Can self-testing be enhanced to hasten safe return of healthcare workers in pandemics? Random order, open label trial using two manufacturers' SARS-CoV-2 lateral flow devices concurrently (preprint)

ObjectivesTo inform management of competing risks from Covid-19 and key-worker absence, we evaluated whether using two manufacturers lateral flow tests (LFTs) concurrently improved SARS-CoV-2 Omicron detection and was acceptable to hospital staff. In a nested study, to understand the risks of return to work after a fixed number of days of isolation or quarantine, we examined virus culture at Days 5-7 after positive test or significant exposure. Methods and Analysis1419 fully-vaccinated Liverpool (UK) University Hospitals staff participated in a random-order, open-label trial testing whether dual LFTs improved SARS-CoV2 detection, and whether dual swabbing was acceptable to users. Main outcome was self-reported LFT result. Staff enrolled via routine testing sites for symptomatic staff and close contacts. Recruitment took place between 7th February and 8th May 2022. Participants employed nose-throat swab Innova and nose-only swab Orient Gene LFTs for 10 days, with daily LFTs taken in random order. A swab for polymerase chain reaction (PCR) analysis was taken at Day-5 and, if positive, Day-10. A questionnaire on acceptability was administered on exit. Selected participants gave swabs for viral culture on Days 5-7; swabs were delivered and returned by courier. Cultures were considered positive if cytopathic effect was apparent or the SARs-COV2 N gene sub-genomic RNA was detected by sequencing. Results226 individuals reported 1466 pairs of LFT results. Tests disagreed in 127 cases (8.7%). Orient Gene was more likely (78 cf. 49, P=0.03) to be positive. Orient Gene positive Innova negative result-pairs became more frequent over time (P<0.001). If Innova was swabbed second, it was less likely to agree with a positive Orient Gene result (P=0.005); swabbing first with Innova made no significant difference (P=0.85). Of 311 individuals completing the exit questionnaire, 90.7% reported dual swabbing was easy, 57.1% said it was no barrier to their daily routine and 65.6% preferred dual testing. Respondents had more confidence in dual c.f. single test results (median 9 cf. 8 on 10-point scale, P<0.001). Viral cultures from swabs taken at Days 5-7 were positive for 6/31 (19.4%, 7.5%-37.5%) and indeterminate for 11/31 (35.5%, 19.2%-54.6%) LFT-positive participants, indicating they were likely still infectious. ConclusionsDual brand testing increased LFT detection of SARS-CoV-2 antigen by a small but meaningful margin and was acceptable to hospital workers. Viral cultures demonstrated that policies recommending safe return to work [~]5 days after Omicron infection/exposure were flawed. Key-workers should be prepared for dynamic self-testing protocols in future pandemics. Trial registrationhttps://www.isrctn.com/ISRCTN47058442 (IRAS Project ID:311842) Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIOmicron BA.1 and BA.2 waves caused large-scale healthcare worker absence in late 2021 - early 2022, risking patient safety from both Covid-19 and reduced care capacity C_LIO_LILateral flow tests (LFTs) reliably detected SARS-CoV-2 antigen, more so with Omicron than prior variants, identifying the most infectious individuals C_LIO_LISelf-testing with LFT SARS-CoV-2 rapid antigen tests reduced Covid-19 transmission, mitigating risks of return to work, including healthcare settings C_LI What this study addsO_LIDual c.f. single brand LFT testing increased SARS-CoV-2 antigen detection marginally, but more than can be explained by extending swabbing from nose-only to nose-throat C_LIO_LINHS deployment of nose-only LFTs in response to compound pressures from Omicron, winter and pandemic burnout was safe and acceptable to most participating hospital staff C_LIO_LICulturable virus was detected confidently in a fifth (and potentially in a further third) of LFT-positive hospital workers 5-7 days after their self-referral for testing, indicating substantial protracted infectiousness C_LI How this study might affect research, practice or policyO_LIThis study shows international Covid-19 policies for return to work after fixed periods (e.g. 5 days after positive test) were flawed: too little emphasis was placed on variation in infectivity between individuals C_LIO_LIFuture pandemic preparedness needs to plan testing quality assurance unified across healthcare and community self-testing contexts, including continuous study of serial daily antigen, nucleic acid and culturable virus test results C_LI

Age- and sex-specific differences in immune responses to BNT162b2 COVID-19 and live-attenuated influenza vaccines in UK adolescents (preprint)

