Tixagevimab-cilgavimab (AZD7442) for the treatment of patients hospitalized with COVID-19 (DisCoVeRy): A phase 3, randomized, double-blind, placebo-controlled trial (preprint)

Background Tixagevimab and cilgavimab (AZD7442) are two monoclonal antibodies developed by AstraZeneca for the pre-exposure prophylaxis and treatment of patients infected by SARS-CoV-2. Its effectiveness and safety in patients hospitalized with COVID-19 was not known at the outset of this trial. Methods DisCoVeRy is a phase 3, adaptive, multicentre, randomized, controlled trial conducted in 63 sites in Europe. Participants were randomly assigned (1:1) to receive placebo or tixagevimab-cilgavimab in addition to standard of care. The primary outcome was the clinical status at day 15 measured by the WHO seven-point ordinal scale. Several clinical, virological, immunological and safety endpoints were also assessed. Findings Due to slow enrolment, recruitment was stopped on July 1st, 2022. The antigen positive modified intention-to-treat population (mITT) was composed of 173 participants randomized to tixagevimab-cilgavimab (n=91) or placebo (n=82), 91.9% (159/173) with supplementary oxygen, and 47.4% (82/173) previously vaccinated at inclusion. There was no significant difference in the distribution of the WHO ordinal scale at day 15 between the two groups (odds ratio (OR) 0.93, 95%CI [0.54-1.61]; p=0.81) nor in any clinical, virological or safety secondary endpoints. In the global mITT (n=226), neutralization antibody titers were significantly higher in the tixagevimab-cilgavimab group/patients compared to placebo at day 3 (Least-square mean differences (LSMD) 1.44, 95% Confidence interval (CI) [1.20-1.68]; p < 10-23) and day 8 (LSMD 0.91, 95%CI [0.64-1.18]; p < 10-8) and it was most important for patients infected with a pre-omicron variant, both at day 3 (LSMD 1.94, 95% CI [1.67-2.20], p < 10-25) and day 8 (LSMD 1.17, 95% CI [0.87-1.47], p < 10-9), with a significant interaction (p < 10-7 and p=0.01 at days 3 and 8, respectively). Interpretation There were no significant differences between tixagevimab-cilgavimab and placebo in clinical endpoints, however the trial lacked power compared to prespecified calculations. Tixagevimab-cilgavimab was well tolerated, with low rates of treatment related events.

Real-world effectiveness of molnupiravir and nirmatrelvir/ritonavir among COVID-19 community, highly vaccinated patients with high risk for severe disease: Evidence that both antivirals reduce the risk for disease progression and death (preprint)

Besides the significant benefits of vaccination against COVID-19, the risk of severe disease and death from COVID-19 among highly vulnerable populations remains of concern. Implementation of oral antiviral treatment has shown significant benefits for outpatients with high risk for severe disease, however, their effectiveness remains to be evaluated in real-life settings and in the presence of new Omicron subvariants. We aimed to investigate the effectiveness of molnupiravir and nirmatrelvir/ritonavir using a retrospective cohort design with outcomes hospital admission and death from COVID-19, in Greece. The effectiveness of each drug was estimated through a comparison of the antiviral's recipients with an age-matched control group of non-recipients, adjusted for age, previous SARS-CoV-2 infection, vaccination status, and vaccination recency. Our analysis showed that molnupiravir significantly reduced the risk for hospitalization (OR = 0.40, p < 0.001) and death from COVID-19 (OR = 0.31, p < 0.001), with the effect being more intense among elderly patients (>=75 years old). The effectiveness was higher among those with full adherence. Nirmatrelvir/ritonavir was found also to significantly reduce the risk of hospital admission (OR = 0.31, p < 0.001) and death (OR = 0.28, p < 0.001) and, similarly to molnupiravir, effectiveness was stronger among elderly patients and those with the highest levels of adherence. Analysis of the relative effectiveness of nirmatrelvir/ritonavir versus molnupiravir suggested that nirmatrelvir/ritonavir was associated with a reduced risk for hospital admission (OR = 0.58, p < 0.001) compared to molnupiravir, adjusted for age, previous SARS-CoV-2 infection, vaccination status, and co-morbidities. Our real-world study provides evidence about the reduced risk of hospitalization and death in highly vaccinated patients with a high risk for severe disease in Greece. These findings highlight that although the hospitalization and mortality risk has been reduced mainly due to vaccination and the emergence of Omicron variants, antivirals provide significant additional benefits in highly vulnerable patients and therefore their use is documented and strongly indicated.

