Advances in genomic technologies have revolutionised practices for hospital infection prevention and control programmes. In this Personal View, we describe a genomic epidemiology service called the Rapid Response (RR) programme at University of California, San Francisco, a large academic medical centre. In collaboration with the hospital infection-prevention team, the RR programme uses whole-genome sequencing and metagenomic next-generation sequencing for outbreak investigations, special interest analyses of emerging pathogens, and surveillance of high-priority microbes. Over 7 years (2017-24), the RR programme conducted a diversity of outbreak investigations and other analyses; most investigations ruled out transmission, and the rapid turnaround of genomic results averted further resource-intensive work. Longitudinal surveillance enabled early detection of changing incidence trends and guided timely infection-prevention responses. Our experiences with the RR programme build upon growing evidence that genomic epidemiology programmes enhance hospital infection prevention and control, augment priority pathogen surveillance, and improve patient safety.

Investigations of wildlife diseases and mortality events can sometimes lead to inconclusive results because of limitations in testing combined with an ever-increasing number of emerging viruses. The use of tools such as unbiased metagenomic next-generation sequencing (mNGS) can facilitate the identification of causative agents when conventional investigation methods fail. We performed a retrospective mNGS analysis on RNA isolated from postmortem samples collected during a mortality event in free-ranging, wild black vultures (Coragyps atratus) that occurred in eastern Pennsylvania and western New Jersey in 2020. We describe the discovery and identification of a novel species of bandavirus (family Phenuiviridae) in case specimens from this die-off, as well as some of the associated pathology findings. The Bandavirus genus comprises tickborne viral species that have been reported across 5 continents. These viruses have been implicated in outbreaks in a variety of mammalian hosts, including humans, and in avian species, making them important potential sources of zoonotic spillover events. Genomic and phylogenetic analyses of the bandavirus that we detected indicate that its closest relative is Hunter Island virus, a bandavirus previously implicated in albatross mortality events off the coast of Tasmania, Australia. Follow-up PCR testing of samples from 16 additional vultures from the same cohort indicate that this new bandavirus was the likely cause of death.

Introduction Rhesus macaques have long been a focus of research for understanding immune responses to human pathogens due to their close phylogenetic relationship with humans. As rhesus macaque antibody germlines show high degrees of polymorphism, the spectrum of database-covered genes expressed in individual macaques remains to be determined.Methods Here, four rhesus macaques infected with SHIVSF162P3N became a study of interest because they developed broadly neutralizing antibodies against HIV-1. To identify the immunoglobulin heavy chain V-gene (IGHV) germlines in these macaques, we applied three sequencing approaches - genomic DNA (gDNA) TOPO sequencing, gDNA MiSeq, and messenger RNA (mRNA) MiSeq inference with IgDiscover, and illustrated the detection power of each method.Results Of the 197 new rhesus IGHV germline sequences identified, 116 (59%) were validated by at least two methods, and 143 (73%) were found in at least two macaques or two sample sources. About 20% of germlines in each macaque are missing from the current database, including a subset frequently expressed. Overall, gDNA MiSeq determined the greatest number of germline sequences, followed by gDNA TOPO sequencing and mRNA MiSeq inference by IgDiscover, with IgDiscover providing direct evidence of allele expression and usage.Discussion Our interdisciplinary study sheds light on germline sequencing, enhances the rhesus IGHV germline database, and highlights the importance of germline sequencing in rhesus immune repertoire studies.

BackgroundGenome streamlining, the process by which genomes become smaller and encode fewer genes over time, is a common phenomenon among pathogenic bacteria. This reduction is driven by selection for minimized energy expenditure in a nutrient-rich environment. As pathogens evolve to become more reliant on the host, metabolic genes and resulting capabilities are lost in favor of siphoning metabolites from the host. Characterizing genome streamlining, gene loss, and metabolic pathway degradation can be useful in assessing pathogen dependency on host metabolism and identifying potential targets for host-directed therapeutics.ResultsPoMeLo (Predictor of Metabolic Loss) is a novel evolutionary genomics-guided computational approach for identifying metabolic gaps in the genomes of pathogenic bacteria. PoMeLo leverages a centralized public database of high-quality genomes and annotations and allows the user to compare an unlimited number of genomes across individual genes and pathways. PoMeLo runs locally using user-friendly prompts in a matter of minutes and generates tabular and visual outputs for users to compare predicted metabolic capacity between groups of bacteria and individual species. Each pathway is assigned a Predicted Metabolic Loss (PML) score to assess the magnitude of genome streamlining. Optionally, PoMeLo places the results in an evolutionary context by including phylogenetic relationships in visual outputs. It can also initially compute phylogenetically-weighted mean genome sizes to identify genome streamlining events. Here, we describe PoMeLo and demonstrate its use in identifying metabolic gaps in genomes of pathogenic Treponema species.ConclusionsPoMeLo represents an advance over existing methods for identifying metabolic gaps in genomic data, allowing comparison across large numbers of genomes and placing the resulting data in a phylogenetic context. PoMeLo is freely available for academic and non-academic use at https://github.com/czbiohub-sf/pomelo.

