitis 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
    • 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is limited to the biomedicine sector and, more specifically, relates to recombinant viruses based on the modified Ankara vaccinia virus (MVA). More specifically, the invention relates to MVA-derived recombinant viruses that act as HCV virus protein expression systems and their use in hepatitis C vaccination.
  • MVA modified Ankara vaccinia virus
  • HCV Hepatitis C virus
  • these vaccines need to generate a potent, broad and functional T-cell response as well as a humoral response against a wide range of HCV antigens.
  • they must be directed against relatively conserved viral regions to cope with the great genetic diversity of the HCV virus both between hosts and within the same host.
  • they must eradicate HCV from the liver without inducing any liver immunopathology so that they can be considered safe vaccines.
  • DNA vaccines with additional techniques that favor their release and the generated immunogenicity, have shown some promising results and have been described as being able to reduce the viral load in some chronically infected patients (Halliday, Klenerman et al. 201 1, Torresi , Johnson et al. 201 1, Ip, Nijman et al. 2012).
  • the most promising observations derive from the use of vaccines based on viral vectors such as replication-defective adenoviruses or vaccinia virus.
  • this strategy does not allow the full HCV genome to be used, given the toxicity of the binding of both molecules (Gómez, Vandermeeren et al. 2005).
  • the vaccine candidates based on the MVA virus currently under test are directed against a limited group of structural and non-structural HCV proteins and have already demonstrated their ability to generate high quality T-cell immune responses in both preclinical studies (Abraham, Himoudi et al. 2004, Rollier, Depla et al. 2004, Fournillier, Gerossier et al. 2007, El-Gogo, Staib et al. 2008) as clinicians (Habersetzer, Honnet et al. 201 1).
  • the most advanced therapeutic studies using MVA have been carried out with the candidate TG4040. It is a recombinant MVA-based T-cell antigenic vaccine that encodes HCV NS3, NS4 and NS5B proteins.
  • HCV-specific T-cell responses were detected in all patients one week after the first vaccination and these responses were maintained during the 6-month follow-up. Vaccination reduced viral loads to 1.5 Iog10 and stronger specific T cell responses were observed in those patients who had the highest levels of viral load reduction (Habersetzer, Honnet et al. 201 1).
  • a randomized phase II study is currently underway involving 153 patients divided into three treatment groups. Preliminary data published on the Transgene website show a reduction in viral load in the pre-vaccinated group with TG4040 prior to the start of the trial one week after the start of the trial. This reduction in viral load occurs more rapidly than in the other groups that had received treatment alone or in combination with TG4040 within the same treatment program (Clinical-Trials.gov, NCT01055821).
  • a first object of the invention is the polynucleotide of the invention comprising the nucleotide sequence corresponding to the MVA virus that acts as an expression vector for HCV genes regulating the transcription, translation and post-translational processing of said HCV genes in most body tissues (MVA-HCV polynucleotide).
  • a particular embodiment of the invention corresponds to the MVA-HCV polynucleotide in which the HCV virus sequence corresponds to the almost complete genome of genotype 1 a and which comprises the structural (Core, E1, E2 and p7) and non-structural ( NS2, NS3, NS4A, NS4B, NS5A plus 201 amino acids in the N-terminal region of NS5B) and whose sequence is SEQ ID No 1.
  • An object of the invention is the process for obtaining the polynucleotide of the invention, which comprises the following steps: (a) Generation of the transfer plasmid pCyA-HCV 7 9 , (b) Construction of the MVA-HCV recombinant virus, (c ) Selection of stable MVA-HCV viruses through successive passes in cell cultures.
  • a particular embodiment of the invention is the method of obtaining the polynucleotide of the invention where the HCV virus sequence corresponds to the almost complete genome of genotype 1 a, with sequence SEQ ID No. 1.
  • Another object of the invention is the recombinant vector MVA-HCV encoded by the polynucleotide of the invention that causes the expression of the viral particle itself and that of the HCV virus proteins to infect the cells.
  • a particular embodiment of the invention is the recombinant vector in which the HCV viral proteins are with SEQ ID No 1.
  • Another object of the invention is the cell containing the polynucleotide of the invention MVA-HCV.
  • a particular embodiment of the invention is the cell containing the MVA-HCV polynucleotide, in which the HCV viral proteins correspond to the structural (Core, E1, E2 and p7) and non-structural (NS2, NS3) protein sequences. , NS4A, NS4B, NS5A and the N-terminal region of NS5B) of HCV genotype 1 to SEQ ID No 1.
  • Another preferred object of the invention is the use of the cell containing the polynucleotide of the invention for obtaining the recombinant vector of the invention.
  • a particular embodiment of the invention is the use of the cell containing the polynucleotide of the invention for obtaining the recombinant vector of the invention, in which the HCV viral proteins correspond to the sequences of the structural proteins (Core, E1 , E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and the N-terminal region of NS5B) of genotype 1 to HCV with SEQ ID No 1.
  • Another object of the invention is the pharmaceutical composition containing the recombinant vector of the invention MVA-HCV, useful as a vaccination mechanism against hepatitis C.
  • a particular embodiment of the invention is the pharmaceutical composition of the invention MVA-HCV useful as a hepatitis C vaccination mechanism containing the recombinant vector of the invention, in which the HCV viral proteins correspond to the structural protein sequences. (Core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and the N-terminal region of NS5B) of HCV genotype 1 to SEQ ID No 1.
  • Another preferred object of the invention is the use of the pharmaceutical composition containing the recombinant vector of the invention MVA-HCV to prevent or treat HCV infection.
  • a particular embodiment of the invention is the use of the pharmaceutical composition of the invention MVA-HCV containing the polynucleotide of the invention for obtaining the recombinant vector of the invention, in which the HCV viral proteins correspond to the sequences of structural (Core, E1, E2 and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and the N-terminal region of NS5B) of HCV genotype 1 to SEQ ID No 1.
  • the authors of the present invention decided to develop a new vaccine candidate based on the attenuated strain MVA constitutively expressing the almost complete genome of the genotype 1 a of HCV.
  • This open reading guideline (ORF) of HCV had previously been used for the generation of the recombinant vaccinia virus vT7-HCV7.9 based on the virulent strain WR, in which the HCV genome is efficiently transcribed resulting in a polyprotein that is correctly processed generating the structural (Core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A plus the 201 amino acids of the N-terminal region of NS5B) mature (Gómez, Vandermeeren et al. 2005 ).
  • HCV genome expression by the WR strain had to be regulated in a controlled manner by the lac I repressor of E.
  • This invention describes the generation, characterization and preclinical evaluation of the MVA-HCV virus constitutively expressing all HCV proteins (except the C-terminal region of NS5B). And more specifically addressing the study of the amplitude, phenotype, polyfunctionality and duration of the generated immune responses. Neither the obtaining of the MVA-HCV vector nor the immune response results were predictable, since a recombinant virus that expressed the entire HCV genome had not previously been achieved due to the toxicity of the viral antigens, nor was it defined in The context of joint expression of all HCV antigens by a viral vector type of immune response induced. Therefore, it has been unexpected to have achieved the recombinant MVA-HCV with the described functionalities.
  • a first object of the invention relates to a polynucleotide, hereinafter polynucleotide of the invention, comprising: i) A transcription regulatory nucleotide sequence corresponding to the MVA virus (sequence deposited in the GenBank with the number of access U94848; http://www.ncbi.nlm.nih.gov/nuccore/U94848) that acts as an expression vector for HCV genes by regulating the transcription, translation and post-translational processing of said HCV genes in most of body tissues, and
  • a particular embodiment of the invention corresponds to the MVA-HCV polynucleotide in which the HCV virus sequence ii) corresponds to the almost complete genome of genotype 1 a and which comprises the structural proteins (Core, E1, E2 and p7) and not Structural (NS2, NS3, NS4A, NS4B, NS5A plus 201 amino acids of the N-terminal region of NS5B), inserted into the thymidine kinase locus (TK) of the MVA genome and whose sequence is SEQ ID No 1.