Transmission potential of human monkeypox in mass gatherings (preprint)

Since May 2022, a large number of monkeypox cases has been reported in non-endemic settings. Taking into account the strict measures implemented due to the COVID-19 pandemic and the desire of people to reclaim what is perceived as lost time, it is anticipated that mass gatherings this summer will be highly attended. Based on data for the secondary attack rate among unvaccinated contacts from endemic countries, we estimate that, on average, more than one secondary case is anticipated per infectious person if he/she has a high number of group contacts (>30) or more than eight close contacts. Although the role of group contacts in mass gatherings is uncertain (less likely to involve physical contact, shorter duration), close contacts associated with the event (e.g. intimate/sexual contact with other attendees) might be the amplifying event. Enforcing awareness, early recognition and engaging affected populations in the monkeypox response are important to control transmission.

Comparative effectiveness of COVID-19 vaccination against death and severe disease in an ongoing nationwide mass vaccination campaign (preprint)

Background: As national COVID-19 mass vaccination campaigns are rolled out, it is important to demonstrate and measure their public health benefit. We aimed to estimate COVID-19 Vaccine Effectiveness (VE) against severe disease and death in the Greek population, for all vaccines in use. Methods: Nationwide active surveillance and vaccination registry data during January-December 2021 were used to estimate VE via quasi-Poisson regression, as one minus the Incidence Rate Ratio, adjusted for age and calendar time. Interaction terms were included to assess VE by age group, against the 'delta' SARS-CoV-2 variant and waning of VE over time. Results: Two doses of BNT162b2, mRNA-1273 or ChAdOx1 nCov-19 vaccines offered very high (>90%) VE against both intubation and death across all age groups, similar against both 'delta' and previous variants, with one-dose Ad26.COV2.S slightly lower. There was some waning over time but VE remained >80% at six months, and three doses increased VE again to near 100%. Vaccination prevented an estimated 19,691 COVID-19 deaths (95% CI: 18,890-20,788) over the study period. Conclusions: All approved vaccines were very highly effective in preventing COVID-19 severe disease and death. Every effort should be made to vaccinate the population with at least two doses, in order to reduce the mortality and morbidity impact of the pandemic.

Autoantibodies targeting cytokines and connective tissue disease autoantigens are common in acute non-SARS-CoV-2 infections. (preprint)

The widespread presence of autoantibodies in acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is increasingly recognized, but the prevalence of autoantibodies in infections with organisms other than SARS-CoV-2 has not yet been reported. We used protein arrays to profile IgG autoantibodies from 317 samples from 268 patients across a spectrum of non-SARS-CoV-2 infections, many of whom were critically ill with pneumonia. Anti-cytokine antibodies (ACA) were identified in > 50% of patients infected with non-SARS-CoV-2 viruses and other pathogens, including patients with pneumonia attributed to bacterial causes. In cell-based functional assays, some ACA blocked binding to surface receptors for type I interferons (Type I IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6). Autoantibodies against traditional autoantigens associated with connective tissue diseases (CTDs) were also commonly observed in these cohorts, including newly-detected antibodies that emerged in longitudinal samples from patients infected with influenza. We conclude that autoantibodies, some of which are functionally active, may be much more prevalent than previously appreciated in patients who are symptomatically infected with diverse pathogens.

