WO2014162031A1 - Recombinant vectors based on the modified vaccinia ankara virus (mva) as preventive and therapeutic vaccines against hepatitis c - Google Patents

Abstract

The recombinant viruses of the invention contain sequences which are inserted at the same site of insertion as that of MVA and allow the simultaneous expression of various antigens of the HCV, concretely 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 proteins. In this way, stable recombinant viruses are obtained, which allow an immune response to be triggered against a large variety of antigens of the HCV, suitable for being used as preventive and therapeutic vaccines against hepatitis C.

Description

 RECOMBINANT VECTORS BASED ON THE MODIFIED ANKARA VIRUS (MVA) AS PREVENTIVE AND THERAPEUTIC VACCINES AGAINST HEPATITIS C SECTOR AND OBJECT OF THE INVENTION

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.

STATE OF THE TECHNIQUE Hepatitis C virus (HCV) infects more than 170 million people worldwide with 3 million new infections every year (Shepard, Finelli et al. 2005). After the acute phase of the infection, 20% of infected people eradicate the virus after weeks or months and are often asymptomatic. The remaining 80% will develop a chronic disease of which approximately 20% will eventually develop liver cirrhosis and 1 to 5% liver cancer (Lauer and Walker 2001, Afdhal 2004).

The current treatment protocol for patients infected with HCV is a combination of pegylated interferon-α and ribavirin. However, this treatment is prolonged over time, has a wide spectrum of side effects, fails frequently and is prohibitively expensive for developing countries (Grieve, Roberts et al. 2006). Because of this, a great effort has been made aimed at the development of new antiviral agents (Pawlotsky 2012). However, due to the cost, side effects and complexity of the treatment as well as the development of resistant HCV mutants and viral heterogeneity, antiviral therapy is not the solution to eradicate HCV infection. Therefore, there is an urgent need to develop a prophylactic vaccine that is effective and reduces the overall incidence of the disease. The generation of vaccines against HCV has proven to be complex. The fact that a significant proportion of patients infected during the acute phase spontaneously eradicate the infection together with a potent antiviral immunity suggests that the development of a prophylactic vaccine is an attainable goal.

The role of the HCV-specific T-cell response in the outcome of primary HCV infection has been extensively studied and although no parameter has been identified that correlates with protection, it is known that this arm of the immune response is decisive in Virus removal Comparative studies in humans have shown that potent CD4 ⁺ and CD8 ⁺ responses against multiple HCV regions and maintained over time are key since they are associated with spontaneous elimination of the virus (Thimme, Oldach et al. 2001, Afdhal 2004, Schulze zur Wiesch, Lauer et al. 2005).

Thus, based on virus-host interactions during HCV infection, there are probably three characteristics that must be shared by both prophylactic and therapeutic vaccine approaches to be successful. First, 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. Second, 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. Third, they must eradicate HCV from the liver without inducing any liver immunopathology so that they can be considered safe vaccines.

Currently, various vaccine candidates are being tested against HCV directed against a limited number of viral antigens, such as peptides, recombinant proteins, DNA and vaccines based on different vectors with different levels of success. Vaccines based on recombinant proteins that induce specific humoral responses to the envelope will hardly provide sterilizing immunity due to the genetic variability of the HCV envelope but may have a role in attenuating the course of the primary infection or serve as an adjuvant for a T-cell-based vaccine. Protein and peptide-based T-cell vaccines have induced weak T-cell responses, so this approach can probably only progress with the development of new adjuvants. 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. However, 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).

These trials demonstrate the viability of an HCV vaccine that produces specific memory responses in the immune system. However, they are limited by the use of a part of the virus genome, which in case of the development of mutations by the virus would limit its scope. Vaccine candidates that expand the intensity and diversity of the immune response generated against multiple regions of HCV are therefore necessary.

EXPLANATION OF THE INVENTION

BRIEF DESCRIPTION OF THE INVENTION 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.

