Different vaccine platforms result in distinct antibody responses to the same antigen in haemodialysis patients (preprint)

Generalised immune dysfunction in chronic kidney disease, especially in patients requiring haemodialysis (HD), significantly enhances the risk of severe infections. Moreover, vaccine-induced immunity is typically reduced in HD populations, but the full mechanisms behind this remain unclear. The SARS-CoV-2 pandemic provided an opportunity to examine the magnitude and functionality of antibody responses in HD patients to a previously unencountered antigen, Spike (S)-glycoprotein, after vaccination with different vaccine platforms (viral vector (VV); mRNA (mRV)). Here, we compared total and functional anti-S antibody responses (cross-variant neutralisation and complement binding) in 187 HD patients and 43 healthy controls 21-28 days after serial immunisation. After 2 doses of the same vaccine, HD patients had anti-S antibody levels and complement binding capacity comparable to controls. However, 2 doses of mRV induced greater polyfunctional antibody responses than VV, yet previous SARS-CoV-2 infection or an mRV boost after 2 doses of VV significantly enhanced antibody functionality in HD patients. Therefore, HD patients can generate near-normal, functional antigen-specific antibody responses following serial vaccination to a novel antigen, suggesting largely intact B cell memory. Encouragingly, exploiting immunological memory by using mRNA vaccines and boosting may improve the success of vaccination strategies in this vulnerable patient population.

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.

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.

PROphylaxis for paTiEnts at risk of COVID-19 infecTion (PROTECT-V) (preprint)

Background: Despite the introduction of vaccination, there remains a need for pre-exposure prophylactic agents against SARS-CoV-2. Several patient groups are more vulnerable to SARS-CoV-2 infection by virtue of underlying health conditions, treatments received or suboptimal responses to vaccination. Methods: PROTECT-V is a platform trial testing pre-exposure prophylactic interventions against SARS- CoV-2 infection in vulnerable patient populations (organ transplant recipients; individuals with oncological/haematological diagnoses, immune deficiency, or autoimmune diseases requiring immunosuppression or on dialysis). Multiple agents can be evaluated across multiple vulnerable populations sharing placebo groups, with the option of adding additional treatments at later time points as these become available. The primary endpoint is symptomatic SARS-CoV-2 infection, and each agent will be independently evaluated in real-time when the required number of events occur. Presently, three agents are approved in the platform: intranasal niclosamide, nasal and inhaled ciclesonide and intravenous sotrovimab. Discussion: Despite the introduction of vaccination, there remains a need for pre-exposure prophylactic agents against SARS-CoV-2. Several patient groups are more vulnerable to COVID-19 disease by virtue of underlying health conditions, treatments received or suboptimal responses to vaccination. Trial registration: Clinicaltrials.gov: NCT04870333; EudraCT: 2020-004144-28.

Systematic review of the clinical effectiveness of Tixagevimab/Cilgavimab for prophylaxis of COVID-19 in immunocompromised patients (preprint)

Background and aims Immunocompromised patients have a reduced ability to generate antibodies after COVID-19 vaccination and are at higher risk of SARS-CoV-2 infection, complications and mortality. Tixagevimab/Cilgavimab (Evusheld) is a monoclonal antibody combination which bind to the SARS-CoV-2 spike protein, preventing the virus entering human cells. The phase III PROVENT trial reported that immunocompromised patients given Tixagevimab/Cilgavimab had a significantly reduced risk of COVID-19 infection. However, PROVENT was conducted before the SARS-CoV-2 Omicron became prevalent. This systematic review provides an updated summary of real-world clinical evidence of Tixagevimab/Cilgavimab effectiveness in immunocompromised patients. Methods Two independent reviewers conducted PubMed and medRxiv searches for the period of 01/01/2021 to 01/10/2022. Clinical studies which reported the primary outcome of breakthrough COVID-19 infections after Tixagevimab/Cilgavimab administration were included in the review. COVID-19-related hospitalisations, ITU admissions and mortality were assessed as secondary outcomes. Clinical effectiveness was determined using the case-control clinical effectiveness methodology. The GRADE tool was used to ascertain the level of certainty for the primary outcome in each study. Results 17 clinical studies were included, comprising 24,773 immunocompromised participants of whom 10,775 received Tixagevimab/Cilgavimab. Most studies reported clinical outcomes during the SARS-CoV-2 Omicron wave. Six studies compared a Tixagevimab/Cilgavimab intervention group to a control group. Overall, the clinical effectiveness of prophylactic Tixagevimab/Cilgavimab against COVID-19 breakthrough infection, hospitalisation and ITU admission were 40.47%, 69.23% and 87.89%, respectively. For prevention of all-cause and COVID-19-specifc mortality, overall clinical effectiveness was 81.29% and 86.36%, respectively. Conclusions There is a growing body of real-world evidence validating the original PROVENT phase III study regarding the clinical effectiveness of Tixagevimab/Cilgavimab as prophylaxis for immunocompromised patients, notably demonstrating effectiveness during the Omicron wave. This review demonstrates the clinical effectiveness of prophylactic Tixagevimab/Cilgavimab at reducing COVID-19 infection, hospitalisation, ITU admission and mortality for immunosuppressed individuals. It is important that ongoing larger-scale and better-controlled real world studies are initiated and evaluated to provide ongoing certainty of the clinical benefit of prophylactic antibody treatment for immunocompromised patients in the face of new variants.