  • TK thymidine kinase locus
  • polynucleotide refers to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides or related structural variants or synthetic analogs thereof) linked through phosphodiester bonds (or related structural variants). in synthetic analogues thereof).
  • the term polynucleotide includes genomic DNA or double or single stranded coding DNA, RNA, any synthetic and genetically manipulated polynucleotide and both both the coding chain and the antisense (although only the coding chain is highlighted herein). This includes single and double stranded molecules, such as DNA-DNA, DNA-RNA and RNA-RNA hybrids.
  • the polynucleotide comprises a nucleotide sequence of the MVA virus that regulates the expression of the almost complete genome of genotype 1 to HCV under the transcriptional control of the early / late synthetic viral promoter and operably linked to said HCV nucleotide sequence.
  • a transcription regulatory sequence refers to a sequence that controls and regulates transcription and, where appropriate, the translation of the HCV polynucleotide.
  • a transcription regulatory sequence includes promoter sequences, sequences encoding transcriptional regulators, ribosome binding sequences (RBS) and / or transcription terminator sequences.
  • Variants according to the present invention include amino acid sequences that are at least 60%, 70%, 80%, 90%, 95% or 96% similar or identical to the recombinant vector sequence and that includes both the corresponding amino acid sequences.
  • the MVA virus as to the amino acid sequences of the HCV virus, in particular structural proteins (Core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A plus 201 amino acids of the NS5B N-terminal region of HCV genotype 1 to) (SEQ ID No 1).
  • structural proteins Core, E1, E2 and p7
  • non-structural NS2, NS3, NS4A, NS4B, NS5A plus 201 amino acids of the NS5B N-terminal region of HCV genotype 1 to
  • SEQ ID No 1 the amino acid sequences of the HCV virus
  • variants or fragments can be generated using conventional techniques, such as mutagenesis, including the creation of discrete point mutation (s), or by truncation.
  • the mutation can give rise to variants that retain substantially the same, or simply a subset, of the biological activity of a polypeptide from which it is derived.
  • vector refers to a nucleic acid molecule that is capable of transferring nucleic acid sequences contained therein to the cell it infects and that is produced by means of techniques. of molecular biology.
  • Some examples of recombinant vectors are linear DNA, plasmid DNA, modified viruses, adenoviruses / adeno-associated viruses, retroviral and viral vectors, etc .; all of them widely described in the literature and that can be used following standard molecular biology techniques or purchased from suppliers.
  • a typical recombinant vector is selected from the group consisting of a lentiviral vector, an adenoviral vector and / or an adeno-associated virus vector.
  • recombinant vector is defined as a vector produced by the binding of different nucleic acid fragments from different sources and whose expression gives rise to a viral particle with compound infective capacity. characteristically of protein capsid, viral genome and proteins associated with the viral genome.
  • a recombinant vector according to the invention can therefore be used both as a biotechnological tool to multiply the virus and be used in pharmaceutical compositions such as vaccines.
  • operably linked means that the nucleotide sequence encoding a polypeptide comprising the genome of the HCV virus, in particular the almost complete genome of genotype 1 to HCV with SEQ ID No.
  • Another object of the invention is a process for obtaining the polynucleotide of the invention, hereinafter the method of the invention, wherein said method generally comprises the following steps: (a) Generation of the transfer plasmid.
  • a DNA fragment of a variable length containing the structural and non-structural proteins of a given HCV genotype would be cleaved from the initial plasmid where it was inserted (pHCV) using the corresponding restriction enzymes and inserted into the plasmid pCyA-20 described below. and previously digested with the same digestion enzymes to generate the corresponding transfer plasmid (pCyA-HCV);
  • HCV is classified into 1 1 genotypes (designated 1 ⁇ 1 1), numerous subtypes (designated a, b, c, ...) and about 100 different strains (numbered 1, 2, 3,).
  • a virus is considered “stable” if it loses less than 50% of infectivity in, for example, a plaque formation assay (PFU), which measures the change in the amount of PFU / ml_ between two anterior and posterior temporal points. .
  • PFU plaque formation assay
  • a particular embodiment of the invention is a method of obtaining the polynucleotide of the invention, where the HCV virus sequence corresponds to SEQ ID No. 1 and comprises the structural (Core, E1, E2 and p7) and non-structural (NS2) proteins. , NS3, NS4A, NS4B, NS5A and the N-terminal region of NS5B) of HCV genotype 1 a.
  • Said method generally comprises the following steps:
  • Plasmid pCyA-20 was generated by inserting a synthetic band containing the early / late viral promoter and a multiple cloning site in the plasmid pLZAWL This synthetic band, obtained by hybridization of two complementary oligonucleotides containing targets for restriction enzymes AscI and Swal, it was digested with Ascl and Swal and inserted into plasmid pLZAWl previously digested with the same restriction enzymes to generate plasmid pCyA-20.
  • HCV genotype 1 a A fragment of 7.9 Kpb DNA containing the proteins Structural (Core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and the N-terminal region of NS5B) of HCV genotype 1 a was cleaved with EcoRI from plasmid pHCVI a (assigned by Charles M. Rice, New York) that contained the complete HCV genome.
  • This DNA fragment was treated with Klenow DNA polymerase to generate blunt ends and inserted into plasmid pCyA-20 previously digested with Pmel and dephosphorylated by incubation with alkaline prawn phosphatase to generate transfer plasmid pCyA-HCV 7 9 (SEQ ID No 2).
  • the plate designated as MVA-HCV-1 .6.1.1 .9.3.2 (stock P1) was grown to generate a crude viral preparation (stock P2: 9.8 x 10 8 PFU / ml) from which a stock was prepared P3 of purified virus from infected BHK-21 cells at a multiplicity of infection of 0.05 PFU / cell through two 36% sucrose mattresses. This P3 stock was the one that was finally selected.
  • a P-2 stock of MVA-HCV was also isolated from the initial P1 after three consecutive passes of plaques obtained in chicken embryonic cells (CEF) and confirmation of protein expression and sterility.
  • CEF chicken embryonic cells
  • plasmids obtained as intermediates in the method of the invention can also be used to obtain variants or derivatives of the polynucleotide of the invention. This will also protect the plasmid pCyA-HCV 7 9 whose sequence corresponds to SEQ ID No 2.
  • the polynucleotide of the invention encodes the structures necessary to generate a viral MVA-HCV particle in CEF and BHK-21 cells and can be used as a recombinant vector to infect and transform those cell cultures into which it is introduced and with this, to produce the expression of viral proteins characteristic of HCV.
  • another object of the invention is the recombinant vector MVA-HCV, or viral particle, which comprises the polynucleotide of the invention.
  • the viral particle or recombinant vector causes cells that are infected by the expression of the viral particle itself and of the HCV virus proteins.
  • the recombinant vector of the invention expresses the HCV virus sequence that corresponds to the sequence SEQ ID No. 1 and that encodes the structural (Core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A) proteins. , NS4B, NS5A and the N-terminal region of NS5B) of HCV genotype 1 a.
  • a culture of host cells encompasses the processes of maintaining and growing said host cells.
  • Cell cultures need controlled conditions of temperature, pH, percentages of gases such as carbon dioxide and oxygen, as well as the presence of adequate nutrients to allow viability and cell division.
