Impact of peptide:HLA complex stability for the identification of SARS-CoV-2-specific CD8+T cells.

Induction of a lasting protective immune response is dependent on presentation of epitopes to patrolling T cells through the HLA complex. While peptide:HLA (pHLA) complex affinity alone is widely exploited for epitope selection, we demonstrate that including the pHLA complex stability as a selection parameter can significantly reduce the high false discovery rate observed with predicted affinity. In this study, pHLA complex stability was measured on three common class I alleles and 1286 overlapping 9-mer peptides derived from the SARS-CoV-2 Spike protein. Peptides were pooled based on measured stability and predicted affinity. Strikingly, stability of the pHLA complex was shown to strongly select for immunogenic epitopes able to activate functional CD8+T cells. This result was observed across the three studied alleles and in both vaccinated and convalescent COVID-19 donors. Deconvolution of peptide pools showed that specific CD8+T cells recognized one or two dominant epitopes. Moreover, SARS-CoV-2 specific CD8+T cells were detected by tetramer-staining across multiple donors. In conclusion, we show that stability analysis of pHLA is a key factor for identifying immunogenic epitopes.

mRNA vaccination drives differential mucosal neutralizing antibody profiles in naïve and SARS-CoV-2 previously-infected individuals.

Two doses of BNT162b2 mRNA vaccine induces a strong systemic SARS-CoV-2 specific humoral response. However, SARS-CoV-2 airborne transmission makes mucosal immune response a crucial first line of defense. Therefore, we characterized SARS-CoV-2-specific IgG responses induced by BNT162b2 vaccine, as well as IgG responses to other pathogenic and seasonal human coronaviruses in oral fluid and plasma from 200 UK healthcare workers who were naïve (N=62) or previously infected with SARS-CoV-2 (N=138) using a pan-coronavirus multiplex binding immunoassay (Meso Scale Discovery®). Additionally, we investigated the impact of historical SARS-CoV-2 infection on vaccine-induced IgG, IgA and neutralizing responses in selected oral fluid samples before vaccination, after a first and second dose of BNT162b2, as well as following a third dose of mRNA vaccine or breakthrough infections using the same immunoassay and an ACE2 inhibition assay. Prior to vaccination, we found that spike-specific IgG levels in oral fluid positively correlated with IgG levels in plasma from previously-infected individuals (Spearman r=0.6858, p<0.0001) demonstrating that oral fluid could be used as a proxy for the presence of plasma SARS-CoV-2 IgG. However, the sensitivity was lower in oral fluid (0.85, 95% CI 0.77-0.91) than in plasma (0.94, 95% CI 0.88-0.97). Similar kinetics of mucosal and systemic spike-specific IgG levels were observed following vaccination in naïve and previously-infected individuals, respectively. In addition, a significant enhancement of OC43 and HKU1 spike-specific IgG levels was observed in previously-infected individuals following one vaccine dose in oral fluid (OC43 S: p<0.0001; HKU1 S: p=0.0423) suggesting cross-reactive IgG responses to seasonal beta coronaviruses. Mucosal spike-specific IgA responses were induced by mRNA vaccination particularly in previously-infected individuals (71%) but less frequently in naïve participants (23%). Neutralizing responses to SARS-CoV-2 ancestral and variants of concerns were detected following vaccination in naïve and previously-infected participants, with likely contribution from both IgG and IgA in previously-infected individuals (correlations between neutralizing responses and IgG: Spearman r=0.5642, p<0.0001; IgA: Spearman r=0.4545, p=0.0001). We also observed that breakthrough infections or a third vaccine dose enhanced mucosal antibody levels and neutralizing responses. These data contribute to show that a previous SARS-CoV-2 infection tailors the mucosal antibody profile induced by vaccination.

Prospective validation of the 4C prognostic models for adults hospitalised with COVID-19 using the ISARIC WHO Clinical Characterisation Protocol.

