Krissey E. Lloyd

Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus

Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

Structural basis for diverse N-glycan recognition by HIV-1–neutralizing V1–V2–directed antibody PG16

HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

Enhanced Potency of a Broadly Neutralizing HIV-1 Antibody In Vitro Improves Protection against Lentiviral Infection In Vivo

ABSTRACT Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope protein was used to engineer a next-generation antibody with 5- to 8-fold increased potency in vitro. When administered to nonhuman primates, this antibody conferred protection at a 5-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate the therapeutic potential of antibodies and their in vivo effectiveness.

Mining the antibodyome for HIV-1–neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains

Next-generation sequencing of antibody transcripts from HIV-1–infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy- and light-chain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy- and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141–145. Heavy- and light-chain phylogenetic trees of PGT141–145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141–145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings.

Diversion of HIV-1 vaccine–induced immunity by gp41-microbiota cross-reactive antibodies

Microbiota can mislead antibodies Unlike the response to many viral infections, most people do not produce antibodies capable of clearing HIV-1. Non-neutralizing antibodies that target HIV-1s envelope glycoprotein (Env) typically dominate the response, which is generated by B cells that cross-react with Env and the intestinal microbiota. Williams et al. analyzed samples from individuals who had received a vaccine containing the Env protein, including the gp41 subunit. Most of the antibodies were non-neutralizing and targeted gp41. The antibodies also reacted to intestinal microbiota, suggesting that preexisting immunity to microbial communities skews vaccineinduced immune responses toward an unproductive target. Science, this issue 10.1126/science.aab1253. The antibody response to an HIV-1 vaccine is dominated by preexisting immunity to microbiota. INTRODUCTION Inducing protective antibodies is a key goal in HIV-1 vaccine development. In acute HIV-1 infection, the dominant initial plasma antibody response is to the gp41 subunit of the envelope (Env) glycoprotein of the virus. These antibodies derive from polyreactive B cells that cross-react with Env and intestinal microbiota (IM) and are unable to neutralize HIV-1. However, whether a similar gp41-IM cross-reactive antibody response would occur in the setting of HIV-1 Env vaccination is unknown. RATIONALE We studied antibody responses in individuals who received a DNA prime vaccine, with a recombinant adenovirus serotype 5 (rAd5) boost (DNA prime–rAd5 boost), a vaccine that included HIV-1 gag, pol, and nef genes, as well as a trivalent mixture of clade A, B, and C env gp140 genes containing both gp120 and gp41 components. This vaccine showed no efficacy. Thus, study of these vaccinees provided an opportunity to determine whether the Env-reactive antibody response in the setting of Env vaccination was dominated by gp41-reactive antibodies derived from Env-IM cross-reactive B cells. RESULTS We found that vaccine-induced antibodies to HIV-1 Env dominantly focused on gp41 compared with gp120 by both serologic analysis and by vaccine-Env memory B cells sorted by flow cytometry (see the figure). Remarkably, the majority of HIV-1 Env-reactive memory B cells induced by the vaccine produced gp41-reactive antibodies, and the majority of gp41-targeted antibodies used restricted immunoglobulin heavy chain variable genes. Functionally, none of the gp41-reactive antibodies could neutralize HIV, and the majority could not mediate antibody-dependent cellular cytotoxicity. Most of the vaccine-induced gp41-reactive antibodies cross-reacted with host and IM antigens. Two of the candidate gp41-intestinal cross-reactive antigens were bacterial RNA polymerase and pyruvate-flavodoxin oxidoreductase, which shared sequence similarities with the heptad repeat 1 region of HIV gp41. Next-generation sequencing of vaccinee B cells demonstrated a prevaccination antibody that was reactive to both IM and the vaccine–Env gp140, which demonstrated the presence of a preexisting pool of gp41-IM cross-reactive B cells from which the vaccine gp41-reactive antibody response was derived. CONCLUSION In this study, we found that the DNA prime–rAd5 boost HIV-1 vaccine induced a gp41-reactive antibody response that was mainly non-neutralizing and derived from an IM-gp41 cross-reactive B cell pool. These findings have important implications for HIV-1 vaccine design. Because IM antigens shape the B cell repertoire from birth, our data raise the hypothesis that neonatal immunization with HIV-1 envelope may be able to imprint the B cell repertoire to respond to envelope antigenic sites that may otherwise be subdominant or disfavored, such as Env broadly neutralizing antibody epitopes. Our data also suggest that deleting or modifying amino acids in the gp41 heptad repeat 1 region of Env-containing vaccine immunogens may avoid IM-gp41 cross-reactivity. Thus, an obstacle that may need to be overcome for development of a successful HIV vaccine is diversion of potentially protective HIV-1 antibody responses by preexisting envelope-IM cross-reactive pools of B cells. Diversion of HIV-1 vaccine–induced immunity by Env gp41–microbiota cross-reactive antibodies. Immunization of humans with a vaccine containing HIV-1 Env gp120 and gp41 components, including the membrane-proximal external region (MPER) of Env, induced a dominant B cell response primarily from a preexisting pool of gp41-IM cross-reactive B cells. This response diverted the vaccine-stimulated antibody response away from smaller subdominant B cell pools capable of reacting with potentially protective epitopes on HIV-1 Env. An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1–reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1–reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy.

Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion

ABSTRACT An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.

HIV-1 Envelope gp41 Antibodies Can Originate from Terminal Ileum B Cells that Share Cross-Reactivity with Commensal Bacteria

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

Toll-Like Receptor 7/8 (TLR7/8) and TLR9 Agonists Cooperate To Enhance HIV-1 Envelope Antibody Responses in Rhesus Macaques

ABSTRACT The development of a vaccine that can induce high titers of functional antibodies against HIV-1 remains a high priority. We have developed an adjuvant based on an oil-in-water emulsion that incorporates Toll-like receptor (TLR) ligands to test whether triggering multiple pathogen-associated molecular pattern receptors could enhance immunogenicity. Compared to single TLR agonists or other pairwise combinations, TLR7/8 and TLR9 agonists combined were able to elicit the highest titers of binding, neutralizing, and antibody-dependent cellular cytotoxicity-mediating antibodies against the protein immunogen, transmitted/founder HIV-1 envelope gp140 (B.63521). We further found that the combination of TLR7/8 and TLR9 agonists was associated with the release of CXCL10 (IP-10), suggesting that this adjuvant formulation may have optimally stimulated innate and adaptive immunity to elicit high titers of antibodies. IMPORTANCE Combining TLR agonists in an adjuvant formulation resulted in higher antibody levels compared to an adjuvant without TLR agonists. Adjuvants that combine TLR agonists may be useful for enhancing antibody responses to HIV-1 vaccines.

Immune perturbations in HIV-1–infected individuals who make broadly neutralizing antibodies

Individuals infected with HIV-1 who produce broadly neutralizing antibodies have a distinct immunological landscape. Setting the stage for HIV vaccines Some HIV-infected individuals produce antibodies that can target multiple HIV strains—broadly neutralizing antibodies. Moody et al. now compare HIV-infected individuals who produce these antibodies with those who do not. They find that broadly neutralizing antibody production associates with particular immune traits, including a higher frequency of autoantibodies, fewer regulatory T cells, and more circulating memory T follicular helper cells. Vaccine protocols that can mimic these immune perturbations may therefore promote induction of broadly neutralizing antibodies and lead to a more successful immune response to HIV. Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. bnAbs occur in some HIV-1–infected individuals and frequently have characteristics of autoantibodies. We have studied cohorts of HIV-1–infected individuals who made bnAbs and compared them with those who did not do so, and determined immune traits associated with the ability to produce bnAbs. HIV-1–infected individuals with bnAbs had a higher frequency of blood autoantibodies, a lower frequency of regulatory CD4+ T cells, a higher frequency of circulating memory T follicular helper CD4+ cells, and a higher T regulatory cell level of programmed cell death–1 expression compared with HIV-1–infected individuals without bnAbs. Thus, induction of HIV-1 bnAbs may require vaccination regimens that transiently mimic immunologic perturbations in HIV-1–infected individuals.

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses.

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.