  • Cell cultures can be grown on solid substrates such as agar or in a liquid medium, allowing large numbers of suspended cells to be cultured.
  • Viral cultures require host cells that provide the cellular and metabolic machinery they lack. This allows the virus not only to be maintained but also to multiply so that the expression of the polynucleotide of the invention in said cell culture can be used to propagate the recombinant vector of the invention.
  • another object of the invention is the cell containing the polynucleotide of the invention MVA-HCV, hereafter referred to as the host cell of the invention.
  • a particular embodiment of the invention relates to the host cell of the invention containing the polynucleotide of the invention MVA-HCV, where the HCV virus sequence corresponds to the sequence SEQ ID No. 1 encoding the structural proteins (Core, E1 , E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and the N-terminal region of NS5B) of HCV genotype 1 a.
  • another object of the invention relates to the use of the host cell of the invention to reproduce and maintain the MVA-HCV virus and to obtain the recombinant vector of the invention.
  • the host cell of the invention is a mammalian cell, more preferably of avian origin and more preferably even chicken embryonic fibroblasts.
  • a particular embodiment of the invention relates to the use of the host cell of the invention to reproduce and maintain the MVA-HCV virus and to obtain the recombinant vector of the invention.
  • the recombinant vector of the invention can transfer the HCV sequence to a cell under the transcriptional control of the early / late synthetic viral promoter of the MVA virus fragment and induce its expression therein.
  • the viral polyprotein generated is processed giving rise to the HCV proteins, particulate to the sequence of the structural proteins, Core, E1, E2 and p7, and non-structural, NS2, NS3, NS4A, NS4B, NS5A plus 201 amino acids from the NS5B N-terminal region, mature of genotype 1 a of the HCV virus.
  • This recombinant vector can, therefore, be used to express HCV genes in an organism and thus induce immune responses against HCV viral proteins in those organisms to which said vector is administered.
  • another object of the invention also relates to a pharmaceutical composition useful for generating a lasting immune response against the HCV virus, hereinafter "pharmaceutical composition of the invention", comprising the vector of the invention and a pharmaceutically carrier. acceptable.
  • said composition may comprise another active and / or adjuvant principle.
  • a particular embodiment of the invention relates to the pharmaceutical composition of the invention in which the vector of the invention contains the sequence SEQ ID No 1 that corresponds to the sequence of the structural proteins, Core, E1, E2 and p7 and not Structural, NS2, NS3, NS4A, NS4B, NS5A plus 201 amino acids in the N-terminal region of NS5B, mature of genotype 1 a of the HCV virus.
  • Another preferred object of the invention relates to the use of the pharmaceutical composition of the invention to generate a lasting and prophylactic immune response for the treatment and prevention of HCV infection.
  • the generation of that protective response can be achieved by administering only the recombinant vector of the invention, in a single dose or in doses administered over time, or as part of an immunization protocol with different vectors expressing HCV antigens, forming part of the recombinant vectors of the invention from the initial dose that triggers the response and / or from one or more subsequent doses intended to enhance the previously generated response.
  • Another particular embodiment of the invention relates to the use of the pharmaceutical composition of the invention to generate a lasting and prophylactic immune response for the treatment and prevention of HCV genotype 1 to infection.
  • the term "medicament or pharmaceutical composition” refers to any substance used for prevention, relief, treatment or cure of diseases in man and / or animals.
  • the pharmaceutical composition or medicament of the invention further comprises a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition or medicament of the invention further comprises an adjuvant.
  • the pharmaceutical composition or medicament of the invention further comprises another active ingredient (additional active ingredient).
  • excipient refers to a substance that helps the absorption of the elements of the composition of the invention, stabilizes said elements and activates or aids the preparation of the composition in the sense of giving it consistency or providing flavors that make it nicer.
  • the excipients could have the function of keeping the ingredients together, such as, for example, starches, sugars or cellulose, the sweetening function, the function as a dye, the protective function of the composition, for example, to isolate it from air and / or moisture, the filling function of a tablet, capsule or any other form of presentation, such as, for example, is the case of dibasic calcium phosphate, the disintegrating function to facilitate the dissolution of the components and its absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
  • vehicle like the excipient, refers to a substance that is used in the pharmaceutical composition or medicament to dilute any of the components of the present invention comprised therein to a certain volume or weight.
  • pharmaceutically acceptable carrier is an inert substance or action analogous to any of the elements of the present invention.
  • the function of the vehicle is to facilitate the incorporation of other elements, allow a better dosage and administration or give consistency and form to the composition.
  • the pharmacologically acceptable carrier is the diluent.
  • adjuvant refers to an agent that increases the formation of antibodies against a certain antigen when it is delivered jointly to it or as part of the same treatment protocol.
  • FIG. 1 Scheme of the construction of the transfer plasmid pCyA-HCV 7 .9.
  • Plasmid pCyA-20 was generated by inserting a synthetic band containing the early / late viral promoter and a multiple cloning site in plasmid pLZAWl. This synthetic band, obtained by hybridizing two complementary oligonucleotides containing targets for the AscI and Swal restriction enzymes, was digested with Ascl and Swal and inserted into the plasmid pLZAWl previously digested with the same restriction enzymes to generate the plasmid pCyA- twenty.
  • a 7.9 kbp DNA fragment containing the structural (Core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and the N-terminal region of NS5B) of HCV genotype 1 a was EcoRI cleaved from plasmid pHCVI a (assigned by Charles M. Rice, New York) containing the complete HCV genome. This DNA fragment was treated with Klenow DNA polymerase to generate blunt ends and inserted into plasmid pCyA-20 previously digested with Pmel and dephosphorylated by incubation with shrimp alkaline phosphatase to generate transfer plasmid pCyA-HCV 7 .9.
  • FIG. 1 In vitro characterization and genetic stability of the MVA-HCV recombinant virus.
  • A Scheme of the organization of the HCV genome in the TK locus of MVA.
  • B Confirmation of HCV genome insertion by PCR analysis. Viral DNA was extracted from BHK-21 cells infected with MVA-WT or MVA-HCV at a multiplicity of infection of 5 PFU / cell. The TK-L and TK-R oligonucleotides that hybridize in the flanking sequences of the locus TK were used for PCR analysis of the TK locus. In the parental MVA virus an 873 bp fragment is obtained while in the recombinant virus a single product of 8393 bp is observed.
  • BHK-21 cells were uninfected or infected with MVA-WT or MVA-HCV at 5 PFU / cell. At 24 hours post-infection, the cells were lysed in the presence of Laemmli buffer, the cell extracts were separated in gels for 12% SDS-PAGE and analyzed by Western-blot using mouse monoclonal antibodies against Core, E1 proteins. , E2, NS4A, NS4B and NS5A.
  • D Analysis of the stability of MVA-HCV along different passages in BHK-21 cells.
  • HCV protein expression was visualized by Western-blot from samples of uninfected or infected BHK-21 cells at 5 PFU / cell with MVA-WT or with the different MVA-HCV passages (from P8 to P1 1 ) using a human serum positive for antibodies to HCV.
  • FIG. 3 (A) Analysis of the growth of the MVA-HCV virus in BHK-21 cells. Monolayers of BHK-21 cells were infected with MVA-WT or MVA-HCV at 0.01 PFU / cell. At different post-infection times (0, 24, 48 and 72 hours), the cells were collected and the presence of infectious viruses was determined by immunostaining of the infected cell foci.
  • C Formation of membranous structures during infection (5 PFU / cell) of HeLa cells by MVA-HCV at 16 h. by electron microscopy.
