Towards a blood-stage vaccine for malaria: Are we following all the leads?

Although the malaria parasite was discovered more than 120 years ago, it is only during the past 20 years, following the cloning of malaria genes, that we have been able to think rationally about vaccine design and development. Effective vaccines for malaria could interrupt the life cycle of the parasite at different stages in the human host or in the mosquito. The purpose of this review is to outline the challenges we face in developing a vaccine that will limit growth of the parasite during the stage within red blood cells - the stage responsible for all the symptoms and pathology of malaria. More than 15 vaccine trials have either been completed or are in progress, and many more are planned. Success in current trials could lead to a vaccine capable of saving more than 2 million lives per year.

Melatonin and IP(3)-induced Ca(2+) Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.

Potencies of human immunodeficiency virus protease inhibitors in vitro against Plasmodium falciparum and in vivo against murine malaria

Parasite resistance to antimalarial drugs is a serious threat to human health, and novel agents that act on enzymes essential for parasite metabolism, such as proteases, are attractive targets for drug development. Recent studies have shown that clinically utilized human immunodeficiency virus (HIV) protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration. The most potent in vitro antimalarial effects have been obtained for parasites treated with saquinavir, ritonavir, or lopinavir, findings confirmed in this study for a genetically distinct P. falciparum line (3D7). To investigate the potential in vivo activity of antiretroviral protease inhibitors (ARPIs) against malaria, we examined the effect of ARPI combinations in a murine model of malaria. In mice infected with Plasmodium chabaudi AS and treated orally with ritonavir-saquinavir or ritonavir-lopinavir, a delay in patency and a significant attenuation of parasitemia were observed. Using modeling and ligand docking studies we examined putative ligand binding sites of ARPIs in aspartyl proteases of P. falciparum (plasmepsins II and IV) and P. chabaudi (plasmepsin) and found that these in silico analyses support the antimalarial activity hypothesized to be mediated through inhibition of these enzymes. In addition, in vitro enzyme assays demonstrated that P. falciparum plasmepsins II and IV are both inhibited by the ARPIs saquinavir, ritonavir, and lopinavir. The combined results suggest that ARPIs have useful antimalarial activity that may be especially relevant in geographical regions where HIV and P. falciparum infections are both endemic.

Wave expansion of CD(34+) progenitor cells in the spleen in rodent malaria

Defense against malaria depends upon amplification of the spleen structure and function for the clearance of parasitized red blood cells (pRBC). We studied the distribution and amount of CD(34+) cells in the spleens of mice infected with rodent malaria. We sought to identify these cells in the spleen and determine their relationship to infection. C57BL/6J mice were infected with self-resolving, Plasmodium chabaudi CR, or one of the lethal rodent malaria strains, P. chabaudi AJ and P. berghei ANKA. We then recorded parasitemia, mortality, and the presence of CD(34+) cells in spleen, as determined by immunohistochemistry and flow cytometry. In the non-lethal strain, the spleen structure was maintained during amplification, but disrupted in lethal models. The abundance of CD(34+) cells increased in the red pulp on the 4th and 6th days p.i. in all models, and subsided on the 8th day p.i. Faint CD(34+) staining on the 8th day p.i., was probably due to differentiation of committed cell lineages. In this work, increase of spleen CD(34+) cells did not correlate with infection control. (c) 2009 Published by Elsevier Inc.

