HIV-1 neutralizing antibodies: understanding nature’s pathways

Structural and genetic features of neutralizing mAbs

Remarkably, investigators have recently solved liganded crystal structures of mAbs bound to each of the four major neutralization epitopes, and the structural features of these mAbs has been recently reviewed in detail (28, 121). Here, we focus on some of the key features of these new mAbs and the implications of this knowledge for vaccine design, including key gaps in our knowledge that still limit our ability to elicit HIV-1 specific BNAbs. A review of the four major neutralization epitopes on the HIV-1 Env reveals some intriguing structural, genetic, and immunological characteristics of the antibodies to each epitope. The CD4-binding site would seem to be an obvious functionally conserved region of Env, yet most of the initial antibodies isolated to this site were poorly neutralizing. Structural studies of neutralizing and non-neutralizing CD4-binding site mAbs revealed that precise targeting of this region by antibody b12 was necessary to avoid steric and conformational clashes that would hinder binding to the native viral spike (122). Hence, many CD4-binding site mAbs display high affinity binding to recombinant gp120 but are unable to access the CD4-binding site on gp120 (123, 124). The isolation and structural characterization of the broadly reactive VRC01 mAb provided a fascinating solution to this problem: the antibody partially mimics binding by cellular receptor CD4 to gp120 (84, 119). Specifically, a region of the heavy chain of VRC01 is arranged in an antiparallel B sheet configuration that is similar to the interaction of domain 1 of CD4 with gp120. Structures of VRC01-like antibodies from a total of six different donors reveal a highly similar mode of recognition of the CD4-binding site (114, 119, 125, P. Kwong, personal communications). Light chain interactions with somewhat more variable regions of V5 and the D-loop regions of gp120 explain the few cases of viral resistance to this class of mAbs. A rather unique feature of VRC01 class antibodies is that heavy chain binding is mediated largely by CDRH2, a region encoded by the V-gene, rather than CDRH3 which is encoded by V-D-J recombination. Interestingly, all known VRC01 type antibodies derive from VH1-2 or the closely related VH1-46 gene, suggesting these two germline genes are required to generate these antibodies (84, 114, 117, 126). Also, these VRC01 class antibodies are highly somatically mutated (~ 20% – 30% nt changes in heavy chain compared to germline), and it is difficult to detect any gp120 binding when the amino acid sequence of the heavy chain V-region is reverted to germline (114, 117, 119, 127). This has raised questions about how naive B cells are triggered to generate VRC01 class antibodies (128-130).

Two additional neutralization epitopes on gp120 were identified when antibodies to conserved glycan-containing regions of V1V2 and V3 respectively were described. mAbs PG9 and PG16, and subsequent mAbs in this category, such as CH01-04 and PGT141-145 (82, 83, 109) bind to relatively conserved residues within V2 and require major interactions with surface glycans, most importantly a N-linked glycan at position 160 (131, 132). Similarly, the PGT mAbs described in 2011 bind to residues at the base of V3 and require specific glycan interactions, particularly at position N332 (83, 133). These glycan reactive antibodies are some of the most potent HIV-1 mAbs isolated and they share a common feature of a long CDRH3 loop that interacts with surface glycans and reaches past these glycans to make key contacts with either V2 or V3 residues on gp120. These antibodies are also highly somatically mutated, and hence, a characteristic of HIV-1 glycan reactive antibodies appears to be a long CDRH3 loop together with a high level of affinity maturation to acquire breadth of reactivity. In the case of V1V2 BNAbs, the germline reverted versions both CH01 and PG16 have been found to neutralize the same few HIV-1 strains, suggesting a common structural requirement for their induction (109, 134). Finally, a new MPER directed mAb, 10e8, was recently isolated and its co-crystal structure solved (110). This antibody binds to a region of the MPER that overlaps with the 4E10 epitope but includes C-terminal residues close to the transmembrane spanning domain. 10e8 is highly somatically mutated, is 5-10 fold more potent than 4E10 and can neutralize ~ 95% of viruses tested. Interestingly, mAb 10e8 does not possess the polyreactivity that is characteristic of mAbs 2F5 and 4E10, but it does have a long HCDR3 and a high number of somatic mutations (110).

