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Neutrophils, pivotal cells of innate and adaptive immune responses, employ reactive oxygen species (ROS) to combat pathogens and control gene expression. Paracetamol (acetaminophen) is widely used as an analgesic and antipyretic medication, yet its precise mechanisms of action are not yet fully understood. Here, we investigate the impact of both ingested and in-vitro paracetamol on neutrophil ROS activity, using flow cytometry and antioxidant assays. Our studies reveal that paracetamol significantly suppresses ROS activity ex-vivo in the short term. Additionally, both paracetamol and its metabolite N-acetyl-p-benzoquinone imine exhibited direct in vitro antioxidant effects, and paracetamol suppressed neutrophil extracellular trap formation ex vivo. These findings suggest a connection between paracetamol use and altered neutrophil responses, with potential implications for use in some patient groups, such as immunocompromised individuals. Further investigation into paracetamol's effects on neutrophil antimicrobial functions is warranted to elucidate possible risks, particularly when taken frequently or in conjunction with other treatments such as vaccinations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: This study investigates the frequency of hospital attendances, emergency care attendances and geographical influences on service interaction in cohorts of patients with primary and secondary antibody deficiency, to inform future service planning and delivery., Methods: The COVID-19 in Antibody Deficiency (COV-AD) study was a United Kingdom study that enrolled 525 participants between April 2021 and September 2022. Data on health care utilisation was extracted from a screening cohort of participants at one participating site (Birmingham, UK). Hospital attendance (i.e. all outpatient and inpatient care episodes, including hospital-based IVIG treatment) and emergency care attendance patterns were analysed. Geographical differences in travel times to hospitals and associated costs were considered for all participants at all recruiting sites., Results: Individuals with antibody deficiency had a median of 7 hospital attendances per year. A diagnosis of secondary antibody deficiency, and antibody deficiency severe enough to require treatment with immunoglobulin replacement were associated with an increased frequency of hospital attendance. 12.7% of the cohort attended the Emergency Department at least once in the preceding twelve months. Individuals with secondary antibody deficiency were at greater risk of requiring emergency care over the preceding one-year and five-year periods. Individuals receiving subcutaneous immunoglobulin lived further from their local immunology centre and were more likely to engage with the COV-AD research study remotely, via dried blood spots sampling., Conclusion: This study highlights the utilisation of emergency and secondary care usage amongst patient with immunodeficiency and may inform service adaptation and development to better accommodate patient needs and circumstances., (© 2024. The Author(s).)

Background: Immunisation is recommended internationally to protect against pneumococcal infections in HIV-infected adults. However, vaccination schedule designs are mostly based on studies of initial rather than long-term antibody responses. This UK observational study investigated the short- and long-term antibody responses to polysaccharide and glycoconjugate pneumococcal vaccines in an adult HIV-infected cohort., Methods: We studied a subgroup of 152 of 839 participants from the AIR (Assessment of Immune Responses to Routine Immunisations in HIV-infected Adults, ISRCTN95588307) study that had received pneumococcal vaccinations and had blood samples collected pre- and post-vaccination, as well as at least annually for four subsequent calendar years. Patients received either Pneumovax-23 (PPV, N = 89) or Prevenar-13 (PCV, N = 63) as their primary vaccine, with immunity assessed by measuring IgG antibody concentrations for 12 pneumococcal polysaccharide serotypes (PnPS). The primary outcome was achieving IgG antibody concentrations above the recommended World Health Organisation (WHO) threshold of 0.35 µg/mL for at least 8/12 of the PnPS assessed (WHO ≥8/12PnPS ). Patients who did not achieve WHO ≥8/12PnPS after the primary vaccination were offered further vaccination with PCV; booster vaccinations with PCV were additionally offered to those where antibody levels subsequently fell below the WHO ≥8/12PnPS threshold., Results: Patients receiving PCV as their primary pneumococcal vaccine were significantly more likely to achieve WHO ≥8/12PnPS after a single vaccine dose than those receiving PPV (54% vs. 33%, p = 0.012). This difference persisted following booster vaccination with PCV, with cumulative rates of WHO ≥8/12PnPS in those receiving PCV vs. PPV as the primary vaccine of 88% vs. 67% and 100% vs. 85% after receiving up to one and two booster vaccinations, respectively. Where WHO ≥8/12PnPS was achieved, this persisted significantly longer in those receiving PCV as their primary vaccine compared to PPV (median: 23.5 vs. 11.1 months; p = 0.010)., Conclusions: Immunisation with PCV resulted in quantitatively greater antibody responses than immunisation with PPV in a cohort of HIV-infected UK adults. Individuals receiving PCV as their primary vaccine required fewer total pneumococcal vaccine doses to achieve WHO ≥8/12PnPS and experienced greater duration of time above this threshold than those with PPV as the primary vaccine. However, the median longevity of both vaccine responses was relatively short, which supports the use of ongoing booster doses using high-valency glycoconjugate vaccines to sustain WHO ≥8/12PnPS threshold antibody levels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: AH, VH, SH, PK, SD, and AR were responsible for the original design and preparation of this Correspondence. AS and NP collated and analysed the secondary immunodeficiency cohort measles and clinical data and contributed to the writing of the Correspondence and figures and table preparation. SF was responsible for the measles testing of the COCO cohort, analysis of birth groups, and figure and table preparation. OG was responsible for the measles testing of the VIBRANT cohort, analysis, and contributed to writing of the Correspondence. AS and AR have accessed and verified all underlying data. All authors had full access to the data in the Correspondence and had final responsibility for the decision to submit for publication. We declare no competing interests related to this Correspondence. AH has received unrelated grant funding from Wellcome Trust, Medical Research Council (MRC), British Society for Antimicrobial Chemotherapy, and Medical Research Foundation. AH acts as a clinical consultant for the WHO Global Influenza Programme, Department of Health and Social Care Scientific Advisory Group for Serology Testing, National Institute for Clinical Excellence, and Scotland Medicines consortium; and is a data monitoring committee member for the BALANCE trial (NCT03005145). AR has received unrelated grant funding from UK Research and Innovation (UKRI) and MRC. SD has received unrelated grant funding from MRC, UKRI, National Institute for Health and Care Research, and Wellcome Trust. VH has received unrelated grant funding from MRC and Health Data Research UK, and is supported by the UK Health Security Agency and the UK Department of Health. The authors welcome applications from researchers looking to use the data published herein. Applications should be directed to the corresponding author and will be reviewed by the authorship group and subject to the establishment of data sharing agreements. This work is a compilation from three different studies and four different funding sources where data on measles immunity has been tested as part of the study and then combined to provide the narrative in this Correspondence. The COCO study was funded by the UK Medical Research Council. The VIBRANT and SIREN studies were funded by UK Research & Innovation COVID-19 Rapid Response Initiative. Laboratory validation and verification of the measles ELISAs was supported by funding from the UK Medical Research Council (MX/X502996/1). Relevant Ethics committee approvals were from Camden and Kings Cross Research Ethics Committee (20/HRA/1817) for the COCO study and the Chelsea Research Ethics Committee (21/HRA/5433) for the VIBRANT study.