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.

Influence of vitamin D supplementation on SARS-CoV-2 vaccine efficacy and immunogenicity (preprint)

Background and Aims: Vitamin D deficiency has been reported to associate with impaired development of antigen-specific responses following vaccination. We aimed to determine whether vitamin D supplements might boost immunogenicity and efficacy of SARS-CoV-2 vaccination. Methods: We conducted three sub-studies nested within the CORONAVIT randomised controlled trial, which investigated effects of offering vitamin D supplements at a dose of 800 or 3200 IU per day vs. no offer on risk of acute respiratory infections, including COVID-19, in UK adults with circulating 25-hydroxyvitamin D concentrations under 75 nmol/L. Sub-study 1 (n=2808) investigated effects of vitamin D supplementation on risk of breakthrough SARS-CoV-2 infection following two doses of SARS-CoV-2 vaccine. Sub-study 2 (n=1853) investigated effects of vitamin D supplementation on titres of combined IgG, IgA and IgM (IgGAM) anti-Spike antibodies in eluates of dried blood spots collected after SARS-CoV-2 vaccination. Sub-study 3 (n=100) investigated effects of vitamin D supplementation on neutralising antibody and cellular responses in venous blood samples collected after SARS-CoV-2 vaccination. Results: 1945/2823 (69.3%) sub-study 1 participants received two doses of ChAdOx1 nCoV-19 (Oxford AstraZeneca); the remainder received two doses of BNT162b2 (Pfizer). Vitamin D supplementation did not influence risk of breakthrough SARS-CoV-2 infection (800 IU per day vs. no offer: adjusted hazard ratio 1.28, 95% CI 0.89 to 1.84; 3200 IU per day vs. no offer: 1.17, 0.81 to 1.70). Neither did it influence IgGAM anti-Spike titres, neutralising antibody titres or interferon-gamma concentrations in supernatants of S peptide-stimulated whole blood. Conclusions: Among adults with sub-optimal baseline vitamin D status, vitamin D replacement at a dose of 800 or 3200 IU per day did not influence protective efficacy or immunogenicity of SARS-CoV-2 vaccination. Clinical Trial Registration: ClinicalTrials.gov NCT04579640.

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.

Incidence, determinants and serological correlates of reactive symptoms following SARS-CoV-2 vaccination: a population-based longitudinal study (COVIDENCE UK) (preprint)

Prospective population-based studies investigating associations between reactive symptoms following SARS-CoV-2 vaccination and serologic responses to vaccination are lacking. We therefore conducted such a study in 9003 adults from the United Kingdom (UK) general population receiving SARS-COV-2 vaccines as part of the national vaccination programme. Data relating to incidence and type of reactive symptoms after vaccination were captured using online questionnaires, along with information on 56 potential determinants of symptom risk. Titres of combined IgG/IgA/IgM responses to SARS-CoV-2 spike (S) glycoprotein were determined in eluates of dried blood spots collected from all participants before and after vaccination. 4262 (47.3%) participants experienced systemic reactive symptoms after a first vaccine dose. Factors associating with lower risk of such symptoms included older age (aOR per additional 10 years of age 0.85, 95% CI 0.81–0.90), male vs. female sex (aOR 0.59, 95% CI 0.53–0.65) and receipt of an mRNA vaccine vs. ChAdOx1 nCoV-19 (0.29, 0.26–0.32 for BNT162b2; 0.06, 0.01–0.26 for mRNA-1273). Higher risk of such symptoms was associated with a history of symptomatic SARS-CoV-2 infection prior to vaccination (2.23, 1.78–2.81) and presence vs. absence of self-rated anxiety or depression at cohort enrolment (1.24, 1.12–1.39). Post-vaccination anti-S titres were higher among participants who experienced reactive symptoms after vaccination vs. those who did not (P < 0.001). We conclude that factors influencing risk of systemic symptoms after SARS-CoV-2 vaccination include demographic characteristics, pre-vaccination SARS-CoV-2 serostatus and vaccine type. Participants experiencing reactive symptoms following SARS-CoV-2 vaccination had higher post-vaccination titres of IgG/A/M anti-S antibodies. Improved public understanding of the frequency of reactogenic symptoms and their positive association with vaccine immunogenicity could potentially increase vaccine uptake.

