WO2013011179A2

WO2013011179A2 - Recombinant vectors based on the modified vaccinia ankara (mva) virus, with deletion in the c6l gene, as vaccines against hiv/aids and other diseases

Info

Publication number: WO2013011179A2
Application number: PCT/ES2012/070521
Authority: WO
Inventors: Juan Francisco GARCÍA ARRIAZA; Carmen Elena Gómez Rodríguez; Mariano ESTEBAN RODRÍGUEZ
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2011-07-19
Filing date: 2012-07-11
Publication date: 2013-01-24
Also published as: ES2401904A2; ES2401904B1; ES2401904R1; WO2013011179A3

Abstract

The invention falls within the fields of molecular biology and biotechnology. It specifically relates to recombinant viruses based on the modified vaccinia ankara (MVA) virus, that express the antigens gp120 and Gag-Pol-Nef of the human immunodeficiency virus (VIH-1) of sub-type B (MVA-B), and wherein the C6L vaccinia gene has been deleted, said recombinant viruses having been designed to be used as vaccines against HIV/AIDS and other diseases.

Description

BINANT RECOMMENDED VECTORS BASED ON THE MODIFIED VIRUS OF ANKARA (MVA), WITH DELETION IN THE C6L GEN, AS VACCINES AGAINST HIV / AIDS AND OTHER TECHNICAL SECTOR DISEASES

The present invention falls within the fields of molecular biology and biotechnology. Specifically it refers to recombinant viruses based on the modified Ankara virus (MVA) that express the gp120 and Gag-Pol-Nef antigens of the human immunodeficiency virus (HIV-1) of subtype B (MVA-B), on which The C6L vaccinia gene has been deleted, and they have been designed to be used as vaccines against HIV / AIDS and other diseases. STATE OF THE PREVIOUS TECHNIQUE

According to WHO data, AIDS, caused by HIV-1, is a globally expanding pandemic, with high impact and severity on human health. Each year the number of new infections and deaths caused by AIDS increases, especially in undeveloped or developing countries. Therefore, the search for an effective vaccine against HIV-1 that can control infection and disease progression is one of the priorities in the scientific field.

One of the most promising vectors to be used as an effective vaccine against HIV-1 is MVA, a highly attenuated strain of vaccinia (Esteban, 2009. Hum. Vaccin. 5: 867-871). MVA has an excellent safety profile, and MVA recombinants expressing HIV-1 antigens induce protection after a challenge with simia / human immunodeficiency virus (SHIV), generating strong, broad, multifunctional and lasting immune responses against HIV-1 antigens in different animal models and clinical trials in humans (García-Ar aza et al., 2010. PLoS One 5: e12395; Gómez et al., 2009. Vaccine 27: 3165-3174; Harah et al., 2008. J Exp Med 205: 63-77; Mooij et al., 2008. J Virol 82: 2975-2988; Gómez et al., 2007. Vaccine 25: 2863-2885; Gomez et al. , 2007. Vaccine 25: 1969-1992; summary in Gómez et al., 2008. Curr Gene Ther 8: 97-120).

Previously, an MVA recombinant that expresses HIV-1 antigens of subtype B, gp120 (SEQ ID No. 15 of PCT / ES2006 / 0701 14) has been constructed in our laboratory as a monomeric protein and Gag-Pol-Nef polyprotein ( SEQ ID No. 16 of PCT / ES2006 / 0701 14) (virus called MVA-B) (Patent: PCT / ES2006 / 0701 14, publication date: 1/2/2007. Authors: Heeney, Jonathan; Mooij, Petra; Gómez Rodríguez, Carmen Elena; Nájera García, José Luis; Jiménez Tentor, Victoria; Esteban Rodríguez, Mariano). In a "DNA prime / MVA boost" immunization protocol in MVA-B mice it induced strong immune responses to HIV-1 antigens (García-Arriaza et al., 2010. PLoS One 5: e12395; Gómez et al., 2009. Vaccine 27: 3165-3174; Gómez et al., 2007. Vaccine 25: 2863-2885). In macaques, a similar construct expressing Env (gp120 of SHIV ₈ 9. ₆ p) and Gag-Pol-Nef (of SIV _maC 239) showed strong specific immune responses of CD4 ⁺ and CD8 ⁺ T cells with a preference for CD8 ⁺ , and high protection after a challenge with SHIV ₈ 9.6P (Mooij et al., 2008. J Virol 82: 2975-2988). In addition, the expression of HIV-1 antigens by MVA-B selectively induces in human dendritic cells the expression of different cellular genes that can act as regulators of immune responses against HIV-1 antigens (Guerra et al., 2010. J Virol 84: 8141-8152). Based on all these previous results, a Phase I clinical trial with MVA-B in healthy volunteers has been carried out in Spain.

However, despite all this background, new, more efficient MVA-B poxviral vectors that may increase the magnitude, amplitude, polyfunctionality and durability of immune responses against HIV-1 antigens are necessary and desirable. This is particularly relevant when a single immunogen is desirable for mass vaccination purposes to simplify immunization protocols and reduce manufacturing costs. Poxviral vectors express numerous genes encoding immunomodulatory proteins that interfere with the host's anti-viral response (Alcamí, 2003. Nat. Rev. Immunol. 3: 36-50). Therefore, deletion in the MVA-B poxviral vector of vaccinia genes that are known or suggest that they may have an immunomodulatory function is a general strategy that can increase the immunogenicity of the vector against HIV-1 antigens. One of these genes whose function is unknown but which we think may have immunomodulatory function is the C6L vaccinia gene, which is present in the genome of MVA vaccinia strains (gene named MVA 019L, SEQ ID No: 1), Western Reserve (WR) (gene called VACV-WR_022, SEQ ID No: 3), and Copenhagen (gene named C6L, SEQ ID No: 5), but absent in the New York Vaccinia Virus (NYVAC) lineage. It is postulated that C6L is an immediate-early gene, according to C6L promoter analysis and transcriptome analysis that detects C6 messenger RNA at 30 minutes post-infection (Assarsson et al., 2008. PNAS 105: 2140-2145 ). C6L encodes a 157 amino acid protein with a predicted molecular weight of 18.2 kDa. Bioinformatic analyzes, without experimental data, have grouped C6L into the family of poxviral genes called "BCL-2-like", which includes A46R, A52R, B15R (called B14R in WR) and K7R (González and Esteban, 2010. Virol. J 7:59), a family of proteins that inhibit at different levels the signaling pathway mediated by "Toll-like receptor (TLR)" (Bowie et al., 2000. PNAS 97: 10162-10167; Chen et al., 2006. J. Gen. Virol. 87: 1451-1458; Chen et al., 2008. PloS Pathog. 4: e22; Graham et al., 2008. PloS Pathog. 4: e1000128; Harte et al., 2003. J Exp. Med. 197: 343-351; Kalverda et al., 2009. J. Mol. Biol. 385: 843-853; Oda et al., 2009. Structure 17: 1528-1537; Schroder et al., 2008 EMBO J. 27: 2147-2157; Stack et al., 2005. J. Exp. Med. 201: 1007-1018). Protein 06 is present, although at low levels, in mature intracellular vaccinia virions (IMV) (Chung et al., 2006. J. Virol. 80: 2127-2140), and binds to KRT4 proteins (keratin 4 ), PDCD6IP and TNNI2 (troponin I) (Zhang et al., 2009. J. Proteome Res. 8: 431 1-4318). In addition, an epitope of 06 (amino acids 74-82 of SEQ ID No: 1 and SEQ ID No: 3) is highly immunogenic in BALB / c mice, and WR induces high levels of IFN-γ-secreting cells specific for C6L in mice. , similar to vaccinia peptides of E3L, F2L and A52R (Oseroff et al., 2008. J. Immunol. 180: 7193-7202). All these characteristics indicate that 06 can have an important immunomodulatory function antagonizing the TLR signaling path.

Therefore, in this invention a new vaccine candidate has been generated against HIV-1, called MVA-B AC6L, which contains a deletion in the MVA-B vector of the C6L vaccinia gene and which we demonstrate acts by inducing the production of type 1 interferon and activating in vivo T cell production of memory, which was not expected, but which represents an attractive alternative to increase the immunogenicity of MVA-based vaccine candidates.

