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.     

Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites.

Immunization of rodents and humans with irradiation-attenuated malaria sporozoites confers preerythrocytic stage-specific protective immunity to challenge infection. This immunity is directed against intrahepatic parasites and involves T cells and interferon gamma, which prevent development of exoerythrocytic stages and subsequent blood infection. The present study was undertaken to determine how protective immunity is achieved after immunization of rodent hosts with irradiated Plasmodium berghei sporozoites. We present evidence that irradiated parasites persist in hepatocytes of rats and mice for up to 6 months after immunization. A relationship between the persistence of parasites and the maintenance of protective immunity was observed. Protective immunity was abrogated in irradiated-sporozoite-immunized rats following the application of chemotherapy to remove preexisting liver parasites. Additionally, protective immunity against sporozoite challenge was established in rats vaccinated with early and late hepatic stages of irradiated parasites. These results show that irradiation-attenuated sporozoites produce persistent intrahepatic stages in vivo necessary for the induction and maintenance of protective immunity.     

Characterization of Plasmodium falciparum sporozoite surface protein 2.

Immunization of mice with Plasmodium yoelii sporozoite surface protein 2 (PySSP2) and circumsporozoite protein protects completely against P. yoelii. The amino acid sequence of PySSP2 suggested that the thrombospondin-related anonymous protein (TRAP) [Robson, K. J. H., Hall, J. R. S., Jennings, M. W., Harris, T. J. R., Marsh, K., Newbold, C. I., Tate, V. E. & Weatherall, D. J. (1988) Nature (London) 335, 79-82] is the Plasmodium falciparum homolog of PySSP2. We report data confirming that TRAP is P. falciparum SSP2 (PfSSP2). Murine antibodies against recombinant PfSSP2 identify a 90-kDa protein in extracts of P. falciparum sporozoites, recognize sporozoites and infected hepatocytes by immunofluorescence, localize PfSSP2 to the sporozoite micronemes by immunoelectron microscopy and to the surface membrane by live immunofluorescence, and inhibit sporozoite invasion and development in hepatocytes in vitro. Human volunteers immunized with irradiated sporozoites and protected against malaria develop antibody and proliferative T-cell responses to PfSSP2, suggesting that, like PySSP2, PfSSP2 is a target of protective immunity, and supporting inclusion of PfSSP2 in a multicomponent malaria vaccine.     

Apoptotic Plasmodium-infected hepatocytes provide antigens to liver dendritic cells

Malaria starts with infection of the host liver by Plasmodium sporozoites. Inoculation with radiation-attenuated Plasmodium sporozoites induces complete protection against malaria. Protection is mediated by dendritic cells (DCs) and CD8(+) T cells, but the source of parasite antigens mediating this response remains unclear. Here, we show that hepatocytes infected with irradiated Plasmodium sporozoites undergo apoptosis shortly after infection. Infection with irradiated sporozoites induces the recruitment of DCs to the liver, where they phagocytose apoptotic infected hepatocytes containing parasite antigens. We propose that apoptotic Plasmodium-infected hepatocytes provide a source of parasite antigens for the initiation of the protective immune response.     

Inhibitory activity against Plasmodium vivax sporozoites induced by plasma from Saimiri monkeys immunized with circumsporozoite recombinant proteins or irradiated sporozoites

Two Saimiri monkey vaccine trials have been conducted comparing four recombinant Plasmodium vivax circumsporozoite proteins and irradiated sporozoites. Only a small number of animals immunized with certain recombinants or irradiated sporozoites became fully protected against sporozoite challenge. Preimmunization and postimmunization plasma samples obtained from 30 monkeys on the day of challenge were tested in an in vitro assay based on sporozoite development into exoerythrocytic stages in primary cultures of Saimiri monkey hepatocytes. The percentage of inhibition was determined by comparison of the number of exoerythrocytic stages developing from sporozoites preincubated with a preimmunization and a postimmunization plasma sample of each animal. The plasma samples of the day of challenge of nearly all the immunized animals had a variable, but significant inhibitory effect, when compared with the corresponding preimmunization sample. We found no correlation between the degree of in vitro inhibition of liver stage development, and the in vivo protection against sporozoite challenge of individual animals. The variable results of the incubation of sporozoites with "normal" plasma of different animals indicates that the in vitro results were affected by plasma factors unrelated to anti-sporozoite antibodies.     

