A Recombinant HIV Envelope Trimer Selects for Quaternary Dependent Antibodies Targeting the Trimer Apex

Abstract

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named "PGDM1400-1412," show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC 50 of 0.003 μg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.HIV | broadly neutralizing antibodies | BG505 SOSIP | B cell | vaccine M ultiple methods have been developed to isolate HIV broadly neutralizing antibodies (bnAbs) (1-12). Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including b12, 2F5, 2G12, 4E10, and Z13 (13-20). These antibodies exhibit a range of neutralization breadth against primary isolates from 30 to 90% but have moderate neutralization potency (median IC 50 of ∼2-4 μg/mL). Access to infected donors who have high serum titers of bnAbs (21,22) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (1-4, 6-8).One of the newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high-throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of the new generation of bnAbs, PG9 and PG16 (1), and since then has revealed several sites of vulnerability to bnAb recognition on HIV envelope (Env) (1-4, 6, 7). An alternative method of bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG + memory B cells of interest by cell sorting (6,8,9,23,24). However, soluble baits have not been successful in isolating antibody responses targeting quaternary epitopes, including the trimer-apex site surrounding the N160 glycan, because the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell-surface Env, called "GFP-293TBaL cells," were used recently to isolate antibodies 3BC176 and 3BC315 (10, 25).These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating the utility of the approach, but the method was reported to be less efficient than the use of soluble protein baits (10,25).The favorable antigenic profile of the solub...

Development of broadly neutralizing antibodies in HIV-1 infected elite neutralizers

“…Bhiman et al [96] described highly mutated “off-track” CAP256-VRC26 mAbs, which have more than 20% SHM but limited neutralization breadth. Similar observations have been made in other bNAb lineages [58, 104, 105], and as mAb isolation methodologies are specifically designed to recover bNAbs, the proportion of bNAb lineages that are off-track is unknown.…”

“…Overall, the low affinity of bNAb precursors for cognate antigens is associated with lower thermodynamic stability, particularly of CDR loops, and SHM leads to epitope focusing [107], improved shape complementarity and increased buried surface area at the interface with the antigen, by conformational re-organization and stabilization of the paratope [47, 97]. Within bNAb lineages, these changes can occur independently within different sublineages maturing to acquire breadth [46, 47, 58, 96, 104, 105]. These varying pathways to breadth strongly demonstrate the plasticity of the immune system, which, even within one antibody lineage, can find multiple structural and genetic solutions to the same immunological problem.…”

The Neutralizing Antibody Response to the HIV-1 Env Protein

“…This was followed by B cell culture and micro-neutralization (functional) screens, and by the use of flow cytometry-based isolation of single B cells using peptide and protein “baits”. Most recently, the design of soluble, recombinant Env trimers and virus-like particles comparable to native Env trimers has provided alternative antigen-specific B cell selection strategies [43–45]. These strategies have enabled the isolation of many dozens of antibodies from elite neutralizers, and some of these have exceptional breadth (approaching 99% of circulating viruses) and potency in the picomolar range [44, 46–48].…”

Free Energy Perturbation Calculation of Relative Binding Free Energy between Broadly Neutralizing Antibodies and the gp120 Glycoprotein of HIV-1

“…Over the past several years, there have been extensive efforts to optimize antibody potency for a number of bNAbs [14], [15], [16]. While experimental optimizations of antibodies can examine a large number of potential candidates, they lack the flexibility to efficiently perform a wide exploration of possible bNAb mutants.…”