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.
Eliciting broadly neutralizing antibodies (bnAbs) is a major goal of HIV-1 vaccine development [1,2]. NAbs block infection by binding to native Env spikes, consisting of trimers of gp120/gp41 heterodimers [2,3]. However, the compact, sequence-diverse, and heavily glycosylated nature of these trimers allows the virus to largely evade neutralization [4,5]. For a neutralizing antibody vaccine to be sufficiently effective, it will have to overcome at least three challenges: i) to consistently induce nAbs in all vaccinees, ii) to induce nAbs that can potently neutralize tier 2 field isolate (s) resembling transmitted strains, and iii) to induce nAbs that are effective against a broad spectrum of tier 2 strains. An ideal vaccine would resolve all these challenges simultaneously. However, most current vaccine candidates usually elicit weak or undetectable autologous tier 2 nAbs, let alone any breadth [1,2, 6]. In natural infection, autologous nAbs typically develop within a few months and invariably precede any bnAb development [7]. This may be a reflection of the unprecedented sequence diversity that makes cross-reactive epitopes extremely rare among the exposed targets available on native trimers. A plausible solution may therefore be to first develop a platform that consistently elicits potent autologous tier 2 nAbs, then to use heterologous boosts to try to recapitulate the steps in nAb breadth development in natural infection [8–11]. In other words, we might implicitly solve the challenges described above in a stepwise manner. Resolving the first challenge (consistent nAb induction) may be facilitated by ensuring that relevant epitope (s) are well-exposed. For example, previous studies have reported that several animals that received JR-FL strain-based immunogens developed modest nAb responses that target the CD4 binding site (CD4bs) [12,13]. To resolve the second challenge (inducing potent tier 2 nAbs), clearly, nAb titers should be sufficient to protect against incident infection. Studies suggest that a ~1: 200 nAb ID50 titer (in the TZM-bl assay) can protect against low dose SHIV challenge [14–19]. However, factors such as the nature of the challenge virus, its dose, and nAb specificity complicate any firm estimates. Conservatively, an ID50 titer >1: 1,000 might be expected to be protective. In one study, rabbits immunized with a JR-CSF gp120 DNA prime-gp120 protein-boost regimen induced exceptional nAb ID50 titers of >1: 10,000 to the tier 2 index virus in the TZM-bl assay, and targeted epitopes involving the gp120 C3/V4 region [20]. Other studies have also shown that DNA prime-soluble protein boost regimens frequently elicit improved nAbs compared to protein-only regimens, although titers usually fall short of what may be protective [13,21,22]. These findings provide reasons to be optimistic that the first two challenges in neutralizing antibody vaccine development can be addressed. Since functional, trimeric Env spikes stringently resist binding by all but the most precisely targeted nAbs, perhaps only these spikes themselves possess the necessary selectivity to elicit nAbs in a vaccine setting. Indeed, the slow progress in nAb vaccine development may derive from the fact that most Env vaccine candidates insufficiently resemble native spikes [23] and consequently elicit largely “off target” (i. e. non-neutralizing) antibodies [1,2, 6]. In an attempt to address this problem, one group generated a" near native" soluble trimer, termed BG505 SOSIP. 664, that elicits largely consistent and potent autologous nAbs [24]. This, and any other vaccine approaches based on authentic Env spikes clearly deserve further attention. To achieve a" near native" conformation, soluble Env vaccine candidates require mutations to increase their stability. However, this comes at a price: to varying extents, these mutations inevitably sacrifice a fully native conformation [23]. In contrast, Env trimers expressed in a natural lipid membrane context do not require trimer-stabilizing mutations, and, unlike their soluble counterparts, fully resemble functional spikes found on infectious virus [25]. Virus-like particles (VLPs) provide one platform for testing membrane-expressed native trimer vaccines. In support of this approach, it is worth noting that all licensed infectious disease vaccines (e. g. HBV, RV, HPV) and many others in development (e. g. influenza, malaria (RTS, S), parvovirus, NDV, RSV, norovirus) are particle-based [26,27]. In the HIV arena, particulate vaccines have so far been explored in the forms of live inactivated viruses, VLPs, liposomes and virosomes (many references are cited in [28]), although none have yet demonstrated a great capacity to elicit tier 2 nAbs. At least two major factors could underlie the lack of progress in developing nanoparticle vaccines to prevent HIV-1 acquisition. First, germline antibody precursors heavily favor protein-based epitopes over glycan epitopes, as glycans are generally considered to be “self” antigens. Given that accessible protein sites on the trimer are protected by a heavy carbohydrate shell [5,29,30], the germline antibody repertoire may therefore have a limited capacity to engage the trimer, negatively impacting nAb development [8,31–34]. This constraint may be particularly relevant in small animal models whose antibody repertoires may not be well equipped to recognize such challenging antigens [35,36]. By comparison, other, simpler forms of Env such as the gp120 monomer are more accessible and can therefore engage antibody germlines more easily. However, an important drawback is that they lack the ability to selectively elicit nAbs. Despite these challenges, a growing portfolio of broadly neutralizing human monoclonal nAbs (mbnAbs) has revealed various new ways that trimer defenses can be breached and provide paradigms for vaccine design. In many cases, these mAbs target recessed protein-based epitopes that are either bordered by glycans or make direct contacts with glycans [37–49]. A second factor that may underlie the lack of progress in particulate HIV-1 vaccine development may be that their surfaces are contaminated with non-functional forms of Env, including uncleaved (UNC) gp160 and gp41 stumps. These aberrant forms of Env may promote the development of non-neutralizing responses, perhaps at the expense of the development of neutralizing responses directed to the more compact native trimer [28,50,51]. In other words, they may act as antigenic decoys. To address this problem, we previously showed that protease treatment can selectively remove