Vitamin D Supplements for Prevention of Covid-19 or other Acute Respiratory Infections: a Phase 3 Randomized Controlled Trial (CORONAVIT) (preprint)

BACKGROUND: Vitamin D metabolites support innate immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory pathogens. Randomized controlled trials of vitamin D to prevent coronavirus disease 2019 (Covid-19) have not yet reported. METHODS: We randomly assigned 6200 U.K. adults to receive an offer of a postal finger-prick 25-hydroxyvitamin D (25[OH]D) test with provision of a 6-month supply of higher-dose vitamin D (3200 IU/d, n=1550) or lower-dose vitamin D (800 IU/d, n=1550) to those with blood 25(OH)D concentration <75 nmol/L, vs. no offer of testing or supplementation (n=3100). The primary outcome was the proportion of participants experiencing at least one swab test- or doctor-confirmed acute respiratory infection (ARI) of any cause at six months. Secondary outcomes included incidence of swab test-confirmed Covid-19. RESULTS: Of 3100 participants offered testing, 2958 (95.4%) accepted, and 2690 (86.8%) had 25(OH)D <75 nmol/L and were sent vitamin D supplements (1356 higher-dose, 1334 lower-dose). 76 (5.0%) vs. 87 (5.7%) vs. 136 (4.6%) participants in higher-dose vs. lower-dose vs. no-offer groups experienced at least one ARI of any cause (odds ratio [OR] for higher-dose vs. no-offer 1.09, 95% CI 0.82-1.46; lower-dose vs. no-offer 1.26, 0.96-1.66). 45 (3.0%) vs. 55 (3.6%) vs. 78 (2.6%) participants in higher-dose vs. lower-dose vs. no-offer groups developed Covid-19 (OR for higher-dose vs. no-offer 1.13, 0.78-1.63; lower-dose vs. no-offer 1.39, 0.98-1.97). CONCLUSIONS: Among adults with a high baseline prevalence of vitamin D insufficiency, implementation of a test-and-treat approach to vitamin D replacement did not reduce risk of all-cause ARI or Covid-19.

Recurrent SARS-CoV-2 Mutations in Immunodeficient Patients (preprint)

Long-term SARS-CoV-2 infections in immunodeficient patients are an important source of variation for the virus but are understudied. Many case studies have been published which describe one or a small number of long-term infected individuals but no study has combined these sequences into a cohesive dataset. This work aims to rectify this and study the genomics of this patient group through a combination of literature searches as well as identifying new case series directly from the COG-UK dataset. The spike gene receptor binding domain (RBD) and N-terminal domains (NTD) were identified as mutation hotspots. Numerous mutations associated with variants of concern were observed to emerge recurrently. Additionally a mutation in the envelope gene, - T30I was determined to be the most recurrent frequently occurring mutation arising in persistent infections. A high proportion of recurrent mutations in immunodeficient individuals are associated with ACE2 affinity, immune escape, or viral packaging optimisation. There is an apparent selective pressure for mutations which aid intra-host transmission or persistence which are often different to mutations which aid inter-host transmission, although the fact that multiple recurrent de novo mutations are considered defining for variants of concern strongly indicates that this potential source of novel variants should not be discounted.