  • Human dendritic cells were uninfected or infected with MVA-WT or MVA-HCV at 0.3 or 1 PFU / cell for 6 hours.
  • the mRNA levels of IFN-a, IFIT1, IFIT2, RIG-I, MDA-5 and IP-10 were quantified by RT-PCR.
  • MRNA levels are expressed as the ratio between the levels of the gene of interest and the levels of Hprt. UA: arbitrary units. * p ⁇ 0.05, ** p ⁇ 0.005, *** p ⁇ 0.001 for all conditions comparing MVA-HCV with MVA-WT at the same multiplicity of infection (MDI).
  • HCV-specific CD8 T cells were measured 10 days after the last immunization by intracellular multiparametric cytokine tracing after stimulation of splenocytes derived from mice immunized with the different mixtures of HCV peptides.
  • the total value in each group represents the sum of the percentages of CD8 + T cells that secrete CD107a and / or IFN- ⁇ and / or IL-2 and / or TNF- ⁇ against all HCV peptide mixtures.
  • the diagrams on the right represent the specific contribution of the different mixtures of HCV peptides to the total CD8 + response in the different immunization groups. The background obtained in the unstimulated samples was subtracted in all cases.
  • FIG. 6 Immune response of HCV-specific T-cells generated by the MVA-HCV recombinant virus in the spleen of C57BL / 6 mice immunized in homologous and heterologous vaccination protocols.
  • A Magnitude of the response of CD4 + or CD8 + T cells.
  • HCV-specific CD4 or CD8 T cells were measured 53 days after the last immunization by intracellular mating of multiparametric cytokines after stimulation of splenocytes derived from mice immunized with the different mixtures of HCV peptides.
  • the total value in each group represents the sum of the percentages of CD4 + or CD8 + T cells that secrete IFN- ⁇ and / or IL-2 and / or TNF-a (CD4) or CD107a and / or IFN- ⁇ and / or IL-2 and / or TNF-a (CD8) vs. all mixtures of HCV peptides.
  • the diagrams on the right represent the specific contribution of the different mixtures of HCV peptides to the total CD4 + or CD8 + response in the different immunization groups.
  • the background obtained in the unstimulated samples was subtracted in all cases. *** p ⁇ 0.001.
  • the p-value indicates significantly higher responses with respect to CD4 + T cell responses or between CD8 + T cell responses obtained in the DNA-HCV / MVA-HCV group compared to those observed in the MVA-HCV / MVA group - HCV.
  • B Functional profile of the memory response of HCV-specific CD8 T cells in the different immunization groups. All possible combinations of the responses are shown on the x-axis while the percentages of the different functional populations within the total population of CD8 T cells are represented on the y-axis. The answers are grouped based on the number of functions. ** p ⁇ 0.005; *** p ⁇ 0.001.
  • C Phenotypic profile of memory CD8 T cells specific for HCV.
  • the upper graphs represent the total percentage of HCV-specific CD8 T cells that have a central memory phenotype (TCM; CD127 + CD62L + ), memory effector (TEM; CD127 + CD62L “ ) or effector (TE; CD127 D62L “ ) .
  • the lower diagrams correspond to representative flow cytometry graphs showing the percentage of specific CD8 T cells versus HCV peptide mixtures p7 + NS2 (left) or NS3 (right) with central memory phenotype, memory effector or effector. ** p ⁇ 0.005.
  • FIG. 7 Immune response of HCV-specific T cells generated by the recombinant MVA-HCV virus in the spleen and in the liver of C57BL / 6 mice immunized in homologous and heterologous vaccination protocols.
  • Flow cytometry profiles showing the response of specific CD8 T cells against mixtures of p7 + NS2 or NS3 peptides in splenocytes and intrahepatic immune cells.
  • FIG. 8 Immune response of HCV-specific T-cells generated by the MVA-HCV recombinant virus in the liver of C57BL / 6 mice immunized in homologous and heterologous vaccination protocols.
  • A Magnitude of the response of CD4 + or CD8 + T cells. HCV-specific CD4 or CD8 T cells were measured in the liver 53 days after the last immunization by intracellular multiparameter cytokine tightening after stimulation of intrahepatic immune cells derived from mice immunized with the different HCV peptide mixtures.
  • the total value in each group represents the sum of the percentages of CD4 + or CD8 + T cells that secrete IFN- ⁇ and / or IL-2 and / or TNF- ⁇ (CD4) or CD107a and / or IFN- ⁇ and / or IL-2 and / or TNF-a (CD8) against all mixtures of HCV peptides.
  • the diagrams on the right represent the specific contribution of the different mixtures of HCV peptides to the total CD4 + or CD8 + response in the different immunization groups.
  • the background obtained in the unstimulated samples was subtracted in all cases. *** p ⁇ 0.001.
  • the p value indicates significantly higher responses with respect to CD4 + T cell responses in different immunization groups.
  • the lower diagrams correspond to representative flow cytometry graphs showing the percentage of specific CD8 T cells versus HCV peptide mixtures p7 + NS2 (left) or NS3 (right) with central memory phenotype, memory effector or effector. ** p ⁇ 0.005.
  • FIG. 9 Adaptive immune response of HCV-specific T cells generated by the MVA-HCV recombinant virus in the spleen of HLA-A2 transgenic mice in a heterologous vaccination protocol.
  • A Magnitude of the response of CD4 + or CD8 + T cells.
  • HCV-specific CD4 or CD8 T cells were measured 10 days after the last immunization by intracellular multiparameter cytokine marking after stimulation of splenocytes derived from mice immunized with the different mixtures of HCV peptides.
  • the total value in each group represents the sum of the percentages of CD4 + or CD8 + T cells that secrete IFN- ⁇ and / or IL-2 and / or TNF-a (CD4) or CD107a and / or IFN- ⁇ and / or IL-2 and / or TNF-a (CD8) against all mixtures of HCV peptides.
  • the diagrams on the right represent the specific contribution of the different mixtures of HCV peptides to the total CD4 + or CD8 + response.
  • the background obtained in the unstimulated samples was subtracted in all cases. *** p ⁇ 0.001. The p value indicates a significantly higher response with respect to the CD4 + T cell response.
  • (B) Flow cytometry profiles showing the response of specific CD4 or CD8 T cells against mixtures of E (CD4) or NS3 (CD8) peptides.
  • FIG. 10 Memory immune response of HCV-specific T cells generated by the recombinant MVA-HCV virus in the spleen of HLA-A2 transgenic mice in a heterologous vaccination protocol.
  • A Magnitude of the response of CD4 + or CD8 + T cells.
  • HCV-specific CD4 or CD8 T cells were measured 53 days after the last immunization by intracellular mating of multiparametric cytokines after stimulation of splenocytes derived from mice immunized with the different mixtures of HCV peptides.
  • the total value in each group represents the sum of the percentages of CD4 + or CD8 + T cells that secrete IFN- ⁇ and / or IL-2 and / or TNF-a (CD4) or CD107a and / or IFN- ⁇ and / or IL-2 and / or TNF-a (CD8) against all mixtures of HCV peptides.
  • the diagram on the right represents the specific contribution of the different mixtures of HCV peptides to the total CD8 + response.
  • the background obtained in the unstimulated samples was subtracted in all cases. *** p ⁇ 0.001. The p value indicates a significantly higher response with respect to the CD4 + T cell response.
  • the graph on the left represents the total percentage of HCV-specific CD8 T cells that have a central memory phenotype (TCM; CD127 + CD62L + ), memory effector (TEM; CD127 + CD62L “ ) or effector (TE; CD127 D62L " ).
