ES2334472B1 - IMMUNO STIMULATOR COMBINATION FOR PROFILAXIS AND HEPATITIS C TREATMENT. - Google Patents

Abstract

Immunostimulatory combination for prophylaxis and hepatitis C treatment.

The present invention relates to a immunostimulatory combination for the prophylaxis and treatment of hepatitis C, characterized in that it comprises: a TLR3 agonist, a CD40 agonist and the hepatitis C virus NS3 protein. On the other hand, the invention relates to the compositions pharmaceuticals comprising said immunostimulatory combination, to the use thereof and to a kit composed of said compositions Pharmaceuticals Finally, the present invention relates to a method to produce an immune response against the virus hepatitis C, and a vaccine against that virus.

Description

Immunostimulatory combination for prophylaxis and hepatitis C treatment.

Technical Field of the Invention

The present invention relates to a immunostimulatory combination for prophylaxis and treatment of hepatitis C, which incorporates HCV NS3 protein, along with adjuvants selected for their ability to induce responses CD8 + and CD4 + powerful and durable specific against the virus HCV

State of the art

With an estimated worldwide prevalence of more than 170 million infected people, virus infection Hepatitis C (HCV) is a heavy health burden today public Prevalence that presumably will remain unchanged in the next years.

HCV infection is characterized by a high tendency to chronicity. HCV persists in 70% of infected individuals, of which 20% develop cirrhosis and a 2.5% evolve to produce liver cancer.

The therapeutic reference tool currently they are the therapeutic protocols based on the Interferon use. However, these antiviral therapies  they are economically expensive, relatively toxic and only effective in 50-60% of patients treated. It is therefore necessary and desirable to develop new therapeutic strategies that are more effective and better tolerated for the patients

An updated review of HCV can found in Nature ("Insights: Hepatitis C". Nature 2005, Supplements; Vol. 436, No. 7053, pp 929-978).

Although unfortunately we still do not have an effective vaccine against hepatitis C virus, there are data and experimental evidence that leads one to think that a vaccine Effective is possible. Although antiviral antibodies are synthesized In response to infection, the chronic state is characterized by the absence of cellular immune responses by T cells cytotoxic (CD8 +) and helper T (CD4 +). Thus, it is postulated that the HCV has developed strategies that allow you to evade specifically antiviral immune responses, where the potency and quality of cytotoxic T and helper T responses determines if patients will recover (either spontaneously or in response to a treatment) or they will develop the infection chronicle.

The main objective of any vaccine is to stimulate the specific acquired immunity of antigen, whose main mediators are B and T lymphocytes. In this context, antigen presenting cells (APCs) play an important role in the initiation of specific immune responses and particularly in the activation of T lymphocytes. APCs, mainly dendritic cells, capture antigens in the peripheral organs and, after receiving an activation stimulus, migrate to the lymphatic organs. There, the dendritic cells present superficially, together with molecules of the major histocompatibility complex MHC, peptide products derived from the degradation of the antigens (epitopes), and simultaneously produce chemokines and cytokines to attract and activate T cells. The activation process Dendritic cell count, also known as maturation, is characterized by high expression of MHC molecules (signal 1), costimulatory molecules (signal 2) and polarizing cytokines such as interleukin-12 (IL-12) (signal 3). Maturation is induced by factors such as components of pathogens or host molecules frequent in the processes of inflammation or cell damage. These factors act on dendritic cells through specific receptors for products derived from microorganisms, such as TLR ( Toll like receptor ) receptors, cytokine receptors (TNF-?, IL-1, IFN-?) Or receptors for ligands on the surface of the cells (eg CD40).

The stimulation and activation of different populations of T cells by APCs is restricted by the type of MHC molecule on the one hand, and on the other by the characteristics of the epitopes that complex with said MHC molecules. Thus, for example, certain fragments of viral proteins have been identified that specifically induce the activation of CD8 + cytotoxic T lymphocytes (CTL), known as lymphocyte epitopes or CD8 + T cells or CD8 + epitopes; or epitopes that specifically induce the activation of CD4 + helper T lymphocytes (HTL), CD4 + epitopes. The "HCV Immunology Database" database ( http://hcv.lanl.gov/content/immuno/
immuno-main.html ) summarizes the epitopes for T lymphocytes, both of CTL CD8 + and HTL CD4 +, identified from viral proteins of different strains and isolated from hepatitis C virus.

The development of immunization protocols based on the use of epitopes in the form of peptides therefore requires prior selection of those peptides that are suitable for each individual, depending on the MHC molecules that Present. This assumes that depending on the MHC of each individual, it would be necessary to select a specific combination of peptides that they could behave as epitopes in that context. The utilization of large antigens allows to obviate this problem, since they are usually polyepitopic, within their sequence they present various epitopes, both for CTL CD8 + and for HTL CD4 +, which They can be presented by MHC molecules from different individuals. Thus, a single antigen can be used as a vaccine in individuals with various MHC.