Key to understanding COVID-19 correlates of protection is assessing vaccine-induced immunity in different demographic groups. Sex- and age-specific immune differences have a wide impact on outcomes from infections and immunisations. Typically, adult females make stronger immune responses and have better disease outcomes but suffer more adverse events following vaccination and are more prone to autoimmune disease. To understand better the mechanisms underlying these differences in vaccine responses, we studied immune responses to two doses of BNT162b2 Pfizer COVID-19 vaccine in an adolescent cohort (n=34, ages 12-16), an age group previously shown to make significantly greater immune responses to the same vaccine compared to young adults. At the same time, we were able to evaluate immune responses to the co-administered live attenuated influenza vaccine, which has been shown to induce stronger immune responses in adult females. Blood samples from 34 adolescents taken pre- and post-vaccination with COVID-19 and influenza vaccines were assayed for SARS-CoV-2-specific IgG and neutralising antibodies, and cellular immunity specific for SARS-CoV-2 and endemic betacoronaviruses. IgG targeting influenza lineages contained in the influenza vaccine was also assessed. As previously demonstrated, total IgG responses to SARS-CoV-2 Spike antigens were significantly higher among vaccinated adolescents compared to adults (aged 32-52) who received the BNT162b2 vaccine (comparing infection-naive, 49,696 vs 33,339; p=0.03; comparing SARS-CoV-2 previously-infected, 743,691 vs 269,985; p<0.0001) by MSD v-plex assay. However, unexpectedly, antibody responses to BNT162b2 and the live-attenuated influenza vaccine were not higher among female adolescents compared to males; among infection-naive adolescents, antibody responses to BNT162b2 were higher in males than females (62,270 vs 36,951 p=0.008). No sex difference was identified in vaccinated adults. These unexpected findings may result from the introduction of novel mRNA vaccination platforms, generating patterns of immunity divergent from established trends, and providing new insights into what might be protective following COVID-19 vaccination.

Enhanced immune ageing associated with increasing COVID-19 disease severity (preprint)

Background The striking increase in COVID-19 severity in older adults provides a clear example of immunesenescence, the age-related remodelling of the immune system. To better characterise the association between convalescent immunesenescence and acute disease severity, we determined the immune phenotype of COVID-19 survivors and non-infected controls.Results We performed detailed immune phenotyping of peripheral blood mononuclear cells isolated from 103 COVID-19 survivors 3–5 months post recovery who were classified as having had severe (n = 56; age 53.12 ± 11.30 years), moderate (n = 32; age 52.28 ± 11.43 years) or mild (n = 15; age 49.67 ± 7.30 years) disease and compared with age and sex-matched healthy adults (n = 59; age 50.49 ± 10.68 years). We assessed a broad range of immune cell phenotypes to generate a composite score, IMM-AGE, to determine the degree of immune senescence. We found increased immunesenescence features in severe COVID-19 survivors compared to controls including: a reduced frequency and number of naïve CD4 and CD8 T cells (p < 0.0001); increased frequency of EMRA CD4 (p < 0.003) and CD8 T cells (p < 0.001); a higher frequency (p < 0.0001) and absolute numbers (p < 0.001) of CD28− ve CD57+ ve senescent CD4 and CD8 T cells; higher frequency (p < 0.003) and absolute numbers (p < 0.02) of PD-1 expressing exhausted CD8 T cells; a two-fold increase in Th17 polarisation (p < 0.0001); higher frequency of memory B cells (p < 0.001) and increased frequency (p < 0.0001) and numbers (p < 0.001) of CD57+ ve senescent NK cells. As a result, the IMM-AGE score was significantly higher in severe COVID-19 survivors than in controls (p < 0.001). Few differences were seen for those with moderate disease and none for mild disease. Regression analysis revealed the only pre-existing variable influencing the IMM-AGE score was South Asian ethnicity (\(\beta\) = 0.174, p= 0.043), with a major influence being disease severity (\(\beta\) = 0.188, p = 0.01).Conclusions Our analyses reveal a state of enhanced immune ageing in survivors of severe COVID-19 and suggest this could be related to SARS-Cov-2 infection. Our data support the rationale for trials of anti-immune ageing interventions for improving clinical outcomes in these patients with severe disease.

Obesity Differs from Diabetes Mellitus in Antibody and T Cell Responses Post COVID-19 Recovery (preprint)

Objective: Obesity and type 2 diabetes (DM) are risk factors for severe COVID-19 outcomes, which disproportionately affect South Asian populations. This study aims to investigate the humoral and cellular immune responses to SARS-CoV-2 in adult COVID-19 survivors with obesity and DM in Bangladesh. Methods: In this cross-sectional study, SARS-CoV-2-specific antibody and T cell responses were investigated in 63 healthy and 75 PCR-confirmed COVID-19 recovered individuals in Bangladesh, during the pre-vaccination first wave of the COVID-19 pandemic in 2020. Results: In COVID-19 survivors, SARS-CoV-2 infection induced robust antibody and T cell responses, which correlated with disease severity. After adjusting for age, sex, DM status, disease severity, and time since onset of symptoms, obesity was associated with decreased neutralising antibody titers, and increased SARS-CoV-2 spike-specific IFN-{gamma} response along with increased proliferation and IL-2 production by CD8+ T cells. In contrast, DM was not associated with SARS-CoV-2-specific antibody and T cell responses after adjustment for obesity and other confounders. Conclusions: Obesity is associated with lower neutralising antibody levels and higher T cell responses to SARS-CoV-2 post COVID-19 recovery, while antibody or T cell responses remain unaltered in DM.

