Daniel Martin Altmann

Plain English Summary The WILCO cohort ('Working out the Immunology of Long Covid'), both adult and paediatric, is novel in terms of being a large, UK cohort of individuals who were largely non-hospitalised during their acute infections and have here been followed longitudinally and in detail, at the level of symptom profile and immunological parameters. Data from this cohort establish a number of key points. This is a large cohort, many of whom have been persistently unwell with multi-system, multi-organ disease for 3-4 years. Longitudinal analysis shows that most symptoms show little improvement across an interval of more than a year. WILCO participants are indicative of Long Covid (LC) trends nationally and globally insofar as many have lost jobs or changed to part-time work as a consequence of their disability. We were able to identify a number of immune correlates of LC, likely contributing to immunopathogenesis: altered immunoglobulin isotype profiles, altered T cell immunity to spike, altered patterns of autoimmunity and altered B cell plasmablast and natural killer cell subsets. Our findings aid in defining the mechanisms underpinning Long Covid, offer potential directions for therapeutic trials and also for diagnostic tests. Executive Summary Background: During the past 4-years, much has been learnt around the world about Long Covid – the chronic, post-viral sequelae persisting more than 12 weeks and, generally, some years, after COVID-19. It is estimated that there are some 400-million sufferers globally and by Office for National Statistics (ONS) Data, 2-million in the UK. These individuals are unable to regain their baseline health, often suffering disability that makes it impossible to return fully to their former employment. This large, new disease burden has profound implications for the workforce, the NHS, and for healthcare planning and provision. This proposal made the point that this is hypothesised to be an immune/inflammatory-mediated disease process, and that we need immune analysis in order to understand the basis of the condition, design diagnostic tests, and build an understanding of the likely approaches for therapeutic drug trials. The work has gone well. The cohort was termed ‘WILCO’ for ‘Working Out the Immunology of Long Covid’ and we were able to recruit nearly 1000 individuals into the cohort. This is a novel and informative Long Covid study cohort since it comprises people with well-characterised Long Covid symptoms, in many cases affected now for 3-4 years, and with their condition deriving from infections across each of the COVID-19 variant waves: ancestral Wuhan Hu-1 (the 2020-wave), Alpha, Delta, and Omicron. Because the analysis is longitudinal, we have been able to understand the persistence of symptoms, their long-term effects and immune correlates. We have also been able to relate symptoms and immune parameters in the adult cohort to our parallel, paediatric Long Covid cohort. Study aims and methods: Our goal was to work towards development of clear, diagnostic tests through high-granularity immunological analysis for biomarker discovery. Analysis would use peripheral blood samples, the study groups comprising: i) Healthy controls, ii) Individuals who had confirmed COVID-19 with rapid recovery and iii) Individuals who meet the criteria for Long Covid - greater than 12 weeks from the start of acute COVID-19 infection without recovery back to baseline health. iv) We would relate findings in this adult, largely non-hospitalised cohort to an additional cohort - a paediatric cohort recruited through referral to the Paediatric Long Covid clinical service at KCL and the Evelina Hospital. The specific questions to be addressed in the lab using cohort blood samples were as follows: • Analysis of white blood cell immune subsets using an approach termed multi-parameter flow cytometry. That is, measuring the specific profile of lymphocyte subsets contributing to immunity using fluorescently labelled antibodies. We would thus compare immune subsets in people with Long Covid relative to people who made a full recovery from acute infection to investigate perturbations in immune subsets. • In related lab analyses, we would make RNA from blood to analyse the differential programmes driving immune subsets in Long Covid and fully recovered controls • We would correlate disease symptoms with specific patterns of antibody and/or T cell autoimmunity, addressing the hypothesis that the origins of Long Covid may be partly through the ability of the virus to drive an autoimmune condition whereby the immune system erroneously attacks cells in the body. Results and key findings: Since the start of the award we have achieved each of our milestones. A cohort of nearly 1000 was consented and recruited at Imperial College London, detailed symptom and clinical history questionnaires were completed at two longitudinal timepoints, analysed and clustered by machine learning, participants gave blood samples which were used in detailed immunological investigations as described above, and these immune parameters have illuminated understanding of the processes underlying Long Covid, offering potential tests and clues to therapeutic approaches. The initial deliverable was to establish the infrastructure to recruit, build and characterise a UK-based, largely non-hospitalised, Long Covid research cohort and recovered controls, representative of the experience of the majority of Long Covid sufferers. Participants were recruited in response to adverts posted through community newsletters, social media, Long Covid support networks, general and Long Covid clinics. All participants completed a detailed, questionnaire-based symptom history using a validated, published, protocol. In most cases this was done at 2 timepoints separated by 12-18 months. This follow-up comprised a second, expanded questionnaire and additional blood sample...

