ES2401904B1 - RECOMBINANT VECTORS BASED ON THE MODIFIED VIRUS OF ANKARA (MVA), WITH DELETION IN THE C6L GEN, AS VACCINES AGAINST HIV / AIDS AND OTHER DISEASES. - Google Patents

Abstract

Recombinant vectors based on the modified Ankara virus (MVA), with deletion in the C6L gene, such as vaccines against HIV / AIDS and other 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.

Description

RECOMBINANT VECTORS BASED ON THE MODIFIED VIRUS OF ANKARA (MVAI, WITH DELETION IN THE GEN C6L, AS VACCINES AGAINST HIV / AIDS and OTHER DISEASES

SECTOR OF THE TECHNIQUE

The present invention falls within the fields of biology

Molecular and biotechnology. Specifically it refers to recombinant viruses based on the modified Ankara virus (MVA) that express the antigens

gp120 and Gag-Pol-Nef of the human immunodeficiency virus (HIV-1) of subtype B (MVA-B), on which the C6L vaccinia gene has been deleted, and which 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 increases the number of new infections and deaths caused by the

AIDS, 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 field

scientific.

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 that express HIV-1 antigens induce protection after a challenge with the simian / 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-Arriaza et aL, 2010. PLoS One

5: e12395; Gómez et al., 2009. Vaccine 27: 3165-3174; Harari 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; Gómez et al., 2007. Vaccine 25: 1969-1992; summary in Gómez et al., 2008. Curr Gene Ther 8: 97-120).

Previously, a recombinant of

MVA expressing HIV-1 antigens of subtype B, gp120 (SEO ID No. 15 of PCT / ES2006 / 070114) as a monomeric protein and Gag-PolNef polyprotein (SEO ID No. 16 of PCT / ES2006 / 070114) (virus denominated MVA-B) (Patent: PCT / ES2006 / 070114, publication date: 1/212007. Authors: Heeney, Jonathan; Mooij, Petra; Gómez Rodríguez, Carmen Elena; Nájera Garcia, José Luis;

Jiménez Tentor, Victoria; Esteban Rodríguez. Mariano). In a protocol of

"DNA prime / MVA boost" immunization in MVA-B mice induced strong immune responses against HIV-1 antigens (Garcia-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 ".6P) and Gag-Pol-Nef (of SIVm • c239) showed strong specific immune responses of CD4" and CD8 "T cells with a preference for CD8", and high protection after a challenge with SHIV89.6P (Mooij et al., 2008. J Viral

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, it has been carried out in

EspaFla a Phase I clinical trial with MVA-B in healthy volunteers. However, despite all this background, they are necessary and

desirable new more efficient MVA-B poxviral vectors that can increase the magnitude, amplitude, polyfunctionality and durability of immune responses against HIV-1 antigens. This is particularly relevant when a single immunogen is desirable for vaccination purposes in

mass to simplify immunization protocols and reduce manufacturing costs. Poxviral vectors express numerous genes encoding immunomodulatory proteins that interfere with the anti-viral response of

host (Alcaml, 2003. Na !. Rev. Immunol. 3: 36-50). Therefore, the deletion in the MVA-B poxviral vector of vaccinia genes that are known or suggest that

can have an immunomodulatory function, it is a general strategy that

It can increase the immunogenicity of the vector against HIV-1 antigens.

One of these genes whose function is unknown but we think may have

Immunomodulatory function is the C6l vaccinia gene, which is present in the genome of MVA vaccinia lines (gene called MVA 019l, SEO ID No: 1), Western Reserve (WR) (gene named VACV-WR_022, SEO ID No : 3), 5 and Copenhagen (gene called C6l, SEO 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 the analysis of the C6l promoter and the transcriptome analysis that detects messenger RNA of C6 at 30 minutes post-infection (Assarsson et al., 2008. P.NAS. 105: 2140- 2145). C6l encodes a protein of 157 10 amino acids 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), one

family of proteins that inhibit the signaling pathway at different levels

15 mediated by "TolI-like receptor (TlR)" (Bowie et al., 2000. P.NAS. 97: 1016210167; 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; 20 Stack et al., 2005. J. Exp. Med. 201: 1007-1018). C6 protein 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 ), PDCD61P and TNNI2 (troponin 1) (Zhang et al., 2009. J. Proteome Res. 8: 4311-4318). In addition, a C6 epltopo (amino acids 74-82 of SEO ID 25 No: 1 and SEO ID No: 3) is highly immunogenic in BAlB / c mice, and WR induces in mice aH IFN-secreting cell levels and specific for C6l, similar to vaccinia peptides of E3l, F2l and A52R (Oseroff et al., 2008. J. Immunol. 180: 7193-7202). All these characteristics indicate that C6 can have an important immunomodulatory function antagonizing the

30 signaling route TlR.

Therefore, in this invention a new vaccine candidate has been generated in front of

to HIV-1, called MVA-B "'C6l, which contains a deletion in the MVA-B vector of the C6l vaccinia gene and which we demonstrate acts by inducing the production of interferon type 1 and activating in vivo the production of T cells from

memory, which was not expected, but that represents an attractive alternative

to increase the immunogenicity of vaccine candidates based on MVA.