Immunological roadblocks to BNAb elicitation

Despite the detailed knowledge of the major neutralizing mAbs described above and the associated atomic level descriptions of their viral epitopes, we are left with rather formidable barriers to their elicitation. Our best attempts to construct vaccine immunogens that present these key epitopes to the immune system have failed to generate antibodies that neutralize most strains of HIV-1 (28, 39, 44, 45, 58). An example of this phenomenon is observed with monomeric recombinant gp120 used as a vaccine. We have known since 1994 that the neutralizing mAb b12 binds with high affinity to the CD4-binding site on recombinant gp120 (64), yet numerous studies of gp120 immunization have shown that this does not result in the generation of NAbs like b12 or newer CD4-binding site antibodies like VRC01 (39, 44, 135, 136). This highlights a long standing dichotomy between antigenicity and immunogenicity that has been described by numerous investigators (24, 27, 63, 137, 138). While additional structural knowledge of new or existing antibodies will likely continue to refine our understanding of these epitopes, we have a wealth of information to provide momentum for our structure-based vaccine design efforts (28, 139, 140). However, a structure-based approach in and of itself will likely not solve the HIV-1 vaccine problem. Rather, the correct antigenic structure must be formulated and delivered in such a way as to overcome the immunological road blocks that appear to limit effective antibody response to the HIV-1 Env (129, 140-142). Thus, the design of improved HIV immunization strategies will likely require a better understanding of epitope dominance and subdominance, immune tolerance and anergy, and the immunogenetics of antibody induction and maturation, including the role of selected germline genes and the process of affinity maturation (Table 2).

Regarding epitope subdominance, we need to understand why certain regions or subregions of the HIV-1 Env are poorly immunogenic and how to engage rare BNAb B-cell precursors and foster their expansion to yield a dominant plasma antibody response (28, 129). As noted previously, the HIV-1 Env is highly immunogenic and induces antibodies to gp41 and gp120 shortly after infection. The hierarchy of the humoral immune response during acute HIV-1 infection has recently been studied in detail (141, 143). The first HIV-1 Env specific antibodies are observed as early as 8 days after plasma viremia is first directed (143). These initial antibodies target immunodominant regions of gp41, followed by the development of non-neutralizing antibodies to gp120, including antibodies to variable loop regions, the CD4-binding site and the CD4-induced co-receptor sites on gp120. Some investigators have speculated that this early antibody response is driven by both surface-exposed immunodominant regions of gp120 and gp41 and by shed gp120 and gp41 stalks that may be a natural part of the viral life cycle (24, 144). Such gp120 and gp41 epitopes may not be accessible on the functional trimeric viral spike, explaining the lack of neutralization by these antibodies.

Over time, virus-neutralizing antibodies develop in most HIV-1 infected individuals, demonstrating that the humoral immune system can target epitopes on the native viral spike. The initial NAb response is directed to the autologous infecting virus and is generally not detected until more than 12 weeks of infection (22, 23, 91, 102, 141). Though these autologous NAbs exert immune pressure on the virus, they are highly strain specific; i.e., the can neutralize the autologous virus potently, but display little breadth of activity against heterologous viruses. Initial cross-reactive NAb may arise after several months of infection but the more broadly reactive NAb that target highly conserved epitopes are generally not found until more than two years of infection (91, 102). The kinetics of the HIV-1 Env antibody response raises numerous questions. Why is there a delay of several months or more before viral NAbs arise? When these antibodies do arise, what accounts for their initial strain-specificity and what characteristics confer neutralization breadth? Do strain-specific NAbs target more variable epitopes in contrast to BNAbs, or does a specific NAb lineage mature over time to gain the necessary affinity and precision to target conserved residues within a neutralizing epitopes? The answers to these questions have clear implications or vaccines design, as discussed below.

