Microbiology & Immunology - Theses
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ItemGenerating CD8+ liver-resident memory T cell immunity against malaria( 2022)Liver resident memory CD8+ T (Trm) cells are attractive vaccine targets for malaria (Plasmodium) liver-stage immunity and can be effectively generated by glycolipid-peptide (GLP) vaccines. To gain insight into underlying mechanisms, we examined the requirements for priming, differentiation, long-term maintenance, and secondary boosting of liver Trm cells. We found that type I conventional dendritic cells (cDC1) were essential for priming CD8+ T cell responses, during which exposure to IL-4, most likely provided by activated type I natural killer T (NKT) cells, enhanced liver Trm cell formation. In addition, optimal generation of liver Trm cells required exposure to a combination of vaccine-derived inflammatory and antigenic signals post-priming, with antigen recognition being associated with enhanced Trm cell longevity. After primary immunisation with GLP vaccines, boosting of liver Trm cells could be achieved with the same GLP vaccine but a substantial delay was required for optimal boosting. This appeared to be due to NKT cell anergy post-priming as NKT cell-independent heterologous boosting could be achieved much earlier. Overall, our study revealed that the generation of liver Trm cells by GLP vaccination is IL-4 and cDC1 dependent, with longevity increased by post-priming antigenic signals and homologous boosting influenced by NKT cell recovery. Like many other malaria subunit vaccines, however, the utility of GLP vaccines is somewhat limited by the scarcity of protective CD8+ T cell epitopes. This issue is particularly prominent in the context of rodent P. berghei ANKA (PbA) infection of B6 mice, an extensively studied model of malaria. Using a combination of mass-spectrometry and in-silico approaches, we generated a library of 400 PbA-derived MHC I-restricted epitopes, from which we identified 4 immunogenic candidates that each reproducibly stimulated CD8+ T cells after pre-erythrocytic and blood-stage infections of B6 mice. Further characterisation of one of these peptide candidates, Db163, revealed cross-reactivity with a known immunogenic, but non-protective peptide PbA GAP5040-48. Targeting two additional epitopes, Db100 and Db177, by GLP vaccines induced substantial CD8+ liver Trm cells but these responses lacked protective efficacy against sporozoite challenge. The fourth epitope is derived from the PbA X, a predominantly late liver-stage antigen. Promisingly, this epitope could be targeted by a GLP vaccine to evoke liver Trm cell-mediated immunity against malaria in B6 mice. This protective immunity was remarkably long-lived with liver Trm cells persisting for at least 210 days. Furthermore, we demonstrated that X-specific liver Trm cells could execute a protective immune response cooperatively with those specific for PbA TRAP130-138, leading to improved sterile immunity even against high-dose sporozoite challenges. Lastly, the discovery of two novel HLA-A 02:01-restricted epitopes within the P. falciparum X proteins provides a future opportunity to dissect their usefulness as human vaccine candidates. Overall, this thesis provides novel mechanistic insights to maximise liver Trm cell formation and longevity after vaccination. Additionally, this thesis identifies novel antigenic targets of liver Trm cells that could be exploited for vaccination to induce immunity against malaria.
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ItemMechanisms that cause functional impairment of dendritic cells following Systemic Inflammatory Response Syndrome( 2022)Dendritic cells (DC) are potent antigen presenting cells which link the adaptive and innate arms of immune response. This normal functioning of DC is severely impaired following recovery from systemic (pro- and anti-)inflammatory response syndrome (SIRS) caused by sepsis or severe trauma, leading to protracted immunosuppression. This so-called “DC paralysis” results in greater risk of secondary infections and higher rates of mortality and morbidity in patients. We modelled SIRS, DC paralysis and immunosuppression in mice after injecting Toll-like receptor (TLR) ligands or malaria infection. Transcriptome and phenotype characterization allowed us to recognize and track paralyzed DC. Functional characterization of paralyzed DC showed normal activity in terms of antigen uptake, T cells priming and cytokine production in vitro. But, paralyzed DC in vivo or ex vivo showed impairments in uptake of antigen, defects in antigen processing and presentation by MHC molecules, altered cytokine production, and elevated production of inhibitory molecules, altogether leading to impaired priming of antigen-specific T cells by paralyzed DC. We were able to improve paralyzed DC function by targeting antigen to a surface receptor or by blocking interferon type I signaling. However, blocking IL-10 signaling, ablation of prominent paralysis marker CD103 or transcription factor Pparg, and depletion of regulatory T cells did not improve function of paralyzed DC. We further showed formation of paralyzed DC did not need DC activation or recognition of TLR ligand, but was instructed by secondary signals after SIRS including TGFb, produced by paralyzed DC themselves. We observed increased number of immediate DC precursors in spleen after SIRS, and performed transfer experiments to understand the developmental stage that paralysis was imprinted on cells of DC lineage. We observed no commitment at any stages of DC development towards paralyzed fate, and that local tissue environment biased the final stages of DC development towards paralysis in situ. We also showed location-specificity of DC paralysis as systemic SIRS caused paralysis in spleen and peripheral lymph nodes but not in lung, and SIRS in lung caused immunosuppression in lung but not in spleen. We finally showed severe trauma (as aseptic cause of SIRS) could lead to DC activation and paralysis. We ultimately aim to translate the understanding from mouse into the clinic towards diagnosis and treatment of DC paralysis in critically-ill patients.
