SARS-CoV-2 convalescence and hybrid immunity elicits mucosal immune responses (preprint)

Mucosal antibodies play a key role in the protection against SARS-CoV-2 infection in the upper respiratory tract, and potentially in limiting virus replication and therefore onward transmission. While systemic immunity to SARS-CoV-2 is well understood, little is known about the antibodies present on the nasal mucosal surfaces. In this study, we evaluated SARS-CoV-2 mucosal antibodies in response to infection, vaccination, or a combination of both. Paired nasal fluid and serum samples were collected from 136 individuals, which include convalescent, vaccinated, or breakthrough infections. We detected a high correlation between IgG responses in serum and nasal fluids, which were higher in both compartments in vaccinated compared to convalescent participants. Contrary, nasal and systemic SARS-CoV-2 IgA responses were weakly correlated, indicating a compartmentalization between the local and systemic IgA responses. SARS-CoV-2 secretory component IgA (s-IgA) antibodies, present exclusively on mucosal surfaces, were detected in the nasal fluid only in a minority of vaccinated subjects and were significantly higher in previously infected individuals. s-IgA binding antibodies showed significant correlation with neutralizing activity of nasal fluids against SARS-CoV-2 ancestral B.1 and Omicron-BA.5 variant, indicating that s-IgA is the crucial contributor to neutralization in the nasal mucosa. Neutralization against both SARS-CoV-2 strains was higher in the mucosa of subjects with previous SARS-CoV-2 infections compared to vaccinated participants. In summary, we demonstrate that currently available vaccines elicit strong systemic antibody responses, but SARS-CoV-2 infection generates more potent binding and neutralizing mucosal antibodies. Our results support the importance to develop SARS-CoV-2 vaccines that elicit mucosal antibodies.

Occupational differences in the prevalence and severity of long-COVID: Analysis of the ONS Coronavirus (COVID-19) Infection Survey (preprint)

Objectives: To establish whether prevalence and severity of long-COVID symptoms vary by industry and occupation. Methods: We utilised ONS Coronavirus Infection Survey (CIS) data (February 2021-April 2022) of working-age participants (16-65 years). Exposures were industrial sector, occupation and major Standard Occupational Classification (SOC) group. Outcomes were self-reported: (1) long-COVID symptoms; and (2) reduced function due to long-COVID. Binary (outcome 1) and ordered (outcome 2) logistic regression were used to estimate odds ratios (OR) and prevalence (marginal means) for all exposures. Results: Public facing industries, including teaching and education, social care, healthcare, civil service, retail and transport industries and occupations had highest odds ratios for long-COVID. By major SOC group, those in caring, leisure and other services (OR 1.44, CIs: 1.38-1.52) had substantially elevated odds than average. For almost all exposures, the pattern of odds ratios for long-COVID symptoms followed that for SARS-CoV-2 infections, except for professional occupations (OR<1 for infection; OR>1 for long-COVID). The probability of reporting long-COVID for industry ranged from 7.7% (financial services) to 11.6% (teaching and education); whereas the prevalence of reduced function by a lot ranged from 17.1% (arts, entertainment and recreation) to 22-23% (teaching and education and armed forces ) and to 27% (those not working). Conclusions: The risk and prevalence of long-COVID differs across industries and occupations. Generally, it appears that likelihood of developing long-COVID symptoms follows likelihood of SARS-CoV-2 infection, except for professional occupations. These findings highlight sectors and occupations where further research is needed to understand the occupational factors resulting in long-COVID.

