Human coronavirus HKU1 recognition of the TMPRSS2 host receptor.

The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. We designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2, providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among orthologous proteases. We identified TMPRSS2 orthologs from five mammalian orders promoting HKU1 S-mediated entry into cells along with key residues governing host receptor usage. Our data show that the TMPRSS2 binding motif is a site of vulnerability to neutralizing antibodies and suggest that HKU1 uses S conformational masking and glycan shielding to balance immune evasion and receptor engagement.

Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein.

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2.

The SARS-CoV-2 spike (S) glycoprotein contains an immunodominant receptor-binding domain (RBD) targeted by most neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite (designated site i) recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge, albeit selecting escape mutants in some animals. Indeed, several SARS-CoV-2 variants, including the B.1.1.7, B.1.351, and P.1 lineages, harbor frequent mutations within the NTD supersite, suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs for protective immunity and vaccine design.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

After the pandemic: perspectives on the future trajectory of COVID-19.

There is a realistic expectation that the global effort in vaccination will bring the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under control. Nonetheless, uncertainties remain about the type of long-term association that the virus will establish with the human population and, in particular, whether coronavirus disease 2019 (COVID-19) will become an endemic disease. Although the trajectory is difficult to predict, the conditions, concepts and variables that influence this transition can be anticipated. Persistence of SARS-CoV-2 as an endemic virus, perhaps with seasonal epidemic peaks, may be fuelled by pockets of susceptible individuals and waning immunity after infection or vaccination, changes in the virus through antigenic drift that diminish protection and re-entries from zoonotic reservoirs. Here we review relevant observations from previous epidemics and discuss the potential evolution of SARS-CoV-2 as it adapts during persistent transmission in the presence of a level of population immunity. Lack of effective surveillance or adequate response could enable the emergence of new epidemic or pandemic patterns from an endemic infection of SARS-CoV-2. There are key pieces of data that are urgently needed in order to make good decisions; we outline these and propose a way forward.

Lectins enhance SARS-CoV-2 infection and influence neutralizing antibodies.

SARS-CoV-2 infection-which involves both cell attachment and membrane fusion-relies on the angiotensin-converting enzyme 2 (ACE2) receptor, which is paradoxically found at low levels in the respiratory tract1-3, suggesting that there may be additional mechanisms facilitating infection. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding immunoglobulin-like lectin 1 (SIGLEC1) function as attachment receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the amino-terminal domain or to the conserved site at the base of the receptor-binding domain, while poorly neutralizing infection of ACE2-overexpressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the receptor binding motif, while potently neutralizing infection of ACE2-overexpressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike, promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.

Broad sarbecovirus neutralization by a human monoclonal antibody.

The recent emergence of SARS-CoV-2 variants of concern1-10 and the recurrent spillovers of coronaviruses11,12 into the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here we describe a human monoclonal antibody designated S2X259, which recognizes a highly conserved cryptic epitope of the receptor-binding domain and cross-reacts with spikes from all clades of sarbecovirus. S2X259 broadly neutralizes spike-mediated cell entry of SARS-CoV-2, including variants of concern (B.1.1.7, B.1.351, P.1, and B.1.427/B.1.429), as well as a wide spectrum of human and potentially zoonotic sarbecoviruses through inhibition of angiotensin-converting enzyme 2 (ACE2) binding to the receptor-binding domain. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses an escape profile that is limited to a single substitution, G504D. We show that prophylactic and therapeutic administration of S2X259 protects Syrian hamsters (Mesocricetus auratus) against challenge with the prototypic SARS-CoV-2 and the B.1.351 variant of concern, which suggests that this monoclonal antibody is a promising candidate for the prevention and treatment of emergent variants and zoonotic infections. Our data reveal a key antigenic site that is targeted by broadly neutralizing antibodies and will guide the design of vaccines that are effective against all sarbecoviruses.

SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.

Transcription Factor-Directed Re-wiring of Chromatin Architecture for Somatic Cell Nuclear Reprogramming toward trans-Differentiation.

MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers, and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs led to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation.

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that is responsible for the current pandemic of coronavirus disease 2019 (COVID-19), which has resulted in more than 3.7 million infections and 260,000 deaths as of 6 May 20201,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe several monoclonal antibodies that target the S glycoprotein of SARS-CoV-2, which we identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. One antibody (named S309) potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2, by engaging the receptor-binding domain of the S glycoprotein. Using cryo-electron microscopy and binding assays, we show that S309 recognizes an epitope containing a glycan that is conserved within the Sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails that include S309 in combination with other antibodies that we identified further enhanced SARS-CoV-2 neutralization, and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and antibody cocktails containing S309 for prophylaxis in individuals at a high risk of exposure or as a post-exposure therapy to limit or treat severe disease.

