؉ T cell decline, viral load, and CD4 ؉ T cell count at 6 months postinfection but not at later time points, suggesting that early events set the stage for the development of breadth. However, in a multivariate analysis, CD4 decline was the major driver of this association, as viral load was not an independent predictor of breadth. Mapping of the epitopes targeted by cross-neutralizing antibodies revealed that in one individual these antibodies recognized the membrane-proximal external region (MPER), while in two other individuals, cross-neutralizing activity was adsorbed by monomeric gp120 and targeted epitopes that involved the N-linked glycan at position 332 in the C3 region. Serum antibodies from the other four participants targeted quaternary epitopes, at least 2 of which were PG9/16-like and depended on the N160 and/or L165 residue in the V2 region. These data indicate that fewer than 20% of HIV-1 subtype C-infected individuals develop antibodies with cross-neutralizing activity after 3 years of infection and that these antibodies target different regions of the HIV-1 envelope, including as yet uncharacterized epitopes.

The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000. CAP256 showed a subtype bias, preferentially neutralizing subtype C and A viruses over subtype B viruses. CAP256 BCN serum targeted a quaternary epitope which included the V1V2 region. Further mapping identified residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRD-K-K motif) in the V2 region as crucial to the CAP256 epitope. However, the fine specificity of the BCN response varied over time and, while consistently dependent on R166 and K169, became gradually less dependent on D167 and K171, possibly contributing to the incremental increase in breadth over 4 years. The presence of an intact FN/LRD-K-K motif in heterologous viruses was associated with sensitivity, although the length of the adjacent V1 loop modulated the degree of sensitivity, with a shorter V1 region significantly associated with higher titers.

We identified three cross-neutralizing plasma samples with high-titer anti-membrane proximal external region (MPER) peptide binding antibodies from among 156 chronically human immunodeficiency virus type 1-infected individuals. In order to establish if these antibodies were directly responsible for the observed neutralization breadth, we used MPER-coated magnetic beads to deplete plasmas of these specific antibodies. Depletion of anti-MPER antibodies from BB34, CAP206, and SAC21 resulted in 77%, 68%, and 46% decreases, respectively, in the number of viruses neutralized. Antibodies eluted from the beads showed neutralization profiles similar to those of the original plasmas, with potencies comparable to those of the known anti-MPER monoclonal antibodies (MAbs), 4E10, 2F5, and Z13e1. The anti-MPER neutralizing antibodies in BB34 were present in the immunoglobulin G3 subclass-enriched fraction. Alanine scanning of the MPER showed that the antibodies from these three plasmas had specificities distinct from those of the known MAbs, requiring one to three crucial residues at positions 670, 673, and 674. These data demonstrate the existence of MPER-specific cross-neutralizing antibodies in plasma, although the ability to elicit such potent antiviral antibodies during natural infection appears to be rare. Nevertheless, the identification of three novel antibody specificities within the MPER supports its further study as a promising target for vaccine design.

dEntry of human immunodeficiency virus type 1 (HIV-1) into cells is mediated by the virion surface envelope (Env) glycoproteins, making it a desirable target for antiretroviral entry inhibitors. We previously isolated a family of gp120 binding RNA aptamers and showed that they neutralized the infectivity of HIV-1. In this study, we assessed the activity of a shortened synthetic derivative of the B40 aptamer, called UCLA1, against a large panel of HIV-1 subtype C viruses. UCLA1 tightly bound to a consensus HIV-1 subtype C gp120 and neutralized isolates of the same subtype with 50% inhibitory concentrations (IC 50 s) in the nanomolar range. The aptamer had little toxicity in tests with cell lines and primary cells. Furthermore, it exhibited high therapeutic indices, suggesting that it may be effective at very low doses. Mapping of UCLA1 binding sites on gp120 revealed eight amino acid residues that modulated neutralization resistance. This included residues within the coreceptor binding site, at the base of the V3 loop, and in the bridging sheet within the conserved V1/V2 stem-loop of gp120. The aptamer was also shown to have synergistic effects with T20, a gp41 fusion inhibitor, and IgG1b12 (b12), an anti-CD4 binding site monoclonal antibody. These results suggest that UCLA1 may be suitable for development as a potent HIV-1 entry inhibitor.

BackgroundIdentification of the epitopes targeted by antibodies that can neutralize diverse HIV-1 strains can provide important clues for the design of a preventative vaccine.MethodsWe have developed a computational approach that can identify key amino acids within the HIV-1 envelope glycoprotein that influence sensitivity to broadly cross-neutralizing antibodies. Given a sequence alignment and neutralization titers for a panel of viruses, the method works by fitting a phylogenetic model that allows the amino acid frequencies at each site to depend on neutralization sensitivities. Sites at which viral evolution influences neutralization sensitivity were identified using Bayes factors (BFs) to compare the fit of this model to that of a null model in which sequences evolved independently of antibody sensitivity. Conformational epitopes were identified with a Metropolis algorithm that searched for a cluster of sites with large Bayes factors on the tertiary structure of the viral envelope.ResultsWe applied our method to ID50 neutralization data generated from seven HIV-1 subtype C serum samples with neutralization breadth that had been tested against a multi-clade panel of 225 pseudoviruses for which envelope sequences were also available. For each sample, between two and four sites were identified that were strongly associated with neutralization sensitivity (2ln(BF) > 6), a subset of which were experimentally confirmed using site-directed mutagenesis.ConclusionsOur results provide strong support for the use of evolutionary models applied to cross-sectional viral neutralization data to identify the epitopes of serum antibodies that confer neutralization breadth.