Establishing SARS-CoV-2 Membrane protein-specific antibodies as a valuable serological target via high-content microscopy

The prevalence and strength of serological responses mounted towards SARS-CoV-2 proteins other than nucleocapsid (N) and spike (S), which may be of use as additional serological markers, remains underexplored. Using high content microscopy to assess antibody responses against full length StrepTagged SARS-CoV-2 proteins, we found that 85% (166/196) of unvaccinated individuals with RT-PCR confirmed SARS-CoV-2 infections and 74% (31/42) of individuals infected after being vaccinated developed detectable IgG against the structural protein M, which is higher than previous estimates. Compared with N antibodies, M IgG displayed a shallower time-dependent decay and greater specificity. Sensitivity for SARS-CoV-2 seroprevalence was enhanced when N and M IgG detection was combined. These findings indicate that screening for M seroconversion may be a good approach for detecting additional vaccine breakthrough infections and highlight the potential to use HCM as a rapidly deployable method to identify the most immunogenic targets of newly emergent pathogens. Graphical

Circulation of respiratory viruses during the COVID-19 pandemic in The Gambia.

Background: In many countries, non-pharmaceutical interventions to limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission resulted in significant reductions in other respiratory viruses. However, similar data from Africa are limited. We explored the extent to which viruses such as influenza and rhinovirus co-circulated with SARS-CoV-2 in The Gambia during the COVID-19 pandemic.  Methods: Between April 2020 and March 2022, respiratory viruses were detected using RT-PCR in nasopharyngeal swabs from 1397 participants with influenza-like illness. An assay to detect SARS-CoV-2 and a viral multiplex RT-PCR assay was used as previously described  to detect influenza A and B, respiratory syncytial virus (RSV) A and B, parainfluenza viruses 1-4, human metapneumovirus (HMPV), adenovirus, seasonal coronaviruses (229E, OC43, NL63) and human rhinovirus. Results: Overall virus positivity was 44.2%, with prevalence higher in children <5 years (80%) compared to children aged 5-17 years (53.1%), adults aged 18-50 (39.5%) and >50 years (39.9%), p<0.0001. After SARS-CoV-2 (18.3%), rhinoviruses (10.5%) and influenza viruses (5.5%) were the most prevalent. SARS-CoV-2 positivity was lower in children <5 (4.3%) and 5-17 years (12.7%) than in adults aged 18-50 (19.3%) and >50 years (24.3%), p<0.0001. In contrast, rhinoviruses were most prevalent in children <5 years (28.7%), followed by children aged 5-17 (15.8%), adults aged 18-50 (8.3%) and >50 years (6.3%), p<0.0001. Four SARS-CoV-2 waves occurred, with 36.1%-52.4% SARS-CoV-2 positivity during peak months. Influenza infections were observed in both 2020 and 2021 during the rainy season as expected (peak positivity 16.4%-23.5%). Peaks of rhinovirus were asynchronous to the months when SARS-CoV-2 and influenza peaked. Conclusion: Our data show that many respiratory viruses continued to circulate during the COVID-19 pandemic in The Gambia, including human rhinoviruses, despite the presence of NPIs during the early stages of the pandemic, and influenza peaks during expected months.

Risk factors for SARS-CoV-2 seroprevalence following the first pandemic wave in UK healthcare workers in a large NHS Foundation Trust.

Background: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. Methods: HCWs at Sheffield Teaching Hospitals NHS Foundation Trust were prospectively enrolled and sampled at two time points. We developed an in-house ELISA for testing participant serum for SARS-CoV-2 IgG and IgA reactivity against Spike and Nucleoprotein. Data were analysed using three statistical models: a seroprevalence model, an antibody kinetics model, and a heterogeneous sensitivity model. Results: Our in-house assay had a sensitivity of 99·47% and specificity of 99·56%. We found that 24·4% (n=311/1275) of HCWs were seropositive as of 12th June 2020. Of these, 39·2% (n=122/311) were asymptomatic. The highest adjusted seroprevalence was measured in HCWs on the Acute Medical Unit (41·1%, 95% CrI 30·0-52·9) and in Physiotherapists and Occupational Therapists (39·2%, 95% CrI 24·4-56·5). Older age groups showed overall higher median antibody titres. Further modelling suggests that, for a serological assay with an overall sensitivity of 80%, antibody titres may be markedly affected by differences in age, with sensitivity estimates of 89% in those over 60 years but 61% in those ≤30 years. Conclusions:  HCWs in acute medical units and those working closely with COVID-19 patients were at highest risk of infection, though whether these are infections acquired from patients or other staff is unknown. Current serological assays may underestimate seroprevalence in younger age groups if validated using sera from older and/or more severe COVID-19 cases.