DETAILED DESCRIPTION OF THE INVENTION

Due to the fact that the current vaccine candidates against HCV are directed against a limited number of viral antigens, 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 ). However, HCV genome expression by the WR strain had to be regulated in a controlled manner by the lac I repressor of E. coli, because when HCV proteins were produced by addition to IPTG cell cultures (which inhibits the repressor lac I), the cells they died by apoptosis and inhibition of protein synthesis, thereby aborting viral replication (Gómez, Vandermeeren et al. 2005). This procedure could not be transferred to an organism due to the impossibility of being able to administer IPTG continuously due to its rapid elimination.

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. Due to its behavior in cells and its immunogenicity in an organism, it is proposed that MVA-HCV can be a good vaccine candidate against HCV. Thus, 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

 ii) A nucleotide sequence corresponding to the HCV genome, inserted into the thymidine kinase (TK) locus of the MVA genome, and with sequence SEQ ID No 1; Y

where the MVA sequence that regulates expression (i) is operatively linked to the HCV nucleotide sequence (ii). 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.

As used herein, the term "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. In the present invention, 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. .

As used herein, the term "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. to 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). As is known in the state of the art, the "similarity" between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of a protein to a sequence of a second protein. The degree of identity between two proteins is determined using computer algorithms and methods that are widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined by the BLASTP algorithm (Altschul, Gish et al. 1990). As those skilled in the art will appreciate, variants or fragments can be generated using conventional techniques, such as mutagenesis, including the creation of discrete point mutation (s), or by truncation. For example, 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.

The term "vector", as used herein, 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.

The term "recombinant vector", as used in the present invention, 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. As used in this description, the term "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. 1 (Structural proteins, Core, E1, E2 and p7, and non-structural proteins, NS2, NS3, NS4A, NS4B, NS5A plus 201 amino acids in the N-terminal region of NS5B) are covalently linked to the nucleotide sequence of the MVA virus and that both are arranged so that the expression of the HCV nucleotide sequence occurs under the control of the transcription regulatory MVA virus sequence.

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);

(b) Construction of the MVA-HCV recombinant virus. BHK-21 cells would be infected with the attenuated MVA-WT virus and subsequently transfected with the plasmid pCyA-HCV obtained in the previous section using a transfectant agent according to the manufacturer's specifications. At 72 hours post-infection, the cells would be collected, frozen / thawed, sonicated and used for the selection of recombinant viruses;

(c) Selection of stable MVA-HCV viruses by successive passes in cell cultures. Recombinant MVA viruses containing the HCV genes of a given genotype and transiently expressing the β-gal marker gene (MVA-HCV (X-Gal ⁺ )) would be selected during Consecutive plate purification passes on BHK-21 cells stained with 5-bromo-4-chloro-3-indolyl-p-galactoside (X-Gal). After a series of consecutive purification passes, a number of recombinant plaques that efficiently express the HCV proteins of a given genotype and that would have lost the marker gene would be isolated. The selected end plate (designated as MVA-HCV stock P1) would be grown to generate a crude viral preparation (stock P2) from which a purified P3 stock of virus would be prepared according to the protocol described below.

Depending on the heterogeneity of the genomic sequence, 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. .

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:

(a) Generation of the transfer plasmid PCVA-HCVT P. 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. 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).

(b) Construction of the MVA-HCV recombinant virus. BHK-21 cells were infected with the attenuated MVA-WT virus and subsequently transfected with the plasmid pCyA-HCV _{7 9} using lipofectamine (Invitrogen) as a transfecting agent. At 72 hours post-infection, the cells were collected, frozen / thawed, sonicated and used for the selection of recombinant viruses.

(c) Selection of stable MVA-HCV viruses by successive passes in cell cultures. Recombinant MVA viruses containing HCV genes and transiently expressing the β-gal marker gene (MVA-HCV (X-Gal ⁺ )) were selected during consecutive plate purification passes on BHK-21 cells stained with 5 - Bromo-4-chloro-3-indolyl-p-galactoside (X-Gal). After 7 consecutive purification passes, 12 recombinant plaques were isolated that efficiently expressed HCV proteins and had lost the marker gene. 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. As is known to one skilled in the art, 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.

