Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/29—Hepatitis virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/24011—Flaviviridae
  • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
  • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/24011—Flaviviridae
  • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
  • C12N2770/24271—Demonstrated in vivo effect

Definitions

• the present invention is related to the branch of medicine and the pharmaceutical industry, particularly to the development of chimeric antigens against hepatitis C virus (HCV), and vaccine compositions containing them.
• HCV hepatitis C virus
• a minimum number of components are used in the invention, since the chimeric antigens are capable of inducing an enhanced and diverse immune response against HCV antigens, by the precise selection of specific regions of said antigens, and the inclusion of specific artificial epitopes. for CD4 + T lymphocytes.
• HCV infects approximately 3% of the world's population (Williams R. Hepatology 2006; 44: 521-526). Most infected individuals move towards chronicity (Amoroso P, et al., J Hepatol 1998; 28: 939-944). Hepatitis C is one of the leading causes of chronic liver damage, cirrhosis, liver failure and liver cancer (Hoofnagle JH. Hepatology 2002; 36 (5 Suppl 1): S21-S29). It is currently the leading cause of liver transplantation in first world countries.
• HCV infection has been linked to extra-hepatic manifestations, such as type II cryoglobulinemia, membranoproliferative glomerulonephritis, cutaneous porphyria, among others. There is no preventive vaccine against this virus, and. Antiviral treatments in use, based on the combination of pegylated interferon (IFN) + ribavirin, are effective in less than 50% of cases. In addition, these cause multiple adverse events (Ghany MG et al., Hepatology. 2009; 49 (4): 1335-74).
• IFN pegylated interferon
• HCV is a virus belonging to the family Flaviviridae, genus hepacivirus. It is an enveloped virus, whose viral particle is between 50 and 70 nm in size, and is associated with very low density lipoproteins (VLDL) (Popescu Cl and Dubuisson J. Biol Cell. 2009 ; 102 (1): 63-74).
• VLDL very low density lipoproteins
• the viral genome is a single stranded, positive polarity ribonucleic acid (RNA) of approximately 9.6 kb.
• the genome encodes a viral polyprotein, which is co-and post-translationally processed into at least 10 viral proteins: Core, E1, E2, p7 (structural proteins) and non-structural proteins: NS2, NS3, NS4A, NS4B, NS5A, NS5B (Bartenschlager R and Lohmann V. 2000. J Gen Virol .81: 1631-48).
• This pathogen is an RNA virus, which can quickly mutate in adaptation to the host environment. This contributes to the high sequence diversity of the multiple viral isolates identified in the world.
• Six major genotypes have been described, which can be differentiated by up to 30% at the nucleotide sequence level (Simmonds P. J Hepatol. 1999; 31 Suppl 1: 54-60).
• the greatest heterogeneity is concentrated in the hypervariable region of HCV E2 protein, where there is precisely a possible target epitope for neutralizing antibodies.
• HCV circulates in the body as a heterogeneous population of viral molecules, a phenomenon known as "quasi-specific" (Simmonds P. J Gen Virol. 2004; 85 (Pt 1 1): 3173-88). It has been shown that the mutations constitute an escape route to the immune response, both humoral and cellular, developed by the host.
• HCV causes persistent infection in immunocompetent individuals, despite the existence of an active immune response (Lechmann et al., Semin Liver Dis 2000, 20, 21 1-226).
• active immune response Lechmann et al., Semin Liver Dis 2000, 20, 21 1-226.
• Currently, several viral effects on the immune system that contribute to viral persistence have been clarified, leading to the development of irrelevant immune responses and preventing the development of truly effective responses to their elimination. These effects are seen in both innate and acquired immunity (Grakoui A et al., Science 2003, 302 (5645): 659-62).
• the ineffective immune response against HCV not only fails to eliminate this pathogen, but also contributes to liver damage.
• Recombinant E1 protein from an isolation of genotype 1b, was purified as homodimers (Maertens et al., Acta Gastroenterol Belg 2000, 63, 203). Two chimpanzees chronically infected with HCV received 9 doses of 50 pg of recombinant E1 protein. Vaccination improved liver histology, determined the disappearance of viral antigens from the liver and decreased alanine aminotransferase (ALT) levels. Although serum viral RNA levels did not change during treatment, liver inflammation and viral antigens reappeared after the end of treatment.
• ALT alanine aminotransferase
• peptides without helper function can be poor immunogens;
• effectiveness of a vaccine is often based on the induction of a broad spectrum multivalent response against different antigens.
• the number of peptides included in the vaccine preparation increases the complexity of the formulation becomes greater, from all points of view.
• These limitations represent weaknesses of this strategy.
• different recombinant viral vectors have been evaluated in. the development of a vaccine against HCV.
