Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

The third variable (V3) loop on the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein trimer is indispensable for virus cell entry. Conformational masking of V3 within the trimer allows efficient neutralization via V3 only by rare, broadly neutralizing glycan-dependent antibodies targeting the closed prefusion trimer but not by abundant antibodies that access the V3 crown on open trimers after CD4 attachment. Here, we report on a distinct category of V3-specific inhibitors based on designed ankyrin repeat protein (DARPin) technology that reinstitute the CD4-bound state as a key neutralization target with up to >90% breadth. Broadly neutralizing DARPins (bnDs) bound V3 solely on open envelope and recognized a four-turn amphipathic α-helix in the carboxy-terminal half of V3 (amino acids 314-324), which we termed 'αV3C'. The bnD contact surface on αV3C was as conserved as the CD4 binding site. Molecular dynamics and escape mutation analyses underscored the functional relevance of αV3C, highlighting the potential of αV3C-based inhibitors and, more generally, of postattachment inhibition of HIV-1

Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new V_H1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC₅₀ = 0.048 μg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505_(SOSIP.664) Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection

HIV-1 superinfection results in broad polyclonal neutralizing antibodies

<div><p>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.</p></div

Bacterial vaginosis and adverse outcomes among full-term infants: a cohort study

Background: Bacterial vaginosis (BV) during pregnancy is a well-established risk factor for preterm birth and other preterm pregnancy complications. Little is known about adverse neonatal outcomes associated with BV exposure in full-term births, nor its influence on adverse outcomes independent of its effect on gestational age. The purpose of this study was to examine the relationship between BV during pregnancy and adverse neonatal outcomes among full-term and preterm infants. Methods: We conducted a retrospective cohort study of Washington State mother/infant pairs from 2003-2013, stratified by full-term (primary outcomes) and preterm births (secondary outcomes). BV-exposed and unexposed women were frequency-matched based on year of delivery. BV exposure and adverse outcomes [assisted ventilation/respiratory distress, neonatal intensive care unit (NICU) admission, neonatal sepsis, fetal mortality, and infant mortality] were identified using birth certificates, ICD-9 codes from linked hospital records, and death certificates. Associations between BV exposure and outcomes were assessed using multivariable Poisson regression, adjusted for maternal demographics, gestational age, and other pregnancy complications, including infections. Results: A total of 12,340 mother/infant pairs were included: 2,468 BV-exposed (2198 term, 267 preterm) and 9,872 BV unexposed (9156 term, 708 preterm). Among full-term infants, BV-exposed mothers were younger, more likely to be Black or Hispanic, more likely to have had a sexually transmitted infection, and less likely to have a college degree than unexposed mothers. Term BV exposed infants were more likely to have meconium at delivery. Following adjustment, BV was associated with an increased risk of assisted ventilation/respiratory distress at birth (aRR = 1.28, 95 % CI 1.02-1.61), NICU admission (aRR = 1.42, 95 % CI 1.11-1.82), and neonatal sepsis (aRR = 1.60, 95 % CI 1.13-2.27) among full-term infants. These associations were independent of the presence of chorioamnionitis or meconium. Among preterm infants, BV-exposure was associated with an increased risk for NICU admissions only (aRR = 1.24, 95 % CI 1.04-1.46). Conclusions: BV exposure during pregnancy is associated with adverse neonatal outcomes even among infants born full-term. These findings amongst full-term infants are novel, and highlight neonatal implications of BV in pregnancy independent of BV’s effect on preterm birth

Massively Parallel Profiling of HIV-1 Resistance to the Fusion Inhibitor Enfuvirtide

Identifying drug resistance mutations is important for the clinical use of antivirals and can help define both a drug&#8217;s mechanism of action and the mechanistic basis of resistance. Resistance mutations are often identified one-at-a-time by studying viral evolution within treated patients or during viral growth in the presence of a drug in cell culture. Such approaches have previously mapped resistance to enfuvirtide, the only clinically approved HIV-1 fusion inhibitor, to enfuvirtide&#8217;s binding site in the N-terminal heptad repeat (NHR) of the Envelope (Env) transmembrane domain as well as a limited number of allosteric sites. Here, we sought to better delineate the genotypic determinants of resistance throughout Env. We used deep mutational scanning to quantify the effect of all single-amino-acid mutations to the subtype A BG505 Env on resistance to enfuvirtide. We identified both previously characterized and numerous novel resistance mutations in the NHR. Additional resistance mutations clustered in other regions of Env conformational intermediates, suggesting they may act during different fusion steps by altering fusion kinetics and/or exposure of the enfuvirtide binding site. This complete map of resistance sheds light on the diverse mechanisms of enfuvirtide resistance and highlights the utility of using deep mutational scanning to comprehensively map potential drug resistance mutations

The amino-acid preferences are modestly correlated among experimental replicates, but the sites tolerate similar numbers of amino acids and prefer similar amino acids across replicates.