Large language models for science and medicine.

Large language models (LLMs) are a type of machine learning model that learn statistical patterns over text, such as predicting the next words in a sequence of text. Both general purpose and task-specific LLMs have demonstrated potential across diverse applications. Science and medicine have many data types that are highly suitable for LLMs, such as scientific texts (publications, patents and textbooks), electronic medical records, large databases of DNA and protein sequences and chemical compounds. Carefully validated systems that can understand and reason across all these modalities may maximize benefits. Despite the inevitable limitations and caveats of any new technology and some uncertainties specific to LLMs, LLMs have the potential to be transformative in science and medicine.

Human gene essentiality.

A gene can be defined as essential when loss of its function compromises viability of the individual (for example, embryonic lethality) or results in profound loss of fitness. At the population level, identification of essential genes is accomplished by observing intolerance to loss-of-function variants. Several computational methods are available to score gene essentiality, and recent progress has been made in defining essentiality in the non-coding genome. Haploinsufficiency is emerging as a critical aspect of gene essentiality: approximately 3,000 human genes cannot tolerate loss of one of the two alleles. Genes identified as essential in human cell lines or knockout mice may be distinct from those in living humans. Reconciling these discrepancies in how we evaluate gene essentiality has applications in clinical genetics and may offer insights for drug development.

Transfer transcriptomic signatures for infectious diseases.

The modulation of the transcriptome is among the earliest responses to infection. However, defining the transcriptomic signatures of disease is challenging because logistic, technical, and cost factors limit the size and representativeness of samples in clinical studies. These limitations lead to a poor performance of signatures when applied to new datasets. Although the study focuses on infection, the central hypothesis of the work is the generalization of sets of signatures across diseases. We use a machine learning approach to identify common elements in datasets and then test empirically whether they are informative about a second dataset from a disease or process distinct from the original dataset. We identify sets of genes, which we name transfer signatures, that are predictive across diverse datasets and/or species (e.g., rhesus to humans). We demonstrate the usefulness of transfer signatures in two use cases: the progression of latent to active tuberculosis and the severity of COVID-19 and influenza A H1N1 infection. This indicates that transfer signatures can be deployed in settings that lack disease-specific biomarkers. The broad significance of our work lies in the concept that a small set of archetypal human immunophenotypes, captured by transfer signatures, can explain a larger set of responses to diverse diseases.

Potent broadly neutralizing antibody VIR-3434 controls hepatitis B and D virus infection and reduces HBsAg in humanized mice.

BACKGROUND & AIMS: Chronic hepatitis B is a global public health problem, and coinfection with hepatitis delta virus (HDV) worsens disease outcome. Here, we describe a hepatitis B virus (HBV) surface antigen (HBsAg)-targeting monoclonal antibody (mAb) with the potential to treat chronic hepatitis B and chronic hepatitis D. METHODS: HBsAg-specific mAbs were isolated from memory B cells of HBV vaccinated individuals. In vitro neutralization was determined against HBV and HDV enveloped with HBsAg representing eight HBV genotypes. Human liver-chimeric mice were treated twice weekly with a candidate mAb starting 3 weeks post HBV inoculation (spreading phase) or during stable HBV or HBV/HDV coinfection (chronic phase). RESULTS: From a panel of human anti-HBs mAbs, VIR-3434 was selected and engineered for pre-clinical development. VIR-3434 targets a conserved, conformational epitope within the antigenic loop of HBsAg and neutralized HBV and HDV infection with higher potency than hepatitis B immunoglobulins in vitro. Neutralization was pan-genotypic against strains representative of HBV genotypes A-H. In the spreading phase of HBV infection in human liver-chimeric mice, a parental mAb of VIR-3434 (HBC34) prevented HBV dissemination and the increase in intrahepatic HBV RNA and covalently closed circular DNA. In the chronic phase of HBV infection or co-infection with HDV, HBC34 treatment decreased circulating HBsAg by >1 log and HDV RNA by >2 logs. CONCLUSIONS: The potently neutralizing anti-HBs mAb VIR-3434 reduces circulating HBsAg and HBV/HDV viremia in human liver-chimeric mice. VIR-3434 is currently in clinical development for treatment of patients with chronic hepatitis B or D. IMPACT AND IMPLICATIONS: Chronic infection with hepatitis B virus and co-infection with hepatitis D virus place approximately 290 million individuals worldwide at risk of severe liver disease and cancer. Available treatments result in low rates of functional cure or require lifelong therapy that does not eliminate the risk of liver disease. We isolated and characterized a potent human antibody that neutralizes hepatitis B and D viruses and reduces infection in a mouse model. This antibody could provide a new treatment for patients with chronic hepatitis B and D.

MP-NeRF: A massively parallel method for accelerating protein structure reconstruction from internal coordinates.