• Defensive recombinant adenoviruses are attractive candidates, due to their hepatotropism, their potency to induce immunity, both humoral and cellular, and the possibility of being administered both parenterally and orally.
• Recombinant adenoviruses containing the genes encoding HCV structural proteins induce an antibody response against each of these proteins (Makimura et al., Vaccine 19 ⁇ 6, 14, 28-36).
• a specific cytotoxic T-type response against these antigens is detected (Bruna-Romero et al., Hepatology 1997, 25, 470-477).
• a modified Ankara vaccinia virus variant, recombinant for non-structural HCV antigens, from NS3 to NS5 has been evaluated in clinical studies in humans (Fournillier et al., Vaccine. 2007; 25 (42): 7339-53 ).
• This candidate has been immunogenic, and well tolerated, in a Phase I clinical study in individuals chronically infected with HCV.
• the impact of immunization with this vaccine candidate on viral load has been observed only in a fraction of those vaccinated, and temporarily. Therefore, in the same way, the clinical impact remains to be demonstrated.
• vaccine variants based on recombinant viruses are affected by safety and regulatory issues related to their application.
• it is a DNA vaccine that expresses a variant NS3 to NS5, which is administered by electroporation (Sallberg M, et al., Expert Opin Biol Ther. 2009; 9 (7): 805-15).
• it is a vaccine composition based on the mixture of a recombinant capsid protein with a plasmid for immunization with DNA, which expresses the structural antigens of HCV (Castellanos M, et al., J Gene Med. 2010; 12 (1): 107-16).
• the present invention is directed precisely to this objective.
• the present invention solves the aforementioned problem by providing a chimeric vaccine antigen against hepatitis C virus (HCV) comprising: a) a first segment consisting of the E2 region (amino acids 408-540) of the HCV polyprotein, b) a second segment consisting of the E1 region (amino acids 190-222) of the HCV polyprotein, and c) a third segment consisting of the Core region (amino acids 1 to 50) of the HCV polyprotein, in that order.
• HCV hepatitis C virus
• the novelty of the invention is given by the specific selection of epitopes and the order in which they are located in the generated protein variants.
• This Vaccine antigen design allows reducing the number of components needed to expand and enhance the immune response spectrum against different HCV antigens.
• a fusion protein is described which comprises in a single chimeric antigen the regions E2 (amino acids 408-540), E1 (amino acids 190-222) and Core (amino acids 1-50) of the HCV polyprotein, in That order particularly.
• the resulting immune response is relevant and is therefore directed against a broad spectrum of viral antigens.
• the selection of specific HCV regions avoids the use of other regions of viral proteins that may have immunosuppressive effects. Likewise, it avoids the use of other regions of viral proteins that could be immunodominant on those selected in the present invention, and that if included in the antigen itself would limit the induction of immune response against the regions selected in the present invention.
• the invention includes the order in which the selected regions are arranged in the artificial protein antigen, since this element significantly influences the induction of immune response against HCV, due to differences in the exposure of epitopes, and in their processing / presentation to the immune system.
• the regions of the Core, E1 and E2 antigens are arranged in reverse order as found in the native viral polyprotein.
• the chimeric vaccine antigen is characterized in that its amino acid sequence is selected from the group consisting of SEQ ID No. 10 (Eq1 antigen) and SEQ ID No. 16 (Eq1 b antigen).
• the chimeric antigens of the invention may comprise in their sequence at least one epitope specific for helper T lymphocytes.
• the specific epitope of helper T lymphocytes that is included within the chimeric antigens is an epitope of HCV non-structural proteins.
• the non-structural protein is the NS3 protein.
• the invention provides a chimeric vaccine antigen that is characterized in that its amino acid sequence is the sequence identified as SEQ ID No. 14 (EqNS3 antigen).
• the specific epitope of helper T lymphocytes that is included within the chimeric antigens is an epitope for artificial CD4 + T lymphocytes.
• artificial epitope is understood as that epitope whose amino acid sequence does not exist naturally, but is designed from bioinformatic elements. The selection and inclusion of artificial epitopes, which are recognized by helper T lymphocytes, contributes to the induction of a specific immune response against the HCV epitopes included in the chimeric antigenic variants.
• the P1 M epitopes SEQ ID No. 17
• P2B SEQ ID No. 18
• the invention provides a specific epitope of helper T lymphocytes whose amino acid sequence corresponds to that identified as SEQ ID No. 17 or SEQ ID No. 18.
• the chimeric antigens have an amino acid sequence that is selected from the group consisting of SEQ ID No. 12 (NSEq2 antigen), SEQ ID No. 13 (EqNSb antigen) and SEQ ID No. 15 (EqP1 antigen), which carry at least one of these helper T cell epitopes.
