Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV-2 infection: a nested, case-control diagnostic accuracy study

Background We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing.Methods We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew’s Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for “viral infection”, “transcriptome”, “biomarker”, and “blood”. We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity.Findings We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27–47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91–0·99), sensitivity 0·84 (0·70–0·93), and specificity 0·95 (0·85–0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91–0·95).Interpretation Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge

Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure

The Omicron, or Pango lineage B.1.1.529, variant of SARS-CoV-2 carries multiple spike mutations with high transmissibility and partial neutralizing antibody (nAb) escape. Vaccinated individuals show protection from severe disease, often attributed to primed cellular immunity. We investigated T and B cell immunity against B.1.1.529 in triple mRNA vaccinated healthcare workers (HCW) with different SARS-CoV-2 infection histories. B and T cell immunity against previous variants of concern was enhanced in triple vaccinated individuals, but magnitude of T and B cell responses against B.1.1.529 spike protein was reduced. Immune imprinting by infection with the earlier B.1.1.7 (Alpha) variant resulted in less durable binding antibody against B.1.1.529. Previously infection-naïve HCW who became infected during the B.1.1.529 wave showed enhanced immunity against earlier variants, but reduced nAb potency and T cell responses against B.1.1.529 itself. Previous Wuhan Hu-1 infection abrogated T cell recognition and any enhanced cross-reactive neutralizing immunity on infection with B.1.1.529

Quantitative, multiplexed, targeted proteomics for ascertaining variant specific SARS-CoV-2 antibody response

Determining the protection an individual has to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VoCs) is crucial for future immune surveillance, vaccine development, and understanding of the changing immune response. We devised an informative assay to current ELISA-based serology using multiplexed, baited, targeted proteomics for direct detection of multiple proteins in the SARS-CoV-2 anti-spike antibody immunocomplex. Serum from individuals collected after infection or first- and second-dose vaccination demonstrates this approach and shows concordance with existing serology and neutralization. Our assays show altered responses of both immunoglobulins and complement to the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.1) VoCs and a reduced response to Omicron (B1.1.1529). We were able to identify individuals who had prior infection, and observed that C1q is closely associated with IgG1 (r > 0.82) and may better reflect neutralization to VoCs. Analyzing additional immunoproteins beyond immunoglobulin (Ig) G, provides important information about our understanding of the response to infection and vaccination

Healthcare Workers Bioresource: Study outline and baseline characteristics of a prospective healthcare worker cohort to study immune protection and pathogenesis in COVID-19

Background: Most biomedical research has focused on sampling COVID-19 patients presenting to hospital with advanced disease, with less focus on the asymptomatic or paucisymptomatic. We established a bioresource with serial sampling of health care workers (HCWs) designed to obtain samples before and during mainly mild disease, with follow-up sampling to evaluate the quality and duration of immune memory.Methods: We conducted a prospective observational study on HCWs from three hospital sites in London, initially at a single centre (recruited just prior to first peak community transmission in London), but then extended to multiple sites 3 weeks later (recruitment still ongoing, target n=1,000). Asymptomatic participants attending work complete a health questionnaire, and provide a nasal swab (for SARS-CoV-2 RNA by RT-PCR tests) and blood samples (mononuclear cells, serum, plasma, RNA and DNA are biobanked) at 16 weekly study visits, and at 6 and 12 months.Results: Preliminary baseline results for the first 731 HCWs (400 single-centre, 331 multicentre extension) are presented. Mean age was 38±11 years; 67% are female, 31% nurses, 20% doctors, and 19% work in intensive care units. COVID-19-associated risk factors were: 37% black, Asian or minority ethnicities; 18% smokers; 13% obesity; 11% asthma; 7% hypertension and 2% diabetes mellitus. At baseline, 41% reported symptoms in the preceding 2 weeks. Preliminary test results from the initial cohort (n=400) are available: PCR at baseline for SARS-CoV-2 was positive in 28 of 396 (7.1%, 95% CI 4.9-10.0%) and 15 of 385 (3.9%, 2.4-6.3%) had circulating IgG antibodies.Conclusions: This COVID-19 bioresource established just before the peak of infections in the UK will provide longitudinal assessments of incident infection and immune responses in HCWs through the natural time course of disease and convalescence. The samples and data from this bioresource are available to academic collaborators by application https://covid-consortium.com/application-for-samples/

Pre-existing polymerase-specific T cells expand in abortive seronegative SARS-CoV-2.

Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1-3. Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs. 4-11), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication-transcription complex (RTC)12,13, in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2 (ref. 14), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae

HLA-DR polymorphism in SARS-CoV-2 infection and susceptibility to symptomatic COVID-19

SARS-CoV-2 infection results in different outcomes ranging from asymptomatic infection to mild or severe disease and death. Reasons for this diversity of outcome include differences in challenge dose, age, gender, comorbidity and host genomic variation. Human leukocyte antigen (HLA) polymorphisms may influence immune response and disease outcome. We investigated the association of HLAII alleles with case definition symptomatic COVID-19, virus-specific antibody and T-cell immunity. A total of 1364 UK healthcare workers (HCWs) were recruited during the first UK SARS-CoV-2 wave and analysed longitudinally, encompassing regular PCR screening for infection, symptom reporting, imputation of HLAII genotype and analysis for antibody and T-cell responses to nucleoprotein (N) and spike (S). Of 272 (20%) HCW who seroconverted, the presence of HLA-DRB1*13:02 was associated with a 6·7-fold increased risk of case definition symptomatic COVID-19. In terms of immune responsiveness, HLA-DRB1*15:02 was associated with lower nucleocapsid T-cell responses. There was no association between DRB1 alleles and anti-spike antibody titres after two COVID vaccine doses. However, HLA DRB1*15:01 was associated with increased spike T-cell responses following both first and second dose vaccination. Trial registration: NCT04318314 and ISRCTN15677965