Mucosal rAd5 Immunization against SARS-CoV-2 Spike Elicits Cross-Reactive Nasal and Serum Neutralizing Antibodies and Protects Against Beta Variant Challenge in African Green Monkeys

Background. Mucosal vaccination may offer increased protection against SARS-CoV-2 compared to parental immunization. Here, we describe immunogenicity and efficacy following viral challenge in non-human primates after intranasal delivery of three unique non-replicating adenoviral vector vaccine (rAd5) candidates. Methods. African green monkeys (AMG) were prime boost immunized 29 days apart with vaccine candidates either expressing the parental spike protein alone (Wuhan-S), spike plus nucleocapsid(Wuhan-S-N), or the spike protein fromthe beta variant (beta-S). Serumand nasal swabs were collected every 14 days and humoral responses to full length spike (S) and receptor binding domain (RBD) were assessed.AllAMGs were challenged with SARS-CoV-2 B.1.351 (beta variant)onday 56. Viral loadsmeasured every two days by TCID50 in nasal washes and bronchial lavage fluid post challenge. Results. Mucosal immunization with Wuhan-S induced significant increases in serum IgG and IgA responses against the homologous parental lineage, as well as beta, delta, and omicron variants. In nasal samples, Wuhan-S immunization elicited over 500-fold increases in in cross-reactive IgA against multiple variants of concern including delta and omicron. While the beta-S rAd5 vaccine candidate induced enhanced serum IgG responses to homologous S and RBD proteins, this approach resulted in less cross-reactive antibodies to other variants compared to Wuhan-S rAd5 vaccine. Despite the differences in the ability to elicit cross-reactive antibody responses, all vaccinated AMGs challenged with SARS-CoV-2 B.1.351 (beta variant), had a significant reduction in viral titers by TCID50 in the nasal passages and reduced viral load in bronchial lavage fluid compared to unvaccinated controls. Conclusion. These results demonstrate mucosal administration of rAd5 clinical candidate vaccine, Wuhan-S, is immunogenic and offers cross-protective humoral responses in both serum and nasal compartments against a mismatched SARS-CoV-2 challenge virus.

Oral Tablet Vaccination Induces Heightened Cross-Reactive CD8 T Cell Responses to SARS-COV-2 in Humans

Background. Covid-19 has accelerated global demand for easily distributed vaccines. Furthermore, as variant SARS-CoV-2 strains that circumvent antibody responses emerge, cross-protective vaccines provide substantial public health benefits. Vaxart is developing a shelf stable oral tablet vaccine that incorporates both the spike (S) and the more conserved nucleocapsid (N) proteins. Vaxart's vaccine platform uses a non-replicating adenovirus and a TLR3 agonist as an adjuvant. Methods. In an open-label phase 1 clinical study, 35 healthy subjects received either a single low (1x1010 IU;n=15) or high (5x1010 IU;n=15) dose of the vaccine candidate VXA-CoV2-1 with a small cohort receiving 2 low doses. PBMCs were taken at pre- and 7 days post-vaccination and restimulated with S and N peptides from SARSCoV-2 or the 4 human endemic coronaviruses (HCoV). Cells were stained for CD4/ CD8/CD107a (surface) and IFNγ/TNFα (intracellular). Subjects that received an intramuscular (i.m.) mRNA vaccine had PBMCs taken at the same timepoints and were compared in the same assay. Results. The study's results indicate that the VXA-CoV2-1 tablet was well tolerated. The majority of subjects had an increase in S-specific anti-viral CD8+ T cell responses. 19/26 (73%) subjects had a measurable CD8+ T cell response on day 8 above baseline, on average 1.5-4.6%. In a comparator experiment with the 2 SARS-CoV-2 i.m. mRNA vaccines, VXA-CoV2-1 outperformed other vaccine candidates with a >3.5-fold increase in S specific antiviral CD8 T cell responses. T cell responses specific to the 4 endemic HCoV were increased by 0.6% in subjects given VXA-CoV2-1. Conclusion. Here we describe a room temperature stable tablet that induces SARS-CoV-2 S specific CD8 T cells of high magnitude after one dose in humans. Overall, the level of antiviral SARS-CoV-2 specific T cells, particularly IFNg-producing CD8s, induced following oral immunization with VXA-CoV2-1 are of higher magnitude than the mRNA vaccines currently in use against COVID-19. T cell responses against 4 endemic HCoV were also induced. Because T cells may be important in protecting against death and severe infection, these results suggest that VXA-CoV2-1 could be cross-protective against a wide array of emerging pandemic coronaviruses.

