Search

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has resulted in viral escape from clinically authorized monoclonal antibodies (mAbs), creating a need for mAbs that are resilient to epitope diversification. Broadly neutralizing coronavirus mAbs that are sufficiently potent for clinical development and retain activity despite viral evolution remain elusive. We identified a human mAb, designated VIR-7229, which targets the viral receptor-binding motif (RBM) with unprecedented cross-reactivity to all sarbecovirus clades, including non-ACE2-utilizing bat sarbecoviruses, while potently neutralizing SARS-CoV-2 variants since 2019, including the recent EG.5, BA.2.86, and JN.1. VIR-7229 tolerates extraordinary epitope variability, partly attributed to its high binding affinity, receptor molecular mimicry, and interactions with RBM backbone atoms. Consequently, VIR-7229 features a high barrier for selection of escape mutants, which are rare and associated with reduced viral fitness, underscoring its potential to be resilient to future viral evolution. VIR-7229 is a strong candidate to become a next-generation medicine., Competing Interests: Declaration of interests L.E.R., A.D.M., D.P., D.B., T.R., J.M.E., K.H., H.V.D., M.G., G.C., C.S., F.Z., E.D., A.R., J.N., Q.T., Y.D., B.T., R.A., S.S., B.G., M.A.S., G. Schnell, J.L.M., F.A.L., N.C., E.C., L.A.P., F.B., J.d.I., M.S.P., A.L., A.T., G. Snell, and D.C. are current or previous employees of Vir Biotechnology and may hold shares in Vir Biotechnology. L.E.R., A.D.M., D.P., E.C., F.B., M.S.P., G. Snell, and D.C. are currently listed as inventors on multiple patent applications that disclose the subject matter described in this paper. J.W.C. is an employee and shareholder of ProtaBody. J.W.C. and ProtaBody have received funding from Vir Biotechnology related to the work described in this paper. I.Z., Z.L., S.P.J.W., G.D.d.M., L.K., H.B., and T.N.S. have received funding through sponsored research awards to their respective institutions from Vir Biotechnology related to the work described in this paper. I.Z. is a current employee of Bristol Myers Squibb. L.A.P. is a former employee and shareholder of Regeneron Pharmaceuticals and is a member of the Scientific Advisory Board AI-driven structure-enabled antiviral platform (ASAP). Regeneron provided no funding for this work. L.A.P. is a current employee of Third Rock Ventures. D.V. is named as inventor on patents for coronavirus vaccines filed by the University of Washington. The lab of T.N.S. has received sponsored research agreements unrelated to the present work from Aerium Therapeutics, Inc. and Invivyd, Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

To date, nearly one-quarter of colorectal cancer (CRC) patients develop liver metastases (CRCLM), and its aggressiveness can be correlated to defined histopathological growth patterns (HGP). From the three main HGPs within CRCLM, the replacement HGP emerges as particularly aggressive, characterized by heightened tumor cell motility and vessel co-option. Here, we investigated the correlation between the expression of calcium- and integrin-binding protein 1 (CIB1), a ubiquitously expressed gene involved in various cellular processes including migration and adhesion, and disease-free (DFS) and overall survival (OS) in primary CRC patients. Additionally, we explored the correlation between CIB1 expression and different HGPs of CRCLM. Proteomic analysis was used to evaluate CIB1 expression in a cohort of 697 primary CRC patients. Additionally, single-cell and spatial RNA-sequencing datasets, along with publicly available bulk sequencing data were used to evaluate CIB1 expression in CRCLM. In silico data were further validated by formalin-fixed paraffin-embedded immunohistochemical stainings. We observed that high CIB1 expression is independently associated with worse DFS and OS, regardless of Union Internationale Contre le Cancer stage, gender, or age. Furthermore, the aggressive replacement CRCLM HGP is significantly associated with high CIB1 expression. Our findings show a correlation between CIB1 levels and the clinical aggressiveness of CRC. Moreover, CIB1 may be a novel marker to stratify HGP CRCLM., Competing Interests: The authors declare no competing interests., (© 2024 Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.)

