Genomic Investigations of Acute Hepatitis of Unknown Aetiology in Children (preprint)

Since the first reports of hepatitis of unknown aetiology occurring in UK children, over 1000 cases have been reported worldwide, including 268 cases in the UK, with the majority younger than 6 years old. Using genomic, proteomic and immunohistochemical methods, we undertook extensive investigation of 28 cases and 136 control subjects. In five cases who underwent liver transplantation, we detected high levels of adeno-associated virus 2 (AAV2) in the explanted livers. AAV2 was also detected at high levels in blood from 10/11 non-transplanted cases. Low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), both of which enable AAV2 lytic replication, were also found in the five explanted livers and blood from 15/17 and 6/9 respectively, of the 23 non-transplant cases tested. In contrast, AAV2 was detected at low titre in 6/100 whole bloods from child controls from cohorts with presence or absence of hepatitis and/or adenovirus infection. Our data show an association of AAV2 at high titre in blood or liver tissue, with unexplained hepatitis in children infected in the recent HAdV-F41 outbreak. We were unable to find evidence by electron microscopy, immunohistochemistry or proteomics of HAdV or AAV2 viral particles or proteins in explanted livers, suggesting that hepatic pathology is not due to direct lytic infection by either virus. The potential that AAV2, although not previously associated with disease, may, together with HAdV-F41 and/or HHV-6, be causally implicated in the outbreak of unexplained hepatitis, requires further investigation.

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks (preprint)

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

The Influence of Time on the Sensitivity of SARS-CoV-2 Serological Testing (preprint)

Background: Serological testing is used to quantify SARS-CoV-2 seroprevalence, guide booster vaccination and select patients for anti-SARS-CoV-2 antibodies therapy. However, our understanding of how serological tests perform as time passes after infection is limited.Methods: Four assays were compared in parallel: 1) the multiplexed spike, nucleoprotein and receptor binding domain Meso Scale Discovery (MSD) assay 2) the Roche Elecsys-Nucleoprotein assay (Roche-N) 3) the Roche Spike assay (Roche-S) and 4) the Abbott Nucleoprotein assay (Abbott-N) on serial positive monthly samples from hospital staff up to 200 days following infection as part of the Co-Stars study.Results: We demonstrate that 50% of the Abbott-N assays give a negative result after 175 days (median survival time 95% CI 168-185 days) while the Roche-N assay (93% survival probability at 200 days, 95% CI 88-97%) maintained seropositivity. The MSD spike (97% survival probability at 200 days, 95% CI 95-99%) and the Roche-S assay (95% survival probability at 200 days, 95% CI 93-97%) also remained seropositive. The best performing quantitative Roche-S assay showed no evidence of waning Spike antibody titres over 200-days.Conclusions: The Abbott-N assay fails to detect SARS-CoV-2 antibodies as time passes since infection. In contrast the Roche and the MSD assays maintained high sensitivity. The limitations of the Abbott assay must be considered in clinical decision making. The long duration of detectable neutralizing spike antibody titres by the quantitative Roche-S assay provides further evidence in support of long-lasting SARS-CoV-2 protection to pre-existing strains of SARS-CoV-2 following natural infection.Trial registration: Co-STARs study was registered with ClinicalTrials.gov on May 8th, 2020, with trial number NCT04380896 (www.clinicaltrials.gov, NCT04380896)

Long-Term Persistence of Spike Antibody and Predictive Modeling of Antibody Dynamics Following Infection with SARS-CoV-2 (preprint)

