RESUMO
In chronic HIV infection a progressive Th1 to Th2/Th0 cytokine-profile shift is related to disease progression. One of the possible benefits of a therapeutic vaccination might be to counterbalance this phenomenon to allow viral replication control under a Th1-type immune response. TERAVAC-HIV-1 is a multiantigenic formulation vaccine candidate against HIV-1 which comprises the recombinant protein CR3 that contains T cell epitopes and the surface and nucleocapsid antigens of Hepatitis B Virus (HBV). Previous studies showed that such virus like particles of the HBV provide a Th1 adjuvant effect. The present studies examined the capacity of TERAVAC to elicit a Th1 response in the presence of an ongoing HIV-specific Th2-type response in Balb/c mice. To examine this issue, we injected subcutaneously the animals with CR3 or viral lysate in alum which resulted in a Th2-type response. The CR3-specific Th2-type response was verified by induction of IL-4 and IL-10 secretion in ex vivo stimulated splenocytes without secretion of IFN-γ and IgG2a antibodies in serum. Further subcutaneous and simultaneous subcutaneous-nasal immunizations of the same mice with TERAVAC promoted IFN-γ secretion and production of IgG2a antibodies in accordance with a Th1-type response. This result suggests a therapeutic benefit of this vaccine candidate in the restoration of the Th1-type HIV-specific cellular response in seropositive patients.
Assuntos
Vacinas contra a AIDS/farmacologia , Citocinas/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Imunoglobulina G/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Citocinas/sangue , Anticorpos Anti-HIV/sangue , HIV-1/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/farmacologia , Células Th1 , Células Th2 , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologiaRESUMO
We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.