Plasmodium Falciparum Sporozoites Research Articles

3D structure and immunogenicity of Plasmodium falciparum sporozoite induced associated protein peptides as components of fully-protective anti-malarial vaccine

SIAP-1 and SIAP-2 are proteins which are implicated in early events involving Plasmodium falciparum infection of the Anopheles mosquito vector and the human host. High affinity HeLa and HepG2 cell binding conserved peptides have been previously identified in these proteins, i.e. SIAP-1 34893 (421KVQGLSYLLRRKNGTKHPVY440) and SIAP-1 34899 (541YVLNSKLLNSRSFDKFKWIQ560) and SIAP-2 36879 (181LLLYSTNSEDNLDISFGELQ200). When amino acid sequences have been properly modified (replacements shown in bold) they have induced high antibody titres against sporozoites in Aotus monkeys (assessed by IFA) and in the corresponding recombinant proteins (determined by ELISA and Western blot). 1H NMR studies of these conserved native and modified high activity binding peptides (HABPs) revealed that all had α-helical structures in different locations and lengths. Conserved and corresponding modified HABPs displayed different lengths between the residues fitting into MHCII molecule pockets 1–9 and different amino acid orientation based on their different HLA-DRβ1∗ binding motifs and binding registers, suggesting that such modifications were associated with making them immunogenic. The results suggested that these modified HAPBs could be potential targets for inclusion as components of a fully-effective, minimal sub-unit based, multi-epitope, and multistage anti-malarial vaccine.

A role for heparan sulfate proteoglycans in Plasmodium falciparum sporozoite invasion of anopheline mosquito salivary glands

HS (heparan sulfate) has been shown to be an important mediator of Plasmodium sporozoite homing and invasion of the liver, but the role of this glycosaminoglycan in mosquito vector host–sporozoite interactions is unknown. We have biochemically characterized the function of AgOXT1 (Anopheles gambiae peptide-O-xylosyltransferase 1) and confirmed that AgOXT1 can modify peptides representing model HS and chondroitin sulfate proteoglycans in vitro. Moreover, we also demonstrated that the mosquito salivary gland basal lamina proteoglycans are modified by HS. We used RNA interference-mediated knockdown of HS biosynthesis in A. gambiae salivary glands to determine whether Plasmodium falciparum sporozoites that are released from mosquito midgut oocysts use salivary gland HS as a receptor for tissue invasion. Our results suggest that salivary gland basal lamina HS glycosaminoglycans only partially mediate midgut sporozoite invasion of this tissue, and that in the absence of HS, the presence of other surface co-receptors is sufficient to facilitate parasite entry.

Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells

Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins’ functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host–pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.

Surveillance of vector populations and malaria transmission during the 2009/10 El Niño event in the western Kenya highlands: opportunities for early detection of malaria hyper-transmission

BackgroundVector control in the highlands of western Kenya has resulted in a significant reduction of malaria transmission and a change in the vectorial system. Climate variability as a result of events such as El Niño increases the highlands suitability for malaria transmission. Surveillance and monitoring is an important component of early transmission risk identification and management. However, below certain disease transmission thresholds, traditional tools for surveillance such as entomological inoculation rates may become insensitive. A rapid diagnostic kit comprising Plasmodium falciparum circumsporozoite surface protein and merozoite surface protein antibodies in humans was tested for early detection of transmission surges in the western Kenya highlands during an El Niño event (October 2009-February 2010).MethodsIndoor resting female adult malaria vectors were collected in western Kenya highlands in four selected villages categorized into two valley systems, the U-shaped (Iguhu and Emutete) and the V-shaped valleys (Marani and Fort Ternan) for eight months. Members of the Anopheles gambiae complex were identified by PCR. Blood samples were collected from children 6-15 years old and exposure to malaria was tested using a circum-sporozoite protein and merozoite surface protein immunchromatographic rapid diagnostic test kit. Sporozoite ELISA was conducted to detect circum-sporozoite protein, later used for estimation of entomological inoculation rates.ResultsAmong the four villages studied, an upsurge in antibody levels was first observed in October 2009. Plasmodium falciparum sporozoites were then first observed in December 2009 at Iguhu village and February 2010 at Emutete. Despite the upsurge in Marani and Fort Ternan no sporozoites were detected throughout the eight month study period. The antibody-based assay had much earlier transmission detection ability than the sporozoite-based assay. The proportion of An. arabiensis among An. gambiae s.l. ranged from 2.9-66.7% indicating a rearrangement of the sibling species of the An. gambiae s.l complex. This is possibly an adaptation to insecticide interventions and climate change.ConclusionThe changing malaria transmission rates in the western Kenya highlands will lead to more unstable transmission, decreased immunity and a high vulnerability to epidemics unless surveillance tools are improved and effective vector control is sustained.