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ItemT cell response to an MHC-II restricted epitope of rodent malaria( 2021)Malaria is caused by different Plasmodium species that can infect a variety of animals including humans and rodents. The life cycle of these parasites is complex and includes a liver stage followed by a blood-stage in their vertebrate hosts. While the host’s immune response against each of these stages is incompletely understood, CD4 T cells are known to play an important role in immunity to Plasmodium infection during both stages. This project aims to examine the specific CD4 T cell response to a novel MHC II-restricted epitope in Plasmodium infection in C57BL/6 mice, and to characterise the protective capacity of these T cells. To this end, we made use of a recently generated TCR transgenic mouse line, termed PbT-II, which responds to a so far unknown Plasmodium derived epitope. In this project, the PbT-II epitope was identified as derived from heat shock protein 90, residues 484 to 496 (Hsp90484-496 or abbreviated DIY). Different priming methods, such as injection of an anti-Clec9A antibody attached to the Hsp90 epitope (aClec9A-DIY), infection with P. berghei ANKA (PbA) infected red blood cells (iRBCs) or immunisation with radiation attenuated PbA sporozoites (RAS), were used to characterise PbT-II memory cell formation. Results revealed the formation of memory PbT-II cells expressing surface markers associated with central memory T cells (TCM), effector memory T cells (TEM) and tissue resident memory T cells (TRM). Given the importance of tissue-resident memory T cells in peripheral immunity, mainly studied in CD8 T cells, we focused our study on the formation and function of CD4 TRM cells in the liver. Parabiosis studies using RAS vaccinated mice confirmed the liver residency of a CD69+ PbT-II cell population. Gene expression profile analysis revealed that these CD4 T cells expressed a core gene signature similar to that of CD8 resident memory T cells. Furthermore, differences in the gene expression profile of PbTII TRM cells generated via different protocols, suggested lineage specific effector mechanisms, such as IL-4 production or perforin expression, for subsets of CD4 TRM cells in the liver. As CD4 T cells can potentially act against both the liver and blood-stage of Plasmodium infection, we sought to investigate the protective potential of PbTII effector and memory cells for both of these stages. While none of the PbT-II priming methods resulted in a reduction of liver parasite burden upon sporozoite infection, mice injected with large numbers of in vitro polarized PbT-II Th1 or Th2 cells showed reduced parasitemia after PbA blood-stage infection. Surprisingly, most of these mice were protected from experimental cerebral malaria (ECM), although they were not able to clear PbA blood-stage infection.
