Comparative study of immune responses induced after immunization with plasmids encoding the HIV-1 Nef protein under the control of the CMV-IE or the muscle-specific desmin promoter.

The objective of this study was to compare immune responses induced against HIV-1 Nef after DNA immunization with Nef encoding plasmids under the control of an ubiquitous or a muscle-specific promoter. To this end, plasmids containing HIV-1 nef under the control of the Cytomegalovirus or the human desmin promoters, specifically expressed in muscle cells, were constructed. Different groups of BALB/c mice were immunized with 10 or 100 microg of both constructs, controls were the pcDNA3 vector or the Nef protein in Freund's adjuvant (Nef-CFA). Our data showed that both plasmids stimulated anti-Nef humoral responses in a dose-dependent manner. The anti-Nef antibody response, however, was earlier and higher with the CMV-IE promoter than with the desmin promoter. We also showed that Nef expressing plasmids induced high titers of anti-Nef antibodies (10(4)), comparable to those obtained in the Nef-CFA group (4x10(4)). Analysis of the specificity of anti-Nef antibodies revealed no influence of the promoter, and in contrast to Nef-CFA, plasmid immunization elicited anti-Nef antibodies directed principally against conformational-dependent epitopes. Data on the lymphoproliferative response showed that the specificity of expression, or the plasmid dose, did not affect the onset-time or the intensity of the response. The predominant IgG2a isotype of anti-Nef antibodies and the cytokine profile, mostly IL-2 and IFN-gamma, produced by Nef-stimulated spleen cells indicated a Th1 response in plasmid-immunized mice, in contrast to mice immunized with Nef-CFA, where a Th2 response was induced. In conclusion, these data indicate that antigen expression by muscle cells is sufficient to stimulate a Th1 immune response.

Characterization of humoral and cellular immune responses in mice induced by immunization with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) microparticles.

We have characterized the humoral and cellular immune responses of BALB/c mice immunized with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) PLG particles. Three groups of mice were immunized with Nef PLG, Nef in the presence of complete Freund's adjuvant (CFA) or Nef alone in PBS. When titers were compared 7 months after the last injection, anti-Nef titers in mice immunized with Nef PLG were still close to the maximum, whereas a significant decrease was observed in mice immunized with Nef alone (five times lower) or with Nef in CFA (three times lower). These results indicate that Nef PLG is at least a similar or better vector/adjuvant than Nef in CFA concerning the duration of the humoral immune response. The analysis of cytokine profiles (IL-5 and IL-10) and the isotypic patterns of anti-Nef antibodies (predominantly IgG1), in the three groups of mice, indicated a predominant Th2 immune response. Using synthetic peptides covering the entire sequence of Nef, we identified at least three linear epitopes within sequences 32-64, 118-167 and 185-205 in the sera of mice immunized with Nef PLG or Nef CFA. In contrast, anti-Nef antibodies against Nef alone failed to recognize synthetic peptides, indicating that the majority of anti-Nef antibodies were primarily directed against conformational epitopes. We then examined the ability of Nef PLG to prime for the antigen-specific proliferative responses in vitro. The data obtained indicate the presence of both B-cell and T-cell epitopes in the C-terminal fragment of the protein after immunization of mice with Nef encapsulated in PLG particles.