Early Transcriptional Responses of HepG2-A16 Liver Cells to Infection by Plasmodium falciparum Sporozoites

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.

Plasmodium falciparum Infection during Suppressive Prophylaxis with Mefloquine Does Not Induce an Antibody Response to Merozoite Surface Protein-1(42)

A sensitive biomarker of malaria infection would obviate the need for placebo control arms in clinical trials of malaria prophylactic drugs. Antibodies to the 42-kDa fragment of merozoite surface protein-1 (MSP1(42)) have been identified as a potential marker of malaria exposure in individuals receiving prophylaxis with mefloquine. We conducted an open-label trial to determine the sensitivity of seroconversion to MSP1(42), defined as a fourfold rise in enzyme-linked immunosorbant assay (ELISA) titer, among 23 malaria naïve volunteers receiving mefloquine prophylaxis and 6 controls after Plasmodium falciparum sporozoite challenge. All members of the control cohort but none of the mefloquine cohort developed patent parasitemia. Four of six controls but zero of the mefloquine cohort seroconverted to MSP1(42). We conclude that malaria infection during suppressive prophylaxis does not induce antibody response to the blood-stage antigen MSP1(42) in a malaria-naïve study population.

Naturally acquired IgG antibodies against the C-terminal part of Plasmodium falciparum sporozoite threonine–asparagine-rich protein in a low endemic area

Plasmodium falciparum sporozoite threonine-asparagine-rich protein (STARP), a 78-kDa surface protein, is considered a potential vaccine candidate. The C-terminal part of STARP has been evolved under positive selection, suggesting the presence of immunodominant epitopes. However, little is known about the immune responses against STARP among individuals upon natural malaria exposure. In this study, we have cloned and expressed in Escherichia coli the recombinant C-terminal part of STARP spanning 118 amino acids in order to examine the humoral immune response against this protein. Blood samples were randomly collected from 74 individuals living in a malaria endemic area of Thailand who were acutely infected with P. falciparum (n = 54) and with Plasmodium vivax (n = 20). Malaria-negative blood samples were also obtained from 27 individuals living in the same endemic area who had experienced prior infection with P. falciparum 6 months to 1 year before sample collection and 20 healthy subjects without history of malaria exposure. Western blot analysis revealed that IgG antibodies against this recombinant peptide were found in 23 of 54 serum samples (42.6%) from P. falciparum-infected individuals. All serum samples from P. vivax-infected cases, non-infected individuals, and those who experienced prior infection with P. falciparum gave negative results, indicating that naturally acquired IgG antibodies against the C-terminal part of STARP are species-specific and short-lived. Provided that antibodies against STARP could confer protection, it is likely that malaria vaccine derived from the C-terminal part of STARP could probably be boosted upon natural exposure to P. falciparum.

Development of an attenuated sporozoite vaccine to prevent and eliminate Plasmodium falciparum malaria