PURPOSE: To prospectively validate two risk scores to predict mortality (4C Mortality) and in-hospital deterioration (4C Deterioration) among adults hospitalised with COVID-19. METHODS: Prospective observational cohort study of adults (age ≥18 years) with confirmed or highly suspected COVID-19 recruited into the International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) WHO Clinical Characterisation Protocol UK (CCP-UK) study in 306 hospitals across England, Scotland and Wales. Patients were recruited between 27 August 2020 and 17 February 2021, with at least 4 weeks follow-up before final data extraction. The main outcome measures were discrimination and calibration of models for in-hospital deterioration (defined as any requirement of ventilatory support or critical care, or death) and mortality, incorporating predefined subgroups. RESULTS: 76 588 participants were included, of whom 27 352 (37.4%) deteriorated and 12 581 (17.4%) died. Both the 4C Mortality (0.78 (0.77 to 0.78)) and 4C Deterioration scores (pooled C-statistic 0.76 (95% CI 0.75 to 0.77)) demonstrated consistent discrimination across all nine National Health Service regions, with similar performance metrics to the original validation cohorts. Calibration remained stable (4C Mortality: pooled slope 1.09, pooled calibration-in-the-large 0.12; 4C Deterioration: 1.00, -0.04), with no need for temporal recalibration during the second UK pandemic wave of hospital admissions. CONCLUSION: Both 4C risk stratification models demonstrate consistent performance to predict clinical deterioration and mortality in a large prospective second wave validation cohort of UK patients. Despite recent advances in the treatment and management of adults hospitalised with COVID-19, both scores can continue to inform clinical decision making. TRIAL REGISTRATION NUMBER: ISRCTN66726260.

SARS-CoV-2 antibody responses associate with sex, age and disease severity in previously uninfected people admitted to hospital with COVID-19: An ISARIC4C prospective study.

The SARS-CoV-2 pandemic enables the analysis of immune responses induced against a novel coronavirus infecting immunologically naïve individuals. This provides an opportunity for analysis of immune responses and associations with age, sex and disease severity. Here we measured an array of solid-phase binding antibody and viral neutralising Ab (nAb) responses in participants (n=337) of the ISARIC4C cohort and characterised their correlation with peak disease severity during acute infection and early convalescence. Overall, the responses in a Double Antigen Binding Assay (DABA) for antibody to the receptor binding domain (anti-RBD) correlated well with IgM as well as IgG responses against viral spike, S1 and nucleocapsid protein (NP) antigens. DABA reactivity also correlated with nAb. As we and others reported previously, there is greater risk of severe disease and death in older men, whilst the sex ratio was found to be equal within each severity grouping in younger people. In older males with severe disease (mean age 68 years), peak antibody levels were found to be delayed by one to two weeks compared with women, and nAb responses were delayed further. Additionally, we demonstrated that solid-phase binding antibody responses reached higher levels in males as measured via DABA and IgM binding against Spike, NP and S1 antigens. In contrast, this was not observed for nAb responses. When measuring SARS-CoV-2 RNA transcripts (as a surrogate for viral shedding) in nasal swabs at recruitment, we saw no significant differences by sex or disease severity status. However, we have shown higher antibody levels associated with low nasal viral RNA indicating a role of antibody responses in controlling viral replication and shedding in the upper airway. In this study, we have shown discernible differences in the humoral immune responses between males and females and these differences associate with age as well as with resultant disease severity.

Implementation of corticosteroids in treatment of COVID-19 in the ISARIC WHO Clinical Characterisation Protocol UK: prospective, cohort study.