• the following chimeric antigens have the characteristics that are summarized below:
• the chimeric antigen Eq1 (SEQ ID No. 10) comprises the regions E2 (amino acids 408-540), E1 (amino acids 190-222) and Core (amino acids 1-50) of HCV polyprotein, in that order in particular.
• the chimeric antigen NSEq2 (SEQ ID No. 12) includes epitopes P2B and P1 M, in that order, inserted within Eq1, between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50).
• the chimeric antigen EqNSb (SEQ ID No.
• the chimeric antigen EqNS3 includes amino acid region 1242-1415 in the HCV polyprotein, inserted into Eq1, between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50).
• the chimeric antigen EqP1 includes the P1 M epitope, inserted into Eq1, between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50).
• the chimeric antigen Eq1 b (SEQ ID No.
• the chimeric antigens thereof were obtained via recombinant DNA, from a bacterium transformed with the plasmids described in Example 1. However, those versed in this branch of the art they know that said antigens can be obtained using other hosts, and purified by widely known techniques, with a view to being used for immunization.
• the invention provides a vaccine composition
• a vaccine composition comprising a chimeric HCV vaccine antigen comprising: a) a first segment consisting of the E2 region (amino acids 408-540) of the HCV polyprotein, b) a second segment consisting of the E1 region (amino acids 190-222) of the HCV polyprotein, and c) a third segment consisting of the Core region (amino acids 1 to 50) of the HCV polyprotein, in that order; and pharmaceutically acceptable excipients and / or adjuvants.
• the vaccine composition comprises the antigens identified as SEQ ID No. 10 (Eq1 antigen), SEQ ID No. 16 (Eq1 b antigen), SEQ ID No. 14 (EqNS3 antigen), SEQ ID No. 12 (NSEq2 antigen), SEQ ID No. 13 (EqNSb antigen) or SEQ ID No. 15 (EqP1 antigen).
• a wide range of pharmaceutically acceptable adjuvants which are commercially available, or those that are in development stages, can be used to enhance the immune response against chimeric antigens, within the vaccine compositions object of the invention.
• the vaccine compositions of the invention may additionally comprise a recombinant protein variant of the structural antigens or the NS3 HCV antigen.
• the vaccine compositions of the invention may additionally comprise a plasmid for immunization with DNA expressing the HCV structural antigens.
• the chimeric antigens can be formulated with a plasmid for immunization with DNA expressing the HCV structural antigens, and a recombinant HCV capsid protein, simultaneously.
• the invention contemplates that the chimeric antigen-based vaccine composition comprising a chimeric HCV vaccine antigen comprising: a) a first segment consisting of the E2 region (amino acids 408-540) of the HCV polyprotein, b) a second segment consisting of the E1 region (amino acids 190-222) of the HCV polyprotein, and c) a third segment consisting of the Core region (amino acids 1 to 50) of the HCV polyprotein, in that order, can be administered in sensitization / recall schemes with Plasmid-based preparations for immunization with DNA, recombinant proteins of HCV structural antigens, or a mixture of both.
• a chimeric HCV vaccine antigen comprising: a) a first segment consisting of the E2 region (amino acids 408-540) of the HCV polyprotein, b) a second segment consisting of the E1 region (amino acids 190-222) of the HCV polyprotein, and c)
• the vaccine compositions object of the present invention have the advantage of inducing an immune response against both humoral and cellular HCV, against various HCV antigens, so that they are active against a broad spectrum of viral isolates, being able to protect in a model of challenge with virus substitute.
• the vaccine compositions can be administered intramuscularly, intradermally, intraperitoneally, subcutaneously, intramucosally, intravenously, sublingually, or others that are known to those skilled in this branch of the art.
• the administration can be by syringes, spray or other administration devices.
• chimeric vaccine antigens comprising a first segment consisting of the E2 region (amino acids 408-540) of the HCV polyprotein, a second segment consisting of the E1 region (amino acids 190- 222) of the HCV polyprotein, and a third segment consisting of the Core region (amino acids 1 to 50) of the HCV polyprotein, in that order, for the manufacture of a vaccine for the induction of immune response against HCV.
• said vaccine is capable of protecting in vivo in a challenge model with substitute virus.
• the vaccines of the invention are capable of inducing response in healthy individuals or in patients infected with HCV. Therefore, it is also an aspect of the invention to provide a method for inducing an immune response against HCV that is characterized in that it is administered to a healthy individual, or to a patient infected with HCV, the chimeric vaccine antigen comprising a first segment consisting of the E2 region (amino acids 408-540) of the HCV polyprotein, a second segment consisting of the E1 region (amino acids 190-222) of the HCV polyprotein, and a third segment consisting of the Core region (amino acids 1 to 50) of the HCV polyprotein, in that order; or a vaccine composition that comprises it.