Oral Epithelial Cells Expressing Low or Undetectable Levels of Human Angiotensin-Converting Enzyme 2 are Susceptible SARS-CoV-2Virus Infection in vitro (preprint)

The oral cavity is thought to be one of the portals for SARS-CoV-2 entry. Because there is limited evidence of active oral infection by SARS-CoV-2 viruses, we assessed the capacity of SARS-CoV-2 to infect and replicate in oral epithelial cells. Oral gingival epithelial cells (hTERT TIGKs), salivary gland epithelial cells (A-253), and oral buccal epithelial cells (TR146), which occupy different regions of the oral cavity, were challenged with replication competent SARS-CoV-2 viruses and with pseudo-typed viruses expressing SARS-CoV-2 spike proteins. All oral epithelial cells expressing undetectable or low levels of human angiotensin-converting enzyme 2 (hACE2) but high levels of the alternative receptor CD147 were susceptible to SARS-CoV-2 infection. Viral dynamics in hTERT TIGKs were different from those in A-253 and TR146 cells. For example, levels of viral transcripts were sustained in hTERT TIGKs but were significantly decreased in A-253 and TR146 cells at day 3 after infection. Analysis of oral epithelial cells infected by replication competent SARS-CoV-2 viruses expressing GFP showed that the signals of GFP and SARS-CoV-2 mRNAs were not evenly distributed. Taken together, our results demonstrated oral epithelial cells were susceptible to SARS-CoV-2 viruses despite of low or undetectable levels of hACE2, suggesting that alternative receptors contribute to SARS-CoV-2 infection and may be considered for development of future vaccines and therapeutics.

Adverse outcomes in SARS-CoV-2 infected pregnant mice are gestational age-dependent and resolve with antiviral treatment (preprint)

SARS-CoV-2 infection during pregnancy is associated with severe COVID-19 and adverse fetal outcomes, but the underlying mechanisms remain poorly understood. Moreover, clinical studies assessing therapeutics against SARS-CoV-2 in pregnancy are limited. To address these gaps, we developed a mouse model of SARS-CoV-2 infection during pregnancy. Outbred CD1 mice were infected at embryonic day (E) 6, E10, or E16 with a mouse adapted SARS-CoV-2 (maSCV2) virus. Outcomes were gestational age-dependent with greater morbidity, reduced pulmonary function, reduced anti-viral immunity, greater viral titers, and more adverse fetal outcomes occurring with infection at E16 (3rd trimester-equivalent) than with infection at either E6 (1st trimester-equivalent) or E10 (2nd trimester-equivalent). To assess the efficacy of ritonavir-boosted nirmatrelvir (recommended for pregnant individuals with COVID-19), we treated E16-infected dams with mouse equivalent doses of nirmatrelvir and ritonavir. Treatment reduced pulmonary viral titers, decreased maternal morbidity, and prevented adverse offspring outcomes. Our results highlight that severe COVID-19 during pregnancy and adverse fetal outcomes are associated with heightened virus replication in maternal lungs. Ritonavir-boosted nirmatrelvir mitigated adverse maternal and fetal outcomes of SARS-CoV-2 infection. These findings prompt the need for further consideration of pregnancy in preclinical and clinical studies of therapeutics against viral infections.

Serum neutralization of SARS-CoV-2 Omicron BA.2, BA.2.75, BA.2.76, BA.5, BF.7, BQ.1.1 and XBB.1.5 in individuals receiving Evusheld (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is undergoing continuous evolution and convergent mutation, which has led to the rapid emergence of several new variants. These new subvariants carry different mutations in theirreceptor-binding domain (RBD), raising concerns that they may evade neutralizing monoclonal antibodies (mAbs). In this study, we investigated the serum neutralization efficacy of Evusheld (cilgavimab and tixagevimab) antibody cocktails against SARS-CoV-2 Omicron sublineages BA.2, BA.2.75, BA.2.76, BA.5, BF.7, BQ.1.1 and XBB.1.5. Our results show that Evusheld retained neutralizing activity against BA.2, BA.2.75 and BA.5, albeit with somewhat reduced titers. However, the neutralizing activity of Evusheld against BA.2.76, BF.7, BQ.1.1 and XBB.1.5 significantly decreased, with XBB.1.5 showing the greatest escape activity among the subvariants, followed by BQ.1.1, BA.2.76 and BF.7. We also observed that recipients of Evusheld displayed elevated antibody levels in their serum, which efficiently neutralized the original variant, and exhibited different characteristics of infection than those who did not receive Evusheld. These findings provide important guidance for the application of Evusheld in treating SARS-CoV-2 subvariant infections.