BACKGROUND: Dexamethasone was the first intervention proven to reduce mortality in patients with COVID-19 being treated in hospital. We aimed to evaluate the adoption of corticosteroids in the treatment of COVID-19 in the UK after the RECOVERY trial publication on June 16, 2020, and to identify discrepancies in care. METHODS: We did an audit of clinical implementation of corticosteroids in a prospective, observational, cohort study in 237 UK acute care hospitals between March 16, 2020, and April 14, 2021, restricted to patients aged 18 years or older with proven or high likelihood of COVID-19, who received supplementary oxygen. The primary outcome was administration of dexamethasone, prednisolone, hydrocortisone, or methylprednisolone. This study is registered with ISRCTN, ISRCTN66726260. FINDINGS: Between June 17, 2020, and April 14, 2021, 47 795 (75·2%) of 63 525 of patients on supplementary oxygen received corticosteroids, higher among patients requiring critical care than in those who received ward care (11 185 [86·6%] of 12 909 vs 36 415 [72·4%] of 50 278). Patients 50 years or older were significantly less likely to receive corticosteroids than those younger than 50 years (adjusted odds ratio 0·79 [95% CI 0·70-0·89], p=0·0001, for 70-79 years; 0·52 [0·46-0·58], p<0·0001, for >80 years), independent of patient demographics and illness severity. 84 (54·2%) of 155 pregnant women received corticosteroids. Rates of corticosteroid administration increased from 27·5% in the week before June 16, 2020, to 75-80% in January, 2021. INTERPRETATION: Implementation of corticosteroids into clinical practice in the UK for patients with COVID-19 has been successful, but not universal. Patients older than 70 years, independent of illness severity, chronic neurological disease, and dementia, were less likely to receive corticosteroids than those who were younger, as were pregnant women. This could reflect appropriate clinical decision making, but the possibility of inequitable access to life-saving care should be considered. FUNDING: UK National Institute for Health Research and UK Medical Research Council.

Evolution of long-term vaccine induced and hybrid immunity in healthcare workers after different COVID19 vaccine regimens: a longitudinal cohort study

Background 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 (Protective immunity from T cells in Healthcare workers (PITCH), within the larger SARS-CoV-2 immunity & 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. Methods 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. Findings 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 and broader T cell responses compared with never-infected people – a feature maintained until 6 months after the third dose. Conclusions 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. Funding Department for Health and Social Care, Medical Research Council Graphical abstract Moore et al. studied antibody and cellular responses to COVID-19 vaccines before and after dose 3. Antibody responses waned, but T cell responses were well maintained. T cells recognised Omicron variants better and for longer than antibodies. Differences due to vaccine regimen and previous infection evened out over time.

Evaluation of eight lateral flow tests for the detection of anti-SARS-CoV-2 antibodies in a vaccinated population.

BACKGROUND: Rapid determination of an individual's antibody status can be beneficial in understanding an individual's immune response to SARS-CoV-2 and for initiation of therapies that are only deemed effective in sero-negative individuals. Antibody lateral flow tests (LFTs) have potential to address this need as a rapid, point of care test. METHODS: Here we present a proof-of-concept evaluation of eight LFT brands using sera from 95 vaccinated individuals to determine sensitivity for detecting vaccination generated antibodies. Samples were analysed on eight different brands of antibody LFT and an automated chemiluminescent microparticle immunoassay (CMIA) that identifies anti-spike antibodies which was used as our reference standard. RESULTS: All 95 (100%) participants tested positive for anti-spike antibodies by the chemiluminescent microparticle immunoassay (CMIA) reference standard post-dose two of their SARS-CoV-2 vaccine: BNT162b2 (Pfizer/BioNTech, n = 60), AZD1222 (AstraZeneca, n = 31), mRNA-1273 (Moderna, n = 2) and Undeclared Vaccine Brand (n = 2). Sensitivity increased from dose one to dose two in six out of eight LFTs with three tests achieving 100% sensitivity at dose two in detecting anti-spike antibodies. CONCLUSIONS: These tests are demonstrated to be highly sensitive to detect raised antibody levels in vaccinated individuals. RDTs are low cost and rapid alternatives to ELISA based systems.

The P323L substitution in the SARS-CoV-2 polymerase (NSP12) confers a selective advantage during infection.

BACKGROUND: The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure. RESULTS: Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323. CONCLUSIONS: These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

Exposed seronegative: Cellular immune responses to SARS-CoV-2 in the absence of seroconversion.