Viral clearance, antibody response and the mutagenic effect of molnupiravir has not been elucidated in at-risk populations. Non-hospitalised participants within 5 days of SARS-CoV-2 symptoms randomised to receive molnupiravir (n = 253) or Usual Care (n = 324) were recruited to study viral and antibody dynamics and the effect of molnupiravir on viral whole genome sequence from 1437 viral genomes. Molnupiravir accelerates viral load decline, but virus is detectable by Day 5 in most cases. At Day 14 (9 days post-treatment), molnupiravir is associated with significantly higher viral persistence and significantly lower anti-SARS-CoV-2 spike antibody titres compared to Usual Care. Serial sequencing reveals increased mutagenesis with molnupiravir treatment. Persistence of detectable viral RNA at Day 14 in the molnupiravir group is associated with higher transition mutations following treatment cessation. Viral viability at Day 14 is similar in both groups with post-molnupiravir treated samples cultured up to 9 days post cessation of treatment. The current 5-day molnupiravir course is too short. Longer courses should be tested to reduce the risk of potentially transmissible molnupiravir-mutated variants being generated. Trial registration: ISRCTN30448031., (© 2024. The Author(s).)

Background and Hypothesis: Use of clozapine in treatment-resistant schizophrenia is often limited due to risk of adverse effects. Cross-sectional associations between clozapine treatment and low immunoglobulin levels have been reported, however prospective studies are required to establish temporal relationships. We tested the hypothesis that reductions in immunoglobulin levels would occur over the first 6 months following initiation of clozapine treatment. Relationships between immunoglobulin levels and symptom severity over the course of clozapine treatment were also explored., Design: This prospective observational study measured immunoglobulin (Ig) levels (A, M and G) in 56 patients with treatment-resistant schizophrenia at 6-, 12- and 24-weeks following initiation with clozapine. Clinical symptoms were also measured at 12 weeks using the positive and negative syndrome scale (PANSS)., Results: IgA, IgG and IgM all decreased during clozapine treatment. For IgA and IgG the reduction was significant at 24 weeks (IgA: β = -32.66, 95% CI = -62.38, -2.93, p = 0.03; IgG: β = -63.96, 95% CI = -118.00, -9.31, p = 0.02). For IgM the reduction was significant at 12 and 24 weeks (12 weeks: β = -23.48, 95% CI = -39.56, -7.42, p = 0.004; 24 weeks: β = -33.12, 95 %CI = -50.30, -15.94, p = <0.001). Reductions in IgA and IgG during clozapine treatment were correlated with reductions in PANSS-total over 12 weeks (n = 32, IgA r = 0.59, p = 0.005; IgG r = 0.48, p = 0.03)., Conclusions: The observed reductions in immunoglobulin levels over six months of clozapine treatment add further evidence linking clozapine to secondary antibody deficiency. Associations between Ig reduction and symptom improvement may however indicate that immune mechanisms contribute to both desirable and undesirable effects of clozapine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Background: Clozapine has unique effectiveness in treatment-resistant schizophrenia and is known to cause immunological side-effects. A transient spike in neutrophils commonly occurs in the first weeks of clozapine therapy. There is contradictory evidence in the literature as to whether neutrophil changes with clozapine are linked to treatment response., Aims: The current study aims to further examine the neutrophil changes in response to clozapine and explore any association between neutrophil trajectory and treatment response., Methods: A retrospective cohort study of patients undergoing their first treatment with clozapine and continuing for at least 2 years identified 425 patients (69% male/31% female). Neutrophil counts at baseline, 3 weeks and 1 month were obtained predominantly by linkage with data from the clozapine monitoring service. Clinical Global Impression- Severity (CGI-S) was rated from case notes at the time of clozapine initiation and at 2 years. Latent class growth analysis (LCGA) was performed to define distinct trajectories of neutrophil changes during the first month of treatment. Logistic regression was then conducted to investigate for association between the trajectory of neutrophil count changes in month 1 and clinical response at 2 years as well as between baseline neutrophil count and response., Results: Of the original cohort, 397 (93%) patients had useable neutrophil data during the first 6 weeks of clozapine treatment. LCGA revealed significant differences in neutrophil trajectories with a three-class model being the most parsimonious. The classes had similar trajectory profiles but differed primarily on overall neutrophil count: with low, high-normal and high neutrophil classes, comprising 52%, 40% and 8% of the sample respectively. Membership of the high-normal group was associated with significantly increased odds of a positive response to clozapine, as compared to the low neutrophil group [Odds ratio (OR) = 2.10, p-value = 0.002; 95% confidence interval (95% CI) = 1.31-3.36]. Baseline neutrophil count was a predictor of response to clozapine at 2 years, with counts of ≥5 × 10 9 /l significantly associated with positive response (OR = 1.60, p-value = 0.03; 95% CI = 1.03-2.49)., Conclusions: Our data are consistent with the hypothesis that patients with low-level inflammation, reflected in a high-normal neutrophil count, are more likely to respond to clozapine, raising the possibility that clozapine exerts its superior efficacy via immune mechanisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The importance of salivary SARS-CoV-2 antibodies, following infection and vaccination, has not been fully established. 875 healthcare workers were sampled during the first wave in 2020 and 66 longitudinally in response to Pfizer BioNTech 162b2 vaccination. We measured SARS-CoV-2 total IgGAM and individual IgG, IgA and IgM antibodies. IgGAM seroprevalence was 39.9%; however, only 34.1% of seropositive individuals also had salivary antibodies. Infection generated serum IgG antibodies in 51.4% and IgA antibodies in 34.1% of individuals. In contrast, the salivary antibody responses were dominated by IgA (30.9% and 12% generating IgA and IgG antibodies, respectively). Post 2nd vaccination dose, in serum, 100% of infection naïve individuals had IgG and 82.8% had IgA responses; in saliva, 65.5% exhibited IgG and 55.2% IgA antibodies. Prior infection enhanced the vaccine antibody response in serum but no such difference was observed in saliva. Strong neutralisation responses were seen for serum 6 months post 2nd-vaccination dose (median 87.1%) compared to low neutralisation responses in saliva (median 1%). Intramuscular vaccination induces significant serum antibodies and to a lesser extent, salivary antibodies; however, salivary antibodies are typically non-neutralising. This study provides further evidence for the need of mucosal vaccines to elicit nasopharyngeal/oral protection. Although saliva is an attractive non-invasive sero-surveillance tool, due to distinct differences between systemic and oral antibody responses, it cannot be used as a proxy for serum antibody measurement., Competing Interests: Declaration of Competing Interest AC is employed by The Binding Site Group Ltd. The SARS-CoV-2 ELISA was developed and commercialised between The Binding Site Group Ltd and the Clinical Immunology Service at University of Birmingham (including AR, SF, MTD, AS, AC). The rest of the authors declared no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Post-acute cardiac sequelae, following SARS-CoV-2 infection, are well recognized as complications of COVID-19. We have previously shown the persistence of autoantibodies against antigens in skin, muscle, and heart in individuals following severe COVID-19; the most common staining on skin tissue displayed an inter-cellular cement pattern consistent with antibodies against desmosomal proteins. Desmosomes play a critical role in maintaining the structural integrity of tissues. For this reason, we analyzed desmosomal protein levels and the presence of anti-desmoglein (DSG) 1, 2, and 3 antibodies in acute and convalescent sera from patients with COVID-19 of differing clinical severity. We find increased levels of DSG2 protein in sera from acute COVID-19 patients. Furthermore, we find that DSG2 autoantibody levels are increased significantly in convalescent sera following severe COVID-19 but not in hospitalized patients recovering from influenza infection or healthy controls. Levels of autoantibody in sera from patients with severe COVID-19 were comparable to levels in patients with non-COVID-19-associated cardiac disease, potentially identifying DSG2 autoantibodies as a novel biomarker for cardiac damage. To determine if there was any association between severe COVID-19 and DSG2, we stained post-mortem cardiac tissue from patients who died from COVID-19 infection. This confirmed DSG2 protein within the intercalated discs and disruption of the intercalated disc between cardiomyocytes in patients who died from COVID-19. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.)