• the invention also contemplates that, in said method, the chimeric vaccine antigen or said vaccine composition comprising it is administered in sensitization / recall schemes with plasmid-based preparations for DNA immunization, recombinant proteins of HCV structural antigens, or A mixture of both.
• the antigens of the invention can be administered simultaneously with the medications that are included within the standard therapy for this type of patients.
• FIG. 1 Plasmid maps containing the coding sequences for the different chimeric antigens.
• E plENSb, plasmid for the expression of the EqNSb antigen.
• FIG. 1 Schematic representation of the different chimeric antigens.
• C: E1q1 antigen comprises E1 (amino acids 190-222), E2 (amino acids 408-540), and Core (amino acids 1-50) of the HCV polyprotein.
• NSEq2 antigen includes epitopes P2B and P1 M, inserted into Eq1 between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50).
• EqP1 antigen includes the P1 M epitope inserted into Eq1, between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50).
• H: Eq b antigen comprises regions E2 (amino acids 408-540), E1 (amino acids 190-222) and Core (amino acids 1-50) of the HCV polyprotein, of the H77 variant of HCV genotype 1a.
• FIG. 3 Antibody response against HCV proteins in immunization scheme with different chimeric antigens.
• the results are shown on the Y axis, as the reciprocal of the antibody titer, which is defined as the maximum dilution of the sera that shows an optical density at 492 nm at least twice the average of the optical density detected in sera from the negative control group, determined by ELISA.
• the different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) as coating antigens in the ELISA.
• FIG. 4 Proliferative response against HCV proteins in immunization scheme with different chimeric antigens.
• the results are shown on the Y axis as the stimulation index, which is defined as the relationship between cells that proliferate in the stimulation condition and the cells that proliferate in the non-stimulation condition, determined in a cytometry test by staining with CFSE ("carboxyfluorescein succinimidyl ester").
• the stimulation index greater than two is considered as a positive response.
• the different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) as antigens for cell stimulation.
• FIG. 5 IFN gamma secretion response against HCV proteins in immunization scheme with different chimeric antigens.
• the results are shown on the Y axis as Net Spots per million cells, which is defined as the number of spots detected in the stimulation condition minus the number of spots detected in the non-stimulation condition, determined in a test of ELISPOT secretion of IFN gamma.
• On the X axis they are shown the different immunogens administered to BALB / c mice. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) as antigens for cell stimulation.
• Figure 6 Response to the viral challenge in immunization scheme with different chimeric antigens.
• the results are shown on the Y axis as the logarithm of the viral titer, which is defined as the logarithm of the number of plaque forming units per mL, detected in ovaries of female mice after the viral challenge.
• vvRE viruses were used, vaccinia virus that expresses the Core, E1 and E2 antigens (amino acid region 1-650 in the HCV polyprotein) and WR, vaccinia virus that does not express HCV antigens.
• the different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• FIG. 7 Antibody response against HCV proteins in immunization scheme with Eq1 mixed with NS3. The results are shown on the Y axis as the reciprocal of the antibody titer, determined by ELISA. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein), E2 (amino acids 384-680 of the HCV polyprotein) and NS3 (amino acids 1192 to 1457 in the HCV polyprotein) as coating antigens in the ELISA.
• FIG. 8 Proliferative response against HCV proteins in immunization scheme with Eq1 mixed with NS3. The results are shown on the Y axis as the stimulation index, determined in a cytometry test by staining with CFSE. The stimulation index greater than two is considered as a positive response. The different immunogens administered to BALB / c mice are shown on the X axis. The error bars show the standard deviation of the average of the animals of the same group.
• the evaluated response was detected using recombinant variants of the Core protein (amino acids 1-120 of the capsid protein), E1 (amino acids 192-340 of HCV polyprotein), E2 (amino acids 384-680 of HCV polyprotein) and NS3 ((amino acids 1192 to 1457 in HCV polyprotein) as antigens for Stimulation of the cells.
• FIG. 9 IFN gamma secretion response against HCV proteins in immunization scheme with Eq1 mixed with NS3. The results are shown on the Y axis as Net Spots per million cells, determined in an ELISPOT assay of IFN gamma secretion. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein), E2 (amino acids 384-680 of the HCV polyprotein) and NS3 (amino acids 1,192 to 1457 in HCV polyprotein) as antigens for cell stimulation.
• Figure 10 Response to the viral challenge in immunization scheme with Eq1 mixed with NS3. The results are shown on the Y axis as the logarithm of the viral titer. The vvRE and WR viruses were used for the viral challenge. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• FIG. 11 Antibody response against HCV proteins in Eq1 immunization scheme combined with plasmid for DNA immunization. The results are shown on the Y axis as the reciprocal of the antibody titer, determined by ELISA. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group. The evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) as coating antigens in the ELISA.