The factors determining whether infection will occur following exposure to SARS-CoV-2 remain elusive. Certain SARS-CoV-2-exposed individuals mount a specific T-cell response but fail to seroconvert, representing a population that may provide further clarity on the nature of infection susceptibility and correlates of protection against SARS-CoV-2. Exposed seronegative individuals have been reported in patients exposed to the blood-borne pathogens Human Immunodeficiency virus and Hepatitis C virus and the sexually transmitted viruses Hepatitis B virus and Herpes Simplex virus. By comparing the quality of seronegative T-cell responses to SARS-CoV-2 with seronegative cellular immunity to these highly divergent viruses, common patterns emerge that offer insights on the role of cellular immunity against infection. For both SARS-CoV-2 and Hepatitis C, T-cell responses in exposed seronegatives are consistently higher than in unexposed individuals, but lower than in infected, seropositive patients. Durability of T-cell responses to Hepatitis C is dependent upon repeated exposure to antigen - single exposures do not generate long-lived memory T-cells. Finally, exposure to SARS-CoV-2 induces varying degrees of immune activation, suggesting that exposed seronegative individuals represent points on a spectrum rather than a discrete group. Together, these findings paint a complex landscape of the nature of infection but provide clues as to what may be protective early on in SARS-CoV-2 disease course. Further research on this phenomenon, particularly through cohort studies, is warranted.

Comparison of UK paediatric SARS-CoV-2 admissions across the first and second pandemic waves.

BACKGROUND: We hypothesised that the clinical characteristics of hospitalised children and young people (CYP) with SARS-CoV-2 in the UK second wave (W2) would differ from the first wave (W1) due to the alpha variant (B.1.1.7), school reopening and relaxation of shielding. METHODS: Prospective multicentre observational cohort study of patients <19 years hospitalised in the UK with SARS-CoV-2 between 17/01/20 and 31/01/21. Clinical characteristics were compared between W1 and W2 (W1 = 17/01/20-31/07/20,W2 = 01/08/20-31/01/21). RESULTS: 2044 CYP < 19 years from 187 hospitals. 427/2044 (20.6%) with asymptomatic/incidental SARS-CoV-2 were excluded from main analysis. 16.0% (248/1548) of symptomatic CYP were admitted to critical care and 0.8% (12/1504) died. 5.6% (91/1617) of symptomatic CYP had Multisystem Inflammatory Syndrome in Children (MIS-C). After excluding CYP with MIS-C, patients in W2 had lower Paediatric Early Warning Scores (PEWS, composite vital sign score), lower antibiotic use and less respiratory and cardiovascular support than W1. The proportion of CYP admitted to critical care was unchanged. 58.0% (938/1617) of symptomatic CYP had no reported comorbidity. Patients without co-morbidities were younger (42.4%, 398/938, <1 year), had lower PEWS, shorter length of stay and less respiratory support. CONCLUSIONS: We found no evidence of increased disease severity in W2 vs W1. A large proportion of hospitalised CYP had no comorbidity. IMPACT: No evidence of increased severity of COVID-19 admissions amongst children and young people (CYP) in the second vs first wave in the UK, despite changes in variant, relaxation of shielding and return to face-to-face schooling. CYP with no comorbidities made up a significant proportion of those admitted. However, they had shorter length of stays and lower treatment requirements than CYP with comorbidities once those with MIS-C were excluded. At least 20% of CYP admitted in this cohort had asymptomatic/incidental SARS-CoV-2 infection. This paper was presented to SAGE to inform CYP vaccination policy in the UK.

Outcome of COVID-19 in hospitalised immunocompromised patients: An analysis of the WHO ISARIC CCP-UK prospective cohort study.

