Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected anopheline mosquitoes

Malaria transmission begins with the injection of Plasmodium sporozoites into the skin of a vertebrate host by infected anopheline mosquitoes. Although the size of the sporozoite inoculum likely affects the course of the disease, the number of sporozoites injected by infected mosquitoes has not been determined in vivo. Using a quantitative PCR assay, we determined the number of sporozoites injected into mice by single mosquitoes. Analysis of 59 mosquito feedings showed that a single infected mosquito injected between 0 and 1,297 sporozoites, with a mean of 123 and a median of 18. Twenty-two percent of infected mosquitoes injected no sporozoites. The number of sporozoites injected was only weakly correlated to the salivary gland load. To better understand the large variability in sporozoite injection among mosquitoes, we quantified the sporozoites injected by individual mosquitoes on three different days. Approximately 20% of moderately to heavily infected mosquitoes injected few to no sporozoites on all 3 days, suggesting that some mosquitoes are poor transmitters of sporozoites. Other mosquitoes injected high numbers of sporozoites on at least one of the days observed and minimal numbers on the other day(s), supporting the hypothesis that sporozoite injection is discontinuous, a pattern that may aid in the establishment of malaria infection.     

Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites

Three monoclonal antibodies generated by immunization of mice withPlasmodium berghei-infected red blood cells were found to react with the 75-kDa heat-shock protein (HSP70) present in liver stages and crythrocytic forms of the parasites. These antibodies were shown to react with a recombinant protein encoding the carboxyl terminal half of PfHSP70 (aa 365–681). Differently from earlier results, we clearly demonstrated that HSP70 was also expressed in the sporozoite stage, using these monoclonal antibodies in an immunofluorescence and Western immunoblot assay. These monoclonal antibodies react not only with sporozoites ofP. berghei, the parasites originally used for the immunization, but also with sporozoites of several other rodent and human plasmodial species. Passive transfer of these monoclonal antibodies into naive mice, simultaneously injected with sporozoites, failed to neutralize the infectivity ofP. berghei sporozoites and to inhibit the development of liver stages ofP. yoelii.     

Neither Mosquito Saliva nor Immunity to Saliva Has a Detectable Effect on the Infectivity of Plasmodium Sporozoites Injected into Mice

Malaria infection is initiated when a female Anopheles mosquito probing for blood injects saliva, together with sporozoites, into the skin of its mammalian host. Prior studies had suggested that saliva may enhance sporozoite infectivity. Using rodent malaria models (Plasmodium berghei and P. yoelii), we were unable to show that saliva had any detectable effect on sporozoite infectivity. This is encouraging for plans to immunize humans with washed, attenuated P. falciparum sporozoites because many individuals develop cutaneous, hypersensitivity reactions to mosquito saliva after repeated exposure. If washed sporozoites have no appreciable loss of infectivity, they likely do not have decreased immunogenicity; thus, vaccinees are unlikely to develop cutaneous reactions against mosquito saliva during attempted immunization with such sporozoites. Earlier studies also suggested that repeated prior exposure to mosquito saliva reduces infectivity of sporozoites injected by mosquitoes into sensitized hosts. However, our own studies show that prior exposure of mice to saliva had no detectable effect on numbers of sporozoites delivered by infected mosquitoes, the rate of disappearance of these sporozoites from the skin or infectivity of the sporozoites. Under natural conditions, sporozoites are delivered both to individuals who may exhibit cutaneous hypersensitivity to mosquito bite and to others who may have not yet developed such reactivity. It was tempting to hypothesize that differences in responsiveness to mosquito bite by different individuals might modulate the infectivity of sporozoites delivered into a milieu of changes induced by cutaneous hypersensitivity. Our results with rodent malaria models, however, were unable to support such a hypothesis.The malaria infection is initiated when a female Anopheles mosquito probing for a blood meal injects saliva, together with sporozoites into the skin of its mammalian host (18, 39). Mosquito saliva is known to enhance the ability of the mosquito to locate a blood source and to inhibit hemostasis by any of several mechanisms. These include injection of an anticoagulant factor (34), inhibition of platelet aggregation by salivary apyrase (29) or a salivary factor that inhibits collagen-induced platelet aggregation (43), inhibition of thrombin activity (14), and vasodilation of host blood vessels (30). Arthropod saliva has been shown to enhance the infectivity of several different pathogens introduced into hosts by arthropods; these include sandfly transmission of Leishmania, tick transmission of viruses and spirochetes, and mosquito transmission of viruses (for a review, see reference 36). Enhancement of Plasmodium sporozoite infectivity by mosquito saliva has also been reported (12, 36) based on a prior study (41), but we felt that this study needed to be reassessed.In addition to these studies on the direct effect of arthropod saliva on infectivity of pathogens injected by arthropods into immunologically naive hosts, studies have also been done on the role of prior exposure of hosts to arthropod saliva in modulating pathogen transmission to immunized hosts. Ever since Trager''s classic study (37) showing that immunity to tick bite can lead to host protection against subsequent feedings by ticks, many workers have studied the role of host immunity to arthropod saliva in interfering with feeding by the arthropod and modulating transmission of pathogens to the host (for reviews, see references 6 and 36). Most of these studies have focused on delayed immune responses that in some cases may enhance and in other cases may control infections with arthropod-transmitted pathogens. This is an appropriate approach in circumstances when a host cellular response may interfere with feeding by the arthropod or may recruit host cells that modulate development of the pathogen at the bite site.Mosquitoes, however, feed relatively rapidly. Thus, only an immediate hypersensitivity response is likely to be able to modulate movement of sporozoites from avascular tissue at the bite site to blood vessels, from which the sporozoite can then reach the liver for further development. Many hosts bitten by mosquitoes over a period of time develop an immediate, cutaneous, hypersensitivity response; it is relevant that this develops at the same site and within the same timeframe during which sporozoites are moving into local blood vessels. We have previously studied the kinetics of P. berghei sporozoite movement out of the skin after deposition by mosquitoes into immunologically naive mice (19). We thus set out to compare this to the kinetics of sporozoites introduced by mosquitoes into mice that we attempted to hyperimmunize against mosquito saliva by repeated mosquito bites. Our results have shown that neither the presence of mosquito saliva nor immediate hypersensitivity to saliva had any detectable effects on deposition of sporozoites by mosquitoes or the movement of these sporozoites from the bite site into the blood to induce infection.     