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.

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.

Rapid escape of new SARS-CoV-2 Omicron variants from BA.2 directed antibody responses

In November 2021 Omicron BA.1, containing a raft of new spike mutations emerged and quickly spread globally. Intense selection pressure to escape the antibody response produced by vaccines or SARS-CoV-2 infection then led to a rapid succession of Omicron sub-lineages with waves of BA.2 then BA.4/5 infection. Recently, many variants have emerged such as BQ.1 and XBB, which carry up to 8 additional RBD amino-acid substitutions compared to BA.2. We describe a panel of 25 potent mAbs generated from vaccinees suffering BA.2 breakthrough infections. Epitope mapping shows potent mAb binding shifting to 3 clusters, 2 corresponding to early-pandemic binding hotspots. The RBD mutations in recent variants map close to these binding sites and knock out or severely knock down neutralization activity of all but 1 potent mAb. This recent mAb escape corresponds with large falls in neutralization titre of vaccine or BA.1, BA.2 or BA.4/5 immune serum. Graphical Dijokaite-Guraliuc et al. analyse potently neutralizing antibodies from vaccinated individuals with BA.2 breakthrough infections. The antibodies bind 3 sites on the receptor binding domain, 2 in common with early pandemic antibodies. Mutations in more recent variants map closely to these sites leading to reduced neutralization in all but one mAb.

Rapid escape of new SARS-CoV-2 Omicron variants from BA.2-directed antibody responses.

In November 2021, Omicron BA.1, containing a raft of new spike mutations, emerged and quickly spread globally. Intense selection pressure to escape the antibody response produced by vaccines or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection then led to a rapid succession of Omicron sub-lineages with waves of BA.2 and then BA.4/5 infection. Recently, many variants have emerged such as BQ.1 and XBB, which carry up to 8 additional receptor-binding domain (RBD) amino acid substitutions compared with BA.2. We describe a panel of 25 potent monoclonal antibodies (mAbs) generated from vaccinees suffering BA.2 breakthrough infections. Epitope mapping shows potent mAb binding shifting to 3 clusters, 2 corresponding to early-pandemic binding hotspots. The RBD mutations in recent variants map close to these binding sites and knock out or severely knock down neutralization activity of all but 1 potent mAb. This recent mAb escape corresponds with large falls in neutralization titer of vaccine or BA.1, BA.2, or BA.4/5 immune serum.

SARS-CoV-2 variant biology: immune escape, transmission and fitness.

In late 2020, after circulating for almost a year in the human population, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited a major step change in its adaptation to humans. These highly mutated forms of SARS-CoV-2 had enhanced rates of transmission relative to previous variants and were termed 'variants of concern' (VOCs). Designated Alpha, Beta, Gamma, Delta and Omicron, the VOCs emerged independently from one another, and in turn each rapidly became dominant, regionally or globally, outcompeting previous variants. The success of each VOC relative to the previously dominant variant was enabled by altered intrinsic functional properties of the virus and, to various degrees, changes to virus antigenicity conferring the ability to evade a primed immune response. The increased virus fitness associated with VOCs is the result of a complex interplay of virus biology in the context of changing human immunity due to both vaccination and prior infection. In this Review, we summarize the literature on the relative transmissibility and antigenicity of SARS-CoV-2 variants, the role of mutations at the furin spike cleavage site and of non-spike proteins, the potential importance of recombination to virus success, and SARS-CoV-2 evolution in the context of T cells, innate immunity and population immunity. SARS-CoV-2 shows a complicated relationship among virus antigenicity, transmission and virulence, which has unpredictable implications for the future trajectory and disease burden of COVID-19.

Genomic epidemiology of SARS-CoV-2 infections in The Gambia: an analysis of routinely collected surveillance data between March, 2020, and January, 2022.