Nanograms of SARS-CoV-2 Spike Protein Delivered by Exosomes Induce Potent Neutralization of Both Delta and Omicron Variants. (preprint)

Exosomes are emerging as potent and safe delivery carriers for use in vaccinology and therapeutics. A better vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to provide improved, broader, longer lasting neutralization of SARS-CoV-2, a more robust T cell response, enable widespread global usage, and further enhance the safety profile of vaccines given the likelihood of repeated booster vaccinations. Here, we use Capricor's StealthXTM platform to engineer exosomes to express native SARS-CoV-2 spike Delta variant (STX-S) protein on the surface for the delivery of a protein-based vaccine for immunization against SARS-CoV-2 infection. The STX-S vaccine induced a strong immunization with the production of a potent humoral immune response as demonstrated by high levels of neutralizing antibody not only against the delta SARS-CoV-2 virus but also two Omicron variants (BA.1 and BA.5), providing broader protection than current mRNA vaccines. Additionally, both CD4+ and CD8+ T cell responses were increased significantly after treatment. Quantification of spike protein by ELISA showed that only nanograms of protein were needed to induce a potent immune response. This is a significantly lower dose than traditional recombinant protein vaccines with no adjuvant required, which makes the StealthXTM exosome platform ideal for the development of multivalent vaccines with a better safety profile. Importantly, our exosome platform allows novel proteins, or variants in the case of SARS-CoV-2, to be engineered onto the surface of exosomes in a matter of weeks, comparable with mRNA vaccine technology, but without the cold storage requirements. The ability to utilize exosomes for cellular delivery of proteins, as demonstrated by STX-S, has enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation at an extremely low dose resulting in a potent, broad antibody response.

COVID-19 Infection and Dementia: Analyses of time-varying risk, subtypes, and subpopulations from the UK Biobank (preprint)

Background Although COVID-19 patients were suggested to experience worse cognitive outcomes, there is a paucity of evidence on time-varying risk of dementia, especially the subtypes, as well as among critical subpopulations.Methods Out of over 50000 individuals from general population in the UK Biobank, SARS-COV-2 infected patients between March 1, 2020, and July 31, 2021 and maximally 5:1 propensity score matched contemporary non-infected individuals were selected, with baseline dementia excluded. Matching was done on demographic characteristics, lifestyle, and comorbidities. Dementia was captured according to primary care, inpatient records, and death registry, with the follow-up ending at the earliest of outcome occurrence, death, or August 31, 2021. Associations were evaluated using time-varying hazard ratios (HRs) and odds ratios (ORs).Results With a mean age of 64.5 years for 18032 COVID-19 patients and 83,008 controls, participants were followed for a median of 247 (IQR: 204–305) days and 255 dementia cases occurred, including 90 Alzheimer’s disease (AD) cases and 42 vascular dementia (VaD) cases. Compared with matched controls, dementia risk declined drastically after COVID-19 infection and sustained for all-cause dementia, VaD, and other dementia. During the acute phase (first 30 days), COVID-19 infection was associated with increased risks of dementia, with HRs (95% CIs) being 12.77 (6.77, 24.08) for all-cause dementia, 9.21 (2.77, 30.59) for AD, 5.53 (1.69, 18.11) for VaD, and 25.35 (8.74, 73.56) for other dementia. Among those not hospitalized within 30 days of enrollment, elevated dementia risk remained for all-cause dementia, VaD, and other dementia, with ORs being 1.82, 4.55, and 1.64, respectively. Among most of the subpopulations classified by demographic characteristics, APOE genotype, and comorbidities (except for those with chronic obstructive pulmonary diseases at enrollment), COVID-19 infection was associated with an elevated all-cause dementia risk and no modification effect was detected.Conclusions Declined yet sustained elevated dementia risk since COVID-19 infection was found and vascular risk factors may need extra attention during the long-term follow-up. Increased dementia risk from COVID-19 infection also applied for the non-hospitalized during the acute phase and most subpopulations. The potential dementia risk associated with Omicron and newer variants warrants further evaluation.

