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Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 Envelope glycoprotein trimers on neutralizing antibody induction.

ABSTRACT: Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits (PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan, again suggesting CD4bs overlap. Heterologous boosts appeared to reduce nAb clashes with the N362 glycan. The fourth serum targeted a N362 glycan-sensitive epitope. VLP manufacture challenges prevented us from immunizing larger rabbit numbers to empower a robust statistical analysis. Nevertheless, trends suggest that targeted glycan removal may improve nAb induction by exposing new epitopes and that it may be possible to modify nAb specificity using rational heterologous boosts.

SUBMITTER: Crooks ET

PROVIDER: S-EPMC5895097 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 Envelope glycoprotein trimers on neutralizing antibody induction.

Crooks Ema T ET Osawa Keiko K Tong Tommy T Grimley Samantha L SL Dai Yang D YD Whalen Robert G RG Kulp Daniel W DW Menis Sergey S Schief William R WR Binley James M JM

Virology 20170306

Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits (PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan ...[more]

PMID: 28279830

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Project description:We covalently attached human immunodeficiency virus type 1 (HIV-1) Env SOSIP trimers to iron oxide nanoparticles (IO-NPs) to create a particulate immunogen for neutralizing antibody (NAb) induction. The attached trimers, ∼20 per particle, retained native-like antigenicity, judged by reactivity with NAbs and non-NAbs. Bivalent (BG505 and B41) trimer IO-NPs were made, as were IO-NPs displaying B41 trimers carrying a PADRE T-cell helper epitope (TCHE). We immunized mice with B41 soluble or IO-NP trimers after PADRE peptide priming. After two immunizations, IO-NP presentation and the TCHE tag independently and substantially increased anti-trimer antibody responses, but titer differences waned after two further doses. Notable and unexpected findings were that autologous NAbs to the N289 glycan hole epitope were consistently induced in mice given soluble but not IO-NP trimers. Various recombinant mannose binding lectins (MBLs) and MBLs in sera of both murine and human origin bound to soluble and IO-NP trimers. MBL binding occluded the autologous NAb epitope on the B41 IO-NP trimers, which may contribute to its poor immunogenicity. The exposure of a subset of broadly active NAb epitopes was also impaired by MBL binding, which could have substantial implications for the utility of trimer-bearing nanoparticles in general and perhaps also for soluble Env proteins.IMPORTANCE Recombinant trimeric SOSIP proteins are vaccine components intended to induce neutralizing antibodies (NAbs) that prevent cells from infection by human immunodeficiency virus type 1 (HIV-1). A way to increase the strength of antibody responses to these proteins is to present them on the surface of nanoparticles (NPs). We chemically attached about 20 SOSIP trimers to NPs made of iron oxide (IO). The resulting IO-NP trimers had appropriate properties when we studied them in the laboratory but, unexpectedly, were less able to induce NAbs than nonattached trimers when used to immunize mice. We found that mannose binding lectins, proteins naturally present in the serum of mice and other animals, bound strongly to the soluble and IO-NP trimers, blocking access to antibody epitopes in a way that may impede the development of NAb responses. These findings should influence how trimer-bearing NPs of various designs are made and used.

Project description:The induction of broadly neutralizing antibodies (bNAbs) is a major goal in vaccine research. HIV-1-infected individuals that develop exceptionally strong bNAb responses, termed elite neutralizers, can inform vaccine design by providing blueprints for the induction of similar bNAb responses. We describe a new recombinant native-like envelope glycoprotein (Env) SOSIP trimer, termed AMC009, based on the viral founder sequences of an elite neutralizer. The subtype B AMC009 SOSIP protein formed stable native-like trimers that displayed multiple bNAb epitopes. Overall, its structure at 4.3-Å resolution was similar to that of BG505 SOSIP.664. The AMC009 trimer resembled one from a second elite neutralizer, AMC011, in having a dense and complete glycan shield. When tested as immunogens in rabbits, the AMC009 trimers did not induce autologous neutralizing antibody (NAb) responses efficiently while the AMC011 trimers did so very weakly, outcomes that may reflect the completeness of their glycan shields. The AMC011 trimer induced antibodies that occasionally cross-neutralized heterologous tier 2 viruses, sometimes at high titer. Cross-neutralizing antibodies were more frequently elicited by a trivalent combination of AMC008, AMC009, and AMC011 trimers, all derived from subtype B viruses. Each of these three individual trimers could deplete the NAb activity from the rabbit sera. Mapping the polyclonal sera by electron microscopy revealed that antibodies of multiple specificities could bind to sites on both autologous and heterologous trimers. These results advance our understanding of how to use Env trimers in multivalent vaccination regimens and the immunogenicity of trimers derived from elite neutralizers.IMPORTANCE Elite neutralizers, i.e., individuals who developed unusually broad and potent neutralizing antibody responses, might serve as blueprints for HIV-1 vaccine design. Here, we studied the immunogenicity of native-like recombinant envelope glycoprotein (Env) trimers based on viral sequences from elite neutralizers. While immunization with single trimers from elite neutralization did not recapitulate the breadth and potency of neutralization observed in these infected individuals, a combination of three subtype B Env trimers from elite neutralizers resulted in some neutralization breadth within subtype B viruses. These results should guide future efforts to design vaccines to induce broadly neutralizing antibodies.