Secondary immunodeficiency (SID), manifesting as increased susceptibility to infection, is an emergent clinical problem in haematoncology. Management of SID includes vaccination, prophylactic antibiotics (pAbx) and immunoglobulin replacement therapy (IgRT). We report clinical and laboratory parameters of 75 individuals, treated for haematological malignancy, who were referred for immunological assessment due to recurrent infections. Forty-five were managed with pAbx while thirty required IgRT after failing to improve on pAbx. Individuals requiring IgRT had significantly more bacterial, viral and fungal infections resulting in hospitalization at least 5 years after their original haemato-oncological diagnosis. Following immunological assessment and intervention, a 4.39-fold reduction in the frequency of hospital admissions to treat infection was observed in the IgRT cohort and a 2.30-fold reduction in the pAbx cohort. Significant reductions in outpatient antibiotic use were also observed in both cohorts following immunology input. Patients requiring IgRT were more hypogammaglobulinaemic and had lower titres of pathogen-specific antibodies and smaller memory B cell populations than those requiring pAbx. Test vaccination with pneumococcal conjugate vaccine discriminated poorly between the two groups. Patients requiring IgRT could be distinguished by combining wider pathogen-specific serology with a frequency of hospital admissions for infection. If validated in larger cohorts, this approach may circumvent the need for test vaccination and enhance patient selection for IgRT., Competing Interests: Mark T. Drayson reports personal fees from Abingdon Health, outside the submitted work. All other authors declare no competing interests., (© 2023 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Both infection and vaccination, alone or in combination, generate antibody and T cell responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the maintenance of such responses-and hence protection from disease-requires careful characterization. In a large prospective study of UK healthcare workers (HCWs) (Protective Immunity from T Cells in Healthcare Workers [PITCH], within the larger SARS-CoV-2 Immunity and Reinfection Evaluation [SIREN] study), we previously observed that prior infection strongly affected subsequent cellular and humoral immunity induced after long and short dosing intervals of BNT162b2 (Pfizer/BioNTech) vaccination., Methods: Here, we report longer follow-up of 684 HCWs in this cohort over 6-9 months following two doses of BNT162b2 or AZD1222 (Oxford/AstraZeneca) vaccination and up to 6 months following a subsequent mRNA booster vaccination., Findings: We make three observations: first, the dynamics of humoral and cellular responses differ; binding and neutralizing antibodies declined, whereas T and memory B cell responses were maintained after the second vaccine dose. Second, vaccine boosting restored immunoglobulin (Ig) G levels; broadened neutralizing activity against variants of concern, including Omicron BA.1, BA.2, and BA.5; and boosted T cell responses above the 6-month level after dose 2. Third, prior infection maintained its impact driving larger and broader T cell responses compared with never-infected people, a feature maintained until 6 months after the third dose., Conclusions: Broadly cross-reactive T cell responses are well maintained over time-especially in those with combined vaccine and infection-induced immunity ("hybrid" immunity)-and may contribute to continued protection against severe disease., Funding: Department for Health and Social Care, Medical Research Council., Competing Interests: Declaration of interests S.J.D. is a Scientific Advisor to the Scottish Parliament on COVID-19, for which she receives a fee. A.J.P. is Chair of UK Department of Health and Social Care’s (DHSC) Joint Committee on Vaccination and Immunisation (JCVI) but does not participate in policy decisions on COVID-19 vaccines. He was previously a member of the WHO’s SAGE. The views expressed in this article do not necessarily represent the views of DHSC, JCVI, or WHO. A.J.P. is chief investigator on clinical trials of Oxford University’s COVID-19 vaccine funded by NIHR. Oxford University has entered a joint COVID-19 vaccine development partnership with AstraZeneca. G.S. sits on the GSK Vaccines Scientific Advisory Board and is a founder member of RQ Biotechnology., (Copyright © 2023. Published by Elsevier Inc.)