• FIG. 12 Proliferative response against HCV proteins in Eq1 immunization scheme combined with plasmid for DNA immunization. The results are shown on the Y axis as the stimulation index, determined in a cytometry test by staining with CFSE. The stimulation index greater than two is considered as a positive response. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) , as antigens for cell stimulation.
• FIG. 13 IFN gamma secretion response against HCV proteins in immunization scheme with Eq1 combined with plasmid for DNA immunization. The results are shown on the Y axis as Net Spots per million cells, determined in an ELISPOT assay of IFN gamma secretion. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) , as antigens for cell stimulation.
• Figure 14 Response to the viral challenge in immunization scheme with Eq1 combined with plasmid for immunization with DNA. The results are shown in the Y axis as the logarithm of the viral titer, detected in ovaries of female mice after the viral challenge. The vvRE and WR viruses were used for the viral challenge. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• FIG. 15 Antibody response against HCV proteins in immunization scheme with Eq1 mixed with recombinant variants of HCV structural proteins. The results are shown on the Y axis as the reciprocal of the antibody titer, determined by ELISA. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group. The evaluated response was detected using variants Core protein recombinants (amino acids 1 -120 of the capsid protein), E1 (amino acids 192-340 of HCV polyprotein) and E2 (amino acids 384-680 of HCV polyprotein), as coating antigens in the ELISA Figure 16.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) , as antigens for cell stimulation.
• FIG. 17 IFN gamma secretion response against HCV proteins in immunization scheme with Eq1 mixed with recombinant variants of HCV structural proteins. The results are shown on the Y axis as Net Spots per million cells, determined in an ELISPOT assay of IFN gamma secretion. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) , as antigens for cell stimulation.
• Figure 18 Response to the viral challenge in immunization scheme with Eq1 mixed with recombinant variants of HCV structural proteins. The results are shown in the Y axis as the logarithm of the viral titer, detected in ovaries of female mice after the viral challenge. The vvRE and WR viruses were used for the viral challenge. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• Figure 19 Antibody response against HCV proteins in immunization scheme with different chimeric antigens that include artificial epitopes and NS3 protein.
• the results are shown on the Y axis as the reciprocal of the antibody titer, determined by ELISA.
• the different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (amino acids 1 -120 of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein), E2 (amino acids 384-680 of the HCV polyprotein) and NS3 ((amino acids 92 to 1457 in HCV polyprotein), as coating antigens in ELISA.
• FIG. 20 Proliferative response against HCV proteins in immunization scheme with different chimeric antigens that include artificial epitopes and NS3 protein. The results are shown on the Y axis as the stimulation index, determined in a cytometry test by staining with CFSE. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein), E2 (amino acids 384-680 of the HCV polyprotein) and NS3 ((amino acids 1 192 to 1457 in HCV polyprotein), as antigens for cell stimulation.
• FIG. 21 IFN gamma secretion response against HCV proteins in immunization scheme with different chimeric antigens including artificial epitopes and NS3 protein. The results are shown on the Y axis as Net Spots per million cells, determined in an ELISPOT assay of IFN gamma secretion. The different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (amino acids 1 -120 of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein), E2 (amino acids 384-680 of the HCV polyprotein) and NS3 ((amino acids 1 192 to 1457 in HCV polyprotein), as antigens for cell stimulation.
• Figure 22 Response to the viral challenge in immunization scheme with different chimeric antigens that include artificial epitopes and NS3 protein.
• the results are shown in the Y axis as the logarithm of the viral titer, detected in ovaries of female mice after the viral challenge.
• the vvRE and WR viruses were used for the viral challenge.
• the different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• FIG 23 Antibody response against HCV proteins in immunization scheme with chimeric antigens Eq1 and Eq1 b. The results are shown on the Y axis as the reciprocal of the antibody titer, determined by ELISA. The different immunogens administered to BALB / c mice are shown on the X axis. The error bars show the standard deviation of the mean of the animals of the same group. The evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) , as coating antigens in the ELISA.
• Figure 24 Antibody response against HCV proteins in immunization scheme with chimeric antigens Eq1 and Eq1 b. The results are shown on the Y axis as the reciprocal of the antibody titer, determined by ELISA. The different immunogens administered to BALB
• Proliferative response against HCV proteins in immunization scheme with chimeric antigens Eq1 and Eq b The results are shown on the Y axis as the stimulation index, determined in a cytometry test by staining with CFSE. The stimulation index greater than two is considered as a positive response.
• the different immunogens administered to BALB / c mice are shown on the X axis. Error bars show the standard deviation of the average of the animals in the same group.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein) , as antigens for cell stimulation.