Reduced control of SARS-CoV-2 infection is associated with lower mucosal antibody responses in pregnant women (preprint)

Importance: Pregnant women are at increased risk of severe COVID-19, but the contribution of viral RNA load, the presence of infectious virus, and mucosal antibody responses remain understudied. Objective: To evaluate the association of COVID-19 outcomes following confirmed infection with vaccination status, mucosal antibody responses, infectious virus recovery and viral RNA levels in pregnant compared with non-pregnant women. Design: A retrospective observational cohort study of remnant clinical specimens from SARS-CoV-2 infected patients between October 2020-May 2022. Setting: Five acute care hospitals within the Johns Hopkins Health System (JHHS) in the Baltimore, MD-Washington, DC area. Participants: Participants included confirmed SARS-CoV-2 infected pregnant women and matched non-pregnant women (matching criteria included age, race/ethnicity, and vaccination status). Exposure: SARS-CoV-2 infection, with documentation of SARS-CoV-2 mRNA vaccination. Main Outcome(s): The primary dependent measures were clinical COVID-19 outcomes, infectious virus recovery, viral RNA levels, and mucosal anti-spike (S) IgG titers from upper respiratory tract samples. Clinical outcomes were compared using odds ratios (OR), and measures of virus and antibody were compared using either Fisher's exact test, two-way ANOVA, or regression analyses. Results were stratified according to pregnancy, vaccination status, maternal age, trimester of pregnancy, and infecting SARS-CoV-2 variant. Results(s): A total of 452 individuals (117 pregnant and 335 non-pregnant) were included in the study, with both vaccinated and unvaccinated individuals represented. Pregnant women were at increased risk of hospitalization (OR = 4.2; CI = 2.0-8.6), ICU admittance, (OR = 4.5; CI = 1.2-14.2), and of being placed on supplemental oxygen therapy (OR = 3.1; CI =1.3-6.9). An age-associated decrease in anti-S IgG titer and corresponding increase in viral RNA levels (P< 0.001) was observed in vaccinated pregnant, but not non-pregnant, women. Individuals in their 3rd trimester had higher anti-S IgG titers and lower viral RNA levels (P< 0.05) than those in their 1st or 2nd trimesters. Pregnant individuals experiencing breakthrough infections due to the omicron variant had reduced anti-S IgG compared to non-pregnant women (P< 0.05). Conclusions and Relevance: In this cohort study, vaccination status, maternal age, trimester of pregnancy, and infecting SARS-CoV-2 variant were each identified as drivers of differences in mucosal anti-S IgG responses in pregnant compared with non-pregnant women. Observed increased severity of COVID-19 and reduced mucosal antibody responses particularly among pregnant participants infected with the Omicron variant suggest that maintaining high levels of SARS-CoV-2 immunity may be important for protection of this at-risk population.

Socio-demographic characteristics associated with COVID-19 vaccination uptake in Switzerland: longitudinal analysis of the CoMix study (preprint)

