Size Doesn’t Matter: Shorter Antibody Loops Can Infiltrate HIV’s Env Apex Defenses
Authors: Jinal N. Bhiman, Penny L. Moore
Journal: Immunity (2017)

An HIV vaccine that elicits broadly neutralizing antibodies, which often have unusual structural features, has not yet been developed. In Immunity this month, Cale et al., 2017Cale E.M. Gorman J. Radakovich N.A. Crooks E.T. Osawa K. Tong T. Li J. Nagarajan R. Ozorowski G. Ambrozak D.R. et al.Immunity. 2017; 46 (this issue): 777-791Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar describe how a new mode of binding allows a conventional antibody to infiltrate HIV’s armor. An HIV vaccine that elicits broadly neutralizing antibodies, which often have unusual structural features, has not yet been developed. In Immunity this month, Cale et al., 2017Cale E.M. Gorman J. Radakovich N.A. Crooks E.T. Osawa K. Tong T. Li J. Nagarajan R. Ozorowski G. Ambrozak D.R. et al.Immunity. 2017; 46 (this issue): 777-791Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar describe how a new mode of binding allows a conventional antibody to infiltrate HIV’s armor. The search for an HIV vaccine has been fraught with many challenges, largely due to the complex nature of the HIV envelope (Env) protein, which mediates viral entry. The Env protein is a master of immunological disguise, with extreme sequence and conformational variability, a heterogeneous glycan shield that occludes protein surfaces, and the co-existence of functional trimeric Env and decoy “junk” Env. Despite this, the human antibody repertoire has unusual ways of subverting these viral defenses, and broadly neutralizing antibodies (bNAbs) develop in many donors, albeit normally after years of infection. The HIV vaccine field has focused on the most broad and potent responses, defining their features and development to provide a template for vaccine design. The isolation of monoclonal bNAbs from these donors has identified five viral vulnerabilities—the V1V2 apex, the N332 supersite, the CD4 binding site, the gp120-gp41 interface, and the membrane proximal external region—which form a continuum of vulnerability spanning the Env trimer (Wibmer et al., 2015Wibmer C.K. Moore P.L. Morris L. Curr. Opin. HIV AIDS. 2015; 10: 135-143Crossref PubMed Scopus (95) Google Scholar). However, these bNAbs, which can in some cases neutralize 99% of circulating viruses from various clades, often possess unusual immunological features, complicating the design of immunogens for their elicitation (McCoy and Burton, 2017McCoy L.E. Burton D.R. Immunol. Rev. 2017; 275: 11-20Crossref PubMed Scopus (144) Google Scholar). In this issue of Immunity, Cale, Gorman et al. describe an antibody that has more conventional features than previously isolated bNAbs and that may represent a more attainable target for vaccine design (Cale et al., 2017Cale E.M. Gorman J. Radakovich N.A. Crooks E.T. Osawa K. Tong T. Li J. Nagarajan R. Ozorowski G. Ambrozak D.R. et al.Immunity. 2017; 46 (this issue): 777-791Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Structural characterization of bNAbs, coupled with studies of their ontogeny, have provided insights into the precursors that initiate bNAb lineages, as well as the features associated with breadth. Many bNAb families exhibit some recognition of self-antigens (autoreactivity), and most have high levels of somatic hypermutation. In addition, structural constraints imposed by the limited accessibility to conserved Env epitopes result in the selection of precursors that have unusually long or short antigen binding loops. For example, many CD4 binding-site bNAbs have rare, short antigen binding loops that avoid steric clashes with hypervariable regions and glycans. In contrast, V1V2-directed bNAbs isolated thus far have unusually long, anionic antigen binding loops formed at the initial V(D)J recombination event in naive B cells (Bonsignori et al., 2011Bonsignori M. Hwang K.K. Chen X. Tsao C.Y. Morris L. Gray E. Marshall D.J. Crump J.A. Kapiga S.H. Sam N.E. et al.J. Virol. 2011; 85: 9998-10009Crossref PubMed Scopus (341) Google Scholar, Doria-Rose et al., 2014Doria-Rose N.A. Schramm C.A. Gorman J. Moore P.L. Bhiman J.N. DeKosky B.J. Ernandes M.J. Georgiev I.S. Kim H.J. Pancera M. et al.NISC Comparative Sequencing ProgramNature. 2014; 509: 55-62Crossref PubMed Scopus (535) Google Scholar, Sok et al., 2014Sok D. van Gils M.J. Pauthner M. Julien J.P. Saye-Francisco K.L. Hsueh J. Briney B. Lee J.H. Le K.M. Lee P.S. et al.Proc. Natl. Acad. Sci. USA. 2014; 111: 17624-17629Crossref PubMed Scopus (233) Google Scholar, Walker et al., 2009Walker L.M. Phogat S.K. Chan-Hui P.Y. Wagner D. Phung P. Goss J.L. Wrin T. Simek M.D. Fling S. Mitcham J.L. et al.Protocol G Principal InvestigatorsScience. 