Adam S. Dingens

The Broad Neutralizing Antibody Responses after HIV-1 Superinfection Are Not Dominated by Antibodies Directed to Epitopes Common in Single Infection.

HIV-1 vaccines designed to date have failed to elicit neutralizing antibodies (Nabs) that are capable of protecting against globally diverse HIV-1 subtypes. One relevant setting to study the development of a strong, cross-reactive Nab response is HIV-1 superinfection (SI), defined as sequential infections from different source partners. SI has previously been shown to lead to a broader and more potent Nab response when compared to single infection, but it is unclear whether SI also impacts epitope specificity and if the epitopes targeted after SI differ from those targeted after single infection. Here the post-SI Nab responses were examined from 21 Kenyan women collectively exposed to subtypes A, C, and D and superinfected after a median time of ~1.07 years following initial infection. Plasma samples chosen for analysis were collected at a median time point ~2.72 years post-SI. Because previous studies of singly infected populations with broad and potent Nab responses have shown that the majority of their neutralizing activity can be mapped to 4 main epitopes on the HIV-1 Envelope, we focused on these targets, which include the CD4-binding site, a V1/V2 glycan, the N332 supersite in V3, and the membrane proximal external region of gp41. Using standard epitope mapping techniques that were applied to the previous cohorts, the present study demonstrates that SI did not induce a dominant Nab response to any one of these epitopes in the 21 women. Computational sera delineation analyses also suggested that 20 of the 21 superinfected women’s Nab responses could not be ascribed a single specificity with high confidence. These data are consistent with a model in which SI with diverse subtypes promotes the development of a broad polyclonal Nab response, and thus would provide support for vaccine designs using multivalent HIV immunogens to elicit a diverse repertoire of Nabs.

HIV-specific CD4-induced Antibodies Mediate Broad and Potent Antibody-dependent Cellular Cytotoxicity Activity and are Commonly Detected in Plasma from HIV-infected Humans

HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specific memory B cells encoding Abs that mediate ADCC from a subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activity when compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donors ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise from unique B-cell lineages in the same individual. Further, these mAbs mediate potent plasma IgG-specific ADCC breadth and potency and contribute to ADCC activity in other HIV-infected individuals.

Experimental Estimation of the Effects of All Amino-Acid Mutations to HIV's Envelope Protein on Viral Replication in Cell Culture.

HIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV’s most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV’s replication in cell culture. We compare our experimental measurements of each site’s preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. Our data enable us to quantify the inherent mutational tolerance of each site in Env. We show that the epitopes of broadly neutralizing antibodies have a significantly reduced inherent capacity to tolerate mutations, rigorously validating a pervasive idea in the field. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env’s evolution.

Cooperation between distinct viral variants promotes growth of H3N2 influenza in cell culture

RNA viruses rapidly diversify into quasispecies of related genotypes. This genetic diversity has long been known to facilitate adaptation, but recent studies have suggested that cooperation between variants might also increase population fitness. Here, we demonstrate strong cooperation between two H3N2 influenza variants that differ by a single mutation at residue 151 in neuraminidase, which normally mediates viral exit from host cells. Residue 151 is often annotated as an ambiguous amino acid in sequenced isolates, indicating mixed viral populations. We show that mixed populations grow better than either variant alone in cell culture. Pure populations of either variant generate the other through mutation and then stably maintain a mix of the two genotypes. We suggest that cooperation arises because mixed populations combine one variant’s proficiency at cell entry with the other’s proficiency at cell exit. Our work demonstrates a specific cooperative interaction between defined variants in a viral quasispecies. DOI: http://dx.doi.org/10.7554/eLife.13974.001

Comprehensive Characterization of Humoral Correlates of Human Immunodeficiency Virus 1 Superinfection Acquisition in High-risk Kenyan Women