In the present invention, 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.

Thus, 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.

In a particular embodiment, 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. Thus, 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.

Thus, 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. Preferably, 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. Thus, 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.

Thus, 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. Optionally, 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.

In the present invention, the term "medicament or pharmaceutical composition" refers to any substance used for prevention, relief, treatment or cure of diseases in man and / or animals.

In a further preferred embodiment, the pharmaceutical composition or medicament of the invention further comprises a pharmaceutically acceptable carrier or excipient. In a more preferred embodiment, the pharmaceutical composition or medicament of the invention further comprises an adjuvant. In an even more preferred embodiment, the pharmaceutical composition or medicament of the invention further comprises another active ingredient (additional active ingredient).

The term "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. Thus, 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.

The term "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. The "pharmacologically 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. When the form of presentation is liquid, the pharmacologically acceptable carrier is the diluent.

Here, the term "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.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and Characteristics of the invention will be apparent in part from the description and in part from the practice of the invention. The following figures and examples are provided by way of illustration and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1.- (A) 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. (B) Analysis of the stability of HCV proteins expressed by MVA-HCV. Thirty individual plates isolated from MVA-HCV infected cells after 1 1 passes were grown in BHK-21 cells, infected lysed cells and proteins separated in gels for 12% SDS-PAGE and analyzed by Western blot using a human serum positive for antibodies to HCV.

Figure 2.- 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. (C) HCV protein expression by Western blot analysis. 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.

Figure 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. (B) Kinetics of HCV protein expression in BHK-21 and HepG2 cells. The expression of HCV proteins at the different post-infection times indicated was determined by Western-blot from samples not infected or infected with MVA-WT or MVA-HCV at 5 PFU / cell using a human serum positive for antibodies against to HCV. (C) Formation of membranous structures during infection (5 PFU / cell) of HeLa cells by MVA-HCV at 16 h. by electron microscopy.

Figure 4.- MVA-HCV infection blocks the innate immune response.

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).

Figure 5.- Adaptive 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. (TO)

Magnitude of the response of CD8 ⁺ T cells. 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-γ and / or IL-2 and / or TNF-α 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. ^*** p <0.001. The p-value indicates significantly higher responses with respect to the parental groups or between the DNA-HCV / MVA-HCV and MVA-HCV / MVA-HCV immunization groups. (B) Flow cytometry profiles showing the response of specific CD8 T cells against mixtures of p7 + NS2 or NS3 peptides. (C) Functional profile of the adaptive 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 <0.001.

Figure 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-γ and / or IL-2 and / or TNF-a (CD4) or CD107a and / or IFN-γ 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. Figure 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.

Figure 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-γ and / or IL-2 and / or TNF-α (CD4) or CD107a and / or IFN-γ 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. (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 <0.001. (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.

Figure 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-γ and / or IL-2 and / or TNF-a (CD4) or CD107a and / or IFN-γ 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. (C) Functional profile of the adaptive response of HCV-specific CD4 or 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 CD4 or 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.

Figure 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-γ and / or IL-2 and / or TNF-a (CD4) or CD107a and / or IFN-γ 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. (B) Flow cytometry profiles showing the response of specific CD8 T cells against mixtures of p7 + NS2 or NS3 peptides. (C) Functional profile of the memory response of specific CD8 T cells for HCV in 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. (D) Phenotypic profile of memory CD8 T cells specific for HCV. 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 ^" ). 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.

EMBODIMENT OF THE INVENTION

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 correct insertion of the HCV genome into the MK TK locus and the purity of the MVA-HCV recombinant virus was confirmed by PCR and sequencing Viral DNA extracted from BHK-21 cells infected with MVA-HCV was amplified using a pair of oligonucleotides that hybridize to the flanking regions of the TK locus. The expected PCR product size is shown in Fig. 2A. DNA extracted from cells infected with the MVA-WT virus was used as a control. As can be seen in Fig. 2B, 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.

