Drug resistance is one of the major impediments to control Plasmodium falciparum malaria worldwide. Heat shock protein 90 (Hsp90) is an essential component of the buffering capacity of eukaryotic cells as a part of the stress response. P. falciparum is no different and requires Hsp90 to chaperone proteins essential for cell cycle progression and drug resistance. Inhibition of P. falciparum Hsp90 (PfHsp90) may be able to not only cripple the parasite but also serve as an adjunctive antimalarial by circumventing drug resistance. The results presented in this thesis identify novel Hsp90 inhibitors that synergize with conventional antimicrobials, such as chloroquine (CQ), when used in combination. The objectives were to identify specific malaria Hsp90 inhibitors, the mechanism of the synergistic phenotype, and whether the strategy translates in vivo. To this end, the antimalarial activity of the purine analog PU-H71, and novel PfHsp90 inhibitors was tested. PU-H71 and the novel inhibitors APPA, harmine, and acrisorcin exhibited antimalarial activity in the nanomolar range and displayed synergistic activity with CQ. PU-H71 was able to reverse CQ resistance in a cell-based assay using the CQ-resistant strain W2. PU-H71 caused ring-stage arrest during the intra-erythrocytic cycle. Co-immunoprecipitation studies revealed that PfHsp90 interacts directly with the CQ resistance transporter (PfCRT). In the P. berghei mouse model of malaria, PU-H71 and harmine were able to reduce parasitemia and synergize with CQ. The interaction of PfHsp90 with PfCRT may underlie the synergistic phenotype. We conclude that PU-H71 and harmine are effective adjunctive antimalarial drugs that may be useful in combination therapies.

As the number of drug-resistant malaria parasites continues to grow, pressure is increasing to find an effective, cross-protective, multi-valent malaria vaccine (32). Expression library immunisation is an un-biased screening technique that leads to the identification of novel, protective antigens that can be administered as components of a multivalent DNA vaccine (9, 50, 75, 86, 92). Here, a P. c. adami DS expression library has been evaluated as a malaria vaccine in mice, and several subpools of cross-protective plasmids have been identified. Upon vaccination with these plasmid subpools, mice demonstrate significantly lower mean cumulative parasitemia values than control vaccinated mice, when challenged with avirulent heterologous P. c. adami DK parasites. These cross-protective responses correlate with the induction of opsonizing antibodies against infected red blood cells and the production of IFN-gamma by T-cells. The determination of P. c. adami antigens capable of inducing strain-transcending immunity implies the identification of orthologues in the P. falciparum genome that may be applied as components of a human malaria vaccine.

Malaria is a widespread parasitic disease, causing 300-500 million infections per year and resulting in over 1 million deaths. There is widespread resistance of the parasite to most of the antimalarial treatments available, indicating the need for a vaccine (http://www.rbm.who.int/wmr2005/). The translationally controlled tumour protein (TCTP) family are highly conserved eukaryotic proteins that have been assigned a variety of functions. While most studies have focused on the intracellular functions of TCTP, human, malarial and other parasitic TCTPs have also been reported to have extracellular functions in the induction of histamine release from immune cells (e.g. MacDonald et al., 2001; Rao et al., 2002). Malarial TCTP has been detected in the sera of malaria-infected individuals (MacDonald et al., 2001) and is also known to bind to the antimalarial drug artemisinin (Bhisutthibhan et al., 1998). In this study, TCTP was investigated as a malarial vaccine candidate due to a previously observed protective effect in mice infected with Plasmodium yoelii YM. In that study, PfTCTP immunisation conferred a significant delay in disease progression, as judged by reduced parasitemia and prolonged survival (Taylor, 2002). It was thought that the protective effect might have been due to the inhibition of the extracellular actions of malarial TCTP by the acquired host immune response. P. falciparum and P. yoelii TCTP were initially expressed in S. cerevisiae, as in the previous study. The recombinant proteins were used to vaccinate mice, which were then challenged with two strains of P. yoelii. No protective effect was observed for either vaccine, and so the previous results using PfTCTP could not be confirmed. The TCTP of P. yoelii and P. berghei were then expressed in E. coli, which increased yield and decreased proteolysis. The recombinant proteins were used as vaccines in mice challenged with P. yoelii YM, P. c. chabaudi AS, or P. berghei ANKA. A significant delay in disease progression was observed in PyTCTP-immunised mice challenged with the non-lethal P.c. chabaudi, as determined by a significantly reduced parasitemia at each day post-infection leading up to a delayed peak parasitemia. A significant reduction in parasitemia was also observed in the early stages of P. yoelii YM infection in PyTCTP-immunised mice. P. berghei ANKA was used to challenge C57BL/6 mice to determine whether PbTCTP immunisation could protect mice from cerebral malaria development, no protective effect was observed. P. berghei ANKA was also used as a second lethal malaria challenge model in BALB/c mice, no significant differences in disease progression were observed in immunised mice. To further assess the functions of malarial TCTP, several attempts were made to create a TCTP-knockout strain of P. berghei ANKA. A TCTP-knockout malaria strain could be assessed for alterations in morphology, infectivity and artemisinin sensitivity compared with wild-type parasites. Initial genotype analysis of parasites resulting from several transfection experiments indicated that TCTP disruption had been successful, however TCTP-disrupted parasites were strongly selected against, and stable knockout strains could not be obtained. This indicates that TCTP performs an important role within the malaria parasite.

Malaria remains a major cause of morbidity and death, especially in developing countries. The effectiveness of conventional antimalarial drugs is waning and there is an urgent need for novel therapeutic approaches. An understanding of malaria parasite biology should facilitate the development of effective therapies that prevent and/or treat malaria. The present studies explore the potential of vitamin A (retinol) as an antimalarial agent. Retinol may act by changing the oxidant milieu within the malaria parasite. Therefore, the nature and consequences of oxidant injury during malaria infection, and its treatment with retinol, have also been explored. The antimalarial potential of retinol was characterised using an established in vitro culture system allowing assessment of efficacy through [3H]-hypoxanthine uptake at different erythrocytic stages of development of Plasmodium falciparum. Retinol losses during culture were significant (>50%). After adjusting for these losses, all parasite stages (early rings to mature trophozoites) showed similar retinol sensitivity, with values of the mean assayed concentration resulting in 50% growth inhibition (IC50) ranging from 10.1 to 21.4 μM. This range was above that in normal human serum (<3 μM) but below that associated with haemolysis in culture (>43 μM). Retinol pre-treatment of uninfected erythrocytes did not inhibit merozoite invasion. Retinol-treated parasites exhibited vacuolisation of the food vacuole and membrane rupture. A P. berghei murine model was used to determine the in vivo preventive and therapeutic efficacy of retinol. Multiple-dose retinol given to healthy Swiss mice before parasite inoculation reduced parasitaemia by 30%, a result comparable to the previously reported reduction in morbidity after vitamin A supplementation in children. A lesser reduction in parasitaemia of 10% was observed when retinol was given after the parasitaemia reached 10-15%. Retinol was ineffective in reducing parasitaemia when given either as single-dose supplementation post-inoculation or at regular intervals before and after infection. Retinol supplementation did not change plasma retinol concentrations during malaria infection whether or not retinol was given, but malaria attenuated the increase in liver retinol content. These data suggest that retinol has most value as prophylaxis. In contrast to published data from humans, previously healthy mice did not develop low plasma retinol concentrations during acute infection

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Met lit. opg., bibliogr. - Met samenvatting in het Nederlands.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.