A pro-inflammatory gut mucosal cytokine response is associated with mild COVID-19 disease and superior induction of serum antibodies.

The relationship between gastrointestinal tract infection, the host immune response, and the clinical outcome of disease is not well understood in COVID-19. We sought to understand the effect of intestinal immune responses to SARS-CoV-2 on patient outcomes including the magnitude of systemic antibody induction. Combining two prospective cohort studies, International Severe Acute Respiratory and emerging Infections Consortium Comprehensive Clinical Characterisations Collaboration (ISARIC4C) and Integrated Network for Surveillance, Trials and Investigations into COVID-19 Transmission (INSTINCT), we acquired samples from 88 COVID-19 cases representing the full spectrum of disease severity and analysed viral RNA and host gut cytokine responses in the context of clinical and virological outcome measures. There was no correlation between the upper respiratory tract and faecal viral loads. Using hierarchical clustering, we identified a group of fecal cytokines including Interleukin-17A, Granulocyte macrophage colony-stimulating factor, Tumor necrosis factorα, Interleukin-23, and S100A8, that were transiently elevated in mild cases and also correlated with the magnitude of systemic anti-Spike-receptor-binding domain antibody induction. Receiver operating characteristic curve analysis showed that expression of these gut cytokines at study enrolment in hospitalised COVID-19 cases was associated negatively with overall clinical severity implicating a protective role in COVID-19. This suggests that a productive intestinal immune response may be beneficial in the response to a respiratory pathogen and a biomarker of a successful barrier response.

Hybrid immunity in older adults is associated with reduced SARS-CoV-2 infections following BNT162b2 COVID-19 immunisation.

BACKGROUND: Older adults, particularly in long-term care facilities (LTCF), remain at considerable risk from SARS-CoV-2. Data on the protective effect and mechanisms of hybrid immunity are skewed towards young adults precluding targeted vaccination strategies. METHODS: A single-centre longitudinal seroprevalence vaccine response study was conducted with 280 LCTF participants (median 82 yrs, IQR 76-88 yrs; 95.4% male). Screening by SARS-CoV-2 polymerase chain reaction with weekly asymptomatic/symptomatic testing (March 2020-October 2021) and serology pre-/post-two-dose Pfizer-BioNTech BNT162b2 vaccination for (i) anti-nucleocapsid, (ii) quantified anti-receptor binding domain (RBD) antibodies at three time-intervals, (iii) pseudovirus neutralisation, and (iv) inhibition by anti-RBD competitive ELISA were conducted. Neutralisation activity: antibody titre relationship was assessed via beta linear-log regression and RBD antibody-binding inhibition: post-vaccine infection relationship by Wilcoxon rank sum test. RESULTS: Here we show neutralising antibody titres are 9.2-fold (95% CI 5.8-14.5) higher associated with hybrid immunity (p < 0.00001); +7.5-fold (95% CI 4.6-12.1) with asymptomatic infection; +20.3-fold, 95% (CI 9.7-42.5) with symptomatic infection. A strong association is observed between antibody titre: neutralising activity (p < 0.00001) and rising anti-RBD antibody titre: RBD antibody-binding inhibition (p < 0.001), although 18/169 (10.7%) participants with high anti-RBD titre (>100BAU/ml), show inhibition <75%. Higher RBD antibody-binding inhibition values are associated with hybrid immunity and reduced likelihood of infection (p = 0.003). CONCLUSIONS: Hybrid immunity in older adults was associated with considerably higher antibody titres, neutralisation and inhibition capacity. Instances of high anti-RBD titre with lower inhibition suggests antibody quantity and quality as independent potential correlates of protection, highlighting added value of measuring inhibition over antibody titre alone to inform vaccine strategy.

