Search

Introduction: Neutralizing antibodies (Abs) are one of the immune components required to protect against viral infections. However, developing vaccines capable of eliciting neutralizing Abs effective against a broad array of HIV-1 isolates has been an arduous challenge., Objective: This study sought to test vaccines aimed to induce Abs against neutralizing epitopes at the V1V2 apex of HIV-1 envelope (Env)., Methods: Four groups of rabbits received a DNA vaccine expressing the V1V2 domain of the CRF01_AE A244 strain on a trimeric 2J9C scaffold (V1V2-2J9C) along with a protein vaccine consisting of an uncleaved prefusion-optimized A244 Env trimer with V3 truncation (UFO-BG.ΔV3) or a V1V2-2J9C protein and their respective immune complexes (ICs). These IC vaccines were made using 2158, a V1V2-specific monoclonal Ab (mAb), which binds the V2i epitope in the underbelly region of V1V2 while allosterically promoting the binding of broadly neutralizing mAb PG9 to its V2 apex epitope in vitro ., Results: Rabbit groups immunized with the DNA vaccine and uncomplexed or complexed UFO-BG.ΔV3 proteins (DNA/UFO-UC or IC) displayed similar profiles of Env- and V1V2-binding Abs but differed from the rabbits receiving the DNA vaccine and uncomplexed or complexed V1V2-2J9C proteins (DNA/V1V2-UC or IC), which generated more cross-reactive V1V2 Abs without detectable binding to gp120 or gp140 Env. Notably, the DNA/UFO-UC vaccine elicited neutralizing Abs against some heterologous tier 1 and tier 2 viruses from different clades, albeit at low titers and only in a fraction of animals, whereas the DNA/V1V2-UC or IC vaccines did not. In comparison with the DNA/UFO-UC group, the DNA/UFO-IC group showed a trend of higher neutralization against TH023.6 and a greater potency of V1V2-specific Ab-dependent cellular phagocytosis (ADCP) but failed to neutralize heterologous viruses., Conclusion: These data demonstrate the capacity of V1V2-2J9C-encoding DNA vaccine in combination with UFO-BG.ΔV3, but not V1V2-2J9C, protein vaccines, to elicit homologous and heterologous neutralizing activities in rabbits. The elicitation of neutralizing and ADCP activities was modulated by delivery of UFO-BG.ΔV3 complexed with V2i mAb 2158., Competing Interests: Author MT was employed by company Molsoft L.L.C. MT, XJ, X-PK, and SZ-P are inventors in the U.S. Patent 10,568,969 for the V1V2-2J9C construct. A provisional patent application is submitted for the UFO-BG.DeltaV3 construct; CEH and MT are listed as inventors in this application. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Hioe, Liu, Banin, Heindel, Klingler, Rao, Luo, Jiang, Pandey, Ordonez, Barnette, Totrov, Zhu, Nádas, Zolla-Pazner, Upadhyay, Shen, Kong and Hessell.)

We studied the contribution of the light chain to functions of human monoclonal antibodies (mAbs) by measuring the relationships between the rate of mutations and cross-reactivity, binding affinity and neutralization activity. We analyzed 12 mAbs of two clonal families specific to the V2 region of HIV-1 derived from two chronically HIV-1 infected individuals. The clonal mAbs exhibited a range of reactivities, and the clones with superior properties were associated with the rate of mutations and the presence of particular mutated residues in the light chains, but not in the heavy chains. Our observations suggest that for some antibodies, the light chains play a vital role in antibody evolution toward more efficient ones and also suggest the importance of optimal residues rather than the rate of mutations in the variable fragment of the antibody., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

The repertoire of antibodies (Abs) produced upon vaccination against a particular antigenic site is rarely studied due to the complexity of the immunogens. We received such an opportunity when one rhesus macaque was immunized six times at 0, 4, 10, 16, 32, and 143 weeks with C4-447 peptide containing the 8-mer epitope for human monoclonal Ab (mAb) 447-52D specific to the V3 region of gp120 HIV-1. Strong anti-V3 antibody responses reached 50% binding titer in serum of 10 -5 at week 10 that declined to 10 -3 by week 70. After an additional boost of C4-447 peptide at week 143, titers rebounded to 10 -5 at week 146, or 2.7 years after the first immunization. Using the blood sample at week 146, we produced 41 V3-specific recombinant mAbs by single B cell isolation and cloning. Sequence analysis revealed 21B cell lineages, single and clonally related, based on immunoglobulin gene usage and CDR3s. The broad repertoire of Abs directed to a small antigenic site shows the targeting potency of a vaccine-elicited immune response in rhesus macaques., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