non-functional Env from VLP surfaces, leaving native trimers intact. The resulting particles are termed “trimer VLPs” [25,52]. Strikingly, the IC50 titers of monoclonal antibody (mAb) binding to trimer VLPs and neutralization correlate well [25]. To evaluate the ability of native trimers to induce nAbs, here we immunized rabbits and guinea pigs with high doses of trimer VLPs. Two rabbits developed remarkably potent serum nAbs. We compared these sera to another rare, potent JR-FL neutralizing serum generated in a rabbit immunized with DNA that expresses native trimers followed by a soluble protein boost. All 3 sera targeted quaternary epitopes that took advantage of holes in the trimer’s carbohydrate shell left by the natural absence of glycans in the C2 domain of JR-FL gp120. The VLP sera were also able to neutralize other clade B tier 2 isolates when the same glycan-deficient gap was introduced, suggesting that they target a conserved site to which access is usually regulated by a glycan. We discuss the impetus of these results for the further development of trimer VLP immunogens.
We previously showed that neutralization exhibits an excellent correlation with the ability of antibodies to recognize native trimers as observed in BN-PAGE-Western blot [3,25,50–54,64,65]. Consistent with their neutralizing activities, mAb b12 and neutralizing sera from animals 613,647,849,2922 and 7672 all bound to the native SOS E168K trimer, as evidenced by depletion of the unliganded trimer (Fig 3A, lanes 2,6 and 8–11). Trimer binding by serum 849 was rather weak, consistent with its modest neutralizing ID50 (Fig 2B). All other samples failed to bind parent JR-FL trimers, consistent with their lack of neutralizing activity (Fig 3A). Recognition of SOS D368R mutant trimers by the 613,647,2922 and 7672 sera suggest that these sera recognize native trimers via D368-independent epitopes. In similar studies, serum 347 also recognized native trimers in a D368-independent manner. In contrast, binding by mAb b12 and serum 849 (SOSIP) was eliminated, consistent with their dependency on residue D368 for trimer binding [12]. It is worth noting here that IgG-trimer complexes are rarely visible in these experiments, as we reported previously [25]. This is likely to be due to IgG bivalency and flexibility. Thus, a single IgG can potentially engage two trimers, two or three individual IgGs can engage a single trimer and higher order IgG-trimer complexes are possible. This plethora of possibilities probably explains the general lack of well-defined trimer-IgG complexes in these experiments. In fact, several products are often observed, reflecting the various complex combinations. This issue may be exacerbated by IgG flexibility, which may cause band smearing. To detect any MPER nAbs, we evaluated sera in a post-CD4CCR5 neutralization assay format we described previously [55], and observed no activity (S4 Table). In contrast, the previously reported BB34 serum (serving here as a reference control) showed some activity [54]. To test whether these sera share contacts on Env with known broadly neutralizing mAbs, we also examined the effects of various knockout mutants on neutralization sensitivity, including N160A, N295Q, and N332Q. None of these mutations eliminated the neutralizing activities of the 613,347 and 647 sera, suggesting that they must contact the trimer via other sites (S4 Table). The poor tier 1 neutralizing activities of our potent vaccine sera (S3 Table) raise the possibility that nAbs may target quaternary tier 2 epitope (s) that are inaccessible on more sensitive HIV-1 strains. To address this possibility, we tested the ability of JR-FL monomeric gp120 and gp140F trimer to interfere with their neutralizing activities (S7 Fig). A D368R mutation was introduced into these soluble Env competitors to prevent them from binding to cellular CD4 and thereby directly inhibiting infection. We already showed that several of our sera (613,347,647,2922, and 7672) recognize native Env trimers independently of the D368R mutation (Fig 3). Therefore, any interference would indicate that nAb epitopes are present on these soluble forms of Env. In S7 Fig, gp120 monomer and gp140F trimer both interfered with mAb 2G12 neutralization, but not with b12 neutralization. The latter was expected, because the D368R mutation is known to ablate b12 recognition. The soluble Envs also interfered with neutralization by group 5 serum 2922, as expected, considering this animal received a gp120 DNA prime, gp120 monomer boost regimen. In contrast, the sera from animals 613,347 or 647 were unaffected (S7A and S7B Fig), suggesting that their neutralizing epitopes are not expressed on soluble forms of Env. This contrasts with the soluble Env interference we observed with VLP sera in our previous study [28]. Given that gp140F trimers were used as a protein boost in animal 647, the lack of D368R gp140F trimer interference was rather surprising. However, since quaternary nAbs developed after only one protein boost (S1 Table), then the boosting may have merely expanded antibodies that had been imprinted by DNA priming. The exceptional MN neutralizing activity present in this animal before protein boosting (S1 Table) suggests an unusually strong response to DNA priming in this animal that could be consistent with such imprinting. Nevertheless, the tier 1 nAbs against the JR-FL A328G were susceptible to monomeric gp120 interference, suggesting they are mediated by antibodies that do not depend on quaternary epitopes, unlike the tier 2 nAbs in this serum (S7C Fig). We next assessed the ability of repeated serum adsorption to densely packed cells expressing native Env trimers to deplete neutralizing activity [44]. Prior to adsorption, pure IgG was extracted from the 613 and 7672 sera (the latter was included as a control). IgG concentrations were then adjusted to match that of their respective parent sera (as verified by ELISA). After adsorption to cells, IgG was again purified and then adjusted back to its pre-adsorption volume. This process resulted in heavily depleted VRC03 and 2G12 neutralization (30- to 100-fold reduction; Fig 4A). NAbs in the 613 and 7672 sera were also dramatically depleted (>100 fold). However, the 613 serum IgG was only modestly depleted by adsorption (~4-fold; Fig 4B). Therefore, the loss in neutralizing activity was due to specific adsorption to the native trimer, rather than non-specific antibody loss during the adsorption process. This data supports the idea that the 613 serum contains nAbs directed to quaternary epitope (s). To further map the potent vaccine sera, we assessed their ability to compete with various mAbs for binding to trimer VLPs [28,60]. Here, competitions reflect direct epitope overlaps or conformational relationships on the native Env trimer. For simplicity, we infer them to indicate epitope overlaps. In addition to our vaccine sera, for reference purposes, three previously mapped HIV-1 plasmas were also analyzed ([54]; see Fig 1). All rabbit serum-mAb competitions were referenced to a rabbit prebleed control. Similarly, HIV-1 donor sera were referenced to the HIV-1 seronegative human plasma from donor 210. Our panel of biotinylated mAbs is organized horizontally in Fig 5, starting with those directed to epitopes at the membrane-distal apex of the trimer on the left and ending on the right with those directed to the membrane-proximal ectodomain region (MPER). Competitions were considered as significant when biontinylated mAb binding was reduced to <50% of control levels (indicated by colored squares). A non-neutralizing reference serum from rabbit 618 did not compete with any mAb to <50%, suggesting that this cutoff is sufficiently stringent to rule out any non-specific inhibition by our vaccine sera. Multiple representatives of major epitope clusters were assayed to ensure the consistency of any inhibitions within these clusters. Each assay was repeated at least 2 times. The generally low standard errors we observed in S8 Fig reveal the consistent patterns in these repeats. In S9A Fig, we show that each mAb inhibited its biotin-labeled counterpart and other mAbs within each epitope cluster to <10% of control levels (diagonal boxes from top left to bottom right). In representative inter-cluster competitions shown in S9A Fig, as expected, 2G12 was unable to inhibit CD4bs nAb binding. Similarly, various gp120-specific nAbs were unable to inhibit the binding of biotinylated 4E10. However, a competitive inter-cluster relationship between PGT125 and CD4bs nAbs was observed, consistent with the known overlap between these epitopes [42,66]. As with the serum-mAb competitions (S8 Fig), the standard error of repeated mAb-mAb competitions (all assays were performed at least twice) were generally low. Representative titrations of biotinylated mAbs in the presence or absence of unlabeled mAb competitors are shown in S10 Fig and exemplify the strong mAb-mAb self-competition in S9 Fig. Of all the polyclonal samples, the potently neutralizing HIV-1-infected donor plasma 1686 showed the highest overall competition, strongly inhibiting binding by all 4 CD4bs mAbs (Fig 5). This agrees with our previous mapping of this sample using other methods [54]. The 1686 plasma also competed with mAb PGT125 (Fig 5), consistent with the PGT125-CD4bs overlap mentioned above (S9 Fig). Competition of most other mAbs we tested was marginal, except for 4E10. In S10 Fig, representative titrations show strong plasma 1686 competition of mAb VRC03 directed to the CD4bs, but little or no competition of mAb PG9. The other 2 more weakly neutralizing human plasmas (BB34 and 1702) showed modest CD4bs competition, as well as 4E10 competition, consistent with their known MPER nAb activities ([54]; S4 Table). The marginal (but consistent) competition of VRC03 and to some extent with PG9 by these two plasmas is shown in S10 Fig. Overall, these findings confirm the specificity and reproducibility of this competition assay. All the neutralizing rabbit sera showed significant competition with CD4bs mAbs VRC03, VRC07, b12 and 1F7 (Figs 5 and S10). Of these, the 347 serum exhibited the strongest activity and, like the human 1686 plasma also competed moderately with mAb PGT125. This serum also competed modestly, but consistently with mAbs PG9 and PG16 (Figs 5 and S10). We suggest that serum 347 binding to trimer has an allosteric effect on the integrity of these V2/quaternary epitopes. The vaccine sera showed little competition with other mAbs. The 613 and 7672 sera showed a similar pattern, competing with all 4 CD4bs nAbs, with possible weak inhibition of PGT125, PG9 and PG16. The 647 and 2922 sera showed weaker competition that was also focused on the CD4bs. In the case of the 647 serum, the unexpectedly weak competition was because limited sample availability meant that we had to use serum from a bleed taken 4 weeks after the immune bleed used elsewhere in this study, by which time the neutralizing ID50 had fallen to ~1: 80. The patterns of inhibition by vaccine sera are represented as raw titrations in S10 Fig, where VRC03 binding is clearly perturbed, but the effects on PG9 and 2G12 are weak or absent, respectively. Although the extent of competition by vaccine sera was in no case as complete as with mAb-mAb competitions (S10 Fig), competition patterns were consistent between assays (S8 Fig). Furthermore, in most cases, inhibition of all four different mAbs within the CD4bs cluster was detected (Fig 5). The competition profiles of our vaccine sera patterns are also strongly supported by our observing expected patterns of competition by HIV-1 donor sera. It may be no coincidence that all vaccine sera overlap with the CD4bs, considering that this is one of the few partially exposed protein patches on the otherwise densely glycan-populated trimer. The observation that the 347 and 7672 sera exhibited stronger competition of this site than the 613 serum, despite having somewhat lower neutralizing ID50s may indicate that their neutralizing activities have a greater overlap with the CD4bs. Like all other vaccine sera described to date, none of our sera significantly neutralized tier 2 viruses, aside from the autologous JR-FL parent virus. The lack of JR-CSF cross-neutralization is sobering, given that this strain was isolated from the same donor as JR-FL and therefore exhibits a degree of sequence homology. This lack of JR-CSF cross-neutralization, however, has the benefit of allowing us to map our sera by measuring their activities against a series of JR-FL Env-based chimeric pseudoviruses with various JR-CSF Env domain swaps [20,67]. Most of these chimeras were infectious (S11A Fig). All of the functional clones resisted neutralization by the V3 loop-specific mAb CO11 [28], suggesting that they retain a tier 2 phenotype. The potent sera from animals 613 and 647 neutralized all of the functional chimeras (S11A Fig). This suggests that they target epitopes involving the JR-FL C1 and/or C2 regions that were not represented in the chimera neutralization analysis, as they were not sufficiently functional (S11A Fig). In contrast, sera 2922 and 7672 targeted epitopes affected by the C-terminal portion of gp120 (S11A Fig). Specifically, the 2922 gp120 DNA prime-protein boost