Older adults continue to be at risk of COVID-19, particularly in residential care home settings. We investigated the effect of infection and vaccination on antibody development and subsequent SARS-CoV-2 infection in older adults. Antibodies are proteins that the immune system produces on infection or vaccination that can help respond to subsequent infection with SARS-CoV-2. We found that older adults produce antibodies to SARS-CoV-2 after 2-doses of Pfizer BioNTech BNT162b2 vaccine. The strongest immune responses were seen among those older adults who also had prior history of infection. The results highlight the importance of both antibody quality and quantity when considering possible indicators of protection against COVID-19 and supports the need for a third, booster, vaccination in this age group..

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.

Risk factors and vectors for SARS-CoV-2 household transmission: a prospective, longitudinal cohort study.

BACKGROUND: Despite circumstantial evidence for aerosol and fomite spread of SARS-CoV-2, empirical data linking either pathway with transmission are scarce. Here we aimed to assess whether the presence of SARS-CoV-2 on frequently-touched surfaces and residents' hands was a predictor of SARS-CoV-2 household transmission. METHODS: In this longitudinal cohort study, during the pre-alpha (September to December, 2020) and alpha (B.1.1.7; December, 2020, to April, 2021) SARS-CoV-2 variant waves, we prospectively recruited contacts from households exposed to newly diagnosed COVID-19 primary cases, in London, UK. To maximally capture transmission events, contacts were recruited regardless of symptom status and serially tested for SARS-CoV-2 infection by RT-PCR on upper respiratory tract (URT) samples and, in a subcohort, by serial serology. Contacts' hands, primary cases' hands, and frequently-touched surface-samples from communal areas were tested for SARS-CoV-2 RNA. SARS-CoV-2 URT isolates from 25 primary case-contact pairs underwent whole-genome sequencing (WGS). FINDINGS: From Aug 1, 2020, until March 31, 2021, 620 contacts of PCR-confirmed SARS-CoV-2-infected primary cases were recruited. 414 household contacts (from 279 households) with available serial URT PCR results were analysed in the full household contacts' cohort, and of those, 134 contacts with available longitudinal serology data and not vaccinated pre-enrolment were analysed in the serology subcohort. Household infection rate was 28·4% (95% CI 20·8-37·5) for pre-alpha-exposed contacts and 51·8% (42·5-61·0) for alpha-exposed contacts (p=0·0047). Primary cases' URT RNA viral load did not correlate with transmission, but was associated with detection of SARS-CoV-2 RNA on their hands (p=0·031). SARS-CoV-2 detected on primary cases' hands, in turn, predicted contacts' risk of infection (adjusted relative risk [aRR]=1·70 [95% CI 1·24-2·31]), as did SARS-CoV-2 RNA presence on household surfaces (aRR=1·66 [1·09-2·55]) and contacts' hands (aRR=2·06 [1·57-2·69]). In six contacts with an initial negative URT PCR result, hand-swab (n=3) and household surface-swab (n=3) PCR positivity preceded URT PCR positivity. WGS corroborated household transmission. INTERPRETATION: Presence of SARS-CoV-2 RNA on primary cases' and contacts' hands and on frequently-touched household surfaces associates with transmission, identifying these as potential vectors for spread in households. FUNDING: National Institute for Health Research Health Protection Research Unit in Respiratory Infections, Medical Research Council.

National and regional prevalence of SARS-CoV-2 antibodies in primary and secondary school children in England: the School Infection Survey, a national open cohort study, November 2021SARS-CoV-2 antibody prevalence in school children.