The V1V2 region of the HIV-1 envelope is the target of several broadly neutralizing antibodies (bNAbs). Antibodies to V1V2 elicited in the RV144 clinical trial correlated with a reduced risk of HIV infection, but these antibodies were without broad neutralizing activity. Antibodies targeting V1V2 also correlated with a reduced viral load in immunized macaques challenged with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV). To focus immune responses on V1V2, we engrafted the native, glycosylated V1V2 domain onto five different multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibodies that targeted structurally distinct V1V2 epitopes. Plasma antibodies displayed limited neutralizing activity but were functionally active for ADCC and phagocytosis, which was detectable 1-2 years after immunizations ended. This study demonstrates that multivalent, glycosylated V1V2-scaffold protein immunogens focus the antibody response on V1V2 and are differentially effective at inducing polyfunctional antibodies with characteristics associated with protection., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: In humans, only one independent immunologic correlate of reduced risk of HIV infection has been identified: a robust antibody (Ab) response to the V1V2 domain of the gp120 envelope (Env) protein. In recent years, the presence and level of V1V2-specific Abs has also been correlated with protection from SIV and SHIV infections. Here, we review the multitude of studies showing the in-vivo protective effects of V1V2 Abs and review their immunologic characteristics and antiviral functions., Recent Findings: Structural and immunologic studies have defined four epitope families in the V1V2 domain: one epitope family, V2q, which preferentially presents as a quaternary structure of the Env trimer, and another epitope family (V2qt) which requires the quaternary trimeric Env structure; these two epitope types are recognized by two families of monoclonal Abs (mAbs)-V2q-specific and V2qt-specific mAbs-which display broad and potent neutralizing activity. A third epitope family, V2i, is present as a discontinuous conformational structure that overlays the α4β7 integrin binding motif, and a fourth epitope family (V2p) exists on V2 peptides. Antibodies specific for V2i and V2p epitopes display only poor neutralizing activity but effectively mediate other antiviral activities and have been correlated with control of and/or protection from HIV, SIV and SHIV. Notably, V2q and V2qt Abs have not been induced by any vaccines, but V2p and V2i Abs have been readily induced with various vaccines in nonhuman primates and humans., Summary: The correlation of vaccine-induced V2p and V2i Abs with protection from HIV, SIV and SHIV suggests that these Ab types are extremely important to induce with prophylactic vaccines.

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1 + Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified post-hoc in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.