BRIEF DESCRIPTION OF THE INVENTION

The present invention represents a new vaccine candidate against HIV-1, called MVA-B AC6L, which contains a deletion in the MVA-B vector of the C6L vaccinia gene. MVA-B AC6L replicates in cell cultures at the same level as the parental MVA-B virus, indicating that C6 is not essential for viral replication. Additionally, MVA-B AC6L induces innate immune responses by increasing IFN-β expression and IFN-α / β-induced genes (IFIT1 and IFIT2) in human THP-1 cells and monocyte-derived dendritic cells (moDCs), suggesting that C6 inhibits the IFN-β signaling pathway by blocking some unknown component involved in the induction of IFN-β. The deletion of the C6L gene in the MVA-B vector increases the humoral and memory T-cell immune response in mice against HIV-1 antigens. Thus, using a "DNA prime / MVA boost" immunization protocol in mice it was shown that, compared to MVA-B, MVA-B AC6L significantly increases the magnitude and polyfunctionality of the immune response of CD4 ⁺ and CD8 ⁺ memory T cells specific against HIV-1, which is mainly mediated by effector phenotype CD8 ⁺ T cells, in both immunization groups. The response of specific CD4 ⁺ memory T cells against HIV-1, induced by MVA-B and MVA-B AC6L was preferentially specific against Env. However, while MVA-B induces specific CD8 ⁺ memory T cell immune responses against Env and Gag, MVA-B AC6L preferentially induces specific CD8 ⁺ memory T cell immune responses against Gag-Pol-Nef (GPN ). In addition, compared to MVA-B, MVA-B AC6L increases antibody levels against Env.

Therefore, MVA-B AC6L represents a new vaccine candidate against HIV-1 that, not being obvious, has an immunological benefit by increasing IFN-β-dependent responses in human cells and increasing the humoral response and the magnitude and quality of the specific immune responses of T-cell memory against HIV-1 antigens. Starting from the examples included herein, where it has been experimented only with MVA-B AC6L, the results obtained would allow to extend the scope of protection to improve the immunogenicity of new recombinant vectors based on MVA, which express other heterologous antigens (examples , malaria, leishmania, hepatitis C virus and prostate cancer) by deleting the C6L gene, in order to be used as vaccines against these diseases. DETAILED DESCRIPTION OF THE INVENTION

The construction of MVA-B AC6L described in the present invention and the tests in which both its in vitro behavior, as well as the innate immune response induced in human cells, and its immunogenic capacity in mice against HIV-1 antigens are evaluated they are described in more detail with the help of the figures and the examples that appear hereinafter.

The present invention refers to a viral vector based on a recombinant MVA virus, characterized in that the nucleotide sequence encoding said vector comprises:

a) at least one mutation in the sequence SEQ ID No: 1 encoding the C6L protein, and

b) at least one nucleotide sequence encoding a heterologous antigen.

In a preferred embodiment of the present invention, reference is made to the viral vector defined above, characterized in that the mutation in the sequence SEQ ID No: 1 is a partial or total deletion. In an even more preferred embodiment, the mutation in the sequence SEQ ID No: 1 is a total deletion.

In another preferred embodiment of the present invention, reference is made to the viral vector defined above, characterized in that the nucleotide sequence encoding said vector comprises at least one nucleotide sequence encoding a heterologous antigen selected from the Next group: HIV antigen, malaria antigen, leishmaniasis antigen, hepatitis C virus antigen and prostate cancer antigen. In an even more preferred embodiment, the nucleotide sequence encoding said vector comprises the nucleotide sequences encoding the following HIV antigens: gp120 antigens (SEQ ID No. 15 of PCT / ES2006 / 0701 14) and Gag-Pol-Nef (SEQ ID No. 16 of PCT / ES2006 / 0701 14) of HIV of subtype B, or of any other subtype, under the control of the early / late viral synthetic promoter inserted into the TK viral locus.

The present invention also refers to the method of manufacturing the viral vector based on a recombinant MVA virus defined above, characterized by comprising the following steps:

a) construct a plasmid comprising in its nucleotide sequence the flanking sequences of recombination of the C6L gene, that is the sequences of the right flank and the left flank of SEQ ID No: 1, b) recombine a recombinant MVA virus, which contains at least a nucleotide sequence encoding a heterologous antigen, with the plasmid generated in a) which directs the mutation of SEQ ID No: 1, preferably said mutation is a partial or total deletion, and more preferably it is a total deletion, and

c) purifying the viral vector based on a recombinant MVA virus defined above, obtained in step b).

The present invention also refers to the use of the viral vector based on a recombinant MVA virus defined above, as an immunogen to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer. In a preferred embodiment, said vector is used as an immunogen to prevent or treat HIV disease.

In another embodiment of the present invention, reference is made to the use of the viral vector based on a recombinant MVA virus defined above, as part of an immunization protocol to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer, in which at least one dose of vaccination is administered to the individual. In a preferred embodiment, said vector is administered to the individual a single dose of vaccination to:

a) induce an immune response of specific CD4 + and CD8 + T cells from specific antigen memory, and / or

b) to induce IFN-β expression in innate immune cells. In another preferred embodiment of the present invention, said vector is used as part of an immunization protocol in which the individual is administered at least one dose of vaccination, to prevent or treat HIV disease.

In another preferred embodiment of the present invention, said vector is used as part of an immunization protocol in which the individual is administered at least one vaccination dose or several vaccination doses of a combination of heterologous vectors (proteins, DNA, VLPs, attenuated viral vectors) to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer.

The present invention also refers to an immunogenic composition or vaccine characterized in that it comprises at least one viral vector based on a recombinant MVA virus defined above and characterized in that the nucleotide sequence coding for said vector comprises:

b) at least one nucleotide sequence encoding a heterologous antigen. The present invention also refers to the use of the immunogenic composition or vaccine defined above, to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer. In a preferred embodiment, said immunogenic composition or vaccine is for preventing or treating HIV. The present invention refers to the following terms:

• The term "MVA recombinant virus" or "MVA" refers interchangeably to: a highly attenuated strain of vaccinia, called Ankara modified virus (MVA), obtained after 576 serial passes in chicken embryonic cell cultures (CEF).

• the term: "MVA-B recombinant virus" or "MVA-B parental virus" or "MVA-B poxviral vector" refers interchangeably to: poxvirus based on the modified Ankara virus (MVA) expressing the gp120 and Gag antigens -Pol- Nef of human immunodeficiency virus (HIV-1) of subtype B (MVA-B). The term "parental" is used when comparing MVA-B with MVA-B AC6L, since the deletion of C6L is made on the genome of MVA-B.

• The term "viral vector" or "recombinant viral vector" refers to: a modified virus that acts as a vehicle for introducing exogenous genetic material into a cell.

• the term: "MVA-B AC6L vector" refers to: an isolated MVA virus, characterized in that it expresses the gp120 and Gag-Pol-Nef antigens of the human immunodeficiency virus (HIV-1) of subtype B, and has a deletion in the polynucleotide sequence encoding an amino acid sequence homologous to that of the C6L gene (SEQ ID No: 1), and is presented for use as a pharmaceutical composition or medicament. The deletion of the C6L gene in MVA-B includes positions 19068 to 19541 of the MVA genome.

• the term "C6L vaccinia gene" refers to: a vaccinia gene, whose function was unknown prior to the presentation of this invention. The C6L gene is present in the genome of MVA vaccinia strains (gene named MVA 019L, positions 19068 to 19541 of the MVA genome, SEQ ID No: 1), Western Reserve (WR) (gene named VACV-WR_022, positions 16401 to 16856 of WR, SEQ ID No: 3), and Copenhagen (gene named C6L, positions 19484 to 19939, SEQ ID No: 5), but absent in the New York Vaccinia Virus (NYVAC) lineage.

• The term "plasmid" or "transfer vector" refers to a circular fragment of extrachromosomal double stranded circular or linear DNA, which is found inside almost all bacteria, and they act and replicate independently to bacterial chromosomal DNA and can be transferred from one bacterium to another. They are used as vectors in genetic manipulation.