• Figure 25 Response to the viral challenge in immunization scheme with chimeric antigens Eq1 and Eq1 b.
• the results are shown in the Y axis as the logarithm of the viral titer, detected in ovaries of female mice after the viral challenge.
• the vvRE and WR viruses were used for the viral challenge.
• the different immunogens administered to BALB / c mice are shown on the X axis.
• the Error bars show the standard deviation of the average of the animals in the same group.
• Example 1 Obtaining different chimeric antigens comprising HCV epitopes
• plMCo64K SEQ ID No. 1
• plME64K SEQ ID No. 2
• plME164K SEQ ID No. 3
• pl NSE64K SEQ ID No. 4
• plENSb obtained (SEQ ID No. 5), plENS3 (SEQ ID No. 6), plMP1 E64K (SEQ ID No. 7), plME64Kb (SEQ ID No. 8).
• These plasmids allow the expression in Escherichia coli of the chimeric antigens Coq1 (SEQ ID No. 9), Eq1 (SEQ ID No. 10), E1q1 (SEQ ID No. 1 1), NSEq2 (SEQ ID No.
• EqNSb SEQ ID No. 13
• EqNS3 SEQ ID No. 14
• EqP1 SEQ ID No. 15
• Eq1 b SEQ ID No. 16
• the Coq 1 chimeric antigen (SEQ ID No. 9) comprises the Core (amino acids 1-50), E1 (amino acids 190-222) and E2 (amino acids 408-540) regions of the polyprotein of the HCV, arranged in that order, particularly.
• the chimeric antigen Eq1 (SEQ ID No. 10) also includes the regions E2 (amino acids 408-540), E1 (amino acids 190-222) and Core (amino acids 1-50) of the HCV polyprotein, but in that order particular.
• the chimeric antigen E1 q1 (SEQ ID No.
• the chimeric antigen NSEq2 (SEQ ID No. 12) includes epitopes P2B and P1 M, in that order, inserted in Eq1, between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50).
• the chimeric antigen EqNSb (SEQ ID No. 13) includes the epitope P2B inserted in Eq1 between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50).
• the chimeric antigen EqNS3 (SEQ ID No. 14) includes amino acid region 1242-1415 in the HCV polyprotein, inserted between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50) of the chimeric antigen Eql El chimeric antigen EqP1 (SEQ ID No. 15) includes the P1 M epitope inserted into Eq1, between the regions of E1 (amino acids 190-222) and Core (amino acids 1-50).
• the chimeric antigen Eq1 b (SEQ ID No.
• the artificial epitopes P1 and P2B included in some of the chimeric antigens, were designed by bioinformatics, to be recognized by human helper T lymphocytes.
• the reasons for binding to HLA-DR13 and HLA-DR1 1 were studied, using the Rankpep, SYFPETHI and ProPred programs, to propose amino acid variants by position in artificial epitopes, mainly according to the frequency of occurrence.
• Artificial T epitopes of 14 amino acids, whose sequence is LPEYVIMVKLPSRA (SEQ ID No. 17), are described as artificial epitopes for helper T lymphocytes.
• P2B of 15 amino acids, whose sequence is GYKVIVLNPRVASTL (SEQ ID No. 18).
• the recombinant protein antigens competent cells of the E. coli strain GC-366 were transformed with the respective plasmids.
• the expression of the recombinant proteins was developed for 12 h at 37 degrees Celsius, using minimal medium for cell cultures. All protein antigens contain at the N-terminus an expression stabilizing fragment, from the P64K protein of Neisser ⁇ a meningitidis, previously known for this function (Yero D et al., Biotechnol Appl Biochem. 2006; 44 (Pt 1) : 27-34).
• the protein vanants comprise at the C-terminal end a tail of 6 Histidines, in order to facilitate their purification.
• the proteins were purified by solubilization of the insoluble fraction from the cell rupture with carbonate-bicarbonate buffer pH 9.6, Urea 8M, and subsequent affinity chromatography by metal chelates.
• mice 8 week old females, weighing 16-18 g, 17 animals per group were immunized.
• the immunization groups were as follows: Group 1, Coq1 chimeric antigen adjuvant in alumina; Group 2, alumina adjuvant E1q1 antigen; Group 3, Eq1 antigen adjuvant in alumina; Group 4, alumina (group control). In all cases, 20 pg of recombinant antigen was administered. Immunizations were performed at weeks 0, 2 and 4, by intramuscular injection. Blood extractions were performed at weeks 0 and 6 to study the antibody response against HCV antigens. In addition, 5 mice were sacrificed per group, at week 6, for the study of the specific cellular response.
