Abstract
ABSTRACTThe conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses. In the absence of the CD4 receptor at the cell surface, primary Envs sample a “closed” conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affect in vitro measurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells in in vitro cultures or ex vivo samples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands.
Highlights
The conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses
Using primary CD4ϩ T cells infected with the NL4.3 ADA GFP WT virus as target cells and autologous peripheral blood mononuclear cells (PBMCs) as effector cells, we found that WT-infected cells were significantly more susceptible to ADCC mediated by PGT126 and 3BNC117 than to that mediated by A32 (Fig. 2A)
In its unliganded form, Env from most primary mbio.asm.org virus samples adopts a “closed” trimeric conformation, preferentially recognized by broadly neutralizing Abs (bNAbs) but not by CD4i Abs, which are abundant in plasma from most HIV-1-infected individuals [2, 4, 10, 15,16,17,18, 23, 59, 60, 86]
Summary
Differential recognition of uninfected bystander cells and infected cells by ADCC-mediating Abs. Similar results were obtained using target cells infected with the transmitted founder CH77 virus (Fig. S4) These results confirm the increased ability of bNAbs to mediate ADCC responses against infected cells compared to CD4i Abs. Assays measuring ADCC activities on the total cell population overestimate the responses mediated by CD4i Abs. The two assays described above are able to distinguish between HIV-1-infected and uninfected bystander cells. Since A32 and HIVϩ sera preferentially recognize uninfected bystander cells (Fig. 1), we hypothesized that most of the ADCC responses detected with the granzyme B and NK activation assays were directed against such cells To test this possibility, uninfected bystander cells (GFPϪ CD4ϩ) were removed from the infected coculture using beads coated with an anti-CD4 antibody that does not compete for gp120 binding (see Materials and Methods) (Fig. 5A). Gp120-coated target cells detected ADCC responses largely mediated by CD4i antibodies and not by bNAbs capable of recognizing functional Env trimers, such as antibodies to the CD4-binding site or to a proteoglycan epitope in the V3 region
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