Down‐selecting circumsporozoite protein‐based malaria vaccine: A comparison of malaria sporozoite challenge model

Plasmodium falciparum circumsporozoite protein (PfCSP) is the main target antigen in development of pre‐erythrocytic malaria vaccines. To evaluate PfCSP vaccines in animal models, challenge by intravenous sporozoite injection is preferentially used. However, in clinical trials, vaccinated human volunteers are exposed to the bites of malaria‐infected mosquitoes. In this study, we down‐selected Escherichia coli‐produced full‐length PfCSP (PfCSP‐F) and its three truncated PfCSPs based on their abilities to elicit immune response and protection in mice against two challenge models. We showed that immunization with three doses of PfCSP‐F elicited high anti‐PfCSP antibody titres and 100% protection against the bites of infected mosquitoes. Meanwhile, three‐dose truncated PfCSP induced 60%‐70% protection after immunization with each truncated PfCSP. Heterologous prime‐boost immunization regimen with adenovirus‐PfCSP‐F and R32LR greatly induced complete protection against intravenous sporozoite injection. Our results suggest that Abs to both anti‐repeat and anti‐nonrepeat regions induced by PfCSP‐F are required to confer complete protection against challenge by the bites of infected mosquitoes, whereas anti‐repeat Abs play an important role in protection against intravenous sporozoite injection. Our findings provide a potential clinical application that PfCSP‐F vaccine induces potent Abs capable of neutralizing sporozoites in the dermis inoculated by infected mosquitoes and subsequently sporozoites in the blood circulation.     

In vivo testing of subunit vaccines against malaria sporozoites using a rodent system.

To test the putative in vivo protective effects of antibodies to circumsporozoite (CS) protein repeats against malarial infection, different strains of mice were immunized against various repetitive regions of the Plasmodium yoelii CS protein in the form of synthetic peptides conjugated to keyhole limpet hemocyanin. Complete Freund's adjuvant or saponin was used as adjuvant. When vaccinated mice were challenged with 500 sporozoites almost all animals became infected. There were no significant protective effects in vaccinated versus unvaccinated mice. Furthermore, there was no correlation between the antibody titer to the CS repeats and infection. The parasites from infected animals were shown to encode a CS protein containing the same repeats as those used for immunization, indicating that the infections were not due to selection for variant parasites. These experiments demonstrate that antibodies to the CS repeats, as derived in vivo with peptides, despite being surface reactive, do not provide protection against sporozoite challenge in vivo. This conclusion is in contrast to previous conclusions based on studies showing protection by way of in vitro sporozoite neutralization procedures and passive transfer of monoclonal antibody.     

Identification of Plasmodium falciparum-infected mosquitoes by a double antibody enzyme-linked immunosorbent assay

A double antibody micro enzyme-linked immunosorbent assay (ELISA) for identifying Plasmodium falciparum sporozoites in mosquitoes is described. Using monoclonal antibodies made against South American P. falciparum sporozoites, the ELISA was able to detect and identify sporozoite antigens of South American and Asian origins in extracts of dried infected mosquitoes.     

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.     

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.     

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)     

Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells

Irradiation-attenuated sporozoite vaccinations confer sterile protection against malaria infection in animal models and humans. Persistent, nonreplicating parasite forms in the liver are presumably necessary for the maintenance of sterile immunity. A novel vaccine approach uses genetically attenuated parasites (GAPs) that undergo arrested development during liver infection. The fate of GAPs after immunization, their persistence in vaccinated animals, and the immune mechanisms that mediate protection are unknown. To examine the developmental defects of genetically attenuated liver stages in vivo, we created deletions of the UIS3 and UIS4 loci in the Plasmodium yoelii rodent malaria model (Pyuis3[-] and Pyuis4[-]). The low 50% infectious dose of P. yoelii in BALB/c mice provides the most sensitive infectivity model. We show that P. yoelii GAPs reach the liver, invade hepatocytes, and develop a parasitophorous vacuole but do not significantly persist 40 h after infection. A single dose of Pyuis4(-) sporozoites conferred complete protection, but full protection by Pyuis3(-) sporozoites required at least 2 immunizations. CD8(+) T cells were essential for protection, but CD4(+) T cells were not. Our results show that genetically distinct GAPs confer different degrees of protective efficacy and that live vaccine persistence in the liver is not necessary to sustain long-lasting protection. These findings have important implications for the development of a P. falciparum GAP 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.     

