A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis

The structural flexibility or 'breathing' of the envelope (E) protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F) of the West Nile virus (WNV) E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV), but not Zika virus (ZIKV), E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing

Whole-Exome Sequencing Identifies Rare and Low-Frequency Coding Variants Associated with LDL Cholesterol

Elevated low-density lipoprotein cholesterol (LDL-C) is a treatable, heritable risk factor for cardiovascular disease. Genome-wide association studies (GWASs) have identified 157 variants associated with lipid levels but are not well suited to assess the impact of rare and low-frequency variants. To determine whether rare or low-frequency coding variants are associated with LDL-C, we exome sequenced 2,005 individuals, including 554 individuals selected for extreme LDL-C (>98th or <2nd percentile). Follow-up analyses included sequencing of 1,302 additional individuals and genotype-based analysis of 52,221 individuals. We observed significant evidence of association between LDL-C and the burden of rare or low-frequency variants in PNPLA5, encoding a phospholipase-domain-containing protein, and both known and previously unidentified variants in PCSK9, LDLR and APOB, three known lipid-related genes. The effect sizes for the burden of rare variants for each associated gene were substantially higher than those observed for individual SNPs identified from GWASs. We replicated the PNPLA5 signal in an independent large-scale sequencing study of 2,084 individuals. In conclusion, this large whole-exome-sequencing study for LDL-C identified a gene not known to be implicated in LDL-C and provides unique insight into the design and analysis of similar experiments

Imputation of coding variants in African Americans: better performance using data from the exome sequencing project

Summary: Although the 1000 Genomes haplotypes are the most commonly used reference panel for imputation, medical sequencing projects are generating large alternate sets of sequenced samples. Imputation in African Americans using 3384 haplotypes from the Exome Sequencing Project, compared with 2184 haplotypes from 1000 Genomes Project, increased effective sample size by 8.3–11.4% for coding variants with minor allele frequency <1%. No loss of imputation quality was observed using a panel built from phenotypic extremes. We recommend using haplotypes from Exome Sequencing Project alone or concatenation of the two panels over quality score-based post-imputation selection or IMPUTE2’s two-panel combination

Defining neutralizing antibody specificities that target diverse HIV-1 transmitted strains

Thesis (Ph.D.)--University of Washington, 2013Studies in animal models have shown that pre-existing neutralizing antibodies (NAbs), which target the human immunodeficiency virus type 1 (HIV-1) envelope (Env) to prevent entry into host cells, can block infection. A limitation of these models is that typically only one challenge virus is used. Given the diversity of HIV-1 in real world settings, a NAb-based vaccine must have broad and potent activity against multiple strains. This thesis describes NAb specificities that target diverse HIV-1 transmitted variants in natural infection within a high-risk population of exposed infants and women. First, we investigated how transmitted viruses `escape' NAbs by comparing neutralization-resistant viruses in infants to neutralization-sensitive maternal variants near the time of transmission in 2 mother-infant pairs. Although the molecular determinants of escape were distinct, NAb escape involved conformational masking of distal epitopes in both pairs. This strategy may allow HIV-1 Env to utilize limited changes to simultaneously evade multiple NAb specificities while preserving the ability to infect a new host. Next, we determined the activity of 7 broadly neutralizing antibodies (bNAbs) against 45 heterosexually transmitted viruses obtained from Kenyan women. NIH45-46W, which targets the CD4 binding site, was most broad, neutralizing 91% of viruses. Viruses resistant to NIH45-46W were neutralized by PGT128, which targets variable loop 3 of Env, suggesting that combining bNAbs with distinct specificities would provide optimal coverage of HIV-1 variants. Finally, we explored the ability of the early immune system to develop bNAbs by screening 28 infant plasma samples against heterologous viruses. Remarkably, as early as 2.5 years of life and of HIV infection, some infants generated NAbs of similar breadth and potency to those found in adults identified as having very broad responses after approximately 5 years of infection. This finding implies that there is sufficient B-cell functionality in early life to generate broad and potent responses against HIV-1. Overall, these studies suggest that for a preventative vaccine, bNAbs targeting distinct conserved epitopes may be required to overcome HIV-1 diversity, and that characterizing the early development of bNAbs in infants may provide insight into how to elicit such NAb responses

Defining the impact of flavivirus envelope protein glycosylation site mutations on sensitivity to broadly neutralizing antibodies