An ideal vaccine for malaria would target all stages of the parasite life cycle, and thereby prevent infection, severe disease and transmission. However, most malariologists agree that if only one stage is to be targeted, it should be the pre-erythrocytic stage, because induction of highly protective immune responses against this stage will prevent blood stage infection and thus disease and parasite transmission. Our consortium is working to develop such a vaccine. The first-generation vaccine is a metabolically active, non-replicating Plasmodium falciparum sporozoite (PfSPZ) vaccine that is attenuated by irradiation. The first major challenge was to manufacture adequate quantities of such a vaccine that met regulatory standards for initial clinical trials, and demonstrate that it was safe, well tolerated and immunogenic in humans. This has been accomplished. The second major challenge is to determine how to administer for the first time in humans an unprecedented, non-replicating and metabolically active (live) whole-organism vaccine formulation composed of PfSPZ that measure 0.5-1.0 μm x 7-10 μm, to induce > 85% protection. Volunteers immunized by intradermal (ID) or subcutaneous (SC) routes were protected in the first clinical trial. However, the number of protected subjects was low, and despite a dose response immunogenicity was sub-optimal. Animal studies show that intravenous (IV) immunization induces much better immunity and protection than immunization by the SC or ID routes. These data have been used to inform the design of the next clinical trial. Parallel efforts are underway to optimize non-IV administration of PfSPZ, and the efficiency and scale-up of manufacture. We are also determining whether genetic targeting techniques can be used to create a parasite clone that will produce attenuated PfSPZ that are not only more potent, but also as safe as radiation-attenuated PfSPZ. Such PfSPZ could be non-replicating (as are radiation-attenuated PfSPZ), replication deficient, or replication competent, but avirulent. Our goal is to develop, license and deploy a highly effective pre-erythrocytic stage PfSPZ vaccine that prevents blood stage infection, disease, and transmission. Such a vaccine could be used at the community level in Pf elimination campaigns, and at the individual level for prevention of Pf malaria in infants, young children, and pregnant women in endemic areas, as well as individuals of all ages who travel to malaria-endemic areas.

Complete abrogation of sporozoite-induced sterile immunity by blood stage parasites of homologous and heterologous malaria species

Immunisation of mice and humans with attenuated sporozoites has been shown to confer sterile immunity against infection. There are ongoing efforts to develop a vaccine based on this system. Attenuation of sporozoites may be achieved via irradiation, genetic modification, or through the use of drugs targeting the blood stage parasite. Recently, it has been shown that the administration of chloroquine, a drug that acts exclusively against the erythrocytic stages of malaria parasites, concurrently with live sporozoites can induce sterile immunity against homologous challenge with Plasmodium falciparum sporozoites in humans. However, it is not known whether the protection achieved against P. falciparum will also protect against other species of human malaria parasites, which are almost always endemic in the same region. Given the high amount of antigen diversity within malaria parasite species, coupled with the large evolutionary distances between the human species, it seems unlikely that immunity to heterologous species would be achieved. Of concern is whether the development of an acute blood stage infection of a heterologous species may abrogate the immunity achieved against the vaccine target species. This phenomenon would have serious consequences for the deployment of an attenuated sporozoite vaccine in a multispecies endemic area. Here, we describe the results of experiments aimed at determining whether immunity achieved against one species of malaria parasite by sporozoite immunisation is protective against a secondary species, and whether the development of acute blood stage infections of the heterologous species abrogates the immunity achieved against the vaccine target species. As such experiments are currently impossible using human malaria parasite species, we have used the rodent malaria parasite species Plasmodium vinckei and Plasmodium yoelii. These two species were selected as they offer the widest possible evolutionary distance between rodent malaria parasites, although they are still much more closely related than any two of the human species. We found that although there was some cross protection between the species following sporozoite immunisation in conjunction with mefloquine treatment, the major component of this immunity was species specific. Mice immunised with P. yoelii sporozoites were completely protected against the development of blood stage infection following P. yoelii sporozoite challenge, whereas they were completely susceptible to infection with P. vinckei. Crucially, we found that the development of a blood stage infection of either species completely removed the sterile protection achieved via sporozoite immunisation.

Ecological transition from natural forest to tea plantations: effect on the dynamics of malaria vectors in the highlands of Cameroon

From October 2002 to September 2003, an entomological survey was carried out in a rural forested fringed village in the highlands of Mount Cameroon region to determine the temporal dynamics of the anopheline population and the intensity of malaria transmission. A total of 2387 Anopheles spp. were collected, with A. funestus predominating (59.9%), followed by A. hancocki (24.4%) and A. gambiae s.l. (15.7%). Considerable differences were observed in the nocturnal biting cycles of parous mosquitoes, with peak activity in the latter part of the night. PCR revealed that all specimens of the A. funestus group were A. funestus s.s. and all specimens from the A. gambiae complex were A. gambiae s.s. of the S molecular form. Plasmodium falciparum sporozoite rates of 17.3% and 8.5% were recorded for A. funestus and A. hancocki, respectively, with an anthropophilic rate of 96.3%. A strong positive correlation ( r = 0.996) was found between the human-biting rate and the entomological inoculation rate (EIR). Malaria transmission was very high and perennial, with an estimated annual EIR of 460.1 infective bites per person per year. These results confirm that in high agricultural activity areas, A. funestus can be by far the major malaria vector responsible for malaria transmission.