Differences in susceptibility among mouse strains to infection with Plasmodium berghei (ANKA clone) sporozoites and its relationship to protection by gamma-irradiated sporozoites

Three inbred mouse strains, C57BL/6 (H-2b), A/J (H-2a), and BALB/c (H-2d), and 1 outbred strain, CD-1, demonstrated differences in susceptibility to iv challenge with the ANKA clone of Plasmodium berghei. Mice were challenged with 100, 1,000, or 10,000 sporozoites, then evaluated daily beginning on day 4 for patency. CD-1 mice were further evaluated at challenge doses of 12,500, 25,000, and 50,000 sporozoites. C57BL/6 mice were the easiest to infect, with 90% becoming infected with 100 sporozoites. The outbred strain CD-1 was the most difficult to infect, requiring a challenge dose of 25,000 sporozoites/mouse in order to achieve a 100% infection rate. Mouse strains also demonstrated differences in their ability to be protected by intravenous immunization with gamma-irradiated sporozoites. A/J mice needed a minimum of 3 doses of irradiated sporozoites for protection against a challenge with 10,000 sporozoites. In contrast, BALB/c mice immunized with a single dose of 1,000 irradiated sporozoites are protected against a 10,000 sporozoite challenge. These data suggest that both infectivity and protection are genetically restricted and that susceptibility to infection may be inversely related to protection.     

Reinforcement of immunity in Saimiri monkeys following immunization with irradiated sporozoites of Plasmodium vivax.

To determine the duration of immunity to Plasmodium vivax following immunization, six Saimiri sciureus boliviensis monkeys were vaccinated with irradiated sporozoites of P. vivax and challenged multiple times with sporozoites. Over a period of almost four years, complete protection from repeated challenge with infective sporozoites was demonstrated in one monkey; protection in two monkeys was obtained on eight of nine occasions, in one monkey on seven of nine occasions, in one monkey on six or nine occasions, and in one monkey on four of eight occasions. Five of six monkeys were protected against infection during the last six challenges. Inoculation with blood-stage parasites at the end of the trial indicated that all animals were susceptible to infection. These results suggest that protection against sporozoite challenge may be strongly reinforced by subsequent exposure to viable sporozoites.     

Cross-protection between attenuated Plasmodium berghei and P. yoelii sporozoites

An attenuated Plasmodium falciparum sporozoite (PfSPZ) vaccine is under development, in part, based on studies in mice with P. berghei. We used P. berghei and P. yoelii to study vaccine-induced protection against challenge with a species of parasite different from the immunizing parasite in BALB/c mice. One-hundred percent of mice were protected against homologous challenge. Seventy-nine percent immunized with attenuated P. berghei sporozoite (PbSPZ) (six experiments) were protected against challenge with P. yoelii sporozoite (PySPZ), and 63% immunized with attenuated PySPZ (three experiments) were protected against challenge with PbSPZ. Antibodies in sera of immunized mice only recognized homologous sporozoites and could not have mediated protection against heterologous challenge. Immunization with attenuated PySPZ or PbSPZ induced CD8+ T cell-dependent protection against heterologous challenge. Immunization with attenuated PySPZ induced CD8+ T cell-dependent protection against homologous challenge. However, homologous protection induced by attenuated PbSPZ was not dependent on CD8+ or CD4+ T cells, and depletion of both populations only reduced protection by 36%. Immunization of C57BL/10 mice with PbSPZ induced CD8+ T cell-dependent protection against P. berghei, but no protection against P. yoelii. The cross-protection data in BALB/c mice support testing a human vaccine based on attenuated PfSPZ for its efficacy against P. vivax.     

