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BR-112017004008-B1 - Nucleic acid sequence, Pichia pastoris host cell, virus-like particle (VLP), method for producing a VLP, vaccine and use thereof to treat or prevent dengue virus infection.

BR112017004008B1BR 112017004008 B1BR112017004008 B1BR 112017004008B1BR-112017004008-B1

Abstract

RECOMBINANT POLYPEPTIDE, NUCLEIC ACID SEQUENCE, HOST CELL, BIONANOPARTICLE, METHODS FOR PRODUCING A BIONANOPARTICLE AND FOR TREATING OR PREVENTING DENGUE VIRUS, AND VACCINE. The invention provides a recombinant polypeptide comprising the EDIII domain of each of the Dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4 bound to the N-terminal of HBsAg.

Inventors

  • Navin Khanna
  • Viswanathan RAMASAMY

Assignees

  • INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY

Dates

Publication Date
20260317
Application Date
20150821
Priority Date
20140901

Claims (13)

  1. 1. Nucleic acid sequence CHARACTERIZED in that it comprises: (i) a first nucleic acid sequence encoding a recombinant protein comprising the EDIII domain of Dengue virus serotypes DENV-1, DENV-2, DENV-3, and DENV-4 bound to the N-terminal end of Hepatitis B virus surface antigen (HBsAg); wherein the EDIII domains are sequentially fused, N-terminal end to C-terminal end, in the sequence DENV-1, DENV-3, DENV-4, DENV-2; and wherein the nucleic acid sequences of the EDIII domains of DENV-1, DENV-2, DENV-3, and DENV-4 are presented in SEQ ID NOs: 5, 6, 7, and 8, respectively; and (ii) a second nucleic acid sequence comprising four expression cassettes encoding HBsAg.
  2. 2. Host cell of Pichia pastoris CHARACTERIZED in that it is transformed or transfected with the nucleic acid sequence as defined in claim 1.
  3. 3. Virus-like particle (VLP) CHARACTERIZED in that it comprises: (i) a recombinant protein comprising the EDIII domain of Dengue virus serotypes DENV-1, DENV-2, DENV-3, and DENV-4 linked to the N-terminal end of Hepatitis B virus surface antigen (HBsAg); wherein the EDIII domains of the recombinant protein are sequentially fused, N-terminal end to C-terminal end, in the sequence DENV-1, DENV-3, DENV-4, DENV-2; and wherein the EDIII domains of DENV-1, DENV-2, DENV-3, and DENV-4 are encoded by the nucleic acid sequences presented in SEQ ID NOS: 5, 6, 7, and 8, respectively; and (ii) HBsAg encoded by four expression cassettes.
  4. 4. Virus-like particle (VLP), according to claim 3, CHARACTERIZED in that it is obtained from the host cell of Pichia pastoris, as defined in claim 2.
  5. 5. Method for producing a virus-like particle (VLP) CHARACTERIZED in that it comprises cultivating the Pichia pastoris host cell, as defined in claim 2, under appropriate conditions and recovering the VLP comprising recombinant protein and HBsAg.
  6. 6. Vaccine CHARACTERIZED in that it comprises a virus-like particle (VLP), as defined in claim 3 or 4.
  7. 7. Use of virus-like particles (VLPs), as defined in claim 3 or 4, CHARACTERIZED in that it is in the manufacture of a medicament for the treatment or prevention of Dengue virus infection.
  8. 8. Use of the vaccine, as defined in claim 6, CHARACTERIZED by the fact that it is in the manufacture of a medicament for the treatment or prevention of Dengue virus infection.
  9. 9. Virus-like particle (VLP), according to claim 3 or 4, CHARACTERIZED in that it is for use in the treatment or prevention of Dengue virus infection.
  10. 10. Vaccine, according to claim 6, CHARACTERIZED in that it is for use in the treatment or prevention of dengue virus infection.
  11. 11. Vaccine, according to claim 6, CHARACTERIZED in that the vaccine generates serotype-specific neutralizing antibodies against each of the four dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4.
  12. 12. Vaccine, according to claim 6, CHARACTERIZED in that the vaccine generates balanced neutralization titers against each of the four serotypes.
  13. 13. Vaccine, according to claim 6, CHARACTERIZED in that the vaccine generates an immune response against all five components, namely EDIII-1 (DENV-1), EDIII-2 (DENV-2), EDIII-3 (DENV-3), EDIII-4 (DENV-4), and HBsAg.