Identification of a major rif transcript common to gametocytes and sporozoites of Plasmodium falciparum

BackgroundThe Plasmodium falciparum parasite is transmitted in its sexual gametocyte stage from man to mosquito and as asexual sporozoites from mosquito to man. Developing gametocytes sequester preferentially in the bone marrow, but mature stage gametocytes are released to the bloodstream. Sexual stage parasite surface proteins are of interest as candidate target antigens for transmission blocking vaccines.MethodsIn this study, the transcript profiles of rif and var genes, known to encode surface antigens in asexual blood stage parasites, were investigated at different stages of 3D7/NF54 gametocytogenesis and in sporozoites.ResultsGametocytes exhibited a rif transcript profile unlinked to the rif and var transcript profile of the asexual progenitors. At stage V, mature gametocytes produced high levels of a single rif gene, PF13_0006, which also dominated the rif transcript profile of sporozoites. All var genes appeared to be silenced in sporozoites.ConclusionsThe most prominent variant surface antigen transcribed in both gametocytes and sporozoites of 3D7/NF54 is a single variant of the RIFIN protein family. This discovery may lead to the identification of the parasites binding ligands responsible for the adhesion during sexual stages and potentially to novel vaccine candidates.

The Ig Domain Protein CD9P-1 Down-regulates CD81 Ability to Support Plasmodium yoelii Infection

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria natural infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that CD81 is required on hepatocytes for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 belongs to the tetraspanin superfamily of transmembrane proteins. By interacting with each other and with other transmembrane proteins, tetraspanins may play a role in the lateral organization of membrane proteins. In this study, we investigated the role of the two major molecular partners of CD81 in hepatocytic cells, CD9P-1/EWI-F and EWI-2, two transmembrane proteins belonging to a novel subfamily of immunoglobulin proteins. We show that CD9P-1 silencing increases the host cell susceptibility to P. yoelii sporozoite infection, whereas EWI-2 knock-down has no effect. Conversely, overexpression of CD9P-1 but not EWI-2 partially inhibits infection. Using CD81 and CD9P-1 chimeric molecules, we demonstrate the role of transmembrane regions in CD81-CD9P-1 interactions. Importantly, a CD9P-1 chimera that no longer associates with CD81 does not affect infection. Based on these data, we conclude that CD9P-1 acts as a negative regulator of P. yoelii infection by interacting with CD81 and regulating its function.

The impact of a small-scale irrigation scheme on malaria transmission in Ziway area, Central Ethiopia

To assess the impact of a small-scale irrigation scheme in Ziway area, a semi-arid area in the Central Ethiopian Rift Valley, on malaria transmission. Parasitological, entomological and socio-economic studies were conducted in a village with and a village without irrigation. Blood smear samples were taken from individuals during the dry and wet seasons of 2005/2006. Socio-economic data were collected from household heads and key agricultural and health informants through interviews and questionnaires. Larval and adult mosquitoes were sampled during the dry and short wet seasons of 2006. Female anopheline mosquitoes were tested by enzyme-linked immunosorbent assay for blood meal sources and sporozoite infections. Malaria prevalence was higher in the irrigated village (19%, P < 0.05) than the non-irrigated village (16%). In the irrigated village, malaria prevalence was higher in the dry season than in the wet season while the reverse occurred in the non-irrigated village. Households with access to irrigation had larger farm land sizes and higher incomes, but also higher prevalence of malaria. Larval and adult abundance of the malaria vectors, Anopheles arabiensis and Anopheles pharoensis, was higher in the irrigated than in the non-irrigated village throughout the study period. Furthermore, the abundance of An. pharoensis was significantly higher than that of An. arabiensis during the dry irrigated period of the year. Canal leakage pools, irrigated fields and irrigation canals were the major breeding habitats of the two vector mosquitoes. Plasmodium falciparum sporozoite infection rates of 1.18% and 0.66% were determined for An. arabiensis and An. pharoensis in the irrigated village. Peak biting activities of the vectors occurred before 22:00 h, which is a source of concern that the effectiveness of ITNs may be compromised as the mosquitoes feed on blood before people go to bed. Irrigation schemes along the Ethiopian Rift Valley may intensify malaria by increasing the level of prevalence during the dry season. To reduce the intensity of malaria transmission in the small-scale irrigation schemes currently in operation in Ethiopia, year-round source reduction by using proper irrigation water management, coupled with health education, needs to be incorporated into the existing malaria control strategies.

