Large clones of pre-existing T cells drive early immunity against SARS-COV-2 and LCMV infection.

We analyzed the dynamics of the earliest T cell response to SARS-COV-2. A wave of TCRs strongly but transiently expand during infection, frequently peaking the same week as the first positive PCR test. These expanding TCR CDR3s were enriched for sequences functionally annotated as SARS-COV-2 specific. Most epitopes recognized by the expanding TCRs were highly conserved between SARS-COV-2 strains, but not with circulating human coronaviruses. Many expanding CDR3s were also present at high precursor frequency in pre-pandemic TCR repertoires. A similar set of early response TCRs specific for lymphocytic choriomeningitis virus epitopes were also found at high frequency in the pre-infection naive repertoire. High frequency naive precursors may allow the T cell response to respond rapidly during the crucial early phases of acute viral infection. One-Sentence SummaryHigh frequency naive precursors underly the rapid T cell response during the crucial early phases of acute viral infection.

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

SUMMARYO_ST_ABSBackground & AimsC_ST_ABSVitamin 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. MethodsWe conducted three sub-studies nested within the CORONAVIT randomised controlled trial, which investigated effects of offering vitamin D supplements at a dose of 800 IU/day or 3200 IU/day vs. no offer on risk of acute respiratory infections, including COVID-19, in UK adults with circulating 25-hydroxyvitamin D concentrations <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. Results1945/2808 (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/day vs. no offer: adjusted hazard ratio 1.28, 95% CI 0.89 to 1.84; 3200 IU/day vs. no offer: 1.17, 0.81 to 1.70). Neither did it influence IgGAM anti-Spike titres, neutralising antibody titres or IFN-{gamma} concentrations in supernatants of S peptide-stimulated whole blood. ConclusionsAmong adults with sub-optimal baseline vitamin D status, vitamin D replacement at a dose of 800 or 3200 IU/day did not influence protective efficacy or immunogenicity of SARS-CoV-2 vaccination. Clinical Trial RegistrationClinicalTrials.gov NCT04579640.

Quantitative, multiplexed, targeted proteomics for ascertaining variant specific SARS-CoV-2 antibody response

Determining the protection an individual has to SARS-CoV-2 variants of concern (VoC) will be crucial for future immune surveillance and understanding the changing immune response. As further variants emerge, current serology tests are becoming less effective in reflecting neutralising capability of the immune system. A better measure of an evolving antigen-antibody immune response is needed. We describe a multiplexed, baited, targeted-proteomic assay for direct detection of multiple proteins in the SARS-CoV-2 anti-spike antibody immunocomplex. This enables a more sophisticated and informative characterisation of the antibody response to vaccination and infection against VoC. Using this assay, we detail different and specific responses to each variant by measuring several antibody classes, isotypes and associated complement binding. Furthermore, we describe how these proteins change using serum from individuals collected after infection, first and second dose vaccination. We show complete IgG1 test concordance with gold standard ELISA (r>0.8) and live virus neutralisation against Wuhan Hu-1, Alpha B.1.1.7, Beta B.1.351, and Delta B.1.617.1 variants (r>0.79). We also describe a wide degree of heterogeneity in the immunocomplex of individuals and a greater IgA response in those patients who had a previous infection. Significantly, our test points to an important role the complement system may play particularly against VoC. Where we observe altered Complement C1q association to the Delta VoC response and a stronger overall association with neutralising antibodies than IgG1. A detailed understanding of an individuals antibody response could benefit public health immunosurveillance, vaccine design and inform vaccination dosing using a personalised medicine approach.

Correlation between post-vaccination titres of combined IgG, IgA, and IgM anti-Spike antibodies and protection against breakthrough SARS-CoV-2 infection: a population-based longitudinal study (COVIDENCE UK)

In this population-based cohort of 7530 adults, combined IgG/A/M anti-Spike titres measured after SARS-CoV-2 vaccination were predictive of protection against breakthrough SARS-CoV-2 infection. Discrimination was significantly improved by adjustment for factors influencing risk of SARS-CoV-2 exposure including household overcrowding, public transport use, and visits to indoor public places.