Large scale phenotyping of long COVID inflammation reveals mechanistic subtypes of disease (preprint)

One in ten SARS-CoV-2 infections result in prolonged symptoms termed "long COVID", yet disease phenotypes and mechanisms are poorly understood. We studied the blood proteome of 719 adults, grouped by long COVID symptoms. Elevated markers of monocytic inflammation and complement activation were associated with increased likelihood of all symptoms. Elevated IL1R2, MATN2 and COLEC12 associated with cardiorespiratory symptoms, fatigue, and anxiety/depression, while elevated MATN2 and DPP10 associated with gastrointestinal (GI) symptoms, and elevated C1QA was associated with cognitive impairment (the proteome of those with cognitive impairment and GI symptoms being most distinct). Markers of neuroinflammation distinguished cognitive impairment whilst elevated SCG3, indicative of brain-gut axis disturbance, distinguished those with GI symptoms. Women had a higher incidence of long COVID and higher inflammatory markers. Symptoms did not associate with respiratory inflammation or persistent virus in sputum. Thus, persistent inflammation is evident in long COVID, distinct profiles being associated with specific symptoms.

CD4+ and CD8+ T cell and antibody correlates of protection against Delta vaccine breakthrough infection: A nested case-control study within the PITCH study (preprint)

T cell correlates of protection against SARS-CoV-2 infection after vaccination ('vaccine breakthrough') are incompletely defined, especially the specific contributions of CD4+ and CD8+ T cells. We studied 279 volunteers in the Protective Immunity from T Cells in Healthcare Workers (PITCH) UK study, including 32 cases (with SARS-CoV-2 positive testing after two vaccine doses during the Delta-dominant era) and 247 controls (no positive test nor anti-nucleocapsid seroconversion during this period). 28 days after second vaccination, before all breakthroughs occurred, cases had lower ancestral S- and RBD-specific immunoglobulin G titres and S1- and S2-specific T cell interferon gamma (IFN{gamma}) responses compared with controls. In a subset of matched cases and controls, cases had lower CD4+ and CD8+ IFN{gamma} and tumour necrosis factor responses to Delta S peptides with reduced CD8+ responses to Delta versus ancestral peptides compared with controls. Our findings support a protective role for T cells against Delta breakthrough infection.

Omicron BA.1/BA.2 infections in triple-vaccinated individuals enhance a diverse repertoire of mucosal and blood immune responses (preprint)

Pronounced immune escape by the SARS-CoV-2 Omicron variant has resulted in large numbers of individuals with hybrid immunity, generated through a combination of vaccination and infection. Based primarily on circulating neutralizing antibody (NAb) data, concerns have been raised that omicron breakthrough infections in triple-vaccinated individuals result in poor induction of omicron-specific immunity, and that a history of prior SARS-CoV-2 in particular is associated with profound immune dampening. Taking a broader and comprehensive approach, we characterized mucosal and blood immunity to both spike and non-spike antigens following BA.1/BA.2 infections in triple mRNA-vaccinated individuals, with and without a history of previous SARS-CoV-2 infection. We find that the majority of individuals increase BA.1/BA.2/BA.5-specific NAb following infection, but confirm that the magnitude of increase and post-omicron titres are indeed higher in those who were infection-naive. In contrast, significant increases in nasal antibody responses are seen regardless of prior infection history, including neutralizing activity against BA.5 spike. Spike-specific T cells increase only in infection-naive vaccinees; however, post-omicron T cell responses are still significantly higher in previously-infected individuals, who appear to have maximally induced responses with a CD8+ phenotype of high cytotoxic potential after their 3rd mRNA vaccine dose. Antibody and T cell responses to non-spike antigens also increase significantly regardless of prior infection status, with a boost seen in previously-infected individuals to immunity primed by their first infection. These findings suggest that hybrid immunity induced by omicron breakthrough infections is highly dynamic, complex, and compartmentalised, with significant immune enhancement that can help protect against COVID-19 caused by future omicron variants.

Nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination. (preprint)

Background Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. Methods Plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. Findings Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months. Nasal and plasma anti-S IgG remained elevated for at least 12 months with high plasma neutralising titres against all variants. Of 180 with complete data, 160 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal. Samples 12 months after admission showed no association between nasal IgA and plasma IgG responses, indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. Interpretation The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity.

The aerobiology of SARS-CoV-2 in UK hospitals and the impact of aerosol generating procedures (preprint)

