Intranasal COVID-19 vaccine induces respiratory memory T cells and protects K18-hACE mice against SARS-CoV-2 infection.

Current COVID-19 vaccines prevent severe disease, but do not induce mucosal immunity or prevent infection with SARS-CoV-2, especially with recent variants. Furthermore, serum antibody responses wane soon after immunization. We assessed the immunogenicity and protective efficacy of an experimental COVID-19 vaccine based on the SARS-CoV-2 Spike trimer formulated with a novel adjuvant LP-GMP, comprising TLR2 and STING agonists. We demonstrated that immunization of mice twice by the intranasal (i.n.) route or by heterologous intramuscular (i.m.) prime and i.n. boost with the Spike-LP-GMP vaccine generated potent Spike-specific IgG, IgA and tissue-resident memory (TRM) T cells in the lungs and nasal mucosa that persisted for at least 3 months. Furthermore, Spike-LP-GMP vaccine delivered by i.n./i.n., i.m./i.n., or i.m./i.m. routes protected human ACE-2 transgenic mice against respiratory infection and COVID-19-like disease following lethal challenge with ancestral or Delta strains of SARS-CoV-2. Our findings underscore the potential for nasal vaccines in preventing infection with SARS-CoV-2 and other respiratory pathogen.

Statistical analyses of ordinal outcomes in randomised controlled trials: protocol for a scoping review.

INTRODUCTION: Randomised controlled trials (RCTs) aim to assess the effect of one (or more) unproven health interventions relative to other reference interventions. RCTs sometimes use an ordinal outcome, which is an endpoint that comprises of multiple, monotonically ordered categories that are not necessarily separated by a quantifiable distance. Ordinal outcomes are appealing in clinical settings as specific disease states can represent meaningful categories that may be of clinical importance to researchers. Ordinal outcomes can also retain information and increase statistical power compared to dichotomised outcomes and can allow multiple clinical outcomes to be comprised in a single endpoint. Target parameters for ordinal outcomes in RCTs may vary depending on the nature of the research question, the modelling assumptions and the expertise of the data analyst. The aim of this scoping review is to systematically describe the use of ordinal outcomes in contemporary RCTs. Specifically, we aim to: [Formula: see text] Identify which target parameters are of interest in trials that use an ordinal outcome, and whether these parameters are explicitly defined. [Formula: see text] Describe how ordinal outcomes are analysed in RCTs to estimate a treatment effect. [Formula: see text] Describe whether RCTs that use an ordinal outcome adequately report key methodological aspects specific to the analysis of the ordinal outcome. Results from this review will outline the current state of practice of the use of ordinal outcomes in RCTs. Ways to improve the analysis and reporting of ordinal outcomes in RCTs will be discussed. METHODS AND ANALYSIS: We will review RCTs that are published in the top four medical journals (British Medical Journal, New England Journal of Medicine, The Lancet and the Journal of the American Medical Association) between 1 January 2012 and 31 July 2022 that use an ordinal outcome as either a primary or a secondary outcome. The review will identify articles through a PubMed-specific search strategy. Our review will adhere to guidelines for scoping reviews as described in the PRISMA-ScR checklist. The study characteristics and details of the study design and analysis, including the target parameter(s) and statistical methods used to analyse the ordinal outcome, will be extracted from eligible studies. The screening, review and data extraction will be conducted using Covidence, a web-based tool for managing systematic reviews. The data will be summarised using descriptive statistics.

Randomized Trial of BCG Vaccine to Protect against Covid-19 in Health Care Workers.