Studies on the genetics of artemisinin resistance in malaria

A existência de estirpes de Plasmodium falciparum resistentes a multiplos fármacos é um dos problemas mais graves no controlo da malária. Novos fármacos, como a artemisinina (ART) e seus derivados são cada vez mais utilizados no tratamento da malaria e muito embora até ao momento não haja registos de fármaco-resistência estável à ART o seu surgimento seria desastroso devido á falta de alternativas. A investigação apresentada nesta tese descreve a selecção de resistência estável à ART e ao artesunato (ATN) utilizando um modelo roedor de malária, o parasita Plasmodium chabaudi chabaudi (Plasmodium chabaudi). Dois clones de Plasmodium chabaudi diferentes, AS-15CQ e AS-30CQ, foram inoculados em murganhos que por sua vez foram tratados na presença de concentrações sucessivamente crescentes de ATN e ART, sendo que no final do processo de seleção de resistência, os parasitas obtidos apresentavam uma resistência de 6 e 15 vezes superior ao ATN e à ART, respectivamente, em relação aos parasitas iniciais. Os clones obtidos foram nomeados respectivamente AS-ATN (obtido a partir de AS-15CQ por seleção com pressão de ATN) e AS-ART (obtido a partir de AS-30CQ por seleção com pressão de ART). A resistência obtida durante o processo de seleção é estável após clonagem, congelamento/descongelamento, passagem sanguínea na ausência de pressão de fármaco e transmissão natural através do mosquito vector. A sequência nucleotídica e o número de cópias dos genes previamente descritos na literatura como moduladores putativos de resistência à ART e seus derivados: mdr1, cg10, tctp e atp6; foi comparada entre parasitas resistentes e sensíveis, não tendo sido encontradas nenhumas alterações, quer na sequência quer no número de cópias destes genes. Posteriormente, numa tentativa de identificar os genes envolvidos na resistância à ART e ao ATN a técnica de Linkage Group Selection (LGS) foi utilizada. Para tal dois cruzamentos genéticos foram realizados. Estes cruzamentos foram realizados entre os clones fármaco-resistentes; AS-ART e AS-ATN e um clone geneticamente distinto dos anteriores e sensível aos fármacos em estudos, AJ. Após realização do LGS quatro loci genéticos; nos cromossomas de P. chabaudi 1, 2, 6 e 8 foram encontrados associados à resistência. Atendendo a que, a selecção no cromossoma 2 era a mais forte, este locus foi submetido a subsequentes análises genéticas, tendo sido encontradas duas mutações diferentes (V739F e V770F) num gene que codifica para um enzima de desubiquitinação (gene ubp-1).

Studies on the genetics of artemisinin resistance in malaria

A existência de estirpes de Plasmodium falciparum resistentes a multiplos fármacos é um dos problemas mais graves no controlo da malária. Novos fármacos, como a artemisinina (ART) e seus derivados são cada vez mais utilizados no tratamento da malaria e muito embora até ao momento não haja registos de fármaco-resistência estável à ART o seu surgimento seria desastroso devido á falta de alternativas. A investigação apresentada nesta tese descreve a selecção de resistência estável à ART e ao artesunato (ATN) utilizando um modelo roedor de malária, o parasita Plasmodium chabaudi chabaudi (Plasmodium chabaudi). Dois clones de Plasmodium chabaudi diferentes, AS-15CQ e AS-30CQ, foram inoculados em murganhos que por sua vez foram tratados na presença de concentrações sucessivamente crescentes de ATN e ART, sendo que no final do processo de seleção de resistência, os parasitas obtidos apresentavam uma resistência de 6 e 15 vezes superior ao ATN e à ART, respectivamente, em relação aos parasitas iniciais. Os clones obtidos foram nomeados respectivamente AS-ATN (obtido a partir de AS-15CQ por seleção com pressão de ATN) e AS-ART (obtido a partir de AS-30CQ por seleção com pressão de ART). A resistência obtida durante o processo de seleção é estável após clonagem, congelamento/descongelamento, passagem sanguínea na ausência de pressão de fármaco e transmissão natural através do mosquito vector. A sequência nucleotídica e o número de cópias dos genes previamente descritos na literatura como moduladores putativos de resistência à ART e seus derivados: mdr1, cg10, tctp e atp6; foi comparada entre parasitas resistentes e sensíveis, não tendo sido encontradas nenhumas alterações, quer na sequência quer no número de cópias destes genes. Posteriormente, numa tentativa de identificar os genes envolvidos na resistância à ART e ao ATN a técnica de Linkage Group Selection (LGS) foi utilizada. Para tal dois cruzamentos genéticos foram realizados. Estes cruzamentos foram realizados entre os clones fármaco-resistentes; AS-ART e AS-ATN e um clone geneticamente distinto dos anteriores e sensível aos fármacos em estudos, AJ. Após realização do LGS quatro loci genéticos; nos cromossomas de P. chabaudi 1, 2, 6 e 8 foram encontrados associados à resistência. Atendendo a que, a selecção no cromossoma 2 era a mais forte, este locus foi submetido a subsequentes análises genéticas, tendo sido encontradas duas mutações diferentes (V739F e V770F) num gene que codifica para um enzima de desubiquitinação (gene ubp-1).

