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Title: Understanding T cell and antibody-mediated immune mechanisms against malaria infection
Author: Thorburn, Samuel Guy
ISNI:       0000 0004 9359 4097
Awarding Body: London School of Hygiene & Tropical Medicine
Current Institution: London School of Hygiene and Tropical Medicine (University of London)
Date of Award: 2020
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CD8+ T cells have been implicated in protective immune responses in pre-erythrocytic malaria and pathogenic responses in blood stage malaria, making them an important factor in determining the outcome of infection. Natural exposure to pre-erythrocytic malaria infection does not elicit sterile protection in humans, whereas multiple immunisations with large numbers of radiation attenuated sporozoites can. This protection is largely due to parasite-specific CD8+ T cells; however, antibody responses have also been shown to be functional in pre-erythrocytic infection. Unfortunately, there is a paucity in known antigens other than the immunodominant protein circumsporozoite protein (CSP) that are recognised by antibodies in the preerythrocytic stages. With regard to blood stage infection, there are a variety of outcomes ranging from non-clinical patent infection to cerebral malaria due to immune-mediated pathology. Indeed, there are several different mouse models of malaria that each produce different outcomes, suggesting there may be subtle differences in immune regulation, which has previously been investigated by our group but is not fully understood. The research presented here attempts to address three hypotheses. First, I hypothesised that the requirement for high doses of antigen at multiple intervals for sterilising protection is at least in part due to inhibition of CD8+ T cell activation via regulatory pathways that are stimulated during exposure to attenuated sporozoites. Secondly, I hypothesised that T cell regulation during blood stage infection through the interleukin 10 receptor (IL-10R) confers resistance in a BALB/c model of Pb ANKA infection, which is normally refractory to experimental cerebral malaria (ECM). Finally, I hypothesise that protective immunisation of C57BL/6 and BALB/c with attenuated Pb ANKA sporozoites leads to the production of non-CSP antibodies that may be viable candidates for further vaccine research. In order to test my first hypothesis I investigated co-inhibitory molecules cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death ligand 1 (PD-L1) and PD-L2 and regulatory cytokines IL-27 and IL-10. Using mouse models, I gave a single non-protective immunisation of γ-radiation attenuated Pb ANKA sporozoites and investigated the CD8+ T cell immune response in the absence of each regulatory pathway. Antibody-mediated blockade of CTLA-4 concurrently with a normally non-protective radiation attenuated Pb ANKA sporozoite immunisation in C57BL/6 mice led to a significant increase in sporozoite antigen-specific interferon gamma (IFNγ) producing CD8+ T cells upon re-stimulation. Additionally, this resulted in sterile protection when challenged 14 days after blockade and immunisation; however, protection was greatly reduced when challenged after 45 days. Using mice deficient in IL-27R or using antibody blockade of PD-L1, PD-L2 and IL-10 with a non-protective single immunisation of radiation attenuated Pb ANKA sporozoites did not lead to significantly increased CD8+ T cell activation upon re-stimulation. Neither did it result in increased protection from parasite load in the liver. These experiments are a proof of principle that certain regulatory pathways can significantly reduce protective CD8+ T cell responses to sporozoite immunisation. For my second hypothesis, I investigated CD4+ and CD8+ T cell responses to Pb ANKA blood stage infection in ECM resistant BALB/c mice treated with antibodies to block the IL-10R. I show that blockade of the IL-10R leads to increased CD4+ and CD8+ T cell production of IFNγ with blood stage Pb ANKA infection. Other work in this chapter shows that IL-10R blockade leads to increased ECM as a result of increased brain pathology. Additionally, I show that IL-10R blockade with Pb ANKA blood stage infection leads to an increase in expression of CTLA-4, but not PD-1 on CD4+ CD8+ T cells. This suggests that there may be a relationship between IL-10- and CTLA-4- mediated regulation in Pb ANKA blood stage infection of BALB/c mice. To address my third hypothesis, I used a novel immunisation schedule using Pb ANKA sporozoites that express Pf CSP rather than Pb ANKA CSP. One immunisation with transgenic parasites followed by a booster immunisation with Pb ANKA was performed using C57BL/6 and BALB/c mice to boost non-CSP responses. This led to antibodies that recognised previously validated pre-erythrocytic antigens and functionally inhibited sporozoite invasion of hepatocytes in vitro. Using high-density microarray technology I investigated all proteins expressed during the preerythrocytic stages of Pb ANKA using serum from these immunised mice. Results show antibody responses to previously unidentified pre-erythrocytic antigens and therefore potential antigens for future vaccine research.
Supervisor: Hafalla, J. C. R. ; Riley, E. M. Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral