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.

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.

Competing interests: The authors have declared that no competing interests exist. Some authors are employed with Altravax Inc, but this does not affect competing interests. No employment, consultancies, patents or products in development or on market represent a competing interest with this article. This does not alter our adherence to all PLOS Pathogens policies on sharing data and materials.

Funding: This work was supported by National Institutes of Health ( www.nih.gov ) grants RO1AI93278, RO1AI58763 and R33AI84714 (JMB); National Institutes of Health grant P01 AI056375 and Department of Defense grant W9113M-08-1-0008 (RGW); the International AIDS Vaccine Initiative ( www.iavi.org ) Intramural Research Program (RTW); supplements provided by the US-China Program in Biomedical Research Cooperation to National Institutes of Health grant AI58763 (JMB) and National Natural Science Foundation of China ( http://www.nsfc.gov.cn/publish/portal1/ ) grant 81261120418 (PZ); and the National Institutes of Health Intramural Research Program (ISG, SO, GSJ, NDR, PDK, and JRM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication

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.

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 ].

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 ].

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.

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.

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.

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.

Results

Our prior work suggested that the ability of VLPs to induce tier 2 nAbs may be improved by eliminating antigenic interference by non-functional forms of Env [50–52], by increasing the immunogen dose, and by use of a model species with a sufficiently complex antibody repertoire to enable responses to the native Env trimer [28]. Fig 1 provides an overview of a panel of reference sera and five groups of small animal vaccine sera. The reference panel includes four HIV-1 donor plasmas (1702, N160, 1686 and BB34) [53,54], an uninfected human control plasma (210), and an anti-JR-FL gp120 monomer serum pool from rabbits (described previously as "R1" in ref. [28]).

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larger image TIFF original image Download: Fig 1. Overview of human plasmas and animal sera. Our plasma and serum panel consisted of human plasmas from 4 HIV-1-infected donors and one uninfected donor, a pool of rabbit sera generated against monomeric JR-FL gp120, groups of rabbit and guinea pig sera generated to various JR-FL Env-VLP-based immunogens. Group 5 animals all received JR-FL-based immunogens via different DNA prime-protein boost protocols. The absolute dose of gp120 and gp140 immunogens is shown. VLP doses are given in estimated gp120 equivalents [28]. Adjuvants and immunization routes are shown (abbreviations are: IM intramuscular, SC subcutaneous and ID intradermal). https://doi.org/10.1371/journal.ppat.1004932.g001

To facilitate comparisons between different vaccine regimens, all animals in groups 1–5 of Fig 1 were immunized with vaccines that present various forms of JR-FL Env. Animals in immunization groups 1–4 were immunized with VLPs bearing gp160∆CT Env in AS01 B adjuvant [28,51,53,55]. An E168K mutation, with or without an additional N189A mutation, was used to partially or completely introduce the broad PG9 mAb epitope [25,52,56]. We used higher VLP doses here than in our previous studies [28,50]. Normalized VLP doses used in guinea pigs (375μg/kg; group 3) were higher than those used in rabbits (150μg/kg; groups 1, 2 and 4), assuming mean masses of 0.8kg for guinea pigs and 4kg for rabbits.

VLP immunogens administered to animal groups 1–3 (Fig 1) were treated with proteases to remove non-functional Env, leaving native Env trimers intact (termed “trimer VLPs”) [25,52]. Groups 2 and 3 received VLPs bearing "SOS" mutant Env that introduces a gp120-gp41 disulfide bond [57]. Previous studies have shown that the various Env modifications in these VLP immunogens (i.e. gp41 tail truncation, E168K, N189A and SOS mutations) all have negligible effects on the tier 2 phenotype, compared to the full-length, unmutated JR-FL parent, thereby justifying their use here [53,55,56]. For reference to the trimer VLPs administered to groups 1–3, undigested VLPs were used to immunize group 4 rabbits. During the course of these immunogenicity studies, the plasmids used to express VLPs changed (Fig 1). Thus, 1st generation VLPs, were expressed using the subgenomic pNL-LucR-E- plasmid (abbreviated as pNL-Luc) to induce budding [28]. Conversely, 2nd generation VLPs were expressed using a plasmid expressing SIV p55. In this case, a Rev-expressing plasmid was also co-expressed to enhance Env mRNA export and thereby boost Env expression (Rev is naturally encoded by pNL-LucR-E- used in 1st generation VLPs). In a BN-PAGE analysis, 2nd generation VLPs exhibited markedly improved trimer expression compared to 1st generation VLPs (S1 Fig compare lanes 7 and 8). Like their predecessors, 2nd generation trimer VLPs were also preferentially recognized by neutralizing mAbs [25]. The SOS mutation led to improved trimer expression compared to WT (S1 Fig, compare lanes 5 and 6 to lane 7; [51,53,55]). Protease digestion substantially (albeit incompletely) cleared non-functional Env, including UNC gp160∆CT monomers and gp41 stumps (S1 Fig, compare lanes 1–4 to lanes 5–8; [52]). The residual undigested monomer (see lanes 7 and 8 in S1 Fig) is probably a minor species of UNC gp160 that bears complex glycans, enabling it to survive protease treatments [52]. Overall, this analysis confirms that animals in groups 1–3 were immunized with VLPs bearing predominantly native trimer (S1 Fig, lanes 6 and 8), whereas group 4 animals received VLPs that bear a higher proportion of non-functional Env (S1 Fig, lane 2).

