An expanding toolkit for preclinical pre-erythrocytic malaria vaccine development: bridging traditional mouse malaria models and human trials.

Malaria remains a significant public health burden with 214 million new infections and over 400,000 deaths in 2015. Elucidating relevant Plasmodium parasite biology can lead to the identification of novel ways to control and ultimately eliminate the parasite within geographic areas. Particularly, the development of an effective vaccine that targets the clinically silent pre-erythrocytic stages of infection would significantly augment existing malaria elimination tools by preventing both the onset of blood-stage infection/disease as well as spread of the parasite through mosquito transmission. In this Perspective, we discuss the role of small animal models in pre-erythrocytic stage vaccine development, highlighting how human liver-chimeric and human immune system mice are emerging as valuable components of these efforts.

Chemical interrogation of the malaria kinome.

Malaria, an infectious disease caused by eukaryotic parasites of the genus Plasmodium, afflicts hundreds of millions of people every year. Both the parasite and its host utilize protein kinases to regulate essential cellular processes. Bioinformatic analyses of parasite genomes predict at least 65 protein kinases, but their biological functions and therapeutic potential are largely unknown. We profiled 1358 small-molecule kinase inhibitors to evaluate the role of both the human and the malaria kinomes in Plasmodium infection of liver cells, the parasites' obligatory but transient developmental stage that precedes the symptomatic blood stage. The screen identified several small molecules that inhibit parasite load in liver cells, some with nanomolar efficacy, and each compound was subsequently assessed for activity against blood-stage malaria. Most of the screening hits inhibited both liver- and blood-stage malaria parasites, which have dissimilar gene expression profiles and infect different host cells. Evaluation of existing kinase activity profiling data for the library members suggests that several kinases are essential to malaria parasites, including cyclin-dependent kinases (CDKs), glycogen synthase kinases, and phosphoinositide-3-kinases. CDK inhibitors were found to bind to Plasmodium protein kinase 5, but it is likely that these compounds target multiple parasite kinases. The dual-stage inhibition of the identified kinase inhibitors makes them useful chemical probes and promising starting points for antimalarial development.

Plasmodium genome in blood donors at risk for malaria after several years of residence in Italy.

BACKGROUND: At present, the main risk of transfusion-transmitted malaria (TTM) in nonendemic countries is chronic, asymptomatic immigrants from malaria-endemic areas. Semi-immune donors may carry undetected parasitemia. This study examines Plasmodium infection in at-risk blood donors in Northern Italy. STUDY DESIGN AND METHODS: Plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using a commercial enzyme immunoassay. The conserved 18S rRNA and the mitochondrial genes of Plasmodium were amplified to detect and quantify parasite genomes (copies/mL). Plasmodium species were identified with a species-specific nested polymerase chain reaction. Parasitemic samples were further tested by amplification of polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) confirmed by sequencing. RESULTS: Three of 83 seropositive (3.6%) and one of 14 seronegative at-risk candidate donors carried Plasmodium genome (4 × 10(3) -8.5 × 10(4) copies/mL): two P. falciparum, one P. malariae (seronegative sample), and one coinfection with P. malariae and P. ovale. Alleles of MSP-1 (MAD20 and K1), MSP-2 (3D7 and FC27), and GLURP were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and GLURP was amplified. No alleles were detected in Samples 283 and 331. CONCLUSIONS: Immigrants from endemic countries might carry infectious Plasmodium after 2 to 5 years of continuous residence in Italy. Serologic screening may miss donors carrying P. malariae. Permanent exclusion or screening for both antibodies and genome are needed to prevent TTM.

Malaria evolution in South Asia: knowledge for control and elimination.

