Inactivation of the Niemann Pick C1 cholesterol transporter 1 (NPC1) restricts SARS-CoV-2 infection (preprint)

The Niemann Pick C1 (NPC1) protein is an intracellular cholesterol transporter located in the late endosome/lysosome (LE/Ly) and is involved in cholesterol mobilization. Loss-of-function mutations of the NPC1 gene lead to accumulation of cholesterol and sphingolipids in LE/Ly, resulting in severe fatal NPC1 disease. Cellular alterations associated with NPC1 inactivation affect both the integrity of lipid rafts and the endocytic pathway. Because the angiotensin-converting enzyme 2 (ACE2) and type 2 serine transmembrane protease (TMPRSS2) of the SARS-CoV-2 Spike (S) protein also localize to lipid rafts, we sought to investigate the hypothesis that NPC1 inactivation would generate an intrinsically unfavorable barrier to SARS-CoV-2 entry. In this study, we demonstrate that NPC1 pharmacological inactivation or CRISP/R-Cas mediated ablation of NPC1 dramatically reduced SARS-CoV-2 infectivity. More specifically, our findings demonstrate that pharmacological inactivation of NPC1 results in massive accumulation of ACE2 in the autophagosomal/lysosomal compartment. A >40-fold decrease in virus titer indicates that this effectively prevents VSV-Spike-GFP infection by impeding virus binding and entry. A similarly marked decrease in viral infectivity is observed in cells that had NPC1 expression genetically abrogated. These observations were further confirmed in a de novo SARS-CoV-2 infection paradigm, where cells were infected with the naturally pathogenic SARS-CoV-2. Overall, this work offers strong evidence that NPC1 function is essential for successful SARS-CoV-2 infection, thus implicating NPC1 as a potential therapeutic target in COVID-19 management.

Changes in Antigen-Reactive Antibody Level at Various Time Spans after COVID-19 Vaccination Using Machine Learning (preprint)

Vaccines trigger a complicated immunological response that includes B and T cells, with B cells producing antibodies. SARS-CoV-2 immunity weakens over time after vaccination. Discovering key changes in antigen-reactive antibodies over time after vaccination could help improve vaccine efficiency. In this study, we collected data on blood antibody levels in a cohort of healthcare workers vaccinated for COVID-19 and obtained 73 antigens in samples from four groups according to the duration after vaccination, including 104 unvaccinated healthcare workers, 534 healthcare workers within 60 days after vaccination, 594 healthcare workers between 60 and 180 days after vaccination, and 141 healthcare workers over 180 days after vaccination. An efficient machine learning based framework containing four feature selection methods (least absolute shrinkage and selection operator, light gradient boosting machine, Monte Carlo feature selection, and maximum relevance minimum redundancy) and four classification algorithms (decision tree, k-nearest neighbor, random forest, and support vector machine) was designed to screen out essential antigens. Several efficient classifiers with weighted F1 value around 0.75 were constructed. This study revealed that S1+S2, S1.mFcTag, S1.HisTag, S1, S2, Spike.RBD.His.Bac, Spike.RBD.rFc, and S1.RBD.mFc were most highly ranked among all features, where S1 and S2 are the subunits of Spike, and the suffixes represent the tagging information of different recombinant proteins. Meanwhile, the classification rules were extracted from the optimal decision tree to explain quantitatively the roles of antigens in the classification. This study identified antibodies associated with decreased clinical immunity based on populations with different time spans after vaccination. These antibodies have important implications for maintaining long-term immunity to SARS-CoV-2.

