Studies On Cellular Immune Responses Of Peripheral Blood Mononuclear Cells From Individuals In An Endemic Rural Community To A Synthetic Plasmodium Falciparum Merozoite Antigen

SUMMARY

Several Plasmodium spp. antigens have been found to be associated with protection against malaria and a few have been tested in the field in human volunteers. However, these efforts have achieved limited success, and as a result the search for additional more potent malaria vaccine components continues. The work reported in this thesis was executed with the objective of determining the suitability of three peptides obtained from the merozoite surface antigen, MSA-2, as putative sub-unit malaria vaccine components. Previous studies have suggested that a malaria vaccine capable of inducing long-lasting protective immune responses would most likely require incorporation of antigens expressing both B-and T-cell epitopes. In this respect the MSA-2 molecule which expresses both B- and T-cell epitopes has been shown to induce immune responses that eliminates mortality due to P. chabaudi malaria and significantly reduces parasitaemia in mice. It is, however, necessary to determine the protective ability of MSA-2 in man, since immunity induced in the animal model is not necessarily identical to the situation in humans. Investigations of this nature in man requires the use of peripheral blood mononuclear cells (PBMC) for in vitro assays. Clinical examination of the subjects included in this study, namely Ghanaian children and Ghanaian adults from a malaria endemic area, and unexposed control Danish adults, revealed no signs of clinical malaria at the time of blood sampling. However, P. falciparum parasites were found in blood samples of some of the Ghanaians with a significantly higher prevalence in the children (49%) than in adults (12%). xiiInitial experiments with fresh and cryo-preserved PBMC showed no significant differences (P< 0.005) in the ability to proliferate and to produce interferon gama (IFN-7 ) upon stimulation with tetanus toxoid (TT) as antigen. Cryo-preserved PBMC were therefore used in the experiments. In vitro lymphoproliferative assays using three MSA-2 peptides, namely GI, G2C and G4 which contain known T-cell epitopes showed significantly higher responses (P0.05). This implies that the observed differences in the response to the MSA-2 peptides may be due to differences in immunological memory related to the level of prior exposure to malaria. Interferon-gamma (IFN-7 ) was detected in 32%, 36% and 29% of PBMC cultures from the Ghanaian adults following stimulation with the GI, G2C and G4 peptides, respectively. In all, 61% of these cultures produced IFN- 7 in response to at least one of the peptides. None of the cultures from Danish adults produced IFN-7 . The difference was statistically significant (P< 0.001), even though using the control antigens, PPD and TT, PBMC cultures from both groups produced similar amounts of IFN-7 . Interleukin-4 production was also detected in response to each of the peptides in PMBC cultures of only the Ghanaian adults. However, the number ofresponding cultures was lower, and there was no statistical difference between the two groups. Similar to the results obtained for IFN-y production, there was no statistical difference between the Ghanaian and Danish adults with respect to IL-4 production in responses to control antigens. With one exception, PMBC cultures from Ghanaian adults produced only IFN-y or IL-4 in response to the MSA-2 peptides. In all, 82% of PMBC cultures from the exposed Ghanaian adults responded to MSA-2 peptides by proliferation or cytokine production. In contrast, only 14% of PMBC cultures from the unexposed Danish adults responded. The ability of the MSA-2 peptides to selectively stimulate PBMC of exposed Ghanaian adults seems to support the immunogenicity of the T-cell epitopes within the MSA-2 peptides in some individuals following natural exposure to malaria parasites. Furthermore, the association of these responses with the expected immunity of Ghanaian adults may suggest that MSA-2 T-cell epitopes could play a role in protective immunity against malaria