  • TCM central memory phenotype
  • TEM memory effector
  • TE effector
  • CD127 D62L effector
  • the diagram on the right shows a representative flow cytometry graph indicating the percentage of specific CD8 T cells versus the mixture of HCV p7 + NS2 peptides with central memory phenotype, memory effector or effector. * p ⁇ 0.05.
  • Example 1 Generation and in vitro characterization of a recombinant MVA virus constitutively expressing the almost complete genome of hepatitis C virus (HCV) of genotype 1 a (MVA-HCV). Purity, expression and genetic stability of HCV proteins expressed by the MVA-HCV recombinant virus
  • the inventors have generated the MVA-HCV virus, a recombinant virus based on the attenuated strain of MVA poxvirus that has the same DNA fragment included in the vT7-HCV 7 9 virus inserted into the TK locus but under the transcriptional control of the viral promoter Synthetic early / late. This promoter therefore directs the constitutive expression of structural and non-structural HCV proteins.
  • a scheme with the different cloning steps carried out for the construction of the transfer plasmid pCyA-HCV 7 9 used for the generation of the MVA-HCV virus and the organization of the HCV genome in the TK locus is shown in Fig. 1A of said recombinant virus is represented in Fig. 2A.
  • the PCR product obtained in cells infected with MVA-HCV is approximately 8 kbp in size, indicating that the HCV genome has been correctly inserted into the TK locus of the MVA virus and not there is contamination with the parental virus in the preparation of the recombinant virus MVA-HCV.
  • the MVA-HCV virus was passed successively in BHK-21 cells from pass 7 (P2 stock) to pass 1 1 (P8 ⁇ P1 1).
  • the expression of HCV proteins in the different countries was analyzed by Western-blot using a human serum positive for antibodies to HCV. As shown in Fig. 2D, the MVA-HCV virus efficiently expresses HCV proteins after 1 1 passes.
  • MVA-HCV infection blocks the innate immune response
  • MVA-HCV administered in homologous combination induces in mice of strain C57BL / 6 a high, broad VHC-specific T cell response , multifunctional and long lasting.
  • mice of strain C57BL / 6 the response of HCV-specific T cells induced using homologous (MVA-HCV / MVA-HCV) or heterologous (DNA-HCV / MVA) immunization protocols -HCV).
  • C57BL / 6 mice (4 in each group) were immunized following the protocol described below.
  • the adaptive immune response was evaluated 10 days after the last dose using a multiparameter cytokine intracellular tick test.
  • Splenocytes isolated from immunized animals were stimulated "ex vivo" for 6 hours with a panel of 457 peptides (from 13 to 19-m overlapping in 1 or 12 amino acids) grouped into 6 mixtures of peptides: Core (28 peptides), E (83 peptides), p7 + NS2 (40 peptides), NS3 (98 peptides), NS4 (47 peptides) and NS5 (161 peptides) and incubated with specific antibodies to identify T-cell lineage (CD3, CD4 and CD8), degranulation (CD107a) and responding cells (IL-2, IFN- ⁇ and TNF- ⁇ ).
  • Animals that received a first dose with parental MVA (MVA-WT) or empty DNA (--DNA
  • the percentages of T cells producing IFN- ⁇ and / or IL-2 and / or TNF-a determined the total response of CD4 + T cells while the percentages of T cells producing CD107a and / or IFN- ⁇ and / or IL-2 and / or TNF- ⁇ determined the total response of CD8 + T cells.
  • the magnitude of the response of CD8 + HCV-specific T cells was significantly higher in animals immunized with the protocols MVA-HCV / MVA-HCV or ADN- HCV / MVA-HCV than in their respective control groups where antigen-specific responses were very low (p ⁇ 0.005) (Fig. 5A). In both groups the immune response induced by vaccination was mediated by CD8 cells while no specific response mediated by CD4 cells was detected (Fig. 5A).
  • the response of CD8 + HCV-specific T cells was significantly higher in animals that received HCV / MVA-HCV DNA compared to those that received M VA-HCV / MVA-HCV (p ⁇ 0.005).
  • 90% of the CD8 + T cell response was directed against the mixture of p7 + NS2 peptides while in animals that received HCV / MVA-HCV DNA 97% of The CD8 + T cell response was directed against the mixture of NS3 peptides (Fig. 5A and B).
  • the quality of the T-cell response can be characterized in part by the cytokine secretion pattern and by its cytotoxic potential.
  • the greater magnitude of the antigen-specific response obtained in the DNA-HCV / MVA-HCV group compared to the MVA-HCV / MVA-HCV group was mainly due to a significant increase in the absolute frequencies of the CD8 T cell populations + which expressed on the surface CD107a, which co-expressed CD107a + TNF-a or the triple producers of CD107a + IL2 + TNF-a.
  • T-cell response induced by immunization. Because of that, We decided to analyze the phenotype of the immune response of MVA-HCV-induced T-cell memory in both the spleen and in the liver of the immunized animals 53 days after the last immunization by intracellular marking of multiparameter cytokines. At this time, splenocytes and intrahepatic immune cells (IHIC) isolated from the spleen and liver, respectively, were stimulated "ex vivo" for 6 hours with the different mixtures of HCV peptides and incubated with specific antibodies to identify cell lineage. T (CD3, CD4 and CD8), degranulation (CD107a), responding cells (IL-2, IFN- ⁇ and TNF- ⁇ ) and memory phenotype (CD127 and CD62L).
  • IHIC intrahepatic immune cells
  • the magnitude of the response of memory-specific CD4 + and CD8 + HCV-T cells was significantly greater in the groups of animals immunized with the MVA-HCV / MVA-HCV or DNA-HCV / MVA-HCV protocols than in their respective control groups, where antigen-specific responses were very low or absent (p ⁇ 0.005).
  • the immune response of memory induced by vaccination in both groups was mainly mediated by CD8 cells.
  • CD8 cells only in the MVA-HCV / MVA-HCV group could a similarly low response of CD4 + T cells be detected against peptide mixtures representing the Core (47%) and E (53% ).
  • the magnitude of the memory response of CD8 + T cells was high in both groups although significantly higher in the DNA-HCV / MVA-HCV group (p ⁇ 0.005).
  • 91% of the CD8 + T cell response detected was directed against the mixture of p7 + NS2 peptides and the rest of the response was distributed against the mixtures of NS3 (7%) and E (2%) peptides.
  • the detected CD8 + T cell response was mainly directed against the mixtures of NS3 (70%) and p7 + NS2 (19%) peptides while the rest of the response It was distributed against the combinations of NS4 (9%) and E (2%) (Fig. 6A and Fig. 7A).
  • CD8 + T cells The memory response of CD8 + T cells induced by both protocols was highly polyfunctional, with more than 90% of cells secreting Simultaneous two, three or four cytokines (Fig. 6B).
  • Fig. 6B The memory response of CD8 + T cells induced by both protocols was highly polyfunctional, with more than 90% of cells secreting Simultaneous two, three or four cytokines (Fig. 6B).
  • Fig. 6B The memory response of CD8 + T cells induced by both protocols was highly polyfunctional, with more than 90% of cells secreting Simultaneous two, three or four cytokines (Fig. 6B).
  • Fig. 6B The memory response of CD8 + T cells induced by both protocols was highly polyfunctional, with more than 90% of cells secreting Simultaneous two, three or four cytokines.
  • Fig. 6B The memory response of CD8 + T cells induced by both protocols was highly polyfunctional, with more than 90% of cells secreting Simultaneous two
  • CD8 + T cells The magnitude of the memory response of CD8 + T cells was high in both groups and very similar. As was the case with splenocytes, the CD8 + T cell response detected in intrahepatic immune cells from the animals of the MVA-HCV / MVA-HCV group was mainly directed against the mixture of p7 + NS2 peptides (97%) while in the animals of the DNA-HCV / MVA-HCV group the response was directed mainly against the peptide mixtures that NS3 (72%) and p7 + NS2 (24%) (Fig. 8A and Fig. 7B).
