Studio di immunogeni per l'induzione di anticorpi neutralizzati ad ampio spettro contro il virus dell'immunodeficienza umana (HIV-1)

AIDS is still representing a pandemic emergency and no cure has yet been found to eradicate it. The recently reported unsuccessful HIV-1 vaccine trials, point to the need of developing new immunogens capable of stimulating humoral immune response towards highly conserved viral epitopes. HIV-1 infects host cells by cellular/viral membrane fusion. During this process, conserved epitopes are exposed on the viral glycoprotein gp120/41 that may provide to be very useful as targets for the induction of antibodies against HIV-1. The study of the formation of fusion complexes between viral gp120/gp41 and cellular CD4-CCR5 molecules has been reported in the first part of this thesis. These complexes were prepared by co-cultivating stably transfected cells at different temperatures (21°, 30°, 37° C), corresponding to intermediate stages of the membrane fusion process, and stabilized using different fixatives (paraformaldehyde, formaldehyde, glutaraldehyde, bis-sulfosuccinimidyl-suberate). Mice were immunized with these fusion complexes to reveal the induction of HIV-1 neutralizing antibodies and murine spleenocytes obtained from immunized mice were used to generate hybridoma clones to be tested for the production of neutralizing monoclonal antibodies. The results obtained indicate that: 1) fusion complexes were immunogenic and induced neutralizing antibodies against R5 and X4 HIV-1 heterologous isolates; 2) extensive purification of antibodies allowed the removal of any unspecific cytotoxic effect; 3) complexes prepared at higher temperatures were more immunogenic and induced higher titers of neutralizing antibodies; 4) titer of neutralizing antibodies was not affected by the fixative used; 5) neutralizing activity was retained after CD4-CCR5 antibody removal; 6) viral neutralization is not directly proportional to the in vitro antibody reactivity against complex purified components; 7) high and broad neutralizing activity against HIV-1 env pseudotyped viruses can be isolated in hybridoma supernatants. The production and characterization of new neutralizing monoclonal antibodies will be useful to identify specific immunogenic structures and epitopes to be used as anti HIV-1 vaccine to induce broad protecting neutralizing antibodies, when administered in a suitable delivery system. The second part of this thesis was focused on the role of host-cell HLA-C chain, acquired by HIV-1 during budding or normally expressed on target cells, during membrane fusion process. It was alredy reported that HLA-C presence enhances the infectivity of many HIV-1 strains and can reduce their susceptibility to neutralizing antibodies. To further understand this mechanism, HLA-C expression was selectively silenced, using the RNA interference technique, in different human cell lines expressing HIV-1 gp120/gp41 of the laboratory strains ADA, LAI and NDK. The fusion efficiency of HLA-C silenced cells was found to be significantly reduced (p<0,01) compared to non-silenced cells when co-cultivated with different target cell lines expressing HIV-1 receptor CD4 and co-receptors CCR5 and/or CXCR4. This observation suggests that HLA-C silencing reduces the fusion efficiency of the gp120/gp41 of many HIV-1 R5 and X4 tropic HIV-1 strains. Conversely, in nonhuman cells, the co-expression of several primary HIV-1 env genes with HLA-C increases significantly (p<0,01) the fusion efficiency with target cells in comparison to cells lacking HLA-C expression. When HIV-1 env pseudotyped viruses were produced in HLA-C silenced 293T cells, a significant reduction (p<0,0001) in their infectivity was observed as compared to HLA-C bearing pseudoviruses. Furthermore, infectivity assays allowed a more detailed analysis of dose dependence of HLA-C sensitive phenotype. However, HIV-1 gp120/gp41 of laboratory strains NDK and J500 appear to be less sensitive to HLA-C presence when co-expressed, in agreement to previous study on viral isolates. In addition, when a lower infectious dose was used, the NDK pseudotyped virus became more sensitive to HLA-C presence. The silencing of HLA-C expression on cells expressing HIV-receptors (CD4, CCR5 and CXCR4) resulted in a decreased fusogenic and infection activity (p<0,0001) with all Env molecules tested, so the mechanism at the basis of HLA-C sensitivity appears to be different when associated to viral envelope or target cellular membrane. HLA-C molecules can be specifically co-purified and detected in fusion complexes when co-expressed with gp120/gp41 or HIV-receptors. Our data indicate that the HLAC conformation responsible for increasing fusion efficiency consists in the heavy " chain associated to the !2-microglobulin. The HLA-C molecule might be able to stabilize the trimeric gp120/gp41 and the fusion complex, by improving the efficiency of the conformational changes, thus enhancing the exposure of the receptor and/or co-receptor binding site on the gp120/gp41. Alternatively, HLA-C might increase the amounts of gp120 on the cell/viral surface, reducing the shedding from gp41 and boosting the efficiency of the fusion process resulting in a greater infectivity of viral particles. Understanding the role of HLA-C in HIV-1 infectivity may provide important new information for the development of an AIDS vaccine. The presence of circulating allogeneic anti-HLA-C antibodies might help in reducing viral infectivity, even for virus strains (as NDK) with an apparent HLA-C insensitivity and could result in a reduced transmission risk and ultimately in an increased protection. The addition of HLA-C molecules to fusion complexes might have potential important implications for the development of new immunogens to elicit broad spectrum HIV-1 neutralizing antibodies.