RESUMEN
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.
Asunto(s)
Vacunas contra el SIDA/farmacología , Citocinas/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Inmunoglobulina G/inmunología , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Animales , Citocinas/sangre , Anticuerpos Anti-VIH/sangre , VIH-1/genética , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/farmacología , Células TH1 , Células Th2 , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/farmacologíaRESUMEN
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.