In this population-based cohort of 7538 adults, combined immunoglobulin (Ig) G, IgA, and IgM (IgG/A/M) anti-spike titers measured after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination were predictive of protection against breakthrough SARS-CoV-2 infection. Discrimination was significantly improved by adjustment for factors influencing risk of SARS-CoV-2 exposure, including household overcrowding, public transport use, and visits to indoor public places. Anti-spike IgG/A/M titers showed positive correlation with neutralizing antibody titers (rs = 0.80 [95% confidence interval, .72-.86]; P < .001) and S peptide-stimulated interferon-γ concentrations (rs = 0.31 [.13-.47]; P < .001)., Competing Interests: Potential conflicts of interest. A. R. M. declares receipt of funding, outside the submitted work, to support vitamin D research from the following companies, which manufacture or sell vitamin D supplements: Pharma Nord, DSM Nutritional Products, Thornton & Ross, and Hyphens Pharma. A. R. M. also declares the following, all outside the submitted work: receipt of funding from the Karl R Pfleger Foundation, the AIM Foundation, the UK National Institute for Health Research Clinical Research Network, Warburtons, and Matthew Isaacs; consulting fees from DSM Nutritional Products and payment from Oregon State University for lectures, presentations, speakers bureaus, manuscript writing, or educational events; support for attending meetings from Pharma Nord and Abiogen Pharma; participation on the data and safety monitoring board for the VITALITY trial (VITamin D for AdoLescents with HIV to reduce musculoskeletal morbidity and ImmunopaThologY); unpaid work as a program committee member for the Vitamin D Workshop; and receipt of vitamin D capsules for clinical trial use from Pharma Nord, Synergy Biologics, and Cytoplan. E. S. C. declares receipt of an honorarium from UCB Pharma for lectures, outside the submitted work, and is an Early Career Trustee for the British Society for Immunology. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In March 2020, the United Kingdom Primary Immunodeficiency Network (UKPIN) established a registry of cases to collate the outcomes of individuals with PID and SID following SARS-CoV-2 infection and treatment. A total of 310 cases of SARS-CoV-2 infection in individuals with PID or SID have now been reported in the UK. The overall mortality within the cohort was 17.7% (n = 55/310). Individuals with CVID demonstrated an infection fatality rate (IFR) of 18.3% (n = 17/93), individuals with PID receiving IgRT had an IFR of 16.3% (n = 26/159) and individuals with SID, an IFR of 27.2% (n = 25/92). Individuals with PID and SID had higher inpatient mortality and died at a younger age than the general population. Increasing age, low pre-SARS-CoV-2 infection lymphocyte count and the presence of common co-morbidities increased the risk of mortality in PID. Access to specific COVID-19 treatments in this cohort was limited: only 22.9% (n = 33/144) of patients admitted to the hospital received dexamethasone, remdesivir, an anti-SARS-CoV-2 antibody-based therapeutic (e.g. REGN-COV2 or convalescent plasma) or tocilizumab as a monotherapy or in combination. Dexamethasone, remdesivir, and anti-SARS-CoV-2 antibody-based therapeutics appeared efficacious in PID and SID. Compared to the general population, individuals with PID or SID are at high risk of mortality following SARS-CoV-2 infection. Increasing age, low baseline lymphocyte count, and the presence of co-morbidities are additional risk factors for poor outcome in this cohort., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology.)

Background: Individuals with primary and secondary immunodeficiency (PID/SID) were shown to be at risk of poor outcomes during the early stages of the SARS-CoV-2 pandemic. SARS-CoV-2 vaccines demonstrate reduced immunogenicity in these patients., Objectives: To understand whether the risk of severe COVID-19 in individuals with PID or SID has changed following the deployment of vaccination and therapeutics in the context of the emergence of novel viral variants of concern., Methods: The outcomes of two cohorts of patients with PID and SID were compared: the first, infected between March and July 2020, prior to vaccination and treatments, the second after these intervention became available between January 2021 and April 2022., Results: 22.7% of immunodeficient patients have been infected at least once with SARS-CoV-2 since the start of the pandemic, compared to over 70% of the general population. Immunodeficient patients were typically infected later in the pandemic when the B.1.1.529 (Omicron) variant was dominant. This delay was associated with receipt of more vaccine doses and higher pre-infection seroprevalence. Compared to March-July 2020, hospitalization rates (53.3% vs 17.9%, p<0.0001) and mortality (Infection fatality rate 20.0% vs 3.4%, p=0.0003) have significantly reduced for patients with PID but remain elevated compared to the general population. The presence of a serological response to vaccination was associated with a reduced duration of viral detection by PCR in the nasopharynx. Early outpatient treatment with antivirals or monoclonal antibodies reduced hospitalization during the Omicron wave., Conclusions: Most individuals with immunodeficiency in the United Kingdom remain SARS-CoV-2 infection naïve. Vaccination, widespread availability of outpatient treatments and, possibly, the emergence of the B.1.1.529 variant have led to significant improvements in morbidity and mortality followings SARS-CoV-2 infection since the start of the pandemic. However, individuals with PID and SID remain at significantly increased risk of poor outcomes compared to the general population; mitigation, vaccination and treatment strategies must be optimized to minimize the ongoing burden of the pandemic in these vulnerable cohorts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shields, Tadros, Al-Hakim, Nell, Lin, Chan, Goddard, Dempster, Dziadzio, Patel, Elkalifa, Huissoon, Duncan, Herwadkar, Khan, Bethune, Elcombe, Thaventhiran, Klenerman, Lowe, Savic, Burns and Richter.)

Background: There is increasing evidence that vitamin D (VD) deficiency may increase individuals' risk of COVID-19 infection and susceptibility. We aimed to determine the relationship between VD deficiency and sufficiency and COVID-19 seropositivity within healthcare workers., Methods: The study included an observational cohort of healthcare workers who isolated due to COVID-19 symptoms from 12 May to 22 May 2020, from the University Hospitals Birmingham National Health Service Foundation Trust. Data collected included SARS-CoV-2 seroconversion status, serum 25(OH)D 3 levels, age, body mass index (BMI), sex, ethnicity, job role and comorbidities. Participants were grouped into four VD categories: (1) Severe VD deficiency (VD<30 nmol/L); (2) VD deficiency (30 nmol/L ≤VD<50 nmol/L); (3) VD insufficiency (50 nmol/L ≤VD<75 nmol/L); (4) VD sufficiency (VD≥75 nmol/L)., Results: When VD levels were compared against COVID-19 seropositivity rate, a U-shaped curve was identified. This trend repeated when participants were split into subgroups of age, sex, ethnicity, BMI and comorbidity status. Significant difference was identified in the COVID-19 seropositivity rate between VD groups in the total population and between groups of men and women; black, Asian and minority ethnic (BAME) group; BMI<30 (kg/m 2 ); 0 and +1 comorbidities; the majority of which were differences when the severely VD deficient category were compared with the other groups. A larger proportion of those within the BAME group (vs white ethnicity) were severely VD deficient (p < 0.00001). A larger proportion of the 0 comorbidity subgroup were VD deficient in comparison to the 1+ comorbidity subgroup (p=0.046)., Conclusions: Our study has shown a U-shaped relationship for COVID-19 seropositivity in UK healthcare workers. Further investigation is required to determine whether high VD levels can have a detrimental effect on susceptibility to COVID-19 infection. Future randomised clinical trials of VD supplementation could potentially identify 'optimal' VD levels, allowing for targeted therapeutic treatment for those at risk., Competing Interests: Competing interests: MH reports personal fees from Thornton Ross, outside the submitted work., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of antibodies to SARS-CoV-2 from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the subject group from whom the sera were obtained. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending the disease status of the subject and on the specific antigen targeted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lamerton, Marcial-Juarez, Faustini, Perez-Toledo, Goodall, Jossi, Newby, Chapple, Dietrich, Veenith, Shields, Harper, Henderson, Rayes, Wraith, Watson, Crispin, Drayson, Richter and Cunningham.)