  • Example 3 The DNA-HCV / MVA-HCV combination induces a high, polyfunctional and long-lasting HCV-specific T cell response in HLA-A2 transgenic mice.
  • mice Because the heterologous DNA-HCV / MVA-HCV immunization protocol induced a greater magnitude of the HCV-specific response in C57BL / 6 mice (Example 2), we decided to evaluate this same approach in the HLA-A2 transgenic mice. These transgenic mice express a chimeric form of the HLA-A2.1 molecule and have previously been shown to enhance a repertoire of HCV-specific responses similar to those detected in the infected human population (23). The adaptive immune response was evaluated 10 days after the last dose using a multiparameter cytokine intracellular tick test.
  • Splenocytes isolated from immunized animals were stimulated "ex vivo" for 6 hours with a panel of 457 peptides (from 13 to 19-m overlapping in 1 or 12 amino acids) grouped into 6 mixtures of peptides: Core (28 peptides), E (83 peptides), p7 + NS2 (40 peptides), NS3 (98 peptides), NS4 (47 peptides) and NS5 (161 peptides) and incubated with specific antibodies to identify T-cell lineage (CD3, CD4 and CD8), degranulation (CD107a) and responding cells (IL-2, IFN- ⁇ and TNF- ⁇ ). Animals that received a first dose with empty DNA (--DNA) followed by a second dose with MVA-WT were used as controls.
  • CD8 T cells In the same way as was observed in mice of strain C57BL / 6, the immune response induced by splenocyte vaccination of transgenic mice was mediated by CD8 T cells (Fig. 9A).
  • the CD4 + T cell response detected had a low magnitude although directed against multiple combinations of peptides representing the different viral proteins, with mixtures of E (80%) and Core (1 1%) peptides being the most recognized.
  • the response of detected CD8 + T cells had a high magnitude and was mainly directed against the mixtures of NS3 (75%) and p7 + NS2 (19%) peptides (Fig. 9A and B). Both responses (CD4 and CD8) were highly polyfunctional with more than 50% of the cells simultaneously secreting two, three or four cytokines (Fig. 9C).
  • Activated CD4 + T cells co-produced mostly IL2 + IFN-y + TNF-a while CD8 + T cells had an enhanced cytotoxic profile represented by a high frequency of activated cells expressing CD107a on their surface.
  • the memory immune response was evaluated 53 days after the last immunization using a multiparameter cytokine intracellular marking test.
  • splenocytes isolated from the spleen of immunized animals were stimulated "ex vivo" for 6 hours with the different combinations of HCV peptides and incubated with specific antibodies to identify T-cell lineage (CD3, CD4 and CD8), degranulation (CD107a), responding cells (IL-2, IFN- ⁇ and TNF- ⁇ ) and memory phenotype (CD127 and CD62L).
  • the immune memory response induced by vaccination was mediated exclusively by CD8 T cells and was primarily directed against the combinations of p7 + NS2 (72%) and NS3 (28%) peptides (Fig. 10A and B).
  • the response of memory-specific CD8 + HCV-specific T cells was highly polyfunctional, with high frequencies of cells expressing simultaneously CD107a + IL2 + IFN-y + TNF-a (Fig. 10C) and with memory effector (TEM, 54%) or central memory (TCM, 34%) phenotypes (Fig. 10D).
  • the BHK-21 cell lines (golden hamster kidney fibroblastoid line, ATCC, Cat. No. CCL-10) and DF-1 (immortalized chicken embryonic fibroblast line, ATCC, Cat. No. CRL-12203) were cultured in minimal essential medium of Eagle modified by Dulbecco (DMEM) (Gibco BRL) supplemented with penicillin (100 U / ml; Sigma), streptomycin (100 ⁇ g / ml; Sigma), fungizone (0.5 U / ml; Gibco), glutamine (2 mM; Merck) and non-essential amino acids (Sigma) (complete DMEM) and 10% (v / v) fetal calf serum (FCS; Sigma).
  • DMEM minimal essential medium of Eagle modified by Dulbecco
  • penicillin 100 U / ml
  • streptomycin 100 ⁇ g / ml
  • fungizone 0.5 U / ml; Gibco
  • HepG2 human hepatocellular carcinoma cells were cultured in complete DMEM medium supplemented with 20 mM N-2- hydroxyethylpiperacin-N ' -2-ethanesulfonic acid buffer, pH 7.4 (HEPES) and 10% (v / v) of FCS).
  • Dendritic cells derived from human monocytes were obtained from peripheral blood lymphocytes (PBMC) previously obtained by Ficoll gradient separation (GE Healthcare) from the buffy coat of a healthy blood donor recruited by the Community Transfusion Center of Madrid.
  • CD14 + monocytes were purified by depletion using the Dynabeads ® Untouched TM human monocyte kit (Invitrogen) following the manufacturer's instructions.
  • the monocytes obtained were cultured for 7 days in 6-well culture plates (3 ⁇ 10 6 cells / well at 1 x 10 6 cells / ml) in complete RPMI 1640 medium supplemented with 50 ng / ml GMCSF, 20 ng / ml of IL-4 (both from Gibco-Life Technologies) and 10% (v / v) of FCS. All cell lines were maintained in an incubator at a temperature of 37 ° C (or 39 ° C for DF-1 cells) and a C0 2 percentage of 5%.
  • Viral infections were performed in the respective media supplemented with 2% (v / v) FCS.
  • the attenuated strain MVA obtained from the Ankara strain after 586 serial passes in chicken embryo fibroblasts (derived from clone F6 of pass 585 and provided by Dr. G. Sutter of the Institute of Molecular Virology in Kunststoff, Germany). Both viruses were grown on BHK-21 cells, purified through two 36% (w / v) sucrose mattresses and titrated by immunostaining according to the methodology previously described (39). The degree was made at least three times. Construction of the transfer plasmid pCyA-HCV 79
  • the transfer plasmid pCyA-HCV 7 9 was constructed for the generation of the MVA-HCV recombinant virus that expresses the structural (Core, E1, E2 and p7) and non-structural (NS3, NS4A, NS4B, NS5A and 201 amino acids of the N-terminal region of NS5B) of the H77 isolate of the HCV virus belonging to genotype 1 a.
  • Plasmid pCyA-HCV 7 9 is derived from plasmid pUC, designed for the selection of blue / white plates.
  • TK-R right and left (TK-L) flanking sequences of the thymidine kinase (TK) viral gene
  • E3L promoter directing the expression of the ⁇ -galactosidase ( ⁇ -Gal) selection marker and the gene of ampicillin resistance (AP).
  • AP ampicillin resistance
  • pE / L early / late synthetic promoter
  • This plasmid was ceded by Linong Zhang, of the Aventis group, Canada. It is a plasmid derived from pUC that contains a left arm of the TK gene, a multiple cloning site for the insertion of exogenous genes, a short repetition of the left arm of the TK gene, the E3L promoter directing the expression of the ⁇ -gal gene, a right arm of the TK gene and the ampicillin resistance gene.
  • - pCyA-20 It was constructed by the inventors from plasmid pLZAWl as depicted in Figure 1A. For this purpose, an 88 bp synthetic DNA band was generated that contained the early / late synthetic promoter sequence followed by a multiple cloning site and at each end contained restriction targets for the AscI enzymes (5 ' end) and Swal (end 3 ' ). Both the synthetic band and the pLZAWl vector were digested with the AscI and Swal enzymes, subsequently ligation being carried out using the enzyme T4 DNA ligase, finally generating the transfer vector pCyA-20.