Project description:In HIV-1 vaccine research, native-like, soluble envelope glycoprotein SOSIP trimers are widely used for immunizing animals. The epitopes of autologous neutralizing antibodies (NAbs) induced by the BG505 and B41 SOSIP trimers in rabbits and macaques have been mapped to a few holes in the glycan shields that cover most of the protein surfaces. For BG505 trimers, the dominant autologous NAb epitope in rabbits involves residues that line a cavity caused by the absence of a glycan at residue 241. Here, we blocked this epitope in BG505 SOSIPv4.1 trimer immunogens by knocking in an N-linked glycan at residue 241. We then opened holes elsewhere on the trimer by knocking out single N-linked glycans at residues 197, 234, 276, 332, and 355 and found that NAb responses induced by the 241-glycan-bearing BG505 trimers were frequently redirected to the newly opened sites. The strongest evidence for redirection of the NAb response to neoepitopes, through the opening and closing of glycan holes, was obtained from trimer immunogen groups with the highest occupancy of the N241 site. We also attempted to knock in the N289-glycan to block the sole autologous NAb epitope on the B41 SOSIP.v4.1 trimer. Although a retrospective analysis showed that the new N289-glycan site was substantially underoccupied, we found some evidence for redirection of the NAb response to a neoepitope when this site was knocked in and the N356-glycan site knocked out. In neither study, however, was redirection associated with increased neutralization of heterologous tier 2 viruses.IMPORTANCE Engineered SOSIP trimers mimic envelope-glycoprotein spikes, which stud the surface of HIV-1 particles and mediate viral entry into cells. When used for immunizing test animals, they elicit antibodies that neutralize resistant sequence-matched HIV-1 isolates. These neutralizing antibodies recognize epitopes in holes in the glycan shield that covers the trimer. Here, we added glycans to block the most immunogenic neutralization epitopes on BG505 and B41 SOSIP trimers. In addition, we removed selected other glycans to open new holes that might expose new immunogenic epitopes. We immunized rabbits with the various glycan-modified trimers and then dissected the specificities of the antibody responses. Thus, in principle, the antibody response might be diverted from one site to a more cross-reactive one, which would help in the induction of broadly neutralizing antibodies by HIV-1 vaccines based on envelope glycoproteins.

Project description:The human immunodeficiency virus type 1 (HIV-1) trimeric envelope glycoprotein (Env) is heavily glycosylated, creating a dense glycan shield that protects the underlying peptidic surface from antibody recognition. The absence of conserved glycans, due to missing potential N-linked glycosylation sites (PNGS), can result in strain-specific, autologous neutralizing antibody (NAb) responses. Here, we sought to gain a deeper understanding of the autologous neutralization by introducing holes in the otherwise dense glycan shields of the AMC011 and AMC016 SOSIP trimers. Specifically, when we knocked out the N130 and N289 glycans, which are absent from the well-characterized B41 SOSIP trimer, we observed stronger autologous NAb responses. We also analyzed the highly variable NAb responses induced in rabbits by diverse SOSIP trimers from subtypes A, B, and C. Statistical analysis, using linear regression, revealed that the cumulative area exposed on a trimer by glycan holes correlates with the magnitude of the autologous NAb response. IMPORTANCE Forty years after the first description of HIV-1, the search for a protective vaccine is still ongoing. The sole target for antibodies that can neutralize the virus are the trimeric envelope glycoproteins (Envs) located on the viral surface. The glycoprotein surface is covered with glycans that shield off the underlying protein components from recognition by the immune system. However, the Env trimers of some viral strains have holes in the glycan shield. Immunized animals developed antibodies against such glycan holes. These antibodies are generally strain specific. Here, we sought to gain a deeper understanding of what drives these specific immune responses. First, we show that strain-specific neutralizing antibody responses can be increased by creating artificial holes in the glycan shield. Second, when studying a diverse set of Env trimers with different characteristics, we found that the surface area of the glycan holes contributes prominently to the induction of strain-specific neutralizing antibodies.

Dataset Information

Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 Envelope glycoprotein trimers on neutralizing antibody induction.

Publications

Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 Envelope glycoprotein trimers on neutralizing antibody induction.

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