The results of the RV144 vaccine clinical trial showed a correlation between high level of anti-V1V2 antibodies (Abs) and a decreased risk of acquiring HIV-1 infection. This turned the focus of HIV vaccine design to the induction of elevated levels of anti-V2 Abs to increase vaccine efficacy. In plasma samples from HIV-1 infected Cameroonian individuals, we observed broad variations in levels of anti-V2 Abs, and 6 of the 79 plasma samples tested longitudinally displayed substantial deficiency of V2 Abs. Sequence analysis of the V2 region from plasma viruses and multivariate analyses of V2 characteristics showed a significant difference in several features between V2-deficient and V2-reactive plasma Abs. These results suggest that HIV vaccine immunogens containing a shorter V2 region with fewer glycosylation sites and higher electrostatic charges can be beneficial for induction of a higher level of anti-V2 Abs and thus contribute to HIV vaccine efficacy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Prophylactic HIV vaccines must elicit antibodies (Abs) against the virus envelope glycoproteins (Env) to effectively prevent HIV infection. We investigated a vaccine platform that utilizes immune complexes made of Env proteins gp120 and monoclonal Abs (mAbs) against different gp120 epitopes. We previously observed alterations in V3 antigenicity upon formation of certain gp120/mAb complexes and demonstrated the ability of these complexes to modulate the elicitation of V3 Ab responses. However, the effects on the V1V2 domain, an important target for Abs that correlate with vaccine-induced protection against HIV, have not been studied, nor have immune complex vaccines made with non-B subtype Env. This study compared subtypes B (JRFL) and CRF_01.AE (A244) Env gp120 proteins in complex with selected gp120-specific mAbs. Allosteric and antigenic changes were detected on these immune complexes, indicating that gp120/mAb interaction induces alterations on the Env surface that may modify the Env immunogenic properties. To evaluate this idea, mice were immunized with gp120/mAb complexes or their uncomplexed gp120 counterparts. The overall serum IgG titers elicited against gp120 were comparable, but a marked skewing toward V1V2 or V3 was evident and dependent on the gp120 strain and the specificity of the mAb used to form the complexes. Compared with uncomplexed gp120 JRFL , gp120 JRFL complexed with CD4bs or V1V2 mAbs, but not with C2 or V3 mAbs, elicited V3 Abs of greater titers and breadth, and Abs more capable of neutralizing tier 1 virus. Epitope mapping revealed a shift to a more conserved site in the V3 crown. However, the complexes did not enhance V1V2 Ab response, and the elicited V1V2 Abs were not cross-reactive. This profile contrasts with Ab responses to gp120 A244 /mAb complexes. Notably, gp120 A244 /mAb complexes induced higher levels of V1V2 Abs with some cross-reactivity, while also stimulating weak or strain-specific V3 Abs. Sera from gp120 A244 /mAb complex-immunized animals displayed no measurable virus neutralization but did mediate Ab-dependent cellular phagocytosis, albeit at levels similar to that induced by gp120 A244 alone. These data indicate the potential utility of immune complexes as vaccines to shape Ab responses toward or away from Env sites of interest.

Elucidating the structural basis of antibody (Ab) gene usage and affinity maturation of vaccine-induced Abs can inform the design of immunogens for inducing desired Ab responses in HIV vaccine development. Analyses of monoclonal Abs (MAbs) encoded by the same immunoglobulin genes at different stages of maturation can help to elucidate the maturation process. We have analyzed four human anti-V3 MAbs with the same VH1-3*01 and VL3-10*01 gene usage. Two MAbs, TA6 and TA7, were developed from a vaccinee in the HIV vaccine phase I trial DP6-001 with a polyvalent DNA prime/protein boost regimen, and two others, 311-11D and 1334, were developed from HIV-infected patients. The somatic hypermutation (SHM) rates in VH of vaccine-induced MAbs are lower than in chronic HIV infection-induced MAbs, while those in VL are comparable. Crystal structures of the antigen-binding fragments (Fabs) in complex with V3 peptides show that these MAbs bind the V3 epitope with a new cradle-binding mode and that the V3 β-hairpin lies along the antigen-binding groove, which consists of residues from both heavy and light chains. Residues conserved from the germ line sequences form specific binding pockets accommodating conserved structural elements of the V3 crown hairpin, predetermining the Ab gene selection, while somatically mutated residues create additional hydrogen bonds, electrostatic interactions, and van der Waals contacts, correlating with an increased binding affinity. Our data provide a unique example of germ line sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection. IMPORTANCE Understanding the structural basis of gene usage and affinity maturation for anti-HIV-1 antibodies may help vaccine design and development. Antibodies targeting the highly immunogenic third variable loop (V3) of HIV-1 gp120 provide a unique opportunity for detailed structural investigations. By comparing the sequences and structures of four anti-V3 MAbs at different stages of affinity maturation but of the same V gene usage, two induced by vaccination and another two by chronic infection, we provide a fine example of how germ line sequence determines the essential elements for epitope recognition and how affinity maturation improves the antibody's recognition of its epitope., (Copyright © 2018 American Society for Microbiology.)