BRIEF DESCRIPTION OF THE INVENTION

The present invention represents a new vaccine candidate against HIV

1, called MVA-B LlC6l, which contains a deletion in the MVA-B vector of the C6L vaccinia gene. MVA-B LlC6l replicates in cell cultures at the same level

that the parental virus MVA-B, indicating that e6 is not essential for replication

viral. Additionally, MVA-B LlC6l induces innate immune responses by increasing IFN-13 expression and IFN-a / (3 (IFIT1 and IFIT2) induced genes in human THP-1 cells and monocyte-derived dendritic cells (moDCs), suggesting that C6 inhibits the IFN- signaling pathway (3 by blocking some unknown component involved in the induction of IFN-3. deletion of the C6l gene in the MVA-B vector increases in mice the humoral and T-cell immune response of memory against HIV-1 antigens. Thus, through

a NONA prime / MVA boost immunization protocol "in mice was shown that,

Compared to MVA-B, MVA-B LlC6l significantly increases the magnitude and polyfunctionality of the immune response of specific CD4 + and CDa + memory T cells against HIV-1, which is mainly mediated by effector phenotype CDa + T cells, in both immunization groups The response of specific CD4 + memory T cells against HIV-1, induced by MVA-B and MVA-B LlC6L was preferentially specific against Env. However, while MVA-B induces specific CDa + memory T immune responses against Env and Gag, MVA-B LlC6L preferentially induces specific CDa + T memory immune responses against Gag-PolNef (GPN). In addition, compared to MVA-B, MVA-B LlC6L increases the

antibody levels against Env.

Therefore, MVA-B LlC6L represents a new vaccine candidate against HIV-1 which, not being obvious, has an immunological benefit by increasing IFN- dependent responses (3 in human cells and increasing the

humoral response and the magnitude and quality of memory immune responses

of specific T cells against HIV-1 antigens.

Starting from the examples included in this report, where

Experienced only with MVA-B LlC6l, the results obtained would broaden the scope of protection to improve the immunogenicity of new 5 recombinant vectors based on MVA, which express other heterologous antigens (examples, malaria, leishmania, hepatitis C virus and cancer of prostate) by deletion of the C6l gene, in order to be used

as vaccines against these diseases.

10 DETAILED DESCRIPTION OF THE INVENTION

the construction of MVA-B LlC6l described in the present invention and the

assays in which both their in vitro behavior, as well as the innate immune response induced in human cells, and their immunogenic capacity are evaluated in

15 mice against HIV-1 antigens are described in more detail with the aid of the figures and examples that appear hereinafter.

The present invention refers to a viral vector based on a virus

MVA recombinant, characterized in that the nucleotide sequence encoding

20 for said vector comprises: a) at least one mutation in the SEO ID No: 1 sequence 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 SEO ID No: 1 sequence 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 sequence

nucleotide coding for said vector comprises at least one sequence

nucleotide that codes for a heterologous antigen selected from the following group: HIV antigen, malaria antigen, leishmaniasis antigen, hepatitis C virus antigen and prostate cancer antigen.

5 In an even more preferred embodiment, the nucleotide sequence

coding for said vector comprises the nucleotide sequences that

code for the following HIV antigens: gp120 antigens (SEO ID No. 15 of PCT / ES2006 / 070114) and Gag-Pol-Nef (SEO ID No. 16 of PCT / ES2000 / 070114) of HIV subtype B, or any other subtype, under the control of the promoter

10 synthetic viral early / late inserted into the viral TK 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) constructing 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 SEO ID No: 1, b) recombine a recombinant MVA virus, which contains at least one nucleotide sequence encoding a heterologous antigen, with the

20 plasmid generated in a) which directs the SEO ID mutation No: 1, preferably said mutation is a partial or total deletion, and more preferably it is a total deletion, and e) purify 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,

30 said vector is used as an immunogen to prevent or treat the disease of the

HIV

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 is given to the individual at least one dose of vaccination. In a preferred embodiment, said vector is administered to the individual a single dose of vaccination to:

a) induce an immune response of memory cells C04 + and C08 +

specific antigen, and / or

b) to induce IFN-13 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 (protelnas, ONA, VLPs, attenuated viral vectors) to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis C, and cancer of prostate.

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 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 an antigen

heterologous

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 e, and cancer of

prostate. In a preferred embodiment, said immunogenic composition or vaccine is for preventing or treating HIV.

In the present invention reference is made to the following terms:

• The term "MVA recombinant virus" or "MVA" refers interchangeably

a: a highly attenuated strain of vaccinia, called a virus

Ankara modified (MVA), obtained after 576 serial passes in chicken embryonic cell cultures (CEF).

• The term: "MVA-S recombinant virus" or "MVA-B parental virus" or "vector

poxviral MVA-B "refers interchangeably to: poxvirus based on the Ankara modified virus (MVA) expressing the gp120 and Gag-PolNef antigens of the human immunodeficiency virus (HIV-1) subtype B (MVAB). The tenin "parental" is used when comparing MVA-B with MVA-B t.C6L, since the deletion of C6L is made on the genome of MVA-B.

• The term "viral vector" or "recombinant viral vector" refers to: a virus

modified that makes vehicle to introduce exogenous genetic material into a cell.

• The term: "MVA-B t.C6L vector" refers to: an isolated MVA virus, characterized by expressing the gp120 and Gag-Pol-Nef antigens of the human immunodeficiency virus (HIV-1) of subtype B, and owns a

deletion in the polynucleotide sequence encoding a sequence

amino acid homologous to that of the C6L gene (SEO ID No: 1), and is presented

for use as a pharmaceutical composition or medication. The deletion

of the C6L gene in MVA-B includes positions 19068 to 19541 of the genome

of MVA.