Two independent temporal-spatial clusters of hospital-onset Rhizopus infections were evaluated using whole-genome sequencing (WGS). Phylogenetic analysis confirmed that isolates within each cluster were unrelated despite epidemiological suspicion of outbreaks. The ITS1 region alone was insufficient for accurate analysis. WGS has utility for rapid rule-out of suspected nosocomial Rhizopus outbreaks.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

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Obligate intracellular bacteria shed essential biosynthetic pathways during their evolution towards host dependency, providing an opportunity for host-directed therapeutics. Using Rickettsiaceae as a model, we employed a novel computational pipeline called PoMeLo to systematically compare this cytosolic family of bacteria to the related Anaplasmataceae , which reside in a membrane-bound vacuole in the host cell. We identified 20 metabolic pathways that have been lost since the divergence of Anaplasmataceae and Rickettsiaceae , corresponding to the latter’s change to a cytosolic niche. We hypothesized that drug inhibition of these host metabolic pathways would reduce the levels of metabolites available to the bacteria, thereby inhibiting bacterial growth. We tested 22 commercially available inhibitors for 14 of the identified pathways and found that the majority (59%) reduced bacterial growth at concentrations that did not induce host cell cytotoxicity. Of these, 5 inhibitors with an IC 50 under 5 μM were tested to determine whether their mode of inhibition was bactericidal or bacteriostatic. Both mycophenolate mofetil, an inhibitor of inosine-5’-monophosphate dehydrogenase in the purine biosynthesis pathway, and roseoflavin, an analog of riboflavin, displayed bactericidal activity. A complementary unbiased mass spectrometry-based metabolomics approach identified 14 pathways impacted by Rickettsia infection based on alterations in metabolite levels. Strikingly, 11 of these (79%) overlapped with those identified by our computational predictions. These in vitro validation studies support the feasibility of a novel evolutionary genomics-guided approach for host-directed antibiotic drug development against obligate pathogens. Importance Many pathogens have evolved to acquire essential metabolites from their host cell, while in turn shedding their own biosynthetic capacities. This leads to an interesting dilemma: on one hand, reduced genomes allow pathogens to save energy and replicate more quickly, while on the other hand, they become more dependent on the host cell for survival. This vulnerability can be exploited by identifying and therapeutically inhibiting the host pathways that are essential for pathogen survival. The significance of our research is in predicting the precise pathways lost during a pathogen’s evolutionary adaptation to parasitism and validating these predictions through targeted in vitro growth assays and an unbiased metabolomic survey of the host-pathogen interface.

The sequencing of antibody repertoires of B-cells at increasing coverage and depth has led to the identification of vast numbers of immunoglobulin heavy and light chains. However, the size and complexity of these Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) datasets makes it difficult to perform exploratory analyses. To aid in data exploration, we have developed AIRRscape, an R Shiny-based interactive web browser application that enables B-cell receptor (BCR) and antibody feature discovery through comparisons among multiple repertoires. Using AIRR-seq data as input, AIRRscape starts by aggregating and sorting repertoires into interactive and explorable bins of germline V-gene, germline J-gene, and CDR3 length, providing a high-level view of the entire repertoire. Interesting subsets of repertoires can be quickly identified and selected, and then network topologies of CDR3 motifs can be generated for further exploration. Here we demonstrate AIRRscape using patient BCR repertoires and sequences of published monoclonal antibodies to investigate patterns of humoral immunity to three viral pathogens: SARS-CoV-2, HIV-1, and DENV (dengue virus). AIRRscape reveals convergent antibody sequences among datasets for all three pathogens, although HIV-1 antibody datasets display limited convergence and idiosyncratic responses. We have made AIRRscape available as a web-based Shiny application, along with code on GitHub to encourage its open development and use by immuno-informaticians, virologists, immunologists, vaccine developers, and other scientists that are interested in exploring and comparing multiple immune receptor repertoires.

Dengue virus (DENV) is a mosquito-borne disease that poses a public health threat to sub/tropical areas worldwide. Vaccination drives require differential diagnosis of serotype-specific DENV exposure to reduce severe dengue risks, yet state-of-the-art DENV serology relies upon short-lived serotype-specific IgM or labor intensive neutralization assays. The need for high-throughput differential diagnosis is met with our multiSero platform (Byrum et al.), a screening technique capable of detecting 48 antigen-antibody pairs simultaneously, demonstrating utility for population-wide screening. Through machine-vision techniques, we quantify and classify antibody-response signals with high sensitivity to develop automated analysis pipelines capable of diagnosing serotype-specific DENV exposure.

Omicron and its subvariants have rendered most authorized monoclonal antibody-based treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ineffective, highlighting the need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective antibodies target variable epitopes. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to known non-neutralizing antibodies that target highly conserved epitopes in the viral spike protein. These inhibitors, called receptor-blocking conserved non-neutralizing antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron. Neutralization potency is lost when the linker joining the binding and inhibitory ReconnAb components is severed. In addition, a bi-functional ReconnAb, made by linking ACE2 to a bi-specific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron and BA.2. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad-spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

All but one of the authorized monoclonal antibody-based treatments for SARS-CoV-2 are largely ineffective against Omicron, highlighting the critical need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective therapeutic antibodies target epitopes that are not highly conserved. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to antibodies that are known to be non-neutralizing, but which target highly conserved epitopes in the viral spike protein. These inhibitors, called Receptor-blocking conserved non-neutralizing Antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOC), including Omicron. Neutralization potency is dependent on both the binding and inhibitory ReconnAb components as activity is lost when the linker joining the two is severed. In addition, a bifunctional ReconnAb, made by linking ACE2 to a bispecific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad- spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL âˆ'1 RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.

