Proteomic analysis of circulating immune cells identifies cellular phenotypes associated with COVID-19 severity.

Certain serum proteins, including C-reactive protein (CRP) and D-dimer, have prognostic value in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonetheless, these factors are non-specific, providing limited mechanistic insight into the peripheral blood mononuclear cell (PBMC) populations that drive the pathogenesis of severe COVID-19. To identify cellular phenotypes associated with disease, we performed a comprehensive, unbiased analysis of total and plasma-membrane PBMC proteomes from 40 unvaccinated individuals with SARS-CoV-2, spanning the whole disease spectrum. Combined with RNA sequencing (RNA-seq) and flow cytometry from the same donors, we define a comprehensive multi-omic profile for each severity level, revealing that immune-cell dysregulation progresses with increasing disease. The cell-surface proteins CEACAMs1, 6, and 8, CD177, CD63, and CD89 are strongly associated with severe COVID-19, corresponding to the emergence of atypical CD3+CD4+CEACAM1/6/8+CD177+CD63+CD89+ and CD16+CEACAM1/6/8+ mononuclear cells. Utilization of these markers may facilitate real-time patient assessment by flow cytometry and identify immune populations that could be targeted to ameliorate immunopathology.

A patient-centric modeling framework captures recovery from SARS-CoV-2 infection.

The biology driving individual patient responses to severe acute respiratory syndrome coronavirus 2 infection remains ill understood. Here, we developed a patient-centric framework leveraging detailed longitudinal phenotyping data and covering a year after disease onset, from 215 infected individuals with differing disease severities. Our analyses revealed distinct 'systemic recovery' profiles, with specific progression and resolution of the inflammatory, immune cell, metabolic and clinical responses. In particular, we found a strong inter-patient and intra-patient temporal covariation of innate immune cell numbers, kynurenine metabolites and lipid metabolites, which highlighted candidate immunologic and metabolic pathways influencing the restoration of homeostasis, the risk of death and that of long COVID. Based on these data, we identified a composite signature predictive of systemic recovery, using a joint model on cellular and molecular parameters measured soon after disease onset. New predictions can be generated using the online tool http://shiny.mrc-bsu.cam.ac.uk/apps/covid-19-systemic-recovery-prediction-app , designed to test our findings prospectively.

Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies.

Transmission of SARS-CoV-2 is uncontrolled in many parts of the world; control is compounded in some areas by the higher transmission potential of the B.1.1.7 variant1, which has now been reported in 94 countries. It is unclear whether the response of the virus to vaccines against SARS-CoV-2 on the basis of the prototypic strain will be affected by the mutations found in B.1.1.7. Here we assess the immune responses of individuals after vaccination with the mRNA-based vaccine BNT162b22. We measured neutralizing antibody responses after the first and second immunizations using pseudoviruses that expressed the wild-type spike protein or a mutated spike protein that contained the eight amino acid changes found in the B.1.1.7 variant. The sera from individuals who received the vaccine exhibited a broad range of neutralizing titres against the wild-type pseudoviruses that were modestly reduced against the B.1.1.7 variant. This reduction was also evident in sera from some patients who had recovered from COVID-19. Decreased neutralization of the B.1.1.7 variant was also observed for monoclonal antibodies that target the N-terminal domain (9 out of 10) and the receptor-binding motif (5 out of 31), but not for monoclonal antibodies that recognize the receptor-binding domain that bind outside the receptor-binding motif. Introduction of the mutation that encodes the E484K substitution in the B.1.1.7 background to reflect a newly emerged variant of concern (VOC 202102/02) led to a more-substantial loss of neutralizing activity by vaccine-elicited antibodies and monoclonal antibodies (19 out of 31) compared with the loss of neutralizing activity conferred by the mutations in B.1.1.7 alone. The emergence of the E484K substitution in a B.1.1.7 background represents a threat to the efficacy of the BNT162b2 vaccine.

Demographic, behavioural and occupational risk factors associated with SARS-CoV-2 infection in UK healthcare workers: a retrospective observational study.