BackgroundSARS-CoV-2 nosocomial transmission to patients and healthcare workers (HCWs) has occurred throughout the COVID-19 pandemic. Aerosol generating procedures (AGPs) seemed particularly risky, and policies have restricted their use in all settings. We examined the prevalence of aerosolized SARS-CoV-2 in the rooms of COVID-19 patients requiring AGP or supplemental oxygen compared to those on room air. MethodsSamples were collected prospectively near to adults hospitalised with COVID-19 at two tertiary care hospitals in the UK from November 2020 - October 2021. The Sartorius MD8 AirPort air sampler was used to collect air samples at a minimum distance of 1.5 meters from patients. RT-qPCR was used following overnight incubation of membranes in culture media and extraction. ResultsWe collected 219 samples from patients rooms: individuals on room air (n=67), receiving oxygen (n=65) or AGP (n=67). Of these, 54 (24.6%) samples were positive for SARS-CoV-2 viral RNA. The highest prevalence was identified in the air around patients receiving oxygen (32.3%, n=21, CI95% 22.2 to 44.3%) with AGP and room air recording prevalence of (20.7%, n=18, CI95% 14.1 - 33.7%) and (22.3%, n=15, CI95% 13.5 - 30.4%) respectively. We did not detect a significant difference in the observed frequency of viral RNA between interventions. InterpretationSARS-CoV-2 viral RNA was detected in the air of hospital rooms of COVID-19 patients, and AGPs did not appear to impact the likelihood of viral RNA. Enhanced respiratory protection and appropriate infection prevention and control measures are required to be fully and carefully implemented for all COVID-19 patients to reduce risk of aerosol transmission.

Outcome of COVID-19 in hospitalised immunocompromised patients: an analysis of the WHO ISARIC CCP-UK prospective cohort study. (preprint)

Background Immunocompromised patients may be at higher risk of mortality if hospitalised with COVID-19 compared with immunocompetent patients. However, previous studies have been contradictory. We aimed to determine whether immunocompromised patients were at greater risk of in-hospital death, and how this risk changed over the pandemic. Methods We included patients >=19yrs with symptomatic community-acquired COVID-19 recruited to the ISARIC WHO Clinical Characterisation Protocol UK. We defined immunocompromise as: immunosuppressant medication preadmission, cancer treatment, organ transplant, HIV, or congenital immunodeficiency. We used logistic regression to compare the risk of death in both groups, adjusting for age, sex, deprivation, ethnicity, vaccination and co-morbidities. We used Bayesian logistic regression to explore mortality over time. Findings Between 17/01/2020 and 28/02/2022 we recruited 156,552 eligible patients, of whom 21,954 (14%) were immunocompromised. 29% (n=6,499) of immunocompromised and 21% (n=28,608) of immunocompetent patients died in hospital. The odds of inhospital mortality were elevated for immunocompromised patients (adjOR 1.44, 95% CI 1.39-1.50, p<0.001). As the pandemic progressed, in-hospital mortality reduced more slowly for immunocompromised patients than for immunocompetent patients. This was particularly evident with increasing age: the probability of the reduction in hospital mortality being less for immunocompromised patients aged 50-69yrs was 88% for men and 83% for women, and for those >80yrs was 99% for men, and 98% for women. Conclusions Immunocompromised patients remain at elevated risk of death from COVID-19. Targeted measures such as additional vaccine doses and monoclonal antibodies should be considered for this group.

Evolution of long-term hybrid immunity in healthcare workers after different COVID-19 vaccination regimens: a longitudinal observational cohort study (preprint)

Both infection and vaccination, alone or in combination, generate antibody and T cell responses against SARS-CoV-2. However, the maintenance of such responses - and hence protection from disease - requires careful characterisation. In a large prospective study of UK healthcare workers (PITCH, within the larger SIREN study) we previously observed that prior infection impacted strongly on subsequent cellular and humoral immunity induced after long and short dosing intervals of BNT162b2 (Pfizer/BioNTech) vaccination. Here, we report longer follow up of 684 HCWs in this cohort over 6-9 months following two doses of BNT162b2 or AZ1222 (Oxford/AstraZeneca) vaccination and following a subsequent BNT162b2 booster vaccination. We make three important observations: Firstly, the dynamics of humoral and cellular responses differ; binding and neutralising antibodies declined whereas T and B cell responses were better maintained after the second vaccine dose. Secondly, vaccine boosting restored IgG levels to post second dose levels and broadened neutralising activity against variants of concern including omicron BA.1, alongside further boosting of T cell responses. Thirdly, prior infection maintained its impact driving larger T cell responses compared to never infected people, including after the third dose. In conclusion, the maintenance of T cell responses in time and against variants of concern may account for continued protection against severe disease.

Changing patterns of SARS-CoV-2 infection through Delta and Omicron waves by vaccination status, previous infection and neighbourhood deprivation: A cohort analysis of 2.7M people. (preprint)

Objective: To examine if SARS-CoV-2 infections vary by vaccination status, if an individual had previously tested positive and by neighbourhood socioeconomic deprivation across the Delta and Omicron epidemic waves of SARS-CoV-2. Design: Cohort study using electronic health records Setting: Cheshire and Merseyside, England (3rd June 2021 to 1st March 2022) Participants: 2.7M residents Main Outcome measure: Registered positive test for SARS-CoV-2 Results: Social inequalities in registered positive tests were dynamic during the study. Originally higher SARS-CoV-2 rates in the most socioeconomically deprived neighbourhoods changed to being higher in the least deprived neighbourhoods from the 1st September 2021. While the introduction of Omicron initially reset inequalities, they continued to be dynamic and inconsistent. Individuals who were fully vaccinated (two doses) were associated with fewer registered positive tests (e.g., between 1st September and 27th November 2021: (i) individuals engaged in testing - Hazards Ratio (HR) = 0.48, 95% Confidence Intervals (CIs) = 0.47-0.50; (ii) individuals engaged with healthcare - HR = 0.34, 95% CIs = 0.33-0.34). Individuals with a previous registered positive test were also less likely to have a registered positive test (e.g., between 1st September and 27th November 2021: (i) individuals engaged in testing - HR = 0.16, 95% CIs = 0.15-0.18; (ii) individuals engaged with healthcare - HR = 0.14, 95% CIs = 0.13-0.16). However, Omicron is disrupting these associations due to immune escape resulting in smaller effect sizes for both measures. Conclusions: Changing patterns of SARS-CoV-2 infections during the Delta and Omicron waves reveals a dynamic pandemic that continues to affect diverse communities in sometimes unexpected ways.