BACKGROUND: Immunocompromised patients may be at higher risk of mortality if hospitalised with Coronavirus Disease 2019 (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 AND FINDINGS: We included patients > = 19 years with symptomatic community-acquired COVID-19 recruited to the ISARIC WHO Clinical Characterisation Protocol UK prospective cohort study. 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 comorbidities. We used Bayesian logistic regression to explore mortality over time. Between 17 January 2020 and 28 February 2022, we recruited 156,552 eligible patients, of whom 21,954 (14%) were immunocompromised. In total, 29% (n = 6,499) of immunocompromised and 21% (n = 28,608) of immunocompetent patients died in hospital. The odds of in-hospital mortality were elevated for immunocompromised patients (adjusted OR 1.44, 95% CI [1.39, 1.50], p < 0.001). Not all immunocompromising conditions had the same risk, for example, patients on active cancer treatment were less likely to have their care escalated to intensive care (adjusted OR 0.77, 95% CI [0.7, 0.85], p < 0.001) or ventilation (adjusted OR 0.65, 95% CI [0.56, 0.76], p < 0.001). However, cancer patients were more likely to die (adjusted OR 2.0, 95% CI [1.87, 2.15], p < 0.001). Analyses were adjusted for age, sex, socioeconomic deprivation, comorbidities, and vaccination status. 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 to 69 years was 88% for men and 83% for women, and for those >80 years was 99% for men and 98% for women. The study is limited by a lack of detailed drug data prior to admission, including steroid doses, meaning that we may have incorrectly categorised some immunocompromised patients as immunocompetent. CONCLUSIONS: Immunocompromised patients remain at elevated risk of death from COVID-19. Targeted measures such as additional vaccine doses, monoclonal antibodies, and nonpharmaceutical preventive interventions should be continually encouraged for this patient group. TRIAL REGISTRATION: ISRCTN 66726260.

SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination.

BACKGROUND: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. METHODS: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FINDINGS: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. INTERPRETATION: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. FUNDING: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript.

Changing patterns of SARS-CoV-2 infection through Delta and Omicron waves by vaccination status, previous infection and neighbourhood deprivation: a cohort analysis of 2.7 M people.

BACKGROUND: Our study examines if SARS-CoV-2 infections varied by vaccination status, if an individual had previously tested positive and by neighbourhood socioeconomic deprivation across the Delta and Omicron epidemic waves of SARS-CoV-2. METHODS: Population cohort study using electronic health records for 2.7 M residents in Cheshire and Merseyside, England (3rd June 2021 to 1st March 2022). Our outcome variable was registered positive test for SARS-CoV-2. Explanatory variables were vaccination status, previous registered positive test and neighbourhood socioeconomic deprivation. Cox regression models were used to analyse associations. RESULTS: Originally higher SARS-CoV-2 rates in the most socioeconomically deprived neighbourhoods changed to being higher in the least deprived neighbourhoods from the 1st September 2021, and were inconsistent during the Omicron wave. Individuals who were fully vaccinated (two doses) were associated with fewer registered positive tests (e.g., individuals engaged in testing between 1st September and 27th November 2021-Hazards Ratio (HR) = 0.48, 95% Confidence Intervals (CIs) = 0.47-0.50. Individuals with a previous registered positive test were also less likely to have a registered positive test (e.g., individuals engaged in testing between 1st September and 27th November 2021-HR = 0.16, 95% CIs = 0.15-0.18. However, the Omicron period saw smaller effect sizes for both vaccination status and previous registered positive test. CONCLUSIONS: Changing patterns of SARS-CoV-2 infections during the Delta and Omicron waves reveals a dynamic pandemic that continues to affect diverse communities in sometimes unexpected ways.

mRNA vaccination drives differential mucosal neutralizing antibody profiles in naïve and SARS-CoV-2 previously-infected individuals