sera targeted the C3V4 region, which is similar to the epitope targets of the sera previously reported using this regimen using the JR-CSF strain [20], suggesting a common nAb development pathway. The 7672 G2C gp120 prime-boost serum was knocked out by a C3-C5 chimera (clone 8070), although its activity could not be further resolved by less substantial domain swaps (S11A Fig). These results are summarized in Fig 6A. Since the competition data in Fig 5 suggested that our potent sera target CD4bs-like epitopes, we first investigated the possible role of the C2 region in the 613 and 647 epitopes, due to its close proximity to the CD4bs. Alignment of JR-FL and JR-CSF C2 regions (Fig 6B) reveals 8 amino acid differences, including 3 additional glycan sequons in JR-CSF. These substantial differences could account for the lack of infectivity of the C2 domain swap mutants (8076 and 8086) in S11A Fig—JR-FL trimers may be unable to accommodate all 3 extra glycans. An analysis of JR-FL-based point mutants at the 8 variant sites revealed that the 613 serum was sensitive to a D197N mutation at the base of the V1V2 loop that introduces one of the three additional JR-CSF glycans (Fig 6B and 6C). Several other D197 mutants (D197N+S199A, D197Q and D197K) that do not introduce a new glycan had no effect, suggesting that the N197 glycan regulates 613 serum neutralization (Fig 6C). None of the other C2 mutants affected neutralization by the 613 serum. It is worth noting that the introduction of the N197 glycan into JR-FL trimer also leads to a ~4 fold reduction in b12 IC50 (Fig 6C), consistent with its role in a putative “glycan fence” that regulates access to the CD4bs [64,67]. In contrast to the 613 serum, the 647 serum was affected by mutations of residues D230, P238 and E269 in loops A and C of the C2 region that straddle a disulfide-bonded hairpin (Fig 6B and 6C). In this case, neutralization was not exclusively eliminated by the introduction of a new glycan at residue 230, but instead depended on the presence of an aspartic acid at this position. The resistance of all these mutants to the CO11 V3 mAb suggests that they retain a tier 2 phenotype. Therefore, we can conclude that these mutants directly affected serum recognition rather than having an adverse effect on trimer folding. Taken together, the above mapping data suggest that both of our potent sera take advantage of the relatively sparse glycan coverage of the JR-FL C2 region. Notably, the N197 glycan is conserved in 98. 6% viruses of a multi-clade panel of >4,000 sequences (Fig 6B). Further experiments using a set of JR-CSF Env-based chimeras (S11B Fig) revealed that two mutants (8073 and 8087) that eliminate the N197 glycan knocked in 613 serum neutralization sensitivity, suggesting that the N197 glycan regulates tier 2 cross-reactivity. These mutants exhibited increased b12 sensitivity [67], consistent with a role of N197 glycan in the glycan fence. In contrast, all JR-CSF-based chimeras remained insensitive to the 647 serum, suggesting that, in this case, neutralization is highly strain-specific and context dependent. An expanded set of JR-CSF-based chimeras similar to the JR-FL chimeras in S11B Fig were also tested, but none were sensitive to the 613 or 647 sera. We next asked if the N197 glycan regulated the sensitivity of other isolates to our sera, as it did for the JR-CSF isolate. To answer this question, we generated N197 glycan-deficient mutants in 28 other tier 2 isolates sampled from several clades, all of which resisted 613 serum neutralization in the presence of this glycan (S5 Table). Remarkably, the removal of the N197 glycan rendered 9 of these viruses (i. e. , 31%) sensitive to the 613 serum (S5A Table and Fig 7). All 9 viruses were from clade B and constituted 50% of the clade B N197 knockout mutants tested (n = 18; Fig 7). Our other neutralizing trimer VLP vaccine serum (from animal 347) neutralized 3 of these 9 viruses (JR-FL, JR-CSF and ADA; S5A Table). Neither serum neutralized any of the 11 non-clade B viruses, which included Envs from clades A, C and several Chinese B' isolates [68]. Not surprisingly, the 647 serum did not neutralize any of the N197 mutants. To properly interpret these findings, it was important to monitor any overt changes associated with the removal of the N197 glycan that might signify a loss of tier 2 phenotype and therefore may impact sensitivity to our sera. Accordingly, our mutants were further characterized using a panel of mAbs (S5 Table). These included 6 non-nAbs, 14e, 39F, b6, F105,17b and 48d, directed to V3, CD4bs and CD4i epitopes. In addition, we monitored sensitivity to a weakly neutralizing HIV+ serum, BB68 [54]. We arbitrarily ascribed global sensitivity to any N197 mutant that was sensitive to at least 2 of the 6 non-nAbs. Six N197Q mutants (JR-CSF, ADA, SC422, YU2, PVO. 4 and BaL) were thus found to be globally sensitive and were therefore excluded from further analysis (S5C Table). Nevertheless, since 5 of these mutants were sensitive to the 613 serum and 4 were sensitive to the 347 serum, we decided to remake 4 of them (JR-CSF, ADA, YU2 and PAVO) as N197D mutants in the hope that the different amino acid exchange would preserve a tier 2 phenotype. A N197D BaL mutant was not made, however, because the parent virus was sensitive to two of our non-nAbs (S5C Table). All 4 new N197D mutants retained a tier 2 phenotype. Moreover, JR-CSF and ADA mutants were sensitive to both the 613 and 347 sera, the YU2 N197D mutant was sensitive to the 613 serum and the PVO. 4 mutant resisted both sera. Thus, all 9 613-sensitive mutants shown in Fig 7A were verified to have a tier 2 phenotype, consistent with modest tier 2 breadth within clade B by recognition of the surface protected by the N197 glycan. The 347 serum also exhibited modest breadth that might have been limited by the fact that it was taken at bleed 3 rather than bleed 4. Although this “breadth” depends on the absence of the N197 glycan, since all the mutant viruses retain a tier 2 phenotype, our data suggest that addressing the key problem of breadth in vaccine design may in future be possible. We next measured the sensitivity of N197 mutant panel to various neutralizing ligands to determine any patterns that might partition with serum 613 sensitivity (S5 Table), using a previously published fingerprinting analysis [69]. Computational analysis suggests that strain selection can improve the predictive accuracy of this approach, i. e. , the ability to discriminate between the different specificities. A recently developed method for virus panel selection was applied to determine any subsets (including all 9 613 serum-sensitive viruses) of the 29 member N197 mutant panel in S5 Table that are more suitable for the fingerprinting analysis compared to the full panel [69]. All possible panels of sizes 20–28 were evaluated. The best panel included 23 N197 mutant strains. Although this was preferable to the full 29-strain panel, neither were optimal and could therefore adversely affect the reliability of any predictions. Our panel size was limited by the practical challenge of making N197 mutants for each Env clone and performing a full neutralization analysis. In the fingerprinting analysis, neutralization via a particular epitope is typically associated with delineation values of >0. 