OBJECTIVE: Healthcare workers (HCWs) are at higher risk of SARS-CoV-2 infection than the general population. This group is pivotal to healthcare system resilience during the COVID-19, and future, pandemics. We investigated demographic, social, behavioural and occupational risk factors for SARS-CoV-2 infection among HCWs. DESIGN/SETTING/PARTICIPANTS: HCWs enrolled in a large-scale sero-epidemiological study at a UK university teaching hospital were sent questionnaires spanning a 5-month period from March to July 2020. In a retrospective observational cohort study, univariate logistic regression was used to assess factors associated with SARS-CoV-2 infection. A Least Absolute Shrinkage Selection Operator regression model was used to identify variables to include in a multivariate logistic regression model. RESULTS: Among 2258 HCWs, highest ORs associated with SARS-CoV-2 antibody seropositivity on multivariate analysis were having a household member previously testing positive for SARS-CoV-2 antibodies (OR 6.94 (95% CI 4.15 to 11.6); p<0.0001) and being of black ethnicity (6.21 (95% CI 2.69 to 14.3); p<0.0001). Occupational factors associated with a higher risk of seropositivity included working as a physiotherapist (OR 2.78 (95% CI 1.21 to 6.36); p=0.015) and working predominantly in acute medicine (OR 2.72 (95% CI 1.57 to 4.69); p<0.0001) or medical subspecialties (not including infectious diseases) (OR 2.33 (95% CI 1.4 to 3.88); p=0.001). Reporting that adequate personal protective equipment (PPE) was 'rarely' available had an OR of 2.83 (95% CI 1.29 to 6.25; p=0.01). Reporting attending a handover where social distancing was not possible had an OR of 1.39 (95% CI 1.02 to 1.9; p=0.038). CONCLUSIONS: The emergence of SARS-CoV-2 variants and potential vaccine escape continue to threaten stability of healthcare systems worldwide, and sustained vigilance against HCW infection remains a priority. Enhanced risk assessments should be considered for HCWs of black ethnicity, physiotherapists and those working in acute medicine or medical subspecialties. Workplace risk reduction measures include ongoing access to high-quality PPE and effective social distancing measures.

Hospitalisation for COVID-19 predicts long lasting cerebrovascular impairment: A prospective observational cohort study.

Human coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has multiple neurological consequences, but its long-term effect on brain health is still uncertain. The cerebrovascular consequences of COVID-19 may also affect brain health. We studied the chronic effect of COVID-19 on cerebrovascular health, in relation to acute severity, adverse clinical outcomes and in contrast to control group data. Here we assess cerebrovascular health in 45 patients six months after hospitalisation for acute COVID-19 using the resting state fluctuation amplitudes (RSFA) from functional magnetic resonance imaging, in relation to disease severity and in contrast with 42 controls. Acute COVID-19 severity was indexed by COVID-19 WHO Progression Scale, inflammatory and coagulatory biomarkers. Chronic widespread changes in frontoparietal RSFA were related to the severity of the acute COVID-19 episode. This relationship was not explained by chronic cardiorespiratory dysfunction, age, or sex. The level of cerebrovascular dysfunction was associated with cognitive, mental, and physical health at follow-up. The principal findings were consistent across univariate and multivariate approaches. The results indicate chronic cerebrovascular impairment following severe acute COVID-19, with the potential for long-term consequences on cognitive function and mental wellbeing.

Brain injury in COVID-19 is associated with dysregulated innate and adaptive immune responses.

COVID-19 is associated with neurological complications including stroke, delirium and encephalitis. Furthermore, a post-viral syndrome dominated by neuropsychiatric symptoms is common, and is seemingly unrelated to COVID-19 severity. The true frequency and underlying mechanisms of neurological injury are unknown, but exaggerated host inflammatory responses appear to be a key driver of COVID-19 severity. We investigated the dynamics of, and relationship between, serum markers of brain injury [neurofilament light (NfL), glial fibrillary acidic protein (GFAP) and total tau] and markers of dysregulated host response (autoantibody production and cytokine profiles) in 175 patients admitted with COVID-19 and 45 patients with influenza. During hospitalization, sera from patients with COVID-19 demonstrated elevations of NfL and GFAP in a severity-dependent manner, with evidence of ongoing active brain injury at follow-up 4 months later. These biomarkers were associated with elevations of pro-inflammatory cytokines and the presence of autoantibodies to a large number of different antigens. Autoantibodies were commonly seen against lung surfactant proteins but also brain proteins such as myelin associated glycoprotein. Commensurate findings were seen in the influenza cohort. A distinct process characterized by elevation of serum total tau was seen in patients at follow-up, which appeared to be independent of initial disease severity and was not associated with dysregulated immune responses unlike NfL and GFAP. These results demonstrate that brain injury is a common consequence of both COVID-19 and influenza, and is therefore likely to be a feature of severe viral infection more broadly. The brain injury occurs in the context of dysregulation of both innate and adaptive immune responses, with no single pathogenic mechanism clearly responsible.

A prospective study of risk factors associated with seroprevalence of SARS-CoV-2 antibodies in healthcare workers at a large UK teaching hospital.

