Medical Biology - Theses
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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.
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ItemStudies on the cellular basis of the immunological defect following thymectomy in the mouse( 1969)Answers have been sought for two key questions relevant to the proposed mode of action of the thymus as a “central” lymphoid organ. Does the peripheral lymphocyte population contain a large number of thymus-derived cells and do these cells respond to antigens by producing a progeny of antibody-forming cells? The number of small lymphocytes emerging from a thoracic duct fistula in young adult neonatally-thymectomized CBA mice was approximately 2% of that in intact mice of the same age and body weight. Multiple injections of chromosomally-marked thymus cells increased the size of the lymphocyte pool and the majority of thoracic duct cells, stimulated into division, carried the chromosome marker of the thymus cell donor. Thymectomy in adult life was not followed by a significant decrease in the output of thoracic duct lymphocytes until many months after the operation. Whole body x-irradiation resulted in a dramatic reduction in the number of cells drained from a thoracic duct fistula and, in mice protected from the lethal effects of haemopoietic failure, the reestablishment of the lymphocyte population was dependent upon the presence of the thymus. The data supports an increasing bulk of indirect evidence which, when taken in toto, strongly suggests that a large proportion of recirculating lymphocytes are thymus -derived cells or their descendants. The number of sheep erythrocyte antigen-reactive cells (ARC) in the thoracic duct lymphocyte population of neonatally-thymectomized mice was markedly reduced when compared with the number in the population from normal mice. The bone marrow did not contain ARC but was a potent source of ARC precursors. Neonatally-thymectomized mice did not lack precursor cells in the bone marrow but apparently lacked the thymus influence necessary for the differentiation of these precursors into ARC. Further studies on the thymus-dependent development of ARC hinted at the possibility that the entity known as "an ARC" required the presence of both thymus- and bone marrow-derived cells to express itself in terms of haemolysin production. Neonatally-thymectomized CBA mice failed to respond in normal fashion to a primary injection of sheep erythrocytes (SRBC) and the peak number of haemolysin plaque-forming cells (PFC) in the spleen was reduced by a factor of 1 log 10. The PFC response was increased by injections of either thymus or thoracic duct lymphocytes from CBA, (CBA x C57BL)F 1 hybrid, and C57BL donor mice. In thymectomized mice reconstituted with semiallogeneic and allogeneic cellular inocula, the PFC carried the immunogenetic characteristics of the host and not those of the inoculated cells. Hence, thymus and thoracic duct cells were not reconstitutive simply by virtue of the ability to transform into PFC. Thymus and thoracic duct cell inocula contained "reactor cells" which responded to SRBC antigens in irradiated mice by undergoing a burst of mitosis. Thoracic duct lymphocytes, unlike thymus and bone marrow cells, were able to produce PFC when injected together with SRBC into irradiated mice. However, the PFC response in irradiated recipients of thoracic duct lymphocytes was increased substantially by a simultaneous injection of bone marrow cells. Combinations of cells from semiallogeneic mice did not interact upon transfer to irradiated recipients but clear evidence of synergism in PFC production was apparent in adult-thymectomized irradiated mice protected with CBA bone marrow cells and injected two weeks later with (CBA x C57BL)F 1 thoracic duct cells. In this case, the vast majority of PFC were derived from the bone marrow inoculum. The results suggest that the bone marrow contains only PFC precursors, the thymus only "reactor cells", but that the thoracic duct lymph contains both cell types. It seems that the normal 19S haemolysin response to SRBC in the CBA mouse requires the collaboration of bone marrow-derived PFC precursors and thymus-derived "reactor cells". The neonatally-thymectomized mouse contains adequate numbers of PFC precursors but, after challenge with SRBC, few are recruited into 19S haemolysin production because of the severe deficiency in thymus-derived ''reactor cells".