Background: Antibodies to Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) have been shown to neutralize the virus in-vitro and prevent disease in animal challenge models upon re-exposure. However, current understanding of SARS-CoV-2 humoral dynamics and longevity is conflicting.Methods: The Co-Stars study prospectively enrolled 3679 healthcare workers to comprehensively characterize the kinetics of SARS-CoV-2 spike (S), receptor-binding-domain (RBD) and nucleoprotein (N) antibodies in parallel. Participants screening seropositive had serial monthly serological testing for maximum 7 months with the Mesoscale Discovery Assay. Survival analysis determined the proportion of sero-reversion while two hierarchical Gamma models predicted the upper- and lower-bounds of long-term antibody trajectory.Results: A total of 1163 monthly samples were provided from 349 seropositive participants. At 200 days post-symptoms, 99% of participants had detectable S-antibodies compared to 75% with detectable N-antibodies. S-antibody was predicted to remain detectable in 95% of participants until 465 days [95%CI 370-575] using a ‘continuous-decay’ model and indefinitely using a ‘decay-to-plateau’ model to account for antibody secretion by long-lived plasma cells. S-antibody titers correlated strongly with surrogate neutralization in-vitro (R2=0.72). N-antibodies, however, decayed rapidly with a half-life of 60 days [95%CI 52-68].Conclusions: The Co-STAR's study data presented here provides evidence for long-term persistence of neutralizing S-antibodies. This has important implications for the duration of functional immunity following SARS-CoV-2 infection. In contrast, the rapid decay of N-antibodies must be considered in future seroprevalence studies and public health decision-making. This is the first study to establish a mathematical framework capable of predicting long-term humoral dynamics following SARS-CoV-2 infection.Trial Registration: NCT04380896.Funding Statement: GOSH charity, Wellcome Trust (201470/Z/16/Z and 220565/Z/20/Z). GOSH NIHR Funded Biomedical Research Centre.Declaration of Interests: The authors have declared that no competing interests exist.Ethics Approval Statement: This study was approved by the UK Health Research Authority (www.hra.nhs.uk). Written informed consent was obtained from all participants before recruitment to the study.

Long-Term Persistence of Spike Antibody and Predictive Modeling of Antibody Dynamics Following Infection with SARS-CoV-2 (preprint)

Background: Antibodies to Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) have been shown to neutralize the virus in-vitro. Similarly, animal challenge models suggest that neutralizing antibodies isolated from SARS-CoV-2 infected individuals prevent against disease upon re-exposure to the virus. Understanding the nature and duration of the antibody response following SARS-CoV-2 infection is therefore critically important. Methods: Between April and October 2020 we undertook a prospective cohort study of 3555 healthcare workers in order to elucidate the duration and dynamics of antibody responses following infection with SARS-CoV-2. After a formal performance evaluation against 169 PCR confirmed cases and negative controls, the Meso-Scale Discovery assay was used to quantify in parallel, antibody titers to the SARS-CoV-2 nucleoprotein (N), spike (S) protein and the receptor-binding-domain (RBD) of the S-protein. All seropositive participants were followed up monthly for a maximum of 7 months; those participants that were symptomatic, with known dates of symptom-onset, seropositive by the MSD assay and who provided 2 or more monthly samples were included in the analysis. Survival analysis was used to determine the proportion of sero-reversion (switching from positive to negative) from the raw data. In order to predict long-term antibody dynamics, two hierarchical longitudinal Gamma models were implemented to provide predictions for the lower bound (continuous antibody decay to zero, 'Gamma-decay') and upper bound (decay-to-plateau due to long lived plasma cells, 'Gamma-plateau') long-term antibody titers. Results: A total of 1163 samples were provided from 349 of 3555 recruited participants who were symptomatic, seropositive by the MSD assay, and were followed up with 2 or more monthly samples. At 200 days post symptom onset, 99% of participants had detectable S-antibody whereas only 75% of participants had detectable N-antibody. Even under our most pessimistic assumption of persistent negative exponential decay, the S-antibody was predicted to remain detectable in 95% of participants until 465 days [95% CI 370-575] after symptom onset. Under the Gamma-plateau model, the entire posterior distribution of S-antibody titers at plateau remained above the threshold for detection indefinitely. Surrogate neutralization assays demonstrated a strong positive correlation between antibody titers to the S-protein and blocking of the ACE-2 receptor in-vitro [R2=0.72, p<0.001]. By contrast, the N-antibody waned rapidly with a half-life of 60 days [95% CI 52-68]. Discussion: This study has demonstrated persistence of the spike antibody in 99% of participants at 200 days following SARS-CoV-2 symptoms and rapid decay of the nucleoprotein antibody. Diagnostic tests or studies that rely on the N-antibody as a measure of seroprevalence must be interpreted with caution. Our lowest bound prediction for duration of the spike antibody was 465 days and our upper bound predicted spike antibody to remain indefinitely in line with the long-term seropositivity reported for SARS-CoV infection. The long-term persistence of the S-antibody, together with the strong positive correlation between the S-antibody and viral surrogate neutralization in-vitro, has important implications for the duration of functional immunity following SARS-CoV-2 infection.

