Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface

Plasmodium parasites of mammals, including the species that cause malaria in humans, infect the liver first and develop there into clinically silent liver stages. Liver stages grow and ultimately produce thousands of first-generation merozoites, which initiate the erythrocytic cycles causing malaria pathology. Here, we present a Plasmodium protein with a critical function for complete liver stage development. UIS4 (up-regulated in infective sporozoites gene 4) is expressed exclusively in infective sporozoites and developing liver stages, where it localizes to the parasitophorous vacuole membrane. Targeted gene disruption of UIS4 in the rodent model malaria parasite Plasmodium berghei generated knockout parasites that progress through the malaria life cycle until after hepatocyte invasion but are severely impaired in further liver stage development. Immunization with UIS4 knockout sporozoites completely protects mice against subsequent infectious WT sporozoite challenge. Genetically attenuated liver stages may thus induce immune responses, which inhibit subsequent infection of the liver with WT parasites.     

Anopheles gambiae salivary gland proteins as putative targets for blocking transmission of malaria parasites

Anopheles gambiae is the primary vector of human malaria in sub-Saharan Africa. Invasion of Anopheles salivary glands by Plasmodium sporozoites is a necessary step in the transmission of malaria and is likely to be mediated by specific receptor-ligand interactions. We are interested in identifying putative an A. gambiae salivary gland receptor or receptors for sporozoite invasion as a possible target for blocking malaria transmission. By using monoclonal antibodies against female-specific A. gambiae salivary gland proteins, two molecules, one of 29 kDa and one of 100 kDa, were identified and characterized with respect to the age and blood-feeding process of mosquitoes. In an in vivo bioassay, the monoclonal antibody against the 100-kDa protein inhibited Plasmodium yoelii sporozoite invasion of salivary glands >/=75%. These results show that A. gambiae salivary gland proteins are accessible to monoclonal antibodies that inhibit sporozoite invasion of the salivary glands and suggest alternate targets for blocking the transmission of malaria by this most competent of malaria vectors.     

Ingestion of Plasmodium falciparum sporozoites during transmission by anopheline mosquitoes.

We investigated the process of sporozoite transmission during blood feeding for Anopheles gambiae and An. stephensi experimentally infected with Plasmodium falciparum. When infective mosquitoes were fed 22-25 days postinfection on an anesthetized rat, sporozoites were detected in the midgut of 96.5% of 57 An. gambiae (geometric mean [GM] = 32.5, range 3-374) and in 96.2% of 26 An. stephensi (GM = 19.5, range 1-345). There were no significant differences between species either in salivary gland sporozoite loads or in the number of ingested sporozoites. There was a significant linear relationship between sporozoite loads and the numbers of ingested sporozoites for both An. gambiae (r = 0.38) and An. stephensi (r = 0.69). Subsequently, An. gambiae were tested for sporozoite transmission by allowing them to feed individually on a suspended capillary tube containing 10 microliters of blood. A total of 83.3% of 18 infective mosquitoes transmitted a GM of 5.9 (range 1-36) sporozoites. The same mosquitoes contained a GM of 23.4 (range 2-165) ingested sporozoites. The number of ingested sporozoites was related to sporozoite loads (r = 0.42) but not to the number of sporozoites ejected into capillary tubes. Ingested sporozoites remained in the midgut up to 10 hr after feeding. The comparable numbers of sporozoites ingested by infective mosquitoes in both experiments indicates that the actual number of sporozoites transmitted to the vertebrate host during blood feeding is significantly reduced by the blood ingestion process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative determination of Plasmodium falciparum sporozoite rates in Afrotropical Anopheles from Kenya by dissection and ELISA.