SARS-CoV-2 infection during pregnancy and the risk of adverse maternal outcomes in the Republic of Georgia: a national birth registry-based cohort study (preprint)

Importance Georgia experienced an increase in maternal mortality (MM) during the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, which warrants further investigation.Objective This study aimed to assess associations between SARS-CoV-2 infection during pregnancy and MM, post-delivery intensive care unit (ICU) admission, and caesarean section (CS) delivery.Methods We performed a national birth registry-based cohort study including pregnant women who delivered between February 28, 2020 and August 31, 2022. Data was linked with Coronavirus disease (COVID-19) testing, vital, and immunization registries. Pregnant women were classified into three groups: confirmed SARS-CoV-2 infection from conception through 31 days before delivery, confirmed infection in the 30 days before or at delivery, and women negative for SARS-CoV-2 infection or without any test results (reference group). Multivariable logistic regression was used to calculate adjusted odds ratios (aOR) and 95% confidence intervals (CIs).Results Among 111,278 pregnant women, 16,661 had confirmed infection during pregnancy; 7,304 were fully vaccinated against COVID-19. Compared to the reference group, those with confirmed infection in the 30 days before or at delivery experienced increased odds of MM (aOR: 45.5, 95% CI, 23.1–89.3), post-delivery ICU admission (aOR: 5.62, 95% CI, 4.37–7.22), and CS delivery (aOR: 1.12, 95% CI, 1.03–1.21).Conclusions Hence, pregnant women in Georgia with confirmed SARS-CoV-2 infection in the 30 days before or at delivery conferred an alarmingly higher risk of MM and post-delivery ICU admission. Additionally, results highlight that most women were not vaccinated against COVID-19. These findings should signal to stakeholders that adherence to public health preventive measures needs to be improved.

Longitudinal Humoral and Cell-Mediated Immune Responses in a Population-Based Cohort in Zurich, Switzerland between March and June 2022 - Evidence for Protection against Omicron SARS-CoV-2 Infection by Neutralizing Antibodies and Spike-specific T cell responses (preprint)

Background: The correlate(s) of protection against SARS-CoV-2 remain incompletely de-fined. Additional information regarding the combinations of antibody and T cell-mediated immunity which can protect against (re)infection are needed. Methods: We conducted a population-based, longitudinal cohort study including 1044 individuals of varying SARS-CoV-2 vaccination and infection statuses. We assessed Spike (S)- and Nucleocapsid (N)-IgG and wildtype, delta, and omicron neutralizing antibodies. In a subset of 328 individuals, we evaluated S, Membrane (M) and N-specific T cells. 3 months later, we reassessed antibody (n=964) and T cell (n=141) responses and evaluated factors associated with protection from (re)infection. Results: At study start, >98% of participants were S-IgG seropositive. N-IgG and M/N-T cell responses increased over time, indicating viral (re)exposure, despite existing S-IgG. Com-pared to N-IgG, M/N-T cells were a more sensitive measure of viral exposure. N-IgG titers in the top 33% of participants, omicron neutralizing antibodies in the top 25%, and S-specific T cell responses were all associated with reduced likelihood of (re)infection over time. Conclusions: Population-level SARS-CoV-2 immunity is S-IgG-dominated, but heterogenous. M/N T cell responses can distinguish previous infection from vaccination, and monitoring a combination of N-IgG, omicron neutralizing antibodies and S-T cell responses may help estimate protection against SARS-CoV-2 (re)infection.

High incidence and mortality diseases control is more imperative apart from COVID-19 in China (preprint)