Western blot analysis of BHK-21 cells infected with MVA-HCV confirmed that the HCV genome is efficiently transcribed during infection producing a viral polyprotein that is properly processed giving rise to mature structural proteins (Core, E1 and E2) and non-structural (NS4A, NS4B and NS5A) of HCV (Fig. 2C).

To confirm that the MVA-HCV recombinant virus can be maintained in cultured cells without losing the inserted HCV fragment an analysis of its stability was performed. 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. In addition, an extract of cells infected with the 1 1 pass of MVA-HCV was used for a new round of plaque purification in BHK-21 cells and the expression of HCV proteins in thirty isolated MVA-HCV plates was analyzed by Western blot. As can be seen in Fig. 1 B, all plaques (100%) express HCV proteins, indicating that the MVA-HCV recombinant virus is genetically stable.

Analysis of viral growth and expression kinetics of MVA-HCV

In order to establish whether the expression of HCV proteins affected viral replication in cultured cells, we analyzed the growth of MVA-HCV and MVA-WT virus in BHK-21 cells. As shown in Fig. 3A, the growth kinetics of both viruses was similar.

In addition, we carried out an expression kinetics of HCV proteins expressed by the MVA-HCV virus in BHK-21 cells and in the HepG2 liver line. Western blot analysis using a human polyclonal antibody revealed that HCV polyprotein is processed and detected at 2-4 hours post-infection in the form of smaller products in both cell lines (Fig. 3B).

Electron microscopy analysis of HeLa cells infected by MVA-HCV

Next, we wanted to determine by electron microscopy the effect of HCV polyprotein expression in the context of an MVA infection. To do this, we infected HeLa cells with the MVA-HCV virus at 5 PFU / cell and at 16 hours post-infection the cells were fixed and processed. As can be seen in Fig. 3C, in the cytoplasm of MVA-HCV infected cells, membranous structures are clearly seen in the cytoplasm and perinuclear region.

MVA-HCV infection blocks the innate immune response

Because it is widely accepted that in natural HCV infection some viral proteins block the innate immune response against the virus, we decided to evaluate the effect of HCV protein expression by the MVA-HCV virus on human dendritic cells. As can be seen in Fig. 4, by real time PCR of infected human dendritic cells for 6 hours, IFN-β expression levels, IFN-β-induced genes (IFIT1, IFIT2) and IP-chemokine 10 were lower than those obtained with the parental virus. The expression of RIG-I and MDA-5 cytosolic RLR receptors was also lower in cells infected with MVA-HCV compared to levels observed with the parental virus. These results indicate that, as occurs in natural infection, the expression of HCV proteins from the MVA-HCV virus significantly blocks the innate immune response. Example 2. MVA-HCV administered in homologous combination (MVA-HCV / MVA-HCV) or heterologous (DNA-HCV / MVA-HCV) induces in mice of strain C57BL / 6 a high, broad VHC-specific T cell response , multifunctional and long lasting.

Adaptive immune response

To characterize the immunogenicity of the MVA-HCV vector, we evaluated in 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).

For this, 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-γ and TNF-α). Animals that received a first dose with parental MVA (MVA-WT) or empty DNA (--DNA) followed by a second dose with MVA-WT were used as controls.

The percentages of T cells producing IFN-γ 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-γ and / or IL-2 and / or TNF-α determined the total response of CD8 ⁺ T cells.