Generation of novel SARS-CoV-2 variants on B.1.1.7 lineage in three patients with advanced HIV disease

The emergence of new SARS-COV-2 variants is of public health concern in case of vaccine escape. Described are three patients with advanced HIV-1 and chronic SARS-CoV-2 infection in whom there is evidence of selection and persistence of novel mutations which are associated with increased transmissibility and immune escape.

The β-NGF/TrkA signalling pathway is associated with the production of anti- nucleoprotein IgG in convalescent COVID-19

BackgroundThe presentation of SARS-CoV-2 infection varies from asymptomatic to severe COVID. Similarly, high variability in the presence, titre and duration of specific antibodies has been reported. While some host factors determining these differences, such as age and ethnicity have been identified, the underlying molecular mechanisms underpinning these differences remain poorly defined. MethodsWe analysed serum and PBMC from 17 subjects with a previous PCR confirmed SARS-CoV-2 infection and 10 unexposed volunteers following the first wave of the pandemic, in the UK. Anti-NP IgG and neutralising antibodies were measured, as well as a panel of infection and inflammation related cytokines. The virus-specific T cell response was determined by IFN-{gamma} ELISPOT and flow cytometry after over-night incubation of PBMCs with pools of selected SARS-CoV-2 specific peptides. ResultsSeven of 17 convalescent subjects had undetectable levels of anti-NP IgG, and a positive correlation was shown between anti-NP IgG levels and the titre of neutralising antibodies (IC50). In contrast, a discrepancy was noted between antibody levels and T cell IFN-{gamma} production by ELISpot following stimulation with specific peptides. Among the analysed cytokines, {beta}-NGF and IL-1 levels were significantly different between anti-NP positive and negative subjects, and only {beta}-NGF significantly correlated with anti-NP positivity. Interestingly, CD4+ T cells of anti-NP negative subjects expressed lower amounts of the {beta}-NGF-specific receptor TrkA. ConclusionsOur results suggest that the {beta}-NGF/TrkA signalling pathway is associated with the production of anti-NP specific antibody in mild SARS-CoV-2 infection and the mechanistic regulation of this pathway in COVID-19 requires further investigation.

Pre-existing polymerase-specific T cells expand in abortive seronegative SARS-CoV-2 infection

Individuals with likely exposure to the highly infectious SARS-CoV-2 do not necessarily develop PCR or antibody positivity, suggesting some may clear sub-clinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1-5. We hypothesised that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-26-12, would expand in vivo to mediate rapid viral control, potentially aborting infection. We studied T cells against the replication transcription complex (RTC) of SARS-CoV-2 since this is transcribed first in the viral life cycle13-15and should be highly conserved. We measured SARS-CoV-2-reactive T cells in a cohort of intensively monitored healthcare workers (HCW) who remained repeatedly negative by PCR, antibody binding, and neutralisation for SARS-CoV-2 (exposed seronegative, ES). 16-weeks post-recruitment, ES had memory T cells that were stronger and more multispecific than an unexposed pre-pandemic cohort, and more frequently directed against the RTC than the structural protein-dominated responses seen post-detectable infection (matched concurrent cohort). The postulate that HCW with the strongest RTC-specific T cells had an abortive infection was supported by a low-level increase in IFI27 transcript, a robust early innate signature of SARS-CoV-2 infection16. We showed that the RNA-polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and was preferentially targeted by T cells from UK and Singapore pre-pandemic cohorts and from ES. RTC epitope-specific T cells capable of cross-recognising HCoV variants were identified in ES. Longitudinal samples from ES and an additional validation cohort, showed pre-existing RNA-polymerase-specific T cells expanded in vivo following SARS-CoV-2 exposure, becoming enriched in the memory response of those with abortive compared to overt infection. In summary, we provide evidence of abortive seronegative SARS-CoV-2 infection with expansion of cross-reactive RTC-specific T cells, highlighting these highly conserved proteins as targets for future vaccines against endemic and emerging Coronaviridae.

Non-severe SARS-CoV-2 infection is characterised by very early T cell proliferation independent of type 1 interferon responses and distinct from other acute respiratory viruses.

