Spike Protein Receptor Binding Research Articles

Differential use of complementarity-determining regions by synthetic nanobodies identifies multiple epitopes on receptor binding domain of SARS-CoV2

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, enters host cells through the receptor angiotensin-converting enzyme 2 (ACE2). Identification of numerous variant strains of SARS-CoV-2 have demanded unprecedented attention in developing rapid diagnostics, effective therapies, and safe vaccines to bring the current pandemic under control. Rapid progress is underway in the characterization of fundamental aspects of the structure and mechanisms of viral adsorption to cells, entry, and replication as well as in studies of the immune response to the virus. Here we report binding studies and crystal structures of the spike protein receptor binding domain (RBD) in complex with two individual synthetic nanobodies (sybodies) (Sb16 and Sb45). These bind the RBD at the previously identified ACE2 interface, positioning their complementarity determining region (CDR)2 and CDR3 in diametrically opposite orientations with large buried surface area. We also solved a structure of the RBD simultaneously bound by two sybodies, Sb45 and Sb68. In this structure, Sb45 binds at the ACE2 interface and Sb68 interacts at a second site. Comparison of structures of Sb16 both free and bound to RBD reveals a large movement of its CDR2 loop. Structural insights gained from these synthetic nanobody complexes will be helpful in designing new vaccines and expand the possibilities of using high affinity antibodies or linked bifunctional antibodies for therapy.

731 FAILURE TO DEVELOP NEUTRALIZING ANTIBODIES FOLLOWING SARS-COV-2 INFECTION IN YOUNG PATIENTS WITH INFLAMMATORY BOWEL DISEASE RECEIVING BIOLOGICS

Objectives and Study: SARS-CoV-2, a novel coronavirus causing the pandemic clinical disease COVID-19, is generally associated with mild disease in most young patients with inflammatory bowel disease (IBD) despite their receiving immunosuppressive therapy. The aim of this study was to longitudinally evaluate characteristics of clinical and serologic response to SARS-CoV-2 infection in a cohort of young patients with IBD receiving biologics. Methods: Starting May 2020 we obtained serum on patients with IBD at the time of infusion with either infliximab or vedolizumab, along with baseline demographic and clinical data, as well possible SARSCoV2 exposure history (patient, family member). To measure antibodies to SARS-CoV-2, we used a fluorescent bead-based immunoassay that takes advantage of the high dynamic range of fluorescent molecules using flow cytometry. We immobilized biotinylated SARS-CoV-2 Spike protein receptor binding domain (S-RBD) or Nucleocapsid protein of SARS-CoV-2 (N) to detect specific IgG antibodies to the virus in patient serum. Spike protein-RBD-specific antibodies were detectable in serial dilutions up to 100,000-fold of serum samples. Titration curves from COVID-19 convalescent and healthy controls were used to normalize the area under the curve (AUC) values to quantitate the antibody levels. Antibody isotypes were measured using anti-Ig (IgG, IgA, IgM, IgG1-4) specific secondary antibodies conjugated to a fluorescent tag. We used a sensitive and high throughput SARS-CoV-2 neutralization assay using a lentivirus that expresses Spike protein to assess specific inhibition of viral entry. The results shown reflect the first serum sample obtained from unique patients. Results: 410 subjects were studied (mean age 17 years, 59% male, 305 (74%) Crohn's disease, 105 (25%) ulcerative colitis/IBD-U, 341 (83%) on infliximab, 69 (17%) vedolizumab, 13% concomitant methotrexate. 27/410 (6.6%) were positive for S-RBD and Nucleocapsid specific IgG. AUC values varied from 3150 to 285724 (Fig 1). S-RBD specific IgA+ 4/27 (15%) and IgG1+ 20/27 (74%) were found. Other isotypes undetectable. Patients' serum efficiently neutralized the virus at up to 10,000-fold serial dilution in only 10/27 (37%). (Fig 2). No differences in age, gender, diagnosis, or specific therapies were noted for (+) vs (-) anti-SARS-CoV-2 antibody status. 13/27 (48%) patients were asymptomatic. Non-exclusive symptoms were rhinorrhea 9 (33%), headache 8 (30%), sore throat 4 (15%). cough 4 (15%), diarrhea 4 (15%), chills 3 (11%), loss of smell/taste 2 (7%), fever 1 (4%). No patient was hospitalized. 4 (15%) had a family member with PCR+ COVID-19. Conclusions: We found a significant prevalence of anti SARS-CoV-2 antibody in our IBD population with the majority of + patients having non-neutralizing antibody. The role of biologics in mitigating clinical and serologic response to SARS-CoV-2 requires exploration.