Relating SARS-CoV-2 shedding rate in wastewater to daily positive tests data: A consistent model based approach (preprint)

During the COVID-19 pandemic, wastewater-based epidemiology (WBE) has been engaged to complement medical surveillance and in some cases to also act as an early diagnosis indicator of viral spreading in the community. Most efforts worldwide by the scientific community and commercial companies focused on the formulation of protocols for SARS CoV-2 analysis in wastewater and approaches addressing the quantitative relationship between WBE and medical surveillance are lacking. In the present study, a mathematical model is developed which uses as input the number of daily positive medical tests together with the highly non-linear shedding rate curve of individuals to estimate the evolution of virus shedding rate in wastewater along calendar days. A comprehensive parametric study by the model using medical surveillance data for the city of Thessaloniki (~700,000 inhabitants, North Greece) reveals the conditions under which WBE can be used as an early warning tool for predicting pandemic outbreaks. It is shown that early warning capacity is not the same along the days of an outbreak and varies strongly with the number of days apart between the day of maximum shedding rate of individuals in their disease cycle and the day of their medical testing. Moreover, the present data show that there exists a proportion between unreported cases (asymptomatic persons and patients with mild symptoms that do not seek medical advice) and reported cases. The proportion is not steady but increases with the number of reported cases. The early detection capacity of WBE improves substantially in the presence of an increasing number of unreported cases. For Thessaloniki at the peak of the pandemic in mid-November 2020, the number of unreported cases reached a maximum around 4 times the number of reported people.

A look into the future of the COVID-19 pandemic in Europe: an expert consultation (preprint)

How will the coronavirus disease 2019 (COVID-19) pandemic develop in the coming months and years? Based on an expert survey, we examine key aspects that are likely to influence COVID-19 in Europe. The future challenges and developments will strongly depend on the progress of national and global vaccination programs, the emergence and spread of variants of concern, and public responses to nonpharmaceutical interventions (NPIs). In the short term, many people are still unvaccinated, VOCs continue to emerge and spread, and mobility and population mixing is expected to increase over the summer. Therefore, policies that lift restrictions too much and too early risk another damaging wave. This challenge remains despite the reduced opportunities for transmission due to vaccination progress and reduced indoor mixing in the summer. In autumn 2021, increased indoor activity might accelerate the spread again, but a necessary reintroduction of NPIs might be too slow. The incidence may strongly rise again, possibly filling intensive care units, if vaccination levels are not high enough. A moderate, adaptive level of NPIs will thus remain necessary. These epidemiological aspects are put into perspective with the economic, social, and health-related consequences and thereby provide a holistic perspective on the future of COVID-19.

Trends in COVID-19 vaccination intent, determinants and reasons for vaccine hesitancy: results from repeated cross-sectional surveys in the adult general population of Greece during November 2020-June 2021 (preprint)

Background: Vaccine hesitancy is a major barrier to achieve large-scale COVID-19 vaccination. We report trends in vaccination intention and associated determinants from surveys in the adult general population in Greece. Methods: Four cross-sectional phone surveys were conducted in November 2020, February, April and May 2021 on nationally representative samples of adults in Greece. Multinomial logistic regression was used on the combined data of the surveys to evaluate independent predictors of vaccination unwillingness/uncertainty. Results: Vaccination intention increased from 67.6% in November 2020 to 84.8%. in May 2021. Individuals aged 65 years or older were more willing to get vaccinated (May 2021: 92.9% vs. 79.5% in 18-39 years, p<0.001) but between age-groups differences decreased over time. Vaccination intention increased substantially in both sexes, though earlier among men than women and was higher in individuals with postgraduate studies (May 2021: 91.3% vs. 84.0% up to junior high). From multivariable analysis, unwillingness and/or uncertainty to get vaccinated was associated with younger age, female gender (in particular in the April 2021 survey), lower educational level and living with a child [≤]12 years old. Among those with vaccine hesitancy, concerns about vaccine effectiveness declined over time (21.6% in November 2020 vs. 9.6% in May 2021, p=0.014) and were reported more often by men; safety concerns remained stable over time (66.3% in November 2020 vs. 62.1% in May 2021, p=0.658) and were reported more often by women. Conclusions: Vaccination intention increased substantially over time. Tailored communication is needed to address vaccine hesitancy and concerns regarding vaccine safety.

