Biology subjects

Bloom, J. D.

Publications and source records attributed to Bloom, J. D..

18 recordsLinked to original sources

bioRxiv2018

Single-cell virus sequencing of influenza infections that trigger innate immunity

The outcome of viral infection is extremely heterogeneous, with infected cells only sometimes activating innate immunity. Here we develop a new approach to assess how the genetic variation inherent in viral populations contributes to this heterogeneity. We do this by determining both the transcriptome and full-length sequences of all viral genes in single influenza-infected cells. Most cells are infected by virions with defects such as amino-acid mutations, internal deletions, or failure to express a gene. We identify instances of each type of defect that increase the likelihood that a cell activates an innate-immune response. However, immune activation remains stochastic in cells infected by virions with these defects, and sometimes occurs even when a cell is infected by a virion that expresses unmutated copies of all genes. Our work shows that viral genetic variation substantially contributes to but does not fully explain the heterogeneity in single influenza-infected cells.

microbiology↗

bioRxiv2018

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

Anti-HIV broadly neutralizing antibodies (bnAbs) have revealed vaccine targets on the viruss 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.

immunology↗

bioRxiv2018

Reconciling disparate estimates of viral genetic diversity during human influenza infections

Deep sequencing can measure viral genetic diversity within human influenza infections, but published studies disagree in their estimates of how much genetic diversity is typically present. One large-scale deep-sequencing study of human influenza reported high levels of shared viral genetic diversity among infected individuals in Hong Kong, but subsequent studies of other cohorts have reported little shared viral diversity. We re-analyze sequencing data from four studies of within-host genetic diversity encompassing more than 500 acute human influenza infections. We identify an anomaly in the Hong Kong data that provides a technical explanation for these discrepancies: read pairs from this study are often split between different biological samples, indicating that some reads are incorrectly assigned. These technical abnormalities explain the high levels of within-host variation and loose transmission bottlenecks reported by this study. Studies without these anomalies consistently report low levels of genetic diversity in acute human influenza infections.

evolutionary biology↗

bioRxiv2018

Characterization of influenza B virus variants with reduced neuraminidase inhibitor susceptibility

Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs) which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyse within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had similar NA enzyme function relative to its wild-type but had slightly reduced replication and transmission efficiency in vivo. The D197N variant had impaired NA enzyme function but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B variant with H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution.

microbiology↗

bioRxiv2018

Comprehensive profiling of translation initiation in influenza virus infected cells

Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. Host transcripts induced by the anti-viral response are enriched for translation initiation at non-canonical start sites and non-AUG start codons. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

microbiology↗

bioRxiv2018

Sera from individuals with narrowly focused influenza virus antibodies rapidly select viral escape mutations in ovo

Influenza viruses use distinct antibody escape mechanisms depending on the overall complexity of the antibody response that is encountered. When grown in the presence of a hemagglutinin (HA) monoclonal antibody, influenza viruses typically acquire a single HA mutation that reduces the binding of that specific monoclonal antibody. In contrast, when confronted with mixtures of HA monoclonal antibodies or polyclonal sera that have antibodies that bind several HA epitopes, influenza viruses acquire mutations that increase HA binding to host cells. Recent data from our laboratory and others suggest that some humans possess antibodies that are narrowly focused on HA epitopes that were present in influenza virus strains that they were likely exposed to in childhood. Here, we completed a series of experiments to determine if humans with narrowly focused HA antibody responses are able to select for influenza virus antigenic escape variants in ovo. We identified three human donors that possessed HA antibody responses that were heavily focused on a single HA antigenic site. Remarkably, sera from all three of these donors selected single HA escape mutations during in ovo passage experiments, similar to what has been previously reported for single monoclonal antibodies. These single HA mutations directly reduced binding of serum antibodies used for selection. We propose that new antigenic variants of influenza viruses might originate in individuals that produce antibodies that are narrowly focused on HA epitopes that were present in viral strains that they encountered in childhood.\n\nImportanceInfluenza vaccine strains must be updated frequently since circulating viral strains continuously change in antigenically important epitopes. Our previous studies have demonstrated that some individuals possess antibody responses that are narrowly focused on epitopes that were present in viral strains that they encountered during childhood. Here, we show that influenza viruses rapidly escape this type of polyclonal antibody response when grown in ovo by acquiring single mutations that directly prevent antibody binding. These studies improve our understanding of how influenza viruses evolve when confronted with narrowly focused polyclonal human antibodies.