Background Viral and infectious illnesses can exert profound and enduring effects on population health and well-being. In the aftermath of SARS-CoV-2 infection, post-acute sequelae, collectively referred to as Long COVID, have emerged as a major global health challenge, affecting more than 400 million people and contributing to estimated annual economic costs exceeding $1 trillion. Scope of the review Long COVID encompasses a wide and heterogeneous spectrum of debilitating symptoms, including cognitive dysfunction, sleep disturbances, severe fatigue, and post-exertional malaise. Despite its substantial burden, fundamental uncertainties remain regarding its underlying pathophysiology, the development of robust diagnostic criteria, and the identification of effective therapeutic options. Key insights This review synthesises current evidence on the biological mechanisms thought to contribute to Long COVID, spanning immune dysregulation, viral persistence, autonomic dysfunction, microvascular pathology, and other emerging hypotheses. We examine advances and limitations in contemporary diagnostic approaches and critically appraise existing treatment strategies, highlighting inconsistencies and gaps that hinder clinical consensus. Implications By integrating interdisciplinary insights, this review underscores the urgent need for mechanistic clarity, validated diagnostic frameworks, and rigorously evaluated treatment pathways. Addressing these gaps will be essential to developing effective, evidence-based management strategies and mitigating the long-term impact of Long COVID on global health.

Objective: Methotrexate is first-line treatment for many immune-mediated inflammatory diseases. However, it inhibits vaccine-induced immunity – a major concern for this vulnerable group of patients. We evaluated if a 2-week interruption of methotrexate treatment immediately after COVID-19 booster improved antibody response against spike protein of the receptor binding domain and live virus neutralisation (ancestral Wuhan and Omicron BA.1) in patients with immune-mediated inflammatory diseases. Design: Open-label, prospective, individually randomised, parallel-group, controlled superiority trial with 1 : 1 randomisation. Setting: Multicentre, secondary-care rheumatology and dermatology outpatient clinics. Participants: Adults with immune-mediated inflammatory diseases attending rheumatology and dermatology clinics taking methotrexate (≤ 25 mg/week) for ≥ 3 months. Intervention: Suspending methotrexate treatment for 2 weeks immediately after COVID-19 booster vaccination. Main outcome(s) and measure(s): The primary outcome was spike protein of the receptor binding domain antibody level 4 weeks after COVID-19 booster vaccination. Secondary outcomes were spike protein of the receptor binding domain antibody levels 12 and 26 weeks after COVID-19 vaccine dose; live virus neutralisation (ancestral Wuhan Hu-1, Omicron BA.1) at weeks 4, 12 and 26; and self-reported inflammatory disease activity, flare-ups, quality of life, global assessment of inflammatory disease and adherence with trial allocation. Results: A total of 383 participants (61% female, average age 59.0 years) were randomised to either suspend or continue methotrexate. The geometric mean (95% confidence interval) spike protein of the receptor binding domain antibody titre was 25,413 (22,227 to 29,056) and 12,326 (10,538 to 14,418) U/ml in those who suspended and continued methotrexate, respectively. The geometric mean ratio (95% confidence interval) was 2.08 (1.59 to 2.70), p < 0.0001. The intervention effect was present across prognostic subgroups, for example, age groups, methotrexate dose, methotrexate administration route, diseases and past severe acute respiratory syndrome coronavirus 2 infection. Enhanced antibody responses were sustained at 12 and 26 weeks with geometric mean ratio (95% confidence interval) 1.88 (1.44 to 2.46) and 1.50 (1.12 to 2.01), respectively. Interruption of treatment improved neutralisation of Wuhan and Omicron BA.1 at 4 weeks with geometric mean ratio (95% confidence interval) 2.56 (1.21 to 5.44) and 2.42 (1.45 to 4.05), respectively. Self-reported inflammatory disease activity initially deteriorated in the suspended methotrexate group, but the groups were comparable at week 12. Conclusion: Two-week interruption of methotrexate treatment for immune-mediated inflammatory diseases enhanced antibody responses after COVID-19 vaccination that were sustained at 12 and 26 weeks. Limitations: Lack of participant masking which could have affected self-reported outcomes. Condition-specific disease activity was not used as we recruited participants with a range of diseases, with many lacking validated outcome measures. We did not have data for memory B-cell and T-cell responses. Some hospitals declined to participate in the 26-week follow-up visit which was added to the study after interim analysis, due to lack of capacity, contributing to increased attrition at week 26. Future work: Future research should evaluate whether interrupting other immune-suppressing treatments soon after vaccination against COVID-19 or other infectious diseases can improve immune responses. Further research should also evaluate whether a shorter hold in methotrexate would improve the immune response elicited by vaccination. Funding: This synopsis presents independent research funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation programme as award number NIHR134607.