Background: Vaccination prevents severe morbidity and mortality from COVID-19 in the general population. The immunogenicity and efficacy of SARS-CoV-2 vaccines in patients with antibody deficiency is poorly understood., Objectives: COVID-19 in patients with antibody deficiency (COV-AD) is a multi-site UK study that aims to determine the immune response to SARS-CoV-2 infection and vaccination in patients with primary or secondary antibody deficiency, a population that suffers from severe and recurrent infection and does not respond well to vaccination., Methods: Individuals on immunoglobulin replacement therapy or with an IgG less than 4 g/L receiving antibiotic prophylaxis were recruited from April 2021. Serological and cellular responses were determined using ELISA, live-virus neutralisation and interferon gamma release assays. SARS-CoV-2 infection and clearance were determined by PCR from serial nasopharyngeal swabs., Results: A total of 5.6% (n = 320) of the cohort reported prior SARS-CoV-2 infection, but only 0.3% remained PCR positive on study entry. Seropositivity, following two doses of SARS-CoV-2 vaccination, was 54.8% (n = 168) compared with 100% of healthy controls (n = 205). The magnitude of the antibody response and its neutralising capacity were both significantly reduced compared to controls. Participants vaccinated with the Pfizer/BioNTech vaccine were more likely to be seropositive (65.7% vs. 48.0%, p = 0.03) and have higher antibody levels compared with the AstraZeneca vaccine (IgGAM ratio 3.73 vs. 2.39, p = 0.0003). T cell responses post vaccination was demonstrable in 46.2% of participants and were associated with better antibody responses but there was no difference between the two vaccines. Eleven vaccine-breakthrough infections have occurred to date, 10 of them in recipients of the AstraZeneca vaccine., Conclusion: SARS-CoV-2 vaccines demonstrate reduced immunogenicity in patients with antibody deficiency with evidence of vaccine breakthrough infection., (© 2022. The Author(s).)

Background: Patients with primary and secondary antibody deficiency are vulnerable to COVID-19 and demonstrate diminished responses following two-dose SARS-CoV-2 vaccine schedules. Third primary vaccinations have been deployed to enhance their humoral and cellular immunity., Objectives: To determine the immunogenicity of the third primary SARS-CoV-2 immunisation in a heterogeneous cohort of patients with antibody deficiency., Methods: Participants enrolled in the COV-AD study were sampled before and after their third vaccine dose. Serological and cellular responses were determined using ELISA, live-virus neutralisation and ELISPOT assays., Results: Following a two-dose schedule, 100% of healthy controls mounted a serological response to SARS-CoV-2 vaccination, however, 38.6% of individuals with antibody deficiency remained seronegative. A third primary SARS-CoV-2 vaccine significantly increased anti-spike glycoprotein antibody seroprevalence from 61.4% to 76.0%, the magnitude of the antibody response, its neutralising capacity and induced seroconversion in individuals who were seronegative after two vaccine doses. Vaccine-induced serological responses were broadly cross-reactive against the SARS-CoV-2 B.1.1.529 variant of concern, however, seroprevalence and antibody levels remained significantly lower than healthy controls. No differences in serological responses were observed between individuals who received AstraZeneca ChAdOx1 nCoV-19 and Pfizer BioNTech 162b2 during their initial two-dose vaccine schedule. SARS-CoV-2 infection-naive participants who had received a heterologous vaccine as a third dose were significantly more likely to have a detectable T cell response following their third vaccine dose (61.5% vs 11.1%)., Conclusion: These data support the widespread use of third primary immunisations to enhance humoral immunity against SARS-CoV-2 in individuals with antibody deficiency., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shields, Faustini, Hill, Al-Taei, Tanner, Ashford, Workman, Moreira, Verma, Wagg, Heritage, Campton, Stamataki, Drayson, Klenerman, Thaventhiran, Elkhalifa, Goddard, Johnston, Huissoon, Bethune, Elcombe, Lowe, Patel, Savic, Richter, Burns and the COV-AD consortium.)

Background: Patients with some types of immunodeficiency can experience chronic or relapsing infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This leads to morbidity and mortality, infection control challenges, and the risk of evolution of novel viral variants. The optimal treatment for chronic coronavirus disease 2019 (COVID-19) is unknown., Objective: Our aim was to characterize a cohort of patients with chronic or relapsing COVID-19 disease and record treatment response., Methods: We conducted a UK physician survey to collect data on underlying diagnosis and demographics, clinical features, and treatment response of immunodeficient patients with chronic (lasting ≥21 days) or relapsing (≥2 episodes) of COVID-19., Results: We identified 31 patients (median age 49 years). Their underlying immunodeficiency was most commonly characterized by antibody deficiency with absent or profoundly reduced peripheral B-cell levels; prior anti-CD20 therapy, and X-linked agammaglobulinemia. Their clinical features of COVID-19 were similar to those of the general population, but their median duration of symptomatic disease was 64 days (maximum 300 days) and individual patients experienced up to 5 episodes of illness. Remdesivir monotherapy (including when given for prolonged courses of ≤20 days) was associated with sustained viral clearance in 7 of 23 clinical episodes (30.4%), whereas the combination of remdesivir with convalescent plasma or anti-SARS-CoV-2 mAbs resulted in viral clearance in 13 of 14 episodes (92.8%). Patients receiving no therapy did not clear SARS-CoV-2., Conclusions: COVID-19 can present as a chronic or relapsing disease in patients with antibody deficiency. Remdesivir monotherapy is frequently associated with treatment failure, but the combination of remdesivir with antibody-based therapeutics holds promise., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

B-cell-depleting agents are among the most commonly used drugs to treat haemato-oncological and autoimmune diseases. They rapidly induce a state of peripheral B-cell aplasia with the potential to interfere with nascent vaccine responses, particularly to novel antigens. We have examined the relationship between B-cell reconstitution and SARS-CoV-2 vaccine responses in two cohorts of patients previously exposed to B-cell-depleting agents: a cohort of patients treated for haematological B-cell malignancy and another treated for rheumatological disease. B-cell depletion severely impairs vaccine responsiveness in the first 6 months after administration: SARS-CoV-2 antibody seroprevalence was 42.2% and 33.3% in the haemato-oncological patients and rheumatology patients, respectively and 22.7% in patients vaccinated while actively receiving anti-lymphoma chemotherapy. After the first 6 months, vaccine responsiveness significantly improved during early B-cell reconstitution; however, the kinetics of reconstitution was significantly faster in haemato-oncology patients. The AstraZeneca ChAdOx1 nCoV-19 vaccine and the Pfizer BioNTech 162b vaccine induced equivalent vaccine responses; however, shorter intervals between vaccine doses (<1 m) improved the magnitude of the antibody response in haeamto-oncology patients. In a subgroup of haemato-oncology patients, with historic exposure to B-cell-depleting agents (>36 m previously), vaccine non-responsiveness was independent of peripheral B-cell reconstitution. The findings have important implications for primary vaccination and booster vaccination strategies in individuals clinically vulnerable to SARS-CoV-2., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Immunology.)

