Characterization of vaccine-vectors expressing Nef of the BF recombinant HIV-1 circulating form and evaluation of the immune response induced in mice

ANA MARÍA RODRIGUEZ; G.SCHULMAN; MF.PACUTTI; F.FERRER; G.TURK; JL.NÁJERA; D.MÓNACO; G.CALAMANTE; M. ESTEBAN; H.SALOMÓN; MM.GHERARDI

Ciudad de México-México

Conferencia; XVII International AIDS Conference; 2008

IAS

The Argentina HIV epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. Nef is an attractive vaccine target because it is synthesized early in the viral life cycle and is one of the most variable proteins of HIV, having 30% of differences between B and BF. In this work, we describe the generation, characterization, and immunogenicity of DNA and MVA vectors expressing Nef of the BF recombinant form of HIV-1, to analyze the impact of BF variants in the design of future vaccines. Nef gene was amplified by PCR from the viral genome from the CRF_12BF of HIV-1, and cloned in a conventional DNA expression vector or in a transference VV plasmid to generate the MVA recombinant virus. Correct expression of NefBF protein from DNA and MVA vectors was evaluated in human and murine cell lines by western blot and confocal microscopy. Growth kinetics of MVA–NefBF and wt MVA were compared in the permissive BHK-21 cell-line, having a similar pattern. NefBF immunogenicity after its delivery from both types of vectors was analyzed in Balb/c mice, administering different prime/boost schedules. Ten days after the last immunization, the specific cellular immune response induced in the spleen was evaluated, incubating the splenocytes with one pool of overlapping 11-15 aa peptides comprising the full NefBF or NefB proteins. Determinations were made by an ELISPOT assay after 24hs of incubation, or quantifying specific IFN-g secreted in the supernatant of the cell-cultures after 72 hs of incubation. Of all the immunization protocols applied the DNA/MVA and the DNA/DNA/DNA were the more immunogenic. Interestingly, in these groups in which we detected a significant cellular response against NefBF peptides, the cross-reactivity against NefB peptides was low or non-detectable. These results are of high relevance in the design of future vaccines for our region.