title: The role of drug-protein haptenation in β-lactam hypersensitivity
Creators: name: Adair, Kareena
Publisher: name: University of Liverpool Repository
Year: 2022

description: β-lactam antibiotics are frequently involved in drug hypersensitivity reactions, mediated by T-cells, and predominantly causing injury to skin and liver. The significant challenge arises from the idiosyncratic nature of these reactions, as the antigens responsible for T-cell activation are yet to be determined. The covalent binding of β-lactam antibiotics to highly abundant circulating proteins has been well-defined. The hapten model of T-cell activation in drug hypersensitivity indicates these protein-adducts may be processed into drug-modified peptides and presented to T-cells. Further, associations with risk HLA alleles for susceptibility to a reaction have been identified. Despite this evidence, the antigenic epitopes involved in the pHLA-TCR interaction has not been defined. The overall aim of this thesis was to investigate the role of β-lactam protein-adduct formation in T-cell activation in β-lactam hypersensitivity. To detect drug-modified proteins, anti-sera against piperacillin- and amoxicillin-modified proteins have been characterised. Synthetic drug-modified proteins have been successfully generated and characterised using mass spectrometry. Antibodies were investigated for their specificity, cross-reactivity and optimised use in immunological detection methods using the drug-modified proteins. A highly specific piperacillin antibody has been generated with no cross-reactivity with other β-lactam antibiotics, indicating the piperacillin side chain is the site of recognition for this antibody, rather than the β-lactam ring. An amoxicillin-specific antibody has also been generated with limited cross-reactivity with piperacillin and ampicillin. Cystic fibrosis patients often undergo piperacillin therapy to help to achieve effective lung function; however, a higher incidence of hypersensitivity reactions have been identified in these patients. The chemical nature of piperacillin haptens involved in T-cell activation has been investigated in patients. PBMC were isolated to investigate the presence of memory drug-specific T-cells. Piperacillin-specific T-cells were characterised revealing the involvement of both CD8+ and CD4+ T-cells. A set of T-cell clones were activated in a processing-dependent manner indicating a hapten mechanism of T-cell activation. Piperacillin-modified protein has been identified in the sera of CF patients, with a dominant hydrolysed hapten occurring in hypersensitive patients. A weak T-cell response to this hapten has been detected in a patient. Further, piperacillin modification has been identified in patient immune cells using an anti-piperacillin antibody. Flucloxacillin and amoxicillin are both common culprits of idiosyncratic DILI showing significant associations to specific HLA class I alleles, HLA-B*57:01 (flucloxacillin) and HLA-A*02:01 (amoxicillin), and HLA class II DRB1-15*01-DQB1-06*02 (amoxicillin). Drug-modified HLA-binding peptides have been generated using three sources including exosomal proteins from drug-treated hepatocytes, the naturally eluted HLA-B*57:01 immunopeptidome and HLA designer peptides. Peptides were purified and characterised using HPLC and mass spectrometric methods, respectively. Amoxicillin-modified HLA peptide-specific T-cell responses have been identified in healthy donors and patients expressing the risk HLA alleles. Following on from the immunogenicity of HLA-A*02:01 binding peptides, the amoxicillin haptenation of naturally HLA class I immunopeptidome was investigated in healthy donors expressing the HLA-A*02:01. Multiple naturally presented HLA class I ligands containing characteristic fragment ions derived from amoxicillin were detected by mass spectrometry analysis, indicating they were amoxicillin-modified peptides. Of these 5 amoxicillin-modified short peptides have been sequenced manually, and sourced to naturally occurring proteins, providing further evidence for the hapten hypothesis. Amoxicillin modification occurred on both lysine and cysteine residues. No major shifts have been identified in the natural HLA class I immunopeptidome in the presence of amoxicillin as seen with abacavir and HLA-B*57:01. In this thesis, a mechanistic role of β-lactam haptenation and HLA associations have been addressed. β-lactam protein haptenation has been detected and characterised in vitro and in vivo, including the detection of drug-protein adducts in patients and the presence of naturally occurring drug-modified HLA peptides. Importantly, the immunogenicity of β-lactam haptenation has been determined at the peptide level. Overall, these studies contribute to defining the pMHC-TCR interactions in β-lactam hypersensitivity, and provide a foundation for determining the antigenic epitopes involved in T-cell activation