Directions of change in intrinsic case severity across successive SARS-CoV-2 variant waves have been inconsistent.

OBJECTIVES: To determine how the intrinsic severity of successively dominant SARS-CoV-2 variants changed over the course of the pandemic. METHODS: A retrospective cohort analysis in the NHS Greater Glasgow and Clyde (NHS GGC) Health Board. All sequenced non-nosocomial adult COVID-19 cases in NHS GGC with relevant SARS-CoV-2 lineages (B.1.177/Alpha, Alpha/Delta, AY.4.2 Delta/non-AY.4.2 Delta, non-AY.4.2 Delta/Omicron, and BA.1 Omicron/BA.2 Omicron) during analysis periods were included. Outcome measures were hospital admission, ICU admission, or death within 28 days of positive COVID-19 test. We report the cumulative odds ratio; the ratio of the odds that an individual experiences a severity event of a given level vs all lower severity levels for the resident and the replacement variant after adjustment. RESULTS: After adjustment for covariates, the cumulative odds ratio was 1.51 (95% CI: 1.08-2.11) for Alpha versus B.1.177, 2.09 (95% CI: 1.42-3.08) for Delta versus Alpha, 0.99 (95% CI: 0.76-1.27) for AY.4.2 Delta versus non-AY.4.2 Delta, 0.49 (95% CI: 0.22-1.06) for Omicron versus non-AY.4.2 Delta, and 0.86 (95% CI: 0.68-1.09) for BA.2 Omicron versus BA.1 Omicron. CONCLUSIONS: The direction of change in intrinsic severity between successively emerging SARS-CoV-2 variants was inconsistent, reminding us that the intrinsic severity of future SARS-CoV-2 variants remains uncertain.

Adeno-associated virus 2 infection in children with non-A-E hepatitis.

An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland1 in April 2022 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4+ T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10-12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a 'helper virus' to support AAV2 replication) and disease susceptibility related to HLA class II status.

The SARS-CoV-2 Alpha variant was associated with increased clinical severity of COVID-19 in Scotland: A genomics-based retrospective cohort analysis.

OBJECTIVES: The SARS-CoV-2 Alpha variant was associated with increased transmission relative to other variants present at the time of its emergence and several studies have shown an association between Alpha variant infection and increased hospitalisation and 28-day mortality. However, none have addressed the impact on maximum severity of illness in the general population classified by the level of respiratory support required, or death. We aimed to do this. METHODS: In this retrospective multi-centre clinical cohort sub-study of the COG-UK consortium, 1475 samples from Scottish hospitalised and community cases collected between 1st November 2020 and 30th January 2021 were sequenced. We matched sequence data to clinical outcomes as the Alpha variant became dominant in Scotland and modelled the association between Alpha variant infection and severe disease using a 4-point scale of maximum severity by 28 days: 1. no respiratory support, 2. supplemental oxygen, 3. ventilation and 4. death. RESULTS: Our cumulative generalised linear mixed model analyses found evidence (cumulative odds ratio: 1.40, 95% CI: 1.02, 1.93) of a positive association between increased clinical severity and lineage (Alpha variant versus pre-Alpha variants). CONCLUSIONS: The Alpha variant was associated with more severe clinical disease in the Scottish population than co-circulating lineages.

SARS-CoV-2 antibody responses associate with sex, age and disease severity in previously uninfected people admitted to hospital with COVID-19: An ISARIC4C prospective study.

The SARS-CoV-2 pandemic enables the analysis of immune responses induced against a novel coronavirus infecting immunologically naïve individuals. This provides an opportunity for analysis of immune responses and associations with age, sex and disease severity. Here we measured an array of solid-phase binding antibody and viral neutralising Ab (nAb) responses in participants (n=337) of the ISARIC4C cohort and characterised their correlation with peak disease severity during acute infection and early convalescence. Overall, the responses in a Double Antigen Binding Assay (DABA) for antibody to the receptor binding domain (anti-RBD) correlated well with IgM as well as IgG responses against viral spike, S1 and nucleocapsid protein (NP) antigens. DABA reactivity also correlated with nAb. As we and others reported previously, there is greater risk of severe disease and death in older men, whilst the sex ratio was found to be equal within each severity grouping in younger people. In older males with severe disease (mean age 68 years), peak antibody levels were found to be delayed by one to two weeks compared with women, and nAb responses were delayed further. Additionally, we demonstrated that solid-phase binding antibody responses reached higher levels in males as measured via DABA and IgM binding against Spike, NP and S1 antigens. In contrast, this was not observed for nAb responses. When measuring SARS-CoV-2 RNA transcripts (as a surrogate for viral shedding) in nasal swabs at recruitment, we saw no significant differences by sex or disease severity status. However, we have shown higher antibody levels associated with low nasal viral RNA indicating a role of antibody responses in controlling viral replication and shedding in the upper airway. In this study, we have shown discernible differences in the humoral immune responses between males and females and these differences associate with age as well as with resultant disease severity.

SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination.

BACKGROUND: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. METHODS: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FINDINGS: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. INTERPRETATION: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. FUNDING: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript.

Viral Coinfections in Hospitalized Coronavirus Disease 2019 Patients Recruited to the International Severe Acute Respiratory and Emerging Infections Consortium WHO Clinical Characterisation Protocol UK Study.

Background: We conducted this study to assess the prevalence of viral coinfection in a well characterized cohort of hospitalized coronavirus disease 2019 (COVID-19) patients and to investigate the impact of coinfection on disease severity. Methods: Multiplex real-time polymerase chain reaction testing for endemic respiratory viruses was performed on upper respiratory tract samples from 1002 patients with COVID-19, aged <1 year to 102 years old, recruited to the International Severe Acute Respiratory and Emerging Infections Consortium WHO Clinical Characterisation Protocol UK study. Comprehensive demographic, clinical, and outcome data were collected prospectively up to 28 days post discharge. Results: A coinfecting virus was detected in 20 (2.0%) participants. Multivariable analysis revealed no significant risk factors for coinfection, although this may be due to rarity of coinfection. Likewise, ordinal logistic regression analysis did not demonstrate a significant association between coinfection and increased disease severity. Conclusions: Viral coinfection was rare among hospitalized COVID-19 patients in the United Kingdom during the first 18 months of the pandemic. With unbiased prospective sampling, we found no evidence of an association between viral coinfection and disease severity. Public health interventions disrupted normal seasonal transmission of respiratory viruses; relaxation of these measures mean it will be important to monitor the prevalence and impact of respiratory viral coinfections going forward.

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway.

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant.

mRNA or ChAd0x1 COVID-19 Vaccination of Adolescents Induces Robust Antibody and Cellular Responses With Continued Recognition of Omicron Following mRNA-1273.

Children and adolescents generally experience mild COVID-19. However, those with underlying physical health conditions are at a significantly increased risk of severe disease. Here, we present a comprehensive analysis of antibody and cellular responses in adolescents with severe neuro-disabilities who received COVID-19 vaccination with either ChAdOx1 (n=6) or an mRNA vaccine (mRNA-1273, n=8, BNT162b2, n=1). Strong immune responses were observed after vaccination and antibody levels and neutralisation titres were both higher after two doses. Both measures were also higher after mRNA vaccination and were further enhanced by prior natural infection where one vaccine dose was sufficient to generate peak antibody response. Robust T-cell responses were generated after dual vaccination and were also higher following mRNA vaccination. Early T-cells were characterised by a dominant effector-memory CD4+ T-cell population with a type-1 cytokine signature with additional production of IL-10. Antibody levels were well-maintained for at least 3 months after vaccination and 3 of 4 donors showed measurable neutralisation titres against the Omicron variant. T-cell responses also remained robust, with generation of a central/stem cell memory pool and showed strong reactivity against Omicron spike. These data demonstrate that COVID-19 vaccines display strong immunogenicity in adolescents and that dual vaccination, or single vaccination following prior infection, generate higher immune responses than seen after natural infection and develop activity against Omicron. Initial evidence suggests that mRNA vaccination elicits stronger immune responses than adenoviral delivery, although the latter is also higher than seen in adult populations. COVID-19 vaccines are therefore highly immunogenic in high-risk adolescents and dual vaccination might be able to provide relative protection against the Omicron variant that is currently globally dominant.

Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of IFN-mediated innate immune defenses.

The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air-liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.

Children develop robust and sustained cross-reactive spike-specific immune responses to SARS-CoV-2 infection.

SARS-CoV-2 infection is generally mild or asymptomatic in children but a biological basis for this outcome is unclear. Here we compare antibody and cellular immunity in children (aged 3-11 years) and adults. Antibody responses against spike protein were high in children and seroconversion boosted responses against seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Neutralization of viral variants was comparable between children and adults. Spike-specific T cell responses were more than twice as high in children and were also detected in many seronegative children, indicating pre-existing cross-reactive responses to seasonal coronaviruses. Importantly, children retained antibody and cellular responses 6 months after infection, whereas relative waning occurred in adults. Spike-specific responses were also broadly stable beyond 12 months. Therefore, children generate robust, cross-reactive and sustained immune responses to SARS-CoV-2 with focused specificity for the spike protein. These findings provide insight into the relative clinical protection that occurs in most children and might help to guide the design of pediatric vaccination regimens.

Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination.