The Plasmodium falciparum rate was determined by microscopical examination of one salivary gland (three lobes) and by enzyme-linked immunosorbent assay (ELISA) of the other salivary gland in each of 1580 Anopheles mosquitoes collected from western Kenya during both the wet and dry seasons. The sporozoite rate in the wet season was much higher than that in the dry season, and the sporozoite rate determined by ELISA was generally lower than that determined by microscopy. The ELISA gave a positive reaction to circumsporozoite protein in some glands whose counterparts did not show the presence of sporozoites by microscopy, thus giving an 'overestimation' of the sporozoite rate. This overestimation was greater in the dry season than in the wet season, and greater in Anopheles gambiae than in An. funestus, but overall it was only 1.2% (19/1580). These results are at variance with reports of other workers, who have shown ELISA overestimation of the sporozoite rate as high as 30%. Our tests indicated that the ELISA sensitivity was 80.6%, its specificity was 98.7%, and its accuracy was 97.5%.     

Field evaluation of an enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum sporozoite detection in anopheline mosquitoes from Kenya

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody that recognizes a repetitive epitope on the circumsporozoite protein of Plasmodium falciparum was used in Kenya to assess malaria infections in Anopheles gambiae s.l. and An. funestus. The ELISA confirmed that 88% of 44 sporozoite-positive gland dissections were P. falciparum. The ELISA infection rate of 18.6% (n = 736) for individually tested mosquitoes for both species was significantly higher than the 10.4% (n = 537) salivary gland sporozoite rate determined by dissection. This difference was due to ELISA detection of medium and large sized oocysts on the midguts of infected mosquitoes which did not contain salivary gland sporozoites. From a series of 379 Anopheles that were cut at the thorax, ELISA tests on "head" and "body" portions showed that 29.5% of 95 positive mosquitoes contained circumsporozoite antigen in the body portion in the absence of salivary gland infections. This field evaluation demonstrates that the ELISA can most accurately be used to estimate sporozoite rates by cutting mosquitoes at the thorax and testing anterior portions.     

Efficient development of plasmodium liver stage-specific memory CD8+ T cells during the course of blood-stage malarial infection

Immunity to Plasmodium liver stages in individuals in malaria-endemic areas is inextricably linked to concomitant blood-stage parasitemia. Although Plasmodium sporozoite infection induces measurable CD8+ T cell responses, the development of memory T cells during active erythrocytic infection remains uncharacterized. Using transgenic T cells, we assessed antigen-specific effector CD8+ T cell responses induced by normal (NorSpz) and radiation-attenuated (IrrSpz) Plasmodium yoelii sporozoites. The magnitude, phenotypic activation, and differentiation pathway of CD8+ T cells were similarly induced by NorSpz and IrrSpz. Moreover, in normal mice, memory T cells elicited after priming with NorSpz and IrrSpz generated identical recall responses after a heterologous boost strategy. Furthermore, these recall responses exhibited comparable in vivo antiparasite activity. Our results indicate that sporozoites that retain their infective capacity induce memory CD8+ T cells that are robustly recalled by secondary immunization. Thus, erythrocytic infection does not preclude the establishment of memory CD8+ T cell responses to malarial liver stages.     

The biotin-streptavidin system in a two-site ELISA for the detection of plasmodial sporozoite antigen in mosquitoes

A two-site ELISA has been designed for the detection of sporozoite antigen in mosquitoes. Biotin-labelled monoclonal antibodies against sporozoites and a streptavidin-biotin-peroxidase complex were used to visualize the antigen. Evaluation of the sensitivity and specificity of the procedure was carried out and background levels of reactivity on the basis of negative mosquitoes were calculated. The test has been deliberately kept as simple as possible for use in the tropics and was designed using Anopheles stephensi infected with in vitro cultivated Plasmodium falciparum gametocytes. A minimum of about 100-350 sporozoites could be detected in mature salivary gland infections; in addition sporozoite antigen was detected in mosquitoes several days before the entry of sporozoites into the salivary glands. No reaction was demonstrable either with bloodstage or ookinete antigens of P. falciparum, or with mosquitoes carrying sporozoites of other plasmodial species. The number of sporozoites in positive mosquitoes and the generating capacity of a single oocyst could be assessed by the use of a calibration curve based on dilution data of a known sporozoite suspension. It was found that a single oocyst can produce about 10,000 sporozoite equivalents.     