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., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The sudden mortality of African elephants (Loxodonta africana) in Botswana and Zimbabwe in 2020 provoked considerable public interest and speculation. Poaching and malicious poisoning were excluded early on in the investigation. Other potential causes included environmental intoxication, infectious diseases, and increased habitat stress due to ongoing drought. Here we show evidence of the mortalities in Zimbabwe as fatal septicaemia associated with Bisgaard taxon 45, an unnamed close relative of Pasteurella multocida. We analyse elephant carcasses and environmental samples, and fail to find evidence of cyanobacterial or other intoxication. Post-mortem and histological findings suggest a bacterial septicaemia similar to haemorrhagic septicaemia caused by P. multocida. Biochemical tests and 16S rDNA analysis of six samples and genomic analysis of one sample confirm the presence of Bisgaard taxon 45. The genome sequence contains many of the canonical P. multocida virulence factors associated with a range of human and animal diseases, including the pmHAS gene for hyaluronidase associated with bovine haemorrhagic septicaemia. Our results demonstrate that Bisgaard taxon 45 is associated with a generalised, lethal infection and that African elephants are susceptible to opportunistically pathogenic Pasteurella species. This represents an important conservation concern for elephants in the largest remaining metapopulation of this endangered species., (© 2023. Springer Nature Limited.)

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain 1 (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting., (© 2023. The Author(s).)

Relative alchemical binding free energy calculations are routinely used in drug discovery projects to optimize the affinity of small molecules for their drug targets. Alchemical methods can also be used to estimate the impact of amino acid mutations on protein:protein binding affinities, but these calculations can involve sampling challenges due to the complex networks of protein and water interactions frequently present in protein:protein interfaces. We investigate these challenges by extending a graphics processing unit (GPU)-accelerated open-source relative free energy calculation package (Perses) to predict the impact of amino acid mutations on protein:protein binding. Using the well-characterized model system barnase:barstar, we describe analyses for identifying and characterizing sampling problems in protein:protein relative free energy calculations. We find that mutations with sampling problems often involve charge-changes, and inadequate sampling can be attributed to slow degrees of freedom that are mutation-specific. We also explore the accuracy and efficiency of current state-of-the-art approaches─alchemical replica exchange and alchemical replica exchange with solute tempering─for overcoming relevant sampling problems. By employing sufficiently long simulations, we achieve accurate predictions (RMSE 1.61, 95% CI: [1.12, 2.11] kcal/mol), with 86% of estimates within 1 kcal/mol of the experimentally determined relative binding free energies and 100% of predictions correctly classifying the sign of the changes in binding free energies. Ultimately, we provide a model workflow for applying protein mutation free energy calculations to protein:protein complexes, and importantly, catalog the sampling challenges associated with these types of alchemical transformations. Our free open-source package (Perses) is based on OpenMM and is available at https://github.com/choderalab/perses.

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 drift 1 and suboptimal immune responses 2 . 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., (© 2023. The Author(s).)

Objective: Effective educational strategies to train and assess residents' skills in behavioral health competencies have been limited. We hypothesized that a training curriculum using virtual reality (VR) simulations would enhance residents' evidence-based skills related to behavioral health anticipatory guidance (BHAG) and motivational interviewing (MI)., Methods: We conducted a randomized controlled pilot trial of our curriculum within an urban academic clinic. Intervention and control groups received four 15-minute, monthly didactics on BHAG and MI followed by VR simulations to 1) deliberately practice delivering BHAG by verbally counseling avatars, who responded real-time (intervention group) or 2) assessing pediatric respiratory distress (control group). Two months post-VR training, all residents participated in a unique VR behavioral health scenario which was recorded and coded by pediatric psychologists via an observation instrument to assess residents' skills. Differences in BHAG and MI competencies were assessed via independent samples t tests for continuous variables and Fisher's exact tests for categorical variables., Results: Sixty-six percent (n = 55) of eligible residents completed all study protocols comprising 35 intervention and 20 control participants. Intervention participants demonstrated an increase in BHAG skills (P = .002), percentage of open-ended questions asked (P = .04), and percentage of MI adherent behaviors (P = .04) when compared to the control group., Conclusions: VR may be an effective educational strategy for residents to acquire BHAG and MI skills though deliberate practice. Next steps will focus on assessing the generalizability of the VR curriculum as well as exploring the opportunities to enhance its scalability through artificial intelligence., (Copyright © 2022 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.