The study of malaria parasites on the Indian subcontinent should help us understand unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and Plasmodium vivax infections. The Malaria Evolution in South Asia (MESA) research program is one of ten International Centers of Excellence for Malaria Research (ICEMR) sponsored by the US National Institutes of Health. In this second of two reviews, we describe why population structures of Plasmodia in India will be characterized and how we will determine their consequences on disease presentation, outcome and patterns. Specific projects will determine if genetic diversity, possibly driven by parasites with higher genetic plasticity, plays a role in changing epidemiology, pathogenesis, vector competence of parasite populations and whether innate human genetic traits protect Indians from malaria today. Deep local clinical knowledge of malaria in India will be supplemented by basic scientists who bring new research tools. Such tools will include whole genome sequencing and analysis methods; in vitro assays to measure genome plasticity, RBC cytoadhesion, invasion, and deformability; mosquito infectivity assays to evaluate changing parasite-vector compatibilities; and host genetics to understand protective traits in Indian populations. The MESA-ICEMR study sites span diagonally across India and include a mixture of very urban and rural hospitals, each with very different disease patterns and patient populations. Research partnerships include government-associated research institutes, private medical schools, city and state government hospitals, and hospitals with industry ties. Between 2012 and 2017, in addition to developing clinical research and basic science infrastructure at new clinical sites, our training workshops will engage new scientists and clinicians throughout South Asia in the malaria research field.

Prospects for malaria elimination in non-Amazonian regions of Latin America.

Latin America contributes 1-1.2 million clinical malaria cases to the global malaria burden of about 300 million per year. In 21 malaria endemic countries, the population at risk in this region represents less than 10% of the total population exposed worldwide. Factors such as rapid deforestation, inadequate agricultural practices, climate change, political instability, and both increasing parasite drug resistance and vector resistance to insecticides contribute to malaria transmission. Recently, several malaria endemic countries have experienced a significant reduction in numbers of malaria cases. This is most likely due to actions taken by National Malaria Control Programs (NMCP) with the support from international funding agencies. We describe here the research strategies and activities to be undertaken by the Centro Latino Americano de Investigación en Malaria (CLAIM), a new research center established for the non-Amazonian region of Latin America by the National Institute of Allergy and Infectious Diseases (NIAID). Throughout a network of countries in the region, initially including Colombia, Guatemala, Panama, and Peru, CLAIM will address major gaps in our understanding of changing malaria epidemiology, vector biology and control, and clinical malaria mainly due to Plasmodium vivax. In close partnership with NMCPs, CLAIM seeks to conduct research on how and why malaria is decreasing in many countries of the region as a basis for developing and implementing new strategies that will accelerate malaria elimination.

A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.

Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.

Malaria infection and human evolution.

During the evolution of the genus Homo, with regard to the species habilis, erectus and sapiens, malaria has played a key biological role in influencing human development. The plasmodia causing malaria have evolved in two ways, in biological and phylogenetic terms: Plasmodium vivax, Plasmodium malariae and Plasmodium ovale appear to have either coevolved with human mankind, or encountered human species during the most ancient phases of Homo evolution; on the other hand, Plasmodium falciparum has been transmitted to humans by monkeys in a more recent period, probably between the end of the Mesolithic and the beginning of the Neolithic age. The authors show both direct and indirect biomolecular evidence of malarial infection, detected in buried subjects, dating to ancient times and brought to light in the course of archaeological excavations in major Mediterranean sites. In this review of the literature the authors present scientific evidence confirming the role of malaria in affecting the evolution of populations in Mediterranean countries. The people living in several different Mediterranean regions, the cradle of western civilization, have been progressively influenced by malaria in the course of the spread of this endemic disease in recent millennia. In addition, populations affected by endemic malaria progressively developed cultural, dietary and behavioural adaptation mechanisms, which contributed to diminish the risk of disease. These habits were probably not fully conscious. Nevertheless it may be thought that both these customs and biological modifications, caused by malarial plasmodia, favoured the emergence of groups of people with greater resistance to malaria. All these factors have diminished the unfavourable demographic impact of the disease, also positively influencing the general development and growth of civilization.

Artesunate + amodiaquine and artesunate + sulphadoxine-pyrimethamine for treatment of uncomplicated malaria in Democratic Republic of Congo: a clinical trial with determination of sulphadoxine and pyrimethamine-resistant haplotypes.