Titers, prevalence and duration of SARS-CoV-2 antibodies in two waves of a local COVID-19 outbreak

Abstract Information concerning the development of neutralizing antibodies and their duration will be critical to establishing herd immunity for COVID-19. We sought to evaluate SARS-CoV-2 spike protein receptor binding domain (RBD)-specific antibodies, their duration and capacity for SARS-CoV-2 neutralization in volunteers while the pandemic spread within our community starting in March 2020. Those participants with the highest starting titers had the longest lasting response, up to 9 months post-diagnosis. SARS-CoV-2 neutralization capacity was correlated with anti-RBD antibody levels. The majority of our participants with confirmed COVID-19 diagnosis had very mild or asymptomatic infections. We also detected low and largely non-neutralizing anti-RBD IgG titers in a few participants with no known COVID-19 diagnosis. Finally, we found that antibody responses induced by vaccination were significantly higher than those induced by natural infection. Thus, our study suggests that vaccination is still critical even for those naturally infected or diagnosed with COVID-19.

Molecular Docking and Dynamics Simulation of a Screening Library from Life Chemicals Database for Potential Protein-Protein Interactions (PPIs) Inhibitors against SARS-CoV-2 Spike Protein

The ongoing pandemic of coronavirus 2 represents a major challenge for global public health authorities. Coronavirus disease 2019 can be fatal especially in elderly people and those with comorbidities. Currently, several vaccines against coronavirus 2 are under application in multiple countries with emergency use authorization. In the same time, many vaccine candidates are under development and assessment. It is worth noting that the design of some of these vaccines depends on the expression of receptor binding domain for viral spike protein to induce host immunity. As such, blocking the spike protein interface with antibodies, peptides or small molecular compounds can impede the ability of coronavirus 2 to invade host cells by intervention with interactions between viral spike protein and cellular angiotensin converting enzyme 2. In this virtual screening study, we have used predictive webservers, molecular docking and dynamics simulation to evaluate the ability of 3000 compounds to interact with interface residues of spike protein receptor binding domain. This library of chemicals was focused by Life Chemicals as potential protein-protein interactions inhibitor. Here, we report that hit compound 7, with IUPAC name of 3‐cyclohexyl‐N‐(4‐{[(1R,9R) ‐6‐oxo‐7,11‐ diazatricyclo [7.3.1.02,7] trideca‐2,4‐dien‐11‐yl] sulfonyl} phenyl) propenamide, may have the capacity to interact with interface of receptor binding domain for viral spike protein and thereby reduce cellular entry of the virus. However, in vitro and in vivo assessments may be required to validate these virtual findings.

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization "Hotspots" in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction.

The novel severe respiratory syndrome-like coronavirus (SARS-CoV-2) causes COVID-19 in humans and is responsible for one of the most destructive pandemics of the last century. At the root of SARS-CoV infection is the interaction between the viral spike protein and the human angiotensin converting enzyme 2 protein, which allows the virus to gain entry into host cells through endocytosis. In this work, we apply hydrogen–deuterium exchange mass spectrometry (HDX-MS) to provide a detailed view of the functional footprint and conformational dynamics associated with this interaction. Our results broadly agree with the binding interface derived from high resolution X-ray crystal structure data but also provide insights into shifts in structure and dynamics that accompany complexation, including some that occur immediately outside of the core binding interface. We propose that dampening of these “binding-site adjacent” dynamic shifts could represent a mechanism for neutralizing activity in a multitude of spike protein-targeted mAbs that have been found to specifically bind these “peripheral” sites. Our results highlight the unique capacity of HDX-MS to detect potential neutralization “hotspots” outside of the core binding interfaces defined by high resolution structural data.

