Abstract
Parasitic infections are a major problem in the world today, especially in developing countries. The antibody isotype immunoglobulin E (IgE) is widely thought to be produced as a natural immune response to parasitic infections, but its effectiveness compared to other antibody isotypes in combatting parasitic infections remains unknown. In this work, we characterised the interactions between parasite antigens and parasite-specific IgE antibodies using biophysical techniques such as surface plasmon resonance (SPR) to determine the affinities, kinetics, and thermodynamics of these interactions, and functional cellular assays to determine the effects of these interactions on the cells.We produced recombinant Plasmodium falciparum circumsporozoite protein (PfCSP), an antigen that is crucial for the survival of Plasmodium falciparum, which is the deadliest species of the malaria parasite. We also produced an IgE variant of CIS43, a previously described human IgG monoclonal antibody that interacts with the PfCSP antigen; we call this construct CIS43ε. We characterised the interaction between CIS43ε and PfCSP using SPR. We observed high-affinity interactions and multivalent interactions between CIS43ε and PfCSP involving the binding of CIS43ε to several regions of the surface of PfCSP. We also observed enhanced affinities under conditions where avidity effects could be exploited.
We produced short peptides of two of these regions, including the Peptide 21 region and the NANP repeats, and compared the interactions between these peptides and CIS43ε. We found that both peptides had biphasic binding with CIS43ε, as a result of monovalent and bivalent interactions and that the Peptide 21 region had the highest affinity for CIS43ε. In addition, we produced different oligomeric forms of the Peptide 21 involving streptavidin-Peptide 21 complexes and performed further characterisation of the interactions involving these oligomers and CIS43ε. We found that while a single CIS43ε was unable to bind to more than one binding site in these oligomers, the interactions under some conditions involved competitive binding. This led to the displacement of the monovalent interactions by the multivalent interactions involving different IgE molecules binding to different regions in a single oligomer. This competitive displacement increased with an increase in the oligomeric state of the streptavidin-Peptide 21 complexes. We also produced a CIS43 Fab and performed initial SPR binding studies involving this Fab fragment and the PfCSP full-length protein and peptides.
Using cellular assays involving RBL-SX38 cells, which express the high-affinity IgE receptor FcεRI, we have determined that the interaction between CIS43ε and the streptavidin-Peptide 21 complexes leads to efficient basophil activation, which is dependent on the oligomeric state of the antigens. We also found efficient and slightly increased levels of basophil activation in similar experiments involving CIS43ε and full-length PfCSP proteins.
This study provides a platform for further studies into elucidating the roles of IgE-mediated immune responses against parasites as part of ongoing efforts in parasitic disease prevention, treatment, and subsequent eradication.
| Date of Award | 1 Jun 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Brian Sutton (Supervisor) & James McDonnell (Supervisor) |