• the term "C6l vaccinia gene" refers to: a vaccinia gene, whose

Function is unknown prior to the presentation of this invention. The C6L gene is present in the genome of MVA vaccinia lines (gene named MVA 019L, positions 19068-19451 of the MVA genome, SEO ID No: 1), Westem Reserve (WR) (gene named VACV-WR_022, positions 16401 to 16856 of WR, SEO ID No: 3), and Copenhagen (gene named C6L, positions 19484 to 19939, SEO ID No: 5), but absent in the New York Vaccinia Virus (NYVAC) lineage.

• The term "plasmid ~ or" transfer vector "refers to 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 only

It can multiply inside the cells of other organisms. Plus

specifically refers to viruses belonging to the family Poxviridae,

which is a family of interrelated DNA viruses called

poxvirus, effective for vertebrate and invertebrate animals.

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

determined.

• The term "heterologous antigen" refers to: a molecule (usually a protein or a polysaccharide), which triggers the formation of

antibodies and may 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 "leishmaniosis antigens" refers to: the antigens of

leishmania, which are expressed from recombinant viruses

MVA that 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. It includes any hepatitis C virus antigen encoded from the hepatitis C virus genome.

o The term "prostate cancer antigens" refers to: the antigens of

5 prostate cancer (including the PSCA and STEAP antigens), which are expressed from the MVA recombinant viruses that contain them.

• The term "vaccine against disease" refers to: a preparation or

immunogenic or antigenic composition used to establish the

immune system response to a disease. They are prepared or

10 combinations of immunogens or antigens that once inside the

organism provoke the response of the immune system, by means of

antibody production, and generate immune memory producing

permanent or transient immunity.

o The term "vaccine against prostate cancer" refers to: a

15 antigen preparation used to establish the immune system 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

20 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 consists of

in the loss of a DNA fragment, which can range from the loss of a single nucleotide (point deletion) to the loss of large regions.

o The term "partial deletion" refers to: the loss of a DNA fragment of a gene, which does not cause the total loss thereof.

o The term "total deletion" refers to: the loss of a DNA fragment

of a gene, which causes the total loss of it. 30 o The term: "immunogen" refers to: those antigens that cause

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 response

host immune generating CD4 + and CD8 + T cells that are capable of specifically recognizing the administered antigen.

• The term: "induce the expression of IFN-j3 in innate immune cells" is

refers to: the ability of the administered vaccine to stimulate the

IFN-j3 production by innate immune cells, such as macrophages and dendritic cells.

• The term: "increase IFN-j3-dependent responses in human cells" refers to: the ability of the administered vaccine to stimulate

to a greater extent those immune responses that occur as

consequence of the production of IFN-j3 by human innate immune cells, such as macrophages and dendritic cells.

• The term: "increase the magnitude and quality of immune responses

memory 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 recognize 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- ')', IL-2, or TNFa.

• The term: "EM and TEMRA phenotypes" refers to: two subpopulations of memory T cells, which are defined based on 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 uhomologla ", as used herein, does

reference 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 l> C6L, although the deletion of the vaccinia C6L gene has been performed on MVA-B, it 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 deletion of the C6L gene,

in order to be used as vaccines against these

infancy. Similarly, although the deletion of C6L has been performed on an MVA vector that expresses HIV-1 antigens of subtype B (MVA

B) it is enough to allow a subject matter expert to obtain new

Recombinant MVA vectors expressing HIV-1 antigens from other subtypes (A, C, D, E, F, G, H, and O). It is also enough to allow

one skilled in the art obtain new poxviral vectors

recombinants based on other strains that fall within the species, such as the Western Reserve and Copenhagen strains of vaccinia, which also have the C6L gene in their genomes (SEO ID No: 3 and SEO 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 l> C6L deletion mutant.

(A) Scheme of the genome of MVA-B ll.C6L, adapted from (Nájera et al., 2006. J Viral 80: 6033-6047; Antaine et al., 1998. Virology 244: 365-396). The different

Viral genome regions are indicated by capital letters. Shows

the right and left terminal regions and below the map the different fragmented or deleted genes are drawn in black. In additional ways

indicates the deleted C6L gene. The sequences of the Gag-Pol-Nef subtype B-type HIV (from isolate IIIB) and gp120 (from isolate BX08) are indicated, under the 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: 28635 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 or OC6L was used for PCR analysis. DNA products corresponding to the parental virus or to the

C6L deletions are indicated by an arrow on the right, including the expected size in base pairs. The molecular weight marker (1 Kb) with

10 corresponding sizes (in base pairs) are indicated on the left. The Mock column represents uninfected cells. (C) Expression of HIV-1 Bxo, gp120 and "'BGPN proteins in infected DF-1 cells (2 PFU / cell) with MVA-B and MVA-B or OC6L, at 24 hours post-infection. (D) Viral growth kinetics of MVAB and MVA-B or OC6L in infected DF-1 cells (0.01 PFU / cell) at different

15 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 or C6L viral growth kinetics in infected HeLa cells

(.01 PFU / cell) at different times and titled by a test of

Immunostaining of plaque with anti-WR antibodies. The extracellular and intracellular viral titer 20 is represented.

Figure 2. Characterization of the expression and location of C6. DF-1 cells were infected with 5 PFU / MVA-B cell and MVA-B or OC6L (A) or WR and MVA (B) in the presence or absence of AraC (B). Cell extracts

25 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 or OC6L for 18 hours. The location of C6 was analyzed by immunofluorescence and the

JO cells were stained with DAPI (stained cell nucleus), purified rabbit polyclonal antibody anti-C6 and anti-14K.