3, and preferably >0. 4. However, we have previously confirmed positive signals of >0. 2 [5,49,69]. In this case, a 23-strain panel and 12 bnAb specificities were used for fingerprint delineation. The 1F7 mAb was included because it is known to be N197-sensitive [67]. The strongest fingerprint signal for the 613 serum was for PG9-like antibodies (Fig 8). It may be no coincidence that all 9 of the 613-sensitive N197 mutants were also sensitive to at least one of the mAbs PG16, PGT145 and VRC26, whose epitopes depend on a compact quaternary conformation (S5 Table). This was not true for 4 of the 9 clade B 613 serum-resistant mutants (1168, QH0515. 01,6101 and BL01). Several non-B clade mutants also resisted these bnAbs. It should be noted that the 613 serum can neutralize the parent JR-FL strain in the absence of a lysine at residue 168 of the V2 loop that is critical for known V2 quaternary-dependent neutralizing mAbs. Therefore, in keeping with the marginal competition in Fig 5, it is unlikely that the 613 serum targets this epitope cluster. Rather, we suggest that trimer compactness is important for neutralization by both the 613 serum and by broadly neutralizing V2 quaternary mAbs. Cumulatively, CD4bs nAbs also showed a strong fingerprint signal; VRC01-like signals were the strongest, along with detectable b12-like and 1F7-like signals (Fig 8). This is consistent with the solid 613 serum-mediated competition of CD4bs mAbs (Fig 5). Overall, N197 mutants exhibited a trend for slightly greater sensitivity to CD4 ligands. Specifically, 84. 9% of virus-ligand combinations in which neutralization was detectable (146 of 172 virus-ligand combinations) exhibited a >2 fold increase in sensitivity. This is consistent with its role in the glycan fence that regulates CD4bs access. There were no consistent effects on several other bnAb epitopes analyzed in S5 Table. Despite these observed PG9-like and CD4bs-like patternsof serum 613, none of the delineation signals were exceptionally strong. This is consistent with a unique epitope that does not fall entirely into any known category. Rather, it suggests a CD4bs-like quaternary epitope that exhibits some characteristics of both clusters. An alignment of the 18 clade B sequences derived from Fig 7 and S5 Table did not reveal any consistent elements (e. g. glycans, V1V2 length, CD4 contact regions) that partitioned with 613 serum sensitivity (S12 Fig). Similarly, an investigation of a variety of phylogenetic tree models (neighbor join, UPGMA, maximum parsimony) and substitution models (Poisson, p-distance) rooted to various reference sequences failed to identify any distinct clustering of 613-sensitive viruses, in part due to the low confidence of bootstrap values. Ultimately, high sequence variability defied any attempt to decipher common element (s) that might predict sensitivity. Perhaps the only clear conclusion we can make is that it is probably no coincidence that all the sensitive viruses were derived from clade B.
Here, for the first time, we showed that authentic trimers can elicit potent nAbs directed to quaternary epitopes that take advantage of strain-specific holes in the glycan shield. This establishes a mechanism by which autologous antibodies can potently neutralize tier 2 isolates in a vaccine setting. The binding sites of two potent sera (from animals 613 and 647) are perhaps best visualized as footprints on the near native BG505. SOSIP. 664 Env trimer model (Fig 9) [5,30]. Given the collective features of the 613 serum nAb—i) CD4bs overlap, ii) N197 glycan-sensitivity, iii) modest PGT cluster II overlap, iv) quaternary dependency, and v) CD4bs/PG9-like fingerprint—we suggest that it targets a CD4bs-like hole in the glycan shield that is accessible only on the trimer, possibly close to the gp120 protomer interface. To reach the CD4bs cavity, antibodies must navigate through a putative" glycan fence" [64], consisting of glycans N197 (in red in Fig 9) [70], N276 [10,32], N362, N386 (all in magenta), and possibly others, depending on the Env strain (Fig 9A). The difficulty of this challenge is illustrated by the exquisite docking of CD4bs mAb VRC01 on the trimer (Fig 9B). The 613 serum nAb must also navigate this fence, albeit with the N197 glycan missing in the JR-FL strain (modeled in Fig 9C and 9D). Serum 647 nAbs make contact with loops A and C of the C2 region that straddle the inner and outer domains (labeled red in Fig 9E). In this BG505 trimer model, we labeled residue 241 in black to signify the presence of a glycan in the JR-FL trimer that happens to be absent in BG505 Env. Conversely, we removed a glycan at residue 234, consistent with its absence in JR-FL Env trimers. Like the 613 serum nAb, the 647 serum appears to take advantage of a rare glycan-deficient patch (modeled in Fig 9F). The position of the 647 epitope on the lower edge of the gp120 outer domain is compatible with a lateral angle of approach similar to that of recently reported mAb 8ANC195, which also targets the C2 hairpin and competes with CD4bs antibodies [47,71]. However, unlike the 647 serum, mAb 8ANC195 recognizes the common N234 glycan that is absent in JR-FL [72]. Even when this glycan was introduced into JR-FL Env, 8ANC195 neutralization remained undetectable and thus prevented us from being able to compete serum 647 and mAb 8ANC195 in the trimer VLP ELISA (as in Fig 5). This difference in specificity might in part explain 8ANC195' s breadth, as compared to the strain-specificity of the 647 serum. The N234 glycan present in BG505 and 79. 