Protective Effects of STI-2020 Antibody Delivered Post-Infection by the Intranasal or Intravenous Route in a Syrian Golden Hamster COVID-19 Model (preprint)

We have previously reported that the SARS-CoV-2 neutralizing antibody, STI-2020, potently inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. We now have extended our in vivo studies of STI-2020 to include disease treatment efficacy, profiling of biodistribution of STI-2020 in mice when antibody is delivered intranasally (IN) or intravenously (IV), as well as pharmacokinetics in mice following IN antibody administration. Importantly, SARS-CoV-2-infected hamsters were treated with STI-2020 using these routes, and treatment effects on severity and duration of COVID-19-like disease in this model were evaluated. In SARS-CoV-2 infected hamsters, treatment with STI-2020 12 hours post-infection using the IN route led to a decrease in severity of clinical disease signs and a more robust recovery during 9 days of infection as compared to animals treated with an isotype control antibody. Treatment via the IV route using the same dose and timing regimen resulted in a decrease in the average number of consecutive days that infected animals experienced weight loss, shortening the duration of disease and allowing recovery to begin more rapidly in STI-2020 treated animals. Following IN administration in mice, STI-2020 was detected within 10 minutes in both lung tissue and lung lavage. The half-life of STI-2020 in lung tissue is approximately 25 hours. We are currently investigating the minimum effective dose of IN-delivered STI-2020 in the hamster model as well as establishing the relative benefit of delivering neutralizing antibodies by both IV and IN routes.

Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables fast and accurate outbreak analysis (preprint)

Current transmission rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still increasing and many countries are facing second waves of infections. Rapid SARS-CoV-2 whole genome sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring and determining transmission links. Here we report a novel reverse complement polymerase chain reaction (RC-PCR) technology for WGS of SARS-CoV-2. This technique is unique as it enables library preparation in a single PCR saving time, resources and enables high throughput screening. A total of 173 samples tested positive for SARS-CoV-2 between March and September 2020 were included. RC-PCR WGS applicability for outbreak analysis in public health service and hospital settings was tested on six predefined clusters containing samples of healthcare workers and patients. RC-PCR resulted in WGS data for 146 samples. It showed a genome coverage of up to 98,2% for samples with a maximum Ct value of 32. Three out of six suspected clusters were fully confirmed, while in other clusters four healthcare workers were not associated. Importantly, a previously unknown chain of transmission was confirmed in the public health service samples. These findings confirm the reliability and applicability of the RC-PCR technology for SARS-CoV-2 sequencing in outbreak analysis and surveillance.

Discovery and Development of Human SARS-CoV-2 Neutralizing Antibodies using an Unbiased Phage Display Library Approach (preprint)

SARS-CoV-2 neutralizing antibodies represent an important component of the ongoing search for effective treatment of and protection against COVID-19. We report here on the use of a naive phage display antibody library to identify a panel of fully human SARS-CoV-2 neutralizing antibodies. Following functional profiling in vitro against an early pandemic isolate as well as a recently emerged isolate bearing the D614G Spike mutation, the clinical candidate antibody, STI-1499, and the affinity-engineered variant, STI-2020, were evaluated for in vivo efficacy in the Syrian golden hamster model of COVID-19. Both antibodies demonstrated potent protection against the pathogenic effects of the disease and a dose-dependent reduction of virus load in the lungs, reaching undetectable levels following a single dose of 500 micrograms of STI-2020. These data support continued development of these antibodies as therapeutics against COVID-19 and future use of this approach to address novel emerging pandemic disease threats.

Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins (preprint)

Membrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

Impact of COVID-19 on Hospital Admission of Acute Stroke patients in Bangladesh (preprint)

Background: With the proposed pathophysiologic mechanism of neurologic injury by SARS COV-2 the frequency of stroke and henceforth the related hospital admissions were expected to rise. In this paper we investigate this presumption by comparing the frequency of admissions of stroke cases in Bangladesh before and during the pandemic. Methods: We conducted a retrospective analysis of stroke admissions in a 100-bed stroke unit at the National Institute of Neurosciences and Hospital (NINS&H) which is considerably a large stroke unit. We considered all the admitted cases from the 1 st January to the 30 th June, 2020. We used Poisson regressions to determine whether statistically significant changes in admission counts can be found before and after 25 March since when there is a surge in COVID-19 infections. Results: A total of 1394 stroke patients got admitted during the study period. Half of the patients were older than 60 years, whereas only 2.6% were 30 years old or younger with a male-female ratio of 1.06:1. From January to March, 2020 the mean rate of admission was 302.3 cases per month which dropped to 162.3 cases per month from April to June with an overall reduction of 46.3% in acute stroke admission per month. In those two periods, reductions in average admission per month for ischemic stroke (IST), intracerebral hemorrhage (ICH), subarachnoid hemorrhage (SAH) and venous stroke (VS) were 45.5%, 37.2%, 71.4% and 39.0%, respectively. Based on weekly data, results of Poisson regressions confirm that the average number of admissions per week dropped significantly during the last three months of the sample period. Further, in the first three months, a total of 22 cases of hyperacute stroke management were done whereas in the last three months there was an 86.4% reduction in the number of hyperacute stroke patients getting reperfusion treatment. Only 38 patients (2.7%) were later found to be RT-PCR for SARS Cov-2 positive based on nasal swab testing. Conclusion: Our study revealed more than fifty percent reduction in acute stroke admission during the COVID-19 pandemic. It is still elusive whether the reduction is related to the fear of getting infected by COVID-19 from hospitalization or the overall restriction on public movement and stay-home measures.