Evaluation of Eight Lateral Flow Tests For The Detection Of Anti-SARS-CoV-2 Antibodies In A Vaccinated Population (preprint)

With the distribution of COVID-19 vaccinations across the globe and the limited access in many countries, quick determination of an individuals antibody status could be beneficial in allocating limited vaccine doses in low- and middle-income countries (LMIC). Antibody lateral flow tests (LFTs) have potential to address this need as a quick, point of care test, they also have a use case for identifying sero-negative individuals for novel therapeutics, and for epidemiology. Here we present a proof-of-concept evaluation of eight LFT brands using sera from 95 vaccinated individuals to determine sensitivity for detecting vaccination generated antibodies. All 95 (100%) participants tested positive for anti-spike antibodies by the chemiluminescent microparticle immunoassay (CMIA) reference standard post-dose two of their SARS-CoV-2 vaccine: BNT162b2 (Pfizer/BioNTech, n=60), AZD1222 (AstraZeneca, n=31), mRNA-1273 (Moderna, n=2) and Undeclared Vaccine Brand (n=2). Sensitivity increased from dose one to dose two in six out of eight LFTs with three tests achieving 100% sensitivity at dose two in detecting anti-spike antibodies. These tests are quick, low-cost point-of-care tools that can be used without prior training to establish antibody status and may prove valuable for allocating limited vaccine doses in LMICs to ensure those in at risk groups access the protection they need. Further investigation into their performance in vaccinated peoples is required before more widespread utilisation is considered.

Comparison of two T cell assays to evaluate T cell responses to SARS-CoV-2 following vaccination in naive and convalescent healthcare workers (preprint)

Background: T cell responses to SARS-CoV-2 following infection and vaccination are less characterised than antibody responses, due to a more complex experimental pathway. Methods: We measured T cell responses in 108 healthcare workers (HCWs) in an observational cohort study, using the commercialised Oxford Immunotec T-SPOT Discovery SARS-CoV-2 assay (OI T-SPOT) and the PITCH ELISpot protocol established for academic research settings. Results: Both assays detected T cell responses to SARS-CoV-2 spike, membrane and nucleocapsid proteins. Responses were significantly lower when reported by OI T-SPOT than by PITCH ELISpot. Four weeks after two doses of either Pfizer/BioNTech BNT162b or ChAdOx1 nCoV-19 AZD1222 vaccine, the responder rate was 63% for OI T-SPOT Panels1+2 (peptides representing SARS-CoV-2 spike protein excluding regions present in seasonal coronaviruses), 69% for OI T-SPOT Panel 14 (peptides representing the entire SARS-CoV-2 spike), and 94% for the PITCH ELISpot assay. The two OI T-SPOT panels correlated strongly with each other showing that either readout quantifies spike-specific T cell responses, although the correlation between the OI T-SPOT panels and the PITCH ELISpot was moderate. Conclusion: The standardisation, relative scalability and longer interval between blood acquisition and processing are advantages of the commercial OI T-SPOT assay. However, the OI T-SPOT assay measures T cell responses at a significantly lower magnitude compared to the PITCH ELISpot assay, detecting T cell responses in a lower proportion of vaccinees. This has implications for the reporting of low-level T cell responses that may be observed in patient populations and for the assessment of T cell durability after vaccination.

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype (preprint)

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

A comprehensive systematic review and meta-analysis of the global data involving 61,532 cancer patients with SARS-CoV-2 infection. (preprint)