Malaria vaccine: a current perspective

The observation that inactivated Plasmodium sporozoites could protect against malaria is about a hundred years old. However, systematic demonstration of protection using irradiated sporozoites occurred in the nineteen-sixties, providing the impetus for the development of a malaria vaccine. In 1983, the circumsporozoite protein (CSP), a major sporozoite surface antigen, became the first Plasmodium gene to be cloned, and a CSP-based vaccine appeared imminent. Today, 25 years later, we are still without an effective malaria vaccine, despite considerable information regarding the genomics and proteomics of the malaria parasites. Although clinical immunity to malaria has been well-documented in adults living in malaria endemic areas, our understanding of the host-immune responses operating in such malaria immune persons remains poor, and limits the development of immune control of the disease. Currently, several antigen and adjuvant combinations have entered clinical trials, in which efficacy against experimental sporozoite challenge and/or exposure to natural infection is evaluated. This review collates information on the recent status of the field. Unresolved challenges facing the development of a malaria vaccine are also discussed.     

Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion

Malaria sporozoites have to cross the layer of sinusoidal liver cells to reach their initial site of multiplication in the mammalian host, the hepatocytes. To determine the sinusoidal cell type sporozoites use for extravasation, endothelia or Kupffer cells, we quantified sporozoite adhesion to and invasion of sinusoidal cells isolated from rat liver. In vitro invasion assays reveal that Plasmodium berghei and P. yoelii sporozoites attach to and enter Kupffer cells, but not sinusoidal endothelia. Unlike hepatocytes and other nonphagocytic cells, which are invaded in vitro only within the first hour of parasite exposure, the number of intracellular sporozoites in Kupffer cells increases for up to 12 hours. By confocal and electron microscopy, sporozoites are enclosed in a vacuole that does not colocalize with lysosomal markers. Inhibition of phagocytosis with gadolinium chloride has no effect on Kupffer cell invasion, but abolishes phagocytosis of inactivated sporozoites. Furthermore, sporozoites traverse in vitro from Kupffer cells to hepatocytes where they eventually develop into exoerythrocytic schizonts. Thus, malaria sporozoites selectively recognize and actively invade Kupffer cells, avoid phagosomal acidification, and safely passage through these phagocytes.     

Strain specificity in the liver-stage development of Plasmodium falciparum in primary cultures of new world monkey hepatocytes

Primary cultures of Aotus and Saimiri monkey hepatocytes were infected with sporozoites of the Plasmodium falciparum NF 54 strain from mosquitoes fed on gametocyte cultures, and with sporozoites of the P. falciparum Santa Lucia strain from mosquitoes fed on an infected Aotus monkey. After 4-8 days, one exoerythrocytic (EE) parasite per 30,000 sporozoites was detected in one of three experiments performed with the P. falciparum NF54 strain. However, numerous EE parasites were detected in Aotus and Saimiri cells infected with sporozoites of the P. falciparum Santa Lucia strain. At day 6, most of the parasites contained several hundred nuclei, and were morphologically similar to those previously described in vivo using light or electron microscopy. A monoclonal antibody directed against the repeat region of the circumsporozoite protein of P. falciparum labeled the plasma and parasitophorous vacuole membrane of five-day-old EE parasites by immunoelectron microscopy, thus supporting previous observations by immunofluorescence indicating that the CS protein persists throughout the EE development of P. falciparum. These results demonstrate that liver stages of P. falciparum can be obtained in Aotus and Saimiri monkey cells, they also suggest a parasite strain specificity for hepatocytes.     

CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites.

In recent malaria sporozoite vaccine trials in humans and mice, antibodies to the sporozoite coat protein have given only modest protection against sporozoite challenge. In contrast, irradiated sporozoites can protect mice against massive sporozoite infections. Evidence suggests that immunity in these mice is mediated by T cells. To identify the mechanism of immunity, we used monoclonal antibodies specific for either the CD4 or CD8 molecule to selectively deplete sporozoite-immunized mice of T-cell subsets. Though in vivo depletion of CD4+ T cells did not reduce immunity, depletion of CD8+ T cells abolished protection. Monoclonal antibody treatment did not affect anti-sporozoite antibody levels. Our data indicate that cytotoxic T cells are critical for immunity to large numbers of sporozoites and suggest that vaccine development should be reoriented toward stimulating cellular as well as humoral immunity.