The magnitude of the response of CD8 ⁺ HCV-specific T cells, determined as the sum of the individual responses obtained against the mixtures of Core, E, p7 + NS2, NS3, NS4 and NS5 peptides, 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). In the MVA-HCV / MVA-HCV group, 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. Based on the analysis of the secretion of IFN-γ, IL-2 and TNF-α, as well as the determination of the expression of CD107a on the surface of activated T cells as an indirect cytotoxicity marker, it is possible to identify 16 populations different from antigen-specific CD8 ⁺ T cells (Fig. 5C). The background value detected in the unstimulated control samples was subtracted for each population. The response of CD8 ⁺ HCV-specific T cells induced by both immunization protocols was highly polyfunctional, with more than 60% of the cells simultaneously secreting two, three or four cytokines. 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.

Memory immune response

An important requirement for prophylactic vaccination is the durability of the 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-γ and TNF-α) and memory phenotype (CD127 and CD62L).

In spleen, 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). As seen in Fig. 6A, the immune response of memory induced by vaccination in both groups was mainly mediated by CD8 cells. At this time, 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). In the splenocytes of the animals of the MVA-HCV / MVA-HCV group, 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. In the animals of the ADN-HCV / MVA-HCV group, 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).

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). Populations of antigen-specific CD8 ⁺ T cells activated after immunization predominantly co-produced CD107a + IFN-y + TNF-a or CD107a + IL2 + IFN-y + TNF-a. Previous studies have shown that the CD127 and CD62L markers define functionally distinct populations of antigen-specific memory T cells (4). Because of this, we have characterized the different stages of differentiation of activated CD8 ⁺ T cells according to the expression levels of CD127 and CD62L in central memory populations (TCM; CD127 ⁺ CD62L ⁺ ), memory effectors (TEM; CD127 ⁺ CD62L ^" ) or effectors (TE; CD127 D62L ^" ). In the splenocytes of animals immunized with both protocols (DNA-HCV / MVA-HCV or MVA-HCV / MVA-HCV), 70% of the activated CD8 ⁺ HCV-specific T cells had a memory effector phenotype (TEM) (Fig. 6C). Since the yield obtained from intrahepatic immune cells (IHIC) from the livers of immunized animals was low, we decided to analyze the memory response of T cells induced by MVA-HCV using only 4 stimuli: p7 + NS2, NS3, mix ( Core + E + NS4 + NS5) and RPMI. The pattern of the T-cell memory response was very similar to that obtained in the spleen. In both groups, the immune memory response induced by vaccination was mediated primarily by CD8 T cells. Only in the DNA-HCV / MVA-HCV group was it possible to detect a low CD4 ⁺ T cell response directed against peptide mixtures representing the Core + E + NS4 + NS5 (66%) and p7 + NS2 (34 %).

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). In liver, the memory response of CD8 ⁺ T cells induced by both protocols was highly polyfunctional, with more than 80% of the cells simultaneously secreting two, three or four cytokines (Fig. 8B). The population of antigen-specific CD8 ⁺ T cells activated after immunization predominantly co-produced CD107a + IFN-y + TNF-a and had a memory effector phenotype (TEM) (Fig. 8C).

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.

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-γ and TNF-α). Animals that received a first dose with empty DNA (--DNA) followed by a second dose with MVA-WT were used as controls.

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. On the other hand, 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. At this time, 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-γ and TNF-α) 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). As with the adaptive phase, 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). Materials and methods

Ethical requirements Animal studies were approved by the Ethical Committee for Animal Experimentation (CEEA-CNB) of the National Center for Biotechnology (CNB-CSIC, Madrid) in accordance with national and international regulations and the Royal Decree (RD 1201/2005) ( Permit number: 1 1048).

Cells and viruses

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 μg / 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). HepG2 human hepatocellular carcinoma cells (ATCC, Cat. No. HB-8065) 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 (moDCs) 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 ™ human monocyte kit (Invitrogen) following the manufacturer's instructions. The monocytes obtained were cultured for 7 days in 6-well culture plates (3 ^χ 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. For the generation of the recombinant MVA-HCV virus, the attenuated strain MVA (Antoine, Scheiflinger et al. 1998) 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 Munich, 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. It contains the right (TK-R) and left (TK-L) flanking sequences of the thymidine kinase (TK) viral gene, the E3L promoter directing the expression of the β-galactosidase (β-Gal) selection marker and the gene of ampicillin resistance (AP). Between the two flanking sequences is the early / late synthetic promoter (pE / L) directing the expression of HCV genes. The position of each of the components included in the plasmid is described in Table 1 below and its sequence is detailed in SEQ ID No 2.

