Ema T. Crooks

Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies

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.

Enzyme Digests Eliminate Nonfunctional Env from HIV-1 Particle Surfaces, Leaving Native Env Trimers Intact and Viral Infectivity Unaffected

ABSTRACT HIV-1 viruses and virus-like particles (VLPs) bear nonnative “junk” forms of envelope (Env) glycoprotein that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a codependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160 that is glycosylated in the endoplasmic reticulum (gp160ER) and then bypasses protein maturation and traffics directly into particles. gp160ER was found to bind two copies of monoclonal antibody (MAb) 2G12, consistent with its exclusively high-mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or near-complete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. “Trimer VLPs” may be useful neutralizing antibody immunogens.

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Nonneutralizing Epitopes

ABSTRACT Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong, K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., “trimer VLPs”). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ∼100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.

Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop

Summary 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, which enable 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, by 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, thereby facilitating V1V2 binding via a non‐protruding loop. The N90‐VRC38 lineage thus identifies a solution for V1V2‐apex binding that provides a more conventional B cell pathway for vaccine design. HighlightsVLPs and stabilized Env trimers identify HIV‐1‐neutralizing N90‐VRC38 Ab lineageCo‐crystal structure of Ab N90‐VRC38.01 with scaffolded V1V2‐Env apexN90‐VRC38 lineage targets the apex of HIV‐1 Env trimer with non‐protruding loopsNew mechanism of Ab:trimer‐apex binding informs V1V2 vaccine strategies &NA; To date, long recognition loops have been a hallmark of apex‐targeting antibodies. Cale et al. identify a lineage of HIV‐1‐neutralizing antibodies that target the envelope trimer apex. The N90‐VRC38 lineage uses a loop of average length—a feature that may make it a useful prototype for vaccine design.

Topological analysis of HIV-1 glycoproteins expressed in situ on virus surfaces reveals tighter packing but greater conformational flexibility than for soluble gp120.

ABSTRACT In natural infection, antibodies interact with HIV-1 primarily through nonfunctional forms of envelope glycoproteins (Env), including uncleaved (UNC) gp160 and gp41 stumps. These antigens are important to fully characterize, as they may be decoys that promote nonneutralizing responses and may also be targets for nonneutralizing effector responses. In this study, we compared the antigenic properties of Env expressed in situ on pseudovirion virus-like particle (VLP) surfaces and soluble gp120 using harmonized enzyme-linked immunosorbent assays (ELISAs) and a panel of 51 monoclonal antibodies (MAbs). Only 32 of 46 soluble gp120-reactive MAbs recognized the primary UNC gp160 antigen of VLPs. Indeed, many epitopes were poorly exposed (C1, V2, C1-C4, C4, C4-V3, CD4 induced [CD4i], and PGT group 3) or obscured (C2, C5, and C1-C5) on VLPs. In further studies, VLP Env exhibited an increased degree of inter-MAb competition, the epicenter of which was the base of the V3 loop, where PGT, 2G12, V3, and CD4 binding site specificities competed. UNC gp160 also underwent more drastic soluble CD4 (sCD4)-induced conformational changes than soluble gp120, exposing CD4i, C1-C4, and V2 epitopes. A greater propensity of UNC gp160 to undergo conformational changes was also suggested by the induction of CD4i MAb binding to VLPs by a V3 MAb as well as by soluble CD4. The same effect was not observed for soluble gp120. Taken together, our data suggest that membrane-expressed UNC gp160 exists in a less “triggered” conformational state than soluble gp120 and that MAb binding to UNC gp160 tends to have greater conformational consequences.

M48U1 CD4 mimetic has a sustained inhibitory effect on cell-associated HIV-1 by attenuating virion infectivity through gp120 shedding

BackgroundHIV-1 infected cells can establish new infections by crossing the vaginal epithelia and subsequently producing virus in a milieu that avoids the high microbicide concentrations of the vaginal lumen.FindingsTo address this problem, here, we report that pretreatment of HIV-infected peripheral blood mononuclear cells (PBMCs) with a 27 amino acid CD4-mimetic, M48U1, causes dramatic and prolonged reduction of infectious virus output, due to its induction of gp120 shedding.ConclusionsM48U1 may, therefore, be valuable for prophylaxis of mucosal HIV-1 transmission.

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 specificity using rational heterologous boosts.