Production of recombinant inmunogens of Hepatitis A virus by the Baculovirus System as a new strategy of vaccine development

SANCHEZ, VICTORIA; AGUIRRE SEBASTIAN; OTERO GABRIELA; CARGNELUTTI, DIEGO; MATTION NORA; SCODELLER EDUARDO

Buenos Aires

Congreso; III International Clinical Virology Symposium and Advances in Vaccines; 2010

Pan American Society for Clinical Virology

III International Clinical Virology Symposium and Advances in Vaccines. December, 2010. Buenos Aires. Poster: “Production of recombinant immunogens of Hepatitis A virus by the Baculovirus System as a new strategy of vaccine development”. Maria Victoria Sanchez, Sebastian Aguirre, Maria Gabriela Otero, Diego Cargnelutti, Nora Mattion, Eduardo Scodeller.     ABSTRACT   Production of recombinant inmunogens of Hepatitis A virus by the Baculovirus System as a new strategy of vaccine development.   Maria Victoria Sanchez1, Sebastian Aguirre2, M. Gabriela Otero2, Diego Cargnelutti1, Nora Mattion2, Eduardo Scodeller 1. 1-Instituto de Medicina y Biología Experimental de Cuyo (IMBECU) CCT-Mendoza, Argentina.  2-Centro de Virología Animal-Centro Cesar Milstein, Buenos Aires, Argentina.   Background: Hepatitis A is a disease caused by the Hepatitis A virus (HAV) which is classified within the Picornaviridae family, in a separate genus (Hepatovirus). Argentina shows areas of high prevalence of infection where low standards of sanitation promote transmission of HAV. However the number of cases has declined dramatically since the vaccine was incorporated into the National Immunization Schedule in 2005. The vaccine is highly effective and has been used for several years already. The price of this vaccine is expensive and the production technology is complex and laborious. The vaccination programs rely entirely on foreign manufacturers.  For these reasons it would be important to develop the technology for HAV vaccine production in our country. The object of this work is the production of a recombinant HAV vaccine by the baculovirus system. Methods: The prototype strain (ATCC M59809) HM-175/43c was grown in FRhK-4 cells and the virus purified by a sucrose gradient.  From the genomic RNA a full length cDNA was obtained by RT-PCR and cloned in the pGEMT vector. The genomic region coding for P1/2A-3C was cloned into the transfer vector pFastBac (under the polyhedrin promoter). A recombinant bacmid containing P1/2A-3C was generated by transformation of E. coli DH10bac with this transfer vector. A recombinant baculovirus was produced by transfection of Sf9 cells. Additionally P1/2A-3C was also cloned into a transfer vector containing a CMV promoter.  With the same procedures described above a recombinant baculovirus was generated containing the HAV genes under the CMV promoter ( BacMam System). Results: Recombinant baculovirus expressing P1/2A-3C of HAV, under the CMV promoter and the polyhedrin promoter, was produced by transfection of Sf9 cells with the purified bacmid. BacMam System can express the HAV genes in mammalian cells in vitro as well as in vivo. Different immunization tests are being performed to evaluate the immunogenicity of these two strategies.Conclusion: Recombinant HAV immunogens were developed using the baculovirus/insect cells system. The suitability of this methodology for production of an HAV vaccine is presently under evaluation.