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Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and -RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin isotypes capable of blocking infection.Methods: We studied spike- and RBD-specific immunoglobulin isotypes in convalescent and acute plasma/serum samples using a multiplex bead assay. We also determined virus neutralization activities in plasma and serum samples, and purified immunoglobulin fractions using a vesicular stomatitis pseudovirus assay.Results: Spike- and RBD-specific immunoglobulin (Ig) M, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency.Conclusion: IgG, IgM, and IgA are critical components of convalescent plasma used for treatment of coronavirus disease 2019 (COVID-19). [ABSTRACT FROM AUTHOR]

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used for clinical trials and therapies across the country. Using this protocol, about 80% of convalescent donor plasmas were potentially suitable for therapies. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs. Author summary: The development of robust and specific serologic assays to detect antibodies to SARS-CoV-2 is essential to understand the pandemic evolution and establish mitigation strategies. Here, we report the emergency development, production and application of a versatile ELISA test for detecting antibodies against the whole spike protein and its receptor binding domain. Over half million tests have been freely distributed in public and private health institutions of Argentina for evaluating immune responses, convalescent plasma programs and for large seroprevalence studies in neighborhoods and health care workers. We are still learning how and when to use serologic testing in different epidemiological settings. This program allowed us to produce large amount of high quality data on antibody levels in symptomatic and asymptomatic SARS-CoV-2 infections and generate relevant information about IgM and IgG seroconversion time and kinetics. We also present standardized protocols for antibody quantification as guidance for convalescent donor plasma selection in hospitals throughout the country for compassionate use and clinical trials. Here, we provide a framework for generating widely available tools, protocols and information of antibody responses for pandemic management. [ABSTRACT FROM AUTHOR]

Nipah virus (NiV) is a highly pathogenic paramyxovirus capable of causing severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, underscoring the urgent need for the development of countermeasures. The NiV surface-displayed glycoproteins, NiV-G and NiV-F, mediate host cell attachment and fusion, respectively, and are heavily targeted by host antibodies. Here, we describe a vaccination-derived neutralizing monoclonal antibody, mAb92, that targets NiV-F. Structural characterization of the Fab region bound to NiV-F (NiV-F-Fab92) by cryo-electron microscopy analysis reveals an epitope in the DIII domain at the membrane distal apex of NiV-F, an established site of vulnerability on the NiV surface. Further, prophylactic treatment of hamsters with mAb92 offered complete protection from NiV disease, demonstrating beneficial activity of mAb92 in vivo. This work provides support for targeting NiV-F in the development of vaccines and therapeutics against NiV. [ABSTRACT FROM AUTHOR]

As SARS-CoV-2 variants continue to emerge capable of evading neutralizing antibodies, it has become increasingly important to fully understand the breadth and functional profile of T cell responses to determine their impact on the immune surveillance of variant strains. Here, sampling healthy individuals, we profiled the kinetics and polyfunctionality of T cell immunity elicited by mRNA vaccination. Modeling of anti-spike T cell responses against ancestral and variant strains of SARS-CoV-2 suggested that epitope immunodominance and cross-reactivity are major predictive determinants of T cell immunity. To identify immunodominant epitopes across the viral proteome, we generated a comprehensive map of CD4 + and CD8 + T cell epitopes within non-spike proteins that induced polyfunctional T cell responses in convalescent patients. We found that immunodominant epitopes mainly resided within regions that were minimally disrupted by mutations in emerging variants. Conservation analysis across historical human coronaviruses combined with in silico alanine scanning mutagenesis of non-spike proteins underscored the functional importance of mutationally-constrained immunodominant regions. Collectively, these findings identify immunodominant T cell epitopes across the mutationally-constrained SARS-CoV-2 proteome, potentially providing immune surveillance against emerging variants, and inform the design of next-generation vaccines targeting antigens throughout SARS-CoV-2 proteome for broader and more durable protection., Competing Interests: Competing interests: CCB is a Bridge Fellow of the Parker Institute of Cancer Immunotherapy (PICI) and received research support. MB is a PICI Scholar. TO is an employee of Imprint Labs and a consultant for CDI Labs, Shennon Biotechnologies, and PopVax. BDG has received honoraria for speaking engagements from Merck, Bristol Meyers Squibb, and Chugai Pharmaceuticals; has received research funding from Bristol Meyers Squibb, Merck, and ROME Therapeutics; and has been a compensated consultant for Darwin Health, Merck, PMV Pharma, Shennon Biotechnologies, and Rome Therapeutics of which he is a co-founder. NB serves as an advisor/board member for Apricity, Break Bio, Carisma Therapeutics, CureVac, Genotwin, Novartis, Primevax, Rome Therapeutics, and Tempest Therapeutics; as a consultant for Genentech, Novartis, and ATP; receives research support from Dragonfly Therapeutics, Harbour Biomed Sciences, Regeneron Pharmaceuticals, and Ludwig Institute for Cancer Research; is an extramural member of PICI and receives research support. The remaining authors did not declare competing interests.