Figure 3. MVA-B "'C6L induces the production of IFN-Il and IFN-induced genes type I in macrophages and dendritic cells. Human macrophages THP-1 (A) and moDCs (B, C, D) were infected with MVA , MVA-B and MVA-B "'C6L (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-Il 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. U.A .: 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 "'C6L. 6 hours later, cell-free supernatants were collected to quantify IFN concentration -Il through EUSA (C) and IFN type I concentration using the line

HL 116 cell, 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 pathway consists of several TLRs located on the cell surface or in intracellular compartments. The activation of these TLRs leads to the recruitment of various adapter proteins and kinases and the subsequent activation of transcription factors (IRFs, NF-KB 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 path (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 IFN via TBK1 / lkk - [-; and B14 inhibits the phosphorylation of IKKf! and therefore prevents the activation of NF-KB.

Figure 5. Interaction between the "retinoic acid-inducible signaling pathway"

gene I (RIG-I) -like receptors "(RLRs) and vaccinia virus (VACV) proteins. The RLR signaling pathway is composed of RIG-I proteins. MDA5 Y

LGP2. The activation of these RlRs leads to the recruitment of several proteins

5 adapters and kinases and the subsequent activation of transcription factors (IRFs and NF-KB) that are translocated to the nucleus and activate the transcription of IFN type I 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 TBK1 / 1kk-E-; and C6

10 inhibits the phosphorylation of IRF3 although its mechanism is still unknown.

Figure 6. MVA-B ~ C6L 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 IIC6L, MVA or with medium (Mock). TO

15 different post-infection times the cells were washed with cold PBS and lysed

for 10 minutes at 4 "C with" Cen Lysis Buffer "(Cen 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 20% polyacrylamide gels (w / v) and transferred to nitrocellulose membranes.The membranes were incubated with antibodies directed against phospho-IRF3 (Cen Signaling) and or -tubulin (Cel! Signaling) After washing, the membranes were incubated with a secondary antibody conjugated with HRP (Pierce). The signal was revealed using the "ECL Westem blotting Analysis System" (GE

25 Healthcare).

Figure 7. Immunization with MVA-B IIC6L increases the magnitude of

specific immune responses of CD4 + and COS + T cells specific against

to HIV-1.

Splenocytes were collected from mice (n = 4 per group) immunized with DNAB / MVA-B, DNA-B / MVA-B IIC6L or DNA-$ / MVA, 53 days after the last immunization. (A) IFN-secreting splenocytes specific to the HIV-1 Gag-B peptide were quantified by an ELlSPOT assay.

Data represent the mean ± standard deviation of triplicates. '** It represents

p <0.005. (B-D) Phenotypic analysis by flow cytometry of C04 'and COS · specific T cells against HIV-1 Env, Gag and GPN antigens. The expression of C044 and C062L was used to identify the sub-populations of memory called "central memory" (CM: C044 '/ C062L'), "effector memory" (EM: C044 '/ C062q and "terminal differentiated effector memory" ( TEMRA: C044 '/ C062L'). The production of IFN-y and IL-2 was analyzed by intracellular labeling (ICS). (B) A representative flow cytometry assay is shown.

Sub-populations of memory T cells are drawn by drawings of

density. The dots represent IFN-y and IL-2 producing T cells. (C) The percentage of C04 'and COS · specific memory T cells versus HIV-1 Env, Gag and GPN antigens is represented. The frequencies were calculated representing the number of memory T cells producing IFNy and / or IL-2 versus the total number of splenocytes CD4 'and CDS ·. The values of the

Un stimulated controls were subtracted in all cases. •• represents p <O.005.

(D) The circulars represent the proportion of CM, MS and TEMRA within the C04 'and COS · specific T cells against HIV-1 Env, Gag and GPN antigens. (A-O) The data are derived from an experiment, representative of 2

Experiments performed

Figure 8. Immunization with MVA-B Ll.C6L increases the polyfunctionality of the immune responses of C04 'and coa' specific T cells against HIV-1. Splenocytes were collected from mice (n; 4 per group) immunized with ONAB / MVA-B, ONA-B / MVA-B Ll.C6L or ONA - $ / MVA, 53 days after the last

immunization and were analyzed by flow cytometry as described

in Figure 7. The polyfunctionality of the C04 '(left part) and CDS' (T) right memory T cells specific to HIV-1 Env + Gag + GPN antigens is defined based on IFN-y production and / or IL-2. 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 C04 'and COS · T cells are shown on the Y axis. •• represents p <O.005. The circles summarize the data. Each part of the circle corresponds to the proportion of C04 'or CD8 + T cells that produce IFN-y, IL-2 or IFN-y + IL-2 within the total of specific CD4 + or CDS + memory T cells against HIV antigens -one . The size of

Each circle represents the magnitude of the immune response of induced memory

specific against HIV-1 antigens.

Figure 9. Immunization with MVA-B .1C6L increases the immune response humoral 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 .1C6L or DNA- ~ IMVA, 53 days after the last immunization. Anti-gp120 antibody titers were determined by ELlSA. The titers represent the last dilution of the serum that gave a signal 3 times higher that the signals obtained with the serum of naNeo mice The dotted line represents the ELLSA detection limit. The horizontal bar represents the value medium and the values obtained by each mouse are represented by circles. * represents p <0.05.

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2S EXAMPLES

The invention will now be illustrated by a series of tests

made 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 herein.

invention.