The term "virus" refers to: a microscopic infectious entity that can only multiply within the cells of other organisms. More specifically, it refers to viruses belonging to the Poxviridae family, which is a family of interrelated DNA viruses called poxviruses, infectious for vertebrate and invertebrate animals,

The term "composition" refers to: those substances that are present in a given sample and in certain quantities.

The term "heterologous antigen" refers to: a molecule (usually a protein or a polysaccharide), which triggers the formation of antibodies and can cause an immune response. It preferably refers to those antigens of a viral species other than vaccinia that are inserted into a viral vaccinia vector.

The term "HIV antigens" refers to: human immunodeficiency virus (HIV) antigens, which are expressed from the recombinant MVA viruses that contain them. It includes any HIV antigen encoded from the HIV genome.

The term "malaria antigens" refers to: malaria antigens, which are expressed from the recombinant MVA viruses that contain them. It includes any malaria antigen encoded from the genome of the pathogen of the genus Plasmodium, which generates the disease, the term "leishmaniasis antigens" refers to: leishmania antigens, which are expressed from the recombinant MVA viruses that They contain them. It includes any leishmania antigen encoded from the genome of the pathogen of the genus Leishmania, which generates the disease.

The term "hepatitis C virus antigens" refers to: hepatitis C virus antigens, which are expressed from the recombinant MVA viruses that contain them. Include any virus antigen of hepatitis C encoded from the hepatitis C virus genome. The term "prostate cancer antigens" refers to: prostate cancer antigens (including PSCA and STEAP antigens), which are expressed as From the recombinant MVA viruses that contain them, the term "disease vaccine" refers to: an immunogenic or antigenic preparation or composition used to establish the immune system's response to a disease. They are prepared or combinations of immunogens or antigens that once inside the body cause the immune system response, through the production of antibodies, and generate immunological memory producing permanent or transient immunity.

The term "vaccine against prostate cancer" refers to: an antigen preparation used to establish the immune system's response to prostate cancer.

The term "mutation" refers to: an alteration or change in the genetic information of a living being and that, therefore, will produce a change of characteristics, which occurs suddenly and spontaneously, and that can be transmitted or inherited To the offspring. The genetic unit capable of mutating is the gene that is the unit of hereditary information that is part of the DNA.

The term "deletion" refers to: a special type of mutation that involves the loss of a DNA fragment, which can range from the loss of a single nucleotide (point deletion) to the loss of large regions, the term "deletion partial "refers to: the loss of a DNA fragment of a gene, which does not cause the total loss thereof,

The term "total deletion" refers to: the loss of a DNA fragment of a gene, which causes the total loss of it.

The term: "immunogen" refers to: those antigens that elicit an immune response.

The term: "immunization protocol" refers to: method used for the administration of an immunogenic agent or a vaccine to an organism to generate an immune response.

the term: "induce an immune response of CD4 + and CD8 + T cells from specific antigen memory "refers to: the ability of the vaccine administered in the immunization protocol to stimulate the host's immune response by generating CD4 + and CD8 + T cells that are capable of specifically recognizing the administered antigen, the term:" inducing the expression of IFN-β in innate immune cells "refers to: the ability of the administered vaccine to stimulate the production of IFN-β by innate immune cells, such as macrophages and dendritic cells.

The term: "Increase IFN-β-dependent responses in human cells" refers to: the ability of the administered vaccine to further stimulate those immune responses that occur as a result of the production of IFN-β by cells Innate human immune systems, such as macrophages and dendritic cells.

The term: "increase the magnitude and quality of memory immune responses of specific T cells against antigens" refers to: the ability of the administered vaccine to further stimulate the number and proportion of memory T cells that are capable of specifically recognizing the administered antigen. The term quality refers to the ability of memory T cells to be polyfunctional, that is, to secrete different cytokines at the same time, such as for example IFN-γ, IL-2, or TNFa.

The term: "EM and TEMRA phenotypes" refers to: two subpopulations of memory T cells, which are defined as a function of the expression of different surface markers such as CD44 and CD62L, and which are called EM ("Effector memory" or effector memory T cells. CD44 ⁺ / CD62L ^" )) and TEMRA (" Effector memory terminally differentiated "or terminally differentiated effector memory T cells. CD44 ^" / CD62L ^" ).

The term "homology", as used herein, refers to the similarity between two structures, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences. Two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure. If Particular of the invention MVA-B AC6L, although the deletion of the C6L vaccinia gene has been performed on MVA-B, is sufficient to allow a person skilled in the art to obtain new recombinant vectors based on MVA, which express other heterologous antigens (malaria , leishmania, hepatitis C virus and prostate cancer) by deleting the C6L gene, in order to be used as vaccines against these diseases. Similarly, although the deletion of C6L has been performed on an MVA vector that expresses HIV-1 antigens of subtype B (MVA-B) is sufficient to allow a person skilled in the art to obtain new recombinant MVA vectors that express antigens of HIV-1 of other subtypes (A, C, D, E, F, G, H and O). It is also sufficient to allow a person skilled in the art to obtain new recombinant poxviral vectors based on other strains that are included within the species, such as the Western Reserve and Copenhagen strains of vaccinia, which also have the C6L gene (SEQ) in their genomes. ID No: 3 and SEQ

ID No: 5, respectively).

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 those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention.

DESCRIPTION OF THE FIGURES

Figure 1. In vitro characterization of the MVA-B AC6L deletion mutant.

(A) MVA-B AC6L genome scheme, adapted from (Nájera et al., 2006. J Virol 80: 6033-6047; Antoine et al., 1998. Virology 244: 365-396). Different regions of the viral genome are indicated by capital letters. The right and left terminal regions are shown and under the map the different fragmented or deleted genes are drawn in black. Additionally, the deleted C6L gene is indicated. The sequences of the Gag-Pol-Nef subtype B-type HIV (from isolate IIIB) and gp120 (from isolate BX08) sequences are indicated under control of the early / late viral synthetic promoter (sE / L) inserted into the TK viral locus (J2R gene) (adapted from Gómez et al., 2007. Vaccine 25: 2863-2885). (B) PCR analysis of the C6L locus. DNA extracted from DF-1 cells infected at 2 PFU / cell with MVA, MVA-B or MVA-B AC6L was used for PCR analysis. DNA products corresponding to the parental virus or C6L deletion are indicated by an arrow on the right, including the expected size in base pairs. The molecular weight marker (1 Kb) with the corresponding sizes (in base pairs) is indicated on the left. The Mock column represents uninfected cells. (C) Expression of HIV-1 Bxo8gp120 and INBGPN proteins in infected DF-1 cells (2 PFU / cell) with MVA-B and MVA-B AC6L, at 24 hours post-infection. (D) MVA-B and MVA-B AC6L viral growth kinetics in infected DF-1 cells (0.01 PFU / cell) at different times and titrated by a plaque immunostaining assay with anti-WR antibodies. The average of 3 independent experiments is represented. (E) WR, MVA-B and MVA-B AC6L viral growth kinetics in infected HeLa cells (0.01 PFU / cell) at different times and titrated by a plaque immunostaining assay with anti-WR antibodies. The extracellular and intracellular viral titer is represented. Figure 2. Characterization of the expression and location of C6.

DF-1 cells were infected with 5 PFU / MVA-B and MVA-B AC6L (A) or WR and MVA (B) cells in the presence or absence of AraC (B). Cell extracts were collected at 24 hours post-infection (A) or at the indicated times (B) and analyzed by SDS-PAGE. C6 vaccinia protein was detected by Western blotting using a polyclonal rabbit serum against C6. (C) DF-1 cells were infected with WR, MVA, MVA-B or MVA-B AC6L for 18 hours. The location of C6 was analyzed by immunofluorescence and the cells were stained with DAPI (stained cell nucleus), purified rabbit polyclonal antibody anti-C6 and anti-14K.