BACKGROUND: SARS-CoV-2 infection rates are likely to be underestimated in children because of asymptomatic or mild infections. We aim to estimate national and regional prevalence of SARS-CoV-2 antibodies in primary (4-11 years old) and secondary (11-18 years old) school children between 10 November and 10 December 2021. METHODS: Cross-sectional surveillance in England using two-stage sampling, firstly stratifying into regions and selecting local authorities, then selecting schools according to a stratified sample within selected local authorities. Participants were sampled using a novel oral fluid-validated assay for SARS-CoV-2 spike and nucleocapsid IgG antibodies. RESULTS: 4980 students from 117 state-funded schools (2706 from 83 primary schools, 2274 from 34 secondary schools) provided a valid sample. After weighting for age, sex, and ethnicity, and adjusting for assay accuracy, the national prevalence of SARS-CoV-2 antibodies in primary school students, who were all unvaccinated, was 40.1% (95% CI 37.3-43.0). Antibody prevalence increased with age (p < 0.001) and was higher in urban than rural schools (p = 0.01). In secondary school students, the adjusted, weighted national prevalence of SARS-CoV-2 antibodies was 82.4% (95% CI 79.5-85.1); including 71.5% (95% CI 65.7-76.8) in unvaccinated and 97.5% (95% CI 96.1-98.5) in vaccinated students. Antibody prevalence increased with age (p < 0.001), and was not significantly different in urban versus rural students (p = 0.1). CONCLUSIONS: In November 2021, using a validated oral fluid assay, national SARS-CoV-2 seroprevalence was estimated to be 40.1% in primary school students and 82.4% in secondary school students. In unvaccinated children, this was approximately threefold higher than confirmed infections highlighting the importance of seroprevalence studies to estimate prior exposure. DATA AVAILABILITY: Deidentified study data are available for access by accredited researchers in the ONS Secure Research Service (SRS) for accredited research purposes under part 5, chapter 5 of the Digital Economy Act 2017. For further information about accreditation, contact Research.support@ons.gov.uk or visit the SRS website.

Plasmodium infection is associated with cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein.

Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.

Simple, sensitive, specific self-sampling assay secures SARS-CoV-2 antibody signals in sero-prevalence and post-vaccine studies.

At-home sampling is key to large scale seroprevalence studies. Dried blood spot (DBS) self-sampling removes the need for medical personnel for specimen collection but facilitates specimen referral to an appropriately accredited laboratory for accurate sample analysis. To establish a highly sensitive and specific antibody assay that would facilitate self-sampling for prevalence and vaccine-response studies. Paired sera and DBS eluates collected from 439 sero-positive, 382 sero-negative individuals and DBS from 34 vaccine recipients were assayed by capture ELISAs for IgG and IgM antibody to SARS-CoV-2. IgG and IgM combined on DBS eluates achieved a diagnostic sensitivity of 97.9% (95%CI 96.6 to 99.3) and a specificity of 99.2% (95% CI 98.4 to 100) compared to serum, displaying limits of detection equivalent to 23 and 10 WHO IU/ml, respectively. A strong correlation (r = 0.81) was observed between serum and DBS reactivities. Reactivity remained stable with samples deliberately rendered inadequate, (p = 0.234) and when samples were accidentally damaged or 'invalid'. All vaccine recipients were sero-positive. This assay provides a secure method for self-sampling by DBS with a sensitivity comparable to serum. The feasibility of DBS testing in sero-prevalence studies and in monitoring post-vaccine responses was confirmed, offering a robust and reliable tool for serological monitoring at a population level.

Detection and quantification of antibody to SARS CoV 2 receptor binding domain provides enhanced sensitivity, specificity and utility.

Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity. The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA), providing the most sensitive format has been exploited in a novel hybrid manner employing a solid-phase S1 preferentially presenting RBD, coupled with a labelled RBD conjugate, used in a two-step sequential assay for detection and measurement of antibody to RBD (anti-RBD). This class and species neutral assay showed a specificity of 100 % on 825 pre COVID-19 samples and a potential sensitivity of 99.6 % on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralization and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine and in humans immunised with both AstraZeneca and Pfizer vaccines. This assay detects anti-RBD at presentation with illness, demonstrates its elevation with disease severity, its sequel to asymptomatic infection and its persistence after the loss of antibody to the nucleoprotein (anti-NP). It also provides serological confirmation of prior infection and offers a secure measure for seroprevalence and studies of vaccine immunisation in human and animal populations. The hybrid DABA also displays the attributes necessary for the detection and quantification of anti-RBD to be used in clinical practice. An absence of detectable anti-RBD by this assay predicates the need for passive immune prophylaxis in at-risk patients.