Two doses of BNT162b2 mRNA vaccine induces a strong systemic SARS-CoV-2 specific humoral response. However, SARS-CoV-2 airborne transmission makes mucosal immune response a crucial first line of defense. Therefore, we characterized SARS-CoV-2-specific IgG responses induced by BNT162b2 vaccine, as well as IgG responses to other pathogenic and seasonal human coronaviruses in oral fluid and plasma from 200 UK healthcare workers who were naïve (N=62) or previously infected with SARS-CoV-2 (N=138) using a pan-coronavirus multiplex binding immunoassay (Meso Scale Discovery®). Additionally, we investigated the impact of historical SARS-CoV-2 infection on vaccine-induced IgG, IgA and neutralizing responses in selected oral fluid samples before vaccination, after a first and second dose of BNT162b2, as well as following a third dose of mRNA vaccine or breakthrough infections using the same immunoassay and an ACE2 inhibition assay. Prior to vaccination, we found that spike-specific IgG levels in oral fluid positively correlated with IgG levels in plasma from previously-infected individuals (Spearman r=0.6858, p<0.0001) demonstrating that oral fluid could be used as a proxy for the presence of plasma SARS-CoV-2 IgG. However, the sensitivity was lower in oral fluid (0.85, 95% CI 0.77-0.91) than in plasma (0.94, 95% CI 0.88-0.97). Similar kinetics of mucosal and systemic spike-specific IgG levels were observed following vaccination in naïve and previously-infected individuals, respectively. In addition, a significant enhancement of OC43 and HKU1 spike-specific IgG levels was observed in previously-infected individuals following one vaccine dose in oral fluid (OC43 S: p<0.0001;HKU1 S: p=0.0423) suggesting cross-reactive IgG responses to seasonal beta coronaviruses. Mucosal spike-specific IgA responses were induced by mRNA vaccination particularly in previously-infected individuals (71%) but less frequently in naïve participants (23%). Neutralizing responses to SARS-CoV-2 ancestral and variants of concerns were detected following vaccination in naïve and previously-infected participants, with likely contribution from both IgG and IgA in previously-infected individuals (correlations between neutralizing responses and IgG: Spearman r=0.5642, p<0.0001;IgA: Spearman r=0.4545, p=0.0001). We also observed that breakthrough infections or a third vaccine dose enhanced mucosal antibody levels and neutralizing responses. These data contribute to show that a previous SARS-CoV-2 infection tailors the mucosal antibody profile induced by vaccination.

Procalcitonin Is Not a Reliable Biomarker of Bacterial Coinfection in People With Coronavirus Disease 2019 Undergoing Microbiological Investigation at the Time of Hospital Admission.

Admission procalcitonin measurements and microbiology results were available for 1040 hospitalized adults with coronavirus disease 2019 (from 48 902 included in the International Severe Acute Respiratory and Emerging Infections Consortium World Health Organization Clinical Characterisation Protocol UK study). Although procalcitonin was higher in bacterial coinfection, this was neither clinically significant (median [IQR], 0.33 [0.11-1.70] ng/mL vs 0.24 [0.10-0.90] ng/mL) nor diagnostically useful (area under the receiver operating characteristic curve, 0.56 [95% confidence interval, .51-.60]).

T-cell and antibody responses to first BNT162b2 vaccine dose in previously infected and SARS-CoV-2-naive UK health-care workers: a multicentre prospective cohort study.