Background: Vaccination is an effective strategy to reduce morbidity and mortality from coronavirus disease 2019 (COVID-19). However, the uptake of COVID-19 vaccination has varied across and within countries. Switzerland has had lower levels of COVID-19 vaccination uptake in the general population than many other high-income countries. Understanding the socio-demographic factors associated with vaccination uptake can help to inform future vaccination strategies to increase uptake. Methods: We conducted a longitudinal online survey in the Swiss population, consisting of six survey waves from June to September 2021. Participants provided information on socio-demographic characteristics, history of testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), social contacts, willingness to be vaccinated, and vaccination status. We used a multivariable Poisson regression model to estimate the adjusted rate ratio (aRR) and 95% confidence intervals (CI) of COVID-19 vaccine uptake. Results: We recorded 6,758 observations from 1,884 adults. For the regression analysis, we included 3,513 observations from 1,883 participants. By September 2021, 600 (75%) of 806 study participants had received at least one vaccine dose. Participants who were older, male, and students, had a higher education, household income, and number of social contacts, and lived in a household with a medically vulnerable person were more likely to have received at least one vaccine dose. Female participants, those who lived in rural areas and smaller households, and people who perceived COVID-19 measures as being too strict were less likely to be vaccinated. We found no significant association between previous SARS-CoV-2 infections and vaccination uptake. Conclusions: Our results suggest that socio-demographic factors as well as individual behaviours and attitudes played an important role in COVID-19 vaccination uptake in Switzerland. Therefore, appropriate communication with the public is needed to ensure that public health interventions are accepted and implemented by the population. Tailored COVID-19 vaccination strategies in Switzerland that aim to improve uptake should target specific subgroups such as women, people from rural areas or people with lower socio-demographic status. Keywords: Vaccine, COVID-19, contact survey, social contact, socio-demographic characteristics, Switzerland

Risk factors for SARS-CoV-2 infection: A test-negative case-control study with additional population controls (preprint)

ABSTRACT Objectives: To assess risk factors for SARS-CoV-2 infection by first comparing positive cases with negative controls as determined by polymerase chain reaction (PCR) testing and then comparing these two groups with an additional population control group. Design and setting: Test-negative design (TND), multicentre case-control study with additional population controls in South Eastern Norway. Participants: Adults who underwent SARS-CoV-2 PCR testing between February and December 2020. PCR-positive cases, PCR-negative controls, and additional age-matched population controls. Primary outcome measures: The associations between various risk factors based on self- reported questionnaire and SARS-CoV-2 infection comparing PCR positive cases and PCR- negative controls. Using subgroup analysis, the risk factors were then compared with a population control group. Univariate and multivariate regression analyses were performed. Results: In total, 400 SARS-CoV-2 PCR-positive cases, 719 PCR-negative controls, and 14,509 population controls were included. Male sex was associated with the risk of SARS- CoV-2 infection when PCR-positive cases were compared with PCR-negative controls (OR 1.9, 95% CI 1.4 to 2.6). Age, education level, comorbidities (asthma, diabetes, hypertension), an exercise were not associated with the risk of SARS-CoV-2 infection when PCR-positive cases were compared with PCR-negative controls. In the subgroup analysis comparing PCR- positive cases with age-matched population controls, asthma was associated with the risk of SARS-CoV-2 infection (OR 1.6, 95% CI 1.1 to 2.1). Daily or occasional smoking was negatively associated with the risk of SARS-CoV-2 infection in both analyses (OR 0.5, 95% CI 0.3 to 0.8 and OR 0.55, 95% CI 0.35, to 0.82, respectively). Conclusions: Male sex was a possible risk factor, whereas smoking was negatively associated with the risk of SARS-CoV-2 infection, when comparing PCR-positive cases and PCR- negative controls. Asthma was associated with the risk of SARS-CoV-2 infection when PCR- positive cases were compared with population controls.

Demographic and co-morbidity characteristics of patients tested for SARS-CoV-2 from March 2020 to January 2022 in a national clinical research network: results from PCORnet(R) (preprint)