ABSTRACTAntibodies targeting an envelope dimer epitope (EDE) cross-neutralize Zika virus (ZIKV) and dengue virus (DENV) and have thus inspired an epitope-focused vaccine design. There are two EDE antibody subclasses (EDE1, EDE2) distinguished by their dependence on viral envelope protein N-linked glycosylation at position N153 (DENV) or N154 (ZIKV) for binding. Here, we determined how envelope glycosylation site mutations affect neutralization by EDE and other broadly neutralizing antibodies. Consistent with structural studies, mutations abolishing the N153/N154 glycosylation site increased DENV and ZIKV sensitivity to neutralization by EDE1 antibodies. Surprisingly, despite their location at predicted contact sites, these mutations also increased sensitivity to EDE2 antibodies. Moreover, despite preserving the glycosylation site motif (N-X-S/T), substituting the threonine at ZIKV envelope residue 156 with a serine resulted in loss of glycan occupancy accompanied with increased neutralization sensitivity to EDE antibodies. For DENV, the presence of a serine instead of a threonine at envelope residue 155 retained glycan occupancy, but nonetheless increased sensitivity to EDE antibodies, in some cases to a similar extent as mutation at N153, which abolishes glycosylation. Envelope glycosylation site mutations also increased ZIKV and DENV sensitivity to other non-EDE broadly neutralizing antibodies, but had limited effects on ZIKV- or DENV-specific antibodies. Thus, envelope protein glycosylation is context-dependent and modulates the potency of broadly neutralizing antibodies in a manner not predicted by existing structures. Manipulating envelope protein glycosylation could be a novel strategy for engineering vaccine antigens to elicit antibodies that broadly neutralize ZIKV and DENV.IMPORTANCEAntibodies that potently cross-neutralize Zika (ZIKV) and dengue (DENV) viruses are attractive to induce via vaccination to protect against these co-circulating flaviviruses. Structural studies have shown that viral envelope protein glycosylation is important for binding by one class of these so-called broadly neutralizing antibodies, but less is known about its effect on neutralization. Here, we investigated how envelope protein glycosylation site mutations impact the potency of broadly neutralizing antibodies against ZIKV and DENV. We found that glycan occupancy was not always predicted by an intact N-X-S/T sequence motif. Moreover, envelope protein glycosylation site mutations alter the potency of broadly neutralizing antibodies in a manner unexpected from their predicted binding mechanism as determined by existing structures. We therefore highlight the complex role and determinants of envelope protein glycosylation that should be considered in the design of vaccine antigens to elicit broadly neutralizing antibodies

Zika Virus Is Not Uniquely Stable at Physiological Temperatures Compared to Other Flaviviruses

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat due in part to its association with congenital abnormalities. Other globally relevant flaviviruses include dengue virus (DENV) and West Nile virus (WNV). High-resolution structures of ZIKV reveal many similarities to DENV and suggest some differences, including an extended glycan loop (D. Sirohi, Z. Chen, L. Sun, T. Klose, T. C. Pierson, et al., 352:467–470, 2016, http://dx.doi.org/10.1126/science.aaf5316) and unique interactions among envelope (E) protein residues that were proposed to confer increased virion stability and contribute mechanistically to the distinctive pathobiology of ZIKV (V. A. Kostyuchenko, E. X. Lim, S. Zhang, G. Fibriansah, T. S. Ng, et al., Nature 533:425–428, 2016, http://dx.doi.org/10.1038/nature17994). However, in the latter study, virus stability was inferred by measuring the loss of infectivity following a short incubation period. Here, we rigorously assessed the relative stability of ZIKV, DENV, and WNV by measuring changes in infectivity following prolonged incubation at physiological temperatures. At 37°C, the half-life of ZIKV was approximately twice as long as the half-life of DENV (11.8 and 5.2 h, respectively) but shorter than that of WNV (17.7 h). Incubation at 40°C accelerated the loss of ZIKV infectivity. Increasing virion maturation efficiency modestly increased ZIKV stability, as observed previously with WNV and DENV. Finally, mutations at E residues predicted to confer increased stability to ZIKV did not affect virion half-life. Our results demonstrate that ZIKV is not uniquely stable relative to other flaviviruses, suggesting that its unique pathobiology is explained by an alternative mechanism

Neutralizing activity of WNV-immune sera.

<p>Sensitivity of WNV WT and T198F RVPs to neutralization by 2-mercaptoethanol (2-ME)-treated or untreated (unt) sera pooled from WT-immune (n = 5) <b>(A and C)</b> or T198F-immune (n = 5) <b>(B and D)</b> five-week old WT C57BL/6J mice. Sera were obtained at 6 <b>(A and B)</b> and 9 <b>(C and D)</b> days post-infection. IgG purified from pooled <b>(E)</b> WT-immune (n = 5) or <b>(F)</b> T198F-immune (n = 5) sera obtained from 9 days post-infection were tested for the ability to neutralize WNV WT and T198F RVPs. Error bars indicate the range of infection from duplicate wells. Data in <b>(A-F)</b> are representative of four independent experiments performed using independent RVP stocks.</p