Background: Previous infection with SARS-CoV-2 affects the immune response to the first dose of the SARS-CoV-2 vaccine. We aimed to compare SARS-CoV-2-specific T-cell and antibody responses in health-care workers with and without previous SARS-CoV-2 infection following a single dose of the BNT162b2 (tozinameran; Pfizer-BioNTech) mRNA vaccine., Methods: We sampled health-care workers enrolled in the PITCH study across four hospital sites in the UK (Oxford, Liverpool, Newcastle, and Sheffield). All health-care workers aged 18 years or older consenting to participate in this prospective cohort study were included, with no exclusion criteria applied. Blood samples were collected where possible before vaccination and 28 (±7) days following one or two doses (given 3-4 weeks apart) of the BNT162b2 vaccine. Previous infection was determined by a documented SARS-CoV-2-positive RT-PCR result or the presence of positive anti-SARS-CoV-2 nucleocapsid antibodies. We measured spike-specific IgG antibodies and quantified T-cell responses by interferon-γ enzyme-linked immunospot assay in all participants where samples were available at the time of analysis, comparing SARS-CoV-2-naive individuals to those with previous infection., Findings: Between Dec 9, 2020, and Feb 9, 2021, 119 SARS-CoV-2-naive and 145 previously infected health-care workers received one dose, and 25 SARS-CoV-2-naive health-care workers received two doses, of the BNT162b2 vaccine. In previously infected health-care workers, the median time from previous infection to vaccination was 268 days (IQR 232-285). At 28 days (IQR 27-33) after a single dose, the spike-specific T-cell response measured in fresh peripheral blood mononuclear cells (PBMCs) was higher in previously infected (n=76) than in infection-naive (n=45) health-care workers (median 284 [IQR 150-461] vs 55 [IQR 24-132] spot-forming units [SFUs] per 10 6 PBMCs; p<0·0001). With cryopreserved PBMCs, the T-cell response in previously infected individuals (n=52) after one vaccine dose was equivalent to that of infection-naive individuals (n=19) after receiving two vaccine doses (median 152 [IQR 119-275] vs 162 [104-258] SFUs/10 6 PBMCs; p=1·00). Anti-spike IgG antibody responses following a single dose in 142 previously infected health-care workers (median 270 373 [IQR 203 461-535 188] antibody units [AU] per mL) were higher than in 111 infection-naive health-care workers following one dose (35 001 [17 099-55 341] AU/mL; p<0·0001) and higher than in 25 infection-naive individuals given two doses (180 904 [108 221-242 467] AU/mL; p<0·0001)., Interpretation: A single dose of the BNT162b2 vaccine is likely to provide greater protection against SARS-CoV-2 infection in individuals with previous SARS-CoV-2 infection, than in SARS-CoV-2-naive individuals, including against variants of concern. Future studies should determine the additional benefit of a second dose on the magnitude and durability of immune responses in individuals vaccinated following infection, alongside evaluation of the impact of extending the interval between vaccine doses., Funding: UK Department of Health and Social Care, and UK Coronavirus Immunology Consortium., Competing Interests: CD worked on the Oxford–AstraZeneca COVID-19 vaccine trial (phase 1–3). AO reports personal fees from Take Two Interactive and personal fees from Genome BC, outside the submitted work. PCM reports grants from the Wellcome Trust during the conduct of the study. SLR-J reports grants from the UK Department of Health and Social Care during the conduct of the study and grants from UK Research and Innovation (UKRI), National Institute for Health Research (NIHR), and Global Challenges Research Fund outside the submitted work. SD reports grants from the UK Department of Health and Social Care, UK Coronavirus Immunology Consortium (UKRI), the Huo Family Foundation, and the NIHR during the conduct of the study. CJAD reports grants from the Wellcome Trust during the conduct of the study. LT reports personal fees from Eisai outside the submitted work. All other authors declare no competing interests., (© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.)

