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...

Identification and specificity of broadly neutralizing antibodies against HIV

“…These bnAbs are also sensitive to natural glycan heterogeneity, which means a fraction of virions may be resistant to neutralization because they contain glycoforms that are not recognized by the Abs, resulting in incomplete neutralization curves 66. We have also observed this phenomenon with additional apex bnAbs such as the potent PGDM140028 and also many other bnAb specificities. This effect varies with different bnAb and viral isolate combinations 70.…”

“…Subsequently, advances in the production of soluble near‐native stabilized Env trimers46 have allowed better selection of Env‐specific B cells, excluding those which bind regions not exposed on the infectious viral spike. Using this method, we isolated PGDM140028 from the same donor that previously yielded the PGT141–145 family of bnAbs via the B‐cell culturing approach 22. Furthermore, stabilized BG505 SOSIP.664 trimers have been used as baits to isolate two bnAbs which occupy overlapping epitopes at the gp120‐gp41 interface and also contact the fusion peptide, ACS20247 and VRC34 48.…”

“…Interestingly, the initial observation that suggested N332 was a key part of this epitope was that only N332 deletion could completely abrogate neutralization, but it did not always do so for all virus strains and N301 was also implicated 22. Further investigation revealed that the high‐mannose patch bnAbs exhibit a degree of promiscuity for different glycan sites across the epitope, allowing them to maintain neutralization breadth in the face of viral changes to glycosylation sites 28. The level of permissiveness for different glycan sites varies among the members of this bnAb class, for example, moving the glycan site from 332 to 334 in a six‐virus panel has no effect on PGT128, prevents neutralization of two of four viruses by PGT121, and renders PGT135 unable to neutralize any virus 28.…”

“…High‐throughput neutralization assays were a major factor in changing that situation. The ability to analyze mAb and serum activity against large panels of viruses was demonstrated20 and subsequently used to evaluate large numbers of HIV‐infected donors in the International AIDS Vaccine Initiative (IAVI) Protocol G and C studies to identify those with exceptionally potent and broad sera,8 map the specificities underlying these responses,7, 21 and then isolate bnAbs from these individuals 22, 23, 24, 25, 26, 27, 28. Independently, the standardization of the TZM‐bl neutralization assay and the definition of neutralization sensitivity tiers29, 30, 31 allowed much more rigorous serum analysis.…”

Antibody‐virus co‐evolution in HIV infection: paths for HIV vaccine development

“…The V3-glycan PCDN, BF520-derived and DH270 bnAb lineages share some common traits: 1) differently from previously described V3-gylcan bnAbs (23, 25), their evolution did not involve insertion/deletion (indel) events, demonstrating that indels are not a universal requirement for the V3-glycan bnAb class to acquire neutralization breadth; 2) neutralization breadth was acquired with relatively modest levels of somatic hypermutation that can be achieved through vaccination (57, 60, 61), and 3) the UCA of the V3-glycan lineages did not neutralize or bind autologous TF suggesting the hypothesis that V3-glycan bnAb lineages may arise in response to altered forms of the Env protein.…”