Objective: In order to enhance the health level, treat and cure well all the diseases, especially the high incidence and mortality diseases. So as to save the life and promote the quality of life. The research has been done to find the facts that that much more death caused diseases apart from the COVID-19 have been killing the people even worse at the COVID-19 pandemic time.  Methods: Summarized the data extracted from three public official internet, the National Bureau of Statistics of China, the WHO and the International Diabetes Federation. Found out the health problems and health influenced diseases data apart from the COVID-19 in China. And analysing the data and facts. Results: Based on the three channels of official internet, check and look up the valuable data, find out the demanded data and reorganize, build new tables. So the concrete facts to show the critical Chinese health problems apart from COVID-19 have been found. The 12 tables have been created and found that in China, the leading causes of death are Cardiovascular diseases, Malignant neoplasms, Respiratory diseases, Diabetes mellitus , injuries. The diseases have not been prevented and cured. The total death in China has been being more and more. The communicable diseases of Viral Hepatitis and Pulmonary Tuberculosis have been being the No.1 and No.2 incidences in Chinese communicable infections. The mortality of Heart Diseases in Chinese rural areas has been being in fast trend of heavier and heavier, from 87.10 in 2008 to 171.36 in 2020 and in recent three years the No. 1 killer in Chinese rural areas. The explosively developed incidence of diabetes mellitus from the year 2000 in 22564.80 cases(in 1000s) exploded into 90045.10 cases(in 1000s) at 2011, and much too more cases developed up to now.  Conclusion: In China, much more works must be done to control the leading causes of death, the diseases incidence and prevalence undermining health, including the Cardiovascular diseases, Malignant neoplasms, Respiratory diseases, Diabetes mellitus, injuries apart from COVID-19, especially the heart diseases in Chinese rural areas has been being the No. 1 killer in recent three years. The communicable diseases of Viral Hepatitis and Pulmonary Tuberculosis must be controlled and prevented. The urgently needed actions to control the leading causes of death, incidence and prevalence apart from COVID-19 in China may be referenced by other countries and international health organizations.

Post-pandemic modeling of COVID-19: Waning immunity determines recurrence frequency (preprint)

There are many factors in the current phase of the COVID-19 pandemic that signal the need for new modeling ideas. In fact, most traditional infectious disease models do not address adequately the waning immunity, in particular as new emerging variants have been able to brake the immune shield acquired either by previous infection by a different strain of the virus, or by inoculation of vaccines not effective for the current variant. Furthermore, in a post-pandemic landscape in which reporting is no longer a default, it is impossible to have reliable quantitative data at the population level. Our contribution to COVID-19 post-pandemic modeling is a simple mathematical predictive model along the age-distributed population framework, that can take into account the waning immunity in a transparent and easily controllable manner. Numerical simulations show that under static conditions, the model produces periodic solutions that are qualitatively similar to the reported data, with the period determined by the immunity waning profile. Evidence from the mathematical model indicates that the immunity dynamics is the main factor in the recurrence of infection spikes, however, irregular perturbation of the transmission rate, due to either mutations of the pathogen or human behavior, may result in suppression of recurrent spikes, and irregular time intervals between consecutive peaks. The spike amplitudes are sensitive to the transmission rate and vaccination strategies, but also to the skewness of the profile describing the waning immunity, suggesting that these factors should be taken into consideration when making predictions about future outbreaks.

Booster immunization with Ad26.COV2.S or Omicron adapted vaccine enhanced immune responses and efficacy against SARS-CoV-2 Omicron in non-human primates (preprint)

Omicron spike (S) encoding vaccines as boosters, are a possible strategy to improve COVID-19 vaccine efficacy against Omicron. Here, non-human primates immunized twenty months earlier with Ad26.COV2.S, were boosted with Ad26.COV2.S, Ad26.COV2.S.529 (encoding Omicron BA.1 S) or a combination of both vaccines. All vaccines elicited a rapid increase in WA1/2020 and Omicron S antibody titers; Omicron BA.1 and BA.2 antibody responses were most effectively boosted by vaccines including Ad26.COV2.S.529. Independent of vaccine used, mostly WA1/2020-reactive or WA1/2020 and Omicron BA.1 cross-reactive B cells were detected. Boosting with vaccines including Ad26.COV2.S.529 provided slightly higher protection of the lower respiratory tract against Omicron BA.1 challenge compared with Ad26.COV2.S. Antibodies and cellular immune responses were identified as complementary correlates of protection. Overall, a booster with an Omicron-spike based vaccine provided moderately improved immune responses and protection compared with the original Wuhan-spike based vaccine, which still provided robust immune responses and protection against Omicron infection.