The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody 1 . Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab 2 , S2X259 3 and S2H97 4 . The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Understanding broadly neutralizing sarbecovirus antibody responses is key to developing countermeasures against SARS-CoV-2 variants and future zoonotic sarbecoviruses. We describe the isolation and characterization of a human monoclonal antibody, designated S2K146, that broadly neutralizes viruses belonging to SARS-CoV- and SARS-CoV-2-related sarbecovirus clades which use ACE2 as an entry receptor. Structural and functional studies show that most of the virus residues that directly bind S2K146 are also involved in binding to ACE2. This allows the antibody to potently inhibit receptor attachment. S2K146 protects against SARS-CoV-2 Beta challenge in hamsters and viral passaging experiments reveal a high barrier for emergence of escape mutants, making it a good candidate for clinical development. The conserved ACE2-binding residues present a site of vulnerability that might be leveraged for developing vaccines eliciting broad sarbecovirus immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission leads to the emergence of variants, including the B.1.617.2 (Delta) variant of concern that is causing a new wave of infections and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing their immune evasion. Mutations in the B.1.617.1 (Kappa) and Delta spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including remodeling of the Delta amino-terminal domain. The angiotensin-converting enzyme 2 binding affinities of the Kappa and Delta receptor binding domains are comparable to the Wuhan-Hu-1 isolate, whereas B.1.617.2+ (Delta+) exhibits markedly reduced affinity.

The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection.

The recent emergence of SARS-CoV-2 variants of concern 1-10 and the recurrent spillovers of coronaviruses 11,12 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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape 1-3 , have activity against diverse sarbecoviruses 4-7 , and be highly protective through viral neutralization 8-11 and effector functions 12,13 . 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 (S2E12 8 ) 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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), which was originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate-based messenger RNA vaccine or from convalescent individuals exhibited neutralizing titers that were reduced 2- to 3.5-fold against the B.1.427/B.1.429 variant relative to wild-type pseudoviruses. The L452R mutation reduced neutralizing activity in 14 of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 of 10 NTD-specific mAbs because the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and the formation of a new disulfide bond, as revealed by mass spectrometry and structural studies., (Copyright © 2021, American Association for the Advancement of Science.)