The conversion of proteins between internal and cartesian coordinates is a limiting step in many pipelines, such as molecular dynamics simulations and machine learning models. This conversion is typically carried out by sequential or parallel applications of the Natural extension of Reference Frame (NeRF) algorithm. This work proposes a massively parallel NeRF implementation which, depending on the polymer length, achieves speedups between 400 and 1200× over the previous state-of-the-art. It accomplishes this by dividing the conversion into three main phases: parallel composition of the monomer backbone, assembly of backbone subunits, and parallel elongation of sidechains; and by batching these computations into a minimal number of efficient matrix operations. Special emphasis is placed on reusability and ease of use. We open source the code (available at https://github.com/EleutherAI/mp_nerf) and provide a corresponding python package.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

Functional characterization of 3D protein structures informed by human genetic diversity.

Sequence variation data of the human proteome can be used to analyze 3D protein structures to derive functional insights. We used genetic variant data from nearly 140,000 individuals to analyze 3D positional conservation in 4,715 proteins and 3,951 homology models using 860,292 missense and 465,886 synonymous variants. Sixty percent of protein structures harbor at least one intolerant 3D site as defined by significant depletion of observed over expected missense variation. Structural intolerance data correlated with deep mutational scanning functional readouts for PPARG, MAPK1/ERK2, UBE2I, SUMO1, PTEN, CALM1, CALM2, and TPK1 and with shallow mutagenesis data for 1,026 proteins. The 3D structural intolerance analysis revealed different features for ligand binding pockets and orthosteric and allosteric sites. Large-scale data on human genetic variation support a definition of functional 3D sites proteome-wide.

Identification of Misclassified ClinVar Variants via Disease Population Prevalence.

There is a significant interest in the standardized classification of human genetic variants. We used whole-genome sequence data from 10,495 unrelated individuals to contrast population frequency of pathogenic variants to the expected population prevalence of the disease. Analyses included the ACMG-recommended 59 gene-condition sets for incidental findings and 463 genes associated with 265 OrphaNet conditions. A total of 25,505 variants were used to identify patterns of inflation (i.e., excess genetic risk and misclassification). Inflation increases as the level of evidence supporting the pathogenic nature of the variant decreases. We observed up to 11.5% of genetic disorders with inflation in pathogenic variant sets and up to 92.3% for the variant set with conflicting interpretations. This improved to 7.7% and 57.7%, respectively, after filtering for disease-specific allele frequency. The patterns of inflation were replicated using public data from more than 138,000 genomes. The burden of rare variants was a main contributing factor of the observed inflation, indicating collective misclassified rare variants. We also analyzed the dynamics of re-classification of variant pathogenicity in ClinVar over time, which indicates progressive improvement in variant classification. The study shows that databases include a significant proportion of wrongly ascertained variants; however, it underscores the critical role of ClinVar to contrast claims and foster validation across submitters.

Comparison of phasing strategies for whole human genomes.

Humans are a diploid species that inherit one set of chromosomes paternally and one homologous set of chromosomes maternally. Unfortunately, most human sequencing initiatives ignore this fact in that they do not directly delineate the nucleotide content of the maternal and paternal copies of the 23 chromosomes individuals possess (i.e., they do not 'phase' the genome) often because of the costs and complexities of doing so. We compared 11 different widely-used approaches to phasing human genomes using the publicly available 'Genome-In-A-Bottle' (GIAB) phased version of the NA12878 genome as a gold standard. The phasing strategies we compared included laboratory-based assays that prepare DNA in unique ways to facilitate phasing as well as purely computational approaches that seek to reconstruct phase information from general sequencing reads and constructs or population-level haplotype frequency information obtained through a reference panel of haplotypes. To assess the performance of the 11 approaches, we used metrics that included, among others, switch error rates, haplotype block lengths, the proportion of fully phase-resolved genes, phasing accuracy and yield between pairs of SNVs. Our comparisons suggest that a hybrid or combined approach that leverages: 1. population-based phasing using the SHAPEIT software suite, 2. either genome-wide sequencing read data or parental genotypes, and 3. a large reference panel of variant and haplotype frequencies, provides a fast and efficient way to produce highly accurate phase-resolved individual human genomes. We found that for population-based approaches, phasing performance is enhanced with the addition of genome-wide read data; e.g., whole genome shotgun and/or RNA sequencing reads. Further, we found that the inclusion of parental genotype data within a population-based phasing strategy can provide as much as a ten-fold reduction in phasing errors. We also considered a majority voting scheme for the construction of a consensus haplotype combining multiple predictions for enhanced performance and site coverage. Finally, we also identified DNA sequence signatures associated with the genomic regions harboring phasing switch errors, which included regions of low polymorphism or SNV density.