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

The function of the C6l vaccinia gene was unknown until the moment of

present this invention, although it was predicted by bioinformatic lodges that

could have an immunomodulatory function, which does not mean that it is

obvious the similarity between prediction and demonstration (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 immunomodulatory role of C6L, we have constructed a deletion mutant that lacks

of the G6l vaccinia gene from the MVA-B recombinant virus, previously

described (Gómez et al., 2007. Vaccine 25: 2863-2885; Patent: PCT / ES2006 / 070114, publication date: 1/212007. Authors: Heeney, Jonathan; Mooij, Petra; Gómez Rodrlguez, Carmen Elena; Nájera García , José Luis; Jiménez Tentar, Victona; Esteban Rodríguez, Mariano) and expressing the HIV-l antigens of subtype B Env, Gag, Poi and Nef. The generated recombinant virus, presented in this invention, has been referred to as MVA-B ~ C6L. A

Schematic diagram of the MVA-B ~ C6L deletion mutant is depicted in Figure lA. The manufacturing method of MVA-B ~ C6L comprises the following steps:

a) construct a plasmid comprising in its nucleotide sequence the

Right flank and left flank sequences of the C6L gene (SEO ID No:

one),

b} recombine the MVA-B recombinant virus, which contains the sequences

nucleotide coding for HIV antigens of subtype B gp120 Y

Gag-Pol-Nef, with the plasmid generated in a} which directs the deletion of the gene

C6L (SEO ID No: 1), and

c} purify the viral vector called MVA-B ~ C6L, based on a virus

MVA-B recombinant 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 ~ C6L deletion mutant, which has a deletion in the vaccinia C6L gene (MVA 019L MVA gene, SEO ID No: 1, is equivalent to the VACV-WR_022 gene of the Western Reserve (WR) strain of

vaccinia, SEa ID No: 3, and the C6l gene of the Copenhagen strain of vaccinia, SEa ID No: S. For simplification, the nomenclature corresponding to the genes of the Copenhagen line is used to refer to the MVA genes). pGem-RGC6l wm was obtained by sequential cloning of five DNA fragments 5 containing the dsRed2, rsGFP genes and flanking sequences (left and right) of recombination of the C6l gene in the plasmid pGem-7Zf (-) (Promega). First, the construction of plasmid pGem-Red-GFP wm (4S40 bp), which contains the dsRed2 and rsGFP genes under the control of a viral promoter, was carried out

Tempranoltardio synthetic (E / L) and that was previously described (García-Arriaza et

10 al., 2010. PloS One S: e1239S). Briefly, the dsRed2 gene under the control of a synthetic viral promoter tempranoltardio (Ell) was amplified by PCR of plasmid pG-dsRed2 using the Red2-B oligonucleotides (SEO ID No: l.S. GAACTAGGATCCTAA CTCGAGAAA-3 ') (comprising the Bam HI restriction site) and Red2-N (SEO ID No: 16. S'-ATTAGTATGCATTTATTTATTTAGG-3 ')

15 (comprises restriction site Nsi 1) (78S bp), digested with Bam HI 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 a synthetic early viral promoter (ElL) was amplified by PCR of plasmid pG-dsRed2 using GFP-X oligonucleotides (SEO ID No:

20 17. S'-CGTTGGTCTAGAGAGAAAA ATTG-3 ') (includes the Xbal restriction site) and GFP-E (SEO ID No: 18. S' -CTATAGAATTCTCAAGCTATGC-3 ') (includes the Eco RI restriction site) (832 pb), digested with Xba I and Eco RI and cloned into the plasmid pGem-Red wm previously digested with the same restriction enzymes to generate pGem-Red-GFP wm (4540 bp).

25 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 (SEO ID No: 7. Nucleotides 18689-18714 in the genome of MVA) (comprising the Aatll restriction site) and RFC6l-Xbal-R (SEO ID No: 8. Nucleotides 19054

30 19079 in the MVA genome) (comprises the Xbal restriction site). 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 (SEO ID No: 9, Nucleotides 18689-18714 in the MVA genome) (comprising Xmal restriction silium) and RF'C6L-Clal -R (SEO ID No: 10, Nucleotides 1905419079 in the MVA genome) (comprising the Clal restriction site), digested with Xmal and Glal and inserted into the plasmid pGem-RG-RFsG6L wm digested with XmallClal to generate pGem-RG -RFdG6L wm (5259 bp), The left flank of the G6L gene (Nucleotides 19530-19942 in the MVA genome. 413 bp) was amplified by PGR from the MVA-B genome with the oligonucleotides LFC6L-Glal-F (SEO ID No: 11, Nucleotides 19530-19555 in the MVA genome) (comprising the Glal restriction site) and LFG6L-BamHI-R (SEO ID No: 12. Nucleotides 19917-19942 in the MVA genome) (comprises the site BamHI restriction), digested with Glal and BamHI and inserted into the plasmid pGemRG-RFdG6L wm digested with GlallBam HI. The resulting plasmid pGem-RG-G6L wm (5642 bp) was confirmed by AON sequence analysis and directs the deletion of the G6L gene from the MVA and MVA-B genome,

Once the plasmid pGem-RG-G6L wm has been generated, obtaining the

invention MVA-B .ó.C6L is carried out in cell cultures by a process of

recombination between the MVA-B virus and the plasmid pGem-RG-G6L wm, which contains the right and left flanks of the G6L gene. Asl, MVA-B LlC6L was

constructed by selecting cell cultures of viral plaques that

coexpress dsRed2 / rsGFP (they 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: eI2395). 3 x 10 'OF-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 AON from plasmid pGem-RG-G6L wm 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 LlG6L deletion mutant was selected from the viral progeny obtained after 6 consecutive rounds of plaque purification in OF-1 cells and during this process the plaques were selected from those with rojalverde fluorescence, Thus, in the first two passes, the virus

were selected from plates that both expressed both proteins

fluorescent (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 5 of the selected plaques do not express any fluorescent marker due to the loss of the dsRed2 and rsGFP genes. Thus, after 6 consecutive rounds of plaque purification in OF-1 cells, the MVA-B "'C6l deletion mutant was obtained and the deletion of the C6l gene was confirmed by PCR amplification of the C6l dellocus, using the oligonucleotides RFC6l-Aatll -F and lFC6l-BamHI-R

10 (previously described) and subsequent analysis by AON sequencing. Thus, the C6l 01er SEO ID No: 1.474 nUcleotides, positions 19068-19541 in the MVA genome) has been completely deleted in the genome of MVA-B "'C6L. The deletion of the C6l gene in MVA-B includes positions 19068 to 19541 of the MVA genome.

