Research Article: A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo

Date Published: October 30, 2015

Publisher: Public Library of Science

Author(s): Natalia T. Freund, Joshua A. Horwitz, Lilian Nogueira, Stuart A. Sievers, Louise Scharf, Johannes F. Scheid, Anna Gazumyan, Cassie Liu, Klara Velinzon, Ariel Goldenthal, Rogier W. Sanders, John P. Moore, Pamela J. Bjorkman, Michael S. Seaman, Bruce D. Walker, Florian Klein, Michel C. Nussenzweig, Ronald C. Desrosiers.


The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans.

Partial Text

Although the envelope glycoproteins (Env) of primate immunodeficiency viruses have extremely variable sequences [1], most of them engage CD4 as the primary cellular receptor to initiate the viral life cycle [2]. The consequence is that the CD4 binding site (CD4bs) is a comparatively well-conserved region of Env that serves as a critical neutralization epitope and an appealing vaccine target. The introduction of single cell antibody cloning techniques [3,4] yielded dozens of broad and potent CD4bs antibodies from infected individuals, some of which neutralize ~90% of HIV-1 strains in vitro [5–7]. Some of these antibodies are also effective at reducing viral load when used to treat infected humanized mice (hu-mice) [8], macaques [9–11] and humans [12].

The CD4bs is a highly conserved epitope on the HIV-1 Env and an important potential target for neutralizing antibodies. Although this site evolved to avoid antibody accessibility, two major groups of CD4bs bNAbs have been discovered [15]. The first group, exemplified by VRC01, is VH-restricted, IGVH1-2 or IGVH1-46, with the heavy chains positioned in a CD4-like orientation and CDRH2 making significant contacts with gp120 [6,7,15]. The CDRL3 [7,21,44], and in some cases also CDRL1 [6], of the corresponding light chains have to be short and compact to minimize potential interference and clashes with the glycans that surround the CD4bs. The emergence of these antibodies involves many somatic hypermutations, some of which are in the framework regions [45]. The second group of CD4bs bNAbs, which includes b12 and HJ16, is far more heterogeneous. These antibodies bind to gp120 via a CDRH3-dominated, loop based mechanism [15]. As might be expected, members of this group of CD4bs bNAbs arise from different VH segments and carry fewer somatic mutations [17–19]. The new antibody described in this study, 179NC75, is a loop binder that is closely related to HJ16. Similarly to HJ16, its Env-binding and virus-neutralizing activities are dependent on the N276 glycan [36]. Consistent with the CDRH3 loop-based mechanism of recognition that was described for antibodies that are not VH-restricted [15], when we generated the predicted germline version of 179NC75, where all mutations were reverted but the CDRH3 was retained, the antibody bound to BG505 SOSIP.664 trimers. This could indicate that any residual mutations present in the CDR3s of the reverted antibody might allow binding. Interestingly, the germline version of HJ16 also had some binding to BG505 SOSIP.664 trimers (Fig 4), however this binding was lower that the one of 179NC75, which could be attributed to a shorter CDRH3 (19 versus 24 residues).




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