This article consists of a citation of a published article describing research funded by the Efficacy and Mechanism Evaluation programme under project number NIHR134607, and is provided as as part of the complete record of research outputs for this project. The original publication is available at: https://doi.org/10.1016/S2665-9913(23)00298-9 Summary: Background: Methotrexate is the first-line treatment for immune-mediated inflammatory diseases and reduces vaccine-induced immunity. We evaluated if a 2-week interruption of methotrexate treatment immediately after COVID-19 booster vaccination improved antibody response against the S1 receptor binding domain (S1-RBD) of the SARS-CoV-2 spike protein and live SARS-CoV-2 neutralisation compared with uninterrupted treatment in patients with immune-mediated inflammatory diseases. Method: We did a multicentre, open-label, parallel-group, randomised, superiority trial in secondary-care rheumatology and dermatology clinics in 26 hospitals in the UK. Adults (aged ≥18 years) with immune-mediated inflammatory diseases taking methotrexate (≤25 mg per week) for at least 3 months, who had received two primary vaccine doses from the UK COVID-19 vaccination programme were eligible. Participants were randomly assigned (1:1) using a centralised validated computer program, to temporarily suspend methotrexate treatment for 2 weeks immediately after COVID-19 booster vaccination or continue treatment as usual. The primary outcome was S1-RBD antibody titres 4 weeks after COVID-19 booster vaccination and was assessed masked to group assignment. All randomly assigned patients were included in primary and safety analyses. This trial is registered with ISRCTN, ISRCTN11442263; following a pre-planned interim analysis, recruitment was stopped early. Finding: Between Sept 30, 2021, and March 7, 2022, we screened 685 individuals, of whom 383 were randomly assigned: to either suspend methotrexate (n=191; mean age 58·8 years [SD 12·5], 118 [62%] women and 73 [38%] men) or to continue methotrexate (n=192; mean age 59·3 years [11·9], 117 [61%] women and 75 [39%] men). At 4 weeks, the geometric mean S1-RBD antibody titre was 25 413 U/mL (95% CI 22 227-29 056) in the suspend methotrexate group and 12 326 U/mL (10 538-14 418) in the continue methotrexate group with a geometric mean ratio (GMR) of 2·08 (95% CI 1·59-2·70; p<0·0001). No intervention-related serious adverse events occurred. Interpretation: 2-week interruption of methotrexate treatment in people with immune-mediated inflammatory diseases enhanced antibody responses after COVID-19 booster vaccination that were sustained at 12 weeks and 26 weeks. There was a temporary increase in inflammatory disease flares, mostly self-managed. The choice to suspend methotrexate should be individualised based on disease status and vulnerability to severe outcomes from COVID-19. Funding: National Institute for Health and Care Research. Funding: This publication was funded by the Efficacy and Mechanism Evaluation programme as a part of award number NIHR134607. This article reports on one component of the research award Vaccine Response On/Off Methotrexate (VROOM): does temporarily suspending methotrexate treatment for two weeks enhance COVID-19 vaccine response? A randomised controlled trial. For more information about this research please view the award page [https://fundingawards.nihr.ac.uk/award/NIHR134607] DOI: https://doi.org/10.1016/S2665-9913(23)00298-9

This article consists of a citation of a published article describing research funded by the Efficacy and Mechanism Evaluation programme under project number NIHR134607, and is provided as as part of the complete record of research outputs for this project. The original publication is available at: https://doi.org/10.1136/bmjopen-2022-062599 Introduction: It is unknown if a temporary break in long-term immune-suppressive treatment after vaccination against COVID-19 improves vaccine response. The objective of this study was to evaluate if a 2-week interruption in low-dose weekly methotrexate treatment after SARS-CoV-2 vaccine boosters enhances the immune response compared with continuing treatment in adults with autoimmune inflammatory