Specific inflammatory cell infiltration of hepatic schizonts in BALB/c mice immunized with attenuated Plasmodium yoelii sporozoites.

We compared immunization of BALB/c mice with radiation-attenuated versus killed sporozoites of the rodent malaria parasite, Plasmodium yoelii. We employed a suboptimal schedule of only two immunizations, in expectation that some parasites might break through the resultant low level immunity and that it might thus be possible to study the response of the host against these 'breakthrough' schizonts. As a measure of protective immunity, we used histological means to determine the percentages of challenge sporozoites prevented from completing development into hepatic schizonts within the liver. Immunization with attenuated sporozoites led to almost complete protection, whereas immunization with similar dosages of killed sporozoites led to approximately a 75% protection. Fluorescent antibody titers against sporozoites were similar in both sets of immunized animals. However, serum from mice immunized with attenuated sporozoites had a protective effect upon passive transfer into immunologically naive mice subsequently challenged with normal sporozoites; serum from mice immunized with killed sporozoites had no such effect. When mice suboptimally immunized with attenuated sporozoites were challenged, we observed breakthrough schizonts being infiltrated with inflammatory cells, primarily mononuclear cells, and neutrophils; partial depletion of CD4+ or CD8+ cells within these mice prior to challenge prevented the infiltration of breakthrough schizonts. Thus, cellular infiltration of schizonts was apparently secondary to earlier action by lymphocytes. This infiltration was also not observed in mice immunized with killed sporozoites. The more effective protective immunity induced by attenuated sporozoites could be due to their ability to release antigen into the cytoplasm of hepatocytes that they invade or their ability to continue differentiating, thereby presenting new antigens that are not seen after immunization with killed sporozoites.     

Susceptibility of different strains of mice to hepatic infection with Plasmodium berghei.

Despite the low susceptibility of BALB/c mice to hepatic infection by Plasmodium berghei, this animal model is routinely used to investigate the basic biology of the malaria parasite and to test vaccines and the immune response against exoerythrocytic (EE) stages derived from sporozoites. A murine model in which a large number of EE parasites are established would be useful for furthering such investigations. Therefore, we assayed six mouse strains for susceptibility to erythrocytic and hepatic infections. The administration of 50 sporozoites by intravenous inoculation was sufficient to establish erythrocytic infections in five of five C57BL/6 mice compared with 10,000 sporozoites required to infect 100% of BALB/c mice. To assay for hepatic infections, mice received an intravenous inoculum of 10(6) sporozoites, and liver sections for light microscopy and histology were obtained at 29 and 44 h postinoculation. EE parasites were visualized by immunofluorescence, using an antibody to a P. falciparum heat shock protein. The mean number of EE parasites per 100 cm2 for C57BL/6 and A/J strains was significantly higher than that for BALB/c (2,190 +/- 260, 88 +/- 38, and 6 +/- 2, respectively). The proportion of inoculated sporozoites transforming into liver schizonts was 8.2% in C57BL/6 and < 1% in C3H/HeJ, DBA/1, and Swiss CD-1/ICR mice. Nonspecific inflammatory infiltrates around EE parasites were less prevalent in liver sections from C57BL/6 mice than in those from BALB/c mice, which contributed to the decrease in developing EE stages in BALB/c mice. These data indicate that the C57BL/6-P. berghei system is preferable for investigating the biology and immunology of liver stage parasites.     

Complete protection against Plasmodium yoelii by adoptive transfer of a CD8+ cytotoxic T-cell clone recognizing sporozoite surface protein 2.