Within the different HCV proteins, core and NS3 have a high immunogenicity, and in those individuals that clear up the infection, powerful CTL responses are detected CD8 + and HTL CD4 + in front of them. However, there are data that show that core can also have deleterious effects for immune system cells when in contact with them, which does not make it advisable as an antigen in strategies of vaccination. On the contrary, NS3 is a protein that has barely showed this kind of effects and could be a good candidate like antigen for induction of CTL CD8 + and HTL CD4 + responses.

HT4 CD4 + play a role in acquired immunity, among other mechanisms by activating APCs, activating CTL and inducing memory. In particular, it has been described that HCV-specific CD4 + cells are necessary for the maintenance of antiviral CTLs (Grakoui A. et al ; "HCV persistence and immune evasion in the absense of memory T cell help"; Science, 2003; 302: 659-662). Therefore, an effective vaccine against the hepatitis C virus should provide maximum power in the induction of not only CT8 + CTL responses, but also of CD4 + HTL responses. Said vaccine will therefore require a selection of specific antigens to provide such responses.

However, it doesn't seem like a combination of antigens alone can provide an effective vaccine against HCV. Since dendritic cell maturation is a requirement for the effective initiation and activation of lymphocytes T, such a vaccine could benefit from inclusion in the immunostimulatory combination of some adjuvants that stimulate maturation of dendritic cells. As adjuvants could used ligands of TLR receptors, of receptors of cytokines or receptors for aforementioned intercellular ligands, or better yet a synergistic combination of said adjuvants.

For example, US2004 / 0141950 describes immunostimulatory combinations that include a TLR agonist and a molecule agonist of the factor superfamilies of tumor necrosis (TNF) or its receptors (TNFR), which may also include an antigen. Among the many combinations possible presents the combination of a CD40 ligand (a anti-CD40 antibody) and poly (I: C), a synthetic ligand of TLR3, combination for which a synergistic effect on the expansion of CD8 + T lymphocytes. Likewise Ahonen et al. (J. Exp. Med. 2004; 199: 775-784) present data on the synergistic capacity of TLR / CD40 agonists to induce the expansion and differentiation of specific CD8 + CTLs of antigen independently of CD4 + T lymphocytes. Though These works describe the capacity of the TLR / CD40 combination to activate antigen-specific memory CD8 + T lymphocytes, these works do not allow to establish if the combination of TLR / CD40 agonists can also enhance HTL responses CD4 +.

In the case of HCV infection, clear differences have been found in the HTL CD4 + responses when comparing infected patients with patients who have been able to eliminate the infection. However, although with less intensity than in cured patients, CD8 + CTL responses are still detectable in infected patients. Therefore, although CTLs behave as an important effector population in the clearance of HCV infection, CD4 + cells also play an important role in disease control. Moreover, it has been described that the induction of specific CD4 + T cells is important for the maintenance of antiviral CTL responses (Grakoui A. et al ; "HCV persistence and immune evasion in the absense of memory T cell help"; Science, 2003 ; 302: 659-662). All these data suggest that for the vaccination and therapy of viral diseases due to HCV it is important to induce both strong and durable CD8 + and CD4 + antiviral responses.

It is therefore. the object of the present invention  select immunostimulatory combinations of antigens and adjuvants suitable for the prophylaxis and treatment of hepatitis C, which provide a stimulation of responses both CD8 + as CD4 + more powerful, complete and durable.

Detailed description of the invention

A first object of the invention relates to an immunostimulatory combination for prophylaxis and treatment of hepatitis C, hereinafter immunostimulatory combination of the invention, comprising a TLR3 agonist, a CD40 agonist or a DNA sequence that encodes it, and a polypeptide that comprises the hepatitis C virus NS3 protein, or a fragment of said NS3 protein capable of inducing CD8 + and CD4 + answers.

A "TLR3 agonist" refers to a ligand that can combine or bind with the TLR3 ("toll like receptor 3") receptors and produce a cellular response. TLR3 is a double-stranded RNA receptor that transmits signals that activate NF-? B and the production of type I interferons (IFN) (IFN-? And IFN-?), And that stimulates dendritic cell maturation . Mice lacking TLR3 expression showed a reduction in their responses to poly (I: C) - a TLR3 ligand similar to double stranded RNA generated during the replication of HCV-type viruses - resistance to the lethal effect of poly (I: C) when they are sensitized with D-galactosamine, and a reduction in the production of inflammatory cytokines (Alexopoulou et al . Nature, 2001, Vol. 413, pp. 732-738). In a particular embodiment of the invention said TLR3 ligand can be a double strand of viral RNA, or a double chain of polyinosinic-polycytidyl acid, poly (I: C).