Circumsporozoite protein of Plasmodium gallinaceum characterized by monoclonal antibodies

Monoclonal antibodies (MoAb) were produced against both salivary gland sporozoites (SGS) and oocyst sporozoites (OS) of Plasmodium gallinaceum, an avian malaria parasite. By indirect immunofluorescence, all of the MoAbs reacted with both SGS and OS of P. gallinaceum and two of the MoAbs cross-reacted weakly with P. berghei sporozoites. None of the MoAbs reacted with sporozoites of six additional species of mammalian plasmodia. In Western blot analysis of extracts of either SGS or OS of P. gallinaceum, these MoAbs identified two polypeptides with molecular weights of approximately 76,000 and 64,000 D. The results of a MoAb inhibition of binding assay and a two-site one-antibody immunoradiometric assay indicate that the circumsporozoite protein of P. gallinaceum, like those of mammalian malaria parasites, contains a repetitive immunodominant epitope. Two of the anti-P. gallinaceum MoAbs were tested in a sporozoite neutralization assay and decreased, but did not abolish, the infectivity of sporozoites for chickens, indicating that the polypeptide of P. gallinaceum identified by immunoblot is probably the protective antigen.     

Identification of malaria species by ELISA in sporozoite and oocyst infected Anopheles from western Kenya

Enzyme-linked immunosorbent assays (ELISAs) for the circumsporozoite (CS) antigens of Plasmodium falciparum, P. malariae, and P. ovale were used to identify species of sporozoite and oocyst infections detected by dissection in Anopheles gambiae s.1. and An. funestus collected in western Kenya. ELISAs identified 92.5% of 1,113 salivary gland infections; Plasmodium species infections included 79.4% P. falciparum, 3.2% P. malariae, 1.7% P. ovale, and 2 or more Plasmodium species were detected in 15.7% of the Anopheles in which the species of parasite was identified. Identification was more likely with greater numbers of sporozoites observed in dissections, increasing from 65% ELISA positivity in mosquitoes with 1-10 sporozoites in their salivary glands to 96% in mosquitoes with over 1,000 sporozoites. ELISAs detected CS antigen in 66% of 294 Anopheles that by dissection had oocysts but uninfected salivary glands. Of 112 Anopheles with a single species of Plasmodium detected in the salivary glands, 29 (25.9%) had 1 or more additional species detected in the midgut, indicating a high potential for multiple infections. Similar proportions of Plasmodium species were found in An. gambiae s.1. and An. funestus.     

Plasmodium vivax and Plasmodium falciparum gametocyte stages are neglected in vaccine development

Plasmodium gametocytes are responsible for transmission from the vertebrate host to the mosquito. Plasmodium gametocytes undergo a complex cycle from asexual stages, through a poorly understood process characterized by expression of stage-specific proteins and adhesion molecules. Gametocytes are capable of inducing specific humoral IgG, and cellular responses, which include induction of TNFalpha, IFNgamma and gammadelta+ lymphocyte proliferation, in addition to immune responses to other stages of the parasite (sporozoite, exo-erythrocytic stages, erythrocytic stages). Although transmission-blocking vaccines against Plasmodium do not currently include components against the gametocytes (rather they focus on gametes, zygotes or ookinetes, stages which occur in the mosquito), further understanding of the mechanisms underlying gametocytogenesis and immune responses against these stages may provide additional strategies for more effective transmission inhibition.     

Strain-specific immunity induced by immunization with pre-erythrocytic stages of Plasmodium chabaudi

One of the most promising approaches in the efforts to produce a malaria vaccine involves the use of attenuated whole sporozoite immunizations. Attenuation may be achieved by the use of genetic modification, irradiation, chemical attenuation, or by the contemporaneous administration of antimalarial drugs that target only the erythrocytic stages of the parasite. Most research to date has focused on the efficacy of these approaches upon challenge with parasites homologous to those used for the initial immunizations. We, as have others, have previously shown that a component of the immunity achieved against the erythrocytic stages of the rodent malaria parasite Plasmodium chabaudi chabaudi is strain-specific, with a stronger immune response targeting the immunizing strain than genetically distinct strains. Here, we show that the immunity induced by infection with the pre-erythrocytic stages of these parasites, achieved via inoculation of sporozoites contemporaneously with mefloquine, also has a strain-specific component.     

