B cell responses to protein adducts

Protein adducts are covalent additions between electrophiles and nucleophilic protein sites, such as amines of some sidechain residues. These adducts may form functional antigenic complex that elicit downstream anti-adduct responses: surfaces encompassing the adduct may be recognised by cognate B cells, while associated peptide may be presented to T cells. In this thesis, I investigate protein adducts in two discrete contexts: 1) as a potential vaccine design tool in overcoming tolerance barriers to an immunorecessive glycan, and 2) the immunogenicity of a protein-reactive small-molecule drug.
Tumour-associated carbohydrate antigens (TACAs) are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessiveness that classical vaccination approaches have failed to overcome. One prominent TACA, the glycan component of ganglioside sugar GM3 (GM3g), is over-expressed on diverse tumours and is notoriously immunorecessive. Weak adaptive responses to GM3 are believed to be associated with both lack of associated T cell help and lack of cognate naïve B cells in the repertoire. Here, I report on the immunogenicity of a novel synthetic protein chemistry pipeline wherein artificial conjugates of GM3g are presented via a short, bespoke synthetic linker, [–amidine-GM3g], on primary amines of lysine sidechains of carrier proteins. These long orthogonal glycoconjugates (LOGs) are highly immunogenic in mice, raising a robust GM3g-specific IgG response. Thorough antigen-specific clonotyping of the B cell response revealed a novel lectin-like clonal class raised against the LOG, with a defined germline configuration dominated by members of the IGHV2 subgroup. Subsequent structural, biophysical and biochemical characterisation of the response demonstrated how the artificial display of GM3g accesses this unique clonotype, but also explains why antibodies do not cross-react with native, ceramide-docked GM3g. These findings represent the first potential step towards a novel germline targeting-like approach to vaccine design against this and other TACAs.
β-lactam antibiotics, such as penicillin G (PenG) are chemically unstable under carbonate buffer systems, such as that in serum. Lactam-based adducts have long been hypothesised as a key antigenic driver of antibody-mediated hypersensitivity, both IgE and IgG, although the molecular basis for these reactions and the determinants of drug adduct immunogenicity are poorly understood. Using a murine model, I investigate both the pharmacokinetic and immunological factors that determine the penicilloyl-specific antibody responses. I demonstrate that free drug availability, innate immune activation and protein carrier factors are critical in determining the serological response. Clonotyping of the anti-PenG response revealed a single dominant clonotype raised against the drug, comprised of phylogeneticially-related IGHV1, IGHV5, IGHV10 and IGHV14 family members. Further structural and biochemical characterisation of the clonal family raised against the drug may inform the design of a PenG analogues that cannot bind this B cell clonal class, representing a candidate no/low allergenic drug alternative in a mouse model.
In summary, protein adducts have broad implications for immunity and pathology. This thesis contributes to how we may utilise these adducts as tools both for vaccine design and to study the role of adducts in disease.

Keywords: B cell
Vaccine
Antibody
Allergy
Glycan
Cancer
Advisor: Sattentau, QJ
Davis, BG