In Vitro Studies of Factors Potentially Affecting Plasmodium Falciparum Infection : (Heparin and Anti-P. falciparum Immune Responses)

Abstract: Plasmodium falciparum malaria is considered one of the major infectious diseases in humans, with regard to mortality and morbidity. Growing resistance of malaria to most anti-malaria drugs and of the Anopheles mosquitoes to insecticides have resulted in a global resurgence of the disease. Therefore, the need to explore drugs with possible antimalarial effects or the development of vaccines against malaria are considered a high priority for control of the disease. In this thesis, the antimalarial effect of heparin and the identification of T- and B-cell epitopes in P. falciparum vaccine candidate antigens, Pf155/RESA and Pf332 have been addressed.Heparin, a drug included in the supportive or ancillary treatment of cerebral malaria, was tested for its anti-parasitic effect on the blood stages of P. falciparum malaria in vitro. Heparin was cleaved into fragments differing in molecular weight and in their affinity for antithrombin III. Both unfractionated heparin and heparin fractions inhibited the merozoite invasion into red blood cells. The mechanism by which heparin acts is not clear. However, the inhibition was reversible by washing heparin-treated P. falciparum cultures indicating a direct effect of heparin. The sensitivity of laboratory strains and/or fresh isolates obtained from individuals residing in malaria endemic areas was compared. Although varying in sensitivity none of the samples tested was found to be resistant to heparin and its derivates. A fraction of heparin with low affinity for antithrombin III was the most potent inhibitor of merozoite invasion. The fraction with low affinity for antithrombin III is devoid of anticoagulant activity, suggesting a potential role of this fraction for the treatment of malaria.For the development of subunit vaccines it is important to identify and characterise epitopes which activate relevant B- and T-cell functions. In this study we have investigated two putative malaria vaccine candidate antigens, Pf155/RESA and Pf332, for T- and B-cell reactive epitopes. A large number of donors had antibodies against the Pf155/RESA sequence 186-206. For the Pf332 derived fragment, EB200, the donors also exhibited high antibody levels in their plasma. In our studies we have measured multi parameters of T-cell activation (proliferation, IFN-g and/or IL-4 production). Peptides corresponding to the N-terminal region of Pf155 or to EB200, stimulated peripheral blood mononuclear cells from P. falciparum-immune donors to proliferate, to induce secretion of IFN-g and/or IL-4. In individual donors the cellular immune responses to the peptides varied considerably. However, there was no clear association between proliferation and production of the cytokines investigated. This lack of association underlines the importance of including multiple parameters when analysing T-cell responses to defined epitopes.In conclusion, the non-repeat region of Pf155/RESA and the EB200 fragment of Pf332 contains several B-cell epitopes as well as several epitopes inducing functionally distinct T-cell responses, which should be a useful tool for inclusion in a subunit malaria vaccine as well as in future immuno-epidemiological studies.