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., Competing Interests: Declaration of interests A.D.M., F.A.L., D.P., M.B., F.Z., J.d.I., M.M.-R., J.Z., L.E.R., S.B., B.G., C.S.F., F.B., E.C., G.S., A.T., H.W.V., D.C., and M.S.P. are employees of Vir Biotechnology Inc. and may hold shares in Vir Biotechnology Inc. D.C. is currently listed as an inventor on multiple patent applications, which disclose the subject matter described in this manuscript. The Neyts laboratories have received sponsored research agreements from Vir Biotechnology Inc. H.W.V. is a founder of PierianDx and Casma Therapeutics. Neither company provided funding for this work or is performing related work. D.V. is a consultant for Vir Biotechnology Inc. The Veesler laboratory has received a sponsored research agreement from Vir Biotechnology Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics., Competing Interests: Declaration of interests L.E.R., R. Spreafico, J.A.W., L.P., J.D., N.C., M.M., A.D.M., J.B., D.P., K.C., F.Z., S.J., M.B., M.P., E.C., J.D.I., H.W.V., A.T., D.C., and G.S. are or were employees of Vir Biotechnology and may hold shares in Vir Biotechnology. C.G. is an external scientific advisor for Humabs BioMed SA. J. Nix and T.I.C. are consultants with Vir Biotechnology. M.G.S. declares interest in Integrum Scientific, Greensboro, NC, outside the scope of this work. J.D.C. is a current member of the Scientific Advisory Board of OpenEye Scientific Software and is a scientific consultant to Foresite Labs. The Chodera laboratory (I.Z., W.G.G., and J.D.C.) receives or has received funding from multiple sources, including the NIH, the National Science Foundation, the Parker Institute for Cancer Immunotherapy, Relay Therapeutics, Entasis Therapeutics, Silicon Therapeutics, EMD Serono (Merck KGaA), AstraZeneca, Vir Biotechnology, XtalPi, the Molecular Sciences Software Institute, the Starr Cancer Consortium, the Open Force Field Consortium, Cycle for Survival, a Louis V. Gerstner Young Investigator Award, and the Sloan Kettering Institute. A complete funding history for the Chodera lab can be found at https://www.choderalab.org/funding. The other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo-electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics., Competing Interests: Declaration of Interests L.P., N.C., M. Beltramello, C.S.-F., D.P., L.E.R., F.Z., N.S., J.B., A.P., S. Jaconi, B.G., A.M., A.D.M., M.S.P., E.C., S.V.G., F.J., C.H.-D., M.S., D.H., G.S., K.F., H.W.V., A.L., and D.C. are employees of Vir Biotechnology Inc. and may hold shares in Vir Biotechnology Inc. D.C. is currently listed as an inventor on multiple patent applications, which disclose the subject matter described in this manuscript. The Veesler laboratory has received a sponsored research agreement from Vir Biotechnology Inc. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bovine tuberculosis (BTB) remains as a costly disease of cattle-herds in the Republic of Ireland (ROI). This persistence is partially attributable to the presence of M. bovis infection in a wildlife reservoir, the European badger (Meles meles). Thus, both area-wide and limited-area targeted-badger-culling have been part of the ROI-BTB control/eradication program to help reduce the future incidence of a cattle-herd BTB breakdown (i.e. a "new herd-level occurrence of BTB"). However, neither badger-culling practice can be sustained as a major component in the ongoing BTB eradication program in the ROI. Vaccination of badgers with Bacille Calmette-Guérin (BCG) has been proposed as an alternative to badger culling. Thus, in 2011, a five-year non-inferiority study was implemented in seven counties in the ROI. This study was designed to compare and contrast the cattle-herd-BTB-incidence in areas where intramuscular badger vaccination would be implemented versus the cattle-herd-BTB-incidence in the remaining area of the same county where targeted-badger-culling was maintained as the standard treatment response to probable badger-sourced BTB breakdowns. Our outcome of interest was a new cattle-herd-BTB-episode (breakdown) with a total of >2 standard skin-test (SICTT) reactors detected during the episode. Treatments (badger vaccination or targeted badger culling) were cluster allocated based on where the majority of the herd owner's land was located. To assess the impact of the two treatments, we compared the incidence-risk, of our defined outcome, for cattle herds in the area under vaccination to the outcome incidence-risk for cattle herds in the remainder of the same county after 4 and 5 years of having implemented badger vaccination. A random-effects logit model with adjustment for clustering by treatment, and statistical control of herd-type, herd-size and five-year prior-BTB-episode history was used for our analyses. Although not included in the logistic model, a relative badger density metric based on the annual number of badgers captured-per-sett-night of capturing effort was developed for each treatment area; this metric indicated that relative badger density was approximately 40 % higher in vaccination areas than in the targeted badger-culling areas during our study. Overall, our study results indicated that vaccination was not inferior to targeted badger-culling in four counties and badger vaccination was deemed to produce ambivalent results in one (County Cork North) of the seven study sites in the ROI. A post-study investigation, in County Galway, where vaccination was deemed inferior to target culling, revealed that widespread purchases of cattle from a nearby cattle mart, by herd owners in the vaccination-area, was associated with the increased herd and vaccination-area risk of BTB. No single "biasing hypothesis" was evident for the apparent vaccine inferiority in the second study site (County Monaghan) where vaccination was deemed inferior to targeted culling; hence no further investigations were conducted., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage.