Figure 3. MVA-B AC6L induces the production of IFN-β and IFN type I-induced genes in macrophages and dendritic cells. Human macrophages THP-1 (A) and moDCs (B, C, D) were infected with MVA, MVA-B and MVA-B AC6L (5 PFU / cell in A, and 0.2, 0.02 or 0.002 PFU / cell in B, C and D). At different post-infection times (1 h, 3 h and 6 h in A, 6 h in B), the RNA was extracted and the levels of IFN-β messenger RNA, genes induced by type I IFN (IFIT1 and IFIT2), Chemokines and HPRT were analyzed by RT-PCR. The results are expressed as the ratio between the levels of messenger RNA of the gene versus the levels of messenger RNA of HPRT. UA: arbitrary units. The data represent the mean ± standard deviation of the samples in duplicate. (C, D) human moDCs were infected with 0.2, 0.02 and 0.002 PFU / cell of MVA, MVA-B and MVA-B AC6L. 6 hours later, cell-free supernatants were collected to quantify IFN-β concentration by ELISA (C) and IFN type I concentration using the HL1 16 cell line, which expresses luciferase under the control of a promoter induced by IFN type I (D). The results are expressed by absorbance values at 450 nm (C), and in luciferase units (D). Data represent the mean ± standard deviation of duplicates and are representative of 2 independent experiments.

Figure 4. Interaction between the "Toll-like receptors" signaling pathway (TLRs) and vaccinia virus (VACV) proteins.

The TLR signaling path is composed of several TLRs located on the cell surface or in intracellular compartments. The activation of these TLRs leads to the recruitment of several adapter proteins and kinases and the subsequent activation of transcription factors (IRFs, NF-κΒ and ATF2 / c-Jun) that are translocated to the nucleus and activate the transcription of type I IFN and cytokines proinflammatory VACV encodes several immunomodulatory proteins that interfere with the TLR signaling pathway (indicated by rectangles): A46 inhibits TLR adapter molecules such as MyD88, TRIF, MAL and TRAM; A52 inhibits molecules such as IRAK-2 and TRAF6; K7 interacts with DDX3 and prevents the induction of I FN via TBKI / lkk-ε-; and B14 inhibits ΙΚΚβ phosphorylation and therefore prevents the activation of N F-KB.

Figure 5. Interaction between the "retinoic acid-inducible gene I (RIG-l) -like receptors" (RLRs) signaling pathway and vaccinia virus (VACV) proteins. The RLR signaling path is composed of the RIG-I, MDA5 and LGP2 proteins. The activation of these RLRs leads to the recruitment of various adapter proteins and kinases and the subsequent activation of transcription factors (IRFs and NF-κΒ) that are translocated to the nucleus and activate the transcription of type I IFN and proinflammatory cytokines. VACV encodes immunomodulatory proteins that interfere with the RLR signaling path (indicated by rectangles): K7 interacts with DDX3 and prevents the induction of IFN via TBKI / lkk-ε-; and C6 inhibits the phosphorylation of IRF3 although its mechanism is still unknown. Figure 6. MVA-B AC6L activates the IRF3 signaling path.

THP-1 cells differentiated with PMA were infected at a multiplicity of infection of 5 PFU / cell with MVA-B, MVA-B AC6L, MVA or with medium (Mock). At different post-infection times the cells were washed with cold PBS and lysed for 10 minutes at 4 ° C with "Cell Lysis Buffer" (Cell Signaling). The reaction mixtures were centrifuged for 10 min at 13,000 rpm. The protein concentration of the supernatants was determined using the "bicinchoninic acid protein assay" (Pierce Biotechnology). 30 g of total protein was loaded into 10% polyacrylamide gels (w / v) and transferred to nitrocellulose membranes. The membranes were incubated with antibodies directed against phospho-IRF3 (Cell Signaling) and α-tubulin (Cell Signaling). After washing, the membranes were incubated with a secondary antibody conjugated to HRP (Pierce). The signal was revealed using the "ECL Western blotting Analysis System" (GE Healthcare). Figure 7. Immunization with MVA-B AC6L increases the magnitude of memory immune responses of specific CD4 ⁺ and CD8 ⁺ T cells against HIV-1.

Splenocytes were collected from mice (n = 4 per group) immunized with DNA-B / MVA-B, DNA-B / MVA-B AC6L or ϋΝΑ-φ / MVA, 53 days after the last immunization. (A) IFN-γ secreting splenocytes specific to the HIV-1 Gag-B peptide were quantified by an ELISPOT assay. Data represent the mean ± standard deviation of triplicates. ^** represents p <0.005. (BD) Phenotypic analysis by flow cytometry of CD4 ⁺ T cells and specific CD8 ⁺ against HIV-1 Env, Gag and GPN antigens. The expression of CD44 and CD62L was used to identify the sub-populations of memory called "central memory" (CM: CD44 ⁺ / CD62L ⁺ ), "effector memory" (MS: CD44 ⁺ / CD62L ^" ) and" terminally differentiated effector memory "(TEMRA: CD447CD62L ^" ). The production of IFN-γ and IL-2 was analyzed by intracellular marking (ICS). (B) A representative flow cytometry test is shown. Sub-populations of memory T cells are drawn by density drawings. The dots represent IFN-y and IL-2 producing T cells. (C) The percentage of specific CD4 ⁺ and CD8 ⁺ memory T cells against HIV-1 Env, Gag and GPN antigens is represented. The frequencies were calculated representing the number of memory T cells producing IFN-y and / or IL-2 versus the total number of splenocytes CD4 ⁺ and CD8 ⁺ . The values of the unstimulated controls were subtracted in all cases. ^** represents p <0.005. (D) The circles represent the proportion of CM, MS and TEMRA within the specific CD4 ⁺ and CD8 ⁺ memory T cells against HIV-1 Env, Gag and GPN antigens. (AD) The data are derived from an experiment, representative of 2 experiments performed.

Figure 8. Immunization with MVA-B AC6L increases the polyfunctionality of specific immune responses of CD4 ⁺ and CD8 ⁺ T cells specific to HIV-1.

Splenocytes were collected from mice (n = 4 per group) immunized with DNA-B / MVA-B, DNA-B / MVA-B AC6L or ϋΝΑ-φ / MVA, 53 days after the last immunization and analyzed by cytometry of flow as described in Figure 7. The polyfunctionality of the specific CD4 ⁺ (left part) and CD8 ⁺ memory T cells (right side) against HIV-1 Env + Gag + GPN antigens is defined based on IFN-y and / or IL-2 production. All possible combinations of responses are shown on the X axis. The percentages of IFN-y and / or IL-2 producing memory T cells among the total CD4 ⁺ and CD8 ⁺ T cells are shown on the Y axis. ^{* *} represents p <0.005. The circles summarize the data. Each part of the circle corresponds to the proportion of CD4 ⁺ or CD8 ⁺ T cells producing IFN-γ, IL-2 or IFN-y + IL-2 within the total of specific CD4 ⁺ or CD8 ⁺ memory T cells versus HIV-1 antigens. The size of Each circle represents the magnitude of the specific induced memory immune response against HIV-1 antigens.

Figure 9. Immunization with MVA-B AC6L increases the humoral immune response induced against HIV-1 gp120 protein.

Serum was collected from mice (n = 3 per group) immunized with DNA-B / MVA-B, DNA-B / MVA-B AC6L or ϋΝΑ-φ / MVA, 53 days after the last immunization. Anti-gp120 antibody titers were determined by ELISA. The titles represent the last dilution of the serum that gave a signal 3 times higher than the signals obtained with the serum of naive mice. The dotted line represents the ELISA detection limit. The horizontal bar represents the average value and the values obtained by each mouse are represented by circles. * represents p <0.05. BIBLIOGRAPHY

Esteban M (2009) Attenuated poxvirus vectors MVA and NYVAC as promising vaccine candidates against HIV / AIDS. Hum Vaccin 5: 867-871. Garcia-Arriaza J, Najera JL, Gómez CE, Sorzano CO, Esteban M (2010) Immunogenic profiling in mice of a HIV / AIDS vaccine candidate (MVA-B) expressing four HIV-1 antigens and potentiation by specific gene deletions. PLoS One 5: e12395. Gómez CE, Najera JL, Sánchez R, Jiménez V, Esteban M (2009) Multimeric soluble CD40 ligand (sCD40L) efficiently enhancing HIV specific cellular immune responses during DNA prime and boost with attenuated poxvirus vectors MVA and NYVAC expressing HIV antigens. Vaccine 27: 3165-3174. Harari A, Bart PA, Stohr W, Tapia G, García M, et al. (2008) An HIV-1 clade C DNA prime, NYVAC boost vaccine regime induces reliable, polyfunctional, and long-lasting T cell responses. J Exp Med 205: 63-77.