Plasmodium falciparum ookinetes migrate intercellularly throughAnopheles stephensi midgut epithelium

The migration ofPlasmodium falciparum andP. berghei ookinetes through the midgut epithelium inAnopheles stephensi was studied by transmission electron microscopy. With ruthenium red (RR) staining, the results of previous studies were confirmed:P. falciparum ookinetes take an intercellular route through the midgut epithelium. In the same mosquito species, the rodent parasiteP. berghei appeared to take an intracellular position, as previously suggested by other authors. The intra- or intercellular ookinete migration ofP. berghei orP. falciparum, respectively, can perhaps be related to the higher mortality ofP. berghei-infected mosquitoes within the first 2 days of infection. Evidence is presented that oocyst capsule formation begins as early as during the migration of the ookinete. After localization between the epithelial cells and the midgut basal lamina, the rapidly expanding oocyst stretches the overlying layer of the latter at the haemocoelic surface while a new basal lamina is generated between the oocyst and epithelial cell.Abbreviations BL basal lamina - CR cristalloid - N nucleus - RER rough endoplasmic reticulum - Mp malarial pigment - M mitochondrion - MV microvillous border - OC oocyst capsule     

A convenient technique for the dissection of mosquito salivary glands

Summary A method is described wherebyP. berghei sprorozoites may be obtained from the salivary glands of infected femaleA. stephensi mosquitoes. The method is relatively easy to master, requires minimal handling of biological material, and is done with ordinary laboratory equipment. In addition, the sporozoites are rendered relatively free of contaminating micro-organisms and the entire operation may be performed aseptically.     

Developmental changes in the circumsporozoite proteins ofPlasmodium berghei andP. gallinaceum in their mosquito vectors

The circumsporozoite (CS) protein covers the surface of the sporozoite of plasmodia. Its role in the development of the malaria parasite in mosquito vectors remains unknown. CS-epitope-containing proteins appear on undifferentiated oocysts on day 7 inPlasmodium berghei and on day 5 inP. gallinaceum as demonstrated by indirect fluorescence antibody tests using monoclonal antibodies directed against the CS-protein repeats. The three-dimensional distribution of the CS-epitope-containing proteins on oocysts was analyzed by confocal scanning laser microscopy. A strong antibody binding was found in patches around the oocysts ofP. berghei andP. gallinaceum, and an accumulation of labeled proteins was found at the base of the oocysts of both species. In Western blots of infected midguts and salivary glands the antibodies recognized two peptides in the salivary glands but up to ten peptides in midgut extracts. The larger number of peptides recognized in midgut preparations might indicate breakdown products during the escape of the sporozoites from the oocyst and their migration on the midgut in the mosquito vector. The data indicate a possible involvement of the CS protein in an active migration process of the sporozoites in the mosquito vector.     