  • plasmid pCyA-HCV 7 9 from the previously described plasmids is depicted in Figure 1A.
  • the DNA fragment containing 7.9 Kpb of the open reading pattern (ORF) of the genome of HCV virus belonging to genotype 1 a was cleaved by digestion with the EcoRI enzyme of plasmid pHCVI b, treated with Klenow DNA polymerase for generate blunt ends and inserted into the vector pCyA-20 previously digested with the restriction endonuclease Pmel and dephosphorylated by incubation with the enzyme alkaline phosphatase, thereby generating the transfer plasmid pCyA-HCV7.9.
  • the generated plasmid directs the insertion of the genes of interest into the TK locus of the genome of the attenuated MVA virus.
  • BHK-21 cells (3 x 10 6 ) were infected with the attenuated MVA-WT virus at a multiplicity of infection of 0.05 PFU / cell and subsequently transfected with 10 ⁇ g of plasmid pCyA-HCV / .g using lipofectamine (Invitrogen) as agent transfectant and following the manufacturer's instructions. At 72 hours post-infection, the cells were collected, frozen / thawed, sonicated and used for the selection of recombinant viruses.
  • lipofectamine Invitrogen
  • Recombinant MVAs containing HCV genes and transiently expressing the ⁇ -gal marker gene were selected during consecutive plaque purification passes on BHK-21 cells stained with 5- Bromo-4- chloro-3-indolyl-p-galactoside (X-Gal) (300 ⁇ g / ml).
  • Recombinant MVAs that contained the HCV genes and that had lost the marker gene were selected as viral stains not stained in BHK-21 cells in the presence of X-Gal.
  • TK-L and TK-R oligonucleotides which hybridize in the flanking sequences of the TK gene, in a reaction mixture containing 0.3 mM dNTPs, 2 mM MgCl 2 and 2.5 U of the enzyme Platinum Taq polymerase (Invitrogen).
  • the program includes an initial cycle of denaturation at 95 ° C for 7 minutes; 30 cycles of denaturation at 95 ° C for 1 minute, hybridization at 62 ° C for 30 seconds and extension at 72 ° C for 4 minutes and a final extension at 72 ° C for 7 minutes.
  • the PCR products were analyzed on a 0.7% agarose gel.
  • BHK-21 cell monolayers were infected with MVA-HCV or MVA-WT at 5 PFU / cell.
  • the cells were lysed in Laemmli buffer and fractionated cell extracts in 12% polyacrylamide denaturing gels (SDS-PAGE), transferred to nitrocellulose membranes and analyzed by Western-blot using polyclonal antibodies against Core (provided by Dr.
  • a stability test was performed by performing several successive passes of the MVA-HCV recombinant virus in BHK-21 cells. Monolayers of BHK-21 cells grown on P100 plates were successively infected at a multiplicity of 0.01 PFU / cell, starting from the P2 stock of the MVA-HCV (pass 7) to pass 1 1 (P1 1). Extracts of BHK-21 cells infected with passes 8, 9, 10 and 1 1 were analyzed by Western-blot.
  • the stability of the recombinant MVA-HCV was also evaluated by the analysis of individual plates. Monolayers of BHK-21 cells grown in 6-well plates were infected with serial dilutions of the lysate obtained in step 1 1. At 48 hours post-infection, the cells were stained with 0.01% neutral red (SIGMA) and 30 individual lysis plates were chopped which were resuspended in 0.5 ml of complete DMEM, frozen / thawed 3 times, sonicated and used (0.2 ml) for the infection of new BHK-21 cells grown in 12-well plates.
  • SIGMA neutral red
  • the cells were lysed in Laemmli buffer and fractionated cell extracts in 12% polyacrylamide denaturing gels (SDS-PAGE), transferred to nitrocellulose membranes and analyzed by Western-blot using a human polyclonal serum anti-HCV (courtesy of Dr. Rafael Fernández of the Ramón y Cajal Hospital, Madrid, diluted 1: 500).
  • a polyclonal antibody generated in goat against total human IgG conjugated to peroxidase (SIGMA) was used as a secondary antibody (diluted 1: 1000).
  • the detection of the protein bands recognized by the corresponding antibodies was carried out by means of the Luminol ECL ® system (GE Healthcare) exposing an X-OMAT autoradiographic film (Kodak). Viral Growth Analysis
  • MVA-HCV monolayers of BHK-21 cells grown in 12-well plates were infected at a multiplicity of 0.01 PFU / cell with MVA-WT or MVA-HCV. After an adsorption of 60 min. at 37 ° C, the inoculum was removed and the cells were incubated at 37 ° C and in a 5% C0 2 atmosphere with fresh DMEM medium enriched with 2% FCS. At different post-infection times (0, 24, 48 and 72 hours), the cells were collected by scraping and subjected to three cycles of freezing / thawing and sonication.
  • the viral titer in the different cell lysates was determined by immunostaining in DF-1 cells using the polyclonal anti-vaccinia antibody (National Biotechnology Center; diluted 1: 1000) followed by an anti-lgG rabbit-peroxidase conjugate (SIGMA; diluted 1: 1000).
  • SIGMA anti-lgG rabbit-peroxidase conjugate
  • HCV proteins To define the expression kinetics of HCV proteins, monolayers of BHK-21 or HepG2 cells grown in 12-well plates were infected at 5 PFU / cell with MVA-HCV or MVA-WT. At different post-infection times, the cells were collected and the cell precipitates fractionated in polyacrylamide denaturing gels (SDS-PAGE), transferred to nitrocellulose membranes and analyzed by Western-blot using a human anti-HCV polyclonal serum (assigned by the Dr. Rafael Fernández of the Ramón y Cajal Hospital, Madrid, diluted 1: 500). A polyclonal antibody generated in goat against total human IgG conjugated to peroxidase (SIGMA) was used as a secondary antibody (1: 1000 dilution).
  • SIGMA polyclonal antibody generated in goat against total human IgG conjugated to peroxidase
  • HeLa cells grown at full confluence with the MVA-HCV virus were infected at a multiplicity of infection of 5 PFU / cell.
  • the culture medium was removed and the cells were fixed for 2 h. to room temperature in a fixation solution containing 2% giufaraidehyde and 1% tannic acid in HEPES buffer.
  • the cells were collected in the presence of the fixative, centrifuged and the pelief was resuspended in 1 ml of HEPES buffer and processed by a conventional inclusion of cells for electron microscopy in the epoxy-resin EML-812 (Taab Laboratories, Adermaston, Berkshire, UK).
  • the pellet was cut with an ultra-microtome in the form of 70 nm thick eitraphine sections that were collected on copper gratings. Sections of cells infected with the MVA-HCV virus were analyzed in a transmission electron microscope model JEOL 101 1.
  • RNA was isolated using the RNeasy Mini kit (Qiagen) from dendritic cells derived from human monocytes infected at 0.3 or 1 PFU / cell with MVA-HCV or MVA-WT for 6 hours.
  • the reverse-transcription of 500 ng of total RNA was performed using the QuantiTect Reverse Transcription kit (Qiagen).
  • Quantitative PCR was carried out using the 7500 Real-Time PCR system (Applied Biosystems) and the Power SYBR Green PCR master mix (Applied Biosystems) as previously described (8).
  • RNAs of the IFN- ⁇ , IFIT1, IFIT2, RIG-I, MDA-5, IP-10 and Hprt genes were determined by RT-PCR using specific oligonucleotides. The expression of each of the genes was referred to relative to the expression of the Hprt gene in arbitrary units (U.A.). The samples were tested in duplicate and two different experiments were performed.