There is also evidence that the BNAb response is limited by self-tolerance and clonal deletion. Structural studies of mAbs 2F5 and 4E10 with respective gp41 peptides show that only a small part of their CDRH3s bind the gp41 epitope (76, 77). Additional studies suggested that remaining hydrophobic CDRH3 regions are important for antibody interactions with the viral membrane, to allow the antibody to lie in close proximity to the membrane. This appears to position the mAbs for high-affinity binding to the gp41 MPER (145-148). Interestingly, both 2F5 and 4E10 react with anionic lipids such as cardiolipin, consistent with the observations that the antibodies bind both to gp41 and to the nearby virion membrane (79, 80, 149). Alam and colleagues (80, 148) demonstrated that both 2F5 and 4E10 encounter the viral membrane followed by induction of a gp41 conformation change that leads to high affinity mAb docking to the MPER epitope on gp41. In this process the ‘hinge’ region of the MPER is extracted from contact with the viral membrane (145, 150). Importantly, in vitro polyreactivity of HIV-1 mAbs may be of clinical significance, as passive infusion of mAbs 4E10, 2F5, and 2G12 resulted in a prolonged partial thromboplastin time (PTT coagulation test) in subjects to whom they were administered (151, 152), although no clinical adverse events were noted. This prolonged PTT test, due to lipid-reactive antibodies that mediate lupus anticoagulant activity, can be detected in vitro for 4E10, but not for 2F5, 2G12, or other mAbs such as b12 or VRC01 (79, 153), suggesting that the prolonged PTT effect was the result of infusion of mAb 4E10.

The polyreactivity of some HIV-1 BNAbs raised the important concept of tolerance and immunoregulatory host controls as one set of factors limiting the frequent elicitation of these antibodies (142). To test this hypothesis, Verkoczy and colleagues (142, 154, 155) made homologous recombinant knock-in mice that expressed the mature and germline versions of the 2F5 antibody heavy and light chains. The polyreactivity of both the germline and mature 2F5 was sufficient to result in deletion of 95% of the antibody precursors in bone marrow at the first tolerance checkpoint. Importantly for vaccine development, not all 2F5 B cells were deleted but could be found in peripheral LN and spleen in an anergic state (155). These anergic BNAb B cells could be awakened by repetitive immunization with the gp41 MPER peptide epitope embedded in lipids (L. Verkoczy and B. Haynes, unpublished data). A similar deletion at the first tolerance check point was found in 4E10 knock-in mice (L. Verkoczy L and B Haynes, unpublished data). Thus, overcoming tolerance controls that limit BNAb induction has emerged as a key concept in HIV-1 vaccine development. Nussenzwieg and colleagues (108, 117, 156, 157) evaluated hundreds of HIV-1 Env specific mAbs from chronically HIV-1 infected donors and showed that these mAb were often highly affinity matured and polyreactive. Interestingly, the plasma cell repertoire of patients acutely infected with HIV-1 also contains autoreactive antibodies. Liao and colleagues found that initial gp41 directed antibodies were often highly mutated and polyreactive, and some were shown to react with gut flora antigens (158). These data raised the hypothesis that B cells were initially activated by gut flora prior and that cross-reactivity with gp41 may further shape the memory B-cell repertoire after HIV-1 infection.

A similar tolerance obstacle may affect BNAbs that interact with peptide-glycan epitopes in the V1V2 and V3 regions. These antibodies have extended CDRH3 structures that are required to reach peptide residues obstructed by glycans (Table 1). Both mouse and human studies have demonstrated a selection against long CDRH3 loops, presumably due to their increased auto or polyreactivity. Mouse studies suggest that many B cells encoding long CDRH3 regions are eliminated before reaching the periphery (159, 160), and human studies show a decreased prevalence of CDRH3 sequences in IgG memory compared to naive B-cell subsets (161) and a selection bias against long CDRH3 loops, particularly those with hydrophobic patches (162). Thus, a key question for eliciting these glycan-reactive BNAb is whether their long CDRH3 loops are encoded during germline recombination or whether an iterative affinity maturation process could result in short insertion events that gradually lengthen the CDRH3. This latter process would require immunization to drive an unprecedented level of affinity maturation. Since we know that a relatively small fraction of B-cell receptors (BCRs) encode for long CDRH3 lengths (161-163), perhaps the more likely scenario is that these long CDRH3 BCRs are selected by specific circulating Env immunogens with high enough affinity to drive their expansion and to overcome their potential autoreactive properties.