BACKGROUND: The bacille Calmette-Guérin (BCG) vaccine has immunomodulatory "off-target" effects that have been hypothesized to protect against coronavirus disease 2019 (Covid-19). METHODS: In this international, double-blind, placebo-controlled trial, we randomly assigned health care workers to receive the BCG-Denmark vaccine or saline placebo and followed them for 12 months. Symptomatic Covid-19 and severe Covid-19, the primary outcomes, were assessed at 6 months; the primary analyses involved the modified intention-to-treat population, which was restricted to participants with a negative test for severe acute respiratory syndrome coronavirus 2 at baseline. RESULTS: A total of 3988 participants underwent randomization; recruitment ceased before the planned sample size was reached owing to the availability of Covid-19 vaccines. The modified intention-to-treat population included 84.9% of the participants who underwent randomization: 1703 in the BCG group and 1683 in the placebo group. The estimated risk of symptomatic Covid-19 by 6 months was 14.7% in the BCG group and 12.3% in the placebo group (risk difference, 2.4 percentage points; 95% confidence interval [CI], -0.7 to 5.5; P = 0.13). The risk of severe Covid-19 by 6 months was 7.6% in the BCG group and 6.5% in the placebo group (risk difference, 1.1 percentage points; 95% CI, -1.2 to 3.5; P = 0.34); the majority of participants who met the trial definition of severe Covid-19 were not hospitalized but were unable to work for at least 3 consecutive days. In supplementary and sensitivity analyses that used less conservative censoring rules, the risk differences were similar but the confidence intervals were narrower. There were five hospitalizations due to Covid-19 in each group (including one death in the placebo group). The hazard ratio for any Covid-19 episode in the BCG group as compared with the placebo group was 1.23 (95% CI, 0.96 to 1.59). No safety concerns were identified. CONCLUSIONS: Vaccination with BCG-Denmark did not result in a lower risk of Covid-19 among health care workers than placebo. (Funded by the Bill and Melinda Gates Foundation and others; BRACE ClinicalTrials.gov number, NCT04327206.).

The causal effect of being born extremely preterm or extremely low birthweight on neurodevelopment and social-emotional development at 2 years.

AIM: To assess the causal effect of being born extremely preterm (EP; <28 weeks' gestation) or extremely low birthweight (ELBW; <1000 g), compared with being born at term, on neurodevelopment and social-emotional development at 2 years' corrected age. METHODS: Prospective geographical cohort study of children born EP/ELBW over 12 months in 2016 from Victoria, Australia, and term-born controls. Children were assessed at 2 years' corrected age with the Bayley Scales of Infant and Toddler Development-3rd edition and the Infant-Toddler Social and Emotional Assessment. Delay was defined as <-1 standard deviation relative to the mean of controls. The estimand of interest was the mean difference/odds ratio (OR) between the EP/ELBW and control groups estimated using linear/logistic regression, adjusted for multiple pregnancy and social risk. RESULTS: A total of 205 EP/ELBW and 201 controls were assessed at 2 years. Delay/concerns were more common in the EP/ELBW group compared with controls, for cognitive (OR 3.7 [95% confidence interval 2.3, 6.0]), language (5.3 [3.1, 9.0]) and motor (3.9 [2.3, 6.3]) development, and social-emotional competence (4.1 [1.6, 10.2]). CONCLUSION: Being born EP/ELBW has an adverse effect on cognitive, language and motor development, and social-emotional competence at 2 years' corrected age. Close developmental surveillance, including social-emotional development, is recommended.

Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants.

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

Early Learning From a Low-Resource COVID-Response Virtual Mental Health Crisis Ward: Mixed Methods Study.