Extension of the interval between vaccine doses for the BNT162b2 mRNA vaccine was introduced in the United Kingdom to accelerate population coverage with a single dose. At this time, trial data were lacking, and we addressed this in a study of United Kingdom healthcare workers. The first vaccine dose induced protection from infection from the circulating alpha (B.1.1.7) variant over several weeks. In a substudy of 589 individuals, we show that this single dose induces severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody (NAb) responses and a sustained B and T cell response to the spike protein. NAb levels were higher after the extended dosing interval (6-14 weeks) compared with the conventional 3- to 4-week regimen, accompanied by enrichment of CD4 + T cells expressing interleukin-2 (IL-2). Prior SARS-CoV-2 infection amplified and accelerated the response. These data on dynamic cellular and humoral responses indicate that extension of the dosing interval is an effective immunogenic protocol., Competing Interests: Declaration of interests A.J.P. is Chair of the United Kingdom Department of Health and Social Care (DHSC) Joint Committee on Vaccination & Immunisation (JCVI) but does not participate in policy decisions on COVID-19 vaccines. He is a member of the WHO’s SAGE. The views expressed in this article do not necessarily represent the views of the DHSC, JCVI, or WHO. A.J.P. is chief investigator on clinical trials of Oxford University’s COVID-19 vaccine funded by NIHR. Oxford University has entered a joint COVID-19 vaccine development partnership with AstraZeneca., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: To determine clinical and ethnodemographic correlates of serological responses against the SARS-CoV-2 spike glycoprotein following mild-to-moderate COVID-19., Design: A retrospective cohort study of healthcare workers who had self-isolated due to COVID-19., Setting: University Hospitals Birmingham NHS Foundation Trust, UK (UHBFT)., Participants: 956 healthcare workers were recruited by open invitation via UHBFT trust email and social media between 27 April 2020 and the 8 June 2020., Intervention: Participants volunteered a venous blood sample that was tested for the presence of anti-SARS-CoV-2 spike glycoprotein antibodies. Results were interpreted in the context of the symptoms of their original illness and ethnodemographic variables., Results: Using an assay that simultaneously measures the combined IgG, IgA and IgM response against the spike glycoprotein (IgGAM), the overall seroprevalence within this cohort was 46.2% (n=442/956). The seroprevalence of immunoglobulin isotypes was 36.3%, 18.7% and 8.1% for IgG, IgA and IgM, respectively. IgGAM identified serological responses in 40.6% (n=52/128) of symptomatic individuals who reported a negative SARS-CoV-2 PCR test. Increasing age, non-white ethnicity and obesity were independently associated with greater IgG antibody response against the spike glycoprotein. Self-reported fever and fatigue were associated with greater IgG and IgA responses against the spike glycoprotein. The combination of fever and/or cough and/or anosmia had a positive predictive value of 92.3% for seropositivity in self-isolating individuals a time when Wuhan strain SARS-CoV-2 was predominant., Conclusions and Relevance: Assays employing combined antibody detection demonstrate enhanced seroepidemiological sensitivity and can detect prior viral exposure even when PCR swabs have been negative. We demonstrate an association between known ethnodemographic risk factors associated with mortality from COVID-19 and the magnitude of serological responses in mild-to-moderate disease., Competing Interests: Competing interests: MTD reports personal fees from Abingdon Health, outside the submitted work., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven relatively straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non-hospitalized SARS-CoV-2-infected subjects. Using trimeric spike glycoprotein, rather than nucleocapsid, enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT-PCR confirmed, non-hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti-spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS-CoV-2 infection., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Coronavirus 19 (COVID-19) has been associated with both transient and persistent systemic symptoms that do not appear to be a direct consequence of viral infection. The generation of autoantibodies has been proposed as a mechanism to explain these symptoms. To understand the prevalence of autoantibodies associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we investigated the frequency and specificity of clinically relevant autoantibodies in 84 individuals previously infected with SARS-CoV-2, suffering from COVID-19 of varying severity in both the acute and convalescent setting. These were compared with results from 32 individuals who were on the intensive therapy unit (ITU) for non-COVID reasons. We demonstrate a higher frequency of autoantibodies in the COVID-19 ITU group compared with non-COVID-19 ITU disease control patients and that autoantibodies were also found in the serum 3-5 months post-COVID-19 infection. Non-COVID patients displayed a diverse pattern of autoantibodies; in contrast, the COVID-19 groups had a more restricted panel of autoantibodies including skin, skeletal muscle and cardiac antibodies. Our results demonstrate that respiratory viral infection with SARS-CoV-2 is associated with the detection of a limited profile of tissue-specific autoantibodies, detectable using routine clinical immunology assays. Further studies are required to determine whether these autoantibodies are specific to SARS-CoV-2 or a phenomenon arising from severe viral infections and to determine the clinical significance of these autoantibodies., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Background: As of November 2020, severe acute respiratory syndrome coronavirus 2 has resulted in 55 million infections worldwide and more than 1.3 million deaths from coronavirus disease 2019 (COVID-19). Outcomes following severe acute respiratory syndrome coronavirus 2 infection in individuals with primary immunodeficiency (PID) or symptomatic secondary immunodeficiency (SID) remain uncertain., Objectives: We sought to document the outcomes of individuals with PID or symptomatic SID following COVID-19 in the United Kingdom., Methods: At the start of the COVID-19 pandemic, the United Kingdom Primary Immunodeficiency Network established a registry of cases to collate the nationwide outcomes of COVID-19 in individuals with PID or symptomatic SID and determine risk factors associated with morbidity and mortality from COVID-19 in these patient groups., Results: A total of 100 patients had been enrolled by July 1, 2020, 60 with PID, 7 with other inborn errors of immunity including autoinflammatory diseases and C1 inhibitor deficiency, and 33 with symptomatic SID. In individuals with PID, 53.3% (32 of 60) were hospitalized, the infection-fatality ratio was 20.0% (12 of 60), the case-fatality ratio was 31.6% (12 of 38), and the inpatient mortality was 37.5% (12 of 32). Individuals with SID had worse outcomes than those with PID; 75.8% (25 of 33) were hospitalized, the infection-fatality ratio was 33.3% (11 of 33), the case-fatality ratio was 39.2% (11 of 28), and inpatient mortality was 44.0% (11 of 25)., Conclusions: In comparison to the general population, adult patients with PID and symptomatic SID display greater morbidity and mortality from COVID-19. This increased risk must be reflected in public health guidelines to adequately protect vulnerable patients from exposure to the virus., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: The COVID-19 pandemic has led to an urgent requirement for novel diagnostic tests that determine infection with SARS-CoV-2 and the development of an immune response against it. The perspective of end users on the characteristics and clinical use of these assays has not been previously considered. Methods: We surveyed 17,186 health care professions (HCPs) in 29 countries to gauge opinion on the design, use, diagnostic impact and diagnostic accuracy of COVID-19 tests. Results were correlated with national statistics on the burden of disease and testing in individual countries. Results: HCPs overwhelmingly recognized the importance of COVID-19 tests but 37.1% were unsure of the appropriate timing of investigations relative to disease symptoms. Confidence in the diagnostic accuracy of assays varied inversely with COVID-19-related mortality in individual countries but had no relationship with the total number of tests performed. There was global consensus that the most important impact of positive antigen and antibody testing was confidence in returning to work following recovery. Saliva was the preferred sampling fluid for COVID-19 diagnostic tests in all groups surveyed. Conclusions: HCP input can ensure novel assays are fit for purpose in varied global health care settings, but HCPs may require support to effectively use novel diagnostics thus minimizing waste when supplies are limited., Competing Interests: MD reports personal fees from Abingdon Health, outside the submitted work. HB, NP, and AAR are employees of M3 Global Research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shields, Brown, Phillips, Drayson, Richter and Richter.)

Background: Understanding the T cell response to SARS-CoV-2 is critical to vaccine development, epidemiological surveillance and disease control strategies. This systematic review critically evaluates and synthesises the relevant peer-reviewed and pre-print literature published from 01/01/2020-26/06/2020., Methods: For this systematic review, keyword-structured literature searches were carried out in MEDLINE, Embase and COVID-19 Primer. Papers were independently screened by two researchers, with arbitration of disagreements by a third researcher. Data were independently extracted into a pre-designed Excel template and studies critically appraised using a modified version of the MetaQAT tool, with resolution of disagreements by consensus. Findings were narratively synthesised., Results: 61 articles were included. 55 (90%) studies used observational designs, 50 (82%) involved hospitalised patients with higher acuity illness, and the majority had important limitations. Symptomatic adult COVID-19 cases consistently show peripheral T cell lymphopenia, which positively correlates with increased disease severity, duration of RNA positivity, and non-survival; while asymptomatic and paediatric cases display preserved counts. People with severe or critical disease generally develop more robust, virus-specific T cell responses. T cell memory and effector function has been demonstrated against multiple viral epitopes, and, cross-reactive T cell responses have been demonstrated in unexposed and uninfected adults, but the significance for protection and susceptibility, respectively, remains unclear., Conclusion: A complex pattern of T cell response to SARS-CoV-2 infection has been demonstrated, but inferences regarding population level immunity are hampered by significant methodological limitations and heterogeneity between studies, as well as a striking lack of research in asymptomatic or pauci-symptomatic individuals. In contrast to antibody responses, population-level surveillance of the T cell response is unlikely to be feasible in the near term. Focused evaluation in specific sub-groups, including vaccine recipients, should be prioritised., Competing Interests: All authors have read the journal's policy and declare: no support from any organisation for the submitted work; JM is chief scientific officer, shareholder and scientific founder of Leucid Bio, a spinout company focused on development of cellular therapeutic agents; no other relationships or activities that could appear to have influenced the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Dried blood spot (DBS) samples can be used for the detection of severe acute respiratory syndrome coronavirus 2 spike antibodies. DBS sampling is comparable to matched serum samples with a relative 98.1% sensitivity and 100% specificity. Thus, DBS sampling offers an alternative for population-wide serologic testing in the coronavirus pandemic.