BALB/c mice immunized with irradiated Plasmodium yoelii sporozoites produce antibodies and cytotoxic T lymphocytes against the circumsporozoite protein and against a 140-kDa protein, sporozoite surface protein 2 (PySSP2). Approximately 50% of mice immunized with P815 cells transfected with the gene encoding PySSP2 are protected against malaria, and this protection is reversed by in vivo depletion of CD8+ T cells. To determine if CD8+ T cells against PySSP2 are adequate to protect against malaria in the absence of other malaria-specific immune responses, we produced three CD8+ T-cell clones by stimulating spleen cells from mice immunized with irradiated P. yoelii sporozoites with a mitomycin-treated P815 cell clone transfected with the PySSP2 gene. Adoptive transfer of clone TSLB7 protected 100% of mice against P. yoelii. The second clone protected 58% of mice, and the third clone provided no protection. Clone TSLB7 protected even when administered 3 h after sporozoite inoculation at a time when sporozoites had entered hepatocytes, suggesting that it is recognizing and eliminating infected hepatocytes. These studies demonstrate that cytotoxic T lymphocytes against PySSP2 can protect against P. yoelii sporozoite challenge in the absence of other parasite-specific immune responses.     

Cytokines inhibit the development of liver schizonts of the malaria parasite Plasmodium berghei in vivo.

The effect of induction of an acute-phase response and its mediators on the development of liver schizonts of the rodent malaria parasite Plasmodium berghei was investigated in Brown Norway rats. Subcutaneous injection of turpentine oil 24 h or 5 min before inoculation of sporozoites resulted in 80% and 35% reduction of schizont development, respectively. Turpentine oil induced high plasma levels of interleukin-6 (IL-6). Intraperitoneal administration of IL-1, IL-6 or both, significantly reduced liver schizont development. This reduction was also present if IL-6 had been administered 24 h after sporozoite inoculation. Inhibition induced by IL-1 could be prevented by simultaneous administration of polyclonal anti-IL-6. Administration of polyclonal anti-IL-6 without IL-1 resulted in a 40% increase of liver schizonts compared to control animals. We conclude that induction of an acute-phase response during experimental Plasmodium berghei infections in Brown Norway rats, strongly inhibits liver schizont development and that IL-6 is a key mediator in this process.     

The Plasmodium falciparum knob-associated PfEMP3 antigen is also expressed at pre-erythrocytic stages and induces antibodies which inhibit sporozoite invasion

The expression of the pfemp3 gene and the corresponding PfEMP3 knob-associated protein in the pre-erythrocytic stages of Plasmodium falciparum was demonstrated by RT-PCR, Western blots, IFAT and IEM. The antigen was found on the surface of the sporozoite and in the cytoplasm of mature hepatic stage parasites. Immunological cross-reactivity was observed with sporozoites from the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei and was exploited to assess a potential role of this protein at the pre-erythrocytic stages. Specific antibodies from immune individuals were found to inhibit P. yoelii yoelii and P. berghei sporozoite invasion of primary hepatocyte cultures. PfEMP3 should now be added to the small list of proteins expressed at the pre-erythrocytic stages of P. falciparum, and its vaccine potential now deserves to be investigated.     

Immunogenicity and protective efficacy of a Plasmodium yoelii Hsp60 DNA vaccine in BALB/c mice

The gene encoding the 60-kDa heat shock protein of Plasmodium yoelii (PyHsp60) was cloned into the VR1012 and VR1020 mammalian expression vectors. Groups of 10 BALB/c mice were immunized intramuscularly at 0, 3, and 9 weeks with 100 microg of PyHsp60 DNA vaccine alone or in combination with 30 microg of pmurGMCSF. Sera from immunized mice but not from vector control groups recognized P. yoelii sporozoites, liver stages, and infected erythrocytes in an indirect fluorescent antibody test. Two weeks after the last immunization, mice were challenged with 50 P. yoelii sporozoites. In one experiment the vaccine pPyHsp60-VR1012 used in combination with pmurGMCSF gave 40% protection (Fisher's exact test; P = 0.03, vaccinated versus control groups). In a second experiment this vaccine did not protect any of the immunized mice but induced a delay in the onset of parasitemia. In neither experiment was there any evidence of a protective effect against the asexual erythrocytic stage of the life cycle. In a third experiment mice were primed with PyHsp60 DNA, were boosted 2 weeks later with 2 x 10(3) irradiated P. yoelii sporozoites, and were challenged several weeks later. The presence of PyHsp60 in the immunization regimen did not lead to reduced blood-stage infection or development of parasites in hepatocytes. PyHsp60 DNA vaccines were immunogenic in BALB/c mice but did not consistently, completely protect against sporozoite challenge. The observation that in some of the PyHsp60 DNA vaccine-immunized mice there was protection against infection or a delay in the onset of parasitemia after sporozoite challenge deserves further evaluation.     