BACKGROUND: COVID-19, caused by SARS-CoV-2, is one of the deadliest pandemics of the past 100 years. Genomic sequencing has an important role in monitoring of the evolution of the virus, including the detection of new viral variants. We aimed to describe the genomic epidemiology of SARS-CoV-2 infections in The Gambia. METHODS: Nasopharyngeal or oropharyngeal swabs collected from people with suspected cases of COVID-19 and international travellers were tested for SARS-CoV-2 with standard RT-PCR methods. SARS-CoV-2-positive samples were sequenced according to standard library preparation and sequencing protocols. Bioinformatic analysis was done using ARTIC pipelines and Pangolin was used to assign lineages. To construct phylogenetic trees, sequences were first stratified into different COVID-19 waves (waves 1-4) and aligned. Clustering analysis was done and phylogenetic trees constructed. FINDINGS: Between March, 2020, and January, 2022, 11 911 confirmed cases of COVID-19 were recorded in The Gambia, and 1638 SARS-CoV-2 genomes were sequenced. Cases were broadly distributed into four waves, with more cases during the waves that coincided with the rainy season (July-October). Each wave occurred after the introduction of new viral variants or lineages, or both, generally those already established in Europe or in other African countries. Local transmission was higher during the first and third waves (ie, those that corresponded with the rainy season), in which the B.1.416 lineage and delta (AY.34.1) were dominant, respectively. The second wave was driven by the alpha and eta variants and the B.1.1.420 lineage. The fourth wave was driven by the omicron variant and was predominantly associated with the BA.1.1 lineage. INTERPRETATION: More cases of SARS-CoV-2 infection were recorded in The Gambia during peaks of the pandemic that coincided with the rainy season, in line with transmission patterns for other respiratory viruses. The introduction of new lineages or variants preceded epidemic waves, highlighting the importance of implementing well structured genomic surveillance at a national level to detect and monitor emerging and circulating variants. FUNDING: Medical Research Unit The Gambia at London School of Hygiene & Tropical Medicine, UK Research and Innovation, WHO.

Circulation of respiratory viruses during the COVID-19 pandemic in The Gambia [version 1, peer review: 2 approved]

Background: In many countries, non-pharmaceutical interventions to limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission resulted in significant reductions in other respiratory viruses. However, similar data from Africa are limited. We explored the extent to which viruses such as influenza and rhinovirus co-circulated with SARS-CoV-2 in The Gambia during the COVID-19 pandemic. Methods: Between April 2020 and March 2022, respiratory viruses were detected using RT-PCR in nasopharyngeal swabs from 1397 participants with influenza-like illness. An assay to detect SARS-CoV-2 and a viral multiplex RT-PCR assay was used as previously described to detect influenza A and B, respiratory syncytial virus (RSV) A and B, parainfluenza viruses 1-4, human metapneumovirus (HMPV), adenovirus, seasonal coronaviruses (229E, OC43, NL63) and human rhinovirus. Results: Overall virus positivity was 44.2%, with prevalence higher in children <5 years (80%) compared to children aged 5-17 years (53.1%), adults aged 18-50 (39.5%) and >50 years (39.9%), p<0.0001. After SARS-CoV-2 (18.3%), rhinoviruses (10.5%) and influenza viruses (5.5%) were the most prevalent. SARS-CoV-2 positivity was lower in children <5 (4.3%) and 5-17 years (12.7%) than in adults aged 18-50 (19.3%) and >50 years (24.3%), p<0.0001. In contrast, rhinoviruses were most prevalent in children <5 years (28.7%), followed by children aged 5-17 (15.8%), adults aged 18-50 (8.3%) and >50 years (6.3%), p<0.0001. Four SARS-CoV-2 waves occurred, with 36.1%-52.4% SARS-CoV-2 positivity during peak months. Influenza infections were observed in both 2020 and 2021 during the rainy season as expected (peak positivity 16.4%-23.5%). Peaks of rhinovirus were asynchronous to the months when SARS-CoV-2 and influenza peaked. Conclusion: Our data show that many respiratory viruses continued to circulate during the COVID-19 pandemic in The Gambia, including human rhinoviruses, despite the presence of NPIs during the early stages of the pandemic, and influenza peaks during expected months.

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.

Effectiveness of rapid SARS-CoV-2 genome sequencing in supporting infection control for hospital-onset COVID-19 infection: Multicentre, prospective study.