The success of trimer VLPs as immunogens could be adversely affected by protease damage. On the other hand, we know from our previous work that, perhaps surprisingly, VLPs remain fully infectious following protease treatment [25,52]. We also know that trimer VLPs remain intact during ELISA analysis [25,52]. To investigate the stability of our VLP immunogens in more detail, we followed their decay over time at 4°C and 37°C using infectivity and BN-PAGE as readouts of trimer function and stability, respectively (S2 Fig). In brief, we found that trimer VLP infectivity decayed more rapidly (t 1/2 of 1.4h) than untreated VLPs (t 1/2 of ~64.5h) at 37°C (S2A Fig). However, residual infectivity (i.e. functional trimer) was nevertheless still detected at 72h. At 4°C full infectivity was retained indefinitely, regardless of protease digestion (S2A Fig). These observations were perfectly complimented by the survival of native trimer under the same conditions, as measured by BN-PAGE-Western blot (S2B Fig). Overall, we conclude that, while trimer VLPs survive protease treatments, they are prone to subsequent decay at physiologic temperatures. Nevertheless, since native trimers and infectivity can still be detected at 72h, they may survive sufficiently long in vivo to be able to induce nAbs.

To provide a comparison to our VLP sera, another group of rabbit sera (group 5 in Fig 1) consisted of the best responders from 4 different DNA prime-soluble Env boost immunogenicity studies. The most potent group 5 serum was from animal 647 that had been immunized with pSVIII SOS gp160∆CT plasmid DNA, followed by a single gp140 foldon (gp140F) trimer boost. Nineteen other rabbits immunized with the same or related DNA prime-boost or gp140F only regimens did not develop potent autologous nAbs (S1 Table). Interestingly, the 647 animal was also the only one to develop high titer nAbs against the tier 1A MN strain after 3 DNA primes, i.e., before it was boosted with gp140F trimers (S1 Table). Thus, a particularly effective response to DNA priming may have imprinted tier 2 JR-FL nAbs that were expanded after a single protein boost. Notably, the neutralizing ID50s (particularly against tier 1 viruses) in many animals decreased following the second protein boost (S1 Table). One explanation might be that the rest period between protein boosts 1 and 2 may have been insufficient for B cells to return to a resting state (4 weeks). Another possibility is that there may be competition between lineages initiated by DNA priming and those initiated by the first protein boost. If the protein-initiated responses predominate, this could explain the transient dip in titers at the second boost. Yet another possibility could be an increasing focus on strain-specific epitopes (i.e. JR-FL-specific epitopes not present on the MN or SF162 strains).

The 2922 serum of group 5 was the most potent of 10 rabbit sera arising from a gp120 DNA-soluble gp120 monomer boost vaccination study (S2 Table; [20]). It is worth noting that the autologous nAb ID50s here were weaker than those observed in a previous study using the same regimen based on the JR-CSF strain [20]. This suggests that the Env clone markedly affects nAb induction by this regimen. Serum 849 was derived from an animal immunized with JR-FL SOSIP gp140 in a DNA prime-protein boost regimen [58]. Finally, the 7672 serum resulted from immunization with a mutant gp120 DNA prime-protein boost regimen that contained a graft of the MPER region within the V2 loop (G2C mutant) [59].

Potently neutralizing sera recognize native trimers in a D368-independent manner 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).

Potently neutralizing sera recognize quaternary epitopes 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). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Adsorption to native Env trimer expressed on cell surfaces removes potent serum neutralizing activities. IgG was first protein A-purified from sera and adjusted to the original serum concentration (~10mg/ml). Samples were then adsorbed repeatedly to native Env trimer-expressing cells. IgG was then re-isolated and adjusted to the original volume. A) Pre- and post-adsorption IgG samples were then assessed for neutralizing activity against JR-FL gp160∆CT WT E168K in the TZM-bl assay. B) Purified IgG from serum 613 was monitored before and after adsorption by silver stain. Each sample was titrated. https://doi.org/10.1371/journal.ppat.1004932.g004

Serum 613 exhibits N197-regulated tier 2 breadth within clade B 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. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 7. Serum 613 neutralization breadth of N197 knockout mutants. 613 serum ID50s against N197 glycan knockout mutants of various clade B (blue symbols) and non-clade B (green symbols) in the TZM-bl assay. N197 glycan knockout mutants sensitive to 613 serum are indicated. Raw data are shown in S5 Table. https://doi.org/10.1371/journal.ppat.1004932.g007 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.