A New SARS-CoV-2 Dual-Purpose Serology Test: Highly Accurate Infection Tracing and Neutralizing Antibody Response Detection.

Many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology tests have proven to be less accurate than expected and do not assess antibody function as neutralizing, correlating with protection from reinfection. A new assay technology measuring the interaction of the purified SARS-CoV-2 spike protein receptor binding domain (RBD) with the extracellular domain of the human angiotensin-converting enzyme 2 (hACE2) receptor detects these important antibodies. The cPass surrogate virus neutralization test (sVNT), compared directly with eight SARS-CoV-2 IgG serology and two live-cell neutralization tests, gives similar or improved accuracy for qualitative delineation between positive and negative individuals in a fast, scalable, and high-throughput assay. The combined data support the cPass sVNT as a tool for highly accurate SARS-CoV-2 immunity surveillance of infected/recovered and/or vaccinated individuals as well as drug and convalescent-phase donor screening. The data also preview a novel application for the cPass sVNT in calibrating the stringency of live-cell neutralization tests and its use in longitudinal testing of recovered and/or vaccinated patients.

Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein.

The outbreak and worldwide pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have a significant impact on global economy and human health. In order to reduce the disease spread, 16 monoclonal antibodies (McAbs) again SARS-CoV-2 were generated by immunized mice with the spike protein receptor binding domain (RBD), which was expressed in Chinese hamster ovary cell (CHO). A colloidal gold-based immunochromatographic strip was developed with two McAbs to detect SARS-CoV-2 spike protein, which can play a potential role in monitoring vaccine quality. The strip is highly specific, detecting only SARS-CoV-2 spike protein, and does not show any non-specific reactions with syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and other coronavirus and influenza viruses. The strip detected subunit vaccine in our laboratory with a detection limit of spike protein of 62.5 ng/mL. This strip provides an effective method in monitoring vaccine quality by detecting the antigen content of spike protein.

Head-to-head comparison of two rapid high-throughput automated electrochemiluminescence immunoassays targeting total antibodies to the SARS-CoV-2 nucleoprotein and spike protein receptor binding domain.

BackgroundAccurate anti-SARS-CoV-2 assays are needed to inform diagnostic, therapeutic, and public health decisions. The first manufacturer-independent head-to-head comparison of two rapid high-throughput automated electrochemiluminescence double-antigen sandwich immunoassays targeting total anti-SARS-CoV-2 antibodies against two different viral proteins, Elecsys Anti-SARS-CoV-2 (Elecsys-N) and Elecsys Anti-SARS-CoV-2 S (Elecsys-S) (Roche Diagnostics), was performed in a routine setting during the exponential growth phase of the epidemic’s second wave.MethodsThe diagnostic specificity of Elecsys-N and Elecsys-S was initially evaluated on a panel of 572 pre−COVID-19 samples, showing 100 % specificity of both assays. Elecsys-N/Elecsys-S head-to-head comparison used 3,416 consecutive blood samples from individuals that were tested for the presence of anti-SARS-CoV-2 within commercial out-of-pocket serologic testing.ResultsElecsys-N/Elecsys-S head-to-head comparison showed overall agreement of 98.68 % (3,371/3,416; 95 % CI, 98.23–99.03 %), positive agreement of 95.16 % (884/929; 95 % CI, 93.52–96.41 %), and a high kappa value of 0.996 (95 % CI, 0.956–0.976). Previous SARS-CoV-2 PCR positivity was identified in 14/24 (58.3 %) Elecsys-N negative/Elecsys-S positive individuals and in 4/21 (19.0 %) Elecsys-N positive/Elecsys-S negative individuals.ConclusionThe first Elecsys-N/Elecsys-S head-to-head comparison showed excellent agreement of two highly specific and rapid high-throughput automated anti-SARS-CoV-2 assays. An important question is whether laboratories offering two different antibody assays could benefit from combining the assays; if so, should use be concomitant or sequential—and, in the latter case, in which order? Based on our results, we favor concomitant over sequential Elecsys-N/Elecsys-S use when testing individuals for anti-SARS-CoV-2 antibodies in high-incidence settings; for example, during the exponential or stationary growth phase of the COVID-19 epidemic.

