Showing papers by "University of Texas Medical Branch published in 2021"

Spike mutation D614G alters SARS-CoV-2 fitness.

Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic. Rapidly spreading SARS-CoV-2 variants may jeopardize newly introduced antibody and vaccine countermeasures. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera and human sera from recipients of the BNT162b2 mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, chimeric strains with South African or Brazilian spike genes and isogenic recombinant viral variants. Many highly neutralizing mAbs engaging the receptor-binding domain or N-terminal domain and most convalescent sera and mRNA vaccine-induced immune sera showed reduced inhibitory activity against viruses containing an E484K spike mutation. As antibodies binding to spike receptor-binding domain and N-terminal domain demonstrate diminished neutralization potency in vitro against some emerging variants, updated mAb cocktails targeting highly conserved regions, enhancement of mAb potency or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera.

Abstract: We engineered three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion + N501Y + D614G from UK; and E484K + N501Y + D614G from SA. Neutralization geometric mean titers (GMTs) of 20 BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses. Human sera from recipients of the BNT162b2 vaccine can neutralize SARS-CoV-2 viruses containing spike mutations present in globally circulating variants of concern.

Neutralizing Activity of BNT162b2-Elicited Serum.

Abstract: Variant SARS-CoV-2 and BNT162b2 Vaccine How well do serum samples obtained from persons injected with the BNT162b2 vaccine neutralize the P.1, B.1.1.7, and B.1.135 lineages of SARS-CoV-2, first ide...

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses.

Abstract: SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-191–5. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. Here we examined antigen-specific B cell responses in peripheral blood (n = 41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene1. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, we identified germinal centre B cells that bound S protein in all participants who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity. Analysis of antigen-specific B cells in lymph nodes of individuals vaccinated with BNT162b2 reveals lasting germinal centre responses, explaining the robust humoral immunity induced by SARS-CoV-2 mRNA-based vaccines.

BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans.

Abstract: BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) that encodes the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy in preventing coronavirus disease-19 (COVID-19)1. Here we extend our previous phase 1/2 trial report2 and present BNT162b2 prime/boost induced immune response data from a second phase 1/2 trial in healthy adults (18-55 years of age). BNT162b2 elicited strong antibody responses, with SARS-CoV-2 serum 50% neutralizing geometric mean titers up to 3.3-fold above those observed in COVID-19 human convalescent samples (HCS) one week post-boost. BNT162b2-elicited sera neutralized 22 pseudoviruses bearing SARS-CoV-2 S variants. Most participants had a strong IFNγ- or IL-2-positive CD8+ and CD4+ T helper type 1 (TH1) T cell response, detectable throughout the full observation period of nine weeks following the boost. pMHC multimer technology identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week post-boost, epitope-specific CD8+ T cells of the early differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes conserved in a broad range of variants at well tolerated doses.

BNT162b vaccines protect rhesus macaques from SARS-CoV-2.

Abstract: A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD–foldon) BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)) The flexibly tethered RBDs of the RBD–foldon bind to human ACE2 with high avidity Approximately 20% of the S(P2) trimers are in the two-RBD ‘down’, one-RBD ‘up’ state In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses Prime–boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 82–182× that of a panel of SARS-CoV-2-convalescent human sera The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement Both candidates are being evaluated in phase I trials in Germany and the USA1–3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728) BNT162b1 and BNT162b2 are two candidate mRNA vaccines against COVID-19 that elicit high virus-entry inhibition titres in mice, elicit high virus-neutralizing titres in rhesus macaques and protect macaques from SARS-CoV-2 challenge

Covid-19 pandemic and the social determinants of health.

Abstract: Lauren Paremoer and colleagues call for action to create a fairer and more sustainable post-covid world

Amyloid oligomers: A joint experimental/computational perspective on Alzheimer's disease, Parkinson's disease, type II diabetes, and amyotrophic lateral sclerosis

Abstract: Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo, and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aβ, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

SARS-CoV-2 Neutralization with BNT162b2 Vaccine Dose 3.

Abstract: Immune Response to a Third Dose of BNT162b2 A third dose of the BNT162b2 vaccine was administered to 23 volunteers 8 to 9 months after the second dose, and the immune response was assessed. Local r...

BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to evolve around the world, generating new variants that are of concern on the basis of their potential for altered transmissibility, pathogenicity, and coverage by vaccines and therapeutic agents1–5. Here we show that serum samples taken from twenty human volunteers, two or four weeks after their second dose of the BNT162b2 vaccine, neutralize engineered SARS-CoV-2 with a USA-WA1/2020 genetic background (a virus strain isolated in January 2020) and spike glycoproteins from the recently identified B.1.617.1, B.1.617.2, B.1.618 (all of which were first identified in India) or B.1.525 (first identified in Nigeria) lineages. Geometric mean plaque reduction neutralization titres against the variant viruses—particularly the B.1.617.1 variant—seemed to be lower than the titre against the USA-WA1/2020 virus, but all sera tested neutralized the variant viruses at titres of at least 1:40. The susceptibility of the variant strains to neutralization elicited by the BNT162b2 vaccine supports mass immunization as a central strategy to end the coronavirus disease 2019 (COVID-19) pandemic globally. Samples of serum from individuals immunized with the BNT162b2 vaccine show neutralization activity against engineered SARS-CoV-2s bearing the spike mutations from B.1.617 and other variants.