Determinants of antibody responses to two doses of ChAdOx1 nCoV-19 or BNT162b2 and a subsequent booster dose of BNT162b2 or mRNA-1273: population-based cohort study (COVIDENCE UK) (preprint)

Summary Background Antibody responses to SARS-CoV-2 vaccination vary for reasons that remain poorly understood. Methods We tested for presence of combined IgG, IgA and IgM (IgGAM) anti-spike antibodies before and after administration of two doses of ChAdOx1 nCoV-19 (ChAdOx1, Oxford-AstraZeneca) or BNT162b2 (Pfizer-BioNTech) in UK adults participating in a population-based longitudinal study who received their first dose of vaccine from December 15, 2020 to July 10, 2021. Information on sixty-six potential sociodemographic, behavioural, clinical, pharmacological and nutritional determinants of serological response to vaccination was captured using serial online questionnaires. We used logistic regression to estimate multivariable-adjusted odds ratios (aORs) for associations between independent variables and risk of seronegativity following two vaccine doses. Participants who were seronegative after receiving two vaccine doses were offered an additional antibody test following subsequent administration of a ‘booster’ dose of BNT162b2 or mRNA-1273 (Moderna) from September 23 to December 12, 2021. Findings Serology results following two vaccine doses were available for 9,101 participants, of whom 5,770 (63.4%) received ChAdOx1 and 3,331 (36.6%) received BNT162b2. Anti-spike IgGAM was undetectable in 378 (4.2%) participants at a median of 8.6 weeks (IQR 6.4-10.7 weeks) after their second dose of vaccine. Seronegativity following two doses of SARS-CoV-2 vaccination was associated with administration of ChAdOx1 vs BNT162b2 (aOR 7.03, 95% CI 4.39-11.24), shorter interval between first and second vaccine doses (aOR 2.37, 1.06-5.26, for <6 weeks vs >10 weeks; aOR 1.59, 1.18-2.13, for 6-10 weeks vs >10 weeks), poorer self-assessed general health (aOR 3.33, 1.49-7.46, for poor vs excellent), immunodeficiencies (aOR 6.75, 2.63-17.35) and prescription of systemic immunosuppressants (aOR 3.76, 2.44-5.78). By contrast, pre-vaccination SARS-CoV-2 seropositivity (aOR 0.16, 0.04-0.70, for symptomatic seropositives vs seronegatives) and supplemental vitamin D intake (aOR 0.73, 0.53-0.99) were associated with reduced risk of post-vaccination seronegativity. 247/378 (65.3%) of participants who were seronegative after two doses of ChAdOx1 vs BNT162b2 provided a third sample at a median of 7.8 weeks (IQR 5.8-10.4) after receiving a booster dose of BNT162b2 or mRNA-1273: eight (3.2%) of them remained seronegative after three vaccine doses, all of whom either had a primary immunodeficiency or were taking systemic immunosuppressant drugs. Interpretation We identify multiple determinants of antibody responses to two doses of ChAdOx1 or BNT162b2, many of which are potentially modifiable. Booster doses of BNT162b2 or mRNA-1273 were highly effective in achieving seroconversion in those who failed to mount antibody responses following two doses of ChAdOx1 or BNT162b2. Study registration https://clinicaltrials.gov/ct2/show/NCT04330599 Funding Barts Charity, Fischer Family Trust, The Exilarch’s Foundation, DSM Nutritional Products, Health Data Research UK Research in context Evidence before this study We searched PubMed, medRxiv, and Google Scholar for papers published from January 1, 2020, to February 1, 2022, using the search terms (antibody OR humoral OR serologic* OR immunogenic*) AND (SARS-CoV-2 vaccine OR ChAdOx1 or BNT162b2 coronavirus), with no language restrictions. Population-based studies investigating multiple potential determinants of vaccine immunogenicity in people with known pre-vaccination SARS-CoV-2 serostatus are lacking. Added value of this study This large population-based study, conducted in a population with known pre-vaccination SARS-CoV-2 serostatus, examines a comprehensive range of potential sociodemographic, behavioural, clinical, pharmacological and nutritional determinants of antibody responses to administration of two major SARS-CoV-2 vaccines (i.e., ChAdOx1 or BNT162b2), many of which have not previously been investigated. It is also the first population-based study to characterise antibody responses to booster doses of SARS-CoV-2 vaccines in adults who were seronegative after their primary course of vaccination. Implications of all the available evidence Increased risk of seronegativity following two doses of SARS-CoV-2 vaccines was associated with administration of ChAdOx1 vs BNT162b2, shorter interval between first and second vaccine doses, poorer self-assessed general health, immunocompromise and SARS-CoV-2 seronegativity pre-vaccination. Regular intake of vitamin D supplements was associated with reduced risk of post-vaccination seronegativity. Randomised controlled trials are now needed to test for causality. Booster doses of BNT162b2 or mRNA-1273 were highly effective in achieving seroconversion in the majority of people who failed to mount antibody responses following a primary course of vaccination, the few exceptions being a subset of those with primary immunodeficiency or systemic immunosuppressant drugs.