A "CD40 agonist" refers to a ligand which can be combined or bound with CD40 receptors by inducing also a cellular response. CD40 is a molecule expressed in the membrane of different cell types, such as B lymphocytes or antigen presenting cells (macrophages, dendritic cells, etc). The natural CD40 ligand (CD40L or CD154) is expressed mainly in T lymphocytes that have been activated after antigen recognition. The interaction of CD40L with CD40 present in the antigen presenting cell induces maturation this. This phenomenon, similar to stimuli from pathogens, it makes the presenting cell of antigen has a greater capacity to induce responses immune Thus, the CD40 agonist of the composition immunostimulatory of the invention, refers on the one hand to ligating CD40L or a fragment of said CD40L that retains the ability to bind to CD40 and induce a cellular response or immune. In a particular embodiment the ligand can be a specific antibody against CD40 (anti-CD40), or a fragment thereof that retains the ability to join CD40. On the other hand, the CD40 ligand or its fragment may be present in the immunostimulatory combination well in the form of protein or also as a recombinant nucleic acid (DNA) that encodes said ligand, for example in a viral vector to gene transfer or therapy.

An "antigen" refers to any substance that is capable of inducing an immune response, both humoral as a cellular, in the organism of a subject (a man or a animal), or that can induce a cellular immune response (expansion, activation and / or maturation of immune cells, cytokine production, or antibodies) when it comes into contact with immune cells. In particular, an antigen can be a viral protein, a peptide or fragment of said viral protein, a  recombinant protein of said viral proteins or even a synthetic peptide capable of inducing the indicated responses.

A "CD8 + inducing epitope" refers to a fragment or partial polypeptide chain of an antigen that is capable of specifically inducing T lymphocyte activation cytotoxic CD8 + (CTL). A "CD4 + inducer epitope" refers to a fragment or partial polypeptide chain of an antigen that is capable of specifically inducing T lymphocyte activation CD4 + helpers (HTL).

The "NS3 protein" refers to the protein NS3 non-structural hepatitis C virus, a protein of 67 kDa that includes 2 domains, a serine proteinase which covers the 189 amino acids of the end N-terminal and a domain with activity helicase nucleoside triphosphatase covering 442 C-terminal end amino acids. The sequence of NS3 protein included in the combination polypeptide immunostimulatory of the invention may correspond to any strain or isolate of hepatitis C virus, in particular any strain or isolate of the human hepatitis C virus. In a particular embodiment, the polypeptide comprising the protein NS3 has been obtained by recombinant technology. In one embodiment non-limiting concrete of the invention an NS3 protein is used recombinant with the sequence SEQ. ID. NO: 1 (corresponding to Genbank Accession numbers DQ068198.1 and AAY84763.1, VRL NOV 28, 2005).

We have also used another protein recombinant SEQ. ID. NO: 2 (corresponding to Genbank Accession number D90208).

In another particular particular embodiment of the  invention it is also possible to use a polypeptide comprising a fragment of the NS3 protein, so that said fragment is capable of inducing CD4 + and CD8 + responses. Therefore, said fragment must include at least one CD8 + inducer epitope and one epitope CD4 + inductor.

In a specific embodiment the combination Immunostimulatory of the invention comprises poly (I: C), a anti-CD40 antibody, and a polypeptide comprising  NS3 protein

In a preferred embodiment of the invention, the  Immunostimulatory combination has all the components forming part of the same pharmaceutical composition, where each of the components is present in quantities pharmaceutically acceptable. On the other hand, the invention also refers to said pharmaceutical composition.

In another specific embodiment of the present invention, the components of the immunostimulatory combination are they are part of at least two compositions Pharmaceuticals Also, the invention relates to the use of said immunostimulatory combination characterized by said Pharmaceutical compositions are administered simultaneously. In other embodiment of the invention, the use of said combination immunostimulatory is characterized in that said compositions pharmaceuticals are administered at different times, for the same route of administration or by different routes. Thus, an embodiment concrete of the invention refers to a kit for administration of the immunostimulatory combination previously described, characterized in that it comprises at least two compositions Different pharmaceuticals.

In another aspect, the invention relates to a method to produce an immune response against the virus hepatitis C characterized in that it comprises administering a stimulator combination defined above, in an amount effective to induce an immune response. In one embodiment preferably the method of the invention constitutes a treatment prophylactic. In a more preferred embodiment the method of The invention constitutes a therapeutic treatment.

Finally, the invention also relates to a vaccine against the hepatitis C virus, characterized in that comprises a previously defined immunostimulatory combination and object of the present invention.