Plasmodium berghei Sporozoites Acquire Virulence and Immunogenicity during Mosquito Hemocoel Transit

Malaria is a vector-borne disease caused by the single-cell eukaryote Plasmodium. The infectious parasite forms are sporozoites, which originate from midgut-associated oocysts, where they eventually egress and reach the mosquito hemocoel. Sporozoites actively colonize the salivary glands in order to be transmitted to the mammalian host. Whether residence in the salivary glands provides distinct and vital cues for the development of infectivity remains unsolved. In this study, we systematically compared the infectivity of Plasmodium berghei sporozoites isolated from the mosquito hemocoel and salivary glands. Hemocoel sporozoites display a lower proportion of gliding motility but develop into liver stages when added to cultured hepatoma cells or after intravenous injection into mice. Mice infected by hemocoel sporozoites had blood infections similar to those induced by sporozoites liberated from salivary glands. These infected mice display indistinguishable systemic inflammatory cytokine responses and develop experimental cerebral malaria. When used as metabolically active, live attenuated vaccine, hemocoel sporozoites elicit substantial protection against sporozoite challenge infections. Collectively, these findings show that salivary gland colonization does not influence parasite virulence in the mammalian host when sporozoites are administered intravenously. This conclusion has important implications for in vitro sporozoite production and manufacturing of whole-sporozoite vaccines.     

expression of members of the heat-shock protein 70 family in the exoerythrocytic stages ofPlasmodium berghei andPlasmodium falciparum

Exoerythrocytic stages ofPlasmodium berghei cultured in HepG2-A16 hepatoma cells and those ofP. falciparum in human hepatocytes transplanted under the kidney capsule of CB-17/ICr scid/scid mice were used to evaluate expression of heat-shock-related stress proteins. Although undetectable in the sporozoites, the expression of proteins similar in sequence of a heat-shock protein of 70 kDa and a glucose-regulated protein of 78 kDa was markedly induced in the hepatic stages of malaria parasites. Expression of these proteins in the exoerythrocytic stages of the malaria parasite warrants a systematic evaluation of their potential role in eliciting cellular immune responses directed against infected hepatocytes.     

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.     

T cell responses in a sporozoite-immunized human volunteer and a chimpanzee

We have recently investigated the specificity of T cells induced in a human volunteer and a chimpanzee immunized by multiple exposures to the bites of large numbers of malaria-infected mosquitoes. T cell lines and clones have been obtained from a human volunteer immunized with Plasmodium falciparum sporozoites. These CD4+ T cell clones specifically recognize the native circumsporozoite (CS) protein expressed on sporozoites, as well as bacteria- and yeast-derived recombinant falciparum CS proteins. The epitope recognized by the sporozoite specific human T cells mapped to the 5' repeat region of the CS protein and was contained in the NANPNVDPNANP sequence. A T cell line has also been isolated from PBL obtained from a chimpanzee immunized by multiple exposures to the bits of P. vivax infected mosquitoes. The CS-specific chimpanzee T cells were used to identify a T cell epitope within a repeat region of the P. vivax CS protein.     

Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations.     

The effect of nosematosis on the development ofPlasmodium falciparum inAnopheles stephensi

To quantify the effect ofNosema algerae (Microsporida, Nosematidae) on the development ofPlasmodium falciparum inAnopheles stephensi (Diptera, Culicidae), we carried out infection experiments under standardized laboratory conditions. Apart from a mean reduction of 69% in oocyst development, smaller numbers of oocysts and fewer sporozoites were found in theNosema-infected mosquitoes. In addition, nosematosis resulted in higher mortality. The potential role ofNosema algerae as a biological control agent is discussed.Supported by a scholarship grant (to G.M.) from the Graduiertenförderung des Landes Nordrhein-westfalen     

Direct microscopic quantification of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice

