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US20040234543A1 - Vaccine formulation potentiated by the combination of a dna and an antigen - Google Patents

Vaccine formulation potentiated by the combination of a dna and an antigen Download PDF

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US20040234543A1
US20040234543A1 US10/484,112 US48411204A US2004234543A1 US 20040234543 A1 US20040234543 A1 US 20040234543A1 US 48411204 A US48411204 A US 48411204A US 2004234543 A1 US2004234543 A1 US 2004234543A1
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hcv
dna
protein
virus
hepatitis
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Inventor
Santiago Carrera
Juan Grillo
Liz Alvarez-Lajonchere de Leon
Alexis Lasa
Rolando Feyt
Ariel Rodriguez
Julio Obregon
Nelson Rivero
Gillian Donato
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Centro de Ingenieria Genetica y Biotecnologia CIGB
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Assigned to CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA reassignment CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CARRERA, SANTIAGO DUENAS, DONATO, GILLIAN MARTINEZ, FEYT, ROLANDO PAJON, GRILLO, JUAN MORALES, PONCE DE LEON, LIZ ALVAREZ-LAJONCHERE, LASA, ALEXIS MUSACCHIO, OBREGON, JULIO C. ALVAREZ, RIVERO, NELSON ACOSTA, RODRIGUEZ, ARIEL VINA
Publication of US20040234543A1 publication Critical patent/US20040234543A1/en
Priority to US12/430,534 priority Critical patent/US8691234B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • 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
    • 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
    • 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
    • A61P31/20—Antivirals for DNA 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—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
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is related with the branch of the medicine, particularly with a new formulation of vaccine antigens.
  • the technical objective of the present invention is the development of new vaccine formulations, minimizing the number of components that are able to induce an enhanced and diverse immune response through the interaction among them. Additionally, the development of combined vaccine formulations is approached in order to increase the immune response induced against the co-administered antigens.
  • the recombinant E1 protein from an isolate of the genotype 1 b was purified as homodimers self-associating in particles of 9 nm diameter, approximately (Maertens et al., Records Gastroenterol Belg 2000, 63, 203). Two chimpanzees chronically infected with HCV received 9 doses of 50 ⁇ g of the recombinant E1 protein. The vaccination improved the hepatic histology and determined the disappearance of the viral antigens of the liver. Vaccination with recombinant E1 protein also reduced the levels of alanine aminotransferase (ALT).
  • ALT alanine aminotransferase
  • virus-like particles from recombinant proteins and their employment as vaccines is very attractive because these structures frequently simulate viral properties.
  • This kind of particles obtained from insect cells infected with a recombinant baculovirus containing the sequence of the HCV structural antigens, have been able to generate both humoral and cellular immune response against these antigens (Baumert et al., Gastroenterology 1999, 117, 1397-407; Lechmann et al., Hepatology 1999, 30, 423-429). Although the results obtained with vaccines based on protein subunits are encouraging, the immune response induced by these variants is mainly humoral, short-term and isolate-specific.
  • recombinant viral vectors have been evaluated in the development of a recombinant vaccine against the HCV.
  • recombinant adenoviral vectors are interesting candidates due to their liver tropism, their power to induce both humoral and cellular immunity, and the feasibility for oral or systemic delivery.
  • Adenoviruses containing the DNA encoding sequence for the HCV structural proteins induce an antibody response against each one of these proteins (Makimura et al., Vaccine 1996, 14, 28-36).
  • recombinant viruses like vaccinia, canary-pox and fowl-pox, containing different HCV genes have induced strong CTL and T-helper immune responses in mice (Shirai et al., J Virol 1994, 68, 3334 -3342; Large et al., J Immunol 1999, 162, 931-938).
  • these recombinant viruses, as well as other variants of alpha virus like the Semliki Forest Virus are also affected by regulatory issues and security concerns related with their application.
  • a DNA vaccine consists on a purified plasmid containing the sequence coding for an antigen of interest, under the control of a functional transcriptional unit in eucariotic cells. After injection of the plasmid in muscle or the skin, the plasmid is taken up by host cells and the antigen is expressed intracellularly. The expression of the encoded antigens in the host cells is one of the major advantages of this methodology because is similar to viral natural infections. The simplicity to manipulate the DNA, together with the DNA stability that makes possible a relatively cheap large-scale production of DNA, is other advantage of DNA vaccination.
  • the immune response induced with this kind of vaccines can be modulated by co-immunization with molecules or genes coding for co-stimulatory molecules like cytokines.
  • the genetic constructs can be modified, by insertions or deletions of transmembrane domains, signal sequences for secretion, or other types of residues affecting the intracellular trafficking and processing of the antigen.