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Brief description of the figures

Figure 1. Immunization with anti-CD40 and poly (I: C) together with the NS3 protein induces multiepitopic CD4 + and CBD T responses . HHD mice (two per group) were injected with 50 g of anti-CD40 (ip). Four hours later, they were injected with 50 g of poly (I: C) (iv) and 500 g of recombinant NS3 protein (ip) (SEQ. ID. NO: 1). Six days later the animals were sacrificed and the splenocytes were extracted for in vitro stimulation with different antigens and the analysis of the induced immune response. (A) The cells were stimulated for five days with the CD8 + 1073, 1406 or 1038 (10 µM) epitopes in the presence of IL-2. Then, in each group of splenocytes, the lytic response was measured against target cells loaded (peptide; black bars) or not (control; white bars) with the corresponding peptide. The results obtained with an effector: target ratio of 100: 1 are shown. B) Similarly, splenocytes were cultured with different concentrations (0.1-10 µM) of peptides 1073 (black circles), 1406 (white triangles) or 1038 (black triangles), and in the culture supernatants obtained After 48 h of stimulation, the IFN-γ content was measured by ELISA. (C) Splenocytes were also stimulated for 48 h with 5 or 1 µg / ml of the NS3 protein used in immunization (SEQ. ID. NO: 1), with 1 µg / ml of NS3 protein produced in bacteria ( SEQ ID NO: 3), or with culture medium (control) to measure the CD4 + response. After this period of time the supernatants were collected and the amount of IFN-γ produced was measured by ELISA.

Figure 2. Titration of the amount of NS3 protein necessary to induce CD4 + and CD8 + T responses in immunization with poly (I: C) and anti-CD40 . HHD mice (two per group) were immunized with NS3 protein (SEQ. ID. NO: 1) (500, 250, 125 or 25 µg / mouse) together with poly (I: C) and anti-CD40 following the protocol that It is described in Figure 1. In addition, an immunized control group was included in the same manner, using 5 µg of NS3 (SEQ. ID. NO: 1) and 50 µg of peptides 1073 and 1038 as antigens, together with poly (I: C) and anti-CD40. Six days later the animals were sacrificed and the splenocytes were stimulated with the different antigens. (A) To measure the induced lytic response, the cells were stimulated for five days with the epitope CD8 + 1073 (10 µM) and IL-2. This response was then measured by facing different amounts of effector cells against a fixed number of target cells loaded with the peptide. On the other hand, the induced CD8 + response was also analyzed, by measuring the production of IFN-γ. For this, the cells were stimulated with different concentrations of peptides 1073 (B) and 1038 (C). Cells were also stimulated with the NS3 protein (SEQ. ID. NO: 1) (D), to quantify the CD4 + response. After 48 h the amount of IFN-γ present in the supernatants was measured.

Figure 3. Immunization with poly (I: C) and anti-CD40 together with the NS3 protein induces CD8 + and CD4 + responses in other strains of mice with different MHC . C57BL6 mice (possessing MHC molecules of type H-2b) (two per group) received one (white squares) or two (black squares) immunizations with 100 µg of NS3 (SEQ. ID. NO: 1) together with poly ( I: C) and anti-CD40 following the protocol indicated in Figure 1. Six days after the last immunization the animals were sacrificed and the splenocytes were cultured with different antigens to measure the induced CD8 + and CD4 + responses. Restriction epitope H-2 Db 1629-1637 (GAVQNEVTL) (SEQ. ID. NO: 7) was used to stimulate splenocytes and measure CD8 + responses (A). The NS3 protein (SEQ. ID. NO: 1) (B) was used as a stimulus to determine the CD4 + responses. After two days of culture, supernatants were collected and the amount of IFN-γ produced was measured.

Figure 4. Immunization with NS3 protein together with poly (I: C) and anti-CD40 induces CD8 + responses capable of recognizing cells that express HCV proteins . (A) HHD mice (two per group) were injected with 100 µg of NS3 protein (SEQ. ID. NO: 2) plus poly (I: C) and anti-CD40 as indicated in Figure 1. Six more days The animals were then sacrificed and their splenocytes were stimulated with T1 / HCVcon cells (T1 cells transfected with a plasmid expressing HCV proteins) treated with mitomycin, in the presence of IL-2. After five days of stimulation, the ability of splenocytes to recognize T1 / HCV cells was measured in lytic activity assays. To do this, different amounts of splenocytes faced a fixed number of T1 / HCVcon cells (black circles) or untransfected T1 control cells (white circles).