Variability in detection of SARS-CoV-2-specific antibody responses following mild infection: a prospective multicentre cross-sectional study, London, United Kingdom, 17 April to 17 July 2020.

IntroductionImmunoassays targeting different SARS-CoV-2-specific antibodies are employed for seroprevalence studies. The degree of variability between immunoassays targeting anti-nucleocapsid (anti-NP; the majority) vs the potentially neutralising anti-spike antibodies (including anti-receptor-binding domain; anti-RBD), particularly in mild or asymptomatic disease, remains unclear.AimsWe aimed to explore variability in anti-NP and anti-RBD antibody detectability following mild symptomatic or asymptomatic SARS-CoV-2 infection and analyse antibody response for correlation with symptomatology.MethodsA multicentre prospective cross-sectional study was undertaken (April-July 2020). Paired serum samples were tested for anti-NP and anti-RBD IgG antibodies and reactivity expressed as binding ratios (BR). Multivariate linear regression was performed analysing age, sex, time since onset, symptomatology, anti-NP and anti-RBD antibody BR.ResultsWe included 906 adults. Antibody results (793/906; 87.5%; 95% confidence interval: 85.2-89.6) and BR strongly correlated (ρ = 0.75). PCR-confirmed cases were more frequently identified by anti-RBD (129/130) than anti-NP (123/130). Anti-RBD testing identified 83 of 325 (25.5%) cases otherwise reported as negative for anti-NP. Anti-NP presence (+1.75/unit increase; p < 0.001), fever (≥ 38°C; +1.81; p < 0.001) or anosmia (+1.91; p < 0.001) were significantly associated with increased anti-RBD BR. Age (p = 0.85), sex (p = 0.28) and cough (p = 0.35) were not. When time since symptom onset was considered, we did not observe a significant change in anti-RBD BR (p = 0.95) but did note decreasing anti-NP BR (p < 0.001).ConclusionSARS-CoV-2 anti-RBD IgG showed significant correlation with anti-NP IgG for absolute seroconversion and BR. Higher BR were seen in symptomatic individuals, particularly those with fever. Inter-assay variability (12.5%) was evident and raises considerations for optimising seroprevalence testing strategies/studies.

Cross-reactive memory T cells associate with protection against SARS-CoV-2 infection in COVID-19 contacts.

Cross-reactive immune responses to SARS-CoV-2 have been observed in pre-pandemic cohorts and proposed to contribute to host protection. Here we assess 52 COVID-19 household contacts to capture immune responses at the earliest timepoints after SARS-CoV-2 exposure. Using a dual cytokine FLISpot assay on peripheral blood mononuclear cells, we enumerate the frequency of T cells specific for spike, nucleocapsid, membrane, envelope and ORF1 SARS-CoV-2 epitopes that cross-react with human endemic coronaviruses. We observe higher frequencies of cross-reactive (p = 0.0139), and nucleocapsid-specific (p = 0.0355) IL-2-secreting memory T cells in contacts who remained PCR-negative despite exposure (n = 26), when compared with those who convert to PCR-positive (n = 26); no significant difference in the frequency of responses to spike is observed, hinting at a limited protective function of spike-cross-reactive T cells. Our results are thus consistent with pre-existing non-spike cross-reactive memory T cells protecting SARS-CoV-2-naïve contacts from infection, thereby supporting the inclusion of non-spike antigens in second-generation vaccines.

Mother to Infant Transmission of Hepatitis B Virus in the Face of Neonatal Immunization Is Not Necessarily Primary Vaccine Failure.