The correlates of natural protective immunity to SARS-CoV-2 in the majority who experience asymptomatic infection or non-severe disease are not fully characterised, and remain important as new variants emerge. We addressed this question using blood transcriptomics, multiparameter flow cytometry and T cell receptor (TCR) sequencing spanning the time of incident infection. We identified a type 1 interferon (IFN) response common to other acute respiratory viruses, and a cell proliferation response that discriminated SARS-CoV-2 from other viruses. These responses peaked by the time the virus was first detected, and in some preceded virus detection. Cell proliferation was most evident in CD8 T cells and associated with rapid expansion of SARS-CoV-2 reactive TCRs. We found an equally rapid increase in immunoglobulin transcripts, but circulating virus-specific antibodies lagged by 1-2 weeks. Our data support a protective role for rapid induction of type 1 IFN and CD8 T cell responses to SARS-CoV-2.

Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV 2 infection

We hypothesised that host-response biomarkers of viral infections may contribute to early identification of SARS-CoV-2 infected individuals, critical to breaking chains of transmission. We identified 20 candidate blood transcriptomic signatures of viral infection by systematic review and evaluated their ability to detect SARS-CoV-2 infection, compared to the gold-standard of virus PCR tests, among a prospective cohort of 400 hospital staff subjected to weekly testing when fit to attend work. The transcriptional signatures had limited overlap, but were mostly co-correlated as components of type 1 interferon responses. We reconstructed each signature score in blood RNA sequencing data from 41 individuals over sequential weeks spanning a first positive SARS-CoV-2 PCR, and after 6-month convalescence. A single blood transcript for IFI27 provided the highest accuracy for discriminating individuals at the time of their first positive viral PCR result from uninfected controls, with area under the receiver operating characteristic curve (AUROC) of 0.95 (95% confidence interval 0.91-0.99), sensitivity 0.84 (0.7-0.93) and specificity 0.95 (0.85-0.98) at a predefined test threshold. The test performed equally well in individuals with and without symptoms, correlated with viral load, and identified incident infections one week before the first positive viral PCR with sensitivity 0.4 (0.17-0.69) and specificity 0.95 (0.85-0.98). Our findings strongly support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection, for screening individuals such as contacts of index cases, in order to facilitate early case isolation and early antiviral treatments as they emerge.

Characterising heterogeneity and sero-reversion in antibody responses to mild SARS-CoV-2 infection: a cohort study using time series analysis and mechanistic modelling