microbiology↗

bioRxiv2018

Complete functional mapping of infection- and vaccine-elicited antibodies against the fusion peptide of HIV

Eliciting broadly neutralizing antibodies (bnAbs) targeting envelope (Env) is a major goal of HIV vaccine development, but cross-clade breadth from immunization has only sporadically been observed. Recently, Xu et al (2018) elicited cross-reactive neutralizing antibody responses in a variety of animal models using immunogens based on the epitope of bnAb VRC34.01. The VRC34.01 antibody, which was elicited by natural human infection, targets the N terminus of the Env fusion peptide, a critical component of the virus entry machinery. Here we precisely characterize the functional epitopes of VRC34.01 and two vaccine-elicited murine antibodies by mapping all single amino-acid mutations to the BG505 Env that affect viral neutralization. While escape from VRC34.01 occurred via mutations in both fusion peptide and distal interacting sites of the Env trimer, escape from the vaccine-elicited antibodies was mediated predominantly by mutations in the fusion peptide. Cryo-electron microscopy of four vaccine-elicited antibodies in complex with Env trimer revealed focused recognition of the fusion peptide and provided a structural basis for development of neutralization breadth. Together, these functional and structural data suggest that the breadth of vaccine-elicited antibodies targeting the fusion peptide can be enhanced by specific interactions with additional portions of Env. Thus, our complete maps of viral escape provide a template to improve the breadth or potency of future vaccine-induced antibodies against Envs fusion peptide.\n\nAuthor summaryA major goal of HIV-1 vaccine design is to elicit antibodies that neutralize diverse strains of HIV-1. Recently, some of us elicited such antibodies in animal models using immunogens based on the epitope of a broad antibody (VRC34.01) isolated from an infected individual. Further improving these vaccine-elicited antibody responses will require a detailed understanding of how the resulting antibodies target HIVs envelope protein (Env). Here, we used mutational antigenic profiling to precisely map the epitope of two vaccine-elicited antibodies and the template VRC34.01 antibody. We did this by quantifying the effect of all possible amino acid mutations to Env on antibody neutralization. Although all antibodies target a similar region of Env, we found clear differences in the functional interaction of Env with the vaccine- and infection-elicited antibodies. We combined these functional data with structural analyses to identify antibody-Env interactions that could improve the breadth of vaccine-elicited antibodies, and thereby help to refine vaccination schemes to achieve broader responses.

immunology↗

bioRxiv2018

Modeling site-specific amino-acid preferences deepens phylogenetic estimates of viral divergence

Molecular phylogenetics is often used to estimate the time since the divergence of modern gene sequences. For highly diverged sequences, such phylogenetic techniques sometimes estimate surprisingly recent divergence times. In the case of viruses, independent evidence indicates that the estimates of deep divergence times from molecular phylogenetics are sometimes too recent. This discrepancy is caused in part by inadequate models of purifying selection leading to branch-length underestimation. Here we examine the effect on branch-length estimation of using models that incorporate experimental measurements of purifying selection. We find that models informed by experimentally measured site-specific amino-acid preferences estimate longer deep branches on phylogenies of influenza virus hemagglutinin. This lengthening of branches is due to more realistic stationary states of the models, and is mostly independent of the branch-length-extension from modeling site-to-site variation in amino-acid substitution rate. The branch-length extension from experimentally informed site-specific models is similar to that achieved by other approaches that allow the stationary state to vary across sites. However, the improvements from all of these site-specific but time-homogeneous and site-independent models are limited by the fact that a proteins amino-acid preferences gradually shift as it evolves. Overall, our work underscores the importance of modeling site-specific amino-acid preferences when estimating deep divergence times--but also shows the inherent limitations of approaches that fail to account for how these preferences shift over time.