BACKGROUND: The COVID-19 pandemic was accompanied by the accelerated uptake of virtual care, leading to a proliferation of virtual ward models as alternatives to facility-based care. Early in the pandemic, our program implemented a virtual mental health crisis ward (vWard) to provide options for individuals requiring intense psychiatric and/or crisis support but who preferred to remain in the community and were deemed safe to do so. OBJECTIVE: The aim of this study was to identify early learnings from the vWard, which was implemented rapidly in a resource-constrained environment, to inform the future state should it be sustained beyond the pandemic. METHODS: Mixed methods of data collection were used to evaluate provider perspectives on the vWard, develop archetypes for individuals who are a good fit for the vWard model, and create a driver diagram. Data sources included an anonymous survey of clinical and managerial staff involved in the vWard, a service planning workshop, and program discharge forms for all individuals admitted between March 2020 and April 2021. Survey responses were coded for themes under categories of "benefits" and "challenges." Discharge forms where the team indicated that the vWard was a good fit for an individual were examined for characteristics common to these admissions. These findings were reviewed in the service planning workshop and refined with input from the participants into patient archetypes. A driver diagram was created for the future state. RESULTS: Survey respondents (N=60) represented diverse roles in crisis services and the vWard team. Ten providers took part in the service planning workshop. A total of 467 discharge forms were reviewed. The vWard was felt to be a model that worked by 39 survey respondents, one respondent felt it did not work, and the remaining participants had no response. Several benefits for the individual and the system were identified alongside challenges, including certain processes and materials related to the nature of rapid implementation during the pandemic, and others due to lack of fit for certain individuals. The model was felt to be a good fit for 67.5% of admissions. Four patient archetypes representing a good fit with the model were developed. The driver diagram connected the program aim with primary drivers of (1) reduce barriers to care; (2) improve outcomes; and (3) provide collaborative, patient- and family-centered care to secondary drivers and interventions that leveraged virtual technology among other crisis care interventions. CONCLUSIONS: Despite some challenges, the vWard demonstrated high levels of provider acceptance and a range of mechanisms by which the model works for a variety of patient archetypes. These early learnings provide a foundation for growth, sustainability, and spread of this model going forward beyond the pandemic.

Passive immunization with equine RBD-specific Fab protects K18-hACE2-mice against Alpha or Beta variants of SARS-CoV-2.

Emergence of variants of concern (VOC) during the COVID-19 pandemic has contributed to the decreased efficacy of therapeutic monoclonal antibody treatments for severe cases of SARS-CoV-2 infection. In addition, the cost of creating these therapeutic treatments is high, making their implementation in low- to middle-income countries devastated by the pandemic very difficult. Here, we explored the use of polyclonal EpF(ab')2 antibodies generated through the immunization of horses with SARS-CoV-2 WA-1 RBD conjugated to HBsAg nanoparticles as a low-cost therapeutic treatment for severe cases of disease. We determined that the equine EpF(ab')2 bind RBD and neutralize ACE2 receptor binding by virus for all VOC strains tested except Omicron. Despite its relatively quick clearance from peripheral circulation, a 100µg dose of EpF(ab')2 was able to fully protect mice against severe disease phenotypes following intranasal SARS-CoV-2 challenge with Alpha and Beta variants. EpF(ab')2 administration increased survival while subsequently lowering disease scores and viral RNA burden in disease-relevant tissues. No significant improvement in survival outcomes or disease scores was observed in EpF(ab')2-treated mice challenged using the Delta variant at 10µg or 100µg doses. Overall, the data presented here provide a proof of concept for the use of EpF(ab')2 in the prevention of severe SARS-CoV-2 infections and underscore the need for either variant-specific treatments or variant-independent therapeutics for COVID-19.

SARS-CoV-2 Delta variant induces enhanced pathology and inflammatory responses in K18-hACE2 mice.