Background Prior studies have documented differences in the age, racial, and ethnic characteristics among patients with SARS-CoV-2 infection. However, little is known about how these characteristics changed over time during the pandemic and whether racial, ethnic, and age disparities evident early in the pandemic were persistent over time. This study reports on trends in SARS-CoV-2 infections among U.S. adults from March 1, 2020 to January, 31 2022, using data from electronic health records. Methods and Findings We captured repeated cross-sectional information from 43 large healthcare systems in 52 U.S. States and territories, participating in PCORnet(R), the National Patient-Centered Clinical Research Network. Using distributed queries executed at each participating institution, we acquired information for all patients [≥] 20 years of age who were tested for SARS-CoV-2 (both positive and negative results), including care setting, age, sex, race, and ethnicity by month as well as comorbidities (assessed with diagnostic codes). During this time period, 1,325,563 patients had positive (13% inpatient) and 6,705,868 patients had negative (25% inpatient) viral tests for SARS-CoV-2. Disparities in testing positive were present across racial and ethnic groups, especially in the inpatient setting. Compared to White patients, Black or African American and other race patients had relative risks for testing positive of 1.5 or greater in the inpatient setting for 12 of the 23-month study period. Compared to nonHispanic patients, Hispanic patients had relative risks for testing positive in the inpatient setting of 1.5 or greater for 16 of 23. Ethnic and racial differences were present in emergency department and ambulatory settings but were less common across time than in inpatient settings. Trends in infections by age group demonstrated higher test positivity for older patients in the inpatient setting only for most months, except for June and July of 2020, April to August 2021, and January 2022. Comorbidities were common, with much higher rates among those hospitalized; hypertension (38% of patients SARS-CoV-2 positive vs. 29% for those negative) and type 2 diabetes mellitus (22% vs. 13%) were the most common. Conclusion and Relevance Racial and ethnic disparities changed over time among persons infected with SARS-CoV-2. These trends highlight potential underlying mechanisms, such as poor access to care and differential vaccination rates, that may have contributed to greater disparities, especially early in the pandemic. Monitoring data on characteristics of patients testing positive in real time could allow public health officials and policymakers to tailor interventions to ensure that patients and communities most in need are receiving adequate testing, mitigation strategies, and treatment.

Serial SARS-CoV-2 antibody titers in vaccinated dialysis patients: prevalence of unrecognized infection and duration of seroresponse (preprint)

Rationale & Objective: SARS-CoV-2 infections are likely underdiagnosed, but the degree of underdiagnosis among maintenance dialysis patients is unknown. Durability of the immune response after third vaccine doses in this population also remains uncertain. This study tracked antibody levels to 1) assess the rate of undiagnosed infections and 2) characterize seroresponse durability after third doses. Study Design: Retrospective observational study Setting & Participants: SARS-CoV-2 vaccinated patients receiving maintenance dialysis through a national dialysis provider. Immunoglobulin G spike antibodies (anti-spike IgG) titers were assessed monthly following vaccination. Exposure(s): Two and three doses of SARS-CoV-2 vaccine Outcome(s): Undiagnosed and diagnosed SARS-CoV-2 infections; anti-spike IgG titers over time Analytical Approach: "Undiagnosed" SARS-CoV-2 infections were identified as an increase in anti-spike IgG titer of [≥]100 BAU/mL, not associated with receipt of vaccine or "diagnosed" SARS-CoV-2 infection (by PCR or antigen test). In descriptive analyses, anti-spike IgG titers were followed over time. Results: Among 2660 patients without prior COVID-19 who received an initial two-dose vaccine series, 371 (76%) SARS-CoV-2 infections were diagnosed and 115 (24%) were undiagnosed. Among 1717 patients without prior COVID-19 who received a third vaccine dose, 155 (80%) SARS-CoV-2 infections were diagnosed and 39 (20%) were undiagnosed. In both cohorts, anti-spike IgG levels declined over time. Of the initial two-dose cohort, 66% had a titer [≥]500 BAU/mL in the first month, with 23% maintaining a titer [≥]500 BAU/mL at six months. Of the third dose cohort, 95% had a titer [≥]500 BAU/mL in the first month after the third dose, with 76% maintaining a titer [≥]500 BAU/mL at six months. Limitations: Assays used had upper limits. Conclusions: Among maintenance dialysis patients, 20-24% of SARS-CoV-2 infections were undiagnosed. Given this population's vulnerability to COVID-19, ongoing infection control measures are needed. A three-dose primary mRNA vaccine series optimizes seroresponse rate and durability.

Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine (preprint)

COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.