Subsequently, the MVA-B "C6l recombinant virus obtained was grown in OF-1 cells to obtain a viral preparation called P2, which was grown in chicken embryonic cells (CEF), and purified by centrifugation through two mattresses. of 36% sucrose (w / v) in 10 mM Tris-HCI pH 9, as previously described (Ramirez et al., 2000. J

20 Virol, 74 (2): 923-933. MVA-B "'C6l was titrated into OF-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: 1 000), as previously described (Antaine et al., 1998. Virology 244: 365-396). The preparation of MVA-B "'C6l is free of

25 mycoplasmas or bacteria.

Starting from this technique of generating recombinant MVA viruses

with deletion in C6L, included herein, and although it has only been

experimented with MVA-B ", C6l, plasmid pGem-RG-C6l wm (or a similar one

30 containing the left and right flanks of the C6l gene) can be used by a technology similar to the one proposed here to delegate the C6l gene on any recombinant MVA virus that expresses other heterologous antigens other than the one presented here 01IH, subtype B), as antigens of malaria, leishmania, hepatitis C virus, prostate cancer, etc; in order to be

used as vaccines against these diseases.

Example 2.- In vitro characterization of the invention MVA-B <lC6L.

The deletion of C6l in MVA · B <1C6l was confirmed by PCR using oligonucleotides capable of amplifying the C6l locus (Figure 1B). C6l deletion in MVA-B <1C6l was also confirmed by DNA sequencing. Additionally, an analysis by Western blot confirmed that MVA-B <lC6l expresses the HIV-1 Bxoagp120 and IIIBGPN antigens at the same level as their parental MVA-B virus (Figure 1C).

Example 3.-C6 is not essential in cell cultures.

The mere isolation of the MVA-B / lC6l deletion mutant demonstrates that C6 protein is not essential for MVA replication. To determine whether the deletion of C6l alters virus replication, the growth of MVAB / lC6l 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 1D). In addition, and similar to the parental virus MVA-B and MVA, MVA-B / lC6l 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 anti-C6 polyclonal antibodies.

The open reading frame (ORF) of C6 (MVA gene 019l, 157 aa, 18.2 kDa. See SEO ID No: 1) was amplified by PCR using the C6l-Nhel-F oligonucleotides (SEO ID No: 13) (comprising the Nhel) and C6lBamHI-R restriction site (SEO 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 pET27b (+) 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 until

reach an optical density of 0.5 to 595 nm. IPTG was added (0.5 mM) and the

5 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

10 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. eluted fractions were grouped, loaded into desalted columns following the manufacturer's instructions (GE-HeaHhcare, Freiburg, Germany), and were collected. the protein was quantified using the assay

Bradford, fractionated by 12% SDS-PAGE and analyzed by Western blot using an anti-His tag antibody (1: 5000) to detect the presence of

C6 vaccinia protein. Fractions containing C6 protein (with a

estimated purity of 90%) were stored in allcuotas at -20'C. C6 protein (1150 ~ g) was injected into "New Zealand Wh ~ e" rabbits to produce anti-C6 serum 20 and rabbit anti-C6 polyclonal antibodies (Biomedal laboratories, Seville).

Example 5.-e6 is expressed early in a viral infection. Western blot analysis using polyclonal antibodies from

25 rabbit against C6 (its generation is described in example 4) allow the identification of an 18.2 kDa protein in DF-1 cells infected with MVA-B, but not with MVA-B "C6l (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

30 strongly for at least 22 hours. The treatment of DF-1 cells, at the beginning of the 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 ilC6L. The reduced fluorescence intensity of C6 indicates the expression of low levels of protein compared to late protein A27.

Example 6.-MVA-B ilC6L increases the expression of IFN-f! in macrophages and

human dendritic cells

As a first step in determining whether C6 blocks the response of innate immune cells to MVA-B, we examine the expression of IFN-f !, real-time genes induced by IFN-f! (IFIT1, IFIT2) And chemokines by THP-1 human macrophages infected for 1, 3 and 6 hours with MVA, MVA-B and MVA-B "C6l (Figure 3A). Compared with MVA and MVA-B, MVA-B ilC6l (5 PFU cell, Figure 3A and 1 PFU cell, data not shown) significantly increases the expression of IFN-f3, as well as IFIT1 and IFIT2 in THP-1 cells. MVA-B "C6l also increases the expression of MIP-1a 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-f! and of IFN-f 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 ilC6l and measure the levels of messenger RNA of IFN- / 3, IFIT1 and IFIT2 at 6 hours post -infection (Figure 3B). We use low doses of virus since MVA induces human moOCs 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 "C6l strongly increases the expression of IFN-f! compared to MVA and MVA-B in moOCs. When inlections are made to

0.2 PFU / ml, the three viruses analyzed similarly stimulate the expression of IFIT1 and IFIT2 messenger RNA in moDCs. However, MVA-B ilC6l was a more potent inducer than MVA and MVA-B at low inlective doses (0.002 PFU / ml, Figure 3B). In addition, MVA-B ilC6l stimulates the release into the environment by moOCs of higher levels of IFN-f! (Figure 3C) and IFN type I that MVA and MVA-B (Figure 3D).