Another potential immunological roadblock arises when considering antibodies such as VRC01 that potently neutralize via binding to a conserved region of the CD4-binding site. As noted above, VRC01-type antibodies from several different donors display a high level of structural similarity and predominant gp120 contacts are made via the heavy chain CDR2 which is encoded by the VH1-2 gene (114, 119, 125). Hence, it appears that this potent class of antibodies derive from a highly restricted set of VH genes and subsequently undergo extensive affinity maturation to achieve effective broad and potent neutralization (121). Investigators have made the rather disconcerting observation that reversion of V-gene mutations to germline VH1-2, even with an intact mature CDRH3, abolished detectable binding to gp120 (117, 119). This appears true even when testing large panels of diverse gp120s (127) and has raised the question about the nature of the antigen that stimulated the naive VH1-2 BCR, e.g. was it an HIV-1 Env antigen or some unrelated antigen (128, 129)? Several groups of investigators have begun to use structural information to design variants of HIV Env immunogens that would bind to germline (or V-gene reverted) versions of VRC01. Finally and has already been noted, BNAbs appear to take months to years to evolve and have high levels of somatic mutation. This suggests a long and potentially tortuous pathway of evolution. Recent studies have shown that vaccination with HIV-1 gp120 induces anti-gp120 antibodies with ~3-5% somatic mutations and an upper range of 8% (164-166). In contrast, the mean level of somatic mutations among BNAbs is 16% with some BNAbs accumulating 32% mutations compared to germline nucleotide sequence (121, 129) (Table 1). Fortunately, newer BNAbs have recently been described that have normal CDRH3 lengths, fewer somatic mutations, and arise much earlier in HIV-1 infection (118). Thus, studies of BNAb lineages and their evolution from early infection through the process of maturation to potent neutralization may provide insights for vaccine immunogen design and immunization strategies.

B-cell lineages from natural infection

The recent isolation and sequencing of numerous BNAbs provides opportunities to study how these antibodies develop and evolve. When more than one BNAb is isolated from an HIV-1 infected donor, either by antigen-specific B-cell sorting or by memory B-cell cultures, the isolated antibodies are often part of the same clonal lineage, suggesting that one lineage can account for a major fraction of the serum NAb response (82-84, 109, 114, 170). Once the genetic sequence of a BNAb is known, next generation sequencing provides a way to dramatically expand the view of an antibody clonal family (114, 162, 171-173). Bioinformatics based analyses allows the identification of intermediate antibody (IA) sequences and the potential to infer the unmutated ancestor (UA) of the BNAb lineage, i.e. the naive BCR sequence. Wu and colleagues (114) used 454 pyrosequencing of antibody gene transcripts to study the heavy and light chain antibody lineages of mAbs VRC01 and VRC-PG04 in their respective donors. Novel bioinformatics analysis allowed the identification of thousands of sequences that formed a specific BNAb lineage. While these studies included a single time point from a chronically infected donor, the investigators were able to identify heavy and light chain clonal relatives with far fewer somatic mutations than the mature isolated antibody. This allowed the expression and testing of antibody intermediates and the demonstration that high levels of affinity maturation were required for the full potency and breadth of the antibody. In spite of the success of elucidating BNAb clonal lineages from chronically infected subjects, these initial studies were limited by lack of samples from the time of transmission and therefore lack of the inciting transmitted-founder virus Env that was involved in stimulating the antibody lineage.

From 2005 to 2012, the Center for HIV/AIDS Vaccine Immunology (CHAVI) enrolled acute HIV-1-infected subjects and followed them for 3-5 years. A subset of these subjects developed cross-reactive Nabs, and their samples are now being studied for NAb and virus co-evolution. Numerous studies have shown that the initial NAb response is directed primarily to the autologous virus and that there is an ongoing pattern of viral escape and further maturation of the NAb response (22, 23, 93, 94, 102, 174, 175). The co-evolution of HIV-1 and the polyclonal antibody response can be thought of as an ‘arms race’, wherein the infecting virus induces an autologous NAb response that is initially limited in breadth. Viral escape mutants appear to drive the antibody response and ~20% of HIV-1 infected subjects develop cross-reactive NAbs (91, 102, 175). In a recent study, Moore and colleagues (175) reported two examples of HIV-1-infected donors whose transmitted-founder viruses did not have an N-linked glycosylation site at position 332 (a critical glycan for binding by the PGT group of mAbs). The transmitted viruses in each donor were initially neutralized by their autologous plasma, but later escaped by adding a glycan at position 332 (175). The development of N332-glycan containing viruses preceded the development of more broadly NAbs that targeted this N332 site. Thus, a BNAb response arose in response to viral escape from earlier autologous NAbs.