Tuberculosis, caused by Mycobacterium tuberculosis , is a disease causing morbidity and mortality in captive elephants ( Elephas maximus and Loxodonta africana ) as well as free-ranging individuals. Elephants in North America diagnosed with tuberculosis are often treated with antituberculosis drugs, unlike livestock species, which has necessitated the development of treatment guidelines adapted from recommendations for humans. There are few published reports describing empirical treatment, which may be complicated by poor patient compliance, interruptions in drug administration, and adverse effects. A survey of elephants in North America was conducted to compile information on treatment protocols, including drugs, dosages, routes of administration, serum drug concentrations, and adverse effects of antituberculosis treatment. Responses were received regarding 182 elephants, 12 of which were treated prophylactically or therapeutically with antituberculosis drugs. Treatment protocols varied among elephants, and included various combinations of isoniazid, rifampin, pyrazinamide, ethambutol, enrofloxacin, levofloxacin, and ethionamide. Serum drug concentrations also varied considerably among and within individuals. Facility staff reported 5 elephants (out of 7 treated elephants with responses) that exhibited clinical signs that may have been associated with antituberculosis drugs or treatment procedures. Anorexia, decreased water intake, constipation, depression, ataxia, limb paresis, and tremors were among the signs observed. Most adverse effects were reported to be moderate or severe, resulting in interruption of the treatment. The results from this survey provide veterinarians and elephant managers with valuable historical data to make informed clinical management decisions regarding antituberculosis therapy in elephants., (© 2019 by American Association of Zoo Veterinarians.)

A recently described nuclear grading system predicted survival in patients with epithelioid malignant pleural mesothelioma. The current study was undertaken to validate the grading system and to identify additional prognostic factors. We analyzed cases of epithelioid malignant pleural mesothelioma from 17 institutions across the globe from 1998 to 2014. Nuclear grade was computed combining nuclear atypia and mitotic count into a grade of I-III using the published system. Nuclear grade was assessed by one pathologist for three institutions, the remaining were scored independently. The presence or absence of necrosis and predominant growth pattern were also evaluated. Two additional scoring systems were evaluated, one combining nuclear grade and necrosis and the other mitotic count and necrosis. Median overall survival was the primary endpoint. A total of 776 cases were identified including 301 (39%) nuclear grade I tumors, 354 (45%) grade II tumors and 121 (16%) grade III tumors. The overall survival was 16 months, and correlated independently with age (P=0.006), sex (0.015), necrosis (0.030), mitotic count (0.001), nuclear atypia (0.009), nuclear grade (<0.0001), and mitosis and necrosis score (<0.0001). The addition of necrosis to nuclear grade further stratified overall survival, allowing classification of epithelioid malignant pleural mesothelioma into four distinct prognostic groups: nuclear grade I tumors without necrosis (29 months), nuclear grade I tumors with necrosis and grade II tumors without necrosis (16 months), nuclear grade II tumors with necrosis (10 months) and nuclear grade III tumors (8 months). The mitosis-necrosis score stratified patients by survival, but not as well as the combination of necrosis and nuclear grade. This study confirms that nuclear grade predicts survival in epithelioid malignant pleural mesothelioma, identifies necrosis as factor that further stratifies overall survival, and validates the grading system across multiple institutions and among both biopsy and resection specimens. An alternative scoring system, the mitosis-necrosis score is also proposed.