Figure 5. Immunization with anti-CD40 and poly (I: C) together with the NS3 protein induces long-lasting CD4 + and CD8 + T responses . HHD mice (two per group) were injected with 100 µg of NS3 protein (SEQ. ID. NO: 2) plus poly (I: C) and anti-CD40 as indicated in Figure 1. Two weeks later, the animals They received a second immunization under the same conditions. Sixty days after the last immunization the animals were sacrificed and their splenocytes were extracted for the study of the long-lasting CD8 + and CD4 + T response. (A) Splenocytes were stimulated with the CD8 + 1073 epitope (10 µM) or in the absence of antigen, and 48 h later culture supernatants were collected to measure the amount of IFN-γ produced. (B) The splenocytes were cultured for 5 days with the 1073 (10 µM) and IL-2 peptide, and then their ability to lyse target cells loaded with the 1073 peptide was studied. To that end, different amounts of effector cells were faced against to a fixed number of target cells loaded with peptide 1073 (black circles) or unloaded with peptide (white circles). (C) The CD4 + response was studied by stimulating splenocytes with the NS3 protein (1 µg / ml) (SEQ. ID. NO: 2) or in the absence of antigen. After 48 h the supernatants were collected and the amount of IFN-γ produced was measured.

Embodiment of the invention

The following examples are intended to illustrate, in in any way limiting, the embodiment of the invention object of This patent application.

Material and relative methods Epitopes, antigens and reagents

The peptides or epitopes used were manually synthesized in a multiple peptide synthesizer using Fmoc chemistry (Wellings DA and Atherton E. Methods Enzymol 1997; 289: 44-67). The test of Kaiser ninhydrin to monitor each step. At the end of the synthesis were cleaved and unprotected with acid trifluoroacetic and washed with diethyl ether. The purity of Peptides were always greater than 90% determined by HPLC.

TABLE 1 Peptides and epitopes synthesized and used in examples

one

The numbering of the peptide or epitope refers to its relative HCVH position, taking as a reference the complete sequence in strain H of human hepatitis C that is usually taken as a prototype (GenBank Accession Number M67463). For example, the database "HCV Immunology Database" (http://hcv.lanl.gov/content/immuno/
immuno-main.html) summarizes the epitopes of T lymphocytes, both cytotoxic T and helper T lymphocytes, identified in viral proteins of different strains and isolated from hepatitis C virus, all of them also ordered according to their relative position to strain H of the virus according to the GeneBank reference indicated.

A 655 amino acid recombinant polypeptide containing the complete sequence of the NS3 protein (SEQ. ID. NO: 1; Genbank accession number AAY84763.1, VRL 28-NOV-2005; 631 amino acids) has been used as an immunogen. In addition to the 631 amino acids of the NS3 protein, the polypeptide also includes a tail with a c-myc sequence, for detection with the anti-cmyc monoclonal antibody, and a Histidine tail. The protein has been produced in Pichia pastoris . It is kept in suspension in a solution of 22.5 mM Tris / 3.76 M Urea / 300 mM NaCl. The protein has been purified by Ni column chromatography.

Another recombinant polypeptide, which contains the 635 amino acids comprising the complete NS3 protein sequence (SEQ. ID. NO: 2; Genbank accession.number D90208), has also been used as an immunogen. In addition to the amino acids corresponding to NS3, the polyprotein contains a histidine tail for purification. The DNA sequence corresponding to NS3 was obtained by digestion with SalI and NotI of plasmid gWIZ containing the NS3 sequence (provided by Dr. G Inchauspe, Lyon, France). The digestion product was cloned between the BsrGI and NotI sites of plasmid pET-45b (+) (Novagen, Madison WI). It was expressed in E. coli and purified by affinity chromatography on a nickel column followed by ion exchange chromatography.

Likewise, a recombinant polypeptide (Mikrogen; Catalog number 94302) containing the last 20 amino acids of the NS2 non-structural protein and the first 508 amino acids of the HCV NS3 protein (SEQ. ID) has been used as in vitro assays. NO: 3).

As agonist TLR3 has been used poly (I: C) obtained from Amersham (catalog number 27-4732-01).

Anti-CD40 antibodies purified from the hybridoma FGK-45 have been used as a CD40 agonist
(Rolink, A., et al . Immunity 1996. 5: 319-330).

All reagents contained <1 unit of endotoxin per mg of product, determined by the QCL-1000 lysate test of limulus amebocyte (Bio Whittaker).

Mice

C57B1 / 6 mice of six to eight weeks were obtained from Harlan. HHD mice, transgenic for human HLA-A2.1 molecules, were also used (Pascolo S et al . J. Exp. Med. 1997. 185: 2043-2051). All animals were kept in pathogen-free conditions and were treated according to the institution's regulations.

Cell lines

T2 cells (Salter R et al . Immunogenetics. 1985 21: 235-246) were used as target cells for chromium release assays with cytotoxic T lymphocytes (CTL) from HHD mice.

T1 cells, transfected with a plasmid carrying the HCV coding region (cells) were used
T1 / HCVcon), for cell recognition assays expressing HCV proteins. These cells were yielded by Dr. D. Moradpour (Freiburg, Germany; Volk B, et al . J Gen Virol. 2005; 86: 1737-1746). Untransfected T1 cells (ATCC, catalog number CRL-1991) were also used as a control.