COMPARATIVE IMMUNOGENICITY OF BNT162b2 mRNA VACCINE WITH NATURAL COVID-19 INFECTION (preprint)

The mRNA vaccine BNT162b2 has proven highly effective and currently many millions are being vaccinated. There are limited and conflicting data from immunogenicity studies on the effects of age, gender, vaccination side effects (VSE), risk factors for severe COVID-19 (RFS-COV), obesity (BMI) and previous SARS-CoV-2 (Pr-CoV) Moreover, immunogenicity data from COVID-19 patients comparing various disease categories of natural infection i.e. asymptomatic vs mild vs moderate vs severe infection are sparse, and include limited number of individuals. This study included 871 vaccinated health care workers (HCW) and 181 patients with natural infection. Immunogenicity was assessed by a quantative assay measuring anti-SARS-CoV-2 against the RBD domain of the spike protein (anti-RBD) and anti-SARS-CoV-2 against nucleocapsid protein (anti-N). Samples were collected 1-2 weeks after completion of the 2nd dose in the vaccinated HCWs and 15-59 days post symptoms onset in patients with natural infection. The concentration of anti-RBD in vaccinated individuals after multivariable analysis was significantly associated with age, gender, VSE and Pr-CoV. Specifically, anti-RBD median levels (95% CI) were lower by 2,466 (651-5,583), 6,228 (3,254-9,203) and 7,651 (4,479-10,823) AU/ml in 35-44, 45-54, 55-70 yrs respectively, compared with 18-34 yrs group. In females, median levels of anti-RBD were higher by 2,823 (859-4,787) compared with males, in individuals with VSE were higher by 5,024 (3,122-6,926) compared with no VSE, and in HCWs with Pr-CoV were higher by 9,971 (5,158-14,783) AU/ml compared with HCWs without Pr-CoV. Among individuals with natural infection, the median anti-RBD levels were 14.8 times higher in patients with critical COVID-19 infection compared with non-hospitalized individuals. The ratio of anti-RBD in vaccinated individuals versus those with natural infection varied from 1.0 up to 19.4 according to the clinical subgroup of natural infection. This study proves the high immunogenicity of BNT162b2 vaccine although its sustainability remains to be seen. The use of comparative data from natural infection serological panels, expressing the clinical heterogeneity of natural infection may facilitate early decisions for vaccine evaluation in clinical trials.

COVID-19 Mortality in Europe, by Latitude and Obesity Status: A Geo-Spatial Analysis in 40 Countries (preprint)

Background: On January 30, 2020, the World Health Organization (WHO) declared the COVID-19 as a public health emergency of international concern and later characterized it as a pandemic. Based on new data, excess body mass and vitamin D deficiency might be related with the disease severity and mortality. Thus, we aimed to evaluate whether latitude, as a proxy of sunlight exposure and Vitamin D synthesis, and prevalent obesity among European populations, is related with COVID-19 spread and severity. Methods: European COVID-19 data (incidence and fatality), including information on the prevalence of obesity, social distancing and others were obtained by the “Our World in Data” website on April 17, 2021.Findings : Adjusted analysis showed that higher COVID-19 incidence and fatality were pictured in countries being in higher latitude, both during the whole period [IRR (95% CI): Incidence= 1·07047 (1·07041, 1·07054), Fatality= 1·08650 (1·08605, 1·08695)], as well as, during the time period 1/11/2020-31/3/2021 [IRR (95% CI): Incidence= 1·07571 (1·07563, 1·07579), Fatality= 1·09202 (1·09145, 1·09258)]. Higher incidence and fatality of COVID-19 were observed where the prevalence of overweight/ obesity was higher during the whole time period [IRR (95% CI): Incidence= 1·01463 (1·01251, 1·01681), Fatality= 1·03000 (1·01438, 1·04587)], whereas during the time period 1/11/2020-31/3/2021, only COVID-19 incidence was higher but not fatality.Interpretation: Excess body mass and high latitude were found to be related with COVID-19 spread and mortality across Europe. These results provide insights for targeted interventions and preventive strategies against COVID-19.Funding: The authors received no specific funding for this work.Declaration of Interest: Declaration of interests: No conflict of interest

Temporal Dominance of B.1.1.7 over B.1.354 SARS-CoV-2 Variant: A Hypothesis Based on Areas of Variant Co-Circulation (preprint)