2009; 326: 285-289Crossref PubMed Scopus (1388) Google Scholar). Long complementarity determining regions in antibody heavy chains (CDRH3s) are generally associated with a higher propensity for autoreactivity and are frequently deleted through tolerance mechanisms. Thus, the pool of potential precursors for V1V2-directed bNAbs is extremely limited, making their selection and expansion by vaccination especially challenging. Despite this restricted precursor pool, V1V2-directed responses are common during infection (Rusert et al., 2016Rusert P. Kouyos R.D. Kadelka C. Ebner H. Schanz M. Huber M. Braun D.L. Hozé N. Scherrer A. Magnus C. et al.Swiss HIV Cohort StudyNat. Med. 2016; 22: 1260-1267Crossref PubMed Scopus (108) Google Scholar), a finding that has focused much attention on this epitope at the apex of the Env trimer. Four prototypic V1V2 bNAb lineages have been isolated from different donors, and these share developmental and structural features (Moore et al., 2017Moore P.L. Gorman J. Doria-Rose N.A. Morris L. Immunol. Rev. 2017; 275: 217-229Crossref PubMed Scopus (21) Google Scholar). These bNAbs have a common mode of binding that relies on their long CDRH3s penetrating and contacting conserved glycans at positions N156 and N160, as well as through main chain-interactions with the C-strand protein core of the V1V2 apex. These trimer-specific V1V2-directed bNAbs were mostly isolated using expensive, labor-intensive B cell culture and micro-neutralization screens, but the design of soluble, recombinant Env proteins comparable to native Env trimers provided an alternative antigen-specific B cell selection strategy (Sok et al., 2014Sok D. van Gils M.J. Pauthner M. Julien J.P. Saye-Francisco K.L. Hsueh J. Briney B. Lee J.H. Le K.M. Lee P.S. et al.Proc. Natl. Acad. Sci. USA. 2014; 111: 17624-17629Crossref PubMed Scopus (233) Google Scholar). Cale and colleagues now describe the use of virus-like particles (VLPs) displaying membrane-bound native Env as a positive-selection “bait” that is more representative of Env spikes on HIV virions. This VLP-based B cell sorting strategy resulted in the identification of the N90-VRC38 lineage of bNAbs, isolated from the chronically infected donor N90 (Figure 1, left). The authors subsequently used soluble Env trimers to isolate additional VRC38 antibodies, but while the soluble trimer sorts yielded a greater number of lineage members, the broadest, with 30% neutralization activity, was selected using VLPs. Furthermore, unlike current soluble trimers, these membrane-bound VLPs retain the membrane proximal external region, an important bNAb epitope at the base of the trimer. Therefore, despite the potentially higher background “stickiness” of VLP baits, they present more biologically relevant Env in the native membrane-associated context, useful for future bNAb studies. Functional antibody mapping and bNAb competition assays suggested that the VRC38 lineage targets the V1V2 region, but in contrast to prototypic V1V2 bNAbs, these antibodies possess CDRs of average length in both the heavy and light chains. VRC38 antigen binding fragments (Fabs) complexed with either scaffolded-V1V2 subunit proteins or soluble native Env trimers were used to generate crystal structures and negative stain electron micrographs, respectively. These data confirmed the V1V2 specificity of the VRC38 antibodies, but also revealed a unique mode of recognition of the V1V2 bNAb epitope. In contrast to known V1V2 bNAbs, which bind directly down the central axis of the Env spike, the VRC38 lineage binds to the apex slightly off center (Figure 1, middle). This alternate angle of approach allows the VRC38 antibodies to bind with two or more Fabs per Env trimer, unlike the conventional 1:1 stoichiometry for the other four V1V2 bNAb lineages. In addition, VRC38 bNAbs contacted the virus through amino acid side-chain interactions using CDRH1, H3, and L1 and did not depend on residues 166 or 169, unlike typical V1V2 bNAbs, which contact residues 167–171 through both backbone and side-chain interactions. This VRC38 reliance only on side-chain interactions reduced the distance required by the CDRs to contact the epitope, providing the first example of V1V2-directed bNAbs that have a mode of recognition independent of a long CDRH3. However, reliance on side-chain interactions also reduces their ability to tolerate viral sequence variation, contributing in part to the limited breadth of the VRC38 lineage. The major limitation of breadth