Multiple functions of human T cells generated by experimental malaria challenge

Protective immunity generated following malaria infection may be comprised of Ab or T cells against malaria Ag of different stages; however, the short-lived immunity that is observed suggests deficiency in immune memory or regulatory activity. In this study, cellular immune responses were investigated in individuals receiving Plasmodium falciparum sporozoite challenge by the natural (mosquito bite) route as part of a malaria vaccine efficacy trial. Parasitemia, monitored by blood film microscopy and PCR, was subsequently cleared with drugs. All individuals demonstrated stable IFN-gamma, IL-2 and IL-4 ex vivo ELISPOT effector responses against P. falciparum-infected RBC (iRBC) Ag, 28 and 90 days after challenge. However, infected RBC-specific central memory responses, as measured by IFN-gamma cultured ELISPOT, were low and unstable over time, despite CD4(+) T cells being highly proliferative by CFSE dilution, and showed an inverse relationship to parasite density. In support of the observation of poor memory, co-culture experiments showed reduced responses to common recall Ag, indicating malaria-specific regulatory activity. This activity could not be accounted for by the expression of IL-10, TGF-beta, FOXP3 or CTLA-4, but proliferating T cells expressed high levels of CD95, indicating a pro-apoptotic phenotype. Lastly, there was an inverse relationship between FOXP3 expression, when measured 10 days after challenge, and ex vivo IFN-gamma measured more than 100 days later. This study shows that malaria infection elicits specific Th1 and Th2 effector cells, but concomitant weak central memory and regulatory activity, which may help to explain the short-lived immunity observed.

Characterization of immunoglobulin G antibodies to Plasmodium falciparum sporozoite surface antigen MB2 in malaria exposed individuals.

BackgroundMB2 protein is a sporozoite surface antigen on the human malaria parasite Plasmodium falciparum. MB2 was identified by screening a P. falciparum sporozoite cDNA expression library using immune sera from a protected donor immunized via the bites of P. falciparum-infected irradiated mosquitoes. It is not known whether natural exposure to P. falciparum also induces the anti-MB2 response and if this response differs from that in protected individuals immunized via the bites of P. falciparum infected irradiated mosquitoes. The anti-MB2 antibody response may be part of a robust protective response against the sporozoite.MethodsFragments of polypeptide regions of MB2 were constructed as recombinant fusions sandwiched between glutathione S-transferase and a hexa histidine tag for bacterial expression. The hexa histidine tag affinity purified proteins were used to immunize rabbits and the polyclonal sera evaluated in an in vitro inhibition of sporozoite invasion assay. The proteins were also used in immunoblots with sera from a limited number of donors immunized via the bites of P. falciparum infected irradiated mosquitoes and plasma and serum obtained from naturally exposed individuals in Kenya.ResultsRabbit polyclonal antibodies targeting the non-repeat region of the basic domain of MB2 inhibited sporozoites entry into HepG2-A16 cells in vitro. Analysis of serum from five human volunteers that were immunized via the bites of P. falciparum infected irradiated mosquitoes that developed immunity and were completely protected against subsequent challenge with non-irradiated parasite also had detectable levels of antibody against MB2 basic domain. In contrast, in three volunteers not protected, anti-MB2 antibodies were below the level of detection. Sera from protected volunteers preferentially recognized a non-repeat region of the basic domain of MB2, whereas plasma from naturally-infected individuals also had antibodies that recognize regions of MB2 that contain a repeat motif in immunoblots. Sequence analysis of eleven field isolates and four laboratory strains showed that these antigenic regions of the basic domain of the MB2 gene are highly conserved in parasites obtained from different parts of the world. Moreover, anti-MB2 antibodies also were detected in the plasma of 83% of the individuals living in a malaria endemic area of Kenya (n = 41).ConclusionA preliminary analysis of the human humoral response against MB2 indicates that it may be an additional highly conserved target for immune intervention at the pre-erythrocytic stage of P. falciparum life cycle.