Mooij P, Balla-Jhagjhoorsingh SS, Koopman G, Beenhakker N, van Haaften P, et to the. (2008) Differential CD4 + versus CD8 + T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates. J Virol 82: 2975-2988. Gómez CE, Najera JL, Jiménez EP, Jiménez V, Wagner R, et al. (2007) Head-to-head comparison on the immunogenicity of two HIV / AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1 BX08 gp120 and HIV-1 (IIIB) Gag- Pol-Nef proteins of clade B. Vaccine 25: 2863-2885.

Gómez CE, Najera JL, Jiménez V, Bieler K, Wild J, et al. (2007) Generation and immunogenicity of novel HIV / AIDS vaccine candidates targeting HIV-1 Env / Gag-Pol-Nef antigens of clade C. Vaccine 25: 1969-1992. Gómez CE, Najera JL, Krupa M, Esteban M (2008) The poxvirus vectors MVA and NYVAC as gene delivery systems for vaccination against infectious diseases and cancer. Curr Gene Ther 8: 97-120.

Guerra S, González JM, Climent N, Reyburn H, Lopez-Fernandez LA, et al. (2010) Selective induction of host genes by MVA-B, a candidate vaccine against HIV / AIDS. J Virol 84: 8141-8152.

Alcami A (2003) Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol 3: 36-50.

Assarsson E, Greenbaum JA, Sundstrom M, Schaffer L, Hammond JA, et al. (2008) Kinetic analysis of a complete poxvirus transcriptome reveal an immediate-early class of genes. Proc Nati Acad Sci USA 105: 2140-2145. González JM, Esteban M (2010) A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history. Virol J 7: 59. Bowie A, Kiss-Toth E, Symons JA, Smith GL, Dower SK, et al. (2000) A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling. Proc Nati Acad Sci USA 97: 10162-10167. Chen RA, Jacobs N, Smith GL (2006) Vaccinia virus strain Western Reserve protein B14 is an intracellular virulence factor. J Gen Virol 87: 1451-1458.

Chen RA, Ryzhakov G, Cooray S, Randow F, Smith GL (2008) Inhibition of IkappaB kinase by vaccinia virus virulence factor B14. PLoS Pathog 4: e22.

Graham SC, Bahar MW, Cooray S, Chen RA, Whalen DM, et al. (2008) Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB rather than apoptosis. PLoS Pathog 4: e1000128. Harte MT, Do IR, Maloney G, Gray P, Reading PC, et al. (2003) The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense. J Exp Med 197: 343-351.

Kalverda AP, Thompson GS, Vogel A, Schroder M, Bowie AG, et al. (2009) Poxvirus K7 protein adopts a Bcl-2 fold: biochemical mapping of its interactions with human DEAD box RNA helicase DDX3. J Mol Biol 385: 843-853.

Oda S, Schroder M, Khan AR (2009) Structural basis for targeting of human RNA helicase DDX3 by poxvirus protein K7. Structure 17: 1528-1537.

Schroder M, Baran M, Bowie AG (2008) Viral targeting of DEAD box protein 3 reveal its role in TBK1 / IKKepsilon-mediated IRF activation. EMBO J 27: 2147-2177.

Stack J, Do IR, Schroder M, Bartlett NW, Maloney G, et al. (2005) Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adapters and contributes to virulence. J Exp Med 201: 1007-1018.

Chung CS, Chen CH, Ho MY, Huang CY, Liao CL, et al. (2006) Vaccinia virus proteome: identification of proteins in vaccinia intracellular virus mature virion Partiols J Virol 80: 2127-2140.

Zhang L, Villa NY, Rahman MM, Smallwood S, Shattuck D, et al. (2009) Analysis of vaccinia virus-host protein-protein interactions: validations of yeast two-hybrid screenings. J Proteome Res 8: 431 1-4318.

Oseroff C, Peters B, Pasquetto V, Moutaftsi M, Sidney J, et al. (2008) Dissociation between epitope hierarchy and immunoprevalence in CD8 responses to vaccinia virus western reserve. J Immunol 180: 7193-7202.

Najera JL, Gómez CE, Domingo-Gil E, Gherardi MM, Esteban M (2006) Cellular and biochemical differences between two attenuated poxvirus vaccine candidates (MVA and NYVAC) and role of the C7L gene. J Virol 80: 6033-6047 Antoine G, Scheiflinger F, Dorner F, Falkner FG (1998) The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses. Virology 244: 365-396.

EXAMPLES

The invention will now be illustrated by a series of tests carried out by the inventors, which show the good immunogenicity of the MVA-B AC6L vaccine. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

Example 1.- Generation of the invention MVA-B AC6L.

The function of the C6L vaccinia gene was unknown until the moment of presenting this invention, although it was predicted by bioinformatic analogies that it could have an immunomodulatory function, which does not mean that the similarity between prediction and demonstration is obvious (González and Esteban, 2010 Virol J 7: 59). In fact we have found that C6L exerts functions different from those postulated, acting on the interferon pathway and inducing specific memory cells. Therefore, to analyze the possible role immunomodulator of C6L, we have constructed a deletion mutant that lacks the vaccinia C6L gene from the recombinant virus MVA-B, previously described (Gómez et al., 2007. Vaccine 25: 2863-2885; Patent: PCT / ES2006 / 0701 14, publication date: 1/2/2007 Authors: Heeney, Jonathan; Mooij, Petra; Gómez Rodríguez, Carmen Elena; Nájera García, José Luis; Jiménez Tentor, Victoria; Esteban Rodríguez, Mariano) and expressing the antigens of HIV-1 subtype B Env, Gag, Pol and Nef. The generated recombinant virus, presented in this invention, has been referred to as MVA-B AC6L. A schematic diagram of the MVA-B AC6L deletion mutant is depicted in Figure 1 A.

The manufacturing method of MVA-B AC6L includes the following eíapas:

a) construct a plasmid comprising in its nucleoid sequence the sequences of the right flank and the left flank of the C6L gene (SEQ ID No: 1),

b) recombine the MVA-B recombinant virus, which contains the nucleotide sequences that code for the HIV antigens of subtype B gp120 and Gag-Pol-Nef, with the plasmid generated in a) which directs the deletion of the C6L gene (SEQ ID No: 1), and

c) purify the viral vector called MVA-B AC6L, based on a recombinant MVA-B virus with deletion of the C6L gene (SEQ ID No: 1), obtained in step b).

The plasmid or transfer vector called pGem-RG-C6L wm was constructed to be able to generate the MVA-B AC6L deletion mutant, which has a deletion in the vaccinia C6L gene (The MVA 019L gene of MVA, SEQ ID No: 1 , is equivalent to the VACV-WR_022 gene of the vaccinia Wesirin Reserve (WR) strain, SEQ ID No: 3, and to the C6L gene of the Copenhagen vaccinia strain, SEQ ID No: 5. For simplification, the corresponding nomenclature is used to the Copenhagen genes to refer to the MVA genes). pGem-RG-C6L wm was obtained by sequential cloning of five DNA fragments that contain the dsRed2, rsGFP and flanking (left and right) recombination sequences of the C6L gene in plasmid pGem-7Zf (-) (Promega). Firstly, the conspiracy of plasmid pGem-Red-GFP wm (4540) was performed pb), which contains the dsRed2 and rsGFP genes under the control of an early / late synthetic viral promoter (E / L) and which was previously described (García-Arriaza et al., 2010. PLoS One 5: e12395). Briefly, the dsRed2 gene under the control of an early / late synthetic viral promoter (E / L) was amplified by PCR of plasmid pG-dsRed2 using Red2-B oligonucleotides (SEQ ID No: 15. 5 ^' - GAACTAGGATCCTAA CTCGAGAAA-3 ^' ) (includes the restriction site Bam Hl) and Red2-N (SEQ ID No: 16. 5 ^' -ATTAGTATG C ATTTATTTATTTAG G-3 ^' ) (comprises the restriction site Nsi I) (785 bp), digested with Bam Hl and Nsi I and cloned into plasmid pGem-7Zf (-) previously digested with the same restriction enzymes to generate pGem-Red wm (3740 bp). The rsGFP gene under the control of an early / late synthetic viral promoter (E / L) was amplified by PCR of plasmid pG-dsRed2 using GFP-X oligonucleotides (SEQ ID No: 17. 5 ^' -C GTTG GTCTAGAG AG AAAA ATTG -3 ^' ) (includes the restriction site Xbal) and GFP-E (SEQ ID No: 18. 5 ^' -CTATAGAATTCTCAAGCTATGC-3 ^' ) (comprises the restriction site Eco Rl) (832 bp), digested with Xba I and Eco Rl and cloned into plasmid pGem-Red wm previously digested with the same restriction enzymes to generate pGem-Red-GFP wm (4540 bp).