Early hepatic stages of Plasmodium berghei: release of circumsporozoite protein and host cellular inflammatory response.

After injection of Plasmodium berghei sporozoites into Norway-Brown rats, we were able to localize these sporozoites and the early hepatic trophozoites developing from them in histological sections of the liver stained with a sensitive immunogold-silver procedure. Sporozoites invading hepatocytes released substantial quantities of circumsporozoite protein into the hepatocyte cytoplasm. This intrahepatic cytoplasmic distribution reached a maximal level at about 4 h post-sporozoite injection. As the hepatic parasites continued to differentiate, circumsporozoite protein became undetectable within the cytoplasm of the hepatocytes and became localized around the periphery of each parasite. There was generalized cellular inflammation within the liver of the host, which first became evident at around 4 h post-sporozoite injection and progressed to the formation of well-defined granulomas by 24 h. Such histopathological changes were not seen in rats injected with killed sporozoites, indicating that the cellular inflammation was induced by viable, infective sporozoites. We did not observe cellular infiltration specifically associated with any of the developing hepatic stages that we observed, even up to 28 h post-sporozoite inoculation. These results indicate that viable sporozoites induced rapid and generalized hepatic inflammation in host rats. However, sporozoites that successfully invaded hepatocytes and then proceeded to develop further did not appear to be the target of inflammatory cells until a period beginning at around 40 h post-sporozoite inoculation.     

Host-parasite interactions and immunity to irradiated sporozoites

We compare and contrast the results of immunizing mice with irradiated sporozoites of Plasmodium berghei and Plasmodium yoelii. Host genetic control of protective immunity is different in the two rodent malarias. Few mouse strains are strongly protected by P. yoelii sporozoites, while all are protected by P. berghei sporozoite immunization. The role of CD8+ T cells in the protective immune response to each of these malarias varies with the strain of mouse. Moreover, a single strain will use a CD8+ T cell-dependent mechanism against one malaria, and a CD8+ independent mechanism against the other. Thus, each host-parasite pairing in these rodent malarias engenders a unique set of immune responses. Such variety should be expected in the immune response to the human malarias, and may complicate the development of universally applicable vaccines.     

Immunization with a recombinant C-terminal fragment of Plasmodium yoelii merozoite surface protein 1 protects mice against homologous but not heterologous P. yoelii sporozoite challenge.

It has been reported previously that immunization with recombinant protein containing the two epidermal growth factor (EGF)-like modules from merozoite surface protein 1 (MSP-1) of Plasmodium yoelii (strain YM) protects mice against a lethal blood-stage challenge with the same parasite strain. Since MSP-1 is expressed in both liver- and blood-stage schizonts and on the surface of merozoites, we evaluated the effectiveness of immunization with recombinant proteins containing either the individual or the two combined EGF-like modules in producing a protective response against a sporozoite challenge. The recombinant protein expressing the combined EGF-like modules of the YM strain protected mice against a homologous sporozoite challenge, and sterile protection, as defined by the absence of detectable blood-stage parasites, was observed in the majority of the mice. In contrast, mice immunized with recombinant P. yoelii YM MSP-1 were not protected against a heterologous challenge with sporozoites from strain 265 BY of P. yoelii. The lack of protection may be explained by differences identified in the amino acid sequences of MSP-1 for the two strains. A recombinant protein containing the two EGF-like modules of MSP-1 from P. yoelii 265 BY was produced and used to immunize mice. These mice were protected against a homologous challenge with sporozoites of P. yoelii 265 BY. The results suggest that a recombinant MSP-1 has potential as a vaccine against malaria, but its efficacy may be limited by sequence polymorphism and selection of variants.     