The number of malaria sporozoites delivered to a host by mosquitoes is thought to have a significant influence on the subsequent course of the infection in the mammalian host. We did studies with Anopheles stephensi mosquitoes with salivary gland infections of Plasmodium berghei sporozoites expressing a red fluorescent protein. After individual mosquitoes fed on an ear pinna or the ventral abdomen of a mouse, fluorescence microscopy was used to count numbers of sporozoites. Mosquitoes allowed to feed on the ear for periods of 3 versus 15 min deposited means of 281 versus 452 sporozoites, respectively, into the skin; this may have epidemiological implications because mosquitoes can feed for longer periods of time on sleeping hosts. Mosquitoes feeding on the ventral abdomen injected sporozoites not only into the skin but also into the underlying peritoneal musculature. Although mosquitoes injected fewer sporozoites into the abdominal tissues, more of these were reingested into the mosquito midgut, probably a consequence of easier access to blood intake from the abdominal area. The most consistent parameter of sporozoite transmission dynamics under all conditions of mosquito probing and feeding was the relatively slow release rate of sporozoites (approximately 1 to 2.5 per second) from the mosquito proboscis. The numbers of sporozoites introduced into the host by mosquitoes and the transmission efficiencies of sporozoite delivery are multifactorial phenomena that vary with length of probing time, skin site being fed upon, and numbers of sporozoites within the salivary glands.     

Growth-inhibitory effect of a fucoidan from brown seaweed <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> on <Emphasis Type="Italic">Plasmodium</Emphasis> parasites

The present study was undertaken to investigate the inhibitory effects of fucoidan, a sulfated polysaccharide isolated from the edible brown seaweed Undaria pinnatifida, on the growth of Plasmodium parasites. In order to assess the anti-malarial activity of fucoidan, growth inhibition activities were evaluated using cultured Plasmodium falciparum parasites in vitro and on Plasmodium berghei-infected mice in vivo. Fucoidan significantly inhibited the invasion of erythrocytes by P. falciparum merozoites, and its 50% inhibition concentration was similar to those for the chloroquine-sensitive P. falciparum 3D7 strain and the chloroquine-resistant K1 strain. Four-day suppressive testing in P. berghei-infected mice with fucoidan resulted in a 37% suppressive effect versus the control group and a delay in death associated with anemia (P < 0.05). In addition, fucoidans had no toxic effect on RAW 264.7 cells. These findings indicate that fucoidans from the Korean brown algae U. pinnatifida inhibits the invasion of Plasmodium merozoites into erythrocytes in vitro and in vivo. J.-H. Chen and J.-D. Lim contributed equally to this work.     

Identification of Plasmodium falciparum-infected mosquitoes using a probe containing repetitive DNA

A cloned repetitive DNA sequence (rep20) was evaluated as a diagnostic probe specific for Plasmodium falciparum sporozoites using experimentally infected mosquitoes squashed directly on nylon filters. Head/thorax portions of mosquitoes, killed 14-16 days after ingesting P. falciparum-infected blood, gave positive signals when examined for the presence of P. falciparum sporozoite DNA by hybridisation. This correlated with the number of oocysts found in a sample of the same batch of mosquitoes examined by dissection. No positive signals were obtained with 50 Plasmodium berghei-infected mosquitoes probed with the rep20 sequence. The results indicate that a probe containing rep20 may be useful in the rapid and specific incrimination of vectors carrying P. falciparum sporozoites. The value of repetitive DNA in the diagnosis of malaria is discussed.     

Disruption of the Plasmodium berghei 2-Cys peroxiredoxin TPx-1 gene hinders the sporozoite development in the vector mosquito

To investigate the physiologic role of cytosolic 2-Cys peroxiredoxin of Plasmodium berghei (PbTPx-1), we infected the vector mosquito Anopheles stephensi with a parasite carrying a targeted knockout of pbtpx-1 (Prx-KO). The number of Prx-KO midgut oocysts at 14–15 days post-feeding (pf) was comparable to that of the parent strain (WT); however, the numbers of sporozoites that formed in midgut oocysts and accumulated in the salivary gland of Prx-KO-infected mosquitoes by 21 days pf were decreased to 10–20% and 3–10%, respectively, of those values in WT-infected mosquitoes. A higher frequency of DNA strand breaks was detected in Prx-KO oocysts than in WT oocysts. Sporozoites carrying the targeted disruption had reduced infectivity in mice; however, the knockout did not affect the ability of the sporozoite to reach the liver parenchyma and initiate exo-erythrocytic form (EEF) development. TPx-1 may be involved in development during exponentially multiplying stages, such as sporozoites and EEF.     

Effect of human circumsporozoite antibodies in Plasmodium-infected Anopheles (Diptera: Culicidae)

Human circumsporozoite (CS) antibodies to Plasmodium falciparum were detected in blood meals from 45.0% of 1,547 field-collected Anopheles gambiae Giles sensu lato and Anopheles funestus Giles from western Kenya. Possible effects on malaria infections within the Anopheles host were investigated. Circumsporozoite antibodies were detected in blood meals up to 36 h after feeding. Antibodies crossing the midgut were detected experimentally in hemolymph from 4 to 36 h after feeding; human IgG also was present in hemolymph from fully gravid field-collected Anopheles. Ingestion of high-titer human CS antibodies or 2A10 monoclonal antibody to P. falciparum sporozoites by P. falciparum-infected An. gambiae, 10 d after feeding on an infected human, had no effect on o?cyst maturation, sporozoite rates, or sporozoite loads. Contact between CS antibodies and sporozoites in the hemocoel did not block sporozoite invasion of salivary glands. Human IgG antibodies were detected by an indirect fluorescent antibody technique on salivary gland sporozoites from 83.3% of 114 field-collected Anopheles. In 65.4% of 26 infections, antibodies persisted on sporozoites for at least three days. Thus, a high proportion of naturally infected An. gambiae s.l. and An. funestus in western Kenya transmit sporozoites that are bound with human IgG acquired during previous blood meals. The infectivity of such sporozoites needs to be determined in relation to natural transmission and to the potential use of malaria sporozoite vaccines.     

Identification of an eosinophil chemotactic factor from anopheline mosquitoes as a chitinase family protein

Anopheline mosquitoes play an essential role in malaria transmission. The mosquito salivates copiously when probing for the location of a blood vessel. We found that the saliva of anopheline mosquitoes has chemotactic activity for naive eosinophils or neutrophils. The major eosinophil chemotactic component in saliva was shown to be one of the chitinase family proteins. A similar chitinase family protein was found also in the midgut of the anopheline mosquito. Production of antibodies to the chitinase family protein was generally observed in the sera of residents of a malaria endemic area. Both Plasmodium falciparum-infected and uninfected individuals had antibodies to chitinases. These results suggest that the chitinase family protein in mosquito saliva contributes to eliciting an inflammatory response of eosinophils in the host skin followed by antibody production in the host.     

Electron microscopic studies on the interaction of rat Kupffer cells andPlasmodium berghei sporozoites

The interactions betweenPlasmodium berghei sporozoites and Kupffer cells in rat liver were studied by transmission electron microscopy. Between 10 and 45 min after inoculation, sporozoites were found in the process of entering Kupffer cells and inside phagolysosomes. The sporozoites entered the Kupffer cells by phagocytosis as determined by the presence of pseudopods and local accumulations of aggregated microfilaments and the resulting exclusion of other organelles in the phagocyte cytoplasm beneath the attached parasite. Sporozoites were taken up either with their anterior end first, or backwards. Scanning electron microscopy of in vitro sporozoite Kupffer cell interaction confirmed these observations. It was concluded that sporozoites are taken up in a normal phagocytic way by the Kupffer cells, regardless of their initial place of contact or position. Thirty min after inoculation sporozoites found in phagolysosomes were still morphologically intact but after 45 min we could encounter completely digested sporozoites.     

TAP‐mediated processing of exoerythrocytic antigens is essential for protection induced with radiation‐attenuated Plasmodium sporozoites

MHC class I dependent CD8⁺ T cells are essential for protection induced by radiation‐attenuated Plasmodium sporozoites (RAS) in murine malaria models. Apart from the mechanism of activation of CD8⁺ T cells specific for the circumsporozoite protein, the major sporozoite antigen (Ag), CD8⁺ T cells specific for other exoerythrocytic Ags that have been shown to mediate protection have not been thoroughly investigated. Specifically, mechanisms of processing and presentation of exoerythrocytic Ags, which includes liver stage (LS) Ags, remain poorly understood. We hypothesize that as exogenous proteins, LS Ags are processed by mechanisms involving either the TAP‐dependent phagosomal‐to‐cytosol or TAP‐independent vacuolar pathway of cross‐presentation. We used TAP‐deficient mice to investigate whether LS Ag mediated induction of naïve CD8⁺ T cells and their recall during sporozoite challenge occur by the TAP‐dependent or TAP‐independent pathways. On the basis of functional attributes, CD8⁺ T cells were activated via the TAP‐independent pathway during immunizations with Plasmodium berghei RAS; however, IFN‐γ⁺CD8⁺ T cells previously induced by P. berghei RAS in TAP‐deficient mice failed to be recalled against sporozoite challenge and the mice became parasitemic. On the basis of these observations, we propose that TAP‐associated Ag processing is indispensable for sterile protection induced with P. berghei RAS.