All cells were grown in medium complete (RPMI 1640 with 10% fetal bovine serum, 100 U / ml of penicillin, 100 µg / ml streptomycin, 2 mM glutamine and 50 µM of 2-mercaptoethanol). The culture medium of The T1 / HCVcon line also contained 2 mg / ml of G418 (Gibco).

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Immunization

Groups of two mice were immunized via i.p. with 50 µg of anti-CD40. Four more hours later, they were injected with 50 µg of poly (I: C) (i.v.) and different amounts of the antigens: NS3 protein or mixtures of NS3 with peptides, (i.p.).

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Spleen cell stimulation for the production of cytokines

Spleen cells were resuspended in complete medium and plated at 8x105 cells / well in 0.2 ml in 96-well plates with a U-bottom, in the absence or presence of peptides or the HCV NS3 recombinant protein.

Two days later the supernatants were collected to measure the presence of IFN-? by ELISA (BD-Pharmingen), following the instructions manufacturer.

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Measurement of the lithic activity of CTL

To measure CTL responses, splenocytes from immunized animals were incubated with peptides (10 µM) for 2h at 37 ° C, washed twice and grown in 24-well plates at a confluence of 7.5x106 cells / well. In the experiments performed to measure the T1 / HCVcon cell recognition, 7.5x106 were cultured Splenocytes of HHD mice with 7.5x105 T1 / HCV cells previously treated with Mitomycin C (Sigma). In all cases, two days later, 2.5 U / ml of IL-2 was added (Boehringer-Mannhein GmbH, Germany) to the wells, and 5 days later the cells were recovered to perform chromium release assays.

Lytic activity was measured by incubating for 4 h different amounts of effector cells with 3000 target cells T2 previously loaded with 51 Cr, with and without peptide (white). In the case of cells stimulated with T1 / HCVcon, the cells effectors faced T1 / HCVcon or Ti cells, previously loaded with 51 Cr. Culture supernatants were collected after 4h of incubation.

The percentage of specific lysis was calculated according to the formula:

(cpm_ {experimental} - cpm_ {spontaneous}) / (cpm_ {maximum} - cpm_ {spontaneous}) x 100

where spontaneous lysis (measured as cpm_ spontaneously) corresponds to target cells incubated in absence of effector cells and the maximum lysis (cpm_ {maximum}) is gets incubating target cells with Tritonx100 at 5%.

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Example 1 Immunization with anti-CD40 and poly (I: C) together with the NS3 protein induces CD4 + and CD8 + T responses multi-epitopic

Immunization with anti-CD40 and poly (I: C) has proven to be very effective for the induction of CD8 + responses by using as immunogens synthetic peptides representing epitopes of CD8 + cells. Although this strategy induces potent responses, it has been shown that when co-immunized with low amounts of NS3 protein (5 µg / mouse), which induces CD4 + responses, it increases the magnitude of the CD8 + response and also increases the high CD8 + response. affinity, that is, one that recognizes low concentrations of antigen. On the other hand, immunization with peptides is only effective in those individuals who possess HLA molecules of the same restriction as the epitopes chosen. In order to address these two points, it was studied whether immunization with larger amounts of NS3 recombinant protein was capable of inducing responses, not only CD4 +, but also CD8 +. To do this, mice were immunized with NS3 together with poly (I: C) and anti-CD40, and the induced responses were studied. Thus, HHD mice (two per group) were injected ip with 50 µg of anti-CD40. Four hours later, they were injected with 50 µg of poly (I: C) (iv) and 500 µg of recombinant NS3 protein (ip) (SEQ. ID. NO: 1). Six days later the animals were sacrificed and the splenocytes were removed. In order to analyze the capacity of NS3, when formulated in the adjuvant poly (I: C) + anti-CD40, to induce CD8 + and CD4 + T responses, splenocytes with different antigens that specifically activate these cell populations were stimulated in vitro . (A) To analyze the CD8 + response, in a first experiment the splenocytes were stimulated for five days with the CD8 + 1073, 1406 or 1038 epitopes, in the presence of IL-2. For each group of cells stimulated with a peptide, their ability to lyse to target cells loaded with the corresponding peptide (black bars) or control target cells without peptide (white bars) was measured. Figure 1A shows the results obtained with an effector: target ratio of 100: 1. (B) The CD8 + response induced after immunization with NS3 was also analyzed by study of IFN-γ production against the same CD8 + epitopes. For this, splenocytes with different amounts of 1073 (black circles), 1406 (white triangles) or 1038 (black triangles) were stimulated. After 48 h of culture, the supernatants were collected and the IFN-γ content was measured. (C) In order to analyze the induced CD4 + response, splenocytes were stimulated with the NS3 protein used in immunization (SEQ. ID. NO: 1). In addition, cells were also stimulated with commercial NS3 protein produced in bacteria (SEQ. ID. NO: 3). In the same way as in the previous point, the degree of activation was measured by the production of IFN-γ.