OBJECTIVES: To describe the risk factors for SARS-CoV-2 infection in UK healthcare workers (HCWs). METHODS: We conducted a prospective sero-epidemiological study of HCWs at a major UK teaching hospital using a SARS-CoV-2 immunoassay. Risk factors for seropositivity were analysed using multivariate logistic regression. RESULTS: 410/5,698 (7·2%) staff tested positive for SARS-CoV-2 antibodies. Seroprevalence was higher in those working in designated COVID-19 areas compared with other areas (9·47% versus 6·16%) Healthcare assistants (aOR 2·06 [95%CI 1·14-3·71]; p=0·016) and domestic and portering staff (aOR 3·45 [95% CI 1·07-11·42]; p=0·039) had significantly higher seroprevalence than other staff groups after adjusting for age, sex, ethnicity and COVID-19 working location. Staff working in acute medicine and medical sub-specialities were also at higher risk (aOR 2·07 [95% CI 1·31-3·25]; p<0·002). Staff from Black, Asian and minority ethnic (BAME) backgrounds had an aOR of 1·65 (95% CI 1·32 - 2·07; p<0·001) compared to white staff; this increased risk was independent of COVID-19 area working. The only symptoms significantly associated with seropositivity in a multivariable model were loss of sense of taste or smell, fever, and myalgia; 31% of staff testing positive reported no prior symptoms. CONCLUSIONS: Risk of SARS-CoV-2 infection amongst HCWs is highly heterogeneous and influenced by COVID-19 working location, role, age and ethnicity. Increased risk amongst BAME staff cannot be accounted for solely by occupational factors.

SARS-CoV-2 spike N-terminal domain modulates TMPRSS2-dependent viral entry and fusogenicity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

Multivariate profile and acute-phase correlates of cognitive deficits in a COVID-19 hospitalised cohort.

Background: Preliminary evidence has highlighted a possible association between severe COVID-19 and persistent cognitive deficits. Further research is required to confirm this association, determine whether cognitive deficits relate to clinical features from the acute phase or to mental health status at the point of assessment, and quantify rate of recovery. Methods: 46 individuals who received critical care for COVID-19 at Addenbrooke's hospital between 10th March 2020 and 31st July 2020 (16 mechanically ventilated) underwent detailed computerised cognitive assessment alongside scales measuring anxiety, depression and post-traumatic stress disorder under supervised conditions at a mean follow up of 6.0 (± 2.1) months following acute illness. Patient and matched control (N = 460) performances were transformed into standard deviation from expected scores, accounting for age and demographic factors using N = 66,008 normative datasets. Global accuracy and response time composites were calculated (G_SScore & G_RT). Linear modelling predicted composite score deficits from acute severity, mental-health status at assessment, and time from hospital admission. The pattern of deficits across tasks was qualitatively compared with normal age-related decline, and early-stage dementia. Findings: COVID-19 survivors were less accurate (G_SScore=-0.53SDs) and slower (G_RT=+0.89SDs) in their responses than expected compared to their matched controls. Acute illness, but not chronic mental health, significantly predicted cognitive deviation from expected scores (G_SScore (p=​​0.0037) and G_RT (p = 0.0366)). The most prominent task associations with COVID-19 were for higher cognition and processing speed, which was qualitatively distinct from the profiles of normal ageing and dementia and similar in magnitude to the effects of ageing between 50 and 70 years of age. A trend towards reduced deficits with time from illness (r∼=0.15) did not reach statistical significance. Interpretation: Cognitive deficits after severe COVID-19 relate most strongly to acute illness severity, persist long into the chronic phase, and recover slowly if at all, with a characteristic profile highlighting higher cognitive functions and processing speed. Funding: This work was funded by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (BRC), NIHR Cambridge Clinical Research Facility (BRC-1215-20014), the Addenbrooke's Charities Trust and NIHR COVID-19 BioResource RG9402. AH is funded by the UK Dementia Research Institute Care Research and Technology Centre and Imperial College London Biomedical Research Centre. ETB and DKM are supported by NIHR Senior Investigator awards. JBR is supported by the Wellcome Trust (220258) and Medical Research Council (SUAG/051 G101400). VFJN is funded by an Academy of Medical Sciences/ The Health Foundation Clinician Scientist Fellowship. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

Evidence of previous SARS-CoV-2 infection in seronegative patients with long COVID.

BACKGROUND: There is currently no consensus on the diagnosis, definition, symptoms, or duration of COVID-19 illness. The diagnostic complexity of Long COVID is compounded in many patients who were or might have been infected with SARS-CoV-2 but not tested during the acute illness and/or are SARS-CoV-2 antibody negative. METHODS: Given the diagnostic conundrum of Long COVID, we set out to investigate SARS-CoV-2-specific T cell responses in patients with confirmed SARS-CoV-2 infection and/or Long COVID from a cohort of mostly non-hospitalised patients. FINDINGS: We discovered that IL-2 release (but not IFN-γ release) from T cells in response to SARS-CoV-2 peptides is both sensitive (75% +/-13%) and specific (88%+/-7%) for previous SARS-CoV-2 infection >6 months after a positive PCR test. We identified that 42-53% of patients with Long COVID, but without detectable SARS-CoV-2 antibodies, nonetheless have detectable SARS-CoV-2 specific T cell responses. INTERPRETATION: Our study reveals evidence (detectable T cell mediated IL-2 release) of previous SARS-CoV-2 infection in seronegative patients with Long COVID. FUNDING: This work was funded by the Addenbrooke's Charitable Trust (900276 to NS), NIHR award (G112259 to NS) and supported by the NIHR Cambridge Biomedical Research Centre. NJM is supported by the MRC (TSF MR/T032413/1) and NHSBT (WPA15-02). PJL is supported by the Wellcome Trust (PRF 210688/Z/18/Z, 084957/Z/08/Z), a Medical Research Council research grant MR/V011561/1 and the United Kingdom Research and a Innovation COVID Immunology Consortium grant (MR/V028448/1).