The COVID-19 pandemic has been fueled by SARS-CoV-2 novel variants of concern (VOC) that have increased transmissibility, receptor binding affinity, and other properties that enhance disease. The goal of this study is to characterize unique pathogenesis of the Delta VOC strain in the K18-hACE2-mouse challenge model. Challenge studies suggested that the lethal dose of Delta was higher than Alpha or Beta strains. To characterize the differences in the Delta strain's pathogenesis, a time-course experiment was performed to evaluate the overall host response to Alpha or Delta variant challenge. qRT-PCR analysis of Alpha- or Delta-challenged mice revealed no significant difference between viral RNA burden in the lung, nasal wash or brain. However, histopathological analysis revealed high lung tissue inflammation and cell infiltration following Delta- but not Alpha-challenge at day 6. Additionally, pro-inflammatory cytokines were highest at day 6 in Delta-challenged mice suggesting enhanced pneumonia. Total RNA-sequencing analysis of lungs comparing challenged to no challenge mice revealed that Alpha-challenged mice have more total genes differentially activated. Conversely, Delta-challenged mice have a higher magnitude of differential gene expression. Delta-challenged mice have increased interferon-dependent gene expression and IFN-γ production compared to Alpha. Analysis of TCR clonotypes suggested that Delta challenged mice have increased T-cell infiltration compared to Alpha challenged. Our data suggest that Delta has evolved to engage interferon responses in a manner that may enhance pathogenesis. The in vivo and in silico observations of this study underscore the need to conduct experiments with VOC strains to best model COVID-19 when evaluating therapeutics and vaccines.

Obesity and metabolic dysfunction drive sex-associated differential disease profiles in hACE2-mice challenged with SARS-CoV-2.

Severe outcomes from SARS-CoV-2 infection are highly associated with preexisting comorbid conditions like hypertension, diabetes, and obesity. We utilized the diet-induced obesity (DIO) model of metabolic dysfunction in K18-hACE2 transgenic mice to model obesity as a COVID-19 comorbidity. Female DIO, but not male DIO mice challenged with SARS-CoV-2 were observed to have shortened time to morbidity compared to controls. Increased susceptibility to SARS-CoV-2 in female DIO was associated with increased viral RNA burden and interferon production compared to males. Transcriptomic analysis of the lungs from all mouse cohorts revealed sex- and DIO-associated differential gene expression profiles. Male DIO mice after challenge had decreased expression of antibody-related genes compared to controls, suggesting antibody producing cell localization in the lung. Collectively, this study establishes a preclinical comorbidity model of COVID-19 in mice where we observed sex- and diet-specific responses that begin explaining the effects of obesity and metabolic disease on COVID-19 pathology.

RBD-VLP Vaccines Adjuvanted with Alum or SWE Protect K18-hACE2 Mice against SARS-CoV-2 VOC Challenge.

The ongoing COVID-19 pandemic has contributed largely to the global vaccine disparity. Development of protein subunit vaccines can help alleviate shortages of COVID-19 vaccines delivered to low-income countries. Here, we evaluated the efficacy of a three-dose virus-like particle (VLP) vaccine composed of hepatitis B surface antigen (HBsAg) decorated with the receptor binding domain (RBD) from the Wuhan or Beta SARS-CoV-2 strain adjuvanted with either aluminum hydroxide (alum) or squalene in water emulsion (SWE). RBD HBsAg vaccines were compared to the standard two doses of Pfizer mRNA vaccine. Alum-adjuvanted vaccines were composed of either HBsAg conjugated with Beta RBD alone (ß RBD HBsAg+Al) or a combination of both Beta RBD HBsAg and Wuhan RBD HBsAg (ß/Wu RBD HBsAg+Al). RBD vaccines adjuvanted with SWE were formulated with Beta RBD HBsAg (ß RBD HBsAg+SWE) or without HBsAg (ß RBD+SWE). Both alum-adjuvanted RBD HBsAg vaccines generated functional RBD IgG against multiple SARS-CoV-2 variants of concern (VOC), decreased viral RNA burden, and lowered inflammation in the lung against Alpha or Beta challenge in K18-hACE2 mice. However, only ß/Wu RBD HBsAg+Al was able to afford 100% survival to mice challenged with Alpha or Beta VOC. Furthermore, mice immunized with ß RBD HBsAg+SWE induced cross-reactive neutralizing antibodies against major VOC of SARS-CoV-2, lowered viral RNA burden in the lung and brain, and protected mice from Alpha or Beta challenge similarly to mice immunized with Pfizer mRNA. However, RBD+SWE immunization failed to protect mice from VOC challenge. Our findings demonstrate that RBD HBsAg VLP vaccines provided similar protection profiles to the approved Pfizer mRNA vaccines used worldwide and may offer protection against SARS-CoV-2 VOC. IMPORTANCE Global COVID-19 vaccine distribution to low-income countries has been a major challenge of the pandemic. To address supply chain issues, RBD virus-like particle (VLP) vaccines that are cost-effective and capable of large-scale production were developed and evaluated for efficacy in preclinical mouse studies. We demonstrated that RBD-VLP vaccines protected K18-hACE2 mice against Alpha or Beta challenge similarly to Pfizer mRNA vaccination. Our findings showed that the VLP platform can be utilized to formulate immunogenic and efficacious COVID-19 vaccines.