The utility of the Edmonton Obesity Staging System for the prediction of COVID-19 outcomes: a multi-centre study.

BACKGROUND: Patients with obesity have an increased risk for adverse COVID-19 outcomes. Body mass index (BMI) does not acknowledge the health burden associated this disease. The performance of the Edmonton Obesity Staging System (EOSS), a clinical classification tool that assesses obesity-related comorbidity, is compared with BMI, with respect to adverse COVID-19 outcomes. METHODS: 1071 patients were evaluated in 11 COVID-19 hospitals in Mexico. Patients were classified into EOSS stages. Adjusted risk factors for COVID-19 outcomes were calculated and survival analysis for mechanical ventilation and death was carried out according to EOSS stage and BMI category. RESULTS: The risk for intubation was higher in patients with EOSS stages 2 and 4 (HR 1.42, 95% CI 1.02-1.97 and 2.78, 95% CI 1.83-4.24), and in patients with BMI classes II and III (HR 1.71, 95% CI 1.06-2.74, and 2.62, 95% CI 1.65-4.17). Mortality rates were significantly lower in patients with EOSS stages 0 and 1 (HR 0.62, 95% CI 0.42-0.92) and higher in patients with BMI class III (HR 1.58, 95% CI 1.03-2.42). In patients with a BMI ≥ 25 kg/m2, the risk for intubation increased with progressive EOSS stages. Only individuals in BMI class III showed an increased risk for intubation (HR 2.24, 95% CI 1.50-3.34). Mortality risk was increased in EOSS stages 2 and 4 compared to EOSS 0 and 1, and in patients with BMI class II and III, compared to patients with overweight. CONCLUSIONS: EOSS was associated with adverse COVID-19 outcomes, and it distinguished risks beyond BMI. Patients with overweight and obesity in EOSS stages 0 and 1 had a lower risk than patients with normal weight. BMI does not adequately reflect adipose tissue-associated disease, it is not ideal for guiding chronic-disease management.

Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein.

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the "pairing potential" of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the "inside" or "outside" of the infected cell. Their therapeutic potential will be discussed.

COVID-19-associated mucormycosis, diabetes and steroid therapy: Experience in a single centre in Western Mexico.

BACKGROUND: COVID-19-associated mucormycosis (CAM) has emerged as a challenging complication as the current pandemic has increased the population requiring treatment with corticosteroids. CAM has caused a massive outbreak in India, reported to be causing cases in Iran, Egypt and The Netherlands. OBJECTIVES: To describe CAM cases occurring in a single centre in Western Mexico. METHODS: Our group carried out a retrospective study from May 2020 to May 2021 to identify CAM cases in patients with previous COVID-19 diagnosis. RESULTS: Six CAM cases occurred in a single centre in Western Mexico during the study period, most of them with diabetes (n = 5/6) and all received corticosteroid therapy even when only three had severe COVID-19. After analysing local COVID-19 burden, it was estimated that in this region, CAM was 300 times more frequent among COVID individuals than the estimates for general population. CONCLUSION: Similar to large reports in India and other countries, CAM cases reported in this study were diagnosed in individuals with diabetes, hyperglycaemic status and with history of previous use of corticosteroids. Identifying these individuals at risk can help the early identification of CAM. In addition, strict glycaemic control and avoidance of unnecessary corticosteroid in non-severe COVID-19 cases could help in preventing this complicated fungal infection.

Neutralisation of SARS-CoV-2 by anatomical embalming solutions.

Teaching and learning anatomy by using human cadaveric specimens has been a foundation of medical and biomedical teaching for hundreds of years. Therefore, the majority of institutions that teach topographical anatomy rely on body donation programmes to provide specimens for both undergraduate and postgraduate teaching of gross anatomy. The COVID-19 pandemic has posed an unprecedented challenge to anatomy teaching because of the suspension of donor acceptance at most institutions. This was largely due to concerns about the potential transmissibility of the SARS-CoV-2 virus and the absence of data about the ability of embalming solutions to neutralise the virus. Twenty embalming solutions commonly used in institutions in the United Kingdom and Ireland were tested for their ability to neutralise SARS-CoV-2, using an established cytotoxicity assay. All embalming solutions tested neutralised SARS-CoV-2, with the majority of solutions being effective at high-working dilutions. These results suggest that successful embalming with the tested solutions can neutralise the SARS-CoV-2 virus, thereby facilitating the safe resumption of body donation programmes and cadaveric anatomy teaching.

