T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations

Investigation of SARS-CoV-2 faecal shedding in the community: a prospective household cohort study (COVID-LIV) in the UK

Background SARS-CoV-2 is frequently shed in the stool of patients hospitalised with COVID-19. The extent of faecal shedding of SARS-CoV-2 among individuals in the community, and its potential to contribute to spread of disease, is unknown. Methods In this prospective, observational cohort study among households in Liverpool, UK, participants underwent weekly nasal/throat swabbing to detect SARS-CoV-2 virus, over a 12-week period from enrolment starting July 2020. Participants that tested positive for SARS-CoV-2 were asked to provide a stool sample three and 14 days later. In addition, in October and November 2020, during a period of high community transmission, stool sampling was undertaken to determine the prevalence of SARS-CoV-2 faecal shedding among all study participants. SARS-CoV-2 RNA was detected using Real-Time PCR. Results A total of 434 participants from 176 households were enrolled. Eighteen participants (4.2%: 95% confidence interval [CI] 2.5–6.5%) tested positive for SARS-CoV-2 virus on nasal/throat swabs and of these, 3/17 (18%: 95% CI 4–43%) had SARS-CoV-2 detected in stool. Two of three participants demonstrated ongoing faecal shedding of SARS-CoV-2, without gastrointestinal symptoms, after testing negative for SARS-CoV-2 in respiratory samples. Among 165/434 participants without SARS-CoV-2 infection and who took part in the prevalence study, none had SARS-CoV-2 in stool. There was no demonstrable household transmission of SARS-CoV-2 among households containing a participant with faecal shedding. Conclusions Faecal shedding of SARS-CoV-2 occurred among community participants with confirmed SARS-CoV-2 infection. However, during a period of high community transmission, faecal shedding of SARS-CoV-2 was not detected among participants without SARS-CoV-2 infection. It is unlikely that the faecal-oral route plays a significant role in household and community transmission of SARS-CoV-2

CHARACTERISATION OF DRUG-SPECIFIC T-CELLS TOWARDS A MECHANISTIC INSIGHT OF DRUG HYPERSENSITIVITY REACTIONS

Drug hypersensitivity reactions are rare but remain a serious problem in clinical practice. Many reactions to a number of drugs are associated with expression of individual HLA class I markers. Carbamazepine (CBZ) is an anticonvulsant which causes severe cutaneous adverse reactions associated with expression of HLA-A*31:01, HLA-B*15:02, HLA-B*15:21, and HLA-B*57:01. Although, in vitro analyses has shown that the CBZ-mediated adverse reaction is associated with activation of drug-specific CD4+ and CD8+ T-cells, the association between drug HLA class I and class II between and T-cell activation has not been fully established. In this thesis, the CBZ-mediated response was evaluated using peripheral blood mononuclear cells from CBZ-naïve healthy donors and CBZ hypersensitive patients from the University of Liverpool, UK, and Prince of Songkla University, Thailand. The in vitro drug response of each participant was evaluated by lymphocyte transformation test and ELISpot and further characterised by T-cell cloning. A total of 76 CBZ-specific CD4+ T-cells were generated by T-cell cloning in 2/5 healthy donors from the University of Liverpool cohort and 4/6 CBZ hypersensitive patients from both cohorts. The generated T-cell clones were polyclonal in terms of CBZ-10,11-epoxide cross-reactivity pattern and T-cell receptor (TCR) Vβ phenotype. Cytokine secretion analysis revealed that the generated CD4+ T-cells secreted IFN-γ and granulysin in response to CBZ. A detailed characterisation showed that the CD4+ CBZ-mediated response was restricted to HLA-DR, particularly HLA-DRB1*07:01. A database review substantiated that this marker was previously reported to be associated with CBZ-induced Stevens-Johnson syndrome and toxic epidermal necrolysis. The HLA-DR allele is also often linked with HLA-B*57:01 as a haplotype. These findings suggested that this HLA class II marker may co-contribute with the HLA class I marker as a haplotype via selective presentation of CBZ to CD4+ T-cells. TCRs that interact with HLAs were characterised by TCR sequencing analyses of drug-specific T-cells. T-cell clones that respond specifically to CBZ, sulfamethoxazole, nitroso-sulfamethoxazole, dapsone, and nitroso-dapsone were included in the sequencing analysis. A remarkable heterogeneity was observed among drug-specific T-cells, notably even among clones that respond to the same drug. No clear relationship between TCR residue expression and T-cell responsiveness (cytokine release, cross-reactivity) was identified. The β-lactamase inhibitors tazobactam and clavulanic acid often cause hypersensitivity when administrated alongside β-lactam antibiotics. Unlike β-lactam hypersensitivity, the cross-reactivity pattern of T-cells responsive to β-lactamase inhibitors is not well understood. Herein, the cross-reactivity of tazobactam- and clavulanate-responsive T-cells was characterised. Clavulanate-specific T-cells showed a total cross-reactivity against tazobactam and the reverse was also true. Mass spectrometric analysis revealed that tazobactam and clavulanate form similar protein adducts, thereby potentially presenting similar modified peptides to T-cells. Collectively, this work showed that there are highly heterogeneous molecular components that underlie drug hypersensitivity reactions. Expression of specific HLA, TCR, and antigenic drug molecule alongside yet to be determined environmental factors all determine whether drug exposure will result in a T-cell response and hypersensitivity