We undertook a trial of artesunate + amodiaquine (AS + AQ) and artesunate + sulphadoxine-pyrimethamine (AS + SP) in 180 children of age 6-59 months with uncomplicated malaria in Democratic Republic of Congo. Children were randomly allocated to receive 3 days observed treatment of AS + AQ (n = 90) or 3 days of AS + SP (n = 90). Primary efficacy outcomes were 28-day parasite recurrence rates, and recrudescence rates were adjusted by genotyping to distinguish new infection and recrudescence. In addition, we determined the prevalence of molecular markers of resistance to sulphadoxine and pyrimethamine. Day 28 parasite recurrence rates were 16.9% (14/83; 95% CI: 9.5-26.7) in the AS + AQ group and 34.6% (28/81; 95% CI: 24.3-46.0) in the AS + SP group (P = 0.009). After PCR correction, recrudescence rates were 6.7% (5/74; 95% CI: 2.2-15.1) for AS + AQ and 19.7% (13/66; 95% CI: 10.9-31.3) for AS + SP (P = 0.02). There was no significant difference between the two arms in time to parasite clearance, fever clearance and gametocyte clearance. Parasite genotyping showed high frequencies of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) molecular SP-resistance markers, with 57% of the samples showing more than three mutations linked to SP resistance, and 27% with triple-dhfr/double-dhps haplotype, confirming that SP treatment failure rates are likely to be high. AS + AQ had significantly higher efficacy than AS + SP. These results contributed to the subsequent change to AS + AQ as first-line regimen in the country. Efforts to properly implement the new protocol and maintain adherence at acceptable levels should include health staff and patient sensitization. The 6.8% recrudescence rate indicates that AS + AQ should be monitored closely until a more effective artemisinin combination therapy regimen is needed and can be introduced.

Chemosensitizers in drug transport mechanisms involved in protozoan resistance.

The emergence and spread of antiparasitic drug resistance pose a severe and increasing public health threat. Failures in prophylaxis or those in treatment with quinolines, hydroxynaphtoquinones, sesquiterpenic lactones, antifolate drugs, arsenic and antimony containing drugs sulfamides induce reemergence of parasitic-related morbidity and mortality. Resistance is often associated with alteration of drug accumulation into parasites, which results from a reduced uptake of the drug, an increased efflux or, a combination of the two processes. Resistance to quinolines, artemisinin derivatives and arsenicals and expression of an active efflux mechanism are more or less correlated in protozoa like Plasmodium spp., Leishmania spp., and Trypanosoma spp. Various parasite candidate genes have been proposed to be involved in drug resistance, each concerned in membrane transport. Genes encoding membrane glycoproteins, orthologue to the P-glycoproteins identified in MDR human cancer cells, have been described in these resistant pathogens in addition to various membrane proteins involved in drug transport. Several compounds have demonstrated, in the past decade, promising capability to reverse the drug resistance in parasite isolates in vitro, in animal models and for human malaria. These drugs belong to different pharmacological classes such as calcium channel blockers, tricyclic antidepressants, antipsychotic calmodulin antagonists, histamine H1-receptor antagonists, analgesic antipyretic drugs, non-steroidal anti-inflammatory drugs, and to different chemical classes such as synthetic surfactants, alkaloids from plants used in traditional medicine, pyrrolidinoaminoalkanes and derivatives, and anthracene derivatives. Here, are summarized the molecular bases of antiparasitic resistance emphasizing recent developments with compounds acting on trans-membrane proteins involved in drug efflux or uptake.

The co-evolution of people, plants, and parasites: biological and cultural adaptations to malaria.