Liao and colleagues (118) recently isolated several closely related CD4-binding site NAbs from a CHAVI cohort seroconverter who had been HIV-1 infected for approximately 2.5 years, and solved the structure of one of these mAbs (CH103) bound to the outer domain of gp120. Using samples derived within several weeks of infection and sequentially thereafter, they were able to establish the initial transmitted-founder viral Env sequence and use antibody gene sequencing to infer the full antibody clonal lineage, including the UA and IA sequences of the CH103 lineage. Notably, the UA of CH103 avidly bound the transmitted-founder Env gp140, and early intermediate antibodies predominantly neutralized the early autologous virus strains. Importantly, the evolution of neutralization breadth was preceded by extensive viral diversification in and near the defined CH103 binding epitope. This was the first demonstration that a specific clonal HIV-1 antibody lineage (in this case to the CD4-binding site) evolves from restricted autologous virus neutralization to broad cross reactive neutralization (Fig. 1). Further studies of the co-evolution of the infecting Env sequences in relation to specific BNAb lineages are likely to provide additional key information on the maturation pathways of BNAbs.

Strategies to elicit BNAbs: guiding the immune response

We have described several major obstacles to the elicitation of broadly reactive NAbs against HIV-1. These include epitope subdominance, tolerance and clonal deletion, gene-restricted antibody lineages, and high levels of affinity maturation. While each of these obstacles is formidable, our recent structural understanding of antibody mode of recognition together with genetic studies of antibody evolution are beginning to elucidate the pathways for BNAb development. This knowledge has raised additional questions to investigate but also provides new approaches to BNAb elicitation (Table 2).

There are at least two related approaches to address epitope subdominance: one is to remove or mask dominant epitopes (28, 176-178), and the other involves the structure-based design of immunogens that present specific conserved epitopes to the immune system (139, 179, 180). Numerous studies with variable region deleted Envs (181-183) or with glycan-masked immunogens (184) suggest that these approaches alone do not solve the problem, though they may have utility as part of a more complex approach. For example, native deglycosylation of gp140 Env exposes gp41 epitopes and enables the UA of some gp41 mAbs to bind HIV-1 Env and has the potential to improve Env immunogenicity (185). It may be possible to focus the immune response by initially immunizing with a restricted epitope and boosting with a native form of gp120 or gp140 (28, 179, 186). This has the potential to stimulate the correct naïve BCR and to affinity mature the response against the same epitope as it exists in its native form on the Env trimer. In this regard, there has been recent progress in the design of protein scaffolds on which specific HIV-1 epitopes can be transplanted and retain their native configuration (187-190), and several studies have shown proof-of-concept that such protein scaffolds can induce an epitope specific antibody response (187, 188). There has also progress in the design of cleaved gp140 molecules that retain antigenic reactivity to BNAbs (191-193). Investigators are just beginning to take advantage of these new immunogens and immunization strategies.

We have already discussed the obstacle of self-tolerance and clonal deletion in some detail. Antibodies with long hydrophobic CDRH3 regions may be disfavored during the process of affinity maturation and this may impact elicitation of glycan reactive BNAbs to the V1V2 and V3 regions (e.g. PG9, PGT128). Further studies should inform our understanding of how these V1V2 and V3 glycan BNAbs arise, if the long CHRH3 is selected at the germline level, and if glycan reactivity results from affinity maturation. This information would inform the design of vaccines to elicit these glycan-reactive antibodies. For example, the PG9 (V1V2-glycan) and PGT128 (V3-glcyan) groups of BNAbs make both protein and glycan contacts on gp120. If the early forms of these antibodies make mainly protein contacts (i.e. glycan reactivity evolves over time with affinity maturation), the approach to elicitation might favor presentation of the appropriate protein contacts surface first. However, if the early antibodies bind both protein and glycan, elicitation may require initial immunization with an appropriately glycosylated native trimer. Self-reactivity may be a particular problem for elicitation of antibodies to the MPER of gp41. BNAbs 2F5 and 4E10 are thought to have hydrophobic CDRH3 regions to close approximation to viral membrane, which favors antibody interaction with the gp41 MPER epitope. However, the recent discovery of broadly neutralizing 10e8 antibody demonstrates that the immune system can target the MPER without engendering substantial lipid reactivity. The isolation and characterization of additional antibodies such as the non-polyreactive 10e8 may offer insights into common features of this immune response. Strategies to overcome these disfavored antibody lineages include structure-based design of immunogens for high affinity binding to the BCR, high avidity multivalent antigen presentation, potent adjuvants and specific modulators (e.g. Toll-like receptor ligands) to enhance the innate and adaptive immune response (28, 129, 139, 140).