Characterization of Plasmodium yoelii monoclonal antibodies directed against stage-specific sporozoite antigens.

A battery of monoclonal antibodies against Plasmodium yoelii sporozoites was produced. Five of these (NYS1 through NYS5) were selected for characterization. All five were positive in the indirect immunofluorescent antibody test with P. yoelii sporozoites; however, each showed a different immunofluorescence pattern. Although NYS1 (immunoglobulin G3 [IgG3]), NYS2 (IgG3), and NYS3 (IgM) were positive in the circumsporozoite precipitation test, only NYS1 and NYS2 were able to neutralize sporozoite infectivity in mice. NYS4 (IgM) and NYS5 (IgG1) were not positive in the precipitation test and did not protect mice from sporozoite infection. All except NYS4 were species as well as stage specific. NYS4 cross-reacted with sporozoites of P. berghei. Electrophoretic immunoblotting analysis showed that these monoclonal antibodies detected sporozoite antigens of various molecular weights. Inhibition enzyme-linked immunosorbent assays indicated that each recognized a different antigenic epitope. The differences in their immunochemical and biological reactivity make them useful for screening a variety of P. yoelii antigens in recombinant DNA libraries. These antigens will be used in an animal model system for vaccine development.     

Acute malaria prolongs susceptibility of mice to Plasmodium berghei sporozoite infection.

The fate of immune response against sporozoite stage in malaria infection was investigated. Two groups (A and B) of mice were inoculated twice with infective sporozoites of Plasmodium berghei. The mice in group A were maintained on chloroquine prophylaxis to prevent the sporozoite infection from causing malaria. Group B animals on the other hand were allowed to develop acute malaria from the infection which was subsequently cured with chloroquine. Upon examination for stage specific immune responses, it was found that the animals in group A produced high antibody titres against sporozoites and none against erythrocytic stages. The mice in group B produced little anti-sporozoite antibodies but had high antibody titres against blood forms. Challenge infection with P. berghei sporozoites showed that group A animals had become resistant against sporozoite-induced parasitaemia, whereas the mice in group B remained susceptible. The possible significance of suppression of protective immunity by malaria in host-parasite relationship is discussed.     

Crossreactivity with sporozoites, exoerythrocytic forms and blood schizonts of Plasmodium berghei in indirect fluorescent antibody tests with sera of rats immunized with sporozoites or infected blood.

IFA studies are reported using plasmodial antigens from three different stages of the life cycle of Plasmodium berghei: sporozoites (SP); exoerythrocytic schizonts in rat liver (EEF); and parasitized rat erythrocytes (SCH = schizonts). Two series of specific sera were applied: sera from adult rats with a blood-induced infection (series A) and sera from rats immunized against sporozoites by mosquito bites and protected against parasitaemia by chloroquine (series B). In series A antibody titres with all three antigens were seen, but those with SCH were generally the highest. Superinfection with parasitized rat blood did not change the titre. In series B sera, collected from rats after a single exposure to infected mosquitoes, showed only titres with SP from day 3 onwards, but after a second exposure titres to all three antigens developed. Crossreactivity with the heterologous antigens in series B was clearly less than in series A. Anti-P. berghei sporozoite antibodies did not crossreact with P. vivax sporozoites. Rats of series A were resistant to a challenge of parasitized blood and could also inhibit the development of sporozoites. Rats of series B were protected against a challenge of sporozoites but not of infected blood. The results are discussed.     

A conserved peptide sequence of the Plasmodium falciparum circumsporozoite protein and antipeptide antibodies inhibit Plasmodium berghei sporozoite invasion of Hep-G2 cells and protect immunized mice against P. berghei sporozoite challenge.