Background: Viral sequencing of SARS-CoV-2 has been used for outbreak investigation, but there is limited evidence supporting routine use for infection prevention and control (IPC) within hospital settings. Methods: We conducted a prospective non-randomised trial of sequencing at 14 acute UK hospital trusts. Sites each had a 4-week baseline data collection period, followed by intervention periods comprising 8 weeks of 'rapid' (<48 hr) and 4 weeks of 'longer-turnaround' (5-10 days) sequencing using a sequence reporting tool (SRT). Data were collected on all hospital-onset COVID-19 infections (HOCIs; detected ≥48 hr from admission). The impact of the sequencing intervention on IPC knowledge and actions, and on the incidence of probable/definite hospital-acquired infections (HAIs), was evaluated. Results: A total of 2170 HOCI cases were recorded from October 2020 to April 2021, corresponding to a period of extreme strain on the health service, with sequence reports returned for 650/1320 (49.2%) during intervention phases. We did not detect a statistically significant change in weekly incidence of HAIs in longer-turnaround (incidence rate ratio 1.60, 95% CI 0.85-3.01; p=0.14) or rapid (0.85, 0.48-1.50; p=0.54) intervention phases compared to baseline phase. However, IPC practice was changed in 7.8 and 7.4% of all HOCI cases in rapid and longer-turnaround phases, respectively, and 17.2 and 11.6% of cases where the report was returned. In a 'per-protocol' sensitivity analysis, there was an impact on IPC actions in 20.7% of HOCI cases when the SRT report was returned within 5 days. Capacity to respond effectively to insights from sequencing was breached in most sites by the volume of cases and limited resources. Conclusions: While we did not demonstrate a direct impact of sequencing on the incidence of nosocomial transmission, our results suggest that sequencing can inform IPC response to HOCIs, particularly when returned within 5 days. Funding: COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) (grant code: MC_PC_19027), and Genome Research Limited, operating as the Wellcome Sanger Institute. Clinical trial number: NCT04405934.

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.

A delicate balance between antibody evasion and ACE2 affinity for Omicron BA.2.75.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have caused successive global waves of infection. These variants, with multiple mutations in the spike protein, are thought to facilitate escape from natural and vaccine-induced immunity and often increase in affinity for ACE2. The latest variant to cause concern is BA.2.75, identified in India where it is now the dominant strain, with evidence of wider dissemination. BA.2.75 is derived from BA.2 and contains four additional mutations in the receptor-binding domain (RBD). Here, we perform an antigenic and biophysical characterization of BA.2.75, revealing an interesting balance between humoral evasion and ACE2 receptor affinity. ACE2 affinity for BA.2.75 is increased 9-fold compared with BA.2; there is also evidence of escape of BA.2.75 from immune serum, particularly that induced by Delta infection, which may explain the rapid spread in India, where where there is a high background of Delta infection. ACE2 affinity appears to be prioritized over greater escape.

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.

Protocol for the COG-UK hospital-onset COVID-19 infection (HOCI) multicentre interventional clinical study: evaluating the efficacy of rapid genome sequencing of SARS-CoV-2 in limiting the spread of COVID-19 in UK NHS hospitals.

OBJECTIVES: Nosocomial transmission of SARS-CoV-2 has been a significant cause of mortality in National Health Service (NHS) hospitals during the COVID-19 pandemic. The COG-UK Consortium Hospital-Onset COVID-19 Infections (COG-UK HOCI) study aims to evaluate whether the use of rapid whole-genome sequencing of SARS-CoV-2, supported by a novel probabilistic reporting methodology, can inform infection prevention and control (IPC) practice within NHS hospital settings. DESIGN: Multicentre, prospective, interventional, superiority study. SETTING: 14 participating NHS hospitals over winter-spring 2020/2021 in the UK. PARTICIPANTS: Eligible patients must be admitted to hospital with first-confirmed SARS-CoV-2 PCR-positive test result >48 hour from time of admission, where COVID-19 diagnosis not suspected on admission. The projected sample size is 2380 patients. INTERVENTION: The intervention is the return of a sequence report, within 48 hours in one phase (rapid local lab processing) and within 5-10 days in a second phase (mimicking central lab), comparing the viral genome from an eligible study participant with others within and outside the hospital site. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcomes are incidence of Public Health England (PHE)/IPC-defined SARS-CoV-2 hospital-acquired infection during the baseline and two interventional phases, and proportion of hospital-onset cases with genomic evidence of transmission linkage following implementation of the intervention where such linkage was not suspected by initial IPC investigation. Secondary outcomes include incidence of hospital outbreaks, with and without sequencing data; actual and desirable changes to IPC actions; periods of healthcare worker (HCW) absence. Health economic analysis will be conducted to determine cost benefit of the intervention. A process evaluation using qualitative interviews with HCWs will be conducted alongside the study. TRIAL REGISTRATION NUMBER: ISRCTN50212645. Pre-results stage. This manuscript is based on protocol V.6.0. 2 September 2021.

Altered subgenomic RNA abundance provides unique insight into SARS-CoV-2 B.1.1.7/Alpha variant infections.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

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.