Virus-like particles identify an HIV V1V2 Apex-1 binding neutralizing antibody that lacks a protruding loop

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, enabl them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain to side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage identifies a solution for V1V2apex binding that provides a more conventional B cell pathway for vaccine design

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

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 speciUcity using rational heterologous boosts

Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

CAPRISA, 2014.Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development

Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis

Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis

Glycoengineering HIV-1 Env creates ‘supercharged’ and ‘hybrid’ glycans to increase neutralizing antibody potency, breadth and saturation

<div><p>The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make <i>some</i> glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing β-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE—thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design.</p></div

Effects of GE on bnAb neutralization IC50s.

<p>Neutralizing IC50s for each control and GE-modified A) JR-FL and B) BG505 PVs are shown in μg/ml. The most sensitive glycoform for each nAb is boxed. JR-FL neutralization assays for mAb 3BC176 used the N88A mutant. Each assay was performed at least in duplicate. In many cases, BG505 neutralization by mAb 35O22 was insufficiently saturating to reach an IC50 and is denoted by a "+". Geometric mean IC50s per treatment are shown, omitting data for 14e, F105 and instances of 35O22 "+". This Fig is linked to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.g003" target="_blank">Fig 3</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.s002" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.s006" target="_blank">S6</a> Figs. Abbreviations: B4G = B4GALT1, ST3 = ST3GAL4, ST6 = ST6GAL1, ST8 = ST8SIA4.</p

Glycan contacts of mature bnAbs are not germline-encoded.

<p>A) GE PVs from donor CAP256 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref035" target="_blank">35</a>] at week 34 were tested for neutralization sensitivity to bnAbs CAP256.09, CAP256.25, intermediate I1 and the inferred unmutated common ancestor (UCA). B) GE-modified 16055 and Q23.17 PVs were tested for sensitivity to PG9 with a reverted heavy chain (gH) mixed with the mature light chain (mL) and fully mature PG9. Results are representative of two replicates; error bars represent SD. C) Nine V2-sensitive strains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref034" target="_blank">34</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref036" target="_blank">36</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref045" target="_blank">45</a>] produced in control, B4GALT1+ST6GAL1 and GNT1- formats were tested for sensitivity to V2 bnAbs and their germline revertants, as indicated. The CH04 UCA (RUA/RUA) was the same as that used in ref [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref033" target="_blank">33</a>]. Mixed mHgL versions of PGT145 and VRC38.01 were used that previously showed the most reactivity of the ancestors tested [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref034" target="_blank">34</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref042" target="_blank">42</a>]. Results are representative of at least two repeats performed in duplicate. Wilcoxon Signed Rank tests were performed on data for each mAb-PV pair, organized into two columns to compare IC50s for control and GE-modified formats. Significant p values are shown.</p

Comparison of the effects of B4GALT1+ST6GAL1 and PBMC passage on PV and IMC sensitivities.

<p>Viruses from 5 HIV-1 strains were produced as PVs or infectious molecular clones (IMCs). Some 293T cell-derived PVs were modified by B4GALT1+ST6GAL1, as indicated. Some IMCs were passaged through PBMCs as indicated. Neutralization assays were performed with the addition of indinavir to assays using IMCs to limit infection to a single round. Results are representative of at least two repeats performed in duplicate.</p