Some emergent SARS-CoV-2 variants raise concerns due to their altered biological properties. For both B.1.1.7 and B.1351 variants, named as variants of concern (VOC), increased transmissibility was reported, whereas B.1.351 was more resistant to multiple monoclonal antibodies (mAbs), as well as convalescent and vaccination sera. To test this hypothesis, we examined the proportion of VOC over time across different geographic areas where the two VOC, B.1.1.7 and B.1.351, co-circulate. Our comparative analysis was based on the number of SARS-CoV-2 sequences on GISAID database. We report that B.1.1.7 dominates over B.1.351 in geographic areas where both variants co-circulate and the B.1.1.7 was the first variant introduced in the population. The only areas where B.1.351 was detected at higher proportion were South Africa and Mayotte in Africa, where this strain was associated with increased community transmission before the detection of B.1.1.7. The dominance of B.1.1.7 over B.1.351 could be important since B.1.351 was more resistant to certain mAbs, as well as heterologous convalescent and vaccination sera, thus suggesting that it may be transmitted more effectively in people with pre-existing immunity to other VOC. This scenario would lessen the effectiveness of vaccine and urge the need to update them with new strains.

Efficient and Targeted COVID-19 Border Testing via Reinforcement Learning (preprint)

Throughout the COVID-19 pandemic, countries relied on a variety of ad-hoc border control protocols to allow for non-essential travel while safeguarding public health: from quarantining all travelers to restricting entry from select nations based on population-level epidemiological metrics such as cases, deaths or testing positivity rates. Here we report the design and performance of a reinforcement learning system, nicknamed “Eva.” In the summer of 2020, Eva was deployed across all Greek borders to limit the influx of asymptomatic travelers infected with SARS-CoV-2, and to inform border policies through real-time estimates of COVID-19 prevalence. In contrast to country-wide protocols, Eva allocated Greece’s limited testing resources based upon incoming travelers’ demographic information and testing results from previous travelers. By comparing Eva’s performance against modeled counterfactual scenarios, we show that Eva identified 1.85 times as many asymptomatic, infected travelers as random surveillance testing, with up to 2-4 times as many during peak travel, and 1.25-1.45 times as many asymptomatic, infected travelers as testing policies that only utilize epidemiological metrics. We demonstrate that this latter benefit arises, at least partially, because population-level epidemiological metrics had limited predictive value for the actual prevalence of SARS-CoV-2 among asymptomatic travelers and exhibited strong country-specific idiosyncrasies in the summer of 2020. Our results raise serious concerns on the effectiveness of country-agnostic internationally proposed border control policies that are based on population-level epidemiological metrics. Instead, our work represents a successful example of the potential of reinforcement learning and real-time data for safeguarding public health.

A Thermostable, Flexible RNA Vaccine Delivery Platform for Pandemic Response (preprint)

Current RNA vaccines against SARS-CoV-2 are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at -20{degrees}C or -70{degrees}C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) RNA vaccine delivery system for use in pandemic preparedness and pandemic response. Liquid NLC is stable at refrigerated temperatures for [≥] 1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC complexed with RNA may be readily lyophilized and stored at room temperature for [≥] 8 months or refrigerated temperature for [≥] 21 months. This thermostable RNA vaccine platform could significantly improve distribution of current and future pandemic response vaccines, particularly in low-resource settings.

Evolution of ACE2 and SARS-CoV-2 Interplay Across 247 Vertebrates (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) cause the most serious pandemics of Coronavirus Disease 2019 (COVID 19), which threatens human health and public safety. SARS-CoV-2 spike (S) protein uses angiotensin-converting enzyme 2 (ACE2) as recognized receptor for its entry into host cell that contributes to the infection of SARS-CoV-2 to hosts. Using computational modeling approach, this study resolved the evolutionary pattern of bonding affinity of ACE2 in 247 jawed vertebrates to the spike (S) protein of SARS-CoV-2. First, high-or-low binding affinity phenotype divergence of ACE2 to the S protein of SARS-CoV-2 has appeared in two ancient species of jawed vertebrates, Scyliorhinus torazame (low-affinity, Chondrichthyes) and Latimeria chalumnae (high-affinity, Coelacanthimorpha). Second, multiple independent affinity divergence events recur in fishes, amphibians-reptiles, birds, and mammals. Third, high affinity phenotypes go up in mammals, possibly implying the rapid expansion of mammals might accelerate the evolution of coronaviruses. Fourth, we found natural mutations at eight amino acid sites of ACE2 can determine most of phenotype divergences of bonding affinity in 247 vertebrates and resolved their related structural basis. Moreover, we also identified high-affinity or low-affinity-associated concomitant mutation group.The group linked to extremely high affinity may provide novel potentials for the development of human recombinant soluble ACE2 (hrsACE2) in treating patients with COVID-19 or for constructing genetically modified SARS-CoV-2 infection models promoting vaccines studies. These findings would offer potential benefits for the treatment and prevention of SARS-CoV-2. Keywords: Vertebrates, ACE2, SARS-CoV-2, Bonding Affinity