Transfrontier conservation areas represent an international effort to encourage conservation and sustainable development. Their success faces a number of challenges, including disease management in wildlife, livestock and humans. Tuberculosis (TB) affects humans and a multitude of non-human animal species and is of particular concern in sub-Saharan Africa. The Kavango-Zambezi Transfrontier Conservation Area encompasses five countries, including Zimbabwe, and is home to the largest contiguous population of free-ranging elephants in Africa. Elephants are known to be susceptible to TB; thus, understanding TB status, exposure and transmission risks to and from elephants in this area is of interest for both conservation and human health. To assess risk factors for TB seroprevalence, a questionnaire was used to collect data regarding elephant management at four ecotourism facilities offering elephant-back tourist rides in the Victoria Falls area of Zimbabwe. Thirty-five working African elephants were screened for Mycobacterium tuberculosis complex antibodies using the ElephantTB Stat-Pak and the DPP VetTB Assay for elephants. Six of 35 elephants (17.1%) were seropositive. The risk factor most important for seropositive status was time in captivity. This is the first study to assess TB seroprevalence and risk factors in working African elephants in their home range. Our findings will provide a foundation to develop guidelines to protect the health of captive and free-ranging elephants in the southern African context, as well as elephant handlers through simple interventions. Minimizing exposure through shared feed with other wildlife, routine TB testing of elephant handlers and regular serological screening of elephants are recommended as preventive measures., (© 2017 Blackwell Verlag GmbH.)

Despite the relatively high prevalence of thyroid cancer, the occurrence of multiple synchronous, distinct subtypes of primary thyroid carcinoma is uncommon. The incidental finding of papillary thyroid microcarcinoma in a gland with a biologically relevant follicular or medullary carcinoma is more frequent than the synchronous occurrence of multiple clinically significant carcinomas. We report a case of synchronous papillary and follicular thyroid carcinomas metastatic to lymph node and bone, respectively. Next generation sequencing showed BRAF V600E mutation in the primary papillary carcinoma and NRAS Q61R mutation in the primary follicular carcinoma and bony metastasis. To our knowledge, this is the first reported case of synchronous and metastatic primary papillary and follicular carcinomas, and the first report of synchronous BRAF V600E mutated papillary and NRAS mutated follicular carcinoma.

Primary neuroendocrine tumors of the breast are a rare and underrecognized subtype of mammary carcinoma. Neuroendocrine tumors of the breast occur predominately in postmenopausal women. The tumors are subclassified into well-differentiated and poorly differentiated neuroendocrine tumors, and invasive breast carcinoma with neuroendocrine features. Well-differentiated tumors show architectural similarity to carcinoids of other sites but lack characteristic neuroendocrine nuclei. Poorly differentiated neuroendocrine tumors are morphologically identical to small cell carcinoma of the lung. Neuroendocrine differentiation, seen in up to 30% of invasive breast carcinomas, is most commonly associated with mucinous and solid papillary carcinomas. The diagnosis of neuroendocrine differentiation requires expression of the neuroendocrine markers synaptophysin or chromogranin. The main differential diagnosis is a metastatic neuroendocrine tumor from an extramammary site. Neuroendocrine tumors of the breast are treated similarly to other invasive breast carcinomas. Although no consensus has been reached on the prognosis, most studies suggest a poor outcome.

Male breast carcinomas (MBCs) are rare neoplasms that account for 0.1% of all male cancers. Still, there are 2000 new cases of MCB diagnosed annually in the United States. Because of its rarity, data regarding the etiology, risk factors, diagnosis, management, and prognosis of MBC are limited. MBC shares some similarities with female breast carcinoma (FBC). This review will address the important clinical, histopathological, immunohistochemical and molecular features, genetics, management, and prognosis of MBC.

Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), is endemic in the Kruger National Park (KNP), South Africa. African lions ( Panthera leo ) are susceptible to BTB, but the impact of the disease on lion populations is unknown. In this study, we used a novel gene expression assay for chemokine (C-X-C motif) ligand 9 (CXCL9) to measure the prevalence of M. bovis infection in 70 free-ranging lions that were opportunistically sampled in the southern and central regions of the KNP. In the southern region of the KNP, the apparent prevalence of M. bovis infection was 54% (95% confidence interval [CI]=36.9-70.5%), compared with 33% (95% CI=18.0-51.8%) in the central region, an important difference (P=0.08). Prevalence of M. bovis infection in lions showed similar patterns to estimated BTB prevalence in African buffaloes ( Syncerus caffer ) in the same areas. Investigation of other risk factors showed a trend for older lions, males, or lions with concurrent feline immunodeficiency virus infection to have a higher M. bovis prevalence. Our findings demonstrate that the CXCL9 gene expression assay is a useful tool for the determination of M. bovis status in free-ranging lions and identifies important epidemiologic trends for future studies.