Correlation of patient serum IgG, IgA and IgM antigen binding with COVID-19 disease severity using multiplexed SARS-CoV-2 antigen microarray and maintained relative IgA and IgM antigen binding over time (preprint)

Zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans in December 2019 caused the coronavirus disease 2019 (COVID-19) pandemic. Serological monitoring is critical for detailed understanding of individual immune responses to infection and protection to guide clinical therapeutic and vaccine strategies. We developed a high throughput multiplexed SARS-CoV-2 antigen microarray incorporating spike (S) and nucleocapsid protein (NP) and fragments expressed in various hosts which allowed simultaneous assessment of serum IgG, IgA, and IgM responses. Antigen glycosylation influenced antibody binding, with S glycosylation generally increasing and NP glycosylation decreasing binding. Purified antibody isotypes demonstrated a binding pattern and intensity that differed from the same isotype in the presence of other isotypes in whole serum, probably due to competition. Using purified antibody isotypes from naive Irish COVID-19 patients, we correlated antibody isotype binding to different panels of antigens with disease severity, with significance for binding to the S region S1 expressed in insect cells (S1 Sf21) for all three antibody isotypes. Assessing longitudinal response for constant concentrations of antibody isotypes for a subset of patients demonstrated that while the relative proportion of antigen-specific IgGs decreased over time for severe disease, the relative proportion of antigen-specific IgA binding remained at the same magnitude at 5 and 9 months post-first symptom onset. Further, the relative proportion of IgM binding decreased for S antigens but remained the same for NP antigens. This may support antigen specific serum IgA and IgM playing a role in maintaining longer-term protection, of importance for developing and assessing vaccine strategies. Overall, these data demonstrate the multiplexed platform as a sensitive and useful platform for expanded humoral immunity studies, allowing detailed elucidation of antibody isotypes response against multiple antigens. This approach will be useful for monoclonal antibody therapeutic studies and screening of donor polyclonal antibodies for patient infusions.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps (preprint)

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells (receptor traps). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta- pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

Vaccine, Booster and Natural Antibody Binding to SARS-CoV-2 Omicron (BA.1) Spike Protein and Vaccine Efficacy (preprint)

The SARS-CoV-2 Omicron variant (BA.1) has 25 unique mutations to the Spike glycoprotein, suggesting the efficacy of current vaccines against the new variant may be seriously degraded. A fully quantitative antibody binding study was performed for Spike Omicron (SO) and original Spike (S) proteins simultaneously on three cohorts of patients: convalescent following RT-PCR-confirmed infection in early 2020, double-vaccinated at [≥]2 weeks, and vaccine boosters. The average (mode) of the booster cohort response distributions were 15.1 mg/L and 13.4 mg/L for S and SO, respectively, compared with the significantly lower double-vaccinated average, S=2.4 mg/L, SO=2.0 mg/L, and natural infections average S=2.0 mg/L, SO = 1.8 mg/L. A preliminary epitope degradation screen was performed for a panel of antibodies raised to the S1 and S2 regions of the original S protein. The panel showed significant degradation to antibody epitopes in the S1 region. Differential antibody binding of the vaccine response to S and SO suggests vaccine efficacy may be reduced by up to 50% against the Omicron variant.

A uniquely stable trimeric model of SARS-CoV-2 spike transmembrane domain (preprint)

The spike (S) protein of SARS-CoV-2 effectuates membrane fusion and virus entry into target cells. Its transmembrane domain (TMD) represents a homotrimer of -helices anchoring the spike in the viral envelope. Although S-protein models available to date include the TMD, its precise configuration was given brief consideration. Understanding viral fusion entails realistic TMD models, while no reliable approaches towards predicting the 3D structure of transmembrane (TM) trimers exist. Here, we propose a comprehensive computational framework to model the spike TMD (S-TMD) based solely on its primary structure. First, we performed amino acid sequence pattern matching and compared molecular hydrophobicity potential (MHP) distribution on the helix surface against TM homotrimers with known 3D structures and thus selected the TMD of the tumour necrosis factor receptor 1 (TNFR-1) for subsequent template-based modelling. We then iteratively built an all-atom homotrimer model of S-TMD based on "dynamic MHP portraits" and residue variability motifs. In this model each helix possessed two overlapping interfaces interacting with either of the remaining helices, which include conservative residues I1216, F1220, I1227, M1229, and M1233. Finally, the stability of this and several alternative models (including a recent NMR structure) and a set of mutant forms was tested in all-atom molecular dynamics (MD) simulations in a POPC bilayer mimicking the viral envelope membrane. Unlike other configurations, our model trimer remained extraordinarily tightly packed over a microsecond-range MD and retained its stability when palmitoylated in accordance with experimental data. Palmitoylation had no significant impact on the TMD conformation nor the way in which the lipid bilayer was perturbed in the presence of the trimer. Overall, the resulting model of S-TMD conforms to known basic principles of TM helix packing and will be further used to explore the complex machinery of membrane fusion from a broader perspective beyond the TMD.