Comparative susceptibility of anopheline mosquitoes in Rondonia, Brazil to infection by Plasmodium vivax

Seven anopheline species from Costa Marques, Rondonia, Brazil were compared with Anopheles darlingi for susceptibility to infection by Plasmodium vivax. Laboratory-reared F1 progeny of field-collected An. darlingi and the test anopheline species were fed at the same time on the same patients, all of whom had gametocytes in peripheral blood before treatment. Mosquitoes were dissected on day 8 after infection for oocysts and on days 14-16 after infection for sporozoites. The mean numbers of P. vivax oocysts and the percent of salivary gland infections for An. darlingi and An. deaneorum were similar and far exceeded those found in the other anopheline species tested. Anopheles albitarsis and An. mediopunctatus were less susceptible to infection by oocyst measurements than An. darlingi. However, for oocyst-infected An. albitarsis and An. mediopunctatus, the percent of mosquitoes with salivary gland infections and the numbers of sporozoites in the salivary glands were similar to An. darlingi. Anopheles triannulatus and An. oswaldoi were both susceptible to P. vivax infection, but the sporozoite infection rates and the numbers of sporozoites observed in the salivary glands were very low. Anopheles braziliensis and An. benarrochi both developed oocysts, but were never observed to have sporozoites in the salivary glands. These studies implicate some anopheline species as potential malaria vectors, but also show that species previously incriminated by ELISA techniques are not vectors of malaria parasites in Costa Marques, Rondonia, Brazil.     

PK4, a eukaryotic initiation factor 2α(eIF2α) kinase, is essential for the development of the erythrocytic cycle of Plasmodium

In response to environmental stresses, the mammalian serine threonine kinases PERK, GCN2, HRI, and PKR phosphorylate the regulatory serine 51 of the eukaryotic translation initiation factor 2α (eIF2α) to inhibit global protein synthesis. Plasmodium, the protozoan that causes malaria, expresses three eIF2α kinases: IK1, IK2, and PK4. Like GCN2, IK1 regulates stress response to amino acid starvation. IK2 inhibits development of malaria sporozoites present in the mosquito salivary glands. Here we show that the phosphorylation by PK4 of the regulatory serine 59 of Plasmodium eIF2α is essential for the completion of the parasite's erythrocytic cycle that causes disease in humans. PK4 activity leads to the arrest of global protein synthesis in schizonts, where ontogeny of daughter merozoites takes place, and in gametocytes that infect Anopheles mosquitoes. The implication of these findings is that drugs that reduce PK4 activity should alleviate disease and inhibit malaria transmission.     

Increased intradermal probing time in sporozoite-infected mosquitoes

Because malaria sporozoites destroy segments of the salivary glands of vector mosquitoes, we determined whether salivary function is impaired. Such pathology would result in a prolonged intradermal probing phase of feeding behavior, because the role of saliva is to help locate blood vessels. Indeed, non-infected Aedes aegypti mosquitoes probed for a shorter period than did either sporozoite-infected or saliva-deprived mosquitoes. Salivary apyrase activity is reduced to a third following maturation of sporozoites. Apyrase activity, normally, is confined to those regions invaded by sporozoites. Sporozoite-infected and non-infected mosquitoes produced equal volumes of saliva. We conclude that sporozoite infection impairs the vector's ability to locate blood vessels by affecting the quality of salivary product, thereby increasing potentially infective host contacts.     

A scanning electron microscopic study of the sporogonic development of Plasmodium falciparum in Anopheles stephensi.