Primary myoepithelial carcinoma of the lung is a rare neoplasm with only 8 cases reported in the English literature to date. Myoepithelial carcinomas of the lung are thought to arise from submucosal bronchial glands and have morphologic features similar to their salivary gland counterparts. The pathologic features and immunohistochemical profile of this tumor have not yet been summarized in the literature. Our objective is to review the clinicopathologic features and immunohistochemistry of these tumors.

Synchronous renal cell neoplasms (RCNs) and angiomyolipomas (AML) occurring in the same kidney are rare. The aim of this retrospective study is to investigate the incidence and clinicopathological features of concurrent AML and RCN in patients without tuberous sclerosis complex (TSC). Partial and radical nephrectomy specimens with a diagnosis of RCN and AML from 1995 to May 2013 were reviewed. Cases were assessed for histological subtype of AML and RCN, topographic relationship between the AML and the RCN, and clinical characteristics. A total of 565 RCNs and 19 AMLs were identified. Of 19 cases of AML, 9 (47%) were associated with renal tumors. Clear-cell renal cell carcinoma (RCC) was the most common malignancy associated with AML (56%). The mean age of patients with concomitant AML and RCN was higher than the mean age of patients with only AML (58.6 vs 55.5 years). The majority of patients with concomitant AML and RCN were female (78%). All concurrent sporadic AMLs were benign. We identified one case of bilateral clear-cell RCC with concomitant multiple bilateral AMLs in a patient with TSC., (© The Author(s) 2015.)

An important approach to understanding how a protein sequence encodes its energy landscape is to compare proteins with different sequences that fold to the same general native structure. In this work, we compare E. coli and T. thermophilus homologs of the protein RNase H. Using protein fragments, we create equilibrium mimics of two different potential partially-folded intermediates (I(core) and I(core+1)) hypothesized to be present on the energy landscapes of these two proteins. We observe that both T. thermophilus RNase H (ttRNH) fragments are folded and have distinct stabilities, indicating that both regions are capable of autonomous folding and that both intermediates are present as local minima on the ttRNH energy landscape. In contrast, the two E. coli RNase H (ecRNH) fragments have very similar stabilities, suggesting that the presence of additional residues in the I(core+1) fragment does not affect the folding or structure as compared to I(core). NMR experiments provide additional evidence that only the I(core) intermediate is populated by ecRNH. This is one of the biggest differences that has been observed between the energy landscapes of these two proteins. Additionally, we used a FRET experiment in the background of full-length ttRNH to specifically monitor the formation of the I(core+1) intermediate. We determine that the ttRNH I(core+1) intermediate is likely the intermediate populated prior to the rate-limiting barrier to global folding, in contrast to E. coli RNase H for which I(core) is the folding intermediate. This result provides new insight into the nature of the rate-limiting barrier for the folding of RNase H.

The folding pathway of Escherichia coli RNase H is one of the best experimentally characterized for any protein. In spite of this, spectroscopic studies have never captured the earliest events. Using continuous-flow microfluidic mixing, we have now observed the first several milliseconds of folding by monitoring the tryptophan fluorescence lifetime (60 μs dead time). Two folding intermediates are observed, the second of which is the previously characterized I(core) millisecond intermediate. The new earlier intermediate is likely on-pathway and appears to have long-range non-native structure, providing a rare example of such non-native structure formation in a folding pathway. The tryptophan fluorescence lifetimes also suggest a deviation from native packing in the second intermediate, I(core). Similar results from a fragment of RNase H demonstrate that only half of the protein is significantly involved in this early structure formation. These studies give us a view of the formation of tertiary structure on the folding pathway, which complements previous hydrogen-exchange studies that monitored only secondary structure and observed sequential native structure formation. Our results provide detailed folding information on both a timescale and a size-scale accessible to all-atom molecular dynamics simulations of protein folding., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.