evolutionary biology↗

bioRxiv2018

Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants

Human influenza virus rapidly accumulates mutations in its major surface protein hemagglutinin (HA). The evolutionary success of influenza virus lineages depends on how these mutations affect HAs functionality and antigenicity. Here we experimentally measure the effects on viral growth in cell culture of all single amino-acid mutations to the HA from a recent human H3N2 influenza virus strain. We show that mutations that are measured to be more favorable for viral growth are enriched in evolutionarily successful H3N2 viral lineages relative to mutations that are measured to be less favorable for viral growth. Therefore, despite the well-known caveats about cell-culture measurements of viral fitness, such measurements can still be informative for understanding evolution in nature. We also compare our measurements for H3 HA to similar data previously generated for a distantly related H1 HA, and find substantial differences in which amino acids are preferred at many sites. For instance, the H3 HA has less disparity in mutational tolerance between the head and stalk domains than the H1 HA. Overall, our work suggests that experimental measurements of mutational effects can be leveraged to help understand the evolutionary fates of viral lineages in nature -- but only when the measurements are made on a viral strain similar to the ones being studied in nature.\n\nSignificance StatementA key goal in the study of influenza virus evolution is to forecast which viral strains will persist and which ones will die out. Here we experimentally measure the effects of all amino-acid mutations to the hemagglutinin protein from a human H3N2 influenza strain on viral growth in cell culture. We show that these measurements have utility for distinguishing among viral strains that do and do not succeed in nature. Overall, our work suggests that new high-throughput experimental approaches may be useful for understanding virus evolution in nature.

evolutionary biology↗

bioRxiv2018

Estimating the frequency of multiplets in single-cell RNA sequencing from cell-mixing experiments

In single-cell RNA-sequencing, it is important to know the frequency at which the sequenced transcriptomes actually derive from multiple cells. A common method to estimate this multiplet frequency is to mix two different types of cells (e.g., human and mouse), and then determine how often the transcriptomes contain transcripts from both cell types. When the two cell types are mixed in equal proportion, the calculation of the multiplet frequency from the frequency of mixed transcriptomes is straightforward. But surprisingly, there are no published descriptions of how to calculate the multiplet frequency in the general case when the cell types are mixed unequally. Here I derive equations to analytically calculate the multiplet frequency from the numbers of observed pure and mixed transcriptomes when two cell types are mixed in arbitrary proportions, under the assumption that the loading of cells into droplets or wells is Poisson.

genomics↗

bioRxiv2017

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.

microbiology↗

bioRxiv2017

Cooperating H3N2 influenza virus variants are not detectable in primary clinical samples

The high mutation rates of RNA viruses lead to rapid genetic diversification, which can enable cooperative interactions between variants in a viral population. We previously described two distinct variants of H3N2 influenza virus that cooperate in cell culture. These variants differ by a single mutation, D151G, in the neuraminidase protein. The D151G mutation reaches a stable frequency of about 50% when virus is passaged in cell culture. However, it is unclear whether selection for the cooperative benefits of D151G is a cell-culture phenomenon, or whether the mutation is also sometimes present at appreciable frequency in virus populations sampled directly from infected humans. Prior work has not detected D151G in unpassaged clinical samples, but these studies have used methods like Sanger sequencing and pyrosequencing that are relatively insensitive to low-frequency variation. We identified nine samples of human H3N2 influenza collected between 2013 to 2015 in which Sanger sequencing had detected a high frequency of the D151G mutation following one to three passages in cell culture. We deep-sequenced the unpassaged clinical samples to identify low-frequency viral variants. The frequency of D151G did not exceed the frequency of library preparation and sequencing errors in any of the sequenced samples. We conclude that passage in cell culture is primarily responsible for the frequent observations of D151G in recent H3N2 influenza strains.\n\nIMPORTANCEViruses mutate rapidly, and recent studies of RNA viruses have shown that related viral variants can sometimes cooperate to improve each others growth. We previously described two variants of H3N2 influenza virus that cooperate in cell culture. The mutation responsible for cooperation is often observed when human samples of influenza virus are grown in the lab before sequencing, but it is unclear whether the mutation also exists in human infections or is exclusively the result of lab passage. We identified nine human isolates of influenza that had developed the cooperating mutation after being grown in the lab, and performed highly sensitive deep-sequencing of the unpassaged clinical samples to determine whether the mutation existed in the original human infections. We found no evidence of the cooperating mutation in the unpassaged samples, suggesting that the cooperation primarily arises in laboratory conditions.

microbiology↗

bioRxiv2017

Quantifying the ease of viral escape from broad and narrow antibodies to influenza hemagglutinin

