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.     

Expression of Plasmodium falciparum circumsporozoite proteins in Escherichia coli for potential use in a human malaria vaccine

The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.     

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.     

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.     

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.     

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.     

Vivax-type malaria parasite of macaques transmissible to man

Transmission of Plasmodium cynomolgi bastianellii from rhesus monkeys to two human subjects by Anopheles freeborni and the occurrence of attacks of malaria in two other laboratory workers not exposed to human malaria suggests the existence of an animal reservoir of infection complicating malaria control and eradication.     

Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum

In a study of recombinant proteins that might be useful in developing a vaccine against malaria, synthetic peptides from the circumsporozoite (CS) protein of Plasmodium falciparum were found to be immunogenic for mice and rabbits. Antibody to peptides from the repeating region of the CS protein recognized native CS protein and blocked sporozoite invasion of human hepatoma cells in vitro. Antibodies to peptides from regions I and II had no biologic activity, although antibody to region I recognized processed CS protein by Western blot analysis. These data support the feasibility of developing a vaccine against the sporozoite stage of the malaria parasite by using synthetic peptides of the repeating region of the CS protein conjugated to a carrier protein.     

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.     

Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae

Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development before invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species.     

Plasmodium knowlesi sporozoite antigen: expression by infectious recombinant vaccinia virus

The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.     

Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria

Protective immunity against malaria can be obtained by vaccination with irradiated sporozoites. The protective antigens known as circumsporozoite (CS) proteins, are polypeptides that cover the surface membrane of the parasite. The CS proteins contain species-specific immunodominant epitopes formed by tandem repeated sequences of amino acids. Here it is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Pro or (NANP)3. Monoclonal antibodies and most or all polyclonal human antibodies to the sporozoites react with (NANP)3, and polyclonal antibodies raised against the synthetic peptide (NANP)3 react with the surface of the parasite and neutralize its infectivity. Since (NANP)3 repeats are present in CS proteins of P. falciparum from many parts of the world, this epitope is a logical target for vaccine development.     

A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis

Exocytosis is essential to the lytic cycle of apicomplexan parasites and required for the pathogenesis of toxoplasmosis and malaria. DOC2 proteins recruit the membrane fusion machinery required for exocytosis in a Ca(2+)-dependent fashion. Here, the phenotype of a Toxoplasma gondii conditional mutant impaired in host cell invasion and egress was pinpointed to a defect in secretion of the micronemes, an apicomplexan-specific organelle that contains adhesion proteins. Whole-genome sequencing identified the etiological point mutation in TgDOC2.1. A conditional allele of the orthologous gene engineered into Plasmodium falciparum was also defective in microneme secretion. However, the major effect was on invasion, suggesting that microneme secretion is dispensable for Plasmodium egress.     

Induction of Plasmodium falciparum transmission-blocking antibodies by recombinant vaccinia virus

Many candidate antigens of malaria vaccines have limited immunological recognition. One exception is Pfs25, a cysteine-rich, 25-kilodalton sexual stage surface protein of Plasmodium falciparum. Pfs25 is a target of monoclonal antibodies that block transmission of malaria from vertebrate host to mosquito vector. The surface of mammalian cells infected with a recombinant vaccinia virus that expressed Pfs25 specifically bound transmission-blocking monoclonal antibodies. Furthermore, major histocompatibility complex-disparate congenic mouse strains immunized with recombinant Pfs25 elicited transmission-blocking antibodies, demonstrating that the capacity to develop transmission-blocking antibodies is not genetically restricted in mice. Live recombinant viruses may provide an inexpensive, easily administered alternative to subunit vaccines prepared from purified recombinant proteins to block transmission of malaria in developing countries.     

Hemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected erythrocytes

The hemoglobins S and C protect carriers from severe Plasmodium falciparum malaria. Here, we found that these hemoglobinopathies affected the trafficking system that directs parasite-encoded proteins to the surface of infected erythrocytes. Cryoelectron tomography revealed that the parasite generated a host-derived actin cytoskeleton within the cytoplasm of wild-type red blood cells that connected the Maurer's clefts with the host cell membrane and to which transport vesicles were attached. The actin cytoskeleton and the Maurer's clefts were aberrant in erythrocytes containing hemoglobin S or C. Hemoglobin oxidation products, enriched in hemoglobin S and C erythrocytes, inhibited actin polymerization in vitro and may account for the protective role in malaria.     

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.     

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.     

Brain capillary blockage produced by a virulent strain of rodent malaria

A sudden enhancement in virulence of a mild Plasmodium berghei yoelii 17 x strain resulted in fulminating and fatal infections in CF1 and A/J mice. The virulent strain has maintained its characteristics after ten cyclical transmissions through Anopheles stephensi. The visible expression of virulence of the mutated strain is its ability to cross the blood-brain barrier and cause intravascular sequestration of injected erythrocytes and blockage of brain capillaries. We, therfore, believe that the virulent line of Plasmodium berghei yoelii 17 x could serve as a useful laboratory model for the study of "cerebral malaria."