for the VRC38 antibodies is their inability to neutralize viruses bearing the N130 glycan, present in >60% of globally circulating viruses. A question for longitudinal follow-up studies will be the potential for VRC38-type antibodies to evolve the capacity to either accommodate or avoid key glycans, a feature that has been observed in other bNAb lineages (McCoy and Burton, 2017McCoy L.E. Burton D.R. Immunol. Rev. 2017; 275: 11-20Crossref PubMed Scopus (144) Google Scholar). Similarly, ascertaining whether the initiation of VRC38-like specificities depends on the absence of this N130 glycan will be of interest. Cale, Gorman et al. propose that immunogens with an engineered N130 “glycan hole” might elicit VRC38-type antibodies, as has been proposed for other specificities, and that bNAb precursors may evolve to tolerate glycans through exposure to immunogens with intact glycan shields. However, the presence of viral variants in donor N90 with an N130 glycan did not support the development of glycan tolerance in the VRC38 lineage. Thus, for this and other bNAb lineages, the question of whether glycan-hole targeting responses are viable bNAb precursors has not yet been addressed experimentally. Neutralization breadth of the best HIV bNAbs approaches 99%, but the combined breadth of the VRC38 antibodies, which did not recapitulate the breadth of the donor plasma, was only 48%. Thus, the VRC38 lineage is on the lower end of the bNAb spectrum, with the broadest member able to neutralize only 30% of a large virus panel. However, Cale et al. show that neutralization breadth within this lineage of just 11 antibodies may be achieved through multiple, subtly different genetic pathways, a phenomenon that has also been observed in bNAb lineages with higher levels of breadth (MacLeod et al., 2016MacLeod D.T. Choi N.M. Briney B. Garces F. Ver L.S. Landais E. Murrell B. Wrin T. Kilembe W. Liang C.H. et al.IAVI Protocol C Investigators & The IAVI African HIV Research NetworkImmunity. 2016; 44: 1215-1226Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The average CDR length and charge of the VRC38 lineage suggests that these kinds of responses should occur more frequently in the naive antibody pool (Figure 1, right). Indeed, computational predictions suggest that 20% of HIV-infected individuals who develop bNAbs may have VRC38-like responses. However, the frequencies of VRC38-type and prototypic V1V2 bNAb responses were similar in this analysis, suggesting that the relatively high level of mutation (15%–20%) of VRC38 antibody members may nonetheless limit their occurrence. Alternatively, previous studies focusing on elite neutralizers may have missed these and other more moderately broad specificities. Thus, the frequency of VRC38-like responses, and how amenable they are to vaccine elicitation, remains to be confirmed by further antibody isolation from human B cell repertoires and by future animal immunization studies. Overall, despite their restricted breadth, the isolation and characterization of the VRC38 lineage has identified a new V1V2 sub-epitope. Three of the other bNAb sites have been classified as supersites, composed of multiple sub-epitopes that differ subtly but share the same core contact residues and can thus be attacked using numerous immunological solutions via multiple angles of approach (Wibmer et al., 2015Wibmer C.K. Moore P.L. Morris L. Curr. Opin. HIV AIDS. 2015; 10: 135-143Crossref PubMed Scopus (95) Google Scholar). The characterization of the VRC38 epitope suggests that the V1V2 apex may be a fourth supersite, providing selection criteria for the isolation of potentially broader VRC38-like antibodies in donor N90 and others. Unlike VRC38, the unusual features of many bNAbs with exceptional breadth present significant hurdles to their elicitation. A polyclonal response of less broad, but more conventional, antibodies may present a practical vaccine strategy, with the moderate breadth of individual antibodies compensated for by a larger pool of potential precursors. Understanding this unique manner in which antibodies can engage the V1V2 bNAb epitope therefore provides additional optimism for the development of an HIV vaccine. Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding LoopCale et al.ImmunityMay 16, 2017In BriefTo date, long recognition loops have been a hallmark of apex-targeting antibodies. Cale et al. identify a lineage of HIV-1-neutralizing antibodies that target the envelope trimer apex. The N90-VRC38 lineage uses a loop of average length—a feature that may make it a useful prototype for vaccine design. Full-Text PDF Open Archive