Table 1.- Components of plasmid pCyA-HCV _{7 9}

 Thread Position Component

 Flank TK-left (TK-L) 410-908 complementary

T5NT signal for complementary β-gal 929-935 complementary β-gal ATG-TAA (936-4079) complementary

Complementary E3L 4080-4140 Promoter

TK- flank repeat

4151 -4498 complementary left (TK-L) Synthetic promoter E / L 4515-4554

 VHC coding region 7.9 ATG-TGA (4578-12470)

 T5NT signal 12480-12486

 Flank TK-right (TK-R) 12494-13239 complementary

Supplemental ATG-TAA ampicillin (14410-15270)

For the construction of this plasmid the following plasmids were used:

- pLZAWl: 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 β-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.

- pHCVI a: This plasmid was ceded by Charles M. Rice, New York

 (Kolykhalov, Agapov et al. 1997).

 It contains the complete genome that codes for structural (Core, E1, E2 and p7) and non-structural (NS3, NS4A, NS4B, NS5A and NS5B) proteins of HCV virus H77 isolate belonging to genotype 1 a with SEQ ID No 1.

The construction of plasmid pCyA-HCV _{7 9} from the previously described plasmids is depicted in Figure 1A. Briefly, 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.

Construction of the MVA-HCV recombinant 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 μg 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. Recombinant MVAs containing HCV genes and transiently expressing the β-gal marker gene (MVA-HCV (X-Gal ⁺ )) were selected during consecutive plaque purification passes on BHK-21 cells stained with 5- Bromo-4- chloro-3-indolyl-p-galactoside (X-Gal) (300 μg / ml). Recombinant MVAs that contained the HCV genes and that had lost the marker gene (MVA-HCV (X-Gal ^" )) were selected as viral stains not stained in BHK-21 cells in the presence of X-Gal. Purification The isolated plates were expanded in BHK-21 for 3 days and the crude viral extract obtained was used for the next plate purification step.After 7 consecutive purification passes 12 recombinant plates were isolated that efficiently expressed HCV proteins and that they had lost the marker gene.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 P3 stock of purified virus was prepared from infected BHK-21 cells at a multiplicity of infection of 0.05 PFU / cell through two 36% sucrose mattresses.This P3 stock, with a titer of 4.75 x 10 ⁹ PFU / ml, was the one used in s different immunization protocols. The sequence of the insert located in the thymidine kinase locus of this recombinant is detailed in SEQ ID No 3. The location of the different elements that make up the insert is indicated in Table 2 below.

Table 2.- Main components of the HCV insert in the MVA-HCV vector

PCR analysis of the MVA-HCV recombinant virus

To confirm the genetic homogeneity of the generated MVA-HCV virus and the integrity of the inserted genes, a PCR analysis of the viral DNA extracted from infected BHK-21 cells at a multiplicity of 5 PFU / cell was performed, using oligonucleotides that hybridize in the flanking TK regions of the insert. The sequence of the oligonucleotides used as primers and the position where they hybridize in the TK locus of the MVA-HCV7.9 virus are detailed in Table 3 and the estimated size of the generated PCR fragment is shown in Figure 2A. Table 3.- Oligonucleotides used for the characterization of the MVA virus-

HCV by PCR

Oligonucleotide Sequence Position TK-L 5 ^' -TGATTAGTTTGATGCGATTC-3 339-358

TK-R 5 ^' -TGTCCTTGATACGGCAG-3 ^' 8716-8732

For PCR analysis, 100 ng of viral DNA extracted from BHK-21 cells infected at a multiplicity of 5 PFU / cell with the MVA-HCV (stock P3) or MVA-WT viruses were used as a template using 100 ng of primers as primers. the 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.