Computational insights into differential interaction of mamalian ACE2 with the SARS-CoV-2 spike receptor binding domain (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causing agent of the COVID-19 pandemic, has spread globally. Angiotensin-converting enzyme 2 (ACE2) has been identified as the host cell receptor that binds to receptor-binding domain (RBD) of the SARS-COV-2 spike protein and mediates cell entry. Because the ACE2 proteins are widely available in mammals, it is important to investigate the interactions between the RBD and the ACE2 of other mammals. Here we analyzed the sequences of ACE2 proteins from 16 mammals and predicted the structures of ACE2-RBD complexes. Analyses on sequence, structure, and dynamics synergistically provide valuable insights into the interactions between ACE2 and RBD. The comparison results suggest that the ACE2 of bovine, cat and panda form strong binding with RBD, while in the cases of rat, least horseshoe bat, horse, pig, mouse and civet, the ACE2 proteins interact weakly with RBD.

Molecular epidemiology of SARS-CoV-2 in Greece reveals low rates of onward virus transmission after lifting of travel restrictions based on risk assessment during summer 2020 (preprint)

Molecular epidemiology has provided an additive value to traditional public health tools by identifying SARS-CoV-2 clusters, or providing evidence that clusters based on virus sequences and contact tracing are highly concordant. Our aim was to infer the levels of virus importation and to estimate the impact of public health measures related to travel restrictions to local transmission in Greece. Our phylogenetic and phylogeographic analyses included 389 SARS-CoV-2 sequences collected during the first 7 months of the pandemic in Greece and a random collection in 5 replicates of 3,000 sequences sampled globally, as well as the best hits to our dataset identified by BLAST. Phylogenetic analyses revealed the presence of 70 genetically distinct viruses identified as independent introductions into Greece. The proportion of imported strains was 41%, 11.5%, and 8.8% during the three periods of sampling, namely, March (no travel restrictions), April to June (strict travel restrictions), and July to September (lifting of travel restrictions based on a thorough risk assessment), respectively. These findings reveal low levels of onward transmission from imported cases during summer and underscore the importance of targeted public health measures that can increase the safety of international travel during a pandemic.

The basis of a more contagious 501Y.V1 variant of SARS-COV-2 (preprint)

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing a world-wide pandemic. A variant of SARS-COV-2 (20I/501Y.V1) recently discovered in the United Kingdom has a single mutation from N501 to Y501 within the receptor binding domain (Y501-RBD), of the Spike protein of the virus. This variant is much more contagious than the original version (N501-RBD). We found that this mutated version of RBD binds to human Angiotensin Converting Enzyme 2 (ACE2) a ~10 times more tightly than the native version (N501-RBD). Modeling analysis showed that the N501Y mutation would allow a potential aromatic ring-ring interaction and an additional hydrogen bond between the RBD and ACE2. However, sera from individuals immunized with the Pfizer-BioNTech vaccine still efficiently block the binding of Y501-RBD to ACE2 though with a slight compromised manner by comparison with their ability to inhibit binding to ACE2 of N501-RBD. This may raise the concern whether therapeutic anti-RBD antibodies used to treat COVID-19 patients are still efficacious. Nevertheless, a therapeutic antibody, Bamlanivimab, still binds to the Y501-RBD as efficiently as its binds to N501-RBD.