Background: Intravascular polymer emboli have been reported in the skin, lungs, and brain following vascular procedures utilizing hydrophilic polymer coated devices. The Cook arterial introducer sheath was the first of these devices to be introduced, after which case reports followed documenting sterile inflammation at the sheath access site, characterized histologically by perivascular granulomas containing hydrophilic polymer. More recently, hydrophilic polymer emboli have been reported in the vessels of the lungs and brain in association with ischemia and infarct following vascular procedures using polymer coated devices., Methods: We report a case of intravascular myocardial emboli associated with acute myocardial infarction following cardiac catheterization. The patient was a 65-year-old man who received cardiac catheterization with placement of a bare metal stent following myocardial infarction. One month later, the patient presented with angina and died shortly after admission., Results: Autopsy revealed hemopericardium with rupture of the left ventricle through an aneurismal defect within the area of prior infarction. Microscopically, an area of acute infarction was present within the aneurismal defect. Numerous small and medium-sized vessels within the left ventricle were occluded by basophilic amorphous granular material with an inflammatory giant cell response., Conclusion: The emboli were most frequent in the area of acute infarction, suggesting that the emboli may have resulted in ischemia leading to the patient's death. This is the third documented case of intramyocardial polymer emboli following cardiac catheterization and the first case to our knowledge to document an association between intravascular myocardial polymer emboli and acute myocardial infarction., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on native-like interfoldon interactions orders the pathway.

We evaluated, refined, and applied well-established polymerase chain reaction (PCR) techniques for detecting Yersinia pestis DNA in fleas (mainly Oropsylla spp.) collected from prairie dog (Cynomys spp.) burrows. Based on results from PCR of avirulent Y. pestis strain A1122 DNA, we used DNA purification and primers for the plasminogen activator gene to screen field-collected fleas. We detected Y. pestis DNA in flea pools from two black-tailed prairie dog (Cynomys ludovicianus) colonies with evidence of recent plague epizootics, and from one of four white-tailed prairie dog (Cynomys leucurus) colony complexes (Wolf Creek) where evidence of epizootic plague was lacking. Relative flea abundance and occurrence of Y. pestis DNA among flea pools appeared to vary over time at Wolf Creek. Both DNA purification and primer sequences appeared to influence the likelihood of detecting Y. pestis DNA by PCR in fleas collected from prairie dog burrows in the absence of observed epizootic plague. Presence of Y. pestis plasmid DNA in fleas collected from prairie dog burrows at Wolf Creek may represent evidence that infected fleas were somehow being maintained in that system between epizootics, consistent with the hypothesized enzootic maintenance of plague in prairie dog colony complexes elsewhere.

Periodic pneumonia outbreaks cause large-scale die-offs that threaten the viability of bighorn sheep (Ovis canadensis) populations. Bighorns are highly susceptible to pneumonia, and in some cases this susceptibility may be exacerbated by trace mineral deficiencies. To evaluate responses to injectable selenium supplementation, eight captive bighorn sheep were treated with either an injectable sodium selenite supplement or a saline control. We collected 6-ml blood aliquots before and at 1, 6, and 12 wk posttreatment. We submitted one set of aliquots immediately to measure selenium (Se) and zinc (Zn) concentrations and glutathione-peroxidase (GSH-Px) activity; additional aliquots were held at about 22 C and then submitted at 1, 3, and 7 days postcollection to assess effects of storage on these measures. Neither Se nor GSH-Px were affected by selenite injections. Both Se and GSH-Px demonstrated small linear decays over the 7-day storage period (0.011 ppm/day [SE=0.0027] and 15.78 mmole/l/sec/day [SE=6.88], respectively); in contrast, Zn concentrations in stored samples increased logarithmically (0.35 ppm/day on the natural log scale). Blood Se and GSH-Px were not correlated in sampled bighorns; however, because all values for both measures were within normal limits, lack of correlation did not affect interpretation of these data in our study.

This document, approved by the Rehabilitation Engineering & Assistive Technology Society of North America (RESNA) Board of Directors in September 2005, shares typical clinical applications and provides evidence from the literature supporting the use of seat-elevating devices for wheelchair