  • the pcDNA-Core, pcDNA-E1, pcDNA-E2 and pcDNA-NS3 plasmid DNAs encoding the Core, E1, E2 and NS3 viral proteins of the H77 isolate, genotype 1 a, were yielded by Dr. Ilkka Julkunen (Health Institute National Public of Finland).
  • the DNAs were purified using the Mega-Prep Endo-Free kit (Qiagen) resuspended in pyrogen-free double-distilled water.
  • peptides obtained through BEI Resources, NIAID, NIH representing the viral proteins Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A were used and NS5B of isolate J4 (genotype 1b; GenPept: AAC15722).
  • the peptides which were between 13 and 19 meters, overlapped in 1 or 12 amino acids and were grouped together to form combinations containing 28 to 53 peptides, depending on the case.
  • the Core protein was represented by the peptide mixture called Core (28 peptides).
  • E The combination of peptides called E represented the proteins E1 (28 peptides) and E2 (55 peptides).
  • the p7 and NS2 proteins were represented by the peptide mixture called p7 + NS2 (40 peptides).
  • the combination of peptides called NS3 represented the NS3 protein and consisted of the mixture NS3-1 (49 peptides) and NS3-2 (49 peptides).
  • the peptide mixture called NS4 represented the NS4A and NS4B proteins (47 peptides).
  • the NS5A and NS5B proteins were represented in the combination of peptides called NS5 and was formed by the mixture NS5-1 (55 peptides), NS5-2 (53 peptides) and NS5-3 (53 peptides).
  • Isolate J4 of genotype 1 b shares 85.7% homology with isolate H77 of genotype 1 a.
  • mice of strain C57BL / 6 were obtained from Jackson laboratories and were between 6 and 8 weeks old when the procedure began.
  • groups of 8 animals were inoculated with a dose of 10 7 PFU / mouse of MVA-WT or MVA-HCV by intraperitoneal route (ip). Two weeks later they received the same dose of the respective virus.
  • mice were inoculated with a dose of 200 ⁇ g of HCV-DNA (50 ⁇ g of pcDNA-Core + 50 ⁇ g of pcDNA-E1 + 50 ⁇ g of pcDNA-E2 + 50 ⁇ g of pcDNA- NS3) or 200 ⁇ g of empty DNA (200 ⁇ g of pcDNA) by intramuscular route (im).
  • a dose of 10 7 PFU / mouse of MVA-HCV or MVA-WT intraperitoneally The animals were sacrificed 10 days after the second dose (day 25) to characterize the adaptive immune response or 53 days after the second dose (day 67) to analyze the immune response of memory.
  • mice of strain C57BL / 6-Tg (HLA-A2.1) 1 Enge / J were obtained from Jackson laboratories and were between 6 and 8 weeks old when the procedure began.
  • This mouse model, transgenic for Tg (HLA-A2.1) 1 Enge expresses significant amounts of the HLA-A2.1 human MHC class I antigen in spleen, bone marrow and thymus cells.
  • Groups of 8 animals were inoculated with a dose of 200 ⁇ g of HCV-DNA (50 ⁇ g of pcDNA-Core + 50 ⁇ g of pcDNA-E1 + 50 ⁇ g of pcDNA-E2 + 50 ⁇ g of pcDNA-NS3) or 200 ⁇ g of DNA vacuum (200 ⁇ g of pcDNA) by intramuscular route (im).
  • the animals were inoculated with a dose of 10 7 PFU / mouse of MVA-HCV or MVA-WT by intraperitoneal route (ip).
  • the animals were sacrificed 10 days after the second dose (day 25) to characterize the adaptive immune response or 53 days after the second dose (day 67) to analyze the memory immune response.
  • Intracellular multiparameter cytokine marking 50 ⁇ g of HCV-DNA (50 ⁇ g of pcDNA-Core + 50 ⁇ g of pcDNA-E1 + 50 ⁇ g of pcDNA-E2 + 50 ⁇
  • the cells were washed, incubated with the different antibodies for surface molecules, fixed and permeabilized using the Cytofix / Cytoperm kit (BD Biosciences) and incubated with the different antibodies specific for molecules intracellular Dead cells were excluded using the Violet LIVE / DEAD stain kit (Invitrogen).
  • the following conjugated antibodies were used to determine cell lineage and cytokine expression: CD3-PE-CF594, CD4-APC-Cy7 or -Alexa 700, CD8-V500, IFN-and-PE-Cy7 or -PerCP-Cy5.5, IL-2-APC and TNF- ⁇ - ⁇ (all from BD Biosciences).
  • CD62L-Alexa 700 or -APC BD Biosciences
  • CD127-PerCP-Cy5.5 eBioscience
  • Cells were acquired using a GALLIOS flow cytometer (Beckman Coulter). Data analysis was performed with the FlowJo program version 8.5.3 (Tree Star, Ashland, OR).

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Abstract

Les virus recombinés de l'invention contiennent des séquences qui sont insérées dans le même site d'insertion du MVA et qui permettent l'expression simultanée de divers antigènes du VHC, en particulier les protéines matures structurales (Core, E1, E2 et p7) et non structurales (NS2, NS3, NS4A, NS4B, NS5A ainsi que les 201 acides aminés de la région N-terminale de NS5B). On obtient ainsi des virus recombinés stables qui permettent le déclenchement d'une réponse immunitaire contre une grande variété d'antigènes du VHC, appropriés pour être utilisés comme vaccins préventifs et thérapeutiques contre l'hépatite C.
PCT/ES2014/070246 2013-04-02 2014-03-31 Vecteurs recombinés dérivés du virus ankara modifié (mva) utilisés comme vaccins préventifs et thérapeutiques contre l'hépatite c WO2014162031A1 (fr)

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EP3928789A1 (fr) 2020-06-24 2021-12-29 Consejo Superior de Investigaciones Científicas (CSIC) Vaccin à base de mva contre le covid-19 exprimant les antigènes du sars-cov-2
WO2021260065A1 (fr) 2020-06-24 2021-12-30 Consejo Superior De Investigaciones Científicas (Csic) Vaccin à base de mva contre la covid-19 exprimant des antigènes de sras-cov-2
EP4108257A1 (fr) 2021-06-23 2022-12-28 Consejo Superior De Investigaciones Científicas Vaccin à base du virus de la vaccine ankara (mva) contre la covid-19 exprimant une protéine sars-cov-2 s stabilisée dans une préfusion
WO2022269003A1 (fr) 2021-06-23 2022-12-29 Consejo Superior De Investigaciones Cientificas Vaccin à base de mva exprimant une protéine s du sars-cov-2 stabilisée par préfusion

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3928789A1 (fr) 2020-06-24 2021-12-29 Consejo Superior de Investigaciones Científicas (CSIC) Vaccin à base de mva contre le covid-19 exprimant les antigènes du sars-cov-2
WO2021260065A1 (fr) 2020-06-24 2021-12-30 Consejo Superior De Investigaciones Científicas (Csic) Vaccin à base de mva contre la covid-19 exprimant des antigènes de sras-cov-2
EP4108257A1 (fr) 2021-06-23 2022-12-28 Consejo Superior De Investigaciones Científicas Vaccin à base du virus de la vaccine ankara (mva) contre la covid-19 exprimant une protéine sars-cov-2 s stabilisée dans une préfusion
WO2022269003A1 (fr) 2021-06-23 2022-12-29 Consejo Superior De Investigaciones Cientificas Vaccin à base de mva exprimant une protéine s du sars-cov-2 stabilisée par préfusion

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