Deficiency of B vitamins leads to cholesterol-independent atherogenic transformation of the aorta (preprint)

Background: Atherosclerosis, the leading cause of cardiovascular disease responsible for the majority of deaths worldwide, cannot be sufficiently explained by established risk factors including hypercholesterolemia. Elevated plasma homocysteine is an independent risk factor for atherosclerosis and is strongly linked to cardiovascular mortality. However, the role of homocysteine in atherosclerosis is still insufficiently understood. Previous research in this area has been also hampered by the lack of reproducible in-vivo models of atherosclerosis that resemble the human situation. Methods: : Aortic specimens obtained from balloon injured rabbits fed either diet deficient in B vitamins, supplemented with 1% cholesterol or combined diet were subjected to ex vivo MRI, myographical, biomechanical, microscopical and histological analysis. Lipoproteins were analyzed by FPLC. Findings: We developed and applied an automated system for vessel wall injury that leads to more homogenous damage and more pronounced atherosclerotic plaque development even at low balloon pressure. Already in the absence of hypercholesterolemia, B vitamin deficiency results in accumulation of macrophages and lipids in the aorta, impairment of its biomechanical properties and disorganization of aortic collagen. Combination of B vitamin deficiency and hypercholesterolemia leads to thickening of the aorta, decreased aortic water diffusion, increased LDL-cholesterol and impaired vascular reactivity of the aorta. Interpretation: Our findings suggest that deficiency of B vitamins leads to atherosclerotic transformation of the aorta already in the absence of hypercholesterolemia and aggravates atherosclerosis development in its presence. Funding This work was funded by the Austrian Science Fund (FWF) (Projects P31105 and P33672), the EU project NanoAthero and BioTechMed-Graz.

Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions (preprint)

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2, and to a lesser extent, the alphacoronavirus HCoV-229E. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting synthesis of full-length and subgenomic HCoV RNAs. Furthermore, consistent with our previous report on the effects of thiopurines on influenza A virus (IAV) glycoproteins, we observed that 6-TG inhibited accumulation of Spike glycoproteins from diverse HCoVs. Specifically, 6-TG treatment decreased the accumulation of Spike proteins and increased their electrophoretic mobility to match the properties of Spike following enzymatic removal of N-linked oligosaccharides with Peptide:N-glycosidase F (PNGaseF). SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on lentiviruses pseudotyped with SARS-CoV-2 Spike; lentiviruses could be harvested from cell supernatants, but they were deficient in Spike and unable to infect human cells bearing ACE2 receptors. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. At low micromolar doses, the primary known mode of action of 6-TG is selective inhibition of the small GTPase Rac1. However, we show that selective chemical inhibitors of the small GTPases Rac1, CDC42 and Rho had no effect on Spike processing and accumulation, whereas the broad GTPase agonist ML099 was able to counter the effects of 6-TG, suggesting that an unknown GTPase could be the relevant 6-TG-target protein involved in regulating Spike processing and accumulation. Overall, these findings provide important clues about the mechanism of action of a candidate antiviral that can broadly target HCoVs and suggest that small GTPases may be promising targets for host-targeted antivirals.