7% of 4,265 published strains is added back in Fig 9G, revealing a possible clash with 647 serum nAb binding. Overall, the epitopes of our 3 potent sera add to previous evidence that the JR-FL Env can quite frequently induce nAbs that overlap the CD4bs [12,13]. This may be a result of strain-specific gaps in its glycan armor of the gp120 C2 region that protects the CD4bs. Similar glycan-deprived sites can also be targets for autologous neutralization in natural infection [9,11]. The potent nAb responses in rabbits 613 and 347 far exceeded any we observed in previous VLP immunogenicity studies [28,50]. This may be in part due to improvements that were suggested by these earlier studies: i) that high doses may be important, ii) that rabbit models may be more competent as nAb vaccine models, and iii) that it may be important to refocus responses on the native trimer by eliminating antigenic interference by non-functional Env. The use of more efficient expression methods should in future help us to ensure that doses are sufficient to induce maximum responses. Our newly adopted Gag-only plasmids (as compared to subgenomic NL-Luc) are also safer because they lack a Ψ packaging signal and therefore do not carry a genetic payload [73]. In contrast to rabbits, the relatively high gp120 binding (Fig 2) and tier 1 nAb titers (S3 Table) but lack of tier 2 nAbs (Fig 2) in guinea pigs suggest that VLPs are immunogenic, but that this species struggles to target the native Env trimer and instead target degraded forms or Env that may appear later. Different stochastic processes might underlie the inconsistent nAb development to the various immunogens we tested. In the case of the gp120 prime-protein boost regimen used in the 2922 animal, since monomeric gp120 is not an authentic target, it may only rarely induce antibodies that happen to cross-react with native trimers and instead largely induces ‘off target’ responses. In the case of regimens that involve authentic trimer as an antigen, different challenges come into play, principally that it is a complex antigen that presents few opportunities for antibody development. For this reason, tier 2 nAbs developed in only 1 of 20 animals that received a DNA prime that expresses largely authentic Env trimer (S1 Table). An additional factor may have been this plasmid DNA expresses only modest amounts of trimer to ensure a mature, authentic product, but that could adversely affect the robustness of priming. Paradoxically, however, the rare tier 2 nAbs observed in animal 647 were associated with unusually effective DNA priming, as evidenced by an exceptional ID50 to the MN strain (S1 Table). The distinct specificities of the tier 1 and 2 nAbs in this animal (S7 Fig) suggest the development of separate antibody lineages: potent tier 1 nAbs that recognize non-native forms of Env are produced during DNA priming, as well as trimer-specific tier 2 nAbs that are expanded upon the first protein boost. Clearly, this effect may be difficult to replicate with any reliability. In contrast, our data show that trimer VLPs induce only limited tier 1 nAbs and generated tier 2 nAbs more frequently, albeit still in a minority of vaccinees. This inconsistency may relate to limited ability of small animal antibody repertoires to find solutions for binding this complex antigen—a problem that may be exacerbated by the low Env spike density on VLP surfaces that may limit crucial signals necessary for affinity selection [31,74]. Overall, these observations suggest that, despite the exposed protein patch in the C2 region, the native JR-FL trimer remains poorly immunogenic. Clearly, it will be important to try to improve the consistency of vaccine nAb responses. One approach may be to combine the approaches used here in DNA prime-VLP boost regimens [13,20,22,75]. A more intriguing approach might be to engineer additional holes in the glycan shield to encourage nAb development [32,76]. For example, CD4bs nAb development might be facilitated by selectively removing glycan fence posts to avoid clashes [32,67,70]. One concern with this strategy is that glycan-depleted trimers may be overtly sensitive to non-neutralizing antibodies, as occurs with the removal of the N301 glycan [64,77]. In addition, some glycans cannot be removed without adversely affecting trimer folding [29]. Nevertheless, in our experience, most single glycans can be removed without global effects on trimer folding and maintain a tier 2 phenotype (i. e. , non-neutralizing epitopes remain occluded). In fact, the conservation and use of glycosylation sites vary considerably between isolates. It is fairly common for tier 2 isolates to lack one or two glycans, like JR-FL, that are present on the majority of other circulating tier 2 viruses. Indeed, modestly glycan-depleted tier 2 viruses can in some scenarios transmit infection, perhaps because these variants can facilitate acquisition in settings where nAb resistance is unimportant [8,78–80]. To our knowledge, our data provide the first clear evidence of potent, vaccine-elicited nAbs directed to a reasonably well-conserved site on tier 2 viruses—albeit one that is camouflaged by a glycan on most strains. In contrast, recent evidence suggests that broad HIV-1 neutralization almost always requires glycan recognition—as opposed to other viruses such as influenza, where, despite high glycan context, no glycan recognition occurs [5]. Our results therefore show how effective the glycan shield is in preventing the development of broad neutralization. With this in mind, how might we try to induce cross-reactive tier 2 nAbs in the future? One strategy might be to evolve breadth from initial autologous nAbs using modified boosts. After all, in both vaccine and natural infection settings, initial nAbs are invariably autologous and there may be no practical way to elicit bnAbs directly. The mechanisms of nAb breadth development in natural infection provide model scenarios that might be mimicked by vaccine strategies [8,9, 11,81–85]. For example, breadth sometimes develops as a result of virus escape from autologous nAbs in which a newly introduced glycan creates a new broad site of vulnerability [8–11]. Indeed, while glycans are largely immunosilent, bnAbs can eventually develop that recognize composite epitopes involving glycan contacts. The evolutionary changes in nAb specificity needed to accommodate a newly added glycan might be either to avoid clashes, perhaps by altering the angle of approach to its epitope (e. g. CD4bs [86]) or to incorporate the glycan as part of its epitope. Either of these scenarios might be a useful first step towards cross-reactivity. For example, it might be possible to evolve changes in neutralizing antibodies like those observed in animal 613 by using appropriate boosts to encourage the development of antibody variants that are no longer sensitive to the N197 glycan. In summary, this study introduces some encouraging new concepts for HIV-1 nAb vaccine development. Specifically that i) native trimers presented in situ can induce tier 2 nAbs, ii) nAbs recognize quaternary epitopes, iii) nAbs target vulnerable gaps in the glycan shield, and iv) these nAbs can neutralize other tier 2 viruses provided that appropriate gaps are artificially introduced in their glycan shell. The advances we made here with VLP immunogens required us to address various limiting factors we identified in preceding studies. A continued commitment to further iterative, rational improvements may in future bring us closer to our goal of inducing tier 2 breadth.
