Developing an effective vaccine against HIV requires eliciting the production of antibodies that will not only recognize and bind proteins present on the virus particle’s surface, but also block viral entry (neutralizing antibodies) and/or trigger antibody-dependent cellular cytotoxicity (ADCC) against HIV-infected cells. To date, most studies have focused on the identification of neutralizing antibodies, while the only clinical vaccine trial that demonstrated protection against HIV infection involved non-neutralizing antibodies mediating ADCC.
The HIV envelope is a complex of two glycoproteins, gp120 is extracellular while gp41 spans the virion membrane. Both glycoproteins are required for HIV infection. While gp120 neutralizing antibodies have been extensively studied, little has been done regarding ADCC mediating antibodies against gp41.
The Overbaugh lab (Human Biology Division)has a strong interest in studying ADCC-mediating antibodies that have unusual targets. Dr. Julie Overbaugh explains the rationale: “There is increasing appreciation for the potential of antibodies that mediate killing of infected cells to contribute to protection for HIV infection and disease. But these types of antibodies have not been as well studied as antibodies that block virus infection- neutralizing antibodies”. Her group has recently isolated antibodies from an infected patient that do not target gp120 while strongly binding virus-like particles. In a work published in PLoS Pathogens last month, they characterized these antibodies and evaluated their capacity to induce ADCC.
The authors performed a Binding Antibody Multiplex Assay (BAMA) to determine the antigen specificity of eight antibodies binding HIV but not gp120. In this assay, different antigens are covalently bound to fluorescent beads (one type of bead per antigen). If an antibody binds to an antigen, the antibody is then specifically marked by the beads and the fluorescence signal for each antibody allows them to determine the level of interaction with each individual antigen. Based on this technique, researchers demonstrated that four out of the eight antibodies specifically bound gp41 antigen but not gp120. The specificity of the binding to the gp41 antigen was confirmed by ELISA (enzyme-linked immunosorbent assay).
In order to assess the capacity of these gp41-specific antibodies to elicit ADCC, the authors performed a Rapid Fluorometric ADCC (RF-ADCC) assay. Target cells are coated with different antigens such as gp41 or gp120 and incubated with the tested antibodies. Peripheral blood mononuclear cells (PBMCs) from a HIV-1 negative individual are then added to the culture and target cell killing is measured by flow cytometry. Williams et al. demonstrated that all four gp41-specific antibodies mediated ADCC when target cells were coated with gp41.
Identification of the specific epitope targeted by these antibodies is essential not only to understand the underlying mechanism but also to design vaccine strategies using similar epitopes. In this context, the researchers conducted an ELISA competition assay where antibodies whose epitope is characterized competed with the four gp41 antibodies to bind gp41 antigen. Only competing antibodies binding the same epitope will result in a decreased interaction between the isolated four antibodies and the antigens. They demonstrated that two of the four antibodies bound a conformation involving the N- and C-terminal heptad repeat regions (NHR and CHR), whereas the two others bound the conserved cysteine loop (C-C loop). These interactions were confirmed and further investigated by phage peptide display, allowing a detailed mapping of the epitopes for each of these antibodies. As Dr. Overbaugh comments: “The antibodies described here mediate ADCC and do so by targeting conserved regions of the virus, which is critical given that HIV is overall quite variable”.
The ability of these gp41-specific antibodies to bind gp41 in its natural conformation is primordial for the potential translation of this work for HIV vaccine strategies. During infection, once gp120 has interacted with CD4 and CCR5, a conformational change leads to folding of gp41 into a stable six-helix bundle. The authors decided to test the ability of the four antibodies to bind this helix structure. Using ELISA, they demonstrated that only the two antibodies that recognize the NHR and CHR were able to bind the helix structure, not the ones specific of the C-C loop, in agreement with the inaccessibility of the C-C loop during the conformational rearrangements. This correlated with an increased ADCC activity of the NHR/CHR specific antibodies as assessed by RF-ADCC assay on gp41 helix-coated target cells, while C-C loop specific antibodies do not mediate ADCC. In addition, three of the four antibodies were capable of binding the gp41 domain when the whole Env protein was used as antigen, independently of the presence of CD4, suggesting that their binding is independent of CD4-induced conformational change of the Env protein.
This work was supported by the National Institute of Health and the Canadian Institutes of Health Research.
Fred Hutch/UW Cancer Consortium member Dr. Julie Overbaugh contributed to this research.
Williams KL, Stumpf M, Naiman NE, Ding S, Garrett M, Gobillot T, Vézina D, Dusenbury K, Ramadoss NS, Basom R, Kim PS, Finzi A, Overbaugh J. 2019. Identification of HIV gp41-specific antibodies that mediate killing of infected cells. PLoS Pathog. 15(2): e1007572. https://doi.org/10.1371/journal.ppat.1007572