Rees-Spear, Chloe Elizabeth; (2024) Investigating the relationship between antibody affinity and B cell activation in HIV. Doctoral thesis (Ph.D), UCL (University College London).

Text ReesSpear_21192549_PhD Thesis.pdf - Accepted Version Access restricted to UCL open access staff until 1 October 2025. Download (20MB)

Abstract

In natural HIV infection, broadly neutralising antibodies (bnAbs) are rare between and within individuals. The gold standard of most HIV vaccine strategies is to induce bnAbs, but this remains a major challenge, with many focussed on improving the affinity of vaccine candidates for bnAbs. Most bnAbs have high affinity for the native HIV Envelope antigen and are thus expected to be preferentially selected in the germinal centre. However, in most natural infections this does not seem to be the case. Here, I investigate whether in vitro assessment of antibody affinity is sufficient to predict subsequent B cell activation. I first assessed the affinity and functionality of a selection of HIV antibodies by ELISA, pseudovirus neutralisation and biolayer interferometry. Then, I generated B cell lines expressing each antibody using an AAV-mediated CRISPR-Cas9 system that replaces the endogenous antibody in situ, enabling assessment of BCR activation in a controlled setting. Subsequent relationships between affinity and antigen-specific B cell activation were then assessed. All bnAbs exhibited high binding affinity (KD <10-9M), above the previously postulated activation affinity ceiling. However, greater affinity did not linearly translate to greater activation of the parent B cell. Conversely, within an epitope, decreases in affinity produced similarly decreased cellular activation. Increasingly higher affinity therefore does not guarantee strong activation, and above the 10-9M ceiling there was no relationship between activation and affinity. Furthermore, I developed an in vitro assay to investigate how this relationship might affect B-to-T cell antigen presentation, as this ultimately determines B cell fate. Together, the data presented here suggests that more complex BCR-antigen interactions than simple affinity, such as BCR:Env binding stoichiometry and epitope availability, may have significant effects on activation of the cell. These insights could be key in understanding bnAb development.

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