Avaliação in vitro e in vivo da atividade antimalárica dos trioxolanos NAC89, LDC67 e LC50 em estirpes sensíveis e resistentes à artemisinina e ao artesunato.

A malária mantem-se como uma das doenças mais importantes do mundo, causando a morte de mais de 1 milhão de pessoas anualmente e elevada morbilidade. Face à propagação da resistência do Plasmodium falciparum à maioria dos medicamentos antimaláricos disponíveis, a Organização Mundial de Saúde (OMS), desde 2006, recomenda a utilização de terapias combinadas com artemisinina (ACTs) como tratamento de primeira linha para a malária não complicada. Em 2008, relatórios clínicos revelaram a falha terapêutica dos ACTs na fronteira Tailândia-Camboja e uma vez que não existem alternativas para o tratamento da malária é fundamental manter linhas de investigação sobre novos e eficazes fármacos. A partir da artemisinina (ART) surgiram novos peróxidos designados trioxolanos que apresentam como farmacóforo a função 1,2,4-trioxano. A acessibilidade, a preparação relativamente económica e a estabilidade da função 1,2,4-trioxano permite a síntese de derivados com estruturas diversas, alargando a possibilidade de desenvolvimento de novos fármacos. Foram realizados testes in vitro de triagem com o MARK III (OMS micro-ensaio), com controlos positivos (artemisinina e dihidroartemisinina) e controlo negativo (sem fármaco). Foram efetuados ensaios diversos com 3 compostos, aqui denominados NAC89, LCD67 e LC50 em culturas da estirpe de P. falciparum (Dd2) para avaliação da atividade antimalárica dos compostos, bem como ensaios utilizando o modelo de malária de murino, Plasmodium chabaudi, com 4 estirpes, denominadas AS-3CQ, AS-ATN, respectivamente sensível e resistente ao artesunato (ATN) e AS-30CQ e AS-ART respectivamente sensível e resistente à (ART). Também foi avaliada a citotoxicidade dos compostos, utilizando células HepG2 de hepatoma humano pelo ensaio com método colorimétrico metil-tiazol-tetrazólico (MTT). No modelo murino compararam-se também duas vias de administração dos novos compostos, sendo uma por via subcutânea nos 3 compostos e outra por via tópica apenas para LC50. A verificação de cura foi efetuada por observação microscópica de esfregaços sanguíneos corados pelo método de Giemsa e determinação da parasitemia. Os resultados observados foram: a) baixa citotoxicidade dos três compostos; b) o composto LC50 eliminou a parasitémia nos ensaios in vitro em cultivos de P. falciparum bem como eliminou P. chabaudi nos tratamentos por via subcutânea e tópica na dose de 50 mg/kg e na dose de 10 mg/Kg na via subcutânea; c) o NAC89 mostrou boa atividade no mesmo ensaio in vivo, na dose de 10 mg/Kg e 50 mg/Kg por via subcutânea; d) fraca atividade para LCD67 na dose de 50 mg/Kg. O LC50 e o NAC89 foram muito eficazes contra parasitas resistentes ao ATN e à ART sugerindo novos mecanismos de ação. Assim, este trabalho de investigação trouxe resultados promissores na àrea de potenciais novos antimaláricos.

The separate and combined effects of MHC genotype, parasite clone, and host gender on the course of malaria in mice.