SARS-CoV-2 variants show a gradual declining pathogenicity and pro-inflammatory cytokine stimulation and an increasing antigenic and anti-inflammatory cytokine induction (preprint)

Hyper-transmissibility with decreased disease severity are typical characteristics of Omicron variant. To understand this phenomenon, we used various bioinformatics approaches to analyze randomly selected genome sequences (one each) of the Gamma, Delta, and Omicron variants submitted to NCBI from 15 to 31 December 2021. We show that: (i) Pathogenicity of SARS-CoV-2 variants decreases in the order: Wuhan > Gamma > Delta > Omicron; however, the antigenic property follows the order: Omicron > Gamma > Wuhan > Delta. (ii) Omicron Spike RBD has lower pathogenicity but higher antigenicity than that of other variants. (iii) Decreased disease severity by Omicron variant may be due to its decreased pro-inflammatory and IL-6 stimulation and increased IFN-{gamma} and IL-4 induction efficacy. (iv) Mutations in N protein are associated with decreased IL-6 induction and human DDX21-mediated increased IL-4 production in Omicron. (v) Due to mutations, the stability of S, M, N, and E proteins decrease in the order: Omicron > Gamma > Delta > Wuhan. (vi) Stronger Spike-hACE2 binding in Omicron is associated with its increased transmissibility. However, the lowest stability of the Omicron Spike protein makes Spike-hACE2 interaction unstable for systemic infection and for causing severe disease. Finally (vii), the highest instability of Omicron E protein may also be associated with decreased viral maturation and low viral load leading to less severe disease and faster recovery. Our method may be used for other similar viruses, and these findings will contribute to the understanding of the dynamics of SARS-CoV-2 variants and the management of emerging variants.

Simultaneous therapeutic targeting of inflammation and virus ameliorates influenza pneumonia and protects from morbidity and mortality (preprint)

Pneumonia is a severe complication caused by inflammation of the lungs following infection with seasonal and pandemic strains of influenza A virus (IAV) that can result in lung pathology, respiratory failure and death. There is currently no treatment available for severe disease and pneumonia caused by IAV. Antivirals are available, but they are far from satisfactory if treatment is not initiated within 48 hours of symptoms onset. Influenza complications and mortality are often associated with high viral load and excessive lung inflammatory cytokine response. Therefore, we simultaneously targeted IAV with the antiviral drug oseltamivir and inflammation with the anti-inflammatory drug etanercept, targeting TNF after the onset of clinical signs to treat IAV pneumonia effectively. The combined treatment effectively reduced lung viral load, lung pathology, morbidity and mortality during respiratory IAV infection in mice, contemporaneous with significant downregulation of the inflammatory cytokines TNF, IL-1β, IL-6, IL-12p40, chemokines CCL2, CCL5 and CXCL10 and dampened STAT3 activation. Consequently, combined therapy with oseltamivir and a STAT3 inhibitor also effectively reduced clinical disease and lung pathology. Combined treatment using either of the anti-inflammatory drugs and oseltamivir dampened an overlapping set of cytokines. Thus, combined therapy targeting a specific cytokine or cytokine signaling pathway plus an antiviral drug provides an effective treatment strategy for ameliorating IAV pneumonia. Effective treatment of IAV pneumonia required multiple doses of etanercept and a high dose of oseltamivir. This approach might apply to the treatment of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Significance Statement Antivirals against influenza A virus (IAV) are ineffective in treating pneumonia if administered 48 h after onset of disease symptoms. The host inflammatory response and tissue damage caused by IAV are responsible for lung pathology. We reasoned that targeting both virus and inflammation would be more effective in reducing lung pathology and pneumonia, morbidity and mortality. The simultaneous treatment with an anti-inflammatory drug targeting TNF or STAT3, combined with the anti-IAV antiviral drug, oseltamivir, significantly improved clinical disease, reduced lung viral load and pathology, and protected mice from severe pneumonia. The combined treatment suppressed multiple pro-inflammatory cytokines and cytokine signaling pathways. Thus, after the onset of disease symptoms, both virus and inflammation must be targeted to treat IAV pneumonia effectively.