Subsequently, the MVA-B genome was used as a template for PCR amplification of the right flank of the C6L gene (Nucleotides 18689-19079 in the MVA genome. 391 bp), using the oligonucleotides RFC6L-Aatll-F (SEQ ID No : 7. Nucleotides 18689-18714 in the MVA genome) (comprising the Aatll restriction site) and RFC6L-Xbal-R (SEQ ID No: 8. Nucleotides 19054-1979 in the MVA genome) (comprises the restriction site Xbal) This right flank was digested with Aatll and Xbal and cloned into the plasmid pGem-Red-GFP wm previously digested with the same restriction enzymes to generate pGem-RG-RFsC6L wm (4898 bp). The repeated right flank of the C6L gene (Nucleotides 18689-19079 in the MVA genome. 391 bp) was amplified by PCR from the MVA-B genome with the oligonucleotides RF ^' C6L-Xmal-F (SEQ ID No: 9. Nucleotides 18689-18714 in the MVA genome) (comprising the Xmal restriction site) and RF ^' C6L-Clal-R (SEQ ID No: 10. Nucleotides 19054-1979 in the MVA genome) (comprises the Clal restriction site ), digested with Xmal and Clal and inserted into the plasmid pGem-RG-RFsC6L wm digested with Xmal / Clal to generate pGem-RG-RFdC6L wm (5259 bp). The left flank of the C6L gene (Nucleotides 19530-19942 in the MVA genome. 413 bp) was PCR amplified from the MVA-B genome with oligonucleotides LFC6L-Clal-F (SEQ ID No: 1 1. Nucleotides 19530-19555 in the MVA genome) (comprising the Clal restriction site) and LFC6L-BamHI- R (SEQ ID No: 12. Nucleotides 19917-19942 in the MVA genome) (comprising the BamH I restriction site), digested with Clal and BamHI and inserted into the plasmid pGem-RG-RFdC6L wm digested with Clal / Bam Hl . The resulting plasmid pGem-RG-C6L wm (5642 bp) was confirmed by DNA sequence analysis and directs the deletion of the C6L gene from the MVA and MVA-B genome.

Once the plasmid pGem-RG-C6L wm has been generated, obtaining the invention MVA-B AC6L is carried out in cell cultures by means of a recombination process between the MVA-B virus and the plasmid pGem-RG-C6L wm, which contains the right and left flanks of the C6L gene. Thus, MVA-B AC6L was constructed by selection in cell cultures of viral plaques that coexpress dsRed2 / rsGFP (express proteins with red and green fluorescence respectively), using the dsRed2 and rsGFP genes as transient selection markers, as previously described. (García-Arriaza et al., 2010. PLoS One 5: e12395). 3 x 10 ⁶ DF-1 cells were infected with MVA-B at a multiplicity of infection of 0.05 PFU / cell and then transfected 1 h later with 6 g of plasmid pGem-RG-C6L wm DNA using Lipofectamine (Invitrogen) according to the manufacturer's instructions. After 72 hours, the cells were collected, lysed by freeze-thaw cycles, sonicated and used for the selection of recombinant viruses. The MVA-B AC6L deletion mutant was selected from the viral progeny obtained after 6 consecutive rounds of plaque purification in DF-1 cells and during this process the plaques were selected from those with red / green fluorescence. Thus, in the first two passes, the viruses were selected from plaques expressing both fluorescent proteins (dsRed2 and rsGFP, red and green fluorescence). In the next two passes, the viral progeny of the selected plaques only expresses a fluorescent marker (Red2 or GFP) and in the last two passes (6 passes in total) the viruses of the selected plaques do not express any fluorescent marker due to loss of the dsRed2 and rsGFP genes. Thus, after 6 consecutive rounds of plaque purification in DF-1 cells, the MVA-B deletion mutant was obtained AC6L and the deletion of the C6L gene was confirmed by PCR amplification of the C6L locus, using oligonucleotides RFC6L-Aatll-F and LFC6L-BamHI-R (described previously) and subsequent analysis by DNA sequencing. Thus, the C6L gene (See SEQ ID No: 1.474 nucleotides, positions 19068-19541 in the MVA genome) has been completely deleted in the MVA-B AC6L genome. The deletion of the C6L gene in MVA-B includes positions 19068 to 19541 of the MVA genome.

Subsequently, the MVA-B AC6L recombinant virus obtained was grown in DF-1 cells to obtain a viral preparation called P2, which was grown in chicken embryonic cells (CEF), and purified by centrifugation through two sucrose mattresses at 36% (w / v) in 10 mM Tris-HCI pH 9, as previously described (Ramírez et al., 2000. J Virol, 74 (2): 923-933. MVA-B AC6L was titled DF-1 cells by an immunostaining assay, using a rabbit polyclonal antibody against the WR vaccinia strain (National Center for Biotechnology; 1: 1000) followed by anti-rabbit-HRP (Sigma; 1: 1000), as It has been previously described (Antoine et al., 1998. Virology 244: 365-396). The preparation of MVA-B AC6L is free of mycoplasmas or bacteria. Starting from this technique of generating recombinant MVA viruses with C6L deletion, included herein, and although it has only been experimented with MVA-B AC6L, plasmid pGem-RG- C6L wm (or a similar one containing the left and right flanks of the C6L gene) can be used by a technology similar to that proposed here to delegate the C6L gene on any recombinant MVA virus that expresses other heterologous antigens other than the one presented here (HIV subtype B), such as antigens of malaria, leishmania, hepatitis C virus, prostate cancer, etc; in order to be able to be used as vaccines against these diseases. Example 2.- In vitro characterization of the invention MVA-B AC6L.

The deletion of C6L in MVA-B AC6L was confirmed by PCR using oligonucleotides capable of amplifying the C6L locus (Figure 1 B). The deletion of C6L in MVA-B AC6L was also confirmed by sequencing of the DNA Additionally, an analysis by Western blot confirmed that MVA-B AC6L expresses HIV-1 _B xo ₈ gp120 and INBGPN antigens at the same level as its parental MVA-B virus (Figure 1 C). Example 3.- C6 is not essential in cell cultures.

The mere isolation of the MVA-B AC6L deletion mutant demonstrates that C6 protein is not essential for MVA replication. To determine if C6L deletion alters virus replication, the growth of MVA-B AC6L and MVA-B in DF-1 cells was compared. Viral kinetics studies revealed that the deletion of C6L in the MVA-B genome does not affect viral replication. Therefore, C6L is not essential for viral propagation in cell cultures (Figure 1 D). In addition, and similar to the MVA-B and MVA parental virus, MVA-B AC6L is an attenuated virus that does not replicate in mammalian cells (Figure 1 E). Example 4.- Expression of the C6 protein in E. coli and production of polyclonal anti-C6 antibodies.