The full development of Plasmodium falciparum in Anopheles stephensi mosquitoes was studied by scanning electron microscopy. Ookinetic development was described from in vitro cultures. Growing oocysts beneath the basal lamina of the midgut wall mechanically stretch this lamina until it is torn and displaced by day 7. In young oocysts the wall appears smooth. In older oocysts wrinkles in the wall are visible after routine fixation. Osmium tetroxide postfixation greatly reduced the occurrence of these wrinkles. Intracapsular development of sporozoites was visualized after mechanical manipulation of the oocysts during sample preparation. In contrast to P. berghei, no ectopic development was seen in P. falciparum in the mosquito midgut. The mechanism of sporozoite escape from the oocyst appears to be similar to that described for rodent malaria. Fracturing of salivary glands provided the first view by scanning electron microscopy of sporozoites located in proximal and distal gland cells and in the draining duct.     

The role of the Kupffer cell in the infection of rodents by sporozoites of Plasmodium: uptake of sporozoites by perfused liver and the establishment of infection in vivo

The uptake of Plasmodium yoelii nigeriensis sporozoites by isolated perfused rat liver was very rapid and efficient. 67% of the initial load was removed from the perfusion media in the first passage through the liver, and 95% after 15 min of perfusion. Much of the uptake was explained by mechanical trapping in the liver. Up to 75% of the sporozoite load was retained after 15 min both by heat killed liver and liver cooled to 4 degrees C, therefore at least 20% of the sporozoite uptake in perfused normal livers was due to a biologically active process. In perfused normal livers, non-infective (heat-killed or trypsin-treated) sporozoites were taken up with an efficiency equal to infective sporozoite controls. However, a reduction in Kupffer cell number and activity, induced by silica treatment, resulted in a very significant decline in uptake of infective sporozoites by the perfused liver--and a parallel fall in the successful infection of the host by inoculated sporozoites in vivo. Since silica treatment produced no significant detectable pathological changes in hepatocytes, and infected blood passage results in a normal parasitaemia in silica treated animals it was concluded that the Kupffer cell was a component of the natural route of infection of the mammalian host by the majority of the infecting population of sporozoites of Plasmodium yoelii nigeriensis.     

Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells

Immunization with Plasmodium sporozoites that have been attenuated by gamma-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and humoral immune responses invoked by infected hepatocytes cells that contain long-lived, partially developed parasites. Here we analyzed sporozoites of Plasmodium berghei that are deficient in P36p (p36p(-)), a member of the P48/45 family of surface proteins. P36p plays no role in the ability of sporozoites to infect and traverse hepatocytes, but p36p(-) sporozoites abort during development within the hepatocyte. Immunization with p36p(-) sporozoites results in a protective immunity against subsequent challenge with infectious wild-type sporozoites, another example of a specifically genetically attenuated sporozoite (GAS) conferring protective immunity. Comparison of biological characteristics of p36p(-) sporozoites with radiation-attenuated sporozoites demonstrates that liver cells infected with p36p(-) sporozoites disappear rapidly as a result of apoptosis of host cells that may potentiate the immune response. Such knowledge of the biological characteristics of GAS and their evoked immune responses are essential for further investigation of the utility of an optimized GAS-based malaria vaccine.     

The intradermal route for inoculation of sporozoites of rodent malaria parasites for immunological studies

Rodent malaria parasites are commonly used for investigations into the immunology of pre-erythrocytic stage malaria infection, as sporozoites can be easily produced in the laboratory. In the majority of past immunological studies using this system, sporozoites are inoculated into mice via the intravenous (IV) route. In natural situations, however, sporozoites are deposited into the skin by the bite of Anopheline mosquitoes, and it is likely that the immunological response to such natural intradermal (ID) inoculation will be different to that achieved through the IV route. Although infected mosquito bites are sometimes used during experimental induction of immunity in mice, this method is problematic because of the low numbers of sporozoites introduced to the skin and the large variation in sporozoite inoculation between individual mosquitoes. Here, we show that ID inoculation of dissected mosquito salivary gland sporozoites of Plasmodium yoelii allows the accurate introduction of known numbers of sporozoites into the skin and that these parasites successfully invade the liver. Furthermore, immunization of mice using ID inoculations of live sporozoites contemporaneously with mefloquine treatment induces an immune response that is protective against the development of liver stage parasites, and this protection does not differ significantly from that achieved with IV immunizations performed in the same manner.