Abstract Background SARS-CoV-2 have been shown to be associated with more severe disease and death in cancer patient. A systematic review and meta-analysis was conducted to determine the risk by age, tumour type and treatment of infection with SARS-CoV-2 in cancer patients. Methods Systematic review by searching PubMed, Web of Science, and Scopus for articles published in English up to June 14, 2021 of SARS-CoV-2 infection in >10 patients with malignant disease. Outcomes included factors in patients with malignant disease that may predict a poor outcome from COVID-19 compared to patients without malignant disease, including patient demographics, tumour subtype and cancer treatments. A meta-analysis was performed using random effects model. Results 81 studies were included, totalling 61,532 cancer patients. Haematological malignancies comprised 22.1% (9,672 of 43,676) of cases. Relative risk (RR) of mortality when age and sex matched was 1.69 (95% CI, 1.46-1.95; p<0.001; I2=51%). RR of mortality, versus non-cancer patients, was associated with decreasing age (exp(b)0.96; 95% CI, 0.922-0.994; p=0.028) but not male sex (exp(b)1.89; 95% CI, 0.222-6.366; p=0.83). RR of mortality in those with haematological malignancies versus non-cancer control was 1.81 (95% CI, 1.53-2.95; I2=0.0%). Compared to other cancers, increased risk of death was seen for lung (RR 1.68, 95% CI, 1.45-1.94; p<0.001), genitourinary (RR 1.11; 95% CI, 1.00-1.24; p=0.059) and haematological malignancies (RR 1.42; 95% CI, 1.31-1.54; p<0.001). Breast (RR 0.51; 95% CI, 0.36-0.71; p<0.001) and gynaecological cancers (RR 0.76; 95% CI, 0.62-0.93; p=0.009) had lower risk of death. Receipt of chemotherapy had greatest overall pooled mortality risk of 30% (95% CI, 25-36%; I2=86.97%) and endocrine therapy the lowest at 11% (95% CI, 6-16%; I2=70.7%). Conclusions Cancer patients, particularly younger cancer patients, appear at increased risk of mortality from COVID-19 compared to non-cancer patients. Differences in outcomes were seen based on tumour types and treatment.

Antiviral activity of molnupiravir precursor NHC against Variants of Concern (VOCs) and its therapeutic window in a human lung cell model (preprint)

Background: The UK Medicines and Regulatory Healthcare Agency (MHRA) have recently licensed the anti-viral drug, molnupiravir, for use in patients with mild-moderate COVID-19 disease with one or more risk factors for serious illness. Treatment with anti-viral drugs is best initiated early to prevent progression to severe disease, although the therapeutic window for intervention has not yet been fully defined. Objectives: This study aimed to determine the activity of the molnupiravir parent drug (NHC) to different SARS-CoV-2 Variants of Concern (VoCs), and to establish the therapeutic window in human lung cell model. Methods: Dose response assays were performed in parallel to determine the IC50 (the concentration of drug required to inhibit virus titre by 50%) of NHC against different variants. Human ACE-2 A549 cells were treated with NHC at different time points either before, during or after infection with SARS-CoV-2. Results: Here we demonstrate that {beta}-D-N4-hydroxycytidine (NHC), the active metabolite of molnupiravir, has equivalent activity against four variants of SARS-CoV-2 in a human lung cell line ranging 0.04-0.16M IC50. Furthermore, we demonstrate that activity of the drug begins to drop after 48 hours post-infection. Conclusions: One of the main advantages of molnupiravir is that it can be administered orally, and thus given to patients in an out-patient setting. These results support giving the drug early on after diagnosis or even in prophylaxis for individuals with high risk of developing severe disease.

Poor Antibody Responses to SARS-CoV-2 Infection or Vaccination Are Associated With High Re-Infection Rates in Haemodialysis and Renal Transplant Patients (preprint)