SARS-CoV-2/COVID-19 and its relationship with NOD2 and ubiquitination.

COVID-19 infection activates the immune system to cause autoimmune and autoinflammatory diseases. We provide a comprehensive review of the relationship between SARS-CoV-2, NOD2 and ubiquitination. COVID-19 infection partly results from host inborn errors and genetic factors and can lead to autoinflammatory disease. The interaction between defective NOD2 and viral infection may trigger NOD2-associated disease. SARS-CoV-2 can alter UBA1 and abnormal ubiquitination leading to VEXAS syndrome. Both NOD2 and ubiquitination play important roles in controlling inflammatory process. Receptor interacting protein kinase 2 is a key component of the NOD2 activation pathway and becomes ubiquitinated to recruit downstream effector proteins. NOD2 mutations result in loss of ubiquitin binding and increase ligand-stimulated NOD2 signaling. During viral infection, mutations of either NOD2 or UBA1 genes or in combination can facilitate autoinflammatory disease. COVID-19 infection can cause autoinflammatory disease. There are reciprocal interactions between SARS-CoV-2, NOD2 and ubiquitination.

Intranasal administration of BReC-CoV-2 COVID-19 vaccine protects K18-hACE2 mice against lethal SARS-CoV-2 challenge.

SARS-CoV-2 is a viral respiratory pathogen responsible for the current global pandemic and the disease that causes COVID-19. All current WHO approved COVID-19 vaccines are administered through the muscular route. We have developed a prototype two-dose vaccine (BReC-CoV-2) by combining the Receptor Binding Domain (RBD) antigen, via conjugation to Diphtheria toxoid (EcoCRM®). The vaccine is adjuvanted with Bacterial Enzymatic Combinatorial Chemistry (BECC), BECC470. Intranasal (IN) administration of BreC-CoV-2 in K18-hACE2 mice induced a strong systemic and localized immune response in the respiratory tissues which provided protection against the Washington strain of SARS-CoV-2. Protection provided after IN administration of BReC-CoV-2 was associated with decreased viral RNA copies in the lung, robust RBD IgA titers in the lung and nasal wash, and induction of broadly neutralizing antibodies in the serum. We also observed that BReC-CoV-2 vaccination administered using an intramuscular (IM) prime and IN boost protected mice from a lethal challenge dose of the Delta variant of SARS-CoV-2. IN administration of BReC-CoV-2 provided better protection than IM only administration to mice against lethal challenge dose of SARS-CoV-2. These data suggest that the IN route of vaccination induces localized immune responses that can better protect against SARS-CoV-2 than the IM route in the upper respiratory tract.

BCG vaccination to reduce the impact of COVID-19 in healthcare workers: Protocol for a randomised controlled trial (BRACE trial).