HIV-1 superinfection, in which an infected individual acquires a second HIV-1 infection from a different partner, is one of the only settings in which HIV acquisition occurs in the context of a pre-existing immune response to natural HIV infection. There is evidence that initial infection provides some protection from superinfection, particularly after 6 months of initial infection, when development of broad immunity occurs. Comparison of the immune response of superinfected individuals at the time of superinfection acquisition to that of individuals who remain singly infected despite continued exposure can shed light on immune correlates of HIV acquisition to inform prophylactic vaccine design. We evaluated a panel of humoral immune responses in the largest published group of superinfected individuals (n = 21), compared to a set of 3:1 matched singly infected controls from the same cohort. The immune functions studied included plasma neutralization, plasma and cervical antibody-dependent cellular cytotoxicity, and plasma IgG and IgA binding to a panel of 18 envelope antigens, including correlates of HIV acquisition in the RV144 vaccine trial, IgG binding to V1V2 and IgA binding to gp140. Association between each immune function and HIV superinfection was evaluated using conditional logistic regression. No significant associations were detected between any of the immune functions and superinfection acquisition. This study constitutes the most comprehensive and detailed characterization of multiple immune correlates of superinfection to date. The results suggest that immune responses not commonly measured in current HIV studies may be important in protection from HIV infection, and these or a more robust humoral response than that seen in naturally infected women may be needed for a protective vaccine.

Mapping mutational effects along the evolutionary landscape of HIV envelope

The immediate evolutionary space accessible to HIV is largely determined by how single amino acid mutations affect fitness. These mutational effects can shift as the virus evolves. However, the prevalence of such shifts in mutational effects remains unclear. Here, we quantify the effects on viral growth of all amino acid mutations to two HIV envelope (Env) proteins that differ at >100 residues. Most mutations similarly affect both Envs, but the amino acid preferences of a minority of sites have clearly shifted. These shifted sites usually prefer a specific amino acid in one Env, but tolerate many amino acids in the other. Surprisingly, shifts are only slightly enriched at sites that have substituted between the Envs—and many occur at residues that do not even contact substitutions. Therefore, long-range epistasis can unpredictably shift Env’s mutational tolerance during HIV evolution, although the amino acid preferences of most sites are conserved between moderately diverged viral strains.

Superinfection Drives HIV Neutralizing Antibody Responses from Several B Cell Lineages that Contribute to a Polyclonal Repertoire

SUMMARY Eliciting broad and potent HIV-specific neutralizing antibody responses represents the holy grail of HIV vaccine efforts. Data from singly infected individuals with broad and potent plasma neutralizing activity targeting one epitope have guided our understanding of how these responses develop. However, far less is known about responses developed by super-infected individuals who acquire two distinct HIV strains. Here, we isolated HIV-specific mAbs from a superinfected individual with a broad plasma response. In this superinfection case, neutralizing activity resulted from multiple distinct B cell lineages that arose in response to either the initial or the superinfecting virus, including an antibody that targets the N332 supersite. This nAb, QA013.2, was specific to the superinfecting virus and was associated with eventual reemergence of the initial infecting virus. The complex dynamic between viruses in superinfection may drive development of a unique collection of polyclonal nAbs that present a higher barrier to escape than monoclonal responses.

An antigenic atlas of HIV-1 escape from broadly neutralizing antibodies

Anti-HIV broadly neutralizing antibodies (bnAbs) have revealed vaccine targets on the virus’s Env protein and are themselves promising immunotherapeutics. The efficacy of bnAb-based therapies and vaccines depends in part on how readily the virus can escape neutralization. While structural studies can define contacts between bnAbs and Env, only functional studies can define mutations that confer escape. Here we map how all single amino-acid mutations to Env affect neutralization of HIV by nine bnAbs targeting five epitopes. For most bnAbs, mutations at only a small fraction of structurally defined contact sites mediated escape, and most escape occurred at sites that are near but do not directly contact the antibody. The mutations selected by two pooled bnAbs were similar to those expected from the combination of the bnAbs’ independent action. Overall, our mutation-level antigenic atlas provides a comprehensive dataset for understanding viral immune escape and refining therapies and vaccines.

Experimental estimation of the effects of all amino-acid mutations to HIV Env

HIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV’s most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV’s growth in cell culture. We compare our experimental measurements of each site’s preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. We show that some regions of Env have a high inherent tolerance to mutation, whereas other regions (such as epitopes of broadly neutralizing antibodies) have a significantly reduced capacity to tolerate mutations. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env’s evolution.