A general computational design strategy for stabilizing viral class I fusion proteins (preprint)

Many pathogenic viruses, including influenza virus, Ebola virus, coronaviruses, and Pneumoviruses, rely on class I fusion proteins to fuse viral and cellular membranes. To drive the fusion process, class I fusion proteins undergo an irreversible conformational change from a metastable prefusion state to an energetically more favorable and stable postfusion state. An increasing amount of evidence exists highlighting that antibodies targeting the prefusion conformation are the most potent. However, many mutations have to be evaluated before identifying prefusion-stabilizing substitutions. We therefore established a computational design protocol that stabilizes the prefusion state while destabilizing the postfusion conformation. As a proof of concept, we applied this principle to the fusion protein of the RSV, hMPV, and SARS-CoV-2 viruses. For each protein, we tested less than a handful of designs to identify stable versions. Solved structures of designed proteins from the three different viruses evidenced the atomic accuracy of our approach. Furthermore, the immunological response of the RSV F design compared to a current clinical candidate in a mouse model. While the parallel design of two conformations allows identifying and selectively modifying energetically less optimized positions for one conformation, our protocol also reveals diverse molecular strategies for stabilization. We recaptured many approaches previously introduced manually for the stabilization of viral surface proteins, such as cavity-filling, optimization of polar interactions, as well as postfusion-disruptive strategies. Using our approach, it is possible to focus on the most impacting mutations and potentially preserve the immunogen as closely as possible to its native version. The latter is important as sequence re-design can cause perturbations to B and T cell epitopes. Given the clinical significance of viruses using class I fusion proteins, our algorithm can substantially contribute to vaccine development by reducing the time and resources needed to optimize these immunogens.

Molecular Neuropathology and Cerebrospinal Fluid Diagnostic Biomarkers of SARS-Cov2 Infection in Central Nervous System: A Scoping Review Protocol (preprint)

Introduction Despite the broad spectrum of neurological symptomatic manifestation in COVID19 patients, the brain tissue susceptibility and permissiveness to SARS-Cov2 infection is yet uncertain. This critical appraisal aims at bridging the gap by consolidating the body of evidence for meticulous evaluation of molecular neuropathological pathways and CSF diagnostic signatures of SARS-Cov2 infection in the central nervous system (CNS) that will underpin further strategic approach for neuroprotection and treatment of neurological COVID19 Methods and Analysis We have developed the protocol of this review according to the provisions of Joanna Briggs Institute Reviewer Manual for Evidence Synthesis ,2015 and Arksey and O Malley Methodological Framewotk ,2005.The articles for this review will be sourced from several electronic databases including EMBASE, PubMed, Scopus, Web of Science (WOS), Cochrane, Crossref Metadata and Semantic scholar. Herein we generated the search strategy using the medical subject headings [ MeSH Terms] , term in all field bibliography at all permutations in conjunctions with boolean operators Ethical Clearance and Dissemination plan Herein the review will not involve the human participants henceforth the ethical clearance approval is not applicable .We will disseminate the final findings of this review to scientific conferences at local and international level. The manuscript for final findings will be published on reputable journal of neuroscience. Keywords: Molecular, Neuropathology, CSF biomarkers, SARS-Cov2

Evidence of Autoinflammation as the Principal Mechanism of Myocardial Injury in SARS-CoV-2 PCR-Positive Autopsy Hearts (preprint)

