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.

Comparative multi-OMICS single-cell atlas of five COVID-19 (rAdVV and mRNA) vaccines describe unique and distinct mechanisms of action

COVID-19 vaccines based on a range of expression platforms have shown considerable protective efficacy, generating antibody and T cell immune responses. However, molecular pathways underpinning COVID-19 vaccine priming of immunity against the SARS-CoV-2 virus have not yet been explored extensively. This analysis is critical to optimization of future vaccination strategies, schedules, and combinations. Thus, we investigated a cohort of individuals pre- and post-vaccination to understand the humoral and cellular immune response against different COVID-19 vaccines, including recombinant adenoviral vector (rAdVV) and mRNA-based vaccines. Single-cell RNA sequencing allowed characterization of monocytes, T, NK and B cell activation at the transcriptomics/proteomic level, in response to different COVID-19 vaccines. Our data revealed that different COVID-19 vaccines elicit a unique and distinct mechanism of action. Specifically, we revealed that rAdVV vaccines negatively regulate CD4+ T cell activation, leukocytes chemotaxis, IL-18 signalling and antigen presentation by monocytes whilst mRNA vaccines positively regulate NKT cell activation, platelets activation and chemokine signalling pathways. An antigen-specific T cell response was already observed following the 1st vaccine dose and was not further augmented after the subsequent 2nd dose of the same vaccine and it was dependent on the type of vaccination used. Our integrated three layered-analyses highlights that COVID-19 vaccines evoke a strong but divergent immune response at the RNA, protein, and cellular levels. Our approach is able to pinpoint efficacy and mechanisms controlling immunity to vaccination and open the door for better vaccination which could induce innate and adaptive immunity equally in the long term. Key findingsO_LIDecrease in major three cell types classical and non-classical monocytes and NK type III cells after COVID-19 vaccination C_LIO_LIIndividual vaccination (AZ, JJ, MD, PB) has differential effect on various immune cell subsets and regulates unique cell populations, whilst no change was observed for CV vaccination C_LIO_LIrAdVV and mRNA vaccines have different mechanism of action for activation of lymphocytes and monocytes, respectively C_LIO_LIrAdVV vaccines negatively regulates CD4+ T cell activation, leukocytes chemotaxis, IL-18 signalling and antigen presentation whilst mRNA vaccines positively regulate NKT cell activation, platelets activation and chemokine signalling pathways. C_LIO_LIAn antigen-specific T cell response was prompted after the 1st vaccine dose and not augmented after the subsequent 2nd dose of the same vaccine. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/507666v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11fd7eborg.highwire.dtl.DTLVardef@198a9c7org.highwire.dtl.DTLVardef@1b28735org.highwire.dtl.DTLVardef@1cadbb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Antibody decay, T cell immunity and breakthrough infections following two SARS-CoV-2 vaccine doses in infliximab- and vedolizumab-treated patients

We report SARS-CoV-2 vaccine-induced immunity and risk of breakthrough infections in patients with inflammatory bowel disease treated with infliximab, a commonly used anti-TNF drug and those treated with vedolizumab, a gut-specific antibody targeting integrin a4b7 that does not impact systemic immunity. In infliximab-treated patients, the magnitude of anti-SARS-CoV2 antibodies was reduced 4-6-fold. One fifth of both infliximab- and vedolizumab-treated patients did not mount a T cell response. Antibody half-life was shorter in infliximab-treated patients. Breakthrough SARS-CoV-2 infections occurred more frequently in infliximab-treated patients and the risk was predicted by the level of antibody response after second vaccine dose. Overall, recipients of two doses of the BNT162b2 vaccine had higher anti-SARS-CoV-2 antibody concentrations, higher seroconversion rates, shorter antibody half-life and less breakthrough infections compared to ChAdOx1 nCoV-19 vaccine recipients. Irrespective of biologic treatment, higher, more sustained antibody levels were observed in patients with a history of SARS-CoV-2 infection prior to vaccination. Patients treated with anti-TNF therapy should be offered third vaccine doses.

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.