Thus, the deletion of C6l in the MVA-B genome promotes the production of IFN-¡3, suggesting that C6 interferes with the signaling pathway that controls the expression of the IFN-¡3 gene in innate immune cells.

Previous studies showed that MVA is capable of inducing IFN-¡3 in

5 THP-1 cells that lack several of the TlRs; and that said induction is dependent on the factors MDA-5 and IPS-l, belonging to the signaling pathway mediated by "retinoic acid-inducible gene I (RIG-l) -Iike receptors (RlRs)" (Delaloye et al., 2009. PloS Pathogens 5: 000480). Therefore, at

contrary to expectations for its sequence homology with vaccinia genes

10 that inhibit the route of the TlRs (Figure 4), C6 acts by inhibiting the signaling pathway mediated by RlRs by blocking some still unknown component (Figure 5).

Example 7.-MVA-B "C6L induces phosphorylation of IRF3 in THP-1 cells.

15 To determine which component of the RlRs-mediated signaling pathway that controls IFN-¡3 gene expression in innate immune cells could be blocked by C6, infections were made in THP-1 cells with MVA, MVA-B and MVA- B "C6l and at different times total extracts were collected and analyzed by Western blot using an anti-phosphorus IRF3 antibody.

20 the results showed that, in comparison with MVA and MVA-B, MVA-B "C6l induces a greater phosphorylation of IRF3, a phenomenon observed mainly at short post-infection times (Figure 6). This result demonstrates that C6 blocks phosphorylation of IRF3, although its target is still unknown.

Example 8.-MVA-B "C6L increases the magnitude and polyfunctionality of the

specific memory T cell response against HIV-1.

Once the immunomodulatory properties of C6, shown

Prior to this invention, we will test whether the deletion of C6 in MVA-B

30 "C6l could increase immunogenicity in vivo by analyzing the immune response of specific T cells against HIV-l in BAlB / c mice immunized with MVA-B or MVA-B" C6l using an immunization protocol '' DNA prime (100 ~ g of DNA-B, intramuscular) I MVA boost (1 x the '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 Viral 76: 7625-7631; Barouch et al., 2000. Science 290: 486-492) Animals inoculated with empty DNA (DNA- ~) 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 : 106-111; Sallusto et al., 1999 Nature 401: 708-712), we analyzed the long-term immunogenic profile (53 days after the last inoculation) induced in splenocytes by vaccination with DNA-B / MVA by IFN- and ELlSPOT and intracellular cytokine labeling (ICS). -B and DNA-B / MVA-B Ll.C6L.

IFN-y ELlSPOT revealed that, compared to MVA-B, MVA-B Ll.C6L increased the response of specific IFN-secreting memory T cells by 2.1 times (p <0.005) against HIV Gag-B peptide- l (an HIV-l peptide representative of the Gag antigen) (Figure 7A). MVA, used as a control, did not induce any specific memory response of HIV-1.

The phenotype of HIV-1-specific memory T cells induced after immunization with DNA-B / MVA-B and DNA-B / MVA-B Ll.C6L was characterized by polychromatic flow cytometry using ICS. Splenic CD4 'and CD8' T cells were coated for the CD44 and CD62L surface markers in order to define the different memory sub-populations: naIve (CD44- / CD62L '), "central memory" (CM: CD44 '/ CD62L'), "effector memory" (MS: CD44 '/ CD62L-) and "terribly differentiated effector memory" (TEMRA: CD44- / CD62L} The production of IFN-y and IL-2 was also evaluated after In vitro stimulation with different "pools" of HIV-1 peptides (Env-pool, Gag-pool and GPN-pool) that cover the entire sequences of HIV-1 present in the poxviral vector (Figure 7B).

The specific total immune response of HIV-1 at 53 days "post-boas" was mainly mediated by CD8 'T cells (70% -85%) (Figure 7C) of EM and TEMRA phenotypes (Figure 7D), in both immunization groups However, unlike immunization with DNA-B / MVA-B, DNA-B / MVA-B

IIC6L induced a greater magnitude of the response of C04 · and C08 · specific memory cells against HIV-1 and IFN-r and IL-2 producers [C04 · T cells: 1.91% in ONA-B / MVA-B IIC6L vs. 1.30% in ONA-B / MVA-B, (p <0.005); C08 T cells: 10.95% in ONA-B / MVA-B IIC6L vs. 3.06% in ONA-B / MVA-B 5 (p <0.005)] (Figure 7C). Both vectors induce a similar pattern of specific memory C04 · T cell responses against HIV-1 (preferably towards Env) (Figure 7B and 7C). Interestingly, the pattern of specific memory C08 · T cells against HIV-1 was different between both vectors: ONA-B / MVA-B IIC6L induced a higher percentage of specific C08 · T cell responses! Or against GPN, while that ONA-B / MVA-B preferentially induced specific COS · T cell responses against Env and Gag (Figure 7B and 7C). In both immunization groups, C08 · T-cells specific to HIV-1 were mainly of the EM (60.5-63%) and TEMRA (37% -39.5%) phenotypes (Figure 70). All C04 · T-cells specific against HIV-1 were of the EM phenotype in

15 the ONA-B / MVA-B group. Although in the ONA-B / MVA-B IIC6L group, most of the C04 · T-cells specific to HIV-1 were of the EM phenotype (S2.S%), a substantial percentage of cells (17.2%) expressed the TEMRA phenotype (Figure 70). No CM phenotype T cells producing IFN-r and / or IL-2 were detected in both immunization groups (Figure 70).