Background Disease from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) remains the third leading cause of death in the United States, after cancer and heart disease. Many patients infected with this virus develop cardiovascular complications including myocardial infarctions, stroke, arrhythmia, heart failure, and sudden cardiac death. Specifically, patients with SARS-CoV-2 have a high prevalence of severe myocardial injury (20–28%). The purpose of this study is to understand the primary mechanism of myocyte injury in patients infected with SARS-CoV-2.Methods We investigated a consecutive cohort of 84 medical examiner cases who died with PCR-positive SARS-CoV-2 (COVpos) infection prior to availability of therapy or vaccines. We compared them to a consecutive cohort of 42 age- and sex-matched controls who were PCR-negative for SARS-CoV-2 (COVneg). Formalin-fixed paraffin embedded sections of left and right ventricle were examined on each case using antibodies directed against CD42 (platelets), CD15 (myeloid cells), CD68 (monocytes), C4d, Fibrin, CD34 (stem cell antigen), CD56 (natural killer cells), and Myeloperoxidase (MPO) (neutrophils and NETs). Slides were scanned using an Aperio slide scanner and viewer and each digital slide was entirely examined at 5x,10x and 20x. Each slide was graded using a 0–3 scale where 3 indicates the marker was present in every field at 20x. We used a Welch 2-sample T-test to determine significance.Results We found a significant difference between COVpos and COVneg samples for all markers, all of which were significant at p < 0.001. The most prominent features were neutrophils (CD15, MPO) and MPO positive debris suggestive of NETS and were located in or around arterioles, venules, and capillaries. A similar distribution of platelets, monocytes, and C4d was seen in COVpos cases. Fibrin was found scattered in arterioles, venules, interstitial regions, and within ventricular cavities. CD34 highlighted vascular alterations of endothelial cells in some but not all cases.Conclusion Autoinflammation is the primary mechanism of myocyte injury observed in COVpos hearts. The significant increase in platelets, monocytes, and neutrophils and the presence of neutrophil NETs, C4d, and fibrin suggest that myocardial injury involves neutrophils, NETosis, coagulation, complement activation, and monocyte accumulation.

An alphacoronavirus polymerase structure reveals conserved co-factor functions (preprint)

Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from the betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine the structure of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. Our structure shows an unexpected nsp8 stoichiometry in comparison to other published coronavirus polymerase structures. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha and betacoronaviruses as previously hypothesized. Our work shows the importance of studying diverse coronaviruses to reveal aspects of coronavirus replication while also identifying areas of conservation to be targeted by antiviral drugs.

The highly conserved stem-loop II motif is dispensable for SARS-CoV-2 (preprint)

The stem-loop II motif (s2m) is a RNA structural element that is found in the 3' untranslated region (UTR) of many RNA viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Though the motif was discovered over twenty-five years ago, its functional significance is unknown. In order to understand the importance of s2m, we created viruses with deletions or mutations of the s2m by reverse genetics and also evaluated a clinical isolate harboring a unique s2m deletion. Deletion or mutation of the s2m had no effect on growth in vitro, or growth and viral fitness in Syrian hamsters in vivo. We also compared the secondary structure of the 3' UTR of wild type and s2m deletion viruses using SHAPE-MaP and DMS-MaPseq. These experiments demonstrate that the s2m forms an independent structure and that its deletion does not alter the overall remaining 3'UTR RNA structure. Together, these findings suggest that s2m is dispensable for SARS-CoV-2.

Development of a Rapid and Specific MALDI-TOF Mass Spectrometric Assay for SARS-CoV-2 Detection (preprint)

We have developed a rapid and highly specific assay for detecting and monitoring SARS-CoV-2 infections by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). As MALDI-TOF mass spectrometers are available in a clinical setting, our assay has the potential to serve as alternative to the commonly used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Sample preparation prior to MALDI-TOF-MS involves the tryptic digestion of SARS-CoV-2 proteins, followed by an enrichment of virus-specific peptides from SARS-CoV-2 nucleoprotein via magnetic antibody beads. Our MALDI-TOF-MS method allows the detection of SARS-CoV-2 nucleoprotein as low as 8 amol/microliter. MALDI-TOF mass spectra are obtained in just a few seconds, which makes our MS-based assay suitable for a high-throughput screening of SARS-CoV-2 in healthcare facilities in addition to PCR. Due to the specific detection of virus peptides, different SARS-CoV-2 variants are readily distinguished from each other. Specifically, we show that our MALDI-TOF-MS assay discriminates SARS-CoV-2 strain B.1.617.2 delta variant from all other variants in patients' samples, making our method highly valuable to monitor the emergence of new virus variants.