  • Plasmids expressing fusion variants to the hepatitis B virus (HBV) surface antigen or other envelope antigens of the HBV have been evaluated (Major et al., J VIROL 1995, 69, 5798-5805). Immunization with these plasmids has generally induced positive CTL and lymphocyte proliferative response.
  • HBV hepatitis B virus
  • the HCV envelope proteins have also constituted targets of interest for this type of technology.
  • the humoral response seems to be mainly directed to the HVR-1 (Lee et al., Mol Cells 1998, 8, 444-451).
  • Immunization with plasmids expressing intracellular or secreted variants of the E1 and E2 proteins has rendered similar immune response (Lee et al., J VIROL 1998, 72, 8430-8436).
  • the inoculation with bicistronic plasmids expressing the GM-CSF and the HCV E1 or E2 proteins increased both humoral and cellular immune response.
  • the non structural proteins have also been evaluated by this technology. Good results were obtained when the region coding for the C-terminal domain of the NS3 protein was included in a vector that allows the simultaneous and independent expression of this domain and the IL-2 (Papa et al., Res Virol 1998, 149, 315-319).
  • the NS4 and NS5 proteins also generate Abs and CTL responses by this immunization strategy (Encke et al., J IMMUNOL 1998, 161, 4917-4923).
  • Dendritic cells derived of former genetically modified mouse bone marrow to express tumor antigens, by using viral vectors (Specht et al., J Exp Med 1997, 186, 1213-1221; Brossart et al., J Immunol 1997, 158, 3270-3276; Song et al., J Exp Med 1997, 186, 1247-1256), or RNA (Boczkowski et to the., J Exp Med 1996, 184, 465-472), have demonstrated their capacity to promote T cell response specific for tumor antigens, and prophylactic immunity mediated by cells against tumors in mouse.
  • a vaccine composition comprising the complex formed by the hepatitis B surface antigen, an antibody specific for this antigen, and a DNA vaccine expressing for this antigen has been evaluated (Wen et al., U.S. Pat. No. 6,221,664, 1998).
  • This formulation allowed the antigen presentation by different means and a quick induction of immune response that resulted superior regarding to the one generated by the individual variants.
  • a vaccine formulation comprising as components only a protein antigen and a plasmid expressing one or several proteins, acting at least one of them as adjuvant of the other one.
  • the capsid antigen of the hepatitis C or B virus, and a plasmid expressing individual or polyprotein variants of the HCV E1 protein are evaluated.
  • the presence of antibodies in the formulation is not required to generate the enhancement of the immune response, thus reducing the number of components required.
  • the biggest flexibility in the vaccine composition also allows generating simultaneously potent immune responses against different antigens.
  • the present invention provides the composition and methods to immunize an individual in a prophylactic or therapeutic way against the HCV and the HBV, as well as their combination. It is reported for the first time a vaccine formulation having as components: (a) a DNA that expresses a protein variant that includes regions of the E1 antigen of the HCV envelope and (b) a protein antigen of the HCV or HBV, in appropriate proportions.
  • the novelty of the invention is given by the adjuvant effect of at least one component on the immune response generated against the other one.
  • Antigens coded by the genetic constructs and expressed by the host cells, as well as the protein antigen comprising the vaccine the formulation are interesting targets to generate an immune response against the HCV and the HBV. Thus, the immune response can be directed against a wide spectrum of important antigens.
  • the vaccine formulation includes a DNA enhancing the immune response generated against a protein antigen mixed with him; being this effect dependent on the expression of one or several proteins coded by the DNA, in the immunized host.
  • the DNA is obtained from a bacterial strain and purified according to traditional procedures (Horn et al., H Gene Ther 1995, 6, 565-573).
  • the vaccine formulation comprises in preferred embodiments at least one of the following plasmids: plDKE1S, plDKE2 and PAEC-ME, whose DNA sequences coding for the protein variants expressed are identified with the number of sequence of 2-4, respectively.
  • the plDKE1S plasmid expressed a protein that comprise the aa from the 176 to the 363 of the HCV E1 (Sec. Id. No. 2).
  • the plDKE2 plasmid expressed a protein encompassing the first 650 aa of the viral polyprotein (C, E1 and a part of the E2) (Sec. Id. No.3).
  • the pAEC-ME plasmid expresses a chimeric protein comprising B and T cells epitopes of different HCV antigens (Sec. Id. No. 4).
  • the coding sequence for the viral antigens was obtained from the cDNA of a HCV cuban isolate (Morales et al., 1998, WO 9825960).
