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Background\ud 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.\ud \ud Methods\ud 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.\ud \ud Results\ud 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.\ud \ud Conclusions\ud 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.

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., Understanding the immune response to SARS-CoV-2 is dependent on being able to distinguish COVID-19 immune responses from cross-reactive immune responses to other coronaviruses. Here the authors show that choice of antigens and whether an ICS, ELISPOT or T cell proliferation assay is used has a major effect on this discriminatory ability.

Typically, drugs are too small to elicit an immune response and therefore the occurrence of drug-mediated reactions is extremely rare. Characteristically these reactions are inherently difficult to predict from only the pharmacology of a drug or from the genotype and phenotype of the patient. Current models such as the lymphocyte transformation cells and drug challenge tests fall short of providing a panacea due to their invasive nature and inaccurate predictive capabilities. The ability to model hypersensitivity reactions in vitro would therefore prove useful in the clinic and during design, preventing morbidity and mortality. An ideal model would mimic the immune microenvironment through incorporating drug, patient, and disease specific factors. In the studies described in this thesis, we aimed to develop tools for future hypersensitivity modelling, encompassing drug, patient and disease specific factors alongside the immune microenvironment. We focused on models which may help predict chemically reactive metabolite formation resulting in T-cell activation, incorporating drug and patient factors. We utilised drug-metabolite specific T-cell clones, donor specific antigen presenting cells and a metabolising system to demonstrate chemically reactive metabolite formation and subsequent T-cell activation. Dapsone-nitroso specific T-cell clones demonstrated strong proliferative responses when cultured in situ dapsone and a metabolising system. Furthermore, through mass spectrometry analysis, we demonstrate the formation of multiple chemically reactive metabolites within the cell culture medium. We next investigated how the tissue microenvironment is related to flucloxacillin accumulation and novel adduct formation in flucloxacillin-mediated drug induced liver injury. Utilising the HepaRG cell line and primary human hepatocytes, evidence suggests that MRP2 and P-gp expression and function may play a role in flucloxacillin accumulation in liver-like cells. Furthermore, utilising membrane transporter inhibitors, we demonstrated how disruption of transporters increases the formation of flucloxacillin modified proteins and peptides for possible T-cell presentation and activation, contributing to the DILI mechanism. We next aimed to incorporate the HLA-predisposition of flucloxacillin in drug induced liver injury alongside the target tissue, creating an autologous T-cell mediated cytotoxicity model. Through use of donor specific induced pluripotent stem cells, T-cell clones and antigen presenting cells, we were able to demonstrate flucloxacillin-specific T-cell mediated killing of HLA-B*57:01 hepatocyte-like cells. Finally, utilising traditional and well-defined T-cell models, we demonstrated how the use of a conventionally safe therapeutic moiety, green tea, may result in a hypersensivity reaction, given the right patient and disease factors. T-cell isolation from HLA-B*35:01 donors demonstrated a strong dose-proliferation relationship upon exposure to epigallocatechin gallate and green tea extract.