Withanone from Withania somnifera Attenuates SARS-CoV-2 RBD and Host ACE2 Interactions to Rescue Spike Protein Induced Pathologies in Humanized Zebrafish Model.

PurposeSARS-CoV-2 engages human ACE2 through its spike (S) protein receptor binding domain (RBD) to enter the host cell. Recent computational studies have reported that withanone and withaferin A, phytochemicals found in Withania somnifera, target viral main protease (MPro) and host transmembrane TMPRSS2, and glucose related protein 78 (GRP78), respectively, implicating their potential as viral entry inhibitors. Absence of specific treatment against SARS-CoV-2 infection has encouraged exploration of phytochemicals as potential antivirals.AimThis study aimed at in silico exploration, along with in vitro and in vivo validation of antiviral efficacy of the phytochemical withanone.MethodsThrough molecular docking, molecular dynamic (MD) simulation and electrostatic energy calculation the plausible biochemical interactions between withanone and the ACE2-RBD complex were investigated. These in silico observations were biochemically validated by ELISA-based assays. Withanone-enriched extract from W. somnifera was tested for its ability to ameliorate clinically relevant pathological features, modelled in humanized zebrafish through SARS-CoV-2 recombinant spike (S) protein induction.ResultsWithanone bound efficiently at the interacting interface of the ACE2-RBD complex and destabilized it energetically. The electrostatic component of binding free energies of the complex was significantly decreased. The two intrachain salt bridge interactions (K31-E35) and the interchain long-range ion-pair (K31-E484), at the ACE2-RBD interface were completely abolished by withanone, in the 50 ns simulation. In vitro binding assay experimentally validated that withanone efficiently inhibited (IC50=0.33 ng/mL) the interaction between ACE2 and RBD, in a dose-dependent manner. A withanone-enriched extract, without any co-extracted withaferin A, was prepared from W. somnifera leaves. This enriched extract was found to be efficient in ameliorating human-like pathological responses induced in humanized zebrafish by SARS-CoV-2 recombinant spike (S) protein.ConclusionIn conclusion, this study provided experimental validation for computational insight into the potential of withanone as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells.

Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

SummarySARS-CoV-2 infection results in viral burden in the respiratory tract, enabling transmission and leading to substantial lung pathology. The 1212C2 fully human monoclonal antibody was derived from an IgM memory B cell of a COVID-19 patient, has high affinity for the Spike protein receptor binding domain, neutralizes SARS-CoV-2, and exhibits in vivo prophylactic and therapeutic activity in hamsters when delivered intraperitoneally, reducing upper and lower respiratory viral burden and lung pathology. Inhalation of nebulized 1212C2 at levels as low as 0.6 mg/kg, corresponding to 0.03 mg/kg lung-deposited dose, reduced the viral burden below the detection limit and mitigated lung pathology. The therapeutic efficacy of an exceedingly low dose of inhaled 1212C2 supports the rationale for local lung delivery for dose-sparing benefits, as compared to the conventional parenteral route of administration. These results suggest that the clinical development of 1212C2 formulated and delivered via inhalation for the treatment of SARS-CoV-2 infection should be considered.

The Influence of Glycosylation on the Interaction of the Sars-Cov-2 Spike Protein Receptor Binding Domain with Therapeutic Candidates.

Glycosylation is an important posttranslational modification of proteins and it often plays a crucial role in protein efficacy and binding. It also needs to be understood for optimization of recombinant protein expression and function. We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. Also we study heparin which has been reported to interact with the spike protein as well. These models are simulated with and without spike protein fragments of different lengths containing the receptor binding domain (RBD) of the SARS-CoV-2 virus spike protein.