Continuing inequalities in COVID-19 mortality in England and Wales, and the changing importance of regional, over local, deprivation (preprint)

Abstract Background: Observational studies have highlighted that where individuals live is far more important for risk of dying with COVID-19, than for dying of other causes. Deprivation is commonly proposed as explaining such differences. During the period of localised restrictions in late 2020, areas with higher restrictions tended to be more deprived. We explore how this impacted the relationship between deprivation and mortality and see whether local or regional deprivation matters more for inequalities in COVID-19 mortality. Methods: We use publicly available population data on deaths due to COVID-19 and all-cause mortality between March 2020 and April 2021 to investigate the scale of spatial inequalities. We use a multiscale approach to simultaneously consider three spatial scales through which processes driving inequalities may act. We go on to explore whether deprivation explains such inequalities. Results Adjusting for population age structure and number of care homes, we find highest regional inequality in October 2020, with a COVID-19 mortality rate ratio of 5.86 (95% CI 3.31 to 19.00) for the median between-region comparison. We find spatial context is most important, and spatial inequalities higher, during periods of low mortality. Almost all unexplained spatial inequality in October 2020 is removed by adjusting for deprivation. During October 2020, one standard deviation increase in regional deprivation was associated with 2.45 times higher local mortality (95% CI, 1.75 to 3.48). Conclusions Spatial inequalities are greatest in periods of lowest overall mortality, implying that as mortality declines it does not do so equally. During the prolonged period of low restrictions and low mortality in summer 2020, spatial inequalities strongly increased. Contrary to previous months, we show that the strong spatial patterning during autumn 2020 is almost entirely explained by deprivation. As overall mortality declines, policymakers must be proactive in detecting areas where this is not happening, or risk worsening already strong health inequalities.

COVID-19 vaccination recruits and matures cross-reactive antibodies to conserved epitopes in endemic coronavirus Spike proteins (preprint)

The COVID-19 pandemic has triggered the first widespread vaccination campaign against a coronavirus. Most vaccinated subjects are na ve to SARS-CoV-2, however almost all have previously encountered other coronaviruses (CoVs) and the role of this immunity in shaping the vaccine response remains uncharacterized. Here we use longitudinal samples and highly-multiplexed serology to identify mRNA-1273 vaccine-induced antibody responses against a range of CoV Spike epitopes and in both phylogenetically conserved and non-conserved regions. Whereas reactivity to SARS-CoV-2 epitopes showed a delayed but progressive increase following vaccination, we observed distinct kinetics for the endemic CoV homologs at two conserved sites in Spike S2: these became detectable sooner, and decayed at later timepoints. Using homolog-specific depletion and alanine-substitution experiments, we show that these distinctly-evolving specificities result from cross-reactive antibodies as they mature against rare, polymorphic residues within these epitopes. Our results reveal mechanisms for the formation of antibodies with broad reactivity against CoVs.

Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 Spike protein (preprint)