The open reading frame (ORF) of C6 (MVA gene 019L, 157 aa, 18.2 kDa. See SEQ ID No: 1) was amplified by PCR using the C6L-Nhel-F oligonucleotides (SEQ ID No: 13) (comprising the Nhel) and C6L-BamHI-R restriction site (SEQ ID No: 14) (comprising the BamHI restriction site), and the MVA DNA as a template. The amplified product (Nucleotides 19068-19541 in the MVA genome. 488 bp) was digested with Nhel and BamHI and cloned into plasmid pET-27b (+) (Novagen). The ligation product was used to transform E.coli strain BL21, and the plasmid of a positive kanamycin resistant colony was sequenced to confirm that it contains the C6L gene sequence. The plasmid generated was called pET-27b-C6L (5837 bp). Plasmid pET-27b (+) provides a tail of 6 histidines at the carboxy terminal end of the C6 protein, generating a recombinant C6 protein of about 24 kDa. Kanamycin-resistant colonies were grown in Luria broth medium to an optical density of 0.5 to 595 nm. IPTG was added (0.5 mM) and the culture was grown for 4 more hours. The cells were centrifuged and for cell lysis the cells were resuspended in 50 mM Tris-HCI, pH 7.5, 0.3 M NaCI, 8 M Urea, and incubated with lysozyme (1 mg / ml) for 30 minutes in the presence of phenylmethylsulfonyl fluoride (1 mM). The suspension was frozen-thawed twice, the cell debris was removed by centrifugation and the supernatant was incubated with Probound resin (Invitrogen). Elution was carried out with different concentrations of imidazole (100 to 500 mM) in 50 mM Tris-HCI, pH 7.5, 0.3 M NaCI. The eluted fractions were grouped, loaded into desalted columns following the manufacturer's instructions (GE-Healthcare, Freiburg, Germany), and were collected. The protein was quantified using the Bradford assay, fractionated by 12% SDS-PAGE and analyzed by Western blot using an anti-His tag antibody (1: 5000) to detect the presence of vaccinia C6 protein. Fractions containing C6 protein (with an estimated purity of 90%) were stored in aliquots at -20 ° C. The C6 protein (1 150 g) was injected into "New Zealand White" rabbits to produce anti-C6 serum and anti-C6 rabbit polyclonal antibodies (Biomedal Laboratories, Seville).

Example 5.- C6 is expressed early in a viral infection.

Western blot analysis using rabbit polyclonal antibodies against C6 (its generation is described in example 4) allows the identification of an 18.2 kDa protein in DF-1 cells infected with MVA-B, but not with MVA-B AC6L (Figure 2A). Subsequently, it was determined how C6 is expressed in DF-1 cells infected with WR and MVA (Figure 2B). C6 expression was detected at 3 hours post-infection, and its expression increases strongly for at least 22 hours. Treatment of DF-1 cells, at the onset of infection, with "cytosine arabinoside (AraC)", an inhibitor of viral DNA replication and therefore of late gene expression, does not inhibit C6 expression suggesting that C6L It is an early gene.

The intracellular location of the C6 protein was examined by immunofluorescence in DF-1 cells infected with different strains of vaccinia (Figure 2C). C6 was detected in the cytoplasm, presumably in viral factories, of DF-1 cells infected with WR, MVA and MVA-B, but not with MVA-B AC6L. The reduced fluorescence intensity of C6 indicates the expression of low protein levels compared to the late A27 protein. Example 6.- MVA-B AC6L increases IFN-β expression in human macrophages and dendritic cells.

As a first step to determine if C6 blocks the response of innate immune cells to MVA-B, we examine by real-time PCR the expression of IFN-β, genes induced by IFN-β (IFIT1, IFIT2) and chemokines by human macrophages THP -1 infected for 1, 3 and 6 hours with MVA, MVA-B and MVA-B AC6L (Figure 3A). Compared with MVA and MVA-B, MVA-B AC6L (5 PFU / cell, Figure 3A and 1 PFU / cell, data not shown) significantly increases IFN-β expression, as well as IFIT1 and IFIT2 in THP cells -one . MVA-B AC6L also increases the expression of MIP-1 a and RANTES, but not that of IL-8 and IP-10 (Figure 3A and data not shown).

To confirm if C6 blocks the expression of IFN-β and IFN-β dependent genes in innate immune cells, we infect human moDCs with increasing doses (0.002, 0.02 and 0.2 PFU / cell) of MVA, MVA-B and MVA-B AC6L and we measured the levels of IFN-β, IFIT1 and IFIT2 messenger RNA at 6 hours post-infection (Figure 3B). We use low doses of virus since MVA induces human moDCs apoptosis (Guerra et al., 2007. J Virol 81: 8707-8721). The results showed that, similar to the results obtained with human THP-1 cells, MVA-B AC6L strongly increases IFN-β expression compared to MVA and MVA-B in moDCs. When infections are performed at 0.2 PFU / ml, the three viruses analyzed similarly stimulate the expression of IFIT1 and IFIT2 messenger RNA in moDCs. However, MVA-B AC6L was a more potent inducer than MVA and MVA-B at low infective doses (0.002 PFU / ml, Figure 3B). In addition, MVA-B AC6L stimulates the release to the medium by moDCs of higher levels of IFN-β (Figure 3C) and IFN type I than MVA and MVA-B (Figure 3D).

Thus, the deletion of C6L in the MVA-B genome promotes the production of IFN-β, suggesting that C6 interferes with the signaling pathway that controls the expression of the IFN-β gene in innate immune cells.

Previous studies showed that MVA is capable of inducing IFN-β in THP-1 cells that lack several of the TLRs; and that said induction is dependent on the factors MDA-5 and IPS-1, belonging to the signaling pathway mediated by "retinoic acid-inducible gene I (RIG-l) -like receptors (RLRs) "(Delaloye et al., 2009. PLoS Pathogens 5: e1000480). Therefore, contrary to what is expected by its sequence homology with vaccinia genes that inhibit the path of TLRs (Figure 4), C6 acts inhibiting the signaling pathway mediated by RLRs by blocking some still unknown component 5 (Figure 5).

Example 7.- MVA-B AC6L induces phosphorylation of IRF3 in THP-1 cells.

To determine which component of the signaling pathway mediated by RLRs that controls IFN-β gene expression in innate immune cells

10 could be blocked by C6, infections were made in THP-1 cells with MVA, MVA-B and MVA-B AC6L and at different times total extracts were collected and analyzed by Western blot using an anti-phosphorus IRF3 antibody. The results showed that, compared to MVA and MVA-B, MVA-B AC6L induces a greater phosphorylation of IRF3, a phenomenon observed mainly at

15 short post-infection times (Figure 6).

This result demonstrates that C6 blocks the phosphorylation of IRF3, although its target is still unknown.

Example 8.- MVA-B AC6L increases the magnitude and polyfunctionality of the response of specific memory T cells against HIV-1.

Once we have shown the immunomodulatory properties of C6, discussed earlier in this invention, we will test whether the deletion of C6 in MVA-B AC6L could increase immunogenicity in vivo by analyzing the immune response of specific T cells against HIV-1 in BALB mice / c immunized

25 with MVA-B or MVA-B AC6L using a "DNA prime (100 pg DNA-B, intramuscular) / MVA boost (1 x 10 ⁷ PFU, intraperitoneal)" (García-Arriaza et al., 2010 PLoS One 5: e12395; Mooij et al., 2008. J Virol 82: 2975-2988; Gómez et al., 2007. CE, Vaccine 25: 2863-2885; Robinson et al., 2007. AIDS Res Hum Retroviruses 23 : 1555-1562; Amara et al., 2002. J Virol 76:

30 7625-7631; Barouch et al., 2000. Science 290: 486-492). Animals inoculated with empty DNA (DNA-φ) and "boosted" with MVA were used as controls. Whereas memory T-cell responses may be critical for protection against HIV-1 infection (Seder et al., 2008. Nat Rev Immunol 8: 247-258; Sallusto et al. , 2004. Annu Rev Immunol 22: 745-763; Champagne et al. , 2001. Nature 410: 106-1 1 1; Sallusto et al., 1999. Nature 401: 708-712), we analyzed the long-term immunogenic profile (53 days after the last inoculation) in splenocytes by vaccination using IFN-γ ELISPOT and intracellular cytokine marking (ICS). with DNA-B / MVA-B and DNA-B / MVA-B AC6L.

IFN-γ ELISPOT revealed that, compared to MVA-B, MVA-B AC6L increased the response of specific IFN-γ secreting memory T cells against the Gag-B peptide of HIV-1 by 2.1 times (p <0.005) ( an HIV-1 peptide representative of the Gag antigen) (Figure 7A). MVA, used as a control, did not induce any specific HIV-1 memory response.