Switched and unswitched memory B cells detected during SARS-CoV-2 convalescence correlate with limited symptom duration

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the pandemic human respiratory illness COVID-19, is a global health emergency. While severe acute disease has been linked to an expansion of antibody-secreting plasmablasts, we sought to identify B cell responses that correlated with positive clinical outcomes in convalescent patients. We characterized the peripheral blood B cell immunophenotype and plasma antibody responses in 40 recovered non-hospitalized COVID-19 subjects that were enrolled as donors in a convalescent plasma treatment study. We observed a significant negative correlation between the frequency of peripheral blood memory B cells and the duration of symptoms for convalescent subjects. Memory B cell subsets in convalescent subjects were composed of classical CD24+ class-switched memory B cells, but also activated CD24-negative and natural unswitched CD27+ IgD+ IgM+ subsets. Memory B cell frequency was significantly correlated with both IgG1 and IgM responses to the SARS-CoV-2 spike protein receptor binding domain (RBD) in most seropositive subjects. IgM+ memory, but not switched memory, directly correlated with virus-specific antibody responses, and remained stable over 3 months. Our findings suggest that the frequency of memory B cells is a critical indicator of disease resolution, and that IgM+ memory B cells may play an important role in SARS-CoV-2 immunity.

Long COVID in the skin: a registry analysis of COVID-19 dermatological duration

Long COVID in the skin: a registry analysis of COVID-19 dermatological duration

Elucidating Interactions Between SARS-CoV-2 Trimeric Spike Protein and ACE2 Using Homology Modeling and Molecular Dynamics Simulations.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). As of October 21, 2020, more than 41.4 million confirmed cases and 1.1 million deaths have been reported. Thus, it is immensely important to develop drugs and vaccines to combat COVID-19. The spike protein present on the outer surface of the virion plays a major role in viral infection by binding to receptor proteins present on the outer membrane of host cells, triggering membrane fusion and internalization, which enables release of viral ssRNA into the host cell. Understanding the interactions between the SARS-CoV-2 trimeric spike protein and its host cell receptor protein, angiotensin converting enzyme 2 (ACE2), is important for developing drugs and vaccines to prevent and treat COVID-19. Several crystal structures of partial and mutant SARS-CoV-2 spike proteins have been reported; however, an atomistic structure of the wild-type SARS-CoV-2 trimeric spike protein complexed with ACE2 is not yet available. Therefore, in our study, homology modeling was used to build the trimeric form of the spike protein complexed with human ACE2, followed by all-atom molecular dynamics simulations to elucidate interactions at the interface between the spike protein and ACE2. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) and in silico alanine scanning were employed to characterize the interacting residues at the interface. Twenty interacting residues in the spike protein were identified that are likely to be responsible for tightly binding to ACE2, of which five residues (Val445, Thr478, Gly485, Phe490, and Ser494) were not reported in the crystal structure of the truncated spike protein receptor binding domain (RBD) complexed with ACE2. These data indicate that the interactions between ACE2 and the tertiary structure of the full-length spike protein trimer are different from those between ACE2 and the truncated monomer of the spike protein RBD. These findings could facilitate the development of drugs and vaccines to prevent SARS-CoV-2 infection and combat COVID-19.

Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals.

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were composed of immunoglobulin heavy variable 3-53 (IGHV3-53) or IGHV3-66 and immunoglobulin heavy joining 6 (IGHJ6) genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different IGHV chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in 6 of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

Phase I Interim Results of a Phase I/II Study of the IgG-Fc Fusion COVID-19 Subunit Vaccine, AKS-452