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

Abstract: The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant

Abstract: SARS-CoV-2 Delta variant has rapidly replaced the Alpha variant around the world. The mechanism that drives this global replacement has not been defined. Here we report that Delta spike mutation P681R plays a key role in the Alpha-to-Delta variant replacement. In a replication competition assay, Delta SARS-CoV-2 efficiently outcompeted the Alpha variant in human lung epithelial cells and primary human airway tissues. Delta SARS-CoV-2 bearing the Alpha-spike glycoprotein replicated less efficiently than the wild-type Delta variant, suggesting the importance of Delta spike in enhancing viral replication. The Delta spike has accumulated mutation P681R located at a furin cleavage site that separates the spike 1 (S1) and S2 subunits. Reverting the P681R mutation to wild-type P681 significantly reduced the replication of Delta variant, to a level lower than the Alpha variant. Mechanistically, the Delta P681R mutation enhanced the cleavage of the full-length spike to S1 and S2, leading to increased infection via cell surface entry. In contrast, the Alpha spike also has a mutation at the same amino acid (P681H), but the spike cleavage from purified Alpha virions was reduced compared to the Delta spike. Collectively, our results indicate P681R as a key mutation in enhancing Delta variant replication via increased S1/S2 cleavage. Spike mutations that potentially affect furin cleavage efficiency must be closely monitored for future variant surveillance.

Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail.

Abstract: Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses.

In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains.

Abstract: Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-21-3, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales

Abstract: In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

The coronavirus proofreading exoribonuclease mediates extensive viral recombination.

Abstract: Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants

Abstract: LY-CoV1404 (also known as bebtelovimab) is a highly potent, neutralizing, SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody identified from a convalescent COVID-19 patient sample, obtained approximately 60 days after symptom onset. In pseudovirus studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.617.2, B.1.1.7, B.1.351, B.1.427/B.1.429, P.1, and B.1.526, binding to these variants in the presence of their underlying RBD mutations (which include K417N, L452R, E484K, and N501Y). LY-CoV1404 also neutralizes multiple isolates of the authentic SARS-CoV-2 virus in two different assays. The RBD positions comprising the LY-CoV1404 epitope are highly conserved, with the exception of N439 and N501; notably the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). New variant-resistant treatments such as LY-CoV1404 are desperately needed, given that some of the existing therapeutic antibodies are less effective or ineffective against certain variants and the impact of variants on vaccine efficacy is still poorly understood. The breadth of variant binding, potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 is one in a panel of well-characterized, clinically developable antibodies that could be deployed as potentially long-term solutions to address current and emerging variants. In Brief LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and all known variants of concern including the B.1.617.2 (Delta) variant No loss of potency against currently circulating variants Binding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID database Breadth of neutralizing activity and potency supports clinical development

LY-CoV1404 potently neutralizes SARS-CoV-2 variants

Abstract: Summary LY-CoV1404 is a highly potent, neutralizing, SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody identified from a convalescent COVID-19 patient approximately 60 days after symptom onset. In pseudovirus studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.427/B.1.429, P.1, and B.1.526 and binds to these variants in the presence of their underlying RBD mutations (which include K417N, L452R, E484K, and N501Y). LY-CoV1404 also neutralizes authentic SARS-CoV-2 in two different assays against multiple isolates. The RBD positions comprising the LY-CoV1404 epitope are highly conserved, with the exception of N439 and N501; notably the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of variant binding, potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 is one in a panel of well-characterized, clinically developable antibodies that could be deployed rapidly to address current and emerging variants. New variant-resistant treatments such as LY-CoV1404 are desperately needed, given that some of the existing therapeutic antibodies are less effective or ineffective against certain variants and the impact of variants on vaccine efficacy is still poorly understood. In Brief LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and all known variants of concern No loss of potency against current variants Binding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID database Breadth of neutralizing activity and potency supports clinical development

Establishment of an African green monkey model for COVID-19 and protection against re-infection.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for an unprecedented global pandemic of COVID-19. Animal models are urgently needed to study the pathogenesis of COVID-19 and to screen vaccines and treatments. We show that African green monkeys (AGMs) support robust SARS-CoV-2 replication and develop pronounced respiratory disease, which may more accurately reflect human COVID-19 cases than other nonhuman primate species. SARS-CoV-2 was detected in mucosal samples, including rectal swabs, as late as 15 days after exposure. Marked inflammation and coagulopathy in blood and tissues were prominent features. Transcriptome analysis demonstrated stimulation of interferon and interleukin-6 pathways in bronchoalveolar lavage samples and repression of natural killer cell- and T cell-associated transcripts in peripheral blood. Despite a slight waning in antibody titers after primary challenge, enhanced antibody and cellular responses contributed to rapid clearance after re-challenge with an identical strain. These data support the utility of AGM for studying COVID-19 pathogenesis and testing medical countermeasures.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.

Abstract: The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

Neutralization of N501Y mutant SARS-CoV-2 by BNT162b2 vaccine-elicited sera

Abstract: Rapidly spreading variants of SARS-CoV-2 that have arisen in the United Kingdom and South Africa share the spike N501Y substitution, which is of particular concern because it is located in the viral receptor binding site for cell entry and increases binding to the receptor (angiotensin converting enzyme 2). We generated isogenic N501 and Y501 SARS-CoV-2. Sera of 20 participants in a previously reported trial of the mRNA-based COVID-19 vaccine BNT162b2 had equivalent neutralizing titers to the N501 and Y501 viruses.

Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape.

Abstract: Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.