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. The region II sequence of Plasmodium falciparum circumsporozoite (CS) protein includes a nonapeptide (WSPCSVTCG) which is highly conserved in all of the CS proteins sequenced to data, including the one from Plasmodium berghei. We have found that two peptides based on the P. falciparum region II sequence, P18 (EWSPCSVTCGNGIQVRIK) and P32 (IEQYLKKIKNS ISTEWSPCSVTCGNGIQVRIK), significantly inhibited P. berghei sporozoite invasion into Hep-G2 cells in vitro. This inhibition was enhanced if either peptide was preincubated with Hep-G2 cells prior to sporozoite invasion. We confirm that region II is a sporozoite ligand for the hepatocyte receptor; moreover, despite the few differences between P. falciparum and P. berghei region II sequences around the nonapeptide sequence (66% homology), the functional characteristics of the motif sequences are not affected. Since the conserved motifs represent a crucial sequence involved in Plasmodium sporozoite invasion of hepatocytes, antibodies to region II should inhibit sporozite invasion into hepatocytes. Indeed, we found that polyclonal antibodies generated to the P. falciparum-based peptide P32 inhibited P. berghei sporozoite invasion of Hep-G2 cells. Furthermore, inbred mice (C57BL/6) immunized with P32 were protected against a lethal challenge of P. berghei sporozoites. Our results suggest that the conserved region II of the CS protein contains crucial B- and T-cell epitopes, that such peptide sequences from the human malaria parasite P. falciparum can be screened in the P. berghei rodent model, and, finally, that region II can be considered useful as one of the components of a malaria vaccine.     

CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria.

Immunization of BALB/c mice with radiation-attenuated Plasmodium yoelii sporozoites induces cytotoxic T lymphocytes (CTL) specific for an epitope located within the amino acid sequence 277-288 of the P. yoelii circumsporozoite (CS) protein. Several CD8+ CTL clones were derived from the spleen cells of sporozoite-immunized mice, all displaying an apparently identical epitope specificity. All the clones induced high levels of cytolysis in vitro upon exposure to peptide-incubated MHC-compatible target cells. The adoptive transfer of two of these clones conferred complete protection against sporozoite challenge to naive mice. This protection is species and stage specific. Using P. yoelii specific ribosomal RNA probes to monitor the in vivo effects of the CTL clones, we found that their target was the intrahepatocytic stage of the parasite. The protective clones completely inhibited the development of the liver stages of P. yoelii. Some CTL clones were only partially inhibitory in vivo, while others failed completely to alter liver stage development and to confer any detectable degree of protection. The elucidation of the effector mechanism of this CTL mediated protection against rodent malaria should facilitate the design of an effective malaria vaccine. From a broader perspective this model may provide further insight into the mechanism(s) of CTL mediated killing of intracellular non-viral pathogens in general.     

Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection

Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver, and infect hepatocytes. The formation of a parasitophorous vacuole (PV) establishes their intracellular niche. Recently, two members of the 6-Cys domain protein family, P52 and P36, were each shown to play an important albeit nonessential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52/p36-deficient Plasmodium yoelii parasites by the simultaneous deletion of both genes using a single genetic manipulation. p52/p36-deficient parasites exhibited normal progression through the life cycle during blood-stage infection, transmission to mosquitoes, mosquito-stage development, and sporozoite infection of the salivary glands. p52/p36-deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52/p36-deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The p52/p36-deficient parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently, they did not cause blood-stage infections even at high sporozoite inoculation doses. Mice immunized with p52/p36-deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of Plasmodium falciparum live attenuated malaria vaccines.     