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4β7, a gut-homing receptor. Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: JA, CC and ASF are inventors of patent 20160075786 submitted by the NIAID, NIH that covers the study of reagents that serve as antagonists of the interaction between HIV gp120 and α4β7 mAb. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The third variable (V3) loop of HIV-1 gp120 is an immunodominant region targeted by neutralizing antibodies (nAbs). Despite limited breadth, better characterization of the structural details of the interactions between these nAbs and their target epitopes would enhance our understanding of the mechanism of neutralization and facilitate designing better immunogens to induce nAbs with greater breadth. Recently, we isolated two anti-V3 neutralizing monoclonal antibodies (MAbs), 10A3 and 10A37, from a rabbit immunized with gp120 of the M group consensus sequence. In this study, crystal structures of these MAbs bound to target epitopes were determined. 10A3 binds to the V3 crown ( 303 TRKSIHIGPGRAF 317 ) using the cradle binding mode, similar to human V3 MAbs encoded by IGHV5-51 germ line genes, and its epitope structure resembles that bound to the human antibodies. In contrast, 10A37, which exhibits greater breadth and potency than 10A3, binds the V3 crown and the succeeding stem region ( 308 HIGPGRAFYTTGEI 323 ). Unexpectedly, the 315 RAFYTT 320 portion of the epitope existed as helical turns, a V3 structure that has not been observed previously. Its main chain-dominated antigen-antibody interactions not only explain the broad neutralization of 10A37 but also show that its epitope is a potential vaccine target to be further evaluated. In conclusion, our study provides novel insights about neutralization-susceptible epitope structures of the V3 loop of HIV-1 gp120 and demonstrates that, despite low amino acid sequence similarity to human antibody germ line genes, rabbits can serve as a useful animal model to evaluate human vaccine candidates. IMPORTANCE The apex crown of V3 of HIV-1 gp120 is the most immunogenic region of the surface glycoprotein, and many MAbs targeting this region have been developed. Structural understanding of V3 crown MAbs not only can help understand how antibody responses target this unique region but also contribute to immunogen design for vaccine development. We present here crystal structures of two neutralizing V3 MAbs, 10A3 and 10A37, developed from a rabbit immunized with gp120. Our analysis of 10A3 in complex with V3 provided a detailed example of how epitope complexity can evolve with affinity maturation, while that of 10A37 revealed a novel V3 binding mode targeting the C-terminal side of the V3 crown and showed that this region can form a helical structure. Our study provides novel insights about neutralization-susceptible V3 epitope structures and demonstrates that rabbits can serve as a useful animal model to evaluate human vaccine candidates., (Copyright © 2018 American Society for Microbiology.)

Background: HIV-1 is known to adapt to the local environment in its usage of receptors, and it can become CD4 independent in the brain where the receptor is scarce. This adaptation is through amino acid variations, but the patterns of such variation are not yet well understood. Given that infection of long-lived CD4-low and CD4-negative cells in anatomical compartments such as the brain expands cell tropism in vivo and may serve as potential viral reservoirs that pose challenge for HIV eradication, understanding the evolution to CD4 independence and envelope conformation associated with infection in the absence of CD4 will not only broaden our insights into HIV pathogenesis but may guide functional cure strategies as well., Methods: We characterize, by site-directed mutagenesis, neutralization assay, and structural analysis, a pair of CD4-dependent (cl2) and CD4-independent (cl20) envelopes concurrently isolated from the cerebral spinal fluid of an SHIV-infected macaque with neurological AIDS and with minimum sequence differences., Results: Residues different between cl2 and cl20 are mapped to the V1V2 and surrounding regions. Mutations of these residues in cl2 increased its CD4 independence in infection, and the effects are cumulative and likely structural., Conclusions: Our data suggested that the determinants of CD4 independence in vivo mapped principally to V1V2 of gp120 that can destabilize the apex of the envelope spike, with an additional change in V4 that abrogated a potential N-linked glycan to facilitate movement of the V1V2 domain and further expose the coreceptor-binding site.