Background: To address the coronavirus disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recombinant subunit vaccine, AKS-452, is being developed comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain (SP/RBD) antigen and human IgG1 Fc emulsified in the water-in-oil adjuvant, Montanide™ ISA 720.Methods: A single-center, open-label, phase I dose-finding and safety study was conducted with 60 healthy adults (18 – 65 years) receiving one or two doses 28 days apart of 22.5 µg, 45 µg, or 90 µg of AKS-452 (i.e., six cohorts, N=10 subjects per cohort). Primary endpoints were safety and reactogenicity and secondary endpoints were immunogenicity assessments.Findings: No AEs ≥ 3, no SAEs attributable to AKS-452, and no SARS-CoV-2 viral infections occurred during the study. Seroconversion rates of anti-SARS-CoV-2 SP/RBD IgG titers in the 22.5, 45, and 90 µg cohorts at day 28 were 70%, 90%, and 100%, respectively, which all increased to 100% at day 56 (except 89% for the single-dose 22.5 µg cohort). All IgG titers were Th1-isotype skewed and efficiently bound mutant SP/RBD from several SARS-CoV-2 variants with strong neutralization potencies of live virus infection of cells (including alpha and delta variants). Interpretation: The favorable safety and immunogenicity profiles of this phase I study (ClinicalTrials.gov: NCT04681092) support phase II initiation of this room-temperature stable vaccine that can be rapidly and inexpensively manufactured to serve vaccination at a global scale without the need of a complex distribution or cold chain. Clinical Trial Registration Details: ClinicalTrials.gov: NCT04681092.Funding Information: This research was not funded by any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Declaration of Interests: The following authors declare the following financial interests/personal relationships which may be considered as potential competing interests: the following authors were employed by and received monetary compensation from Akston Biosciences, Inc.: DGA, ARD,MMS, SM, RR, EKG, TS, SK, JRH, NJS, VR, SN, TML, TZ. No other authors were personally compensated by Akston Biosciences.Ethics Approval Statement: The trial was reviewed and approved by the Central Committee on esearch involving Humans in The Hague, together with a marginal review by the competent authority (Ministry of Health, Welfare and Sport (VWS)). Local feasibility was assessed and approved by the UMCG Institutional Review Board. An informed consent form was signed voluntarily before any study-related procedure was performed, indicating that the subject understood the purpose and procedures required for the study and was willing to participate.

512. Kinetics of SARS-CoV-2 IgG responses among hospitalized patients with COVID-19

BackgroundThe kinetics of antibody responses to SARS-CoV-2 infection are not fully understood. We analyzed IgG responses to the SARS-CoV-2 Spike protein receptor binding domain (RBD) in COVID-19 patients admitted to VA Greater Los Angeles (VAGLA) and correlated with clinical outcomes.MethodsSerially admitted patients from March 20-May 10, 2020 with at least one available residual serum specimen were included in this analysis. Serum samples selected for analysis included first, last, and intermediaries spaced ≥ 5 days apart, as available. Anti-RBD IgG was detected with an enzyme immunoassay (EIA) using recombinant RBD protein. Serum from an uninfected individual collected April 2019 was used as control. The average optical density of the control in triplicate plus 3 standard deviations was considered the threshold positive/negative value. The highest dilution above the threshold value was considered the IgG titer. Clinical groups were defined as asymptomatic, moderate/severe (no ICU) or critical (mechanical ventilation, cytokine storm and/or death).ResultsOf the 43 consecutive patients admitted to VAGLA with COVID-19 in this analysis, 40 developed detectable RBD IgG responses with maximum inverse titers (MIT) ranging 100-819,200, geometric mean 12,152. Five patients remained asymptomatic but had positive EIAs with median MIT 3200 (IQR 800–3200). Twenty-five had moderate-severe illness with median MIT 25600 (IQR 6400–102400). Ten patients with critical disease had median MIT 38400 (IQR 8800–51200). The median time to positive IgG was 10 days for asymptomatic (IQR 10,10), 4 days for moderate-severe (IQR 3,15), and 7 days for critical (IQR 3.5,14.5). The figure depicts RBD IgG titers over time after onset of symptoms. Asymptomatic patients had a more gradual rate of increase and lower peak titers, while critical patients had the fastest rate of rise and the highest peak titers. Of the 21 patients with samples > 30 days after symptom onset (range 31–67 days), there was no evidence for decrease in anti-RBD IgG.Kinetics of IgG to SARS-CoV-2 receptor binding domain by clinical severityConclusionFollowing infection with SARS-CoV-2, disease severity correlates with both the rate of increase and peak in antibody titers. Anti-RBD IgG titers did not decrease over the observation period. Disclosures All Authors: No reported disclosures

Cross-species recognition of SARS-CoV-2 to bat ACE2

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2-related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 from Rhinolophus macrotis (bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.

A Potent SARS-CoV-2 Neutralizing Human Monoclonal Antibody That Reduces Viral Burden and Disease Severity in Syrian Hamsters.

The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro. Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12–15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1–5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.