Objective: To determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers., Design: A cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020., Setting: University Hospitals Birmingham NHS Foundation Trust (UHBFT), UK., Participants: 545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded., Intervention: Participants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked., Main Outcome Measure: Proportion of participants demonstrating infection and positive SARS-CoV-2 serology., Results: The point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ 2 =21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%), with lower rates observed in participants working in intensive care (14.8%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02)., Conclusions and Relevance: We identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices., Competing Interests: Competing interests: MTD reports personal fees from Abingdon Health, outside the submitted work. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: To determine whether optimal use of serial measurements of serum levels of soluble cell adhesion molecules (CAM) can improve monitoring of disease activity in SLE., Methods: Serum levels of soluble CAM and conventional SLE biomarkers were measured in serial samples (n = 80) from 21 SLE patients during and after flare and correlated in longitudinal analysis with disease activity determined by ECLAM score. Blood samples from a second cohort of 34 SLE patients were subject to flow cytometry to correlate serum biomarkers with B cell subsets., Results: By adjusting for the baseline level (at the first visit), delta soluble vascular cell adhesion molecule-1 (sVCAM-1) showed stronger correlation with changes in ECLAM score and improved sensitivity and specificity for identifying SLE responders versus non-responders compared to conventional SLE biomarkers including anti-dsDNA antibody titre and complement C3. Multiple regression analysis identified delta sVCAM-1 as the best marker of SLE clinical response. sVCAM-1 levels were significantly correlated with CD95(+)CD27(+) activated memory B cells, CD95(+) plasmablasts and circulating plasma cell numbers in SLE patients., Conclusion: Subtracting a baseline level of sVCAM-1 for each individual substantially improved its utility as a biomarker. Delta sVCAM-1 was superior to conventional SLE biomarkers for monitoring changes in disease activity. This suggests that serial monitoring of serum sVCAM-1 trends should be considered in SLE patients to document responses to treatment. We hypothesise that the correlation between activated B cell subsets and circulating plasma cell numbers with soluble VCAM-1 serum levels in SLE may relate to the important role of VCAM-1 in B lymphocyte survival and maturation in bone marrow and secondary lymphoid tissues.

Background: A single risk haplotype across UBE2L3 is strongly associated with systemic lupus erythematosus (SLE) and many other autoimmune diseases. UBE2L3 is an E2 ubiquitin-conjugating enzyme with specificity for RING-in-between-RING E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC), which has a pivotal role in inflammation, through crucial regulation of NF-κB. We aimed to determine whether UBE2L3 regulates LUBAC-mediated activation of NF-κB, and determine the effect of UBE2L3 genotype on NF-κB activation and B-cell differentiation., Methods: UBE2L3 genotype data from SLE genome-wide association studies was imputed by use of 1000 Genomes data. UBE2L3 function was studied in a HEK293-NF-κB reporter cell line with standard molecular biology techniques. p65 NF-κB translocation in ex-vivo B cells and monocytes from genotyped healthy individuals was quantified by imaging flow cytometry. B-cell subsets from healthy individuals and patients with SLE, stratified by UBE2L3 genotype, were determined by multicolour flow cytometry., Findings: rs140490, located at -270 base pairs of the UBE2L3 promoter, was identified as the most strongly associated single nucleotide polymorphism (p=8·6 × 10(-14), odds ratio 1·30, 95% CI 1·21-1·39). The rs140490 risk allele increased UBE2L3 expression in B cells and monocytes. Marked upregulation of NF-κB was observed with combined overexpression of UBE2L3 and LUBAC, but abolished by dominant-negative mutant UBE2L3 (C86S), or UBE2L3 silencing. The rs140490 genotype correlated with basal NF-κB activation in ex-vivo human B cells and monocytes, as well as NF-κB sensitivity to CD40 or tumour necrosis factor (TNF) stimulation. UBE2L3 expression was 3-4 times higher in circulating plasmablasts and plasma cells than in other B-cell subsets, with higher levels in patients with SLE than in controls. The rs140490 genotype correlated with increasing plasmablast and plasma cell differentiation in patients with SLE., Interpretation: This study shows that NF-κB activation mediated by LUBAC is exquisitely sensitive to the expression level of UBE2L3. The UBE2L3 risk haplotype is correlated with TNF and CD40 induced NF-κB activation in primary human cells, and with plasmablast and plasma cell expansion in SLE, consistent with the dependence of these cells on NF-κB as a survival factor. Since UBE2L3 is highly expressed in plasma cells, UBE2L3 could be a novel therapeutic target in SLE., Funding: Arthritis Research UK, Wellcome Trust, George Koukis Foundation, European Community's Seventh Framework Programme., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

UBE2L3 is associated with increased susceptibility to numerous autoimmune diseases, but the underlying mechanism is unexplained. By using data from a genome-wide association study of systemic lupus erythematosus (SLE), we observed a single risk haplotype spanning UBE2L3, consistently aligned across multiple autoimmune diseases, associated with increased UBE2L3 expression in B cells and monocytes. rs140490 in the UBE2L3 promoter region showed the strongest association. UBE2L3 is an E2 ubiquitin-conjugating enzyme, specially adapted to function with HECT and RING-in-between-RING (RBR) E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC). Our data demonstrate that UBE2L3 is the preferred E2 conjugating enzyme for LUBAC in vivo, and UBE2L3 is essential for LUBAC-mediated activation of NF-κB. By accurately quantifying NF-κB translocation in primary human cells from healthy individuals stratified by rs140490 genotype, we observed that the autoimmune disease risk UBE2L3 genotype was correlated with basal NF-κB activation in unstimulated B cells and monocytes and regulated the sensitivity of NF-κB to CD40 stimulation in B cells and TNF stimulation in monocytes. The UBE2L3 risk allele correlated with increased circulating plasmablast and plasma cell numbers in SLE individuals, consistent with substantially elevated UBE2L3 protein levels in plasmablasts and plasma cells. These results identify key immunological consequences of the UBE2L3 autoimmune risk haplotype and highlight an important role for UBE2L3 in plasmablast and plasma cell development., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)