Expression of HCV proteins from MVA-HCV

To determine the correct expression of HCV proteins from the MVA-HCV virus, BHK-21 cell monolayers were infected with MVA-HCV or MVA-WT at 5 PFU / cell. At 24 hours post-infection, 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. Ilkka Julkunen, National Public Health Institute of Finland, diluted 1: 1000), monoclonal antibodies against E1 (Acris Antibodies, diluted 1: 1000), E2 (GenWay Biotech, diluted 1: 500), NS4A, NS4B and NS5A (all from GenWay Biotech, diluted 1: 1000). The polyclonal antibodies generated in goat against total rabbit or mouse IgG conjugated with peroxidase (SIGMA) were used as secondary antibodies. The detection of the protein bands recognized by the specific antibodies was carried out by means of the Luminol ECL ^{® system} (GE Healthcare) exposing an X-OMAT autoradiographic film (Kodak).

Genetic stability of the MVA-HCV recombinant virus

To verify that the MVA-HCV recombinant virus could be passed successively without losing the expression of the inserted genes, 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. To perform these tests, monolayers of BHK-21 cells grown in 12-well plates were infected with 5 PFU / cell of the different viral extracts obtained in passes 8, 9, 10 and 1 1 (P8 → P1 1) of the recombinant virus MVA-HCV. The extracts were collected at 24 hours post-infection, fractionated into denaturing polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes and analyzed by Western-blot using a human anti-HCV polyclonal serum (provided by Dr. Rafael Fernández del 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. The detection of the protein bands recognized by the specific antibodies was carried out by means of the Luminol ECL ^{® system} (GE Healthcare) exposing an X-OMAT autoradiographic film (Kodak).

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. At 72 hours post-infection, 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

To analyze the growth profile of 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). Expression kinetics of HCV proteins from MVA-HCV

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). 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). Electron microscopy analysis of HeLa cells infected by MVA-HCV

HeLa cells grown at full confluence with the MVA-HCV virus were infected at a multiplicity of infection of 5 PFU / cell. At 16 hours post-infection, 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. After the fixation time, 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). Next, 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.

Analysis of RNA expression by quantitative RT-PCR

Total 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). The expression levels of the RNAs of the IFN-β, 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.

DNA vectors

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

For the evaluation of the immune response generated by MVA-HCV, mixtures of 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). 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.

Immunization scheme in mice of strain C57BU6

The mice of strain C57BL / 6 were obtained from Jackson laboratories and were between 6 and 8 weeks old when the procedure began. In the homologous immunization protocol 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. In the heterologous immunization protocol, groups of 8 animals were inoculated with a dose of 200 μg of HCV-DNA (50 μg of pcDNA-Core + 50 μg of pcDNA-E1 + 50 μg of pcDNA-E2 + 50 μg of pcDNA- NS3) or 200 μg of empty DNA (200 μg of pcDNA) by intramuscular route (im). Two weeks later the animals were inoculated with 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.

Immunization scheme in HLA-A2 transgenic mice

The 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 μg of HCV-DNA (50 μg of pcDNA-Core + 50 μg of pcDNA-E1 + 50 μg of pcDNA-E2 + 50 μg of pcDNA-NS3) or 200 μg of DNA vacuum (200 μg of pcDNA) by intramuscular route (im). Two weeks later 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

To determine the magnitude, polyfunctionality and phenotype of the antigen-specific T-cell response, a multiparameter intracellular marking test was used. After being isolated, both splenocytes and intrahepatic immune cells were allowed to stand overnight in complete RPMI 1640 medium supplemented with 10% FCS. The next day, they were seeded in a 96-well conical bottom plate (4 x 10 ⁶ cells / condition) and stimulated for 6 hours using a mixture of complete RPMI 1640 supplemented with 10% FCS containing 1 μΙ / ml of the GolgiPlug reagent (BD Biosciences), monesin, anti-CD107a-Alexa 488 (BD Biosciences) and 1 μg / ml of the different mixtures of HCV peptides. At the end of the stimulation period, 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-α-ΡΕ (all from BD Biosciences). The following conjugated antibodies were included for the analysis of the memory response phenotype: CD62L-Alexa 700 or -APC (BD Biosciences) and 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).

