Faculty Bibliography

Simian-human immunodeficiency virus (SHIV) infection provides a relevant animal model to study HIV-1 neutralization breadth. With previously identified SHIV_SF162P3N infected rhesus macaques that did or did not develop neutralization breadth, we characterized the transmitted/founder viruses and initial autologous/homologous neutralizing antibodies in these animals. The plasma viral load and blood CD4 count did not distinguish macaques with and without breadth, and only one tested homologous envelope clone revealed a trend for macaques with breadth to favor an early homologous response. In two macaques with breadth, GB40 and FF69, infected with uncloned SHIV_SF162P3N, multiple viral variants were transmitted, and the transmitted variants were not equal in neutralization sensitivity. The targets of initial autologous neutralizing antibodies, arising between 10 and 20 weeks post infection, were mapped to N462 glycan and G460a in gp120 V5 in GB40 and FF69, respectively. Although it is unclear whether these targets are related to later neutralization breadth development, the G460a target but not N462 glycan appeared more common in macaques with breadth than those without. Longitudinal plasmas revealed 2⁻3 sequential waves of neutralizing antibodies in macaques with breadth, implicating that 3 sequential envelope variants, if not more, may be required for the broadening of HIV-1 neutralizing antibodies.

Tau antibodies have shown therapeutic potential for Alzheimer's disease and several are in clinical trials. As a microtubule-associated protein, tau relies on dynamic phosphorylation for its normal functions. In tauopathies, it becomes hyperphosphorylated and aggregates into toxic assemblies, which collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser396 has received particular attention because of its prominence and stability in tauopathies. Here we report the first structure of a monoclonal tau antibody in complex with the pathologically important phospho-Ser396 residue. Its binding region reveals tau residues Tyr394 to phospho-Ser396 stabilized in a β-strand conformation that is coordinated by a phospho-specific antigen binding site. These details highlight a molecular switch that defines this prominent conformation of tau and ways to target it. Overall, the structure of the antibody-antigen complex clarifies why certain phosphorylation sites in tau are more closely linked to neurodegeneration than others.

The 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 (303TRKSIHIGPGRAF317), using the cradle binding mode similar to human V3 mAbs encoded by IGHV5-51 germline 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 (308HIGPGRAFYTTGEI323). Unexpectedly, the 315RAFYTT320 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 from human antibody germline genes, rabbits can serve as a useful animal model to evaluate human vaccine candidates.IMPORTANCE The apex crown of the third variable loop (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 targeting 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 rabbits 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 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.

Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 β-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.

Background: 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 in different stages of maturation can help to understand the maturation process.
Method(s): 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.
Result(s): The somatic hypermutation (SHM) rates in VH of the vaccine-induced mAbs are lower than that of the 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 the V3 beta hairpin lies along the antigen-binding groove, which consists of residues of both heavy and light chains. Residues conserved from the germline 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.
Conclusion(s): Our data provide a unique example of germline sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection

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.

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.