  • the pAEC-ME, plDKE1S and plDKE2 plasmids contain the coding sequence for the HCV antigens inserted into the multiple cloning site of the pAEC-K6 plasmid (Herrera et al., Biochem Biophys Res Commun . 2000, 279, 548-551).
  • the plasmids included in the present invention have the regulatory elements able to direct the antigen expression in human cells.
  • These regulatory elements include a transcriptional unit functional in mammals, integrated for example by the human cytomegalovirus promoter and the polyadenilation signal of the simian virus 40.
  • These plasmids also contain a replication origin in bacteria and a selection marker for the resistance to kanamyicin.
  • the protein component of the formulation can be a soluble viral antigen able to form particles, with a purity superior to 90%.
  • the present invention also contemplates the procedure for the mixture of the DNA with the antigen.
  • the mixture is prepared by addition of components, DNA and antigen, dissolved in an appropriate buffer.
  • the formulation can be prepared by the combination of both components, dissolved in saline phosphate, in 101 (ww) proportion.
  • the mixture is incubated at least 2 h between 26° C. and 30° C., with shaking, before administration to the individuals.
  • This formulation can be administered by intramuscular, subcutaneous, intraperitoneal, intramucosal, intravenous sublingual way, or others.
  • the immunization can be performed by means of syringes, gene gun, sprays or other delivery devices.
  • Each individual receives a dose ranging from 0.001 to 10 mg of each component in a volume determined by the animal species and the immunization method employed.
  • a superior product can be obtained compared with each one of the individual components due to:
  • the immunization with a DNA that expresses a protein variant that includes regions of the HCV E1 protein increased the immunogenicity of HBV protein antigens, present in the formulation.
  • the mixture with the HBsAg or the HBcAg allows superior results to those obtained with this antigens due to:
  • FIG. 1 Schematic representation of the plasmids pAEC-ME, plDKE1S and plDKE2.
  • FIG. 2 E 1 ectron microscopy of the particles of the hepatitis C virus capsid (A), of the hepatitis B virus surface antigen (B) and of the hepatitis B virus capsid (C).
  • FIG. 3 Immunization schedule with the plDKE2 plasmid and the Core protein. The animals were immunized intramuscularly with 50 ⁇ g of DNA and 5 ⁇ g of protein.
  • FIG. 4 Immunization schedule with different plasmids and the protein HBcAg. The animals were immunized intramuscularly with 50 ⁇ g of DNA and 5 ⁇ g of protein.
  • FIG. 5 Immunization schedule with different plasmids and the protein HBsAg. The animals were immunized intramuscularly with 50 ⁇ g of DNA and 5 ⁇ g of protein.
  • the plasmid contains the sequence coding for the first 650 aa of the viral polyprotein, Sec. Id. No.3).
  • the group 2 was inoculated with 5 ⁇ g of the Core protein (comprising the first 173 aa of the HCV capsid protein).
  • the group 3 received a first dose with 5 ⁇ g of the Core protein and a second one with 50 ⁇ g of the plDKE2 (CoreplDKE2).
  • the group 4 was inoculated under similar conditions to the group 3 but in inverse order (plDKE2Core).
  • the group 5 was inoculated with the mixture of 50 ⁇ g of the plDKE2 and 5 ⁇ g of the Core protein in the days 0 and 21 (CoreplDKE2).
  • the group 6 was inoculated in the same way that the group 5 but only in the day 0 (CoreplDKE2 (1)). Additionally, a seventh group, negative control, was immunized with 50 ⁇ g of the plasmid pAEC-K6 (it doesn't contain sequences coding for the HCV antigens).
  • the antibody response was determined by ELISA to detect the Ab response against the HCV structural proteins.
  • the Student T test was employed to analyze the results, statistical differences were considered for p ⁇ 0.05.
  • FIG. 3 shows that it is possible to increase the immune response against the HCV structural antigens by the administration of two doses of the mixture of the plDKE2 with the Core protein with respect to the individual components.
  • This formulation in two doses
  • These Ab titers were also statistically higher to the levels of Abs against the HCV capsid protein, generated by the plDKE2-Core mixture administered in a single dose (FIG. 3A).
  • the inoculation of the mixture in a single dose always induced the lower levels of Abs among the immunized groups.
  • FIG. 3B The evaluation of the lymphoproliferative response against the HCV structural antigens (FIG. 3B) indicated a significantly superior response against the capsid in the group of animals immunized with the plDKE2-core in 2 doses, with respect to the remaining groups. The results are shown as the stimulation index of spleen cells obtained from immunized animals. The stimulation index was determined by the (H 3 ) Thymidine uptake. It is possible to conclude that the immunization with the mixture of plDKE2 and the Core protein generates a synergic stimulation of the immune response induced against the HCV structural antigens.