Vaccines are proving to be highly effective in controlling hospitalisation and deaths associated with SARS-CoV-2 infection but the emergence of viral variants with novel antigenic profiles threatens to diminish their efficacy. Assessment of the ability of sera from vaccine recipients to neutralise SARS-CoV-2 variants will inform the success of strategies for minimising COVID19 cases and the design of effective antigenic formulations. Here, we examine the sensitivity of variants of concern (VOCs) representative of the B.1.617.1 and B.1.617.2 (first associated with infections in India) and B.1.351 (first associated with infection in South Africa) lineages of SARS-CoV-2 to neutralisation by sera from individuals vaccinated with the BNT162b2 (Pfizer/BioNTech) and ChAdOx1 (Oxford/AstraZeneca) vaccines. Across all vaccinated individuals, the spike glycoproteins from B.1.617.1 and B.1.617.2 conferred reductions in neutralisation of 4.31 and 5.11-fold respectively. The reduction seen with the B.1.617.2 lineage approached that conferred by the glycoprotein from B.1.351 (South African) variant (6.29-fold reduction) that is known to be associated with reduced vaccine efficacy. Neutralising antibody titres elicited by vaccination with two doses of BNT162b2 were significantly higher than those elicited by vaccination with two doses of ChAdOx1. Fold decreases in the magnitude of neutralisation titre following two doses of BNT162b2, conferred reductions in titre of 7.77, 11.30 and 9.56-fold respectively to B.1.617.1, B.1.617.2 and B.1.351 pseudoviruses, the reduction in neutralisation of the delta variant B.1.617.2 surpassing that of B.1.351. Fold changes in those vaccinated with two doses of ChAdOx1 were 0.69, 4.01 and 1.48 respectively. The accumulation of mutations in these VOCs, and others, demonstrate the quantifiable risk of antigenic drift and subsequent reduction in vaccine efficacy. Accordingly, booster vaccines based on updated variants are likely to be required over time to prevent productive infection. This study also suggests that two dose regimes of vaccine are required for maximal BNT162b2 and ChAdOx1-induced immunity.

De Novo Powered Air-Purifying Respirator Design and Fabrication for Pandemic Response.

The rapid spread of COVID-19 and disruption of normal supply chains has resulted in severe shortages of personal protective equipment (PPE), particularly devices with few suppliers such as powered air-purifying respirators (PAPRs). A scarcity of information describing design and performance criteria for PAPRs represents a substantial barrier to mitigating shortages. We sought to apply open-source product development (OSPD) to PAPRs to enable alternative sources of supply and further innovation. We describe the design, prototyping, validation, and user testing of locally manufactured, modular, PAPR components, including filter cartridges and blower units, developed by the Greater Boston Pandemic Fabrication Team (PanFab). Two designs, one with a fully custom-made filter and blower unit housing, and the other with commercially available variants (the "Custom" and "Commercial" designs, respectively) were developed; the components in the Custom design are interchangeable with those in Commercial design, although the form factor differs. The engineering performance of the prototypes was measured and safety validated using National Institutes for Occupational Safety and Health (NIOSH)-equivalent tests on apparatus available under pandemic conditions at university laboratories. Feedback was obtained from four individuals; two clinicians working in ambulatory clinical care and two research technical staff for whom PAPR use is standard occupational PPE; these individuals were asked to compare PanFab prototypes to commercial PAPRs from the perspective of usability and suggest areas for improvement. Respondents rated the PanFab Custom PAPR a 4 to 5 on a 5 Likert-scale 1) as compared to current PPE options, 2) for the sense of security with use in a clinical setting, and 3) for comfort compared to standard, commercially available PAPRs. The three other versions of the designs (with a Commercial blower unit, filter, or both) performed favorably, with survey responses consisting of scores ranging from 3 to 5. Engineering testing and clinical feedback demonstrate that the PanFab designs represent favorable alternatives to traditional PAPRs in terms of user comfort, mobility, and sense of security. A nonrestrictive license promotes innovation in respiratory protection for current and future medical emergencies.

Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2.

Although two-dose mRNA vaccination provides excellent protection against SARS-CoV-2, there is little information about vaccine efficacy against variants of concern (VOC) in individuals above eighty years of age1. Here we analysed immune responses following vaccination with the BNT162b2 mRNA vaccine2 in elderly participants and younger healthcare workers. Serum neutralization and levels of binding IgG or IgA after the first vaccine dose were lower in older individuals, with a marked drop in participants over eighty years old. Sera from participants above eighty showed lower neutralization potency against the B.1.1.7 (Alpha), B.1.351 (Beta) and P.1. (Gamma) VOC than against the wild-type virus and were more likely to lack any neutralization against VOC following the first dose. However, following the second dose, neutralization against VOC was detectable regardless of age. The frequency of SARS-CoV-2 spike-specific memory B cells was higher in elderly responders (whose serum showed neutralization activity) than in non-responders after the first dose. Elderly participants showed a clear reduction in somatic hypermutation of class-switched cells. The production of interferon-γ and interleukin-2 by SARS-CoV-2 spike-specific T cells was lower in older participants, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high-risk population and that specific measures to boost vaccine responses in this population are warranted, particularly where variants of concern are circulating.

Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data.

We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5' UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5' end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity.

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.