Recombinant HIV type 1 gp120 (CAP45.2.00) (a.a. 34-518) [His]

To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here, we demonstrate that this mechanism is specific for the Env conformation and affects the efficiency of antibody-dependent cellular cytotoxicity (ADCC). Using flow cytometry and confocal microscopy, we show that broadly neutralizing antibodies (bNAbs) targeting the "closed" conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibilities of infected cells to ADCC. Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the "closed" conformation of Env expressed on HIV-1-infected cells. IMPORTANCE HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). cc) Thus, a better understanding of this mechanism might help develop antibodies with improved capacities to mediate ADCC.

The HIV-1 envelope glycoprotein (Env) is present on the surface of the virion at a very low density compared to most other enveloped viruses. Substitution of various parts of the stalk domain of Env (gp41) with the corresponding elements from other viral glycoproteins has been shown to increase Env spike density on the cell membrane and surface of virus-like particles (VLPs). In this study, chimeric Env antigens were generated by replacing the transmembrane and cytoplasmic domains of HIV-1 Env with the corresponding regions from the influenza H5 hemagglutinin (HA) (gp140HA(2)tr) and by replacing the entire gp41 region of Env with the HA(2) subunit of HA (gp120HA(2)). Recombinant DNA and modified vaccinia Ankara (MVA) vaccines expressing HIV-1 subtype C mosaic Gag and gp150 Env or either of the chimeras were generated. Surprisingly, no significant differences were found in the levels of expression of gp150 Env or either of the chimeras on the surface of cells or on Gag VLPs. Differences were, however, observed in the binding of different monoclonal antibodies to the HIV-1 Env. Monoclonal antibodies, which recognized a V1 / V2 quaternary epitope at the tip of the native Env trimer, bound gp150 and gp140HA(2)tr chimera but failed to bind to the gp120HA(2) chimera. Autologous Tier 2 neutralizing antibodies (NAbs) were produced by rabbits inoculated with DNA and MVA vaccines expressing the gp140HA(2)tr chimera or gp150 Env, but not those immunized with the gp120HA(2) Env. These results showed that the addition of an HA(2) stalk to HIV-1 gp120 did not improve immunogenicity, but rather that the full-length gp150 was required for optimal presentation of epitopes for the elicitation of a neutralizing antibody response to HIV-1.