BACKGROUND: Surveillance programs undertaken in infants born to mothers with hepatitis B virus (HBV) provide an opportunity to analyze virological markers from the neonate and early infancy. These data inform on mechanisms of HBV transmission and how available interventions can be better used for control of HBV infections arising at the mother/child interface. METHODS: Retrospective analysis of HBV serological markers was undertaken in dried blood spots collected from infants born to mothers infected with HBV. In addition, molecular analysis was performed in newborn blood spot cards, collected after birth, from infants identified as infected with HBV despite receiving prophylaxis. RESULTS: Perinatal exposure could not account for all transmissions, with at least one-quarter (22%) of infants already infected in utero. All harbored a wild-type hepatitis B surface antigen (HBsAg), with identical sequences noted in the neonatal and early infancy samples. In contrast, in infants infected perinatally (43%), selection of viruses harboring amino acid changes in the HBsAg were common (80% of sequences) and divergent from the linked maternal sample. CONCLUSION: Currently considered to represent vaccine failure, it is likely that a proportion of HBV infections result from in utero acquisition. These infections are unlikely to be susceptible to postnatal prophylaxis, and current recommendations for maternal antiviral treatment may be too late to prevent transmission. Consideration should be given to the earlier use of antivirals during gestation to reduce the risk of intrauterine transmission together with completion of the immunization schedule also to reduce the perinatal risk of HBV transmission.

Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific real time reverse transcription PCR.

BACKGROUND: The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in many parts of the world. To help guide public health policies it is important to monitor efficiently its spread. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. OBJECTIVE: To develop and evaluate a rapid, simple and inexpensive alternative to sequencing for Delta identification. METHODS: A double-mismatch allele-specific RT-PCR (DMAS-RT-PCR) was developed. The technique exploits allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer was enhanced by an additional mismatch located at the fourth nucleotide from the 3' end. Specificity was assessed by testing well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. RESULTS: In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants (Alpha and Beta), and 'wild-type' SARS-CoV-2. Influenza A and RSV were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay. Several samples that could not be sequenced due to insufficient virus were successfully genotyped by DMAS-RT-PCR. CONCLUSION: The method we describe would be simple to establish in any laboratory that can conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant globally.

Broadening symptom criteria improves early case identification in SARS-CoV-2 contacts.

BACKGROUND: The success of case isolation and contact tracing for the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission depends on the accuracy and speed of case identification. We assessed whether inclusion of additional symptoms alongside three canonical symptoms (CS), i.e. fever, cough and loss or change in smell or taste, could improve case definitions and accelerate case identification in SARS-CoV-2 contacts. METHODS: Two prospective longitudinal London (UK)-based cohorts of community SARS-CoV-2 contacts, recruited within 5 days of exposure, provided independent training and test datasets. Infected and uninfected contacts completed daily symptom diaries from the earliest possible time-points. Diagnostic information gained by adding symptoms to the CS was quantified using likelihood ratios and area under the receiver operating characteristic curve. Improvements in sensitivity and time to detection were compared with penalties in terms of specificity and number needed to test. RESULTS: Of 529 contacts within two cohorts, 164 (31%) developed PCR-confirmed infection and 365 (69%) remained uninfected. In the training dataset (n=168), 29% of infected contacts did not report the CS. Four symptoms (sore throat, muscle aches, headache and appetite loss) were identified as early-predictors (EP) which added diagnostic value to the CS. The broadened symptom criterion "≥1 of the CS, or ≥2 of the EP" identified PCR-positive contacts in the test dataset on average 2 days earlier after exposure (p=0.07) than "≥1 of the CS", with only modest reduction in specificity (5.7%). CONCLUSIONS: Broadening symptom criteria to include individuals with at least two of muscle aches, headache, appetite loss and sore throat identifies more infections and reduces time to detection, providing greater opportunities to prevent SARS-CoV-2 transmission.

Upregulation of Human Endogenous Retroviruses in Bronchoalveolar Lavage Fluid of COVID-19 Patients.