conditions. Methods and analysis: An open-label, pragmatic, prospective, parallel group, randomised controlled superiority trial with internal feasibility assessment and nested mechanistic substudy will be conducted in rheumatology and dermatology clinics in approximately 25 UK hospitals. The sample size is 560, randomised 1:1 to intervention and usual care arms. The main outcome measure is anti-spike receptor-binding domain (RBD) antibody level, collected at prebooster (baseline), 4 weeks (primary outcome) and 12 weeks (secondary outcome) post booster vaccination. Other secondary outcome measures are patient global assessments of disease activity, disease flares and their treatment, EuroQol 5- dimention 5-level (EQ-5D-5L), self-reported adherence with advice to interrupt or continue methotrexate, neutralising antibody titre against SARS-CoV-2 (mechanistic substudy) and oral methotrexate biochemical adherence (mechanistic substudy). Analysis of B-cell memory and T-cell responses at baseline and weeks 4 and 12 will be investigated subject to obtaining additional funding. The principal analysis will be performed on the groups as randomised (ie, intention to treat). The difference between the study arms in anti-spike RBD antibody level will be estimated using mixed effects model, allowing for repeated measures clustered within participants. The models will be adjusted for randomisation factors and prior SARS-CoV-2 infection status. Ethics and dissemination: This study was approved by the Leeds West Research Ethics Committee and Health Research Authority (REC reference: 21/HRA/3483, IRAS 303827). Participants will be required to give written informed consent before taking part in the trial. Dissemination will be via peer review publications, newsletters and conferences. Results will be communicated to policymakers. Trial registration number: ISRCTN11442263. Funding: This publication was funded by the Efficacy and Mechanism Evaluation programme as a part of award number NIHR134607. This article reports on one component of the research award Vaccine Response On/Off Methotrexate (VROOM): does temporarily suspending methotrexate treatment for two weeks enhance COVID-19 vaccine response? A randomised controlled trial. For more information about this research please view the award page [https://fundingawards.nihr.ac.uk/award/NIHR134607] DOI: https://doi.org/10.1136/bmjopen-2022-062599

This article consists of a citation of a published article describing research funded by the Efficacy and Mechanism Evaluation programme under project number NIHR134607, and is provided as as part of the complete record of research outputs for this project. The original publication is available at: https://doi.org/10.1016/S2213-2600(22)00186-2 Background: Immunosuppressive treatments inhibit vaccine-induced immunity against SARS-CoV-2. We evaluated whether a 2-week interruption of methotrexate treatment immediately after the COVID-19 vaccine booster improved antibody responses against the S1 receptor-binding domain (S1-RBD) of the SARS-CoV-2 spike protein compared with uninterrupted treatment in patients with immune-mediated inflammatory diseases. Methods: We did an open-label, prospective, two-arm, parallel-group, multicentre, randomised, controlled, superiority trial in 26 hospitals in the UK. We recruited adults from rheumatology and dermatology clinics who had been diagnosed with an immune-mediated inflammatory disease (eg, rheumatoid arthritis, psoriasis with or without arthritis, axial spondyloarthritis, atopic dermatitis, polymyalgia rheumatica, and systemic lupus erythematosus) and who were taking low-dose weekly methotrexate (≤25 mg per week) for at least 3 months. Participants also had to have received two primary vaccine doses from the UK COVID-19 vaccination programme. We randomly assigned the participants (1:1), using a centralised validated computer randomisation program, to suspend methotrexate treatment for 2 weeks immediately after their COVID-19 booster (suspend methotrexate group) or to continue treatment as usual (continue methotrexate group). Participants, investigators, clinical research staff, and data analysts were unmasked, while researchers doing the laboratory analyses