Induction Of Hiv-1 Neutralizing Antibodies In The Optimization Of The Membrane Protein Antigen, And Transformation

Human immunodeficiency virus type 1 (HIV-1) is a major public health concern facing a WHO estimation of at least 42 million people infected worldwide and the emergence of drug resistant strains. It is now well accepted that vaccine is an urgent need, more especially for developing countries. An efficient vaccine should target both cellular and humoral arms of the immune system at peripheral as well as mucosal sites. The induction of potent and broadly NAb against HIV remains a major goal of current AIDS vaccine research.The failure to induce broadly neutralizing antibodies (NAbs) against primary isolates of human immunodeficiency virus (HIV) remains a major impediment to the development of effective vaccines against AIDS. Although HIV type 1 (HIV-1) envelope proteins have been targeted for vaccine development for over two decades, immunity induced by early vaccines has been effective only against laboratory-adapted isolates or chimeric viruses (SHIV) bearing the homologous env genes.In this study, A CRF₀7BC Env S939 which was sensitive to Mab b12,4E10 and some HIV-1 positive sera was chose as the wildtype Env. The S939 Env was optimized according to the epitope recogined by four broadly and potent Mabs, specific amino acid on Env which was reported that can increase the viral neutralizing sensitivity, and elimination of the N-linked glycosylation sites. Env mutants were produced by site-directed mutagenesis. Pseudoviruses were produced to compare viral infectivity, virion production, and antibody-mediated neutralization of mutants to wild-type Env.The mutant FE which contains the epitope recognized by 2F5 and 4E10 showed a at least 11-fold increase in susceptibility to neutralization by 2F5. 8 specific amino acid which were reported that can enhance the viral neutralizing sensitivity were mutated on FE Env individually, 6 mutants showed an significant reduction of viral infectivity, 2 of the 8 mutants showed no effect in viral infectivity and antibody-mediated neutralization compare to FE. 25 potential N-linked glycosylation sites located on FE were eliminated individually. Five glycosylation mutants located in the V1/V2 and C1/C2 domains showed no detectable infectivity. Removal of PNLG located in the immunologically-silent face domains (V4, C4, and V5), except N392 (V4), were shown to increase viral infectivity. Other glycosylation mutants showed a reduction in viral infectivity compared to the wild-type. Elimination of glycans at N197 in C2, N301 in V3, N442 in C4 and N625 in the gp41 were shown to render FE more susceptible to neutralization by HIV-1 positive sera, anti-CD4 binding site and anti-gp41 antibodies.The mutation in our experiment, which showed a significant increase in susceptibility to neutralization by Mabs and HIV-1 positive sera, were chosed to mutate with the N-glycans located on C3, C4 and V5 in combination. The 7 combinatorial PNLG site eliminated mutants were showed more susceptible to neutralization than wild-type FE and individually N-glycan mutants. These studies are likely to contribute to the design of more effective envelope-based immunogens.