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Antibodies can have beneficial, neutral, or harmful effects so resolving an antibody repertoire to its target epitopes may explain heterogeneity in susceptibility to infectious disease. However, the three-dimensional nature of antibody-epitope interactions limits discovery of important targets. We describe and experimentally validated a computational method and synthetic biology pipeline for identifying structurally stable and functionally important epitopes from the SARS-CoV-2 proteome. We identify patterns of epitope-binding antibodies associated with immunopathology, including a non-isotype switching IgM response to a membrane protein epitope which is the strongest single immunological feature associated with severe COVID-19 to date (adjusted OR 72.14, 95% CI: 9.71 - 1300.15). We suggest the mechanism is T independent B cell activation and identify persistence (> 1 year) of this response in individuals with long COVID particularly affected by fatigue and depression. These findings highlight a previously unrecognized coronavirus host:pathogen interaction which is potentially an upstream event in severe immunopathology and this may have implications for the ongoing medical and public health response to the pandemic. The membrane protein epitope is a promising vaccine and monoclonal antibody target which may complement anti-spike vaccination or monoclonal antibody therapies broadening immunological protection. One-Sentence SummaryUsing a novel B cell epitope discovery method we have identified antibody signatures strongly associated with SARS-CoV-2 immunopathology and suggest the membrane protein is a pathological T independent antigen.
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Both infection and vaccination, alone or in combination, generate antibody and T cell responses against SARSCoV2. However, the maintenance of such responses, and hence protection from disease, requires careful characterisation. In a large prospective study of UK healthcare workers (Protective immunity from T cells in Healthcare workers (PITCH), within the larger SARSCoV2 immunity and reinfection evaluation (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 AZD1222 (Oxford/AstraZeneca) vaccination and up to 6 months following a subsequent mRNA booster vaccination. We make three observations: Firstly, the dynamics of humoral and cellular responses differ; binding and neutralising antibodies declined whereas T and memory B cell responses were maintained after the second vaccine dose. Secondly, vaccine boosting restored IgG levels, broadened neutralising activity against variants of concern including omicron BA.1, BA.2 and BA.5, and boosted T cell responses above the 6 month level post dose 2. Thirdly, prior infection maintained its impact driving larger as well as broader T cell responses compared with never-infected people, a feature maintained until 6 months after the third dose. In conclusion, broadly cross-reactive T cell responses are well maintained over time, especially in those with combined vaccine and infection-induced immunity (hybrid immunity), and may contribute to continued protection against severe disease.
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BackgroundT cell responses to SARS-CoV-2 following infection and vaccination are less characterised than antibody responses, due to a more complex experimental pathway. MethodsWe 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. ResultsBoth 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. ConclusionThe 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.
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On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.
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BackgroundAlthough 6 COVID-19 vaccines have been approved by the World Health Organisation as of 16th June 2021, global supply remains limited. An understanding of the immune response associated with protection could facilitate rapid licensure of new vaccines. MethodsData from a randomised efficacy trial of ChAdOx1 nCoV-19 (AZD1222) vaccine in the UK was analysed to determine the antibody levels associated with protection against SARS-CoV-2. Anti-spike and anti-RBD IgG by multiplex immunoassay, pseudovirus and live neutralising antibody at 28 days after the second dose were measured in infected and non-infected vaccine recipients. Weighted generalised additive models for binary data were applied to symptomatic and asymptomatic SARS-CoV-2 infection data from ChAdOx1 nCoV-19 recipients. Cubic spline smoothed log antibody levels, and weights were applied to account for potential selection bias in sample processing. Models were adjusted for baseline risk of exposure to SARS-CoV-2 infection. ResultsHigher levels of all immune markers were correlated with a reduced risk of symptomatic infection. Vaccine efficacy of 80% against primary symptomatic COVID-19 was achieved with an antibody level of 40923 (95% CI: 16748, 125017) and 63383 (95% CI: 16903, not computed (NC)) for anti-spike and anti-RBD, and 185 (95% CI: NC, NC) and 247 (95% CI: 101, NC) for pseudo- and live-neutralisation assays respectively. Antibody responses did not correlate with overall protection against asymptomatic infection. ConclusionsCorrelates of protection can be used to bridge to new populations using validated assays. The data can be used to extrapolate efficacy estimates for new vaccines where large efficacy trials cannot be conducted. More work is needed to assess correlates for emerging variants.