First it was possible to verify that this antigen was able to induce CD8 + responses, which could be detected in both chromium release assays (Figure 1A) and by inducing the production of IFN-? (Figure 1B). Also, this answer was  multi-epitope, being directed against several CD8 + epitopes that have been characterized within the sequence of NS3 (eg peptides 1073, 1406 and 1038). Finally, it was also proven that he was capable of inducing CD4 + responses, which recognized the NS3 protein used in immunization and commercial NS3 protein produced in bacteria (Figure 1C). The answer to the latter was more low, presumably because there were some changes in the sequence of both proteins, and to which the protein expressed in bacteria was shorter, so you could lose some epitopes recognized by CD4 + T lymphocytes.

Example 2 Administration of 25 µg of recombinant NS3 together with poly (I: C) and anti-CD40 is enough to induce CD4 + and CD8 + T responses

From previous experiments we knew that with 5 NS3 \ CD4 + responses were induced but not CD8 +, so we wanted to find out the minimum amount of NS3 enough to induce CD8 + T responses. For this, HHD mice were immunized with 500, 250, 125 and 25 µg of NS3 (SEQ. ID. NO: 1). I also know included as control a group immunized with peptides corresponding to CD8 + epitopes, which would induce CD8 + responses, plus 5 \ mug of NS3 (SEQ. ID. NO: 1), which would induce responses CD4 +. To do this, in each group of animals immunized with a dose of NS3 the CD8 + response and the CD4 + response were analyzed. The CD8 + response was analyzed as the ability to lyse cells target loaded with the epitope CD8 + 1073 (Fig 2A), as well as the ability to produce IFN-? versus different concentrations of the CD8 + 1073 epitopes (Fig 2B) and 1038 (Fig 2C). The CD4 + response was measured by the ability to produce IFN-? versus different NS3 concentrations (SEQ. ID. NO: 1) (Fig 2D). This experiment showed that all quantities of NS3 tested were able of inducing CD8 + responses, when the lytic response was studied against peptide 1073 (Fig 2A), the dose being 25 µg the which induced lower intensity responses. In addition all doses were able to induce the production of IFN-? Versus epitopes 1073 (Fig 2B) and 1038 (Fig 2C), which indicated that even reducing the dose maintains the ability to induce multi-epitope responses. Finally, and as expected, all of them induced responses CD4 +. Since in the experiments shown, in most of cases the induced response was lower when 25 were used ? of NS3, for subsequent experiments a dose was chosen 100 µg / mouse, dose from which it was no longer observed an increase in the induction of responses.

Example 3 Immunization with poly (I: C) and anti-CD40 together with the NS3 protein induces CD8 + and CD4 + responses in others strains of mice with different MHC

Since in an antigen as large as the NS3 protein is the possibility that CD4 + epitopes are found and CD8 + that can be presented by different MHC molecules, the ability of this immunization protocol to study induce CD4 + and CD8 + responses in another mouse strain with molecules from different MHC. For this, C57 / B16 mice were immunized, which they have H-2b restriction MHC molecules, with 100 \ mug of NS3 (SEQ. ID. NO: 1). In order to improve responses, one group received a single immunization and another group He received a second immunization of remembrance. First of all it measured the CD8 + response, as production of IFN-? Versus the peptide 1629-1637 (SEQ. ID. NO: 7), which contains an epitope CD8 + presented by MHC class I molecules H-2 D b. As seen in Figure 3A, in both groups of mice a detectable response was induced, although the levels were considerably higher in the group that had received two immunizations (black squares) that in which received an immunization (white squares). The CD4 + answer, measured as IFN-γ production versus NS3 recombinant protein (SEQ. ID. NO: 1), was also detected in the two groups (Figure 3B), and again showed that two immunizations (black squares) induced more potent responses than a single immunization (white squares).