INTRODUCTION: BCG vaccination modulates immune responses to unrelated pathogens. This off-target effect could reduce the impact of emerging pathogens. As a readily available, inexpensive intervention that has a well-established safety profile, BCG is a good candidate for protecting healthcare workers (HCWs) and other vulnerable groups against COVID-19. METHODS AND ANALYSIS: This international multicentre phase III randomised controlled trial aims to determine if BCG vaccination reduces the incidence of symptomatic and severe COVID-19 at 6 months (co-primary outcomes) compared with no BCG vaccination. We plan to randomise 10 078 HCWs from Australia, The Netherlands, Spain, the UK and Brazil in a 1:1 ratio to BCG vaccination or no BCG (control group). The participants will be followed for 1 year with questionnaires and collection of blood samples. For any episode of illness, clinical details will be collected daily, and the participant will be tested for SARS-CoV-2 infection. The secondary objectives are to determine if BCG vaccination reduces the rate, incidence, and severity of any febrile or respiratory illness (including SARS-CoV-2), as well as work absenteeism. The safety of BCG vaccination in HCWs will also be evaluated. Immunological analyses will assess changes in the immune system following vaccination, and identify factors associated with susceptibility to or protection against SARS-CoV-2 and other infections. ETHICS AND DISSEMINATION: Ethical and governance approval will be obtained from participating sites. Results will be published in peer-reviewed open-access journals. The final cleaned and locked database will be deposited in a data sharing repository archiving system. TRIAL REGISTRATION: ClinicalTrials.gov NCT04327206.

Impact of moderate and late preterm birth on neurodevelopment, brain development and respiratory health at school age: protocol for a longitudinal cohort study (LaPrem study).

INTRODUCTION: Children born moderate to late preterm (MLP, 32-36 weeks' gestation) account for approximately 85% of all preterm births globally. Compared with children born at term, children born MLP are at increased risk of poor neurodevelopmental outcomes. Despite making up the largest group of preterm children, developmental outcomes of children born MLP are less well studied than in other preterm groups. This study aimed to (1) compare neurodevelopmental, respiratory health and brain magnetic resonance imaging (MRI) outcomes between children born MLP and term at 9 years of age; (2) examine the differences in brain growth trajectory from infancy to 9 years between children born MLP and term; and in children born MLP; (3) examine the relationship between brain development and neurodevelopment at 9 years; and (4) identify risk factors for poorer outcomes at 9 years. METHODS AND ANALYSIS: The "LaPrem" (Late Preterm MRI Study) study is a longitudinal cohort study of children born MLP and term controls, born at the Royal Women's Hospital in Melbourne, Australia, between 2010 and 2013. Participants were recruited in the neonatal period and were previously followed up at 2 and 5 years. This 9-year school-age follow-up includes neuropsychology, motor and physical activities, and lung function assessments, as well as brain MRI. Outcomes at 9 years will be compared between birth groups using linear and logistic regressions. Trajectories of brain development will be compared between birth groups using mixed effects models. The relationships between MRI and neurodevelopmental outcomes, as well as other early predictors of poor 9-year outcomes, will be explored using linear and logistic regression. ETHICS AND DISSEMINATION: This study was approved by the human research ethics committee at the Royal Children's Hospital, Melbourne, Australia. Study outcomes will be disseminated through peer-reviewed publications, conference presentations and social media.

Disease Risk from Human-Environment Interactions: Environment and Development Economics for Joint Conservation-Health Policy.

Emergence of COVID-19 joins a collection of evidence that local and global health are influenced by human interactions with the natural environment. Frameworks that simultaneously model decisions to interact with natural systems and environmental mechanisms of zoonotic disease spread allow for identification of policy levers to mitigate disease risk and promote conservation. Here, we highlight opportunities to broaden existing conservation economics frameworks that represent human behavior to include disease transmission in order to inform conservation-disease risk policy. Using examples from wildlife markets and forest extraction, we call for environment, resource, and development economists to develop and analyze empirically-grounded models of people's decisions about interacting with the environment, with particular attention to LMIC settings and ecological-epidemiological risk factors. Integrating the decisions that drive human-environment interactions with ecological and epidemiological research in an interdisciplinary approach to understanding pathogen transmission will inform policy needed to improve both conservation and disease spread outcomes.