• mice were challenged with vvRE recombinant vaccinia virus (Alvarez-Lajonchere et al., Applied Biotechnology 2007; 24 (3- 4): 246-253), which expresses the HCV structural antigens, and another 5 animals with WR control vaccinia virus, at week 6. Five days later, the mice were sacrificed, and the viral titer in ovaries was determined, as previously described (Alvarez-Lajonchere et al., Biotechnol Appl Biochem. 2008; 51 ( Pt 2): 97-105).
• the specific immune response against HCV antigens is shown in Figures 3 through 5.
• the evaluated response was detected using recombinant variants of the Core protein (amino acids 1-120 of the capsid protein, Alvarez-Obregon JC and cois. Vaccine 2001; 19: 3940-3946), ET (amino acids 192-340 of the HCV polyprotein, Lorenzo LJ et al., Biotechnol Appl Biochem 2000; 32 (2): 137-143), and E2 (amino acids 384-680 HCV polyprotein, Mart ⁇ nez-Donato et al., Mol Biotechnol. 2007; 35 (3): 225-36) as coating antigens in ELISA, or as stimulation antigens in cell response assays.
• the Core protein amino acids 1-120 of the capsid protein, Alvarez-Obregon JC and cois. Vaccine 2001; 19: 3940-3946
• ET amino acids 192-340 of the HCV polyprotein, Lorenzo L
• mice 8 week old females, 16-18 g in weight, 17 animals per group were immunized.
• the immunization groups were the following: Group 1, Eq1 antigen adjuvant in alumina; Group 2, Eq1 antigen mixed with recombinant NS3 protein (Palenzuela D et al., Applied Biotechnology 2006; 23: 94-98) alumina adjuvants; Group 3, recombinant protein NS3 adjuvant in alumina; Group 4, alumina (control group). 20 pg of Eq1 recombinant chimeric antigen and 10 pg of NS3 recombinant protein were administered in the corresponding groups. Immunizations were performed at weeks 0, 2 and 4, by intramuscular injection.
• the specific immune response against HCV antigens is shown in Figures 7 through 9.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein, E1 (amino acids 192-340 of HCV polyprotein), E2 (amino acids 384-680 of HCV polyprotein), and NS3 (amino acids 1 192 to 1457 in HCV polyprotein), as coating antigens in ELISA, or as stimulation antigens in Cellular response assays
• E1 amino acids 192-340 of HCV polyprotein
• E2 amino acids 384-680 of HCV polyprotein
• NS3 amino acids 1 192 to 1457 in HCV polyprotein
• mice 8 week old females, 16-18 g in weight, 17 animals per group were immunized.
• the immunization groups were the following: Group 1, Eq1 antigen adjuvant in alumina; Group 2, Eq1 antigen mixed with plasmid for immunization with plDKE2 DNA (Due ⁇ as-Carrera et al., Biotechnol Appl Biochem. 2004; 39: 249-55) in saline solution; Group 3, Eq1 antigen mixed with plasmid plDKE2 and with recombinant protein Co.
• the specific immune response against HCV antigens is shown in Figures 1 through 13.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein) and E2 (amino acids 384-680 of the HCV polyprotein), as coating antigens in the ELISA, or as antigens for stimulation in cell response assays.
• antibody response against HCV structural antigens is induced in all immunized groups, with the exception of controls.
• the group immunized with the mixture of Eq1 and Co.120 proteins with plasmid plDKE2 induced a response proliferative against the Ei and E2 antigens significantly higher than that induced by the rest of the groups (p ⁇ 0.05, ANOVA and Newman-Keuls multiple comparisons test), with the exception of the immunized in sensitization / recall scheme (Group 4) .
• mice 8 week old females, 16-18g in weight, 17 animals per group were immunized.
• the immunization groups were as follows: Group 1, Eq1 antigen adjuvant in alumina; Group 2, mixture of proteins Co.120, E1.340 (Lorenzo LJ et al., Biotechnol Appl Bioc em 2000; 32 (2): 137-143) and E2.680 (Mart ⁇ nez-Donato et al., Mol Biotechnol.
• Eq1 chimeric antigen 20 pg of Eq1 chimeric antigen were administered; 16.7 pg of E1 and E2 proteins, as well as 0.1 pg of Co.120 protein, in the corresponding groups. Immunizations were performed at weeks 0, 2 and 4 by intramuscular injection.
• the specific immune response against HCV antigens is shown in Figures 15 through 17.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of HCV polypyrotein) and E2 (amino acids 384-680 of HCV polyprotein), as coating antigens in ELISA, or as stimulation antigens in cell response assays.