20 To have a detailed measure of the quality of the T-cell memory response, we next evaluate the production of IFN-r and / or IL-2 by C04 · and COS · T-specific memory cells against HIV-1 ( Figure 8). ONAB / MVA-B IIC6L increased the polyfunctionality of specific CD4 · and COS · T memory cells against HIV-1, consisting of cells that produce both

25 IFN-r and IL-2 [T cells C04 ·: 34% in ONA-B / MVA-B IIC6L vs. 16% in ONAB / MVA-B, (p <O.005); T COS cells: 29% in ONA-B / MVA-B IIC6L vs. 16% in ONA-B / MVA-B, (p <0.005)] (Figure S).

All these results, together, establish that immunization

with ONA-B / MVA-B IIC6L significantly increases the magnitude and

30 polyfunctionality of the response of memory cells C04 + and specific COS · against HIV-1, with the majority of the response mediated by EM and TEMRA T cells. After vaccination with ONA-BIMVA-B and ONA-B / MVA-B IIC6L, the responses of C04 · T memory cells specific to HIV-1 were preferentially specific against Env. ONA-B / MVA-B or OC6L induced immunodominance against C08 "specific memory cells against GPN, while ONA-BIMVA-B preferentially induced C08" specific T memory cells against Env and Gag.

Example 9.-MVA-B or OC6L increases antibody levels against the 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 assess whether

10 immunization with ONA-B / MVA-B and ONA-B / MVA-B or OC6L stimulated the production of antibodies against HIV-l Env protein. Serum was extracted from individual mice at 53 post-boost days and anti-gpl20 antibodies were quantified by ELlSA, measuring the levels of specific antibodies that react against the gp160 protein of the HIV-1 LAV isolate (subtype B),

15 which includes gp120. Compared to ONA-B / MVA-B, immunization with ONAB / MVA-B tlC6L increased reagent antibody levels 44 times against the gp120 protein (Figure 9). Therefore, MVA-B or C6L increases humoral immune responses against HIV-l Env protein.

Claims (15)

1. A viral vector based on a recombinant MVA virus. characterized in that the nucleotide sequence coding for said vector comprises: a) at least one mutation in the SEO ID No: 1 sequence that codes for the C6l protein, and b) at least one nucleotide sequence encoding an antigen heterologous 2. The viral vector based on a recombinant MVA virus according to claim 1, characterized in that the mutation in the SEO ID sequence No: 1 It is a partial or total deletion. 3. The viral vector based on a recombinant MVA virus according to the claim 2, characterized in that the mutation in the SEO ID sequence No: 1 It is a total deletion. 4. The viral vector based on a recombinant MVA virus according to the claims 1-3, characterized in that the nucleotide sequence encoding said vector comprises at least one nucleotide sequence encoding for a heterologous antigen selected from the following group: antigen of HIV, malaria antigen, leishmaniosis antigen, virus antigen of hepatitis e and prostate cancer antigen. 5. The viral vector based on an MVA recombinant virus according to claims 1-4, characterized in that the nucleotide sequence encoding said vector comprises the nucleotide sequences encoding the following HIV antigens: gp120 and Gag-Pol-Nef antigens HIV subtype B. or of any air subtype, under the control of the early viral synthetic promoter inserted into the viral TK locus. 6. Method of manufacturing the viral vector based on a recombinant virus MVA defined in claims 1-5, characterized by comprising the following steps:  a) construct a plasmid comprising in its nucleotide sequence the Right flank and left flank sequences of SEO ID No: 1, b) recombining a recombinant MVA virus, which contains at least one nucleotide sequence encoding a heterologous antigen, with the plasmid generated in a) which directs the SEO ID mutation 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 in the manufacture of a medicament or composition immunogenic 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 the claim 7. characterized in that said medicament or composition Immunogenic is to prevent or treat HIV disease. 9. Use of the viral vector based on a recombinant MVA virus defined in claims 1-5 in the manufacture of a medicament or immunogemca composition for use as part of an immunization protocol for prevent or treat one of the following diseases: HIV, malaria, leishmaniosis, hepatitis C, and prostate cancer, in which the individual is administered at least  A dose of vaccination. 10. Use of the viral vector based on a recombinant MVA virus according to claim 9 characterized in that said medicament or composition  Immunogenic is given to the individual at least one dose of vaccination to: a) induce an immune response of specific CD4 + and CD8 + T cells from antigen specific memory, and b) to induce IFN-j3 expression in innate immune cells. 11. Use of the viral vector based on a recombinant MVA virus according to the claim 10, characterized in that said immunogenic drug or composition is used as part of an immunization protocol in which the individual is administered at least one dose of vaccination, to prevent or treat the HIV disease 12. Use of the viral vector based on a recombinant MVA virus according to the Claim 10, characterized in that said medicament or immunogenic composition is used as part of an immunization protocol in which administers to the individual at least one dose of vaccination of a combination of heterogygous vectors to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis e, and prostate cancer. 13. An immunogenic composition characterized by comprising the minus a viral vector defined in claims 1-5. 14. Use of the immunogenic composition defined in claim 13, in the 15 manufacture of a vaccine to prevent or treat one of the following diseases: HIV, malaria, leishmaniasis, hepatitis e, and prostate cancer. 15. Use of the immunogenic composition according to claim 14, characterized in that said vaccine is for preventing or treating HIV.