Monoclonal antibodies (mAbs) were obtained from their producers, the AIDS reagent repositories of the UK Medical Research Council and the NIH, or were purchased from commercial suppliers. Further information on these mAbs can be found at the web link: (www. hiv. lanl. gov). The mAb panel included the following (originators given in parentheses): 2G12 (Katinger), directed to a unique glycan-dependent epitope of gp120 [87]; 14e, 39F and CO11 (J. Robinson), directed to the gp120 V3 loop [25,28,53]; b12 and b6 (Burton), VRC01 and VRC03 (Mascola), 8ANC131 and 3BNC117 (Nussenzweig), CH103 (Haynes), HJ16 (Lanzavecchia), F105 (Posner) 15e (J. Robinson) and 1F7 (Katinger), directed to epitopes that overlap the CD4bs [25,38,40,67,72,83]; 8ANC195 (Nussenzweig), directed to the C2 region [47,71]; 17b and 48d (J. Robinson), directed to CD4-induced (CD4i) epitopes of gp120; PGT121, PGT125 and PGT128 (Burton) directed to epitopes involving the base of the V3 loop of gp120 and the N332 glycan [42]; PG9, PG16 and PGT145 (Burton), and CAP256-VRC26. 08 (abbreviated as VRC26; directed to quaternary, glycan-dependent epitopes that involve the V2 loop [37,56,84]; 35O22 (Connors), directed to a quaternary epitope of the gp120-gp41 interface [49]; PGT151 (D. Burton), directed to a quaternary gp41 epitope [43]; 7B2 and 2. 2B (J. Robinson), directed to the gp41 cluster I and II epitopes, respectively [11]; 4E10,2F5 (Katinger) and 10e8 (Connors), directed to the gp41 membrane-proximal ectodomain region (MPER) [45]. Recombinant monomeric JR-FL gp120 produced in CHO cells and soluble CD4 (sCD4) consisting of its 4 outer domains were gifts from Progenics Pharmaceuticals (Tarrytown, NY). 2-domain CD4-Ig was a gift from Marie Pancera (NIH). Recombinant HXB2 gp41 was obtained from Meridian Life Science (Catalog#VTI310; residues 546–682, Saco, Maine). Plasmid pCAGGS was used to express JR-FL gp160∆CT on VLP surfaces [51,55]. Gp160∆CT is truncated at amino acid 709, leaving a 3 amino acid gp41 cytoplasmic tail. This increases native trimer expression and can be used to produce pseudoviruses with similar neutralization sensitivity profiles compared to their full-length gp160 counterparts [53]. The use of the JR-FL Env strain has several rare advantages, including efficient expression and gp120/gp41 processing [51,53]. Mutants were generated by Quikchange (Agilent Technologies) and were numbered according to the HXB2 reference strain [54]." SOS" mutations (A501C and T506C) introduce an intermolecular disulfide bond between gp120 and gp41 [57]. E168K and N189A mutations knock in the broadly neutralizing “PG” epitopes that are normally absent in the JR-FL isolate and increases trimer expression [25]. The A328G mutation dramatically enhances neutralization sensitivity, otherwise known as" global" sensitivity [25]. The D368R mutation abrogates CD4 binding capability [13]. Various plasmids expressing other Env gp160s were obtained from the NIH AIDS repository, including clade B and C virus panels. We also obtained a set of Chinese B' Env-expressing plasmids from Dr. Zhiwei Chen [68]. Mutant versions of these sequences in which the N197 glycan is eliminated were used for the fingerprinting analysis. Codon optimized soluble JR-FL D368R gp140 foldon (gp140F) was expressed from a pCDNA3. 1 (-) vector using a CD5 leader and CMV promoter [13]. Another set of plasmids encoded a series of domain-exchanged hybrid gp160s using sequences from the JR-FL and JR-CSF isolates [20,67]. Other plasmids used to make VLPs included Env-deficient sub-genomic plasmids pNL4-3. Luc. R-E- and pSG3∆Env that have been described previously [50]. Plasmids pMV-2024 and pMV-0932 express full-length SIVmac251 (BK28) Gag and codon-optimized HIV-1 Rev, respectively, both under the control of a CMV promoter. First generation VLPs were produced by co-transfecting 293T cells with an Env-expressing plasmid and pNL4-3. Luc. R-E-, using polyethyleneimine, as described previously [25]. Second generation VLPs were produced using plasmids pMV-2024 and pMV-0932 in place of pNL4-3. Luc. R-E-. Two days later, supernatants were collected, precleared by low speed centrifugation and pelleted at 50,000 x g in a Sorvall SS34 rotor. To remove residual medium, VLP pellets were diluted with 1ml of PBS, then re-centrifuged at 15,000 rpm and resuspended in PBS at 1,000 x the original concentration. VLPs were referred to as WT-VLPs or SOS-VLPs, depending on the form of Env displayed on their surfaces or as bald-VLPs, bearing no Env, produced by transfecting pNL4-3. Luc. R-E- alone [50]." Trimer-VLPs" were made by digestion using a cocktail of proteases including proteinase K, subtilisin, trypsin and chymotrypsin, as previously described [25,52]. VLPs were inactivated using aldrithiol (AT-2) [50]. Reference controls included a pooled serum generated from six rabbits that were immunized with monomeric gp120 in AS01B, as described previously [28]. HIV-1-infected donor plasmas BB34, BB68,1686,1702 and N160 and uninfected control plasma 210 were also described previously [28,54]. ELISAs were used to measure serum binding to various antigens and were also used to determine their specificities [25,28,60]. Briefly, Immulon II plates were coated with 20x concentrated VLPs, recombinant gp120 or gp41 at 5μg/ml overnight at 4°C. Following a PBS wash and blocking, sera were titrated against each antigen in blocking buffer. Species-specific alkaline phosphatase anti-Fc conjugates (Accurate, Westbury, NY) and SigmaFAST p-nitrophenyl phosphate tablets (Sigma) were then used to detect binding. Plates were read at 405nm. Titers are taken when ELISA signals exhibited an optical density of 0. 5 (approximately 3x background). In competitive ELISAs, we determined the ability of sera at a 1: 10 dilution to inhibit binding of graded doses of various biotinylated mAbs to SOS E168K trimer VLPs. MAbs were biotinylated using NHS-X-biotin reagent (Calbiochem). Biotinylated mAb binding was detected using streptavidin-alkaline phosphatase (Vector, Burlingame, CA), and developed as above. A prebleed rabbit serum or an HIV-1-seronegative plasma (donor 210) were used as reference controls. Titers of biotinylated mAbs (in μg/ml) measured at OD = 0. 