Immunity to Plasmodium berghei exoerythrocytic forms derived from irradiated sporozoites

 The nature of immunity generated by Plasmodium berghei exoerythrocytic (EE) stages developing from irradiated sporozoites was studied using in vivo parameters of host protection on immunization with irradiated sporozoites and in vitro parameters of inhibition of sporozoite invasion and EE form development by serum antibodies from immunized mice. On in vivo challenge of immunized mice by sporozoites, protection was observed in an irradiation-dose-dependent manner. This finding stresses that protection is dependent on the irradiation dose of sporozoites that allows sporozoite penetration yet controls EE form development within the liver. Using the human hepatoma line Hep G2 as host cells in vitro, we observed that serum antibodies raised in mice immunized with irradiated sporozoites reacted with sporozoite- and hepatic-stage parasites in an immunofluorescent antibody test (IFAT). No reactivity was observed with blood-stage parasites. Serum antibodies from mice immunized with 6- to 18-krad-irradiated sporozoites inhibited sporozoite invasion and caused severe inhibition of EE form development in hepatoma cells, pointing to the antigenic content of EE forms developing from irradiated sporozoites (irra EE forms) as critical immunogens. Moreover, in an enzyme-linked immunosorbent assay (ELISA), serum antibodies raised to 12-krad-irradiated sporozoites showed reactivity to synthetic peptides representing the conserved Region II sequences of the P. falciparum circumsporozoite (CS) protein as well as the P. falciparum liver-stage-specific antigen (LSA-1)-based repeat sequences, thus implicating an important role for both the sporozoite and the hepatic stage in protection. Received: 21 June 1995 / Accepted: 27 Oktober 1995     

Multiple T helper cell epitopes of the circumsporozoite protein of Plasmodium berghei

The present findings establish the lack of genetic restriction of the humoral immune response to sporozoites of Plasmodium berghei, corraborating earlier observations that mice of different strains can be protected by immunization with irradiated sporozoites. Most, if not all, anti-sporozoite antibodies are directed against the repetitive B cell epitope of the circumsporozoite (CS) protein. However, neither a peptide containing a dimer of this repeat (17.1), nor a peptide polymer containing multiple repeats induced an antibody response in mice of different H-2 and different genetic backgrounds. A yeast-derived recombinant, containing the repeat domain and part of the surrounding amino and carboxy-terminal regions of the P. berghei CS protein, induces very different levels of antibody in mice of diverse H-2 haplotypes. H-2j mice are high responders and the immunized mice are extensively protected against sporozoite challenge. The lymph node cells of the H-2j mice (but not from other strains) proliferated in the presence of peptide N, contained in the amino terminal region of the CS recombinant. Additional H-2-restricted T cell epitopes have been identified in amino and carboxy-terminal regions of the CS protein, and mice of most of the strains recognized multiple T cell epitopes. Two peptides representing T cell epitopes were synthesized in tandem with a peptide representing the B cell epitope, and were assayed for T helper activity in vivo. The antibody response of mice, primed by a single injection of sporozoites, was boosted very effectively by the administration of peptide N + 17.1 or peptide B-4 + 17.1. The B-4 T cell epitope is located in the carboxy-terminal region of the CS protein and is recognized by mice of at least four different H-2 haplotypes. These observations demonstrate that the immune response to the CS protein of P. berghei is not genetically restricted and that it contains several T cell epitopes, some of which can function as helper epitopes. In addition, they show that a synthetic sporozoite vaccine can boost the immune response to sporozoites.     