BackgroundSARS-CoV-2 serology is used to identify prior infection at individual and at population level. Extended longitudinal studies with multi-timepoint sampling to evaluate dynamic changes in antibody levels are required to identify the time horizon in which these applications of serology are valid, and to explore the longevity of protective humoral immunity. MethodsHealth-care workers were recruited to a prospective cohort study from the first SARS-CoV-2 epidemic peak in London, undergoing weekly symptom screen, viral PCR and blood sampling over 16-21 weeks. Serological analysis (n=12,990) was performed using semi-quantitative Euroimmun IgG to viral spike S1 domain and Roche total antibody to viral nucleocapsid protein (NP) assays. Comparisons were made to previously reported pseudovirus neutralising antibody measurements. FindingsA total of 157/729 (21.5%) participants developed positive SARS-CoV-2 serology by one or other assay, of whom 31.0% were asymptomatic and there were no deaths. Peak Euroimmun anti-S1 and Roche anti-NP measurements correlated (r=0.57, p<0.0001) but only anti-S1 measurements correlated with near-contemporary pseudovirus neutralising antibody titres (measured at 16-18 weeks, r=0.57, p<0.0001). By 21 weeks follow-up, 31/143 (21.7%) anti-S1 and 6/150 (4.0%) anti-NP measurements reverted to negative. Mathematical modelling suggested faster clearance of anti-S1 compared to anti-NP (median half-life of 2.5 weeks versus 4.0 weeks), earlier transition to lower levels of antibody production (median of 8 versus 13 weeks), and greater reductions in relative antibody production rate after the transition (median of 35% versus 50%). InterpretationMild SARS-CoV-2 infection is associated with heterogenous serological responses in Euroimmun anti-S1 and Roche anti-NP assays. Anti-S1 responses showed faster rates of clearance, more rapid transition from high to low level production rate and greater reduction in production rate after this transition. The application of individual assays for diagnostic and epidemiological serology requires validation in time series analysis. FundingCharitable donations via Barts Charity Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed, medRxiv, and bioRxiv for ["antibody" OR "serology"] AND ["SARS-CoV-2" OR "COVID-19"]. The available literature highlights widespread use of serology to detect recent SARS-CoV-2 infection in individual patients and in population epidemiological surveys. Antibody to virus spike protein S1 domain is widely reported to correlate with neutralising antibody titres. The existing assays have good sensitivity to detect seroconversion within 14 days of incident infection, but the available longitudinal studies have reported variable rates of decline in antibody levels and reversion to undetectable levels in some people over 3 months. High frequency multi-time point serology data for different antibody targets or assays in longitudinal cohorts from the time of incident infection to greater than 3 months follow up are lacking. Added value of this studyWe combine detailed longitudinal serology using the Euroimmun anti-S1 and Roche anti-nucleocapsid protein (NP) assays in 731 health care workers from the time of the first SARS-CoV-2 epidemic peak in London, UK. In 157 seroconverters (using either assay) we show substantial heterogeneity in semiquantitative antibody measurements over time between individuals and between assays. Mathematical modelling of individual participant antibody production and clearance rates in individuals with at least 8 data points over 21 weeks showed anti-S1 antibodies to have a faster clearance rate, earlier transition from the initial antibody production rate to lower rates, and greater reduction in antibody production rate after this transition, compared to anti-NP antibodies as measured by these assays. As a result, Euroimmun anti-S1 measurements peaked earlier and then reduced more rapidly than Roche anti-NP measurements. In this study, these differences led to 21% anti-S1 sero-reversion, compared to 4% anti-NP sero-reversion over 4-5 months. Implications of all of the available evidenceThe rapid decline in anti-S1 antibodies measured by the Euroimmun assay following infection limits its application for diagnostic and epidemiological screening. If generalisable, these data are consistent with the hypothesis that anti-S1 mediated humoral immunity may not be sustained in some people beyond the initial post-infective period. Further work is required to understand the mechanisms behind the heterogeneity in antibody kinetics between individuals to SARS-CoV-2. Our data point to differential mechanisms regulating humoral immunity against these two viral targets.

Healthcare workers with mild / asymptomatic SARS-CoV-2 infection show T cell responses and neutralising antibodies after the first wave

Studies of adaptive immunity to SARS-CoV-2 include characterisation of lethal, severe and mild cases1-8. Understanding how long immunity lasts in people who have had mild or asymptomatic infection is crucial. Healthcare worker (HCW) cohorts exposed to and infected by SARS-CoV-2 during the early stages of the pandemic are an invaluable resource to study this question9-14. The UK COVIDsortium is a longitudinal, London hospital HCW cohort, followed from the time of UK lockdown9,10 ; weekly PCR, serology and symptom diaries allowed capture of asymptomatic infection around the time of onset, so duration of immunity could be tracked. Here, we conduct a cross-sectional, case-control, sub-study of 136 HCW at 16-18 weeks after UK lockdown, with 76 having had laboratory-confirmed SARS-CoV-2 mild or asymptomatic infection. Neutralising antibodies (nAb) were present in 90% of infected HCW sampled after the first wave; titres, likely to correlate with functional protection, were present in 66% at 16-18 weeks. T cell responses tended to be lower in asymptomatic infected HCW than those reporting case-definition symptoms of COVID-19, while nAb titres were maintained irrespective of symptoms. T cell and antibody responses were discordant. HCW lacking nAb also showed undetectable T cells to Spike protein but had T cells of other specificities. Our findings suggest that the majority of HCW with mild or asymptomatic SARS-CoV-2 infection carry nAb complemented by multi-specific T cell responses for at least 4 months after mild or asymptomatic SARS-CoV-2 infection.