Severe COVID-19 pneumonia has been associated with the development of intense inflammatory responses during the course of infections with SARS-CoV-2. Given that human endogenous retroviruses (HERVs) are known to be activated during and participate in inflammatory processes, we examined whether HERV dysregulation signatures are present in COVID-19 patients. By comparing transcriptomes of bronchoalveolar lavage fluid (BALF) of COVID-19 patients and healthy controls, and peripheral blood monocytes (PBMCs) from patients and controls, we have shown that HERVs are intensely dysregulated in BALF of COVID-19 patients compared to those in BALF of healthy control patients but not in PBMCs. In particular, upregulation in the expression of specific HERV families was detected in BALF samples of COVID-19 patients, with HERV-FRD being the most highly upregulated family among the families analyzed. In addition, we compared the expression of HERVs in human bronchial epithelial cells (HBECs) without and after senescence induction in an oncogene-induced senescence model in order to quantitatively measure changes in the expression of HERVs in bronchial cells during the process of cellular senescence. This apparent difference of HERV dysregulation between PBMCs and BALF warrants further studies in the involvement of HERVs in inflammatory pathogenetic mechanisms as well as exploration of HERVs as potential biomarkers for disease progression. Furthermore, the increase in the expression of HERVs in senescent HBECs in comparison to that in noninduced HBECs provides a potential link for increased COVID-19 severity and mortality in aged populations. IMPORTANCE SARS-CoV-2 emerged in late 2019 in China, causing a global pandemic. Severe COVID-19 is characterized by intensive inflammatory responses, and older age is an important risk factor for unfavorable outcomes. HERVs are remnants of ancient infections whose expression is upregulated in multiple conditions, including cancer and inflammation, and their expression is increased with increasing age. The significance of this work is that we were able to recognize dysregulated expression of endogenous retroviral elements in BALF samples but not in PBMCs of COVID-19 patients. At the same time, we were able to identify upregulated expression of multiple HERV families in senescence-induced HBECs in comparison to that in noninduced HBECs, a fact that could possibly explain the differences in disease severity among age groups. These results indicate that HERV expression might play a pathophysiological role in local inflammatory pathways in lungs afflicted by SARS-CoV-2 and their expression could be a potential therapeutic target.

Hepatitis E virus: Whole genome sequencing as a new tool for understanding HEV epidemiology and phenotypes.

Hepatitis E Virus (HEV) is emerging as a public health concern across Europe and tools for complete genome data to aid epidemiological and virulence analysis are needed. The high sequence heterogeneity observed amongst HEV genotypes has restricted most analyses to subgenomic regions using PCR-based methods, which can be unreliable due to poor primer homology. We designed a panel of custom-designed RNA probes complementary to all published HEV full genome NCBI sequences. A target enrichment protocol was performed according to the NimbleGen® standard protocol for Illumina® library preparation. Optimisation of this protocol was performed using 40 HEV RNA-positive serum samples and the World Health Organization International Reference Panel for Hepatitis E Virus RNA Genotypes for Nucleic Acid Amplification Technique (NAT)-Based Assays and related reference materials. Deep sequencing using this target enrichment protocol resulted in whole genome consensus sequences from samples with a viral load range of 1.25 × 104-1.17 × 107 IU/mL. Phylogenetic analysis of these sequences recapitulated and extended the partial genome results obtained from genotyping by Sanger sequencing (genotype 1, ten samples and genotype 3, 30 samples). The protocol is highly adaptable to automation and could be used to sequence full genomes of large sample numbers. A more comprehensive understanding of hepatitis E virus transmission, epidemiology and viral phenotype prediction supported by an efficient method of sequencing the whole viral genome will facilitate public health initiatives to reduce the prevalence and mitigate the harm of HEV infection in Europe.

SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity.

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.

The Association Between Antibody Response to Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Post-COVID-19 Syndrome in Healthcare Workers.

It is currently unknown how post-COVID-19 syndrome (PCS) may affect those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This longitudinal study includes healthcare staff who tested positive for SARS-CoV-2 between March and April 2020, with follow-up of their antibody titers and symptoms. More than half (21 of 38) had PCS after 7-8 months. There was no statistically significant difference between initial reverse-transcription polymerase chain reaction titers or serial antibody levels between those who did and those who did not develop PCS. This study highlights the relative commonality of PCS in healthcare workers and this should be considered in vaccination scheduling and workforce planning to allow adequate frontline staffing numbers.