The V3 loop in the HIV envelope gp120 is one of the immunogenic sites targeted by Abs. The V3 crown in particular has conserved structural elements recognized by cross-reactive neutralizing Abs, indicating its potential contribution in protection against HIV. Crystallographic analyses of anti-V3 crown mAbs in complex with the V3 peptides have revealed that these mAbs recognize the conserved sites on the V3 crown via two distinct strategies: a cradle-binding mode (V3C) and a ladle-binding (V3L) mode. However, almost all of the anti-V3 crown mAbs studied in the past were isolated from chronically HIV-infected individuals. The extents to which the two types of anti-V3 crown Abs are generated by vaccination are unknown. This study analyzed the prevalence of V3C-type and V3L-type Ab responses in HIV-infected individuals and in HIV envelope-immunized humans and animals using peptide mimotopes that distinguish the two Ab types. The results show that both V3L-type and V3C-type Abs were generated by the vast majority of chronically HIV-infected humans, although the V3L-type were more prevalent. In contrast, only one of the two V3 Ab types was elicited in vaccinated humans or animal models, irrespective of HIV-1 envelope clades, envelope constructs (oligomeric or monomeric), and protocols (DNA plus protein or protein alone) used for vaccinations. The V3C-type Abs were produced by vaccinated humans, macaques, and rabbits, whereas the V3L-type Abs were made by mice. The V3C-type and V3L-type Abs generated by the vaccinations were able to mediate virus neutralization. These data indicate the restricted repertoires and the species-specific differences in the functional V3-specific Ab responses induced by the HIV envelope vaccines. The study implies the need for improving immunogen designs and vaccination strategies to broaden the diversity of Abs in order to target the different conserved epitopes in the V3 loop and, by extension, in the entire HIV envelope., (Published by Elsevier Ltd.)

The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing monoclonal antibodies (MAbs) such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic as it can also form a helical conformation recognized by RV144 vaccine-induced MAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific antibody (Ab) responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by enzyme-linked immunosorbent assay (ELISA) and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with unconstrained V1V2 antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with constrained V1V2 antigens, suggesting recognition of the β-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope., Importance: The correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The isolation of HIV-1 broadly neutralizing antibodies (bnAbs) has demonstrated the ability of the human immune system to mount effective antibody responses against the virus. To harness this immune potential to elicit similar antibody responses by vaccination, it is important to understand the immunological processes that produce them. Here we review recent advances in crystal structural determinations of HIV-1 bnAb epitopes that directly portray the antigenic landscape of the HIV-1 envelope glycoprotein. We also summarize new immunological concepts implicated in bnAb sequences and their lineage studies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4β7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1.

Unlabelled: The region consisting of the first and second variable regions (V1V2) of gp120 plays vital roles in the functioning of the HIV-1 envelope (Env). V1V2, which harbors multiple glycans and is highly sequence diverse, is located at the Env apex and stabilizes the trimeric gp120 spike on the virion surface. It shields V3 and the coreceptor binding sites in the prefusion state and exposes them upon CD4 binding. Data from the RV144 human HIV-1 vaccine trial suggested that antibody responses targeting the V1V2 region inversely correlated with the risk of infection; thus, understanding the antigenic structure of V1V2 can contribute to vaccine design. We have determined a crystal structure of a V1V2 scaffold molecule (V1V2ZM109-1FD6) in complex with 830A, a human monoclonal antibody that recognizes a V1V2 epitope overlapping the integrin-binding motif in V2. The structure revealed that V1V2 assumes a five-stranded beta barrel structure with the region of the integrin-binding site (amino acids [aa] 179 to 181) included in a "kink" followed by an extra beta strand. The complete barrel structure naturally presents the glycans on its outer surface and packs into its core conserved hydrophobic residues, including the Ile at position 181 which was highly correlated with vaccine efficacy in RV144. The epitope of monoclonal antibody 830A is discontinuous and composed of three segments: (i) Thr175, Tyr177, Leu179, and Asp180 at the kink overlapping the integrin-binding site; (ii) Arg153 and Val154 in V1; and (iii) Ile194 at the C terminus of V2. This report thus provides the atomic details of the immunogenic "V2i epitope.", Importance: Data from the RV144 phase III clinical trial suggested that the presence of antibodies to the first and second variable regions (V1V2) of gp120 was associated with the modest protection afforded by the vaccine. V1V2 is a highly variable and immunogenic region of HIV-1 surface glycoprotein gp120, and structural information about this region and its antigenic landscape will be crucial in the design of an effective HIV-1 vaccine. We have determined a crystal structure of V1V2 in complex with human MAb 830A and have shown that MAb 830A recognizes a region overlapping the α4β7 integrin-binding site. We also showed that V1V2 forms a 5-stranded beta barrel, an elegant structure allowing sequence variations in the strand-connecting loops while preserving a conserved core., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The widespread use of highly effective, combination antiretroviral therapy (cART) has led to a significant reduction in the incidence of HIV-associated dementia (HAD). Despite these advances, the prevalence of HIV-1 associated neurocognitive disorders (HANDs) has been estimated at approximately 40%-50%. In the cART era, the majority of this disease burden is represented by asymptomatic neurocognitive impairment and mild neurocognitive disorder (ANI and MND respectively). Although less severe than HAD, these diagnoses carry with them substantial morbidity., Results: In this cross-sectional study, single genome amplification (SGA) was used to sequence 717 full-length HIV-1 envelope (env) clade B variants from the paired cerebrospinal fluid (CSF) and blood plasma samples of fifteen chronically infected HIV-positive individuals with normal neurocognitive performance (NCN), ANI and MND. Various degrees of compartmentalization were found across disease states and history of cART utilization. In individuals with compartmentalized virus, mean HIV-1 env population diversity was lower in the CSF than plasma-derived variants. Overall, mean V1V2 loop length was shorter in CSF-derived quasispecies when compared to contemporaneous plasma populations, and this was found to correlate with a lower mean number of N-linked glycosylation sites in this region. A number of discrete amino acid positions that correlate strongly with compartmentalization in the CSF were identified in both variable and constant regions of gp120 as well as in gp41. Correlated mutation analyses further identified that a subset of amino acid residues in these compartmentalization "hot spot" positions were strongly correlated with one another, suggesting they may play an important, definable role in the adaptation of viral variants to the CSF. Analysis of these hot spots in the context of a well-supported crystal structure of HIV-1 gp120 suggests mechanisms through which amino acid differences at the identified residues might contribute to viral compartmentalization in the CSF., Conclusions: The detailed analyses of SGA-derived full length HIV-1 env from subjects with both normal neurocognitive performance and the most common HAND diagnoses in the cART era allow us to identify novel and confirm previously described HIV-1 env genetic determinants of neuroadaptation and relate potential motifs to HIV-1 env structure and function.