During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21–5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml−1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective. Although rare, antibodies against the receptor-binding domain of SARS-CoV-2 that showed potent antiviral activity were obtained from all tested convalescent individuals, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

We describe an adaptation of conventional ELISA methods to an ELISA-Array format using non-contact Piezo printing of up to 30 spots of purified recombinant viral fusion proteins, vaccine and virus on 96 well high-protein binding plates. Antigens were printed in 1 nanoliter volumes of protein stabilizing buffer using as little as 0.25 nanograms of protein, 2000-fold less than conventional ELISA. The performance of the ELISA-Array was demonstrated by serially diluting n=8 human post-flu vaccination plasma samples starting at a 1/1000 dilution and measuring binding to the array of Influenza antigens. Plasma polyclonal antibody levels were detected using a cocktail of biotinylated anti-human kappa and lambda light chain antibodies, followed by a Streptavidin-horseradish peroxidase conjugate and the dose-dependent signal was developed with a precipitable TMB substrate. Intra- and inter-assay precision of absorbance units among the eight donor samples showed mean CVs of 4.8% and 10.8%, respectively. The plasma could be differentiated by donor and antigen with titer sensitivities ranging from 1 × 10 3 to 4 × 10 6 , IC 50 values from 1 × 10 4 to 9 × 10 6 , and monoclonal antibody sensitivities in the ng/mL range. Equivalent sensitivities of ELISA versus ELISA-Array, compared using plasma and an H1N1 HA trimer, were achieved on the ELISA-Array printed at 0.25ng per 200um spot and 1000ng per ELISA 96-well. Vacuum-sealed array plates were shown to be stable when stored for at least 2 days at ambient temperature and up to 1 month at 4-8°C. By the use of any set of printed antigens and analyte matrices the methods of this multiplexed ELISA-Array format can be broadly applied in translational research.

The HIV-1 envelope (Env) undergoes conformational changes during infection. Broadly neutralizing antibodies (bNAbs) are typically isolated by using soluble Env trimers, which do not capture all Env states. To address these limitations, we devised a vesicular stomatitis virus (VSV)-based probe to display membrane-embedded Env trimers and isolated five bNAbs from two chronically infected donors, M4008 and M1214. Donor B cell receptor (BCR) repertoires identified two bNAb lineages, M4008_N1 and M1214_N1, that class-switched to immunoglobulin G (IgG) and IgA. Variants of these bNAbs reconstituted as IgA demonstrated broadly neutralizing activity, and the IgA fraction of M1214 plasma conferred neutralization. M4008_N1 epitope mapping revealed a glycan-independent V3 epitope conferring tier 2 virus neutralization. A 4.86-Å-resolution cryogenic electron microscopy (cryo-EM) structure of M1214_N1 complexed with CH505 SOSIP revealed another elongated epitope, the V2V5 corridor, extending from V2 to V5. Overall, the VSVENV probe identified bNAb lineages with neutralizing IgG and IgA members targeting distinct sites of HIV-1 Env vulnerability.

The Arctic climate was warmer than today at the last interglacial and the Holocene thermal optimum. To reveal the impact of past climate-warming events on the demographic history of an Arctic specialist, we examined both mitochondrial and nuclear genomic variation in the collared lemming (Dicrostonyx torquatus, Pallas), a keystone species in tundra communities, across its entire distribution in northern Eurasia. The ancestral phylogenetic position of the West Beringian group and divergence time estimates support the hypothesis of continental range contraction to a single refugial area located in West Beringia during high-magnitude warming of the last interglacial, followed by westward recolonization of northern Eurasia in the last glacial period. The West Beringian group harbors the highest mitogenome diversity and its inferred demography indicates a constantly large effective population size over the Late Pleistocene to Holocene. This suggests that northward forest expansion during recent warming of the Holocene thermal optimum did not affect the gene pool of the collared lemming in West Beringia but reduced genomic diversity and effective population size in all other regions of the Eurasian Arctic. Demographic inference from genomic diversity was corroborated by species distribution modeling showing reduction in species distribution during past climate warming. These conclusions are supported by recent paleoecological evidence suggesting smaller temperature increases and moderate northward forest advances in the extreme northeast of Eurasia during the Late Pleistocene-to-Holocene warming events. This study emphasizes the importance of West Beringia as a potential refugium for cold-adapted Arctic species under ongoing climate warming.

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To define bNAb targets, we characterized 28 antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified J9 and J8, two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies showed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against DENV with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested that J9 and J8 followed divergent somatic hypermutation pathways, and that a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a new epitope that can be exploited for vaccine design.