5 (approximately 3 x background) were determined in the presence of competitor and control samples. Then, competition was expressed as the % residual binding = [ (titer in the presence of the control sample) / (titer in the presence of competitor) x 100]. Blue native PAGE (BN-PAGE) was performed as described previously [3,50,51,53]. Briefly, VLPs were solubilized in 0. 12% Triton X-100 in 1 mM EDTA. An equal volume of 2x sample buffer (100 mM morpholinepropanesulfonic acid (MOPS), 100 mM Tris HCl, pH 7. 7,40% glycerol, and 0. 1% Coomassie blue) was added. Samples were then loaded onto a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and separated at 4°C for 3 hours at 100V. The gel was then blotted onto polyvinylidene difluoride membrane, destained, immersed in blocking buffer (4% nonfat milk in PBS) and probed with an anti-gp120 cocktail (mAb b12 and 39F at 1μg/ml) and/or a anti-gp41 cocktail (mAb 2F5,4E10,7B2,2. 2B at 1μg/ml). Blots were then probed by an anti-human Fc alkaline phosphatase conjugate (Accurate Chemicals) and developed using SigmaFast BCIP/NBT substrate (Sigma). BN-PAGE “shift” assays were used to measure the ability of antibodies to bind and deplete the unliganded trimer [3,25,50,51,53]. VLPs were incubated with mAb or serum for 1h at 37°C, then washed with PBS and resolved by BN-PAGE-Western blot, as above. Experiments were conducted to determine the stability of VLPs over time after digestion with proteases. Heat-inactivated sera and protein A-purified serum IgGs were analyzed for neutralization of various pseudoviruses produced by co-transfecting either 293T cells [64] with an Env plasmid and pNL4-3. Luc. R-E- (CF2 assays) or pSG3∆Env (TZM-bl assays). Data is representative of at least 3 repeat assays performed in duplicate. Mann-Whitney and ANOVA two-tailed tests were used to determine any statistically significant differences between study groups. The neutralization sensitivities of a panel of N197 glycan knockout mutants to a vaccine serum and a panel of neutralizing antibodies was used as a basis for a bioinformatics-based approach to inferring the neutralizing specificities in the serum, according to a previously published protocol [5,49,69,72]. A cutoff of ID50 = 50 (since input data was ‘<50’ in most cases) was used to denote the presence or absence of neutralization. A reference set of 12 antibody specificities was used in the analysis, with one or more representative antibodies included for each specificity: VRC01-like (VRC01,3BNC117,8ANC131, CH103), b12-like (b12), CD4 (sCD4, CD4-Ig), HJ16-like (HJ16), 1F7-like (1F7), 8ANC195-like (8ANC195), PG9-like (PG9, PGT145), PGT128-like (PGT121, PGT128), 2G12-like (2G12), 2F5-like (2F5), 10E8-like (4E10,10E8), 35O22-like (35O22). The recently reported structure of BG505 SOSIP gp140 trimer ([5]; PDB id: 4TVP) was used to generate a visual model for the distribution of glycans on the native spike. First, atomic clashes present in the 4TVP crystal structure were relieved and missing side-chains rebuilt, by executing 1,000 symmetric ROSETTA-fixbb simulations, selecting the lowest scoring model, and then running a constrained ROSETTA-relax simulation. Each N-linked glycosylation motif for each of the 15 models was decorated with Man8GlcNAc2 glycans at sites of predicted oligomannose-type glycans and with Man5GlcNAc2 glycans at remaining sites. GlycanRelax [88] was used to approximate the conformational behavior of glycans in a glycoprotein context. For each model, 10 separate GlycanRelax trajectories of 10,000 cycles of MonteCarlo trials were carried out. Each glycan on the gp120 was allowed to move independently throughout the GlycanRelax minimization. A single low energy model was chosen for Figs. All Figs were generated in PyMOL Molecular Modeling Software (Version 1. 5. 0. 4 Schrödinger, LLC). The archived adult human plasmas used in this study have previously been described [28,54]. All donors provided written consent for the use of these samples. Institutional Review Board (IRB) approval for this project was obtained through the San Diego Biomedical Research Institute IRB Committee (approval number: IRB-14-04-JB; Federal Wide Assurance number: 00021327). All immunization protocols for rabbits and guinea pigs were approved (protocol PRF2A) by the Explora Biolabs Animal Care and Use Committee (IACUC). Explora Biolabs’ animal welfare assurance (AWA) number is A4487-01. Pocono Rabbit Farm, Covance and Aldevron are all approved for rabbit and guinea pig immunizations by the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC). The AWA number Pocono Rabbit Farm is A-3886-01. The protocol for rabbits immunized at Covance (Denver, PA), was approved (protocol 0310–11) by the Covance IACUC Committee (AWA number A-3850-01; NIH profile number 9640807). Protocols for rabbits immunized at Aldevron LLC (Fargo, ND) were approved (protocols 2-04-007-09-2005,1-06-001-08-2007, and 1-07-005-10-2008) by the Aldevron IACUC Committee; AWA number A4422-01). All animals were fed, housed and handled in strict accordance with the recommendations of the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act and Regulations and guidelines established in 1993 by the American Veterinary Medical Association Panel on Euthanasia. Two rabbit sera (7672 and 849) were archived from previous studies in which animal welfare information was documented [12,58,59].
Here we show that native HIV-1 Env spikes expressed in a natural membrane context can induce potent tier 2 nAbs in rabbits. These antibodies reacted exclusively with epitopes present on these trimers and not with isolated Env subunits. Intriguingly, the neutralizing sera were found to take advantage of natural gaps in the carbohydrate defenses of Env spikes of the vaccine strain. Some sera were able to neutralize heterologous isolates, provided that a key, regulating glycan was removed. Overall, these findings suggest that native, membrane-expressed trimers hold promise for further development as vaccine candidates. In the future, by adapting our current findings, we might be able to encourage nAb development to key conserved sites by introducing additional, targeted gaps in the trimer' s glycan shell. We suggest that the rare ability to predictably induce potent autologous neutralizing antibodies to field isolates, as we report here, provides a foundation for exploring new strategies aimed at inducing neutralization breadth which is widely expected to be essential for vaccine-induced protection.
Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site