Background: Patients with end-stage renal disease (ESRD) are vulnerable to SARS-CoV-2 infection and mount poor antibody responses to standard vaccines. We addressed whether ESRD patients could mount immune responses that protected against re-infection following natural SARS-CoV-2 infection or 2-dose vaccination.Methods: Haemodialysis (HD and renal transplant patients were recruited following SARS-CoV-2 infection (n=46) or before SARS-CoV-2 vaccination (n=94). SARS-CoV-2 IgG responses, surrogate neutralising antibody (NAb) titres to wildtype and VOCs, T cell responses and viral sequencing in the vaccine-naïve convalescent cohort were serially assessed following infection. Surrogate NAb titres were measured pre-vaccination and 33 days after 2nd vaccine. Incidence of breakthrough infection was assessed 180 days following 1st vaccination. Findings: 22% of vaccine-naive HD (n=9/36) and transplant patients (n=1/10) demonstrated PCR-positive re-infection (RI) at median 212 days (IQR 140-239) post 1st infection. Prior to RI episodes, RI patients demonstrated poor IgG Spike and RBD responses which were equivalent to levels in pre-pandemic sera (median RI titres: Spike 187 AU/ml, IQR 143-3432, p=0.96; RBD 145 AU/ml, IQR 85-938, p>0.99), unlike patients who developed a single infection only (SI) when compared to pre-pandemic sera (median SI titres: Spike 22826 AU/ml, IQR 1255-63811, p<0.0001; RBD 9588 AU/ml, IQR 270-21616, p=0.001). IgG Spike and RBD titres increased following RI compared to pre-pandemic sera (median RI titres: Spike 22611 AU/ml, IQR 4488-75509, p=0.0006; RBD 6354 AU/ml, IQR 1671-20962, p=0.01). T cell analysis revealed no differences between RI and SI cohorts. Following 2-dose vaccination, 5% of the HD cohort who received AZD1222 (n=3/61) developed breakthrough infection at 6 months following 1st vaccination, unlike those who received BNT162b2 (n=0/16). AZD1222-vaccinated, infection-naïve (I-N) HD patients (n=32) and immunosuppressed transplant recipients (n=17) made poor NAb responses to wildtype, alpha, beta and gamma when compared to infection-experienced (I-E) HD patients (n=29) (I-N vs I-E HD wildtype p<0.0001, alpha p=0.0007, beta p<0.0001, gamma p=0.002). NAb responses improved with BNT162b2 vaccination (n=16); RI patients mounted larger NAb responses to AZD1222 vaccination than SI patients (wildtype p=0.01, alpha p=0.02, beta p<0.02). Interpretation: ESRD patients are highly susceptible to SARS-CoV-2 re-infection, or breakthrough infection following vaccination, associated with poor protective antibody responses. SARS-CoV-2-specific IgG and surrogate NAb responses increase with repeated exposure (infection experience and/or vaccination) in patients who survive infections. Our findings support the case for specific booster regimens in such immune-incompetent patients. Funding Information: Oxford Transplant Foundation, Oxfordshire Health Services Research Committee, UK Department of Health and Social Care, Huo Family Foundation, NIHR (COV19-RECPLAS), UK Coronavirus Immunology Consortium, NIHR Oxford Biomedical Research Centre, WT109965MA.Declaration of Interests: We declare no competing interestsEthics Approval Statement: Haemodialysis (HD) and transplant cohorts: In this prospective, observational cohort study, HD and transplant patients within Oxford University Hospitals NHS Foundation Trust(OUH) were recruited under Oxford Radcliffe Biobank approved studies, “Biomarkers to stratify risk in Renal Transplant Recipients and Dialysis Patients with Covid-19” (ref: ORB 20/A056), and “Immunological responses to COVID-19 vaccines in transplant and haemodialysis patients” (ref: ORB 21/A014). The Oxford Radcliffe Biobank has a favorable ethics opinion from the South Central Oxford Committee C (REC: 19/SC/0173). Healthcare Worker cohort (HC, PITCH study): PITCH is a sub-study of the SIREN study which was approved by the Berkshire Research Ethics Committee, Health Research 250 Authority (IRAS ID 284460, REC reference 20/SC/0230), with PITCH recognised as a sub-study on 2 December 2020. SIREN is registered with ISRCTN (Trial ID:252 ISRCTN11041050)The study was conducted in compliance with all relevant ethical regulations for work with human participants, and according to the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines. Written informed consent was obtained for all patients enrolled in the study.

Comparison of children and young people admitted with SARS-CoV-2 across the UK in the first and second pandemic waves: prospective multicentre observational cohort study (preprint)

BackgroundChildren and young people (CYP) were less affected than adults in the first wave of SARS-CoV-2 in the UK. We test the hypothesis that clinical characteristics of hospitalized CYP with SARS-CoV-2 in the UK second wave would differ from the first due to the combined impact of the alpha variant, school reopening and relaxation of shielding. MethodsPatients <19 years hospitalised in the UK with clinician-reported SARS-CoV-2 were enrolled in a prospective multicentre observational cohort study between 17th January 2020 and 31st January 2021. Minimum follow up time was two weeks. Clinical characteristics were compared between the first (W1) and second wave (W2) of infections. Findings2044 CYP aged <19 years were reported from 187 hospitals. 427/2044 (20.6%) had asymptomatic/incidental SARS-CoV-2 infection and were excluded from main analysis. 16.0% (248/1548) of symptomatic CYP were admitted to critical care and 0.8% (12/1504) died. 5.6% (91/1617) of symptomatic CYP had Multisystem Inflammatory Syndrome in Children (MIS-C). Patients in W2 were significantly older (median age 6.5 years, IQR 0.3-14.9) than W1 (4.0 (0.4-13.6, p 0.015). Fever was more common in W1, otherwise presenting symptoms and comorbidities were similar across waves. After excluding CYP with MIS-C, patients in W2 had lower PEWS at presentation, lower antibiotic use and less respiratory and cardiovascular support compared to W1. There was no change in the proportion of CYP admitted to critical care between W1 and W2. 58.0% (938/1617) of symptomatic CYP had no reported comorbidity. Patients without co-morbidities were younger (42.4%, 398/938, <1 year old), had lower Paediatric Early Warning Scores (PEWS) at presentation, shorter length of hospital stay and received less respiratory support. MIS-C was responsible for a large proportion of critical care admissions, invasive and non-invasive ventilatory support, inotrope and intravenous corticosteroid use in CYP without comorbidities. InterpretationSevere disease in CYP admitted with symptomatic SARS-CoV-2 in the UK remains rare. One in five CYP in this cohort had asymptomatic/incidental SARS-CoV-2 infection. We found no evidence of increased disease severity in W2 compared with W1. FundingShort form: National Institute for Health Research, UK Medical Research Council, Wellcome Trust, Department for International Development and the Bill and Melinda Gates Foundation. Long form: This work is supported by grants from the National Institute for Health Research (award CO-CIN-01) and the Medical Research Council (grant MC_PC_19059) and by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford (NIHR award 200907), Wellcome Trust and Department for International Development (215091/Z/18/Z), and the Bill and Melinda Gates Foundation (OPP1209135). Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research (grant reference: C18616/A25153). JSN-V-T is seconded to the Department of Health and Social Care, England (DHSC). The views expressed are those of the authors and not necessarily those of the DHSC, DID, NIHR, MRC, Wellcome Trust, or PHE.

