Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae

The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.     

Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region

We surveyed an Anopheles gambiae population in a West African malaria transmission zone for naturally occurring genetic loci that control mosquito infection with the human malaria parasite, Plasmodium falciparum. The strongest Plasmodium resistance loci cluster in a small region of chromosome 2L and each locus explains at least 89% of parasite-free mosquitoes in independent pedigrees. Together, the clustered loci form a genomic Plasmodium-resistance island that explains most of the genetic variation for malaria parasite infection of mosquitoes in nature. Among the candidate genes in this chromosome region, RNA interference knockdown assays confirm a role in Plasmodium resistance for Anopheles Plasmodium-responsive leucine-rich repeat 1 (APL1), encoding a leucine-rich repeat protein that is similar to molecules involved in natural pathogen resistance mechanisms in plants and mammals.     

Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population

Successful propagation of the malaria parasite Plasmodium falciparum within a susceptible mosquito vector is a prerequisite for the transmission of malaria. A field-based genetic analysis of the major human malaria vector, Anopheles gambiae, has revealed natural factors that reduce the transmission of P. falciparum. Differences in P. falciparum oocyst numbers between mosquito isofemale families fed on the same infected blood indicated a large genetic component affecting resistance to the parasite, and genome-wide scanning in pedigrees of wild mosquitoes detected segregating resistance alleles. The apparently high natural frequency of resistance alleles suggests that malaria parasites (or a similar pathogen) exert a significant selective pressure on vector populations.     

Malaria parasite development in a Drosophila model

Malaria is a devastating public health menace, killing over one million people every year and infecting about half a billion. Here it is shown that the protozoan Plasmodium gallinaceum, a close relative of the human malaria parasite Plasmodium falciparum, can develop in the fruit fly Drosophila melanogaster. Plasmodium gallinaceum ookinetes injected into the fly developed into sporozoites infectious to the vertebrate host with similar kinetics as seen in the mosquito host Aedes aegypti. In the fly, a component of the insect's innate immune system, the macrophage, can destroy Plasmodia. These experiments suggest that Drosophila can be used as a surrogate mosquito for defining the genetic pathways involved in both vector competence and part of the parasite sexual cycle.     

Effects of mosquito genes on Plasmodium development

Malaria parasites must complete a complex developmental cycle in an Anopheles mosquito vector before transmission to a vertebrate host. Sexual development of the parasite in the midgut is initiated in the lumen immediately after the mosquito ingests infected blood, and the resulting ookinetes must traverse the surrounding epithelial layer before transforming into oocysts. The innate immune system of the mosquito is activated during midgut invasion, but to date, no evidence has been published identifying mosquito immune genes that affect parasite development. Here, we show by gene silencing that an Anopheles gambiae leucine rich-repeat protein acts as an antagonist and two C-type lectines act as protective agonists on the development of Plasmodium ookinetes to oocysts.     

Early origin and recent expansion of Plasmodium falciparum

The emergence of virulent Plasmodium falciparum in Africa within the past 6000 years as a result of a cascade of changes in human behavior and mosquito transmission has recently been hypothesized. Here, we provide genetic evidence for a sudden increase in the African malaria parasite population about 10,000 years ago, followed by migration to other regions on the basis of variation in 100 worldwide mitochondrial DNA sequences. However, both the world and some regional populations appear to be older (50,000 to 100,000 years old), suggesting an earlier wave of migration out of Africa, perhaps during the Pleistocene migration of human beings.     

Plasmodium malariae: transmission from monkey to man by mosquito bite

Anopheles freeborni mosquitoes were infected by feeding on New World monkeys, Aotus trivirgatus, infected with a Nigerian strain of Plasmodium malariae. The infection was passed to human volunteers through the bites of these mosquitoes, demonstrating the practicability of using a simian host for infection of mosquitoes with a third species of human malaria parasite and of the use of such mosquitoes to transmit the infection from monkey to man.     

Genome sequencing. Closing in on a deadly parasite's genome

Extremely difficult to decipher, the genome of Plasmodium falciparum, the most virulent malaria parasite, is already providing targets for new drugs. Next project: the mosquito genome.     

Erythrocyte G protein-coupled receptor signaling in malarial infection

Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte beta2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Galphas) regulated the entry of the human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic adenosine 3',5'-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Galphas protein function reduced parasitemia in P. falciparum cultures in vitro, and beta-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte beta2-adrenergic receptor and Galphas may regulate malarial infection across parasite species.     

Malaria. A renewed assault on an old and deadly foe

After languishing for decades in the scientific backwaters, malaria research is suddenly being swept into the mainstream. Money is beginning to pour in from international finance and aid organizations, giving researchers who have been doggedly pursuing an intractable foe with limited resources the means to follow new leads. But on the ground, the disease is unyielding, and the current weapons are losing their effectiveness. In a series of related stories, Science explores the World Health Organization's crusade that aims to cut malaria mortality in half over the next 10 years, conditions on the front lines of clinical research in Africa, the challenges that have slowed development of a so-far elusive vaccine, renewed interest in a Chinese herbal remedy that could aid in the fight against drug-resistant malaria, progress in attacking the Plasmodium parasite through its genome, and the dream of building a malaria-proof mosquito.     

Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications

The first human vaccines against the malaria parasite have been designed to elicit antibodies to the circumsporozoite protein of Plasmodium falciparum. However, it is not known whether any level of naturally acquired antibodies to the circumsporozoite protein can predict resistance to Plasmodium falciparum malaria. In this study, 83 adults in a malaria-endemic region of Kenya were tested for circumsporozoite antibodies and then treated for malaria. They were monitored for the development of new malaria infections for 98 days. Antibody levels, as determined by four assays in vitro, were indistinguishable between the 60 individuals who did and the 23 who did not develop parasitemia during follow-up, and there was no apparent relation between day of onset of parasitemia and level of antibodies to circumsporozoite protein. Unless immunization with sporozoite vaccines induces antibodies that are quantitatively or qualitatively superior to the circumsporozoite antibodies in these adults, it is unlikely that such antibodies will prevent infection in areas with as intense malaria transmission as western Kenya.     

Circumsporozoite protein of Plasmodium vivax: gene cloning and characterization of the immunodominant epitope

The gene encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium vivax has been cloned. The deduced sequence of the protein consists of 373 amino acids with a central region of 19 tandem repeats of the nonapeptide Asp-Arg-Ala-Asp/Ala-Gly-Gln-Pro-Ala-Gly. A synthetic 18-amino acid peptide containing two tandem repeats binds to a monoclonal antibody directed to the CS protein of Plasmodium vivax and inhibits the interaction of this antibody with the native protein in sporozoite extracts. The portions of the CS gene that do not contain repeats are closely related to the corresponding regions of the CS genes of two simian malarias, Plasmodium cynomolgi and Plasmodium knowlesi. In contrast, the homology between the CS genes of Plasmodium vivax and Plasmodium falciparum, another malaria parasite of humans, is very limited.     

Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity

Plasmodium ookinetes traverse midgut epithelial cells before they encounter the complement system in the mosquito hemolymph. We identified a heme peroxidase (HPX2) and NADPH oxidase 5 (NOX5) as critical mediators of midgut epithelial nitration and antiplasmodial immunity that enhance nitric oxide toxicity in Anopheles gambiae. We show that the two immune mechanisms that target ookinetes-epithelial nitration and thioester-containing protein 1 (TEP1)-mediated lysis-work sequentially, and we propose that epithelial nitration works as an opsonization-like system that promotes activation of the mosquito complement cascade.     

Discovery of gene function by expression profiling of the malaria parasite life cycle

The completion of the genome sequence for Plasmodium falciparum, the species responsible for most malaria human deaths, has the potential to reveal hundreds of new drug targets and proteins involved in pathogenesis. However, only approximately 35% of the genes code for proteins with an identifiable function. The absence of routine genetic tools for studying Plasmodium parasites suggests that this number is unlikely to change quickly if conventional serial methods are used to characterize encoded proteins. Here, we use a high-density oligonucleotide array to generate expression profiles of human and mosquito stages of the malaria parasite's life cycle. Genes with highly correlated levels and temporal patterns of expression were often involved in similar functions or cellular processes.     

Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.     

Inhibition of development of exoerythrocytic forms of malaria parasites by gamma-interferon

A specific DNA probe was used to study the effect of recombinant rat, mouse, and human gamma-interferon (gamma-IFN) on the course of sporozoite-induced malaria infections. In mice and rats infected with sporozoites of Plasmodium berghei, mouse and rat gamma-IFN's strongly inhibited the development of the exoerythrocytic forms in the liver liver cells of the hosts, but not the development of the erythrocytic stages. The degree of inhibition of the exoerythrocytic forms was proportional to the dose of gamma-IFN administered, but was independent of the number of sporozoites used for challenge. A 30 percent reduction in the development of exoerythrocytic forms in rat liver was achieved when 150 units (about 15 nanograms of protein) of rat gamma-IFN were injected a few hours before sporozoite challenge; the reduction was 90 percent or more with higher doses of gamma-IFN. The effect was less pronounced if the gamma-IFN was administered 18 hours before or a few hours after challenge. Human gamma-IFN also diminished the parasitemia in chimpanzees infected with sporozoites of the human malaria parasite Plasmodium vivax. The target of gamma-IFN activity may be the infected hepatocytes themselves, as shown by in vitro experiments in which small doses of the human lymphokine inhibited the development of exoerythrocytic forms of Plasmodium berghei in a human hepatoma cell line. These results suggest that immunologically induced interferon may be involved in controlling malaria infection under natural conditions.     

DNA cloning of Plasmodium falciparum circumsporozoite gene: amino acid sequence of repetitive epitope

A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS beta-lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.     

Glucose-6-phosphate dehydrogenase deficient red cells: resistance to infection by malarial parasites

Erythrocyte mosaicism occurs in females heterozygous for glucose-6-phosphate dehydrogenase deficiency. In blood from female children with acute Plasmodium falciparum malaria the parasite rate was 2 to 80 times higher in normal than in deficient erythrocytes. This may be the mechanism whereby the gene for glucose-6-phosphate dehydrogenase deficiency confers selective advantage against malaria to heterozygous females, and thus may have attained the polymorphic frequency occurring in populations living in areas with endemic malaria.     

Targeting malaria virulence and remodeling proteins to the host erythrocyte

To establish infection in the host, malaria parasites export remodeling and virulence proteins into the erythrocyte. These proteins can traverse a series of membranes, including the parasite membrane, the parasitophorous vacuole membrane, and the erythrocyte membrane. We show that a conserved pentameric sequence plays a central role in protein export into the host cell and predict the exported proteome in Plasmodium falciparum. We identified 400 putative erythrocyte-targeted proteins corresponding to approximately 8% of all predicted genes, with 225 virulence proteins and a further 160 proteins likely to be involved in remodeling of the host erythrocyte. The conservation of this signal across Plasmodium species has implications for the development of new antimalarials.