The high level of affinity maturation of most BNAbs presents a major potential obstacle to their elicitation by traditional immunization methods. It is clear from over 20 years of study of HIV-1 Env immunogenicity that immunization with a current Env vaccines will not induce broad NAbs (39, 194). In addition, only modest levels of affinity maturation have been reported with subunit protein immunization in humans (164-166). To overcome these limitations, we may have to employ new approaches to stimulate suitable B-cell lineages and guide affinity maturation. Studies such as those discussed above, have begun to trace the co-evolution of virus and BNAb lineages to carefully define the Envs immunogen associated with BNAb development (118) (Fig. 1). For vaccine design, the expression and study of these unmutated and early ancestor antibodies may be critical for defining Env immunogens with binding affinity for the appropriate naive B-cell receptor. This approach of B-cell lineage immunogen design has three steps (Fig. 2). The first step is the isolation of one or more clonally related broadly neutralizing mAbs from an HIV-1 infected donor. Second, using the sequences of the known mAb and next generation sequencing to expand the clonal family, one reconstructs the antibody lineage back to the UA. Third, using recombinant antibody technology, the members of the BNAb lineage are expressed and Env constructs are selected that optimally bind to each clonal lineage member at each stage of the lineage. These specific Env immunogens can be used in a prime and boost fashion to engage the naïve BCR and to further stimulate the evolution of the NAb response. (Fig. 2). Recently, Env antigens that bind to the unmutated and early intermediate antibodies of a number of B-cell lineages have been defined (109, 195), and in one case, those Envs with higher binding to the 2F5 UA have proved to induce the highest MPER antibodies (185). Importantly, detailed mapping of the co-evolution of a BNAb lineage and Env escape provides key information for informed design of either sequential or polyvalent mixtures of Envs or Env subunits. Thus, in addition to the recently defined CH103 BNAb linages, it will be important to define additional antibody lineages, including those directed to other conserved neutralization epitopes such as the MPER, V1V2-glycan, and V3-glycan sites. In addition to a highly lineage directed approach, it is also likely that the most effective vaccine-induced antibody response will be polyclonal and be directed to multiple neutralizing epitopes, thus future vaccine strategies may also include polyvalent immunogens as a way to stimulate multiple B-cell lineages or as a means to focus the response on common conserved regions (196-198).

The observation that some HIV-1 BNAbs are restricted to BCRs that derive from specific VH genes has raised the additional question of how to stimulate these specific B cells. VRC01 type antibodies engage the CD4-binding site mainly via CDRH2 interactions, which is encoded by the VH gene. Thus, it is interesting that the UA (or even just V-gene reverted) version of VRC01 antibodies do not bind to heterologous Envs (117, 119, 127). This has led to the hypothesis that stimulation of such antibodies will require the rational design of modified Env immunogens to engage the appropriate BCR (121, 128-130, 139, 199). Several groups of investigators are pursuing variants of the core of gp120 (78, 179, 186, 200) or the more restricted outer domain of gp120 to stimulate CD4-binding site antibodies (201-203). Schief and colleagues (204) have recently reported on the design of an outer domain construct that can bind the V-gene reverted versions of VRC01 and related antibodies. These highly specific germline binding immunogens can be tested empirically for elicitation of VRC01 like antibodies. However, mice and presumably most other small animal species do not have a close homologue to the human VH1-2 gene. Even rhesus macaques that share a similar IgG locus and an average 93% genetic homology to humans (136) may not be the ideal model to test such immunogens, since small changes in the germline sequence may alter key contact sites (114, 119, 126). It may be possible to test such immunogens in mice engineered to have a human IgG locus, though we do not know if these mice can generate the level of affinity maturation required to achieve detectable HIV-1 neutralization (205). In contrast to the VRC01 class of antibodies, HIV-1 Env constructs have been found to bind the germline reverted versions of V1V2 (109, 134) and gp41 membrane proximal external region BNAbs (195). Likewise, the UA of the recently isolated CD4-binding site CH103 mAb can bind its donor autologous transmitted-founder Env with high affinity (118). Thus, lack of reported Env binding by germline reverted HIV-1 specific NAb appears to be a particular characteristic of the highly somatically mutated VRC01 class of CD4-binding site antibodies that bind predominantly via the V-gene encoded CDRH2. Even for these antibodies, investigators have not yet studied their development in enough detail to know if the germline antibodies can bind to the transmitted-founder Env.