The urgency generated by drug-resistant strains of malaria has accelerated anti-malarial drug research over the last two decades. While synthetic pharmaceutical agents continue to dominate research, attention increasingly has been directed to natural products. The present paper explores the larger context in which plant use occurs and considers how the selection of medicinal plants has evolved over millennia as part of the larger human effort to mediate illness. First attention is directed to indigenous medicinal plants whose anti-malarial activity is based on an oxidant mode of action, by which intracellular constituents lose electrons (become more electropositive). Next, parallels are drawn between these plant substances and a suite of malaria-protective genetic traits: glucose-6-phosphate dehydrogenase deficiency; haemoglobins S, C and E; alpha- and beta-thalassemias. These erythrocyte anomalies are classic examples of Darwinian evolution, occurring in high frequency in populations who have experienced considerable selective pressure from malaria. Characterized by discrete loci and pathophysiologies, they are united through the phenomenon of increased erythrocyte oxidation. In this model, then, oxidant anti-malarial plants are culturally constructed analogues, and molecular mimics, of these genetic adaptations. To further reinforce the scheme, it is noted that the anti-malarial action of pharmaceutical agents such as chloroquine and mefloquine duplicates both the genetic anomalies and the folk therapeutic models based in oxidant plants. This discussion coheres around a theoretical foundation that relates plant secondary metabolites (oxidants) to plasmodial biochemistry and human biological and cultural adaptations to malaria. Co-evolution provides a theoretical link that illuminates how medical cultures manage the relationships among humans, plants, herbivores and their respective pathogens.

Antimalarial drug resistance: surveillance and molecular methods for national malaria control programmes.

National malaria control programmes have the responsibility to develop a policy for malaria disease management based on a set of defined criteria as efficacy, side effects, costs and compliance. These will fluctuate over time and national guidelines will require periodic re-assessment and revision. Changing a drug policy is a major undertaking that can take several years before being fully operational. The standard methods on which a decision can be taken are the in vivo and the in vitro tests. The latter allow a quantitative measurement of the drug response and the assessment of several drugs at once. However, in terms of drug policy change its results might be difficult to interpret although they may be used as an early warning system for 2nd or 3rd line drugs. The new WHO 14-days in vivo test addresses mainly the problem of treatment failure and of haematological parameters changes in sick children. It gives valuable information on whether a drug still 'works'. None of these methods are well suited for large-scale studies. Molecular methods based on detection of mutations in parasite molecules targeted by antimalarial drugs could be attractive tools for surveillance. However, their relationship with in vivo test results needs to be established.

Factors regulating natural transmission of Plasmodium berghei to the mosquito vector, and the cloning of a transmission-blocking immunogen.

Naturally occurring factors that regulate the infectivity of P. berghei infected rodent hosts to the mosquito vector in vivo have been compared in T.O., Balb/C and immunodeficient SCID mice. No detectable differences in infectivity were observed suggesting B and T cell mediated factors are not involved. Further studies investigated roles for macrophage colony stimulating factors, the cytokines IFN gamma and TNF alpha, of neutrophils, and of nitric oxide in the SCID mouse, but have failed to demonstrate an important role in vivo for any factor examined. Differences between these results and those obtained in vitro on the human and primate parasites must therefore be explained by biological differences between the parasite/host combinations, or by technical differences in experimental designs. Induced immunity to the ookinete surface antigen Pbs 21 of P. berghei can totally block the transmission of the parasite from the gametocyte infected host to the vector. We have cloned the gene encoding Pbs 21 and shown it bears striking structural similarities to Pfs 25, Pgs 25 and more particularly Pgs 28 in that it has a high cysteine content (9.5%), 4 EGF-like domains and hydrophobic amino-'signal'--and carboxyl-'anchor' sequences. The encoding gene is on chromosome 5 and is found also in P. chabaudi, P. yoelii and P. vinckei.

Chromosomal rearrangements and point mutations in the DHFR-TS gene of Plasmodium chabaudi under antifolate selection.

Selection of the rodent malaria Plasmodium chabaudi with low levels of the antifolate drug pyrimethamine has previously been shown by us to result in duplication of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene by a duplication of chromosome 7 and subsequent rearrangements. We have selected this resultant parasite line with large doses of pyrimethamine and analysed the DHFR-TS gene and chromosomes for any changes. Increased drug pressure has resulted in reappearance of a chromosome with the same structure as chromosome 7 from DS the parent line. Sequencing of the DHFR gene from each of the chromosomes has identified a single point mutation that results in a serine to asparagine change at position 106. This is the equivalent mutation that has been identified as the key residue in the mechanism of resistance to pyrimethamine in Plasmodium falciparum. There is no apparent increase in transcription of the DHFR-TS gene and the large increase in resistance is most likely a result of the mutation in the DHFR gene.