Examining the Immunological Effects of COVID-19 Vaccination in Patients with Conditions Potentially Leading to Diminished Immune Response Capacity – The OCTAVE Trial (preprint)

SARS-COV-2 vaccines have been shown to be efficacious primarily in healthy volunteer populations and population level studies. Immune responses following SARS-CoV-2 vaccination are less well characterised in potentially immune vulnerable patient groups, including those with immune-mediated inflammatory and chronic diseases (inflammatory arthritis [IA] incorporating rheumatoid arthritis [RA] and psoriatic arthritis [PsA]; ANCA-Associated Vasculitis [AAV]; inflammatory bowel disease [IBD]); hepatic disease (HepD), end stage kidney disease requiring haemodialysis (HD) without or with immunosuppression (HDIS); solid cancers (SC) and haematological malignancies (HM), and those that have undergone haemopoietic stem cell transplant (HSCT). The OCTAVE trial is a multi-centre, multi-disease, prospective cohort that will comprehensively assess SARS-CoV-2 vaccine responses within and between the abovementioned disease cohorts using common analytical platforms in patients recruited across the United Kingdom (UK). The majority of subjects received either COVID-19 mRNA Vaccine BNT162b2 (Pfizer/BioNTech) or ChAdOx1 Vaccine (AstraZeneca formerly AZD1222) as part of the UK National COVID19 vaccination programme. As of 13 th August 2021; 2,583 patients have been recruited. We report herein the humoral and T cell immune response results from the first 600 participants recruited where serology data are available at baseline, pre-second vaccine dose (boost) and/or 4 weeks post second dose. We also include in the analysis, data obtained from 231 healthy individuals from the PITCH (Protective Immunity from T cells in Healthcare workers) study. Overall, in comparison to PITCH where 100% of tested individuals (n=93) generated anti-Spike antibodies after vaccine doses, 89% of patients within OCTAVE seroconverted 4 weeks after second vaccine dose. By corollary, approximately 11% of patients across all disease cohorts fail to generate antibodies that react to SARS-CoV-2 spike 4 weeks after two vaccines. Failure to generate spike reactive antibodies was found at a higher proportion in some specific patient subgroups, particularly AAV (72.4%), HD-IS (16.7%) and HepD (16.7%). Importantly, all recruited AAV patients had received Rituximab; a targeted B cell depletion therapy. Furthermore, even in those who seroconverted, 40% of patients across disease cohorts generate lower levels of SARS-CoV-2 antibody reactivity compared to healthy subjects after two SARS-CoV-2 vaccines; the functional significance of these findings in providing protection from subsequent SARS-CoV-2 exposure is not currently known. In contrast to the observed serological response, evaluation of the Spike-specific T cell response revealed that across all patient sub-groups (including AAV) a response similar to healthy individuals was generated. Our data argue strongly for further vaccination strategies to optimise humoral immune responses against SARS-CoV-2 in patients with chronic diseases and/or patients on immune suppressive therapies. Trial Registration: The trial is registered on ISRCTN 12821688.Funding: This work was supported by the Medical Research Council COVID-19 Immunity – National Core Study (IMM-NCS) [grant number MC-PC-20031]. Staff at the Cancer Research UK Clinical Trials Unit (CRCTU) are supported by a core funding grant from Cancer Research UK (C22436/A25354). PK and EB are supported by the NIHR Birmingham Biomedical Research Centres at the University Hospitals Birmingham NHS Foundation Trust and the University of Birmingham Biomedical Research Centres. EB and PK are supported by an NIHR Senior Investigator award. PK is funded by WT109965MA. SJD is funded by an NIHR Global Research Professorship (NIHR300791). TdS is funded by a Wellcome Trust Intermediate Clinical Fellowship (110058/Z/15/Z). DS is supported by the NIHR Academic Clinical Lecturer programme in Oxford. LT is supported by the Wellcome Trust (grant number 205228/Z/16/Z), the U.S. Food and Drug Administration Medical Countermeasures Initiative contract 75F40120C00085. and the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections (NIHR200907) at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford. The PITCH (Protective Immunity from T cells to Covid-19 in Health workers) Consortium, is funded by the UK Department of Health and Social Care with contributions from UKRI/NIHR through the UK Coronavirus Immunology Consortium (UKCIC), the Huo Family Foundation and The National Institute for Health Research (UKRIDHSC COVID-19 Rapid Response Rolling Call, Grant Reference Number COV19-RECPLAS).Declaration of Interest: None to declare. Ethical Approval: This study was approved by the UK Medicines and Healthcare Products Regulatory Agency on the 5th February 2021 and the London and Chelsea Research Ethics Committee (REC Ref:21/HRA/0489) on 12th February 2021, with subsequent amendments approved on 3rd March 2021, 19th April 2021 and 26th April 2021).