Unlabelled: Data from the RV144 HIV vaccine trial indicated that gp120 V2 antibodies were associated with a lower risk of infection; thus, the mapping of V2 epitopes can contribute to the design of an effective HIV vaccine. We solved the crystal structure of human monoclonal antibody (MAb) 2158, which targets a conformational V2 epitope overlapping the α4β7 integrin binding site, and constructed a full-length model of V1V2. Comparison of computational energy stability to experimental enzyme-linked immunosorbent assay (ELISA) results identified a hydrophobic core that stabilizes the V2 region for optimal 2158 binding, as well as residues that directly mediate side chain interactions with MAb 2158. These data define the binding surface recognized by MAb 2158 and offer a structural explanation for why a mismatched mutation at position 181 (I181X) in the V2 loop was associated with a higher vaccine efficiency in the RV144 clinical vaccine trial., Importance: Correlate analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the second variable region (V2) of HIV-1 gp120 was responsible for the modest protection observed in the trial. V2 is a highly variable and immunogenic region, and structural information on its antigenic landscape will be important for rational design of an effective HIV-1 vaccine. Using X-ray crystallography, computational design tools, and mutagenesis assays, we carried out a detailed and systematic investigation of the epitope recognition of human V2 MAb 2158 and demonstrated that its epitope region overlaps the integrin binding site within V2. In addition, we propose a structure-based mechanism for mismatching of the isoleucine at position 181 and the increased vaccine efficacy seen in the RV144 vaccine trial.

Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.

The recent analysis of the first successful RV144 vaccine trial revealed that a high titer of plasma anti-V2 antibodies (Abs) correlated with a decreased risk of HIV-1 infection in vaccine recipients. To understand the mechanism of immune correlates, we studied seven anti-V2 monoclonal Abs (mAbs) developed from HIV-1 infected individuals. The V2 mAbs target conserved epitopes, including the binding site for α4β7 integrin, and are broadly cross-reactive with various gp120 proteins. Preferential usage of the VH1-69 gene by V2 mAbs may depend on selection by the same antigenic structure. Six of seven V2 mAbs weakly neutralized four to eight of the 41 pseudoviruses tested and resistance to neutralization was correlated with longer V2 domains. The data suggest the presence of shared, conserved structural elements in the V2 loop, and these can be used in the design of vaccine immunogens inducing broadly reactive Abs with anti-viral activities., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 Å in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)