The phenotype of HIV-1-specific memory T cells induced after immunization with DNA-B / MVA-B and DNA-B / MVA-B AC6L was characterized by polychromatic flow cytometry using ICS. Splenic CD4 ⁺ and CD8 ⁺ T cells were co-stained for surface markers CD44 and CD62L in order to define the different memory sub-populations: naive (CD447CD62L ⁺ ), "central memory" (CM: CD44 ⁺ / CD62L ⁺ ), "effector memory" (EM: CD44 ⁺ / CD62L ^" ) and" effector terminal memory differentiated "(TEMRA: CD447CD62L ^" ). The production of IFN-γ and IL-2 was also evaluated after in vitro stimulation with different "poles" of HIV-1 peptides (Env-pool, Gag-pool and GPN-pool) that cover the entire HIV sequences -1 present in the poxviral vector (Figure 7B).

The specific total immune response of HIV-1 at 53 days "post-boost" was mediated mainly by CD8 ⁺ T cells (70% -85%) (Figure 7C) of EM and TEMRA phenotypes (Figure 7D), in both groups of immunization. However, unlike immunization with DNA-B / MVA-B, DNA-B / MVA-B AC6L induced a greater magnitude of the response of specific CD4 ⁺ and CD8 ⁺ memory cells against HIV-1 and producing of IFN-γ and / or IL-2 [CD4 ⁺ T cells: 1.91% in DNA-B / MVA-B AC6L vs. 1.30% in DNA-B / MVA-B, (p <0.005); CD8 ⁺ T cells: 10.95% in DNA-B / MVA-B AC6L vs. 3.06% in DNA-B / MVA-B (p <0.005)] (Figure 7C). Both vectors induce a similar pattern of specific memory CD4 ⁺ T cell responses against HIV-1 (preferably towards Env) (Figure 7B and 7C). Interestingly, the memory CD8 ⁺ T cell pattern specific against HIV-1 was different between both vectors: DNA-B / MVA-B AC6L induced a higher percentage of specific CD8 ⁺ T cell responses against GPN, while DNA-B / MVA-B preferentially induced cell responses Specific CD8 ⁺ T versus Env and Gag (Figure 7B and 7C). In both immunization groups, the specific CD8 ⁺ T cells against HIV-1 were mainly of the EM (60.5-63%) and TEMRA (37% -39.5%) phenotypes (Figure 7D). All specific CD4 ⁺ T cells against HIV-1 were of the EM phenotype in the DNA-B / MVA-B group. Although in the DNA-B / MVA-B AC6L group, the majority of CD4 ⁺ T cells specific to HIV-1 were of the EM phenotype (82.8%), a substantial percentage of cells (17.2%) expressed the TEMRA phenotype (Figure 7D). No CM phenotype T cells producing IFN-y and / or IL-2 were detected in both immunization groups (Figure 7D).

To have a detailed measure of the quality of the T-cell memory response, we next evaluate the production of IFN-γ and / or IL-2 by specific CD4 ⁺ and CD8 ⁺ T memory cells against HIV-1 (Figure 8). DNA-B / MVA-B AC6L increased the polyfunctionality of specific CD4 ⁺ and CD8 ⁺ T memory cells against HIV-1, consisting of cells that produce both IFN-γ and IL-2 [CD4 ⁺ T cells: 34 % in DNA-B / MVA-B AC6L vs. 16% in DNA-B / MVA-B, (p <0.005); CD8 ⁺ T cells: 29% in DNA-B / MVA-B AC6L vs. 16% in DNA-B / MVA-B, (p <0.005)] (Figure 8).

All these results, together, establish that immunization with DNA-B / MVA-B AC6L significantly increases the magnitude and polyfunctionality of the response of specific CD4 ⁺ and CD8 ⁺ T memory cells against HIV-1, with the majority of the response mediated by EM and TEMRA T cells. After vaccination with DNA-B / MVA-B and DNA-B / MVA-B AC6L, the responses of specific CD4 ⁺ T memory cells against HIV-1 were preferentially specific against Env. DNA-B / MVA-B AC6L induced immunodominance against specific CD8 ⁺ T memory cells against GPN, while DNA-B / MVA-B preferentially induced specific CD8 ⁺ T memory cells against Env and Gag.

Example 9.- MVA-B AC6L increases antibody levels against HIV-1 gp120 protein. Since cells infected with MVA-B release the gp120 monomeric protein (Gómez et al., 2007. CE, Vaccine 25: 2863-2885), we evaluate whether immunization with DNA-B / MVA-B and DNA-B / MVA -B AC6L stimulated the production of antibodies against HIV-1 Env protein. Serum was extracted from individual mice at 53 days "post-boost" and anti-gp120 antibodies were quantified by ELISA, measuring the levels of specific antibodies that react against the gp160 protein of the HIV-1 LAV isolate (subtype B), which includes gp120. Compared with DNA-B / MVA-B, immunization with DNA-B / MVA-B AC6L increased the levels of reactive antibodies against the gp120 protein 44 times (Figure 9). Therefore, MVA-B AC6L increases humoral immune responses against HIV-1 Env protein.

Claims

one . A viral vector based on a recombinant MVA virus, characterized in that the nucleotide sequence encoding said vector comprises:

b) at least one nucleotide sequence encoding a heterologous antigen.

2. The viral vector based on a recombinant MVA virus according to claim 1, characterized in that the mutation in the sequence SEQ ID No: 1 is a partial or total deletion.

3. The viral vector based on a recombinant MVA virus according to claim 2, characterized in that the mutation in the sequence SEQ ID No: 1 is a total deletion.

4. The viral vector based on a recombinant MVA virus according to claims 1-3, characterized in that the nucleotide sequence encoding said vector comprises at least one nucleotide sequence encoding a heterologous antigen selected from the following group: HIV antigen, malaria antigen, leishmaniosis antigen, hepatitis C virus antigen and prostate cancer antigen.

5. The viral vector based on a recombinant MVA virus according to claims 1 -4, characterized in that the nucleotide sequence encoding said vector comprises the nucleotide sequences encoding the following HIV antigens: HIV gp120 and Gag-Pol-Nef antigens of subtype B, or any other subtype, under the control of the early / late viral synthetic promoter inserted into the TK viral locus.

6. Method of manufacturing the viral vector based on a recombinant MVA virus defined in claims 1-5, characterized by comprising the following steps: a) construct a plasmid comprising in its nucleotide sequence the sequences of the right flank and the left flank of SEQ ID No: 1, b) recombine a recombinant MVA virus, which contains at least one nucleotide sequence encoding a heterologous antigen, with the plasmid generated in a) which directs the mutation of SEQ ID No: 1, and c) purifying the viral vector based on a recombinant MVA virus defined in claims 1-5, obtained in step b).

7. Use of the viral vector based on a recombinant MVA virus defined in claims 1-5 as an immunogen to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer.

8. Use of the viral vector based on a recombinant MVA virus according to claim 7, characterized in that said vector is used as an immunogen to prevent or treat HIV disease.

9. Use of the viral vector based on a recombinant MVA virus defined in claims 1-5 as part of an immunization protocol to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer, in which the individual is administered at least one dose of vaccination.

10. Use of the viral vector based on a recombinant MVA virus according to claim 9 characterized in that at least one vaccination dose is administered to the individual for:

b) to induce IFN-β expression in innate immune cells.

eleven . Use of the viral vector based on a recombinant MVA virus according to claim 10, characterized in that said vector is used as part of an immunization protocol in which at least one vaccination dose is administered to the individual, to prevent or treat HIV disease. .

12. Use of the viral vector based on a recombinant MVA virus according to claim 10, characterized in that said vector is used as part of an immunization protocol in which at least one vaccination dose of a combination of heterologous vectors is administered to the individual for prevent or treat one of the following diseases: HIV, malaria, leishmaniosis, hepatitis C, and prostate cancer.

13. An immunogenic composition characterized in that it comprises at least one viral vector defined in claims 1-5.

14. Use of the immunogenic composition defined in claim 13, to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and prostate cancer.

15. Use of the immunogenic composition according to claim 14, to prevent or treat HIV.