Decrease in Pediatric Invasive Pneumococcal Disease During the COVID-19 Pandemic.

Measures to limit SARS-CoV-2 transmission in 2020 reduced other viral infections. Among 7 US children's hospitals, invasive pneumococcal disease cumulative incidence decreased by 46% in 2020 vs 2017-2019. Limited droplet transmission of pneumococci and preceding viral pathogens may be responsible.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Using trained dogs and organic semi-conducting sensors to identify asymptomatic and mild SARS-CoV-2 infections: an observational study.

BACKGROUND: A rapid, accurate, non-invasive diagnostic screen is needed to identify people with SARS-CoV-2 infection. We investigated whether organic semi-conducting (OSC) sensors and trained dogs could distinguish between people infected with asymptomatic or mild symptoms, and uninfected individuals, and the impact of screening at ports-of-entry. METHODS: Odour samples were collected from adults, and SARS-CoV-2 infection status confirmed using RT-PCR. OSC sensors captured the volatile organic compound (VOC) profile of odour samples. Trained dogs were tested in a double-blind trial to determine their ability to detect differences in VOCs between infected and uninfected individuals, with sensitivity and specificity as the primary outcome. Mathematical modelling was used to investigate the impact of bio-detection dogs for screening. RESULTS: About, 3921 adults were enrolled in the study and odour samples collected from 1097 SARS-CoV-2 infected and 2031 uninfected individuals. OSC sensors were able to distinguish between SARS-CoV-2 infected individuals and uninfected, with sensitivity from 98% (95% CI 95-100) to 100% and specificity from 99% (95% CI 97-100) to 100%. Six dogs were able to distinguish between samples with sensitivity ranging from 82% (95% CI 76-87) to 94% (95% CI 89-98) and specificity ranging from 76% (95% CI 70-82) to 92% (95% CI 88-96). Mathematical modelling suggests that dog screening plus a confirmatory PCR test could detect up to 89% of SARS-CoV-2 infections, averting up to 2.2 times as much transmission compared to isolation of symptomatic individuals only. CONCLUSIONS: People infected with SARS-CoV-2, with asymptomatic or mild symptoms, have a distinct odour that can be identified by sensors and trained dogs with a high degree of accuracy. Odour-based diagnostics using sensors and/or dogs may prove a rapid and effective tool for screening large numbers of people.Trial Registration NCT04509713 (clinicaltrials.gov).

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Coagulation Factor V is a T cell inhibitor expressed by leukocytes in COVID-19

Clotting Factor V (FV) is primarily synthesised in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes and T regulatory cells as sources of increased FV in hospitalised patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system. Graphical

Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19.

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

The impact of hypoxia on B cells in COVID-19.

BACKGROUND: Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This presentation resembles the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. METHODS: Detailed B cell phenotyping was undertaken by flow-cytometry on longitudinal samples from patients with COVID-19 across a range of severities (NIHR Cambridge BioResource). The impact of hypoxia on the transcriptome was assessed by single-cell and whole blood RNA sequencing analysis. The direct effect of hypoxia on B cells was determined through immunisation studies in genetically modified and hypoxia-exposed mice. FINDINGS: We demonstrate the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes also observed in B cell VHL-deficient mice. These findings were associated with hypoxia-related transcriptional changes in COVID-19 patient B cells, and similar B cell abnormalities were seen in mice kept in hypoxic conditions. INTERPRETATION: Hypoxia may contribute to the pronounced and persistent B cell pathology observed in acute COVID-19 pneumonia. Assessment of the impact of early oxygen therapy on these immune defects should be considered, as their correction could contribute to improved outcomes. FUNDING: Evelyn Trust, Addenbrooke's Charitable Trust, UKRI/NIHR, Wellcome Trust.

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination

Kotagiri et al. find that SARS-CoV-2 infection versus vaccination induces distinct changes in the B cell receptor repertoire, including prominent clonal expansion in IgA and IgM after infection, but IgG after vaccination. A broad anti-spike response to infection contrasts with a narrower RBD-focused one after vaccination, potentially informing vaccination strategies.