Example 4 Immunization with NS3 protein together with poly (I: C) and anti-CD40 induces CD8 + responses capable of recognize cells that express HCV proteins

In order to study whether immunization using NS3 protein together with poly (I: C) and anti-CD40 was able to induce responses that could potentially kill HCV-infected cells, an in vitro model of target cells transfected with a plasmid expressing HCV proteins (T1 / HCVcon). These cells express in their MHC class I molecules the same peptides that a HCV-infected cell would express, so a response against them would be assimilable to a response against an infected cell. The NS3 protein (SEQ. ID. NO: 1) used in the experiments of Figures 1 to 3 corresponds to a viral strain different from the viral strain that is present in T1 / HCVcon cells. These two strains have some differences in the CD8 + epitopes studied so far. In order to optimize the ability to recognize the CD8 + epitopes present in T1 / HCVcon cells, an NS3 protein (SEQ. ID. NO: 2) was used as an immunogen whose sequence had a higher degree of homology with the protein present in T1 / HCVcon cells. Six days after immunization of HHD mice with 100 µg of NS3, splenocytes were stimulated with T1 / HCVcon cells. The ability to recognize T1 / HCVcon cells was analyzed in lytic activity assays. For this, the stimulated splenocytes were confronted with T1 / HCVcon cells and with control T1 cells. As shown in Figure 4, immunization with NS3 induced responses with a greater ability to lyse T1 cells expressing HCV proteins (black circles) than T1 control cells (white circles).

Example 5 Immunization with anti-CD40 and poly (I: C) along with the NS3 protein induces long CD4 + and CD8 + T responses duration

One of the main properties that you should owning a vaccination protocol is its ability to induce long-lasting immune responses, so that the protection conferred by long-term immunization. To study whether immunization with anti-CD40 and poly (I: C) together with the NS3 protein was able to induce such responses, HHD mice were immunized with 100 µg of NS3 according to the protocol described in Example 1. In order to to reinforce the response, at 15 days, the animals received a dose of memory in the same conditions. Sixty days after the last immunization, the animals were sacrificed and their splenocytes were stimulated with different antigens to analyze the CD8 + and CD4 + T responses that remained in that moment. To study the CD8 + T response, the cells with epitope 1073, and the production of IFN-? And lytic activity. As shown in Figure 5A, sixty days after the last immunization, splenocytes from mice immunized with anti-CD40 and poly (I: C) along with the protein NS3, were able to produce large amounts of IFN-? When stimulated with the peptide 1073, but not in the absence of antigen. In addition, these cells were capable of lysing target cells pulsed with the 1073 peptide (black circles), but not target cells that did not contain antigen (white circles) (Figure 5B). Finally, we also studied the CD4 + response, using as an antigen the NS3 protein used in immunization Figure 5C shows that this protocol of immunization also induces potent long CD4 + responses duration that specifically recognize NS3.

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twenty

Claims (17)

1. An immunostimulatory combination for prophylaxis and treatment of hepatitis C, characterized in that it comprises:

to)

a TLR3 agonist,

b)

a CD40 agonist or a DNA sequence that encodes it, and

C)

a polypeptide comprising the NS3 protein, or a fragment of said NS3 protein with the ability to induce CD8 + responses and CD4 +.

2. An immunostimulatory combination according to claim 1, characterized in that the TLR3 agonist is poly (I: C). 3. An immunostimulatory combination according to claim 1 or 2, characterized in that the CD40 agonist is selected from an anti-CD40 and / or CD40L antibody. 4. An immunostimulatory combination according to claim 3, characterized in that the CD40 agonist is an anti-CD40 antibody. 5. An immunostimulatory combination according to claim 1, characterized in that it comprises:

to)

poly (I: C),

b)

a anti-CD40 antibody, and

C)

a polypeptide comprising the NS3 protein.

6. An immunostimulatory combination according to claim 4 or 5, characterized in that the polypeptide comprising the NS3 protein is a polypeptide with the SEQ. ID. NO .: 1. 7. An immunostimulatory combination according to one of claims 1 to 6, characterized in that all components are part of the same pharmaceutical composition. 8. An immunostimulatory combination according to one of claims 1 to 6, characterized in that the components are part of at least two different pharmaceutical compositions. 9. Use of an immunostimulatory combination according to claim 8, characterized in that said pharmaceutical compositions are administered simultaneously. 10. Use of an immunostimulatory combination according to claim 9, characterized in that said pharmaceutical compositions are administered at different times. 11. Use of an immunostimulatory combination according to claim 9, characterized in that said pharmaceutical compositions are administered by different routes of administration. 12. A pharmaceutical composition containing an immunostimulatory combination described in one of claims 1 to 8, characterized in that each of the components is present in pharmaceutically acceptable amounts. 13. A pharmaceutical composition characterized by comprising an immunostimulatory combination defined in one of claims 1 to 8, administered in an amount effective to induce an immune response against the hepatitis C virus. 14. A pharmaceutical composition according to claim 13, characterized in that said composition is employed in a prophylactic treatment. 15. A pharmaceutical composition according to claim 13, characterized in that said composition is employed in a therapeutic treatment. 16. A kit for the administration of an immunostimulatory combination defined in one of claims 1 to 6, characterized in that it comprises at least two different pharmaceutical compositions. 17. A vaccine against the hepatitis C virus, characterized in that it comprises an immunostimulatory combination defined in one of claims 1 to 8.