Medical Biology - Theses
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ItemThe Transcription Factor T-bet in the Control of Germinal Centre Dynamics in Malaria( 2019)With reductions in the global malaria burden stalled, this preventable and curable infectious disease caused by the Plasmodium parasites, remains a public health challenge that affects the world’s most vulnerable populations. Naturally acquired immunity plays an important role in protection from disease; however, there is long-standing evidence that it requires years of repeated infections to develop. The reasons for this are largely elusive, but immuno-epidemiological studies support that protective antibodies and memory B cells are short-lived and inefficiently generated to infection. Moreover, recurrent infections are associated with an expansion of atypical memory B cells that may have impaired function. Histological analyses revealed significant disorganisation of the spleen in severe malaria patients, which led to the concept that acute infection may undermine the acquisition of B cell memory. T helper 1 pro-inflammatory responses induced by blood-stage infection were subsequently shown to compromise the induction of humoral immunity by inhibiting effective T follicular helper (Tfh) cell differentiation and germinal centre (GC) reactions. The relative contribution of the T helper 1 lineage-defining transcription factor, T-bet, in CD4+ T cells and B cells to GC development in malaria, was investigated using the P. berghei ANKA blood-stage infection model. T-bet expression in CD4+ T cells limited the differentiation of Tfh cells that supported GC development in the spleen. This led to an impaired generation of antibody-secreting cells and memory B cells following infection. In addition to its impact on CD4+ T cells, T-bet was highly up-regulated in GC B cells elicited by infection, and limited the magnitude of the GC response in a B cell-intrinsic manner. Strikingly, T-bet expression in the B cell compartment modulated the transcriptional landscape of GC B cells to promote the GC dark zone program but constrained light zone development. In particular, T-bet suppressed expression of the regulator of G-protein signaling 13, which down-regulates the responsiveness of B cells to migrate towards the chemokine CXCL12, for effective dark and light zone transition within the GC. T-bet-driven dark zone skewing of the GC reaction following malaria infection associated with enhanced somatic hypermutation of GC B cells, and improved the avidity of antibodies against the parasite. Therefore, this thesis supports a model in which malaria-elicited inflammation mediated by T-bet, exquisitely modulates the dynamics of the GC reaction, promoting GC B cell dark zone polarization that promotes the generation of B cells with increased affinity for antigen, consequently enhancing affinity maturation. This provides novel insight into the cellular mechanisms that underlie the development of humoral immune responses in malaria, and has implications for other chronic infections and autoimmune disease that are characterised by a similarly potent inflammatory milieu.
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ItemB cell responses during severe malaria: the impact of inflammation on T follicular helper cell and germinal centre responses( 2015)Despite many advances in malaria control and elimination, infection by Plasmodium remains a significantly widespread cause of morbidity and mortality worldwide. Naturally acquired immunity to the parasite plays an important role in protection against malaria infection and the development of symptomatic disease. However, no evidence exists of sterile immunity to the disease and the development of sustained clinically protective antibody responses has been shown to require repeated infections. While many studies have focused on the complex nature of these responses against the antigenically diverse parasite, few have addressed the effect of malaria infection on the generation of memory B cell responses. A study of children in areas of high seasonal malaria transmission revealed a delay in malaria-specific MBC generation despite continual exposure to the parasite. In contrast, in a low transmission setting, lasting memory B cell responses were detected in adults following a single exposure to the parasite. These data indicate clinical malaria infections may hinder the generation and maintenance of malaria-specific memory B cell populations. Long-lived populations of B cells, including memory B cells and long-lived plasma cells, are generated during the germinal centre (GC) reaction in secondary lymphoid organs, such as the spleen. In support of the notion that clinical malaria episodes hinder the induction of humoral memory, histological studies revealed that human fatal malaria infections are accompanied by dramatic changes in splenic architecture, including impaired GC formation. The bulk of studies examining the induction of GC responses following malaria infection have made use of self-resolving infection models in mice. To specifically address the impact of severe malaria infections on these processes, the development of GC responses was assessed using the P. berghei ANKA model of severe malaria in comparison to immunisation with an equivalent antigenic load of attenuated parasites. This model permitted the uncoupling of the effects of severe malaria infection and parasite exposure, and demonstrated that severe malaria infections profoundly impede the correct generation of GC structures. Further, compared to immunised control animals, infected animals had reduced numbers of GC B cells. Critically, the excessive inflammatory processes caused by severe malaria infection directly impaired T follicular helper cell differentiation and lead to the preferential accumulation of Tfh precursors. As a consequence of impaired GC induction, memory responses were not efficiently generated following severe malaria. Collectively, the data presented in this thesis demonstrate a novel role for inflammation in the control of Tfh and GC responses and provide valuable insight into the mechanisms underlying inefficient B cell responses following clinical malaria infections in humans.
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ItemPlasmodium chabaudi adami: vaccine antigens and antigenic variation( 2003)There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studi Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.