Background: SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper- proinflammatory state involves activation of signaling by NF{kappa}B and ENaC, and expression of high levels of cytokines and chemokines. Post-infection inflammation may contribute to "Long COVID", and there are also other long term consequences of acute severe COVID-19, which double or triple the chances of dying from any cause within a year. Enhanced signaling by NF{kappa}B and ENaC also marks the airway of patients suffering from cystic fibrosis, a lethal proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 Spike protein. Methods: This hypothesis was tested using the hTERT-transformed BCi-NS1.1 basal stem cell, previously derived from small airway epithelia, which were differentiated into a model of small airway epithelia on an air-liquid-interface (ALI). Cyclic AMP-activated CFTR chloride channel activity was measured using an Ussing Chamber. Cell surface-CFTR was labeled with the impermeant biotin method. Results: Exposure of differentiated airway epithelia to SARS-CoV-2 Spike protein resulted in loss of CFTR protein expression. As hypothesized, TNF/NF{kappa}B signaling was activated, based on increased protein expression of TRADD, the first intracellular adaptor for the TNF/TNFR1 complex, TNFR1, the TNF receptor, phosphorylated I{kappa}B, and the chemokine IL8. ENaC activity was also activated, based on specific changes in molecular weights for and{gamma}ENaC. Exposure of the epithelia to viral Spike protein suppressed cAMP-activated CFTR chloride channel activity. However, addition of 30 nM concentrations of cardiac glycoside drugs ouabain, digitoxin and digoxin, prevented loss of channel activity. ACE2 and CFTR were found to co-immunoprecipitate (co-IP) in both basal cells and epithelia, suggesting that the mechanism for Spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, Spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane, suggesting that failure of CFTR recovery from endosomal recycling might be a mechanism for Spike-dependent loss of CFTR. Conclusion: Based on experiments with this model of small airway epithelia, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by SARS-CoV-2 Spike protein, thus inducing a CFTR-null, cystic fibrosis-like clinical phenotype. Descriptions of COVID-19 in CF carriers with only one copy of wildtype CFTR suggest that this model-based conclusion might be consistent with patient-based experience.

Estimation of excess all-cause mortality due to COVID-19 in Thailand (preprint)

Thailand has experienced the most prominent COVID-19 outbreak, resulting in a new record for COVID-19 cases and deaths in 2021. To assess the influence of the COVID-19 outbreak on mortality, we estimated excess all-cause and pneumonia mortality in Thailand during the COVID-19 outbreak from April to October 2021. We used the previous five years’ mortality to estimate the baseline number of deaths using generalized linear mixed models (GLMMs). The models were adjusted for seasonality and demographics. We found that the estimated cumulative excess death was 14.3% (95% CI: 8.6%-18.8%) higher than the baseline. The results also showed that the excess deaths in males were higher than in females by approximately 26.3%. The excess deaths directly caused by the COVID-19 infections accounted for approximately 75.0% of the all-cause excess deaths. Furthermore, excess pneumonia deaths were also found to be 26.2% (95% CI: 4.8%-46.0%) above baseline. There was a significant rise in excess fatalities, especially in the older age groups. Therefore, the age and sex structure of the population are essential to assessing the mortality impact of COVID-19. Our modeling results could potentially provide insights into the COVID-19 outbreaks and provide a guide for outbreak control and intervention.

Development and Characterization of Recombinant Vesicular Stomatitis Virus (rVSV)-based Bivalent Vaccine Against COVID-19 Delta Variant and Influenza Virus (preprint)

COVID-19 and influenza are both highly contagious respiratory diseases with a wide range of severe symptoms and cause great disease burdens globally. It has become very urgent and important to develop a bivalent vaccine that is able to target these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant VSV (rVSV) vaccine candidates. These rVSV-based vaccines co-express SARS-CoV-2 Delta variant spike protein (SP) or the receptor binding domain (RBD) and four copies of the highly conserved M2 ectodomain (M2e) of influenza A fused with the Ebola glycoprotein DC-targeting/activation domain. Animal studies have shown that immunization with these bivalent rVSV vaccines induced efficient but variable levels of humoral and cell-mediated immune responses against both SARS-CoV-2 and influenza M2e protein. Significantly, our vaccine candidates induced production of high levels of neutralizing antibodies that protected cells against SARS-CoV-2 Delta and other SP-pseudovirus infections in culture. Furthermore, vaccination with the bivalent VSV vaccine via either intramuscular or intranasal route efficiently protected mice from the lethal challenge of H1N1 and H3N2 influenza viruses and significantly reduced viral load in the lungs. These studies provide convincing evidence for the high efficacy of this bivalent vaccine to prevent influenza replication and initiate robust immune responses against SARS-CoV-2 Delta variants. Further investigation of its efficacy to protect against SARS-CoV-2 Delta variants will provide substantial evidence for new avenues to control two contagious respiratory infections, COVID-19 and influenza.