were masked to group assignment. The primary outcome was S1-RBD antibody titres 4 weeks after receiving the COVID-19 booster vaccine dose, assessed in the intention-to-treat population. This trial is registered with ISRCT, ISRCTN11442263; following the pre-planned interim analysis, recruitment was stopped early. Findings: Between Sept 30, 2021 and March 3, 2022, we recruited 340 participants, of whom 254 were included in the interim analysis and had been randomly assigned to one of the two groups: 127 in the continue methotrexate group and 127 in the suspend methotrexate group. Their mean age was 59·1 years, 155 (61%) were female, 130 (51%) had rheumatoid arthritis, and 86 (34%) had psoriasis with or without arthritis. After 4 weeks, the geometric mean S1-RBD antibody titre was 22 750 U/mL (95% CI 19 314-26 796) in the suspend methotrexate group and 10 798 U/mL (8970-12 997) in the continue methotrexate group, with a geometric mean ratio (GMR) of 2·19 (95% CI 1·57-3·04; p<0·0001; mixed-effects model). The increased antibody response in the suspend methotrexate group was consistent across methotrexate dose, administration route, type of immune-mediated inflammatory disease, age, primary vaccination platform, and history of SARS-CoV-2 infection. There were no intervention-related serious adverse events. Interpretation: A 2-week interruption of methotrexate treatment for people with immune-mediated inflammatory diseases resulted in enhanced boosting of antibody responses after COVID-19 vaccination. This intervention is simple, low-cost, and easy to implement, and could potentially translate to increased vaccine efficacy and duration of protection for susceptible groups. Funding: National Institute for Health and Care Research. Funding: This publication was funded by the Efficacy and Mechanism Evaluation programme as a part of award number NIHR134607. This article reports on one component of the research award Vaccine Response On/Off Methotrexate (VROOM): does temporarily suspending methotrexate treatment for two weeks enhance COVID-19 vaccine response? A randomised controlled trial. For more information about this research please view the award page [https://fundingawards.nihr.ac.uk/award/NIHR134607] DOI: https://doi.org/10.1016/S2213-2600(22)00186-2

Epidemiological, histological, and immunogenetic studies suggest that podoconiosis (a non-infectious tropical lymphoedema affecting approximately 4 million people globally) is an HLA class II-associated inflammatory condition that develops in response to an unknown trigger found in volcanic red clay soils. Silicate particles of the kaolinite and aluminum types have been identified in femoral lymph node biopsy samples from endemic area residents, suggesting a possible role in the pathogenesis of podoconiosis. We measured in vitro peripheral blood mononuclear cell cytokine responses (TNF-α, IL-1β, and IFN-γ) to stimulation with the minerals kaolinite, chlorite, and beryllium sulfate (all at 100 µM) using ELISA. Real time PCR was used to measure gene expression of signature cytokines in fresh whole blood, comparing podoconiosis patients and endemic healthy controls. Our results showed that the levels of TNF-α and IL-1β from in vitro cell cultures were significantly higher in unstimulated samples from patients compared to controls (p = 0.04 and p = 0.005, respectively). The minerals kaolinite and chlorite induced two and three-fold higher levels of IL-1β following 24 h of stimulation in healthy controls compared to patients, respectively. We did not find significant differences in mRNA expression of the cytokine genes assayed, though a slight fold increment in IL-1β and TGF-β was observed. In conclusion, our data suggest that the immune system is in a state of persistent activation in vivo in podoconiosis patients, and additional studies of immune regulation and exhaustion are needed to further characterize immune dysfunction in the pathogenesis of the disease. A better understanding of the underlying processes could lead to the development of a ‘biosignature’ detectable in the early reversible stages that could ultimately contribute to the elimination of this preventable, disabling, neglected tropical disease.