CD4 downregulation precedes Env expression and protects HIV-1-infected cells from ADCC mediated by non-neutralizing antibodies

Abstract

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion closed conformation, which is targeted by broadly-neutralizing antibodies (bnAbs). CD4 binding drives Env into more open conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA-flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively-infected cells that already downmodulated CD4. This suggest that CD4 downmodulation precedes env mRNA expression. Consequently, productively-infected cells express closed Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1 infected humanized mice with the ADCC mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively-infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy.

Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in presence of CD4-mimetics

“…However, upon addition of the indoline CD4mc CJF-III-288, recognition by all plasma was significantly enhanced. This enhanced recognition translated into a marked increase in ADCC activity (Figure 1B), as measured with a previously described FACS-based ADCC assay (11,13,27). As expected, recognition of infected cells by plasma samples was significantly associated with their ADCC activity (Figure 1C).…”

“…Anti-human IgG Fc secondary antibodies (Thermo Fisher Scientific) were used when cell surface binding was performed in presence of Fab fragment blockade. The PE-conjugated anti-HIV p24 Ab (Clone KC57-RD1, Beckman Coulter) and the FITC anti-human CD4 OKT4 Antibody (Biolegend) were used to detect productively HIV-1 infected cells in flow cytometry cell-surface staining and ADCC assays (27).…”