• E1 amino acids 192-340 of HCV polypyrotein
• E2 amino acids 384-680 of HCV polyprotein
• proliferative response against HCV antigens was induced in all groups, with the exception of control, as shown in Figure 16. There were no statistically significant differences between the groups that received the immunogenic variants with respect to the response. proliferative against the E1 and E2 antigens. In contrast, all groups that received chimeric Eq1 antigen, in any of the combinations (Groups 1 to 4), induced a proliferative response against Core significantly higher than that induced by the HCV structural protein mixture (Group 5) (p ⁇ 0.05, ANOVA and Newman-Keuls multiple comparisons test).
• the IFN gamma secretory cell response had a similar behavior to that observed for the proliferative response.
• group immunized with the HCV structural protein mixture (group 5) had an IFN gamma secreting cell response against Core significantly lower than that induced in the groups immunized with the chimeric antigen Eq1 alone (Group 1) and the mixture of Eq1 with the HCV structural proteins (Group 4) (p ⁇ 0.05, ANOVA and Newman-Keuls multiple comparisons test).
• a vaccine composition based on the administration of the chimeric antigen Eq1 mixed with a preparation comprising recombinant protein variants of the structural antigens Core, E1 and E2, allows the induction of a specific increased immune response both humoral and cellular against the antigens HCV structure, with functional activity in vivo, as it is able to protect against the challenge model with substitute virus.
• Example 6 Immunogenicity study in mice of different chimeric antigens comprising HCV epitopes and artificial epitopes specific for helper T lymphocytes BALB / c mice, 8 week old females, 16-18 g in weight, 17 animals per group were immunized.
• the immunization groups were as follows: Group 1, chimeric antigen Eq 1 adjuvant in alumina; Group 2, chimeric antigen NSEq2 adjuvant in alumina; Group 3, chimeric EqNSb antigen adjuvant in alumina; Group 4, chimeric antigen EqNS3 adjuvant in alumina; Group 5, chimeric EqP1 antigen adjuvant in alumina; Group 6, alumina (control). 20 g of the recombinant chimeric antigens were administered in the corresponding groups. Immunizations were performed at weeks 0, 2 and 4, by intramuscular injection.
• mice were sacrificed per group at week 6, for the study of the specific cellular response. Additionally, 5 animals per group were challenged with vvRE recombinant vaccinia virus, which expresses the HCV structural antigens, and another 5 animals with the WR control vaccinia virus, at week 6. Five days later, the mice were sacrificed, and it was determined viral titer in ovaries as previously described.
• the specific immune response against HCV antigens is shown in Figures 19 through 21.
• the evaluated response was detected using recombinant variants of the Core protein (1-120 amino acids of the capsid protein), E1 (amino acids 192-340 of the HCV polyprotein), E2 (amino acids 384-680 of the HCV polyprotein) and NS3 (amino acids 1 192 to 1457 in the HCV polyprotein), as coating antigens in the ELISA, or as stimulation antigens in the assays of cellular response.
• results show that the insertion of epitopes or regions of NS3 in the Eq1 sequence allows the induction of a specific immune response against this viral antigen without affecting the immune response against HCV structural antigens.
• the inclusion of the artificial epitopes P1 M and P2B in the sequence of the Eq1 protein makes it possible to significantly increase the proliferative immune response against the Core and E1 antigens, without affecting the ability to induce humoral or cellular immune response against those same antigens. or E2, maintaining functional activity in vivo, as it is able to protect against the challenge model with substitute virus.
• mice 8 week old females, 16-18 g in weight, 17 animals per group were immunized.
• the immunization groups were as follows: Group 1, chimeric antigen Eq1 b adjuvant in alumina; Group 2, chimeric antigen Eq1 adjuvant in alumina; Group 3, alumina (control). 20 pg of the recombinant chimeric antigens were administered in the corresponding groups. Immunizations were performed at weeks 0, 2 and 4, by intramuscular injection. Blood extractions were performed at weeks 0 and 6 to study the antibody response against HCV antigens. In addition, 5 mice were sacrificed per group at week 6, for the study of the specific cellular response.
• mice were challenged with vvRE recombinant vaccinia virus, which expresses the HCV structural antigens, and another 5 animals with the WR control vaccinia virus, at week 6. Five days later the mice were sacrificed, and the viral titer in ovaries as previously described.
• the specific immune response against HCV antigens is shown in Figures 23 and 24.
• the evaluated response was detected using recombinant variants of the Core protein (amino acids 1 -120 of the capsid protein), E1 (amino acids 192-340 of HCV polyprotein) and E2 (amino acids 384-680 of HCV polyprotein), as coating antigens in ELISA, or as stimulation antigens in cell response assays.
• the Core protein amino acids 1 -120 of the capsid protein
• E1 amino acids 192-340 of HCV polyprotein
• E2 amino acids 384-680 of HCV polyprotein

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