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Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete understanding of potentially druggable immune mediators of disease. To advance this, we present a comprehensive multi-omic blood atlas in patients with varying COVID-19 severity and compare with influenza, sepsis and healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity revealed cells, their inflammatory mediators and networks as potential therapeutic targets, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Tensor and matrix decomposition of the overall dataset revealed feature groupings linked with disease severity and specificity. Our systems-based integrative approach and blood atlas will inform future drug development, clinical trial design and personalised medicine approaches for COVID-19.
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It is unclear whether prior endemic coronavirus infections affect COVID-19 severity. Here, we show that in cases of fatal COVID-19, antibody responses to the SARS-COV-2 spike are directed against epitopes shared with endemic beta-coronaviruses in the S2 subunit of the SARS-CoV-2 spike protein. This immune response is associated with the compromised production of a de novo SARS-CoV-2 spike response among individuals with fatal COVID-19 outcomes.
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Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.
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Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, detection of seroconversion after vaccination, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests have a long history in blood typing, and general serology through linkage of reporter molecules to the red cell surface. They do not require special equipment, are read by eye, have short development times, low cost and can be applied as a Point of Care Test (POCT). We describe a red cell agglutination test for the detection of antibodies to the SARS-CoV-2 receptor binding domain (RBD). We show that the Haemagglutination Test ("HAT") has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. The HAT can be titrated, detects rising titres in the first five days of hospital admission, correlates well with a commercial test that detects antibodies to the RBD, and can be applied as a point of care test. The developing reagent is composed of a previously described nanobody to a conserved glycophorin A epitope on red cells, linked to the RBD from SARS-CoV-2. It can be lyophilised for ease of shipping. We have scaled up production of this reagent to one gram, which is sufficient for ten million tests, at a cost of [~]0.27 UK pence per test well. Aliquots of this reagent are ready to be supplied to qualified groups anywhere in the world that need to detect antibodies to SARS-CoV-2, but do not have the facilities for high throughput commercial tests.
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A major issue in identification of protective T cell responses against SARS-CoV-2 lies in distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity generated by exposure to other coronaviruses. We characterised SARS-CoV-2 T cell immune responses in 168 PCR-confirmed SARS-CoV-2 infected subjects and 118 seronegative subjects without known SARS-CoV-2 exposure using a range of T cell assays that differentially capture immune cell function. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) were found in those who had been infected by SARS-CoV-2 but were rare in pre-pandemic and unexposed seronegative subjects. However, seronegative doctors with high occupational exposure and recent COVID-19 compatible illness showed patterns of T cell responses characteristic of infection, indicating that these readouts are highly sensitive. By contrast, over 90% of convalescent or unexposed people showed proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on the choice of assay and antigen. Memory responses to specific non-spike proteins provides a method to distinguish recent infection from pre-existing immunity in exposed populations.
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COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFN{gamma} based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4+ and/or CD8+ epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8+ T cells than spike-specific CD8+ T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8+ to CD4+ T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.
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BackgroundLaboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood. MethodsWe undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=111 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples. ResultsWe identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected [≥]28 days post symptom onset, 0/143 (0%, 95%CI 0.0-2.5%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA. ConclusionsvRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19.
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BackgroundThe COVID-19 pandemic caused >1 million infections during January-March 2020. There is an urgent need for reliable antibody detection approaches to support diagnosis, vaccine development, safe release of individuals from quarantine, and population lock-down exit strategies. We set out to evaluate the performance of ELISA and lateral flow immunoassay (LFIA) devices. MethodsWe tested plasma for COVID (SARS-CoV-2) IgM and IgG antibodies by ELISA and using nine different LFIA devices. We used a panel of plasma samples from individuals who have had confirmed COVID infection based on a PCR result (n=40), and pre-pandemic negative control samples banked in the UK prior to December-2019 (n=142). ResultsELISA detected IgM or IgG in 34/40 individuals with a confirmed history of COVID infection (sensitivity 85%, 95%CI 70-94%), vs. 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 COVID-positive individuals tested [≥]10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. ConclusionsCurrently available commercial LFIA devices do not perform sufficiently well for individual patient applications. However, ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following first symptoms.