<p><b>(A)</b> Correlations between the site-specific amino-acid preferences from each replicate. <b>(B)</b> Correlations between the effective number of amino acids tolerated per site. For each site <i>r</i>, the effective number of tolerated amino acids is , where <i>H</i>_<i>r</i> is the Shannon entropy of that site’s amino-acid preferences. This number ranges between 1 and 20, with 20 indicating all amino acids are preferred equally and 1 indicating only a single amino acid is preferred. <b>(C)</b> Correlations between the preference-weighted hydrophobicities. For each site <i>r</i>, the preference-weighted hydrophobicity is ∑_<i>a</i> <i>π</i>_{<i>r</i>,<i>a</i>} × <i>X</i>_<i>a</i> where <i>π</i>_{<i>r</i>,<i>a</i>} is the preference of <i>r</i> for amino acid <i>a</i>, and <i>X</i>_<i>a</i> is the Kyte-Doolittle hydropathy [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.ref090" target="_blank">90</a>] of <i>a</i>. The fact that both the effective number of tolerated amino acids and the hydrophobicities are more correlated than the amino-acid preferences means that when different amino acids are preferred at a site in different experimental replicates, the number and chemical properties of the preferred amino acids are similar. Each plot shows the Pearson correlation coefficient and associated P-value. Similar data for replicates 3b-1 and 3b-2 are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.s009" target="_blank">S6 Fig</a>. The plots in this and subsequent figures show all 20 amino-acid preferences for each site; although only 19 of these preferences are independent parameters, all 20 values are shown because otherwise the correlation will depend on which value is excluded.</p

The correlation between the experimentally measured preferences and amino-acid frequencies in natural sequences is low at glycosylation sites, but high at disulfide-bonded cysteines.

<p><b>(A)</b> The logo plots show the frequencies of amino acids in the group-M alignment or the amino-acid preferences from our experiments at a subset of potential N-linked glycosylation sites (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.s010" target="_blank">S7 Fig</a> for all 30 sites). The glycosylation sites are conserved in nature, but tolerant of mutations in our experiment. The scatter plot shows that there is a poor correlation between the preferences and natural amino-acid frequencies at all 22 alignable glycosylation sites: red triangles represent the first position in each glycosylation site, whereas gray circles represent all other sites. <b>(B)</b> There is much better concordance between the preferences and natural amino-acid frequencies for Env’s disulfide-bonded cysteines. The logo plots show each pair of cysteines for a subset of disulfides (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.s010" target="_blank">S7 Fig</a> for all 10 disulfides). The scatter plot shows that there is a strong correlation between the preferences and natural amino-acid frequencies at all disulfide-bonded cysteines.</p

Data from: 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

Selection purged mutations in most of <i>env</i>, but favored mutations at a few sites.

<p><b>(A)</b> For each replicate, we deep sequenced the initial plasmids (DNA) and the viruses after two rounds of passaging (P2). Bars show the per-codon mutation frequency averaged across sites after subtracting error rates determined from the wildtype controls (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.s005" target="_blank">S2 Fig</a>). When mutation frequencies are averaged across all sites, selection purged stop codons to <1% of their frequency in the initial DNA. Selection only slightly reduced the average frequency of nonsynonymous mutations; however, this average results from two distinct trends. For ≈4% of sites, the frequency of nonsynonymous mutations in the twice-passaged viruses (<i>f</i>^<i>P</i>2) increased >3-fold relative to the frequency in the initial plasmid DNA (<i>f</i>^<i>DNA</i>). For all other sites, the frequency of nonsynonymous mutations decreased substantially after selection. <b>(B)</b> The sites at which the error-corrected mutation frequency increased >3-fold are similar between replicates, indicating consistent selection for tissue-culture adaptation at a few positions. The left Venn diagram shows the overlap among replicates in the sites with a >3-fold increase. The right Venn diagram shows the expected overlap if the same number of sites per replicate are randomly drawn from Env’s primary sequence. This difference is statistically significant, with <i>P</i> < 10⁻⁴ when comparing the actual overlap among all three replicates to the random expectation. Another summary view of selection on <i>env</i> is provided by <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.s007" target="_blank">S4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.s008" target="_blank">S5</a> Figs.</p