Gene targeting in the rodent malaria parasite Plasmodium yoelii

It is anticipated that the sequencing of Plasmodium falciparum genome will soon be completed. Rodent models of malaria infection and stable transformation systems provide powerful means of using this information to study gene function in vivo. To date, gene targeting has only been developed for one rodent malaria species, Plasmodium berghei. Another rodent species, Plasmodium yoelii, however, is favored to study the mechanisms of protective immunity to the pre-erythrocytic stages of infection and vaccine development. In addition, it offers the opportunity to investigate unique aspects of pathogenesis of blood stage infection. Here, we report on the stable transfection and gene targeting of P. yoelii. Purified late blood stage schizonts were used as targets for electroporation with a plasmid that contains a pyrimethamine-resistant form of the P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) fused to green fluorescent protein (gfp) gene. After drug selection, fluorescent parasites contained intact, non-rearranged plasmids that remain stable under drug-pressure. In addition, we used another dhfr-ts/gfp based plasmid to disrupt the P. yoelii trap (thrombospondin-related anonymous protein) locus by site-specific integration. The phenotype of P. yoelii TRAP knockout was identical to that previously reported for the P. berghei TRAP knockout. In the absence of TRAP, the erythrocytic cycle, gametocyte and oocyst development of the mutant parasites were indistinguishable from wild type (WT). Although the sporozoites appeared morphologically normal, they failed to glide and to invade the salivary glands of mosquitoes.     

Linear and multiple antigen peptides containing defined T and B epitopes of the Plasmodium yoelii circumsporozoite protein: antibody- mediated protection and boosting by sporozoite infection

We previously reported the identification of a T cell epitope in the N- terminal part of the circumsporozoite protein (CSP) of Plasmodium yoelii yoelii (Pyy). CD4+ T cell clones derived from mice immunized with a 21-mer peptide (amino acids 59-79, referred to as Py1) containing this epitope confer complete protection after passive transfer in mice. These clones proliferate in vitro in the presence of a 13-mer peptide (amino acids 59-71, referred to as Py1T). This shorter peptide was found to behave as a Th epitope in vivo, allowing overcoming of the genetic restriction for production of anti-repeat antibodies in BALB/c mice, when cross-linked to three (QGPGAP) repeats of the Pyy CSP. In this study, we report protection in BALB/c mice, against a challenge with Pyy sporozoites after immunization with linear and multiple antigen peptides containing Py1T as T epitope and three repeats QGPGAP (Py3) as B epitope. Multiple antigen peptide (MAP4-Py1T- Py3)-induced immunity was shown to be more effective than immunity induced by the linear form of the conjugate (Py1T-Py3), protecting against challenges with higher numbers of sporozoites. In both cases, levels of anti-repeat antibodies were strongly correlated with anti- parasite antibodies and protection. When tested in vitro, sera from mice immunized with the protective constructs strongly inhibited Pyy liver stages, while lymph node T cells displayed no cytotoxicity. In vivo, depletion of CD4+ or CD8+ T cells did not affect protection. Furthermore, MAP4-Py1T-Py3-immunized mice were not protected against a challenge with P. yoelii nigeriensis sporozoites, a parasite which has the same Py1T sequence but differs from Pyy in its repeated sequence. These results demonstrate that anti-repeat antibodies raised by immunization with the linear or the MAP form are exclusively responsible for the protection. Furthermore, this antibody response is boosted by a sporozoite challenge, allowing protection against a second challenge.      

Class II-restricted protective immunity induced by malaria sporozoites

The irradiated-sporozoite vaccine elicits sterile immunity against Plasmodium parasites in experimental rodent hosts and human volunteers. Based on rodent malaria models, it has been proposed that CD8+ T cells are the key protective effector mechanism required in sporozoite-induced immunity. To investigate the role of class II-restricted immunity in protective immunity, we immunized beta2-microglobulin knockout (beta2M-/-) mice with irradiated Plasmodium yoelii or P. berghei sporozoites. Sterile immunity was obtained in the CD8+-T-cell-deficient mice immunized with either P. berghei or P. yoelii sporozoites. beta2M-/- mice with the BALB/c (H-2d) genetic background as well as those with the C57BL (H-2b) genetic background were protected. Effector mechanisms included CD4+ T cells, mediated in part through the production of gamma interferon, and neutralizing antibodies that targeted the extracellular sporozoites. We conclude that in the absence of class I-restricted CD8+ T cells, sporozoite-induced protective immunity can be effectively mediated by class II-restricted immune effector mechanisms. These results support efforts to develop subunit vaccines that effectively elicit high levels of antibody and CD4+ T cells to target Plasmodium pre-erythrocytic stages.