Description

FIELD OF THE INVENTION [001] The present invention relates to a recombinant subunit vaccine against dengue fever, against the four dengue serotypes - DENV-1, DENV-2, DENV-3 and DENV-4. The present invention also relates to a quadrivalent dengue vaccine candidate based on VLP (virus-like particle), comprising tetravalent EDIII-T molecule and Hepatitis B virus surface antigen (HBsAg). The present invention also relates to a process for producing a quadrivalent dengue vaccine candidate based on VLP (virus-like particle). FUNDAMENTALS OF THE INVENTION [002] Dengue disease, caused by four antigenically distinct dengue viruses (DENVs), is a serious health concern in more than 150 countries worldwide, especially in highly endemic countries like India. This disease has been on the rise over the last decade and has become a global public health threat due to the lack of an effective vaccine or antiviral therapies. Dengue disease is a global challenge for health systems, particularly during outbreaks, and millions of dollars are spent every year on vector control. An efficient, safe, and cost-effective vaccine could address the burden that the dengue virus imposes on affected countries. Despite intensive efforts over the last three decades to develop a prophylactic vaccine to limit the spread of the disease, there is still no licensed vaccine on the market. Research groups/companies around the world are making efforts to develop an effective tetravalent vaccine against all serotypes of the dengue virus. Previously, most vaccines developed were based on live, attenuated chimeric viruses, and some of these are currently under clinical trials. However, due to limitations such as viral interference, the research focus has shifted to subunit vaccines using, in particular, domain III of the envelope protein (E) of the dengue virus. Numerous patents/publications exploring this domain have also been reported. [003] Although a live flavivirus-based dengue vaccine has entered phase III clinical trials, problems have been reported due to viral interference. Viral interference presumably arises due to differences in the replicative potential and immunogenicity of the four vaccine virus strains. [004] Non-replicating subunit vaccines have the potential to overcome the risk of viral interference associated with live virus vaccines [Swaminathan, Khana, N. (2009), Dengue: Recent advances in biology and current status of translational research, Current Mol. Med. 9:152-173]. Several approaches using recombinant DNA and protein-based subunit vaccines are being explored. Most such recombinant subunit vaccines focus on the major envelope protein (E). A number of evidences have additionally shown that many of the vaccine properties of the E protein are associated with domain III (EDIII). [005] It has been shown that the DENV envelope domain III (EDIII) is responsible for host cell receptor recognition and generation of neutralizing antibodies [Swaminathan, Khana, N. (2009), Dengue: Recent advances in biology and current status of translational research, Current Mol. Med. 9:152-173; Guzman, M.G. Hermida, L., Bernardo, L., Ramirez, R. Guillen, G. (2010). Domain II of the envelope protein as a dengue vaccine target]. Furthermore, EDIII has been reported as having only a very low intrinsic potential to induce cross-reacting antibodies [Simmons, M., Nelson, W.M., Wu, S.J., Hayes, C.G. (1998). Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice; Am. J. Trop. Med. Hyg. 58: 655-662; Simmons, M. Murphy, G.S. Hayes, C.G. (2001). Short report: antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine, Am. J. Trop. Med. Hyg. 65: 159-161]. These attributes make EDIII an excellent vaccine candidate. The efficacy of EDIII as a potential dengue vaccine antigen, in the form of a tetravalent protein, has already been established by the inventors [Etemad, B., Batra, G. Raut, R., Dahiya, S., Khanam, S., Swaminathan, S., Khana, N. (2008)]. [006] Numerous patents/publications exploring domain III of the envelope protein (E) of the dengue virus have been reported, namely, Suzarte, E., Gil, L., Valdés, I., Marcos, E., Lazo, L., Izquierdo, A., ... & Hermida, L. (2015), International Immunology, dxv011 describes a novel tetravalent formulation, combining the four aggregated proteins in domain III-capsid from dengue virus, which induces a functional immune response in mice and monkeys. This reference teaches a candidate vaccine against the dengue virus based on two different viral regions, domain III of the envelope protein and the capsid protein, in which the tetravalent formulation of DIIIC proteins was used. The novel chimeric protein derived from dengue virus-2 (domain III-capsid (DIIIC-2)), when presented as aggregates incorporating oligodeoxynucleotides, induced antiviral and neutralizing antibodies, a cellular immune response, and conf