  • the group 1 was inoculated with the plasmid pAEC-K6 (negative control).
  • the group 2 was administered with the HBcAg protein.
  • the group 3 was vaccinated with plDKE2.
  • the groups 4 and 5 were vaccinated with the mixture of the HBcAg with the plasmids plDKE2 and pAEC-K6, respectively.
  • the Student T test was employed to analyze the results statistically, a significant difference was considered for p ⁇ 0.05.
  • FIG. 4 shows the antibody response induced in mice 19 weeks after primary immunization.
  • FIG. 4A shows that the mixture of the plDKE2 plasmid with the HBcAg induced Ab titers against the HBcAg, statistically higher to the observed in the rest of the vaccinated animals. No statistical differences were detected between the groups immunized with HBcAg alone or mixed with the pAEC-K6. Therefore it is possible to conclude that the plasmid plDKE2 enhance the immune response against the HBcAg.
  • FIG. 4B shows that the mixture of the plDKE2 plasmid with the HBcAg induces antibody titers against the HCV structural antigens higher to those generated in the animals immunized with the plDKE2 alone. Therefore, the HBcAg is also capable of enhance the immune response induced against the HCV structural antigens induced after the administration of the plDKE2.
  • the group 1 was inoculated with the mixture of 50 ⁇ g of the plasmid plDKCo, containing the sequence coding for the first 176 aa of the HCV capsid protein (Due ⁇ as-Carrera et al., Vaccine 2000;19(7):992-997), and 5 ⁇ g of the HBsAg (plDKCo-HBsAg).
  • the groups 2 to 7 were inoculated with mixtures of DNA and HBsAg in same quantities but using the following plasmids: group 2 (plDKE1S-HBsAg), the plasmid plDKE1S (FIG. 1, containing the sequence coding for the aa 176-363 of the HCV polyprotein, Sec. Id.
  • group 3 group 3 (pAEC-ME-HBsAg), the plasmid pAEC-ME (FIG. 1, containing the sequence coding for a protein that includes different epitopes of the HCV antigens, Sec. Id. No.4); group 4 (plDKE2-HBsAg), the plasmid plDKE2 (FIG. 1) containing the sequence coding for the aa 1-650 of the HCV polyprotein, Sec. Id.
  • the plasmid plDKE1Sm is identical to the plDKE1S except that it includes 2 nucleotide insertions in the region coding for the HCV E1 that changes the open reading frame and impedes the expression of this protein (Sec. Id.
  • group 6 pAEC-d2-HBsAg-HBsAg
  • the plasmid pAEC-d2-HBsAg contains the sequence coding for the HBV HBsAg (Musacchio et al., Biochem Bioph Res Commun 2001, 282, 442446)
  • group 7 pAEC-K6-HBsAg
  • the plasmid pAEC-K6 negative control, doesn't contain coding sequence under the control of the transcriptional unit.
  • the groups 8 and 9 were inoculated with 5 ⁇ g of HBsAg formulated in Aluminum hydroxide or alone, respectively. The Student T test was employed to analyze the results statistically, a significant difference was considered for p ⁇ 0.05.
  • FIG. 5 shows the Abs titers generated against the HBsAg, 16 weeks after primary immunization.
  • the levels of Abs induced by the HBsAg alone in PBS were statistically inferior to the rest of the variants evaluated except for the mixture formed by the HBsAg and the pAEC-K6.
  • the mixtures of HBsAg with the plasmids plDKCo, plDKE1S, pAEC-ME and plDKE2 induced Ab titres against the HBsAg statistically higher to those induced by the immunization with the HBsAg formulated in Aluminum hydroxide or mixed with the pAEC-K6.
  • the immunization with the HBsAg formulated with aluminum hydroxide or mixed with pAEC-K6, plDKE1Sm and pAEC-d2-HBsAg induced similar levels of Ab titers against the HBsAg. It is possible to conclude that the expression in the host cells of protein variants that include the amino acid regions of the HCV E1 antigen, from the plasmids administered, enhance the immune response generated against the protein antigen mixed with the DNA construct.

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WO2006078979A2 (en) 2005-01-20 2006-07-27 Nature Technology Corp. Vectors and methods for genetic immunization
US20100303859A1 (en) * 2007-05-29 2010-12-02 Nature Technology Corporation Vectors and method for genetic immunization

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CU23496A1 (es) * 2004-09-03 2010-02-23 Ct Ingenieria Genetica Biotech Composición vacunal contra el virus de la hepatitis c
CN104830789A (zh) * 2015-05-05 2015-08-12 杨光华 基于hcv1抗原的dc细胞、靶向性免疫细胞群及其制备方法和用途

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