Remdesivir induced viral RNA and subgenomic RNA suppression, and evolution of viral variants in SARS-CoV-2 infected patients. (preprint)

While changes in SARS-CoV-2 viral load over time have been documented, detailed information on the impact of remdesivir and how it might alter intra-host viral evolution is limited. Sequential viral loads and deep sequencing of SARS-CoV-2 recovered from the upper respiratory tract of hospitalised children revealed that remdesivir treatment suppressed viral RNA levels in one patient but not in a second infected with an identical strain. Evidence of drug resistance to explain this difference was not found. Reduced levels of subgenomic (sg) RNA during treatment of the second patient, suggest an additional effect of remdesivir on viral replication that is independent of viral RNA levels. Haplotype reconstruction uncovered persistent SARS-CoV-2 variant genotypes in four patients. We conclude that these are likely to have arisen from within-host evolution, and not co-transmission, although superinfection cannot be excluded in one case. Sample-to-sample heterogeneity in the abundances of variant genotypes is best explained by the presence of discrete viral populations in the lung with incomplete population sampling in diagnostic swabs. Such compartmentalisation is well described in serious lung infections caused by influenza and Mycobacterium tuberculosis and has been associated with poor drug penetration, suboptimal treatment and drug resistance. Our data provide evidence that remdesivir is able to suppress SARS-CoV-2 replication in vivo but that its efficacy may be compromised by factors reducing penetration into the lung. Based on data from influenza and Mycobacterium tuberculosis lung infections we conclude that early use of remdesivir combined with other agents should now be evaluated. Summary SentenceDeep sequencing of longitudinal samples from SARS-CoV-2 infected paediatric patients identifies evidence of remdesivir-associated inhibition of viral replication in vivo and uncovers evidence of within host evolution of distinct viral genotypes.

Humoral Response Dynamics Following Infection with SARS-CoV-2 (preprint)

Introduction: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) specific antibodies have been shown to neutralize the virus in-vitro. Understanding antibody dynamics following SARS-CoV-2 infection is therefore crucial. Sensitive measurement of SARS-CoV-2 antibodies is also vital for large seroprevalence surveys which inform government policies and public health interventions. However, rapidly waning antibodies following SARS-CoV-2 infection could jeopardize the sensitivity of serological testing on which these surveys depend. Methods: This prospective cohort study of SARS-CoV-2 humoral dynamics in a central London hospital analyzed 137 serial samples collected from 67 participants seropositive to SARS-CoV-2 by the Meso-Scale Discovery assay. Antibody titers were quantified to the SARS-CoV-2 nucleoprotein (N), spike (S-)protein and the receptor-binding-domain (RBD) of the S-protein. Titers were log-transformed and a multivariate log-linear model with time-since-infection and clinical variables was fitted by Bayesian methods. Results: The mean estimated half-life of the N-antibody was 52 days (95% CI 42-65). The S- and RBD-antibody had significantly longer mean half-lives of 81 days (95% CI 61-111) and 83 days (95% CI 55-137) respectively. An ACE-2-receptor competition assay demonstrated significant correlation between the S and RBD-antibody titers and ACE2-receptor blocking in-vitro. The time-to-a-negative N-antibody test for 50% of the seropositive population was predicted to be 195 days (95% CI 163-236). Discussion: After SARS-CoV-2 infection, the predicted half-life of N-antibody was 52 days with 50% of seropositive participants becoming seronegative to this antibody at 195 days. Widely used serological tests that depend on the N-antibody will therefore significantly underestimate the prevalence of infection following the majority of infections.