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ItemNo Preview AvailableElucidating the essential role of PTEX during the asexual blood stage of Plasmodium falciparum( 2021)The human malaria parasite Plasmodium falciparum is renowned for its ability to modify its host red blood cell (RBC), during the asexual blood stage of its life cycle. Many host cell modifications are involved in immune evasion and are therefore of clinical importance. These modifications are established by parasite effector proteins, exported across a vacuole membrane that envelops the intracellular parasite. Protein export is performed by an ATP-driven translocon termed the Plasmodium translocon of exported proteins (PTEX). PTEX is a parasite-derived protein complex and constitutes the sole pore for these exported effector proteins to gain access into the RBC compartment. Importantly, PTEX has been found to be essential for blood stage growth, likely as a result of it exporting essential effector proteins into the RBC. The parasite is predicted to export around 500 effector proteins into the RBC, however, most of these proteins have yet to be studied or allocated specific roles, and therefore a great deal remains to be discovered about these proteins in Plasmodium species. In this thesis I employed three approaches to study why PTEX is essential. In the first Aim, I attempted to derive the functions of 13 exported proteins previously shown to physically associate with the essential RhopH complex that the parasite installs in the RBC membrane to import vital nutrients from the blood plasma. Although none of the 13 proteins appeared important for nutrient uptake, five of the proteins studied interacted with the RhopH complex, potentially to help traffic or stabilise the complex to the iRBC membrane. I also confirmed the localisation of six new exported proteins and the interactome of 10 of the 13 proteins studied and the complexes they form. In my second Aim, I defined the essential exportome in P. falciparum and studied five proteins identified in my analysis and characterised them further. Three of the five proteins showed reduced growth when they were conditionally depleted indicating they might be important for blood stage growth. One of the proteins was not exported- but identified here as a novel protein of the parasite vacuole membrane. In my third and final Aim, I investigated metabolic changes in parasites following knockdown of PTEX, where preliminary data had indicated that knocking down PTEX disrupts the haemoglobin digestion pathway. To investigate this further I studied the association of PTEX with two haemoglobin proteases, plasmepsin II (PMII) and falcipain 2a (FP2a). I found that knocking down PTEX affected the trafficking of these two proteases. Furthermore, through the use of protein-binding and inducible folding assays, I determined that the FP2a reporter protein directly associated with a subunit of PTEX, HSP101, which unfolds protein cargo prior to export. These results indicate that PTEX may not only be required to help deliver important proteins into the RBC compartment but also to correctly traffic proteins at the parasite surface that are important for intracellular functions such as haemoglobin digestion. Taken together, this research greatly expands our knowledge of the diverse roles that both PTEX and its various cargo proteins perform in different compartments of the infected RBC.
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ItemInducing immunity to liver stage malaria through endogenous tissue resident memory cells( 2020)Tissue resident memory CD8 T (TRM) cells provide effective tissue surveillance and can respond rapidly to infection due to their strategic location. Within the liver, TRM cells can induce effective protection against liver-stage Plasmodium infection. Recently, members from our group identified a highly immunogenic peptide (named Pb 1) within the putative 60S ribosomal protein L6 of P. berghei ANKA. Experiments conducted and presented in this thesis aimed to assess the suitability of Pb 1 for the induction of endogenous liver TRM cells that confer sterilizing protection in B6 mice. To this end, a series of different immunisation strategies targeting the Pb 1 epitope were implemented and specific CD8 T cell responses were assessed. Results revealed that the number of naive specific CD8 T cell precursors for the Pb 1 epitope was very large. Substantial expansion and formation of specific liver TRM cells was achieved by two different immunisation strategies: i) Single injection with Clec9A mAb plus adjuvant and ii) Prime and trap, both targeting the Pb 1 epitope. While mice vaccinated with Clec9A mAb developed partial protection, almost all mice vaccinated with prime-and-trap targeting Pb 1 were sterilely protected against liver stage challenge. Inflammation favours the formation TRM cells and adjuvants can affect their numbers. Accordingly, a second focus of this thesis sought to investigate how to enhance liver TRM cell formation by using TLR and RIG I like receptors agonists as adjuvants. For this, eight different agonists were assessed for the generation of liver TRM cells induced by Clec9A targeted immunisation with the Pb 1 epitope. Data from this screen showed that CpG based adjuvants were most effective at inducing the formation of TRM cells in the livers of vaccinated mice and that the transfection reagent DOTAP enhanced this effect. Based on this understanding, we then investigated the potential of CpG and its encapsulation in DOTAP to improve TRM cell generation by other vaccination strategies. Surprisingly, these studies revealed that CpG based adjuvants did not improve liver TRM cell generation by vaccination with radiation attenuated sporozoites. The basis for this outcome is discussed. Altogether, these findings provide insights into elements that favour the generation of protective liver TRM cells; information that can be used for the design of TRM cell based subunit vaccines against Plasmodium infection.