SARS-CoV-2 testing in the community: Testing positive samples with the TaqMan SARS-CoV-2 Mutation Panel to find variants in real-time (preprint)

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages, however there can be limitations due to sequence drop-out when used to identify specific key mutations. Recently, Thermo Fisher Scientific have developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the Thermo Fisher TaqMan Mutation Panel Genotyping Assay, initially for three mutations of concern and then an additional two mutations of concern, against SARS-CoV-2 positive clinical samples and the corresponding COG-UK sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, an excellent complement to genome sequencing and has real clinical health value potential allowing laboratories to report and action variants of concern much quicker.

Determinants of Pre-Vaccination Antibody Responses to SARS-CoV-2: A Population-Based Longitudinal Study (COVIDENCE UK) (preprint)

Background: Prospective population-based studies investigating multiple determinants of pre-vaccination antibody responses to SARS-CoV-2 are lacking.Methods: We did a prospective population-based study in SARS-CoV-2 vaccine-naive UK adults between May 1 and Nov 2, 2020. Information on 88 potential risk factors was obtained through online questionnaires, and combined IgG/IgA/IgM responses to SARS-CoV-2 spike glycoprotein were determined in dried blood spots. We used logistic and linear regression to estimate adjusted odds ratios (aORs) and adjusted geometric mean ratios (aGMRs) for potential determinants of SARS-CoV-2 seropositivity (all participants) and antibody titres (seropositive participants only), respectively.Findings: 1696 (15.2%) of 11,130 participants were seropositive. Factors independently associated with increased risk included frontline health/care occupation (aOR 1.86, 95% CI 1.49–2.33), international travel (1.22, 1.08–1.37), BMI >30 vs <25 kg/m² (1.22, 1.05–1.42), Asian/Asian British vs White ethnicity (1.65, 1.10–2.47), and alcohol consumption ≥15 vs 0 units/week (1.26, 1.06–1.49). Light physical exercise associated with decreased risk (0.80, 0.69–0.93, for ≥10 vs 0–4 h/week). Higher titres associated with frontline health/care occupation (aGMR 1.26, 95% CI 1.13–1.41), international travel (1.10, 1.04–1.16), BMI >30 vs <25 kg/m² (1.09, 1.01–1.17), and Asian/Asian British vs White ethnicity (1.23, 1.03–1.46); these associations were not substantially attenuated by adjustment for disease severity.Interpretation: Higher alcohol consumption and reduced physical exercise represent new modifiable risk factors for SARS-CoV-2 infection. Recognised associations between Asian/Asian British ethnic origin and obesity and increased risk of SARS-CoV-2 seropositivity were independent of other sociodemographic, clinical, or behavioural factors investigated.Funding: Barts Charity, Health Data Research UK.Declaration of Interest: JS declares receipt of payments from Reach plc for news stories written about recruitment to, and findings of, the COVIDENCE UK study. AS is a member of the Scottish Government Chief Medical Officer’s COVID-19 Advisory Group and its Standing Committee on Pandemics. He is also a member of the UK Government’s NERVTAG’s Risk Stratification Subgroup. ARM declares receipt of funding in the last 36 months to support vitamin D research from the following companies who manufacture or sell vitamin D supplements: Pharma Nord Ltd, DSM Nutritional Products Ltd, Thornton & Ross Ltd, Cytoplan Ltd and Hyphens Pharma Ltd. ARM also declares support for attending meetings from the following companies who manufacture or sell vitamin D supplements: Pharma Nord Ltd and Abiogen Pharma Ltd. ARM also declares participation on the Data and Safety Monitoring Board for the Chair, DSMB, VITALITY trial (Vitamin D for Adolescents with HIV to reduce musculoskeletal morbidity and immunopathology). ARM also declares unpaid work as a Programme Committee member for the Vitamin D Workshop. ARM also declares receipt of vitamin D capsules for clinical trial use from Pharma Nord Ltd, Synergy Biologics Ltd and Cytoplan Ltd.Ethical Approval: COVIDENCE UK was sponsored by Queen Mary University of London and approved by Leicester South Research Ethics Committee (ref 20/EM/0117). It is registered withClinicalTrials.gov (NCT04330599).

