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Unlabelled: The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55-62, 2014, http://dx.doi.org/10.1038/nature13036). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 μg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent., Importance: Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 μg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.

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.

Serum characterization and antibody isolation are transforming our understanding of the humoral immune response to viral infection. Here, we show that epitope specificities of HIV-1-neutralizing antibodies in serum can be elucidated from the serum pattern of neutralization against a diverse panel of HIV-1 isolates. We determined "neutralization fingerprints" for 30 neutralizing antibodies on a panel of 34 diverse HIV-1 strains and showed that similarity in neutralization fingerprint correlated with similarity in epitope. We used these fingerprints to delineate specificities of polyclonal sera from 24 HIV-1-infected donors and a chimeric siman-human immunodeficiency virus-infected macaque. Delineated specificities matched published specificities and were further confirmed by antibody isolation for two sera. Patterns of virus-isolate neutralization can thus afford a detailed epitope-specific understanding of neutralizing-antibody responses to viral infection.

Antibody PG9 is a prototypical member of a class of V1/V2-directed antibodies that effectively neutralizes diverse strains of HIV-1. We analyzed strain-specific resistance to PG9 using sequence and structural information. For multiply resistant strains, mutations in a short segment of V1/V2 resulted in gain of sensitivity to PG9 and related V1/V2 neutralizing antibodies, suggesting both a common mechanism of HIV-1 resistance to and a common mode of recognition by this class of antibodies.