BACKGROUND: The link between host MHC (major histocompatibility complex) genotype and malaria is largely based on correlative data with little or no experimental control of potential confounding factors. We used an experimental mouse model to test for main effects of MHC-haplotypes, MHC heterozygosity, and MHC x parasite clone interactions. We experimentally infected MHC-congenic mice (F2 segregants, homo- and heterozygotes, males and females) with one of two clones of Plasmodium chabaudi and recorded disease progression. RESULTS: We found that MHC haplotype and parasite clone each have a significant influence on the course of the disease, but there was no significant host genotype by parasite genotype interaction. We found no evidence for overdominance nor any other sort of heterozygote advantage or disadvantage. CONCLUSION: When tested under experimental conditions, variation in the MHC can significantly influence the course of malaria. However, MHC heterozygote advantage through overdominance or dominance of resistance cannot be assumed in the case of single-strain infections. Future studies might focus on the interaction between MHC heterozygosity and multiple-clone infections.

Antimalarial drugs disrupt ion homeostasis in malarial parasites

Plasmodium chabaudi malaria parasite organelles are major elements for ion homeostasis and cellular signaling and also target for antimalarial drugs. By using confocal imaging of intraerythrocytic parasites we demonstrated that the dye acridine orange (AO) is accumulated into P. chabaudi subcellular compartments. The AO could be released from the parasite organelles by collapsing the pH gradient with the K+/H+ ionophore nigericin (20 µM), or by inhibiting the H+-pump with bafilomycin (4 µM). Similarly, in isolated parasites loaded with calcium indicator Fluo 3-AM, bafilomycin caused calcium mobilization of the acidic calcium pool that could also be release with nigericin. Interestingly after complete release of the acidic compartments, addition of thapsigargin at 10 µM was still effective in releasing parasite intracellular calcium stores in parasites at trophozoite stage. The addition of antimalarial drugs chloroquine and artemisinin resulted in AO release from acidic compartments and also affected maintenance of calcium in ER store by using different drug concentrations.

The course of malaria in mice: major histocompatibility complex (MHC) effects, but no general MHC heterozygote advantage in single-strain infections.

A general MHC-heterozygote advantage in parasite-infected organisms is often assumed, although there is little experimental evidence for this. We tested the response of MHC-congenic mice (F2 segregants) to malaria and found the course of infection to be significantly influenced by MHC haplotype, parasite strain, and host gender. However, the MHC heterozygotes did worse than expected from the average response of the homozygotes.

No impact of malaria infection or immunisation on the expression of the immune GTPase GIMAP1 at the protein or mRNA levels (Article no. 53)

GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. Methods A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. Results GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK), in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. Conclusion The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

Anti-IL-2 Treatment Impairs the Expansion of T-reg Cell Population during Acute Malaria and Enhances the Th1 Cell Response at the Chronic Disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T-reg) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T-reg cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4+ T cell activation and IFN-gamma production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-alpha and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T-reg cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.

Intravital microscopy technique to study parasite dynamics in the labyrinth layer of the mouse placenta

Intravital imaging techniques are the best approach to investigate in situ cellular behavior under physiological conditions. Many techniques have emerged during these last few years for this purpose. We recently described an intravital imaging technique that allows for the observation of placenta physiological responses at the labyrinth layer of this tissue. This technique will be very useful to study many placental opportunistic infections and in this article we reinforce its usefulness by analyzing placental physiological entrapment of beads and parasites. In particular, our results show that small beads (1.0 μm) or Plasmodium chabaudi-GFP-infected-Red Blood Cells (Pc-GFP-iRBCs) cannot get trapped inside small or large blood vessels of popliteal lymph nodes (PLNs). Inside the placenta, clusters of beads could only be found inside the maternal blood vessels. However, Pc-GFP-iRBCs were found inside and outside the maternal blood vessels. We observed that trophoblasts can ingest infected-Red Blood Cells (iRBCs) in vitro and immunofluorescence of placenta revealed Pc-GFP-iRBCs inside and outside the maternal blood vessels. Taken together, we conclude that fast deposition of particles inside blood vessels seems to be an intrinsic characteristic of placenta blood flow, but iRBCs could be internalized by trophoblast cells. Thus these results represent one of the many possible uses of our intravital imaging technique to address important questions inside the parasitological field.