Statistic analysis

For the statistical analysis of the cytometry data we use an approach described above that corrects the response obtained in the stimulation control group (RPMI) and at the same time allows the calculation of confidence intervals and p values of the different hypotheses (14, 34 ). Only values significantly higher than the RPMI control are represented. The background values for the different intracellular cytokines in the non-stimulated controls never exceeded 0.05%. The analysis and presentation of the distribution of the different functional subpopulations was carried out using the SPICE program version 5.1, downloaded from itpj / exon.r-lai i. niH. ov (Roederer, Nozzi et al. 201 1). Significant p values of less than 0.05 were considered: ^* p <0.05; ^** p <0.01; ^*** p <0.005.

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Claims

1. - A polynucleotide characterized by comprising:

 i. a nucleotide sequence corresponding to the MVA virus that acts as an expression vector for HCV genes by regulating the transcription, translation and post-translational processing of said HCV genes in most tissues of the body, and i. a nucleotide sequence corresponding to the HCV genome, inserted into the thymidine kinase (TK) locus of the MVA genome; and where the MVA sequence that regulates expression (i) is operably linked to the HCV nucleotide sequence (ii).

2. - A polynucleotide according to claim 1 characterized in that the HCV virus sequence ii) corresponds to SEQ ID No. 1.

3. - A process for obtaining the polynucleotide according to claims 1 and 2 characterized in that it generally comprises the following steps:

 to. Generate the transfer plasmid pCyA-HCV,

 b. Build the MVA-HCV recombinant virus, and

 C. Select stable MVA-HCV viruses by successive passes in cell cultures.

4. - A method of obtaining the polynucleotide according to claim 3, characterized in that the transfer plasmid of a) is the plasmid pCyA-HCV7.9 whose sequence corresponds to SEQ ID No 2 and the recombinant virus MVA-HCV of b) is the MVA-HCV genotype 1 recombinant virus that contains SEQ ID No 1.

5. - Plasmid pCyA-HCV _{7 9} useful for obtaining variants of the polynucleotide of the invention according to claim 4 characterized in that its sequence corresponds to SEQ ID No 2.

6. A MVA-HCV recombinant vector, characterized in that it comprises the polynucleotide according to claims 1 and 2.

7. Host cell characterized in that it contains the polynucleotide of the invention according to claims 1 and 2.

8. - Host cell according to claim 7 characterized in that it is a cell belonging to the following group: mammalian cell, cell of avian origin.

9. - Host cell according to claim 10 characterized in that the cell of avian origin consists of a chicken embryonic fibroblast.

10. Use of the host cell to obtain the recombinant vector according to claim 6.

eleven . - Pharmaceutical composition characterized in that it comprises the recombinant vector according to claim 6.

12. - Pharmaceutical composition according to claim 1 characterized in that it comprises a pharmaceutically acceptable carrier or excipient.

13. - Pharmaceutical composition according to claims 1 1 and 12 characterized by comprising an adjuvant.

14. - Pharmaceutical composition according to claims 1 to 13 characterized by comprising another active ingredient.

15. Use of the recombinant vector according to claim 5 or the pharmaceutical composition according to any of claims 1 to 14 for the preparation of a medicament.

16. Use of the recombinant vector according to claim 5 or of the pharmaceutical composition according to any one of claims 1 to 14 for the preparation of a medicament for the prevention and / or treatment of an HCV infection.

17. Use according to any of claims 15 or 16 wherein the medicament is a vaccine.