Number of cases eligible for statistical analysis in each age group.

<p>Observed numbers of cases with common autosomal trisomies (trisomies 21, 18 and 13) and predicted maternal age-specific rates in each age group (at the time of amniocentesis).</p

Results of AS-PCR and DDB.

<p><b>(a)</b> Electrophoresis was applied by using 2.5% agarose gel with 100V for 30 minute. A control product was 301 bp long while the specific PCR product was 137 bp long. The alleles with PCR product included <i>HLA-B*15</i>:<i>02</i> (No. 1 and 8), <i>B*15</i>:<i>21</i> (No. 2), <i>B*15</i>:<i>25</i> (No. 3), <i>B*15</i>:<i>11</i> (No. 4), <i>B*15</i>:<i>32</i> (No. 5), <i>B*15</i>:<i>01</i> (No. 6), <i>B*15</i>:<i>31</i> (No. 7), and <i>B*46</i>:<i>01</i> (No. 4, 8). <i>HLA-B*15</i>:<i>13</i> (No. 9) and other common alleles (No. 10–16) gave negative results in the PCR step. <b>(b)</b> The samples analyzed in the PCR step (No. 1–9) were subsequently examined by DDB. (NTC: “no template control”).</p

Maternal Age-Specific Rates for Trisomy 21 and Common Autosomal Trisomies in Fetuses from a Single Diagnostic Center in Thailand

<div><p>To provide maternal age-specific rates for trisomy 21 (T21) and common autosomal trisomies (including trisomies 21, 18 and 13) in fetuses. We retrospectively reviewed prenatal cytogenetic results obtained between 1990 and 2009 in Songklanagarind Hospital, a university teaching hospital, in southern Thailand. Maternal age-specific rates of T21 and common autosomal trisomies were established using different regression models, from which only the fittest models were used for the study. A total of 17,819 records were included in the statistical analysis. The fittest models for predicting rates of T21 and common autosomal trisomies were regression models with 2 parameters (Age and Age²). The rate of T21 ranged between 2.67 per 1,000 fetuses at the age of 34 and 71.06 per 1,000 at the age of 48. The rate of common autosomal trisomies ranged between 4.54 per 1,000 and 99.65 per 1,000 at the same ages. This report provides the first maternal age-specific rates for T21 and common autosomal trisomies fetuses in a Southeast Asian population and the largest case number of fetuses have ever been reported in Asians.</p></div