Vitamin D Status: A U-shaped relationship for SARS-CoV-2 seropositivity in UK healthcare workers (preprint)

Background Vitamin D has numerous mechanistic roles within the immune system. There is increasing evidence to suggest Vitamin D deficiency may increase individuals’ risk of COVID-19 infection and susceptibility. We aimed to determine the relationship between severity of vitamin D deficiency and sufficiency and COVID-19 infection within healthcare workers. Methods The study included an observational cohort of healthcare workers who isolated due to COVID-19 symptoms from 12th to 22nd May 2020, from the University Hospitals Birmingham NHS Foundation Trust (UHBFT). This was part of the COVID-19 convalescent immunity study (COCO). Data collected included SARS-CoV-2 seroconversion status, serum 25(OH)D 3 levels as well as age, body mass index (BMI), sex, ethnicity, job role, and co-morbidities. Participants were grouped into four vitamin D (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 in the total population. This trend repeated when split into subgroups of age, sex, ethnicity, BMI, and co-morbidity status. Significant difference was identified in the COVID-19 seropositivity rate between VD groups between multiple VD groups in the total population, males, females, BAME, BMI<30 (kg/m 2 ), 0 and +1 comorbidities; the majority of which were differences when the severely VD deficient category were compared to the other group. A significantly larger proportion of those within the Black, Asian, minority ethnic (BAME) group ( vs . white ethnicity) were severely vitamin D deficient ( P < 0.00001). A significantly higher proportion of the 0-comorbidity subgroup were vitamin D deficient in comparison to the 1+ comorbidity subgroup ( P = 0.046). Conclusions Further investigation of the U-shaped curves 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. This would allow for targeted therapeutic treatment for those at-risk such as in the BAME group.

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.

Impaired Neutralisation of SARS-CoV-2 Delta Variant in Vaccinated Patients With B Cell Chronic Lymphocytic Leukaemia (preprint)

Background: Immune suppression is a clinical feature of chronic lymphocytic leukaemia (CLL) and patients show increased vulnerability to SARS-CoV-2 infection and suboptimal antibody responses.Method: We studied antibody responses in 500 patients following dual COVID-19 vaccination to assess the magnitude, correlates of response, stability and functional activity of the spike-specific antibody response with 2 different vaccine platforms.Results: Spike-specific seroconversion post-vaccine was seen in 67% of patients compared to 100% of age-matched controls. Amongst responders, titres were 3.7 times lower than the control group. Antibody responses showed a 33% fall over the next 4 months. The use of an mRNA (n=204) or adenovirus-based (n=296) vaccine platform did not impact on antibody response. Male gender, BTKi therapy, prophylactic antibiotics use and low serum IgA/IgM were predictive of failure to respond. Antibody responses after CD20-targeted immunotherapy recovered 12 months-post treatment. Post-vaccine sera from CLL patients with Spike-specific antibody response showed markedly reduced neutralisation of the SARS-CoV-2 delta variant compared to healthy controls. Patients with previous natural SARS-CoV-2 infection showed equivalent antibody levels and function as healthy donors after vaccination.Interpretation: These findings demonstrate impaired antibody responses following dual COVID-19 vaccination in patients with CLL and further define patient risk groups. Furthermore, humoral protection against the globally-dominant delta variant is markedly impaired in CLL patients and indicates the need for further optimisation of immune protection in this patient cohort.Funding Information: This work was partially supported by the UK Coronavirus Immunology Consortium (UK-CIC) funded by DHSC/UKRI and the National Core Studies Immunity programme.Declaration of Interests: The authors declare no conflicts of interest.Ethics Approval Statement: Informed consent was obtained by remote consultation and work performed under the CIA UPH IRAS approval (REC 20\NW\0240) from North-West and Preston ethics committee and conducted according to the Declaration of Helsinki.

Outcomes Following SARS-CoV-2 Infection in Patients With Primary and Secondary Immunodeficiency in The United Kingdom (preprint)

Purpose: To define the burden of morbidity and mortality arising from COVID-19 in individuals with primary (PID) and secondary immunodeficiency (SID) in the United Kingdom. Methods 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. Anonymised demographic data, pre-SARS-CoV-2 infection lymphocyte counts, co-morbidities, targeted treatments and outcomes were collected. Three groups were analysed in further detail: individuals with common variable immunodeficiency (CVID), individuals with any PID, including CVID, receiving immunoglobulin replacement therapy (IgRT) and individuals with secondary immunodeficiency. Results 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 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. Conclusion 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.

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).