Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains (preprint)

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

Emerging SARS-CoV-2 Variants of Concern: Spike Protein Mutational Analysis and Epitope for Broad Neutralization (preprint)

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we report the discovery of a novel antibody fragment (V H ab6) that neutralizes all major variants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

Chest X-ray Severity and its Association with Outcomes in Patients with COVID-19 Presenting to the Emergency Department (preprint)

Background Severity of radiographic abnormalities on chest X-ray (CXR) in patients with COVID-19 has been shown to be associated with worse outcomes, but studies are limited by different scoring systems, sample size, patient age and study duration. Data regarding the longitudinal evolution of radiographic abnormalities and its association with outcomes is scarce. We sought to evaluate these questions using a well-validated scoring system (the Radiographic Assessment of Lung Edema [RALE] score) using data over 6 months from a large, multi-hospital healthcare system. Methods We collected clinical and demographic data and quantified radiographic edema on CXRs obtained in the emergency department (ED) as well as on days 1-2 and 3-5 (in those admitted) in patients with a nasopharyngeal swab positive for SARS-CoV-2 PCR visiting the ED for COVID-19-related complaints between March and September 2020. We examined the association of baseline and longitudinal evolution of radiographic edema with severity of hypoxemia and clinical outcomes. Results 870 patients were included (median age 53.6, 50.8% female). Inter-rate agreement for RALE scores was excellent (ICC = 0.84, 95% CI 0.82 - 0.87, p < 0.0001). RALE scores correlated with hypoxemia as quantified by SpO2-FiO2 ratio (r = -0.42, p < 0.001). Admitted patients had higher RALE scores than those discharged (6 [2, 11] vs 0 [0, 3], p < 0.001). An increase of RALE score of 4 or more was associated with worse 30-day survival (p < 0.01). Larger increases in the RALE score were associated with worse survival. Conclusions The RALE score is reproducible and easily implementable in adult patients presenting to the ED with COVID-19. Its association with physiologic parameters and outcomes at baseline and longitudinally makes it a readily available tool for prognostication and early ICU triage, particularly in patients with worsening radiographic edema.

Structural and Biochemical Rationale for Enhanced Spike Protein Fitness in Delta and Kappa SARS-CoV-2 Variants (preprint)

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a novel head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding (preprint)

The recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and cryo-EM structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

Neutralization of European, South African, and United States SARS-CoV-2 mutants by a human antibody and antibody domains (preprint)

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission with several emerging variants remain uncontrolled in many countries, indicating the pandemic remains severe. Recent studies showed reduction of neutralization against these emerging SARS-CoV-2 variants by vaccine-elicited antibodies. Among those emerging SARS-CoV-2 variants, a panel of amino acid mutations was characterized including those in the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. In the present study, we evaluated our previously identified antibody and antibody domains for binding to these RBD variants with the emerging mutations, and neutralization of pseudo typed viruses carrying spike proteins with such mutations. Our results showed that one previously identified antibody domain, ab6, can bind 32 out of 35 RBD mutants tested in an ELISA assay. All three antibodies and antibody domains can neutralize pseudo typed B.1.1.7 (UK variant), but only the antibody domain ab6 can neutralize the pseudo typed virus with the triple mutation (K417N, E484K, N501Y). This domain and its improvements have potential for therapy of infections caused by SARS-CoV-2 mutants.

Change-Point Analysis of Cyberbullying-Related Twitter Discussions During COVID-19 (preprint)

Due to the outbreak of COVID-19, users are increasingly turning to online services. An increase in social media usage has also been observed, leading to the suspicion that this has also raised cyberbullying. In this initial work, we explore the possibility of an increase in cyberbullying incidents due to the pandemic and high social media usage. To evaluate this trend, we collected 454,046 cyberbullying-related public tweets posted between January 1st, 2020 -- June 7th, 2020. We summarize the tweets containing multiple keywords into their daily counts. Our analysis showed the existence of at most one statistically significant changepoint for most of these keywords, which were primarily located around the end of March. Almost all these changepoint time-locations can be attributed to COVID-19, which substantiates our initial hypothesis of an increase in cyberbullying through analysis of discussions over Twitter.

Evaluation of the commercially available LightMix® Modular E-gene kit using clinical and proficiency testing specimens for SARS-CoV-2 detection.

BACKGROUND: Rapid and sensitive diagnostic assays for SARS-CoV-2 detection are required for prompt patient management and infection control. The analytical and clinical performances of LightMix® Modular SARS and Wuhan CoV E-gene kit, a widely used commercial assay for SARS-CoV-2 detection, have not been well studied. OBJECTIVE: To evaluate the performance characteristics of the LightMix® E-gene kit in comparison with well-validated in-house developed COVID-19 RT-PCR assays. STUDY DESIGN: Serial dilutions of SARS-CoV-2 culture isolate extracts were used for analytical sensitivity evaluation. A total of 289 clinical specimens from 186 patients with suspected COVID-19 and 8 proficiency testing (PT) samples were used to evaluate the diagnostic performance of the LightMix® E-gene kit against in-house developed COVID-19-RdRp/Hel and COVID-19-N RT-PCR assays. RESULTS: The LightMix® E-gene kit had a limit of detection of 1.8 × 10-1 TCID50/mL, which was one log10 lower than those of the two in-house RT-PCR assays. The LightMix® E-gene kit (149/289 [51.6%]) had similar sensitivity as the in-house assays (144/289 [49.8%] for RdRp/Hel and 146/289 [50.5%] for N). All three assays gave correct results for all the PT samples. Cycle threshold (Cp) values of the LightMix® E-gene kit and in-house assays showed excellent correlation. Reproducibility of the Cp values was satisfactory with intra- and inter-assay coefficient of variation values <5%. Importantly, the LightMix® E-gene kit, when used as a stand-alone assay, was equally sensitive as testing algorithms using multiple COVID-19 RT-PCR assays. CONCLUSIONS: The LightMix® E-gene kit is a rapid and sensitive assay for SARS-CoV-2 detection. It has fewer verification requirements compared to laboratory-developed tests.