The development and evaluation of a single step multiplex PCR for simultaneous detection of Anopheles annularis group mosquitoes, human host preference and Plasmodium falciparum sporozoite presence

The Anopheles annularis group mosquitoes, subgenus Cellia Theobald (Diptera: Culicidae), includes five recognized species: An. annularis Van der Wulp, An. nivipes Theobald, An. pallidus Theobald, An. philippinensis Ludlow and An. schueffneri Stanton. From these five, the three most common species found in Orissa were considered for this study because of their remarkable vectorial and behavioral variation and the important role they play in malaria transmission. To identify and understand their role in malaria transmission we developed a single multiplex PCR-based assay. This assay included the detection of human blood feeding habit and Plasmodium falciparum sporozoite presence. Of the 186 An. annularis mosquitoes collected, morphological character-based identification showed that 94 were An. annularis, 54 were An. philippinensis and 38 were An. pallidus. However, the multiplex PCR assay confirmed that 91 were An. annularis, 56 were An. philippinensis and 39 were An. pallidus individuals after adjustments were made for misidentified specimens in the morphological method. Anopheles annularis and An. philippinensis were found positive for human blood, and two samples of An. annularis species were positive for P. falciparum sporozoites. This one-step PCR-based method constitutes a very powerful tool in large surveys of anopheline populations.

Phase I/II Evaluation of the Prophylactic Antimalarial Activity of Pafuramidine in Healthy Volunteers Challenged with Plasmodium falciparum Sporozoites

We evaluated the causal prophylactic antimalarial activity of a single oral dose of pafuramidine (DB289), an experimental prodrug of active metabolite DB75, in a randomized, double-blind, placebo-controlled, outpatient study. Sixteen healthy volunteers were dosed and challenged in a single cohort. Subjects were randomly assigned to one of three treatment arms: 100 mg pafuramidine eight days before challenge, 100 mg pafuramidine the day before challenge, or placebo. Challenge was by the bites of Plasmodium falciparum-infected Anopheles gambiae. Malaria developed in 15 persons but did not develop in one person in the day -8 pafuramidine treatment arm. Plasma levels of DB75 were lower than expected, and as intended were too low to provide suppressive prophylaxis at the earliest appearance of erythrocytic parasites. We conclude that a single dose of 100 mg pafuramidine does not adequately protect non-immune individuals against P. falciparum and shows no clinically or statistically significant evidence of causal prophylactic activity.

The Effects of radiation on the safety and protective efficacy of an attenuated Plasmodium yoelii sporozoite malaria vaccine

We are developing a radiation attenuated Plasmodium falciparum sporozoite (PfSPZ) malaria vaccine. An important step was to determine the minimum dose of irradiation required to adequately attenuate each sporozoite. This was studied in the Plasmodium yoelii rodent model system. Exposure to 100 Gy completely attenuated P. yoelii sporozoites (PySPZ). Next we demonstrated that immunization of mice intravenously with 3 doses of 750 PySPZ that had received 200 Gy, double the radiation dose required for attenuation, resulted in 100% protection. These results support the contention that a radiation attenuated sporozoite vaccine for malaria will be safe and effective at a range of radiation doses.

Proteomic Profiling of Plasmodium Sporozoite Maturation Identifies New Proteins Essential for Parasite Development and Infectivity

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito—early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.

Scavenger Receptor BI Boosts Hepatocyte Permissiveness to Plasmodium Infection

Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as specific SR-BI-blocking antibodies, we demonstrate that SR-BI significantly boosts hepatocyte permissiveness to P. falciparum, P. yoelii, and P. berghei entry and promotes parasite development. We show that SR-BI, but not the low-density lipoprotein receptor, acts as a major cholesterol provider that enhances Plasmodium infection. SR-BI regulates the organization of CD81 at the plasma membrane, mediating an arrangement that is highly permissive to penetration by sporozoites. Concomitantly, SR-BI upregulates the expression of the liver fatty-acid carrier L-FABP, a protein implicated in Plasmodium liver-stage maturation. These findings establish the mechanistic basis of the CD81-dependent Plasmodium sporozoite invasion pathway.