BACKGROUND: Previous infection with SARS-CoV-2 affects the immune response to the first dose of the SARS-CoV-2 vaccine. We aimed to compare SARS-CoV-2-specific T-cell and antibody responses in health-care workers with and without previous SARS-CoV-2 infection following a single dose of the BNT162b2 (tozinameran; Pfizer-BioNTech) mRNA vaccine. METHODS: We sampled health-care workers enrolled in the PITCH study across four hospital sites in the UK (Oxford, Liverpool, Newcastle, and Sheffield). All health-care workers aged 18 years or older consenting to participate in this prospective cohort study were included, with no exclusion criteria applied. Blood samples were collected where possible before vaccination and 28 (±7) days following one or two doses (given 3-4 weeks apart) of the BNT162b2 vaccine. Previous infection was determined by a documented SARS-CoV-2-positive RT-PCR result or the presence of positive anti-SARS-CoV-2 nucleocapsid antibodies. We measured spike-specific IgG antibodies and quantified T-cell responses by interferon-γ enzyme-linked immunospot assay in all participants where samples were available at the time of analysis, comparing SARS-CoV-2-naive individuals to those with previous infection. FINDINGS: Between Dec 9, 2020, and Feb 9, 2021, 119 SARS-CoV-2-naive and 145 previously infected health-care workers received one dose, and 25 SARS-CoV-2-naive health-care workers received two doses, of the BNT162b2 vaccine. In previously infected health-care workers, the median time from previous infection to vaccination was 268 days (IQR 232-285). At 28 days (IQR 27-33) after a single dose, the spike-specific T-cell response measured in fresh peripheral blood mononuclear cells (PBMCs) was higher in previously infected (n=76) than in infection-naive (n=45) health-care workers (median 284 [IQR 150-461] vs 55 [IQR 24-132] spot-forming units [SFUs] per 106 PBMCs; p<0·0001). With cryopreserved PBMCs, the T-cell response in previously infected individuals (n=52) after one vaccine dose was equivalent to that of infection-naive individuals (n=19) after receiving two vaccine doses (median 152 [IQR 119-275] vs 162 [104-258] SFUs/106 PBMCs; p=1·00). Anti-spike IgG antibody responses following a single dose in 142 previously infected health-care workers (median 270 373 [IQR 203 461-535 188] antibody units [AU] per mL) were higher than in 111 infection-naive health-care workers following one dose (35 001 [17 099-55 341] AU/mL; p<0·0001) and higher than in 25 infection-naive individuals given two doses (180 904 [108 221-242 467] AU/mL; p<0·0001). INTERPRETATION: A single dose of the BNT162b2 vaccine is likely to provide greater protection against SARS-CoV-2 infection in individuals with previous SARS-CoV-2 infection, than in SARS-CoV-2-naive individuals, including against variants of concern. Future studies should determine the additional benefit of a second dose on the magnitude and durability of immune responses in individuals vaccinated following infection, alongside evaluation of the impact of extending the interval between vaccine doses. FUNDING: UK Department of Health and Social Care, and UK Coronavirus Immunology Consortium.

Analysis of SARS-CoV-2 known and novel subgenomic mRNAs in cell culture, animal model, and clinical samples using LeTRS, a bioinformatic tool to identify unique sequence identifiers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgmRNAs has a unique 5' sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS junction), that can be identified using sequencing. High-resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture and animal models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS junctions and can be used as a proxy to quantify sgmRNAs for understanding virus biology. LeTRS is readily adaptable for other coronaviruses such as Middle East respiratory syndrome coronavirus or a future newly discovered coronavirus. LeTRS was tested on published data sets and novel clinical samples from patients and longitudinal samples from animal models with coronavirus disease 2019. LeTRS identified known leader-TRS junctions and identified putative novel sgmRNAs that were common across different mammalian species. This may be indicative of an evolutionary mechanism where plasticity in transcription generates novel open reading frames, which can then subject to selection pressure. The data indicated multiphasic abundance of sgmRNAs in two different animal models. This recapitulates the relative sgmRNA abundance observed in cells at early points in infection but not at late points. This pattern is reflected in some human nasopharyngeal samples and therefore has implications for transmission models and nucleic acid-based diagnostics. LeTRS provides a quantitative measure of sgmRNA abundance from sequencing data. This can be used to assess the biology of SARS-CoV-2 (or other coronaviruses) in clinical and nonclinical samples, especially to evaluate different variants and medical countermeasures that may influence viral RNA synthesis.

Methods of SARS-CoV-2 Inactivation.

Inactivation methods allow for hazard group 3 (HG3) pathogens to be disposed of and used safely in downstream experiments and assays to be carried out at lower containment levels. Commonly used viral inactivation methods include heat inactivation, fixation methods, ultraviolet (UV) light and detergent inactivation. Here we describe known methods used to inactivate SARS-CoV-2 for safe downstream biological assays.