PIN112 Geographical Inequalities of the Incidence of Coronavirus Disease 2019 (COVID-19) Caused By Sars-COV-2 VIRUS in the European Union

Objectives: The occurrence of coronavirus disease 2019 (COVID-19) showed different patterns in different countries. The aim of our study is to analyse the geographical inequalities in the occurrence of COVID-19 caused by SARS-CoV-2 virus in member states of the European Union (EU). Methods: Data derived from the European Centre for Disease Prevention and Control (ECDC) of European Union. Member states of the European Union were classified according to their geographical location: Western-European (Austria, Belgium, France, Germany, Luxembourg, Netherlands), Eastern-European (Bulgaria, Croatia, Czechia, Estonia, Hungary, Latvia, Lithuania, Poland, Romania, Slovakia, Slovenia), Mediterranean (Cyprus, Greece, Italy, Malta, Portugal, Spain) and Nordic (Denmark, Finland, Ireland, Sweden) countries. Total number of COVID-19 cases per 1 million population and standard deviation (SD) was calculated with weighted averages. Period up to 7 July 2020 was covered. Single factor analysis of variance (ANOVA) was performed. Results: Up to 07.07.2020 in the European Union 1,313,317 COVID-19 cases were identified resulting in an incidence of 2.950 cases/1 million population. We found the highest occurrence in Luxembourg (7,352 cases/1 million population), Sweden (7,262), Spain (6,400), Belgium (5,354), Ireland (5,171), while the lowest occurrence was observed in Lithuania (678), Latvia (601), Hungary (435), Greece (344), Slovakia (324). We found 19.8-fold differences in the incidence of COVID-19 cases among EU countries with the lowest (Slovakia: 324) and highest (Luxembourg: 7,352) occurrence. Mediterranean countries had the highest incidence 4,563 (SD:+/-2,378), followed by Nordic: 4,514 (SD:+/-2,732), Western-European: 2.683 (SD:+/-2,114) and Eastern-European: 986 (SD:+/-395) countries. The relative risk of COVID-19 occurrence was 4.63-times higher in Mediterranean, 4.58-times higher in Nordic and 2.72-times higher in Western-European countries compared to Eastern-European countries. Single factor analysis of variance (ANOVA) showed significant differences among country groups (P<0.008). Conclusions: Eastern-European countries had significantly lower incidence of COVID-19 cases compared to Western-European, Mediterranean and Nordic countries in the European Union.

PIN81 Within Country Regional Inequalities of the Incidence of Coronavirus Disease 2019 (COVID-19) Caused By Sars-COV-2 VIRUS in Hungary

Objectives: The first patient with coronavirus disease 2019 (COVID-19) was diagnosed on the 4th of March 2020 in Hungary. The aim of our study is to analyse the regional inequalities in the occurrence of the coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 virus in Hungary. Methods: Data derived from the National Surveillance System (OSZIR) of the National Public Health Center (Nemzeti Népegészségügyi Központ) of Hungary. Patients diagnosed with coronavirus disease 2019 were confirmed by reverse transcription polymerase chain reaction (RT-PCR) in highly specialised laboratories designated for SARS-CoV-2 virus diagnostic. Data of laboratory confirmed cases were reported to the National Surveillance System. The period from the onset of first COVID-19 case up to 7th July 2020 was covered. Data were analysed according to 20 counties of Hungary. Results: Altogether 4,205 laboratory confirmed COVID-19 cases were identified in Hungary resulting in an incidence of 4.35 cases per 10,000 population. The number of novel coronavirus daily cases reached its peak in Hungary between 10-23 April with higher than 100 novel cases per day (0.102 new cases per 10,000 population). There was a 28.88 times higher incidence of COVID-19 in the county with the lowest (Békés 0.39) and with the highest (Budapest 11.36) occurrence. We found 4 counties with very high COVID-19 incidence (cases per 10,000 population): Budapest (11.36), Komárom-Esztergom county (10.20), Zala county (9.8) and Fejér county (9.05). The lowest frequency of COVID-19 was observed in the following counties (cases per 10,000 population): Jász-Nagykun-Szolnok (0.46), Hajdú-Bihar (0.44), Bács-Kiskun (0.42) and Békés (0.39). Conclusions: We found 28.88-fold differences in the incidence of COVID-19 cases among Hungarian counties with the lowest and highest occurrence. The highest incidence was observed in the capital city (Budapest) and in counties characterized by either nosocomial infections or cumulative cases in social institutions.