Rationale: SARS-CoV-2 entry into host cells is facilitated by endogenous and exogenous proteases that proteolytically activate the spike glycoprotein and antiproteases inhibiting this process. Understanding the key actors in viral entry is crucial for advancing knowledge of virus tropism, pathogenesis, and potential therapeutic targets., Objectives: We aimed to investigate the role of naïve serum and alpha-1-antitrypsin (AAT) in inhibiting protease-mediated SARS-CoV-2 entry and explore the implications of AAT deficiency on susceptibility to different SARS-CoV-2 variants., Findings: Our study demonstrates that naïve serum exhibits significant inhibition of SARS-CoV-2 entry, with AAT identified as the major serum protease inhibitor potently restricting entry. Using pseudoparticles, replication-competent pseudoviruses, and authentic SARS-CoV-2, we show that AAT inhibition occurs at low concentrations compared with those in serum and bronchoalveolar tissues, suggesting physiological relevance. Furthermore, sera from subjects with an AAT-deficient genotype show reduced ability to inhibit entry of both Wuhan-Hu-1 (WT) and B.1.617.2 (Delta) but exhibit no difference in inhibiting B.1.1.529 (Omicron) entry., Conclusions: AAT may have a variant-dependent therapeutic potential against SARS-CoV-2. Our findings highlight the importance of further investigating the complex interplay between proteases, antiproteases, and spike glycoprotein activation in SARS-CoV-2 and other respiratory viruses to identify potential therapeutic targets and improve understanding of disease pathogenesis.

Bats are significant reservoir hosts for many viruses with zoonotic potential1. SARS-CoV-2, Ebola virus, and Nipah virus are examples of such viruses that have caused deadly epidemics and pandemics when spilled over from bats into human and animal populations2,3. Careful surveillance of viruses in bats is critical for identifying potential zoonotic pathogens. However, metagenomic surveys in bats often do not result in full-length viral sequences that can be used to regenerate such viruses for targeted characterization4. Here, we identify and characterize a novel morbillivirus from a vespertilionid bat species (Myotis riparius) in Brazil, which we term myotis bat morbillivirus (MBaMV). There are 7 species of morbilliviruses including measles virus (MeV), canine distemper virus (CDV) and rinderpest virus (RPV)5. All morbilliviruses cause severe disease in their natural hosts6-10, and pathogenicity is largely determined by species specific expression of canonical morbillivirus receptors, CD150/SLAMF111 and NECTIN412. MBaMV used Myotis spp CD150 much better than human and dog CD150 in fusion assays. We confirmed this using live MBaMV that was rescued by reverse genetics. Surprisingly, MBaMV replicated efficiently in primary human myeloid but not lymphoid cells. Furthermore, MBaMV replicated in human epithelial cells and used human NECTIN4 almost as well as MeV. Our results demonstrate the unusual ability of MBaMV to infect and replicate in some human cells that are critical for MeV pathogenesis and transmission. This raises the specter of zoonotic transmission of a bat morbillivirus.

The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales suggests herd immunity may fail to eliminate the virus. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC 90 ) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines., Competing Interests: Competing interests: B.L. and K.Y.O. are named inventors on a patent filed by the Icahn School of Medicine for some of the materials used in this work. J.P.K. is a consultant for BioNTech (advisory panel on coronavirus variants).

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) plays critical roles in host cell entry. Non-synonymous substitutions affecting S are not uncommon and have become fixed in a number of SARS-CoV-2 lineages. A subset of such mutations enable escape from neutralizing antibodies or are thought to enhance transmission through mechanisms such as increased affinity for the cell entry receptor, angiotensin-converting enzyme 2 (ACE2). Independent genomic surveillance programs based in New Mexico and Louisiana contemporaneously detected the rapid rise of numerous clade 20G (lineage B.1.2) infections carrying a Q677P substitution in S. The variant was first detected in the US on October 23, yet between 01 Dec 2020 and 19 Jan 2021 it rose to represent 27.8% and 11.3% of all SARS-CoV-2 genomes sequenced from Louisiana and New Mexico, respectively. Q677P cases have been detected predominantly in the south central and southwest United States; as of 03 Feb 2021, GISAID data show 499 viral sequences of this variant from the USA. Phylogenetic analyses revealed the independent evolution and spread of at least six distinct Q677H sub-lineages, with first collection dates ranging from mid-August to late November 2020. Four 677H clades from clade 20G (B.1.2), 20A (B.1.234), and 20B (B.1.1.220, and B.1.1.222) each contain roughly 100 or fewer sequenced cases, while a distinct pair of clade 20G clusters are represented by 754 and 298 cases, respectively. Although sampling bias and founder effects may have contributed to the rise of S:677 polymorphic variants, the proximity of this position to the polybasic cleavage site at the S1/S2 boundary are consistent with its potential functional relevance during cell entry, suggesting parallel evolution of a trait that may confer an advantage in spread or transmission. Taken together, our findings demonstrate simultaneous convergent evolution, thus providing an impetus to further evaluate S:677 polymorphisms for effects on proteolytic processing, cell tropism, and transmissibility., Competing Interests: Conflicts of Interest. V.S.C. and D.J.S. are co-founders of Microbial Genome Sequencing Center, LLC. All other authors declare no conflicts of interest.

Background: SARS-CoV-2 has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and - RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin (Ig) isotypes capable of blocking infection., Methods: We studied spike- and RBD-specific Ig isotypes in convalescent and acute plasma/sera using a multiplex bead assay. We also determined virus neutralization activities in plasma, sera, and purified Ig fractions using a VSV pseudovirus assay., Results: Spike- and RBD-specific IgM, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency., Conclusion: IgG, IgM and IgA are critical components of convalescent plasma used for COVID-19 treatment., Competing Interests: The authors declare no competing interests.

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVΔG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.