Influenza virus can completely escape most antibodies with single mutations. However, rare antibodies broadly neutralize many viral strains. It is unclear how easily influenza virus might escape such antibodies if it was under strong pressure to do so. Here we map all single amino-acid mutations that increase resistance to broad antibodies targeting an H1 hemagglutinin. Crucially, our approach not only identifies antigenic mutations but also quantifies their effect sizes. All antibodies select mutations, but the effect sizes vary widely. The virus can escape a broad antibody that targets residues in hemagglutinins receptor-binding site the same way it escapes narrow strain-specific antibodies: via single mutations with huge effects. In contrast, broad antibodies targeting hemagglutinins stalk only select mutations with small effects. Therefore, among the antibodies we have examined, breadth is an imperfect indicator of the potential for viral escape via single mutations. Broadly neutralizing antibodies targeting the H1 hemagglutinin stalk are quantifiably harder to escape than the other antibodies tested here.

microbiology↗

bioRxiv2017

Extreme heterogeneity of influenza virus infection in single cells

Viral infection can dramatically alter a cells transcriptome. However, these changes have mostly been studied by bulk measurements on many cells. Here we use single-cell mRNA sequencing to examine the transcriptional consequences of Influenza virus infection. We 1nd extremely wide cell-to-cell variation in production of viral gene transcripts - viral transcripts compose less than a percent of total mRNA in many infected cells, but a few cells derive over half their mRNA from virus. Some infected cells fail to express at least one viral gene, and this gene absence partially explains variation in viral transcriptional load. Despite variation in total viral load, the relative abundances of viral mRNAs are fairly consistent across infected cells. Activation of innate immune pathways is rare, but some cellular genes co-vary in abundance with the amount of viral mRNA. Overall, our results highlight the complexity of viral infection at the level of single cells.

genomics↗

bioRxiv2017

Expanding the Atlas of Functional Missense Variation for Human Genes

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon-mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features, and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

molecular biology↗

bioRxiv2017

phydms: Software for phylogenetic analyses informed by deep mutational scanning

BackgroundThe evolution of protein-coding genes can be quantitatively modeled using phylogenetic methods. Recently, it has been shown that high-throughput experimental measurements of mutational effects made via deep mutational scanning can inform site-specific phylogenetic substitution models of gene evolution. However, there is currently no software tailored for such analyses.\n\nResultsWe describe software that efficiently performs phylogenetic analyses with substitution models informed by deep mutational scanning. This software, phydms, is [~]100-fold faster than existing programs that accommodate such substitution models. It can be used to compare the results of deep mutational scanning experiments to the selection on genes in nature. For instance, phydms enables rigorous comparison of how well different experiments on the same gene describe natural selection. It also enables the re-scaling of deep mutational scanning data to account for differences in the stringency of selection in the lab and nature. Finally, phydms can identify sites that are evolving differently in nature than expected from experiments in the lab.\n\nConclusionsThe phydms software makes it easy to use phylogenetic substitution models informed by deep mutational scanning experiments. As data from such experiments becomes increasingly widespread, phydms will facilitate quantitative comparison of the experimental results to the actual selection pressures shaping evolution in nature.

evolutionary biology↗

bioRxiv2017

Comprehensive mapping of HIV-1 escape from a broadly neutralizing antibody

Precisely defining how viral mutations affect HIVs sensitivity to antibodies is vital to the development and evaluation of vaccines and antibody immunotherapeutics. But despite great effort, a full map of escape mutants has not yet been delineated for even a single anti-HIV antibody. Here we describe a massively parallel experimental approach to quantify how all single amino-acid mutations to Envelope (Env) affect HIVs sensitivity to a neutralizing antibody. We applied this approach to PGT151 and identified novel sites of escape in addition to those previously defined by structural and functional studies, such as glycans at sites 611 and 637, residue 647, and sites in the fusion peptide. Evaluating the effect of each amino acid at each site lends insight into the biochemical basis of escape throughout the epitope. Thus, comprehensive mapping of HIV antibody escape gives a quantitative, mutation-level view of the ways that Env can evade neutralization.

microbiology↗

bioRxiv2016

Complete mapping of viral escape from neutralizing antibodies

Identifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. Here we quantify how every amino-acid mutation to influenza hemagglutinin affects neutralization by monoclonal antibodies targeting several antigenic regions. Our approach involves creating all replication-competent protein variants of the virus, selecting these variants with antibody, and using deep sequencing to identify enriched mutations. These high-throughput measurements are predictive of the effects of individual mutations in traditional neutralization assays. At many residues, only some of the possible mutations escape from an antibody. For instance, at a single residue targeted by two different antibodies, we identify some mutations that escape both antibodies and other mutations that escape only one or the other. Therefore, our approach maps how viruses can escape antibodies with mutation-level sensitivity, and shows that only some mutations at antigenic residues actually alter antigenicity.

microbiology↗