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     

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.     

Host-parasite interactions in sympatric and allopatric populations of European bitterling

Susceptibility to parasite infection was examined in a field experiment for four populations of 0+ juvenile European bitterling (Rhodeus amarus): one sympatric to local parasite fauna, one allopatric, and two hybrid populations. Significantly higher parasite abundance was recorded in the allopatric bitterling population, suggesting a maladaptation of parasites to their sympatric host. Type of parasite life cycle played an important role in host–parasite interactions. While the abundance of allogenic species between populations was comparable, a significant difference was found in abundance of autogenic parasite species between fish populations, with the allopatric population more infected. These results correspond with a prediction of higher dispersion probability and higher gene flow among geographically distant populations of allogenic species as compared to autogenic species. Increased susceptibility to parasites that do not occur within the natural host's geographical distribution was found in the allopatric host, but only for autogenic species. A difference in infection susceptibility was detected among populations of early-hatched bitterling exposed to infection during a period of high parasite abundance and richness in the environment. Differences in parasite abundance and species diversity among populations diminished, however, with increasing time of exposure. No difference was found within late-hatched populations, probably due to a lower probability of infection in late-hatched cohorts.     

Host-parasite interactions in the pathogenesis of experimental renal candidiasis

To study the pathogenesis of renal candidiasis, viable Candida albicans blastospores were injected directly into the left renal artery of New Zealand white rabbits. The progression of the disease was followed by light and electron microscopy over a 6-day period. Within 5 minutes after injection of the yeasts, the organisms localized within glomerular and peritubular capillaries of the cortex. Localization of yeasts within the capillaries occurred through adherence, demonstrated by the presence of surface fibrils originating from the yeast cells. Two to 10 hours later, inflammatory nodules comprised of polymorphonuclear leukocytes formed within capillary lumina. Many of the entrapped yeasts remained viable and extended through adjacent endothelium and epithelium by the formation of germ tubes which penetrated between or directly through intact host cells. After 24 hours, numerous hyphal forms were observed within tubules of the cortex, and some necrotic host cells were noted at sites of penetration. Abscesses replaced renal parenchyma in focal areas during subsequent time intervals. These studies indicate that attachment of Candida albicans to endothelium within capillaries of the cortex is a key event in the disease process. Also, growth of germ tubes into renal tubules provides a temporal advantage for amplification of Candida organisms.     

First clinical trial of purified, irradiated malaria sporozoites in humans

Purified, irradiated, metabolically active sporozoites of Plasmodium falciparum (PfSPZ) have been administered to 80 volunteers through subcutaneous or intradermal routes. This PfSPZ vaccine has been shown to be safe but of low immunogenicity and hence protective efficacy as shown after challenge with infected mosquitoes. Intravenous delivery of PfSPZ in nonhuman primates induced memory CD8(+) and CD4(+) T cells specific for PfSPZ that reside in the liver. Further studies using PfSPZ and the intravenous route in humans are warranted to fully explore the potential of purified, attenuated sporozoites for malaria vaccine development.     

In vitro interactions between murine macrophages and Eimeria falciformis sporozoites

A short-term (2 h) assay was used to investigate the in vitro fate of Eimeria falciformis sporozoites in murine peritoneal macrophages. In minimal medium, uptake of sporozoites was low by both normal (naive) and immune macrophages. However, when heat-inactivated serum from immune mice was added to the incubation medium, sporozoite uptake was much more efficient. Sporozoite lysis was observed only in immune macrophages and required both antibodies and complement. Pretreatment of immune macrophages with chloroquine inhibited sporozoite lysis and resulted in an accumulation of sporozoites within the cells. Immunoabsorption assays revealed that IgG2a was the major isotype mediating entry of sporozoites into macrophages, both in early (6 days post-primary) and late (second) infections.     

Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells.

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites.     

Synthetic peptides as antigens for the detection of humoral immunity to Plasmodium falciparum sporozoites

The presence of antibodies against P. falciparum sporozoites in humans living in malaria-endemic areas was measured using as antigen the synthetic peptide (NANP)3, which represents the immunodominant region of the circumsporozoite (CS) protein. By using a competitive binding assay it was determined that antibodies which recognize (NANP)3 do not react with a 22-Mer synthetic peptide representing a cross-reacting epitope present in an antigen (5.1) from the blood stages of the parasite. Antibodies present in human sera which react with the 5.1 peptide did not react with (NANP)3. This strongly suggests that antibodies to (NANP)3 found in sera of individuals living in endemic areas are a reflection of exposure to P. falciparum sporozoites. These results validate the use of (NANP)3 for epidemiological studies to detect and measure humoral immunity to P. falciparum sporozoites.     

In vitro interactions between murine neutrophils and Eimeria falciformis sporozoites.

The in vitro interactions between elicited mouse peritoneal neutrophils, antibodies and newly excysted sporozoites of Eimeria falciformis resulted in lysis of the parasite. This lysis required the presence of a heat-labile component of normal mouse serum, and was antibody- and cell-concentration-dependent. Under optimal conditions (serum dilution = 1/192, effector cell/sporozoite = 10/1) this lysis, which began after incubation at 37 degrees C for 4 h, was nearly complete after 18 h. It began by opsonization of the sporozoites by antibodies and complement. Inhibition studies performed with inhibitors of neutrophil function did not enable us to determine the mechanism of this extracellular lysis (oxidative respiratory burst or enzyme release), since only metal chelators, lysosomotropic reagents and compounds known to interfere with adenylate cyclase activity were truly inhibitory.     

Chemical attenuation of Plasmodium berghei sporozoites induces sterile immunity in mice

Radiation and genetic attenuation of Plasmodium sporozoites are two approaches for whole-organism vaccines that protect against malaria. We evaluated chemical attenuation of sporozoites as an alternative vaccine strategy. Sporozoites were treated with the DNA sequence-specific alkylating agent centanamycin, a compound that significantly affects blood stage parasitemia and transmission of murine malaria and also inhibits Plasmodium falciparum growth in vitro. Here we show that treatment of Plasmodium berghei sporozoites with centanamycin impaired parasite function both in vitro and in vivo. The infection of hepatocytes by sporozoites in vitro was significantly reduced, and treated parasites showed arrested liver stage development. Inoculation of mice with sporozoites that were treated in vitro with centanamycin failed to produce blood stage infections. Furthermore, BALB/c and C57BL/6 mice vaccinated with treated sporozoites were protected against subsequent challenge with wild-type sporozoites. Our findings demonstrate that chemically attenuated sporozoites could be a viable alternative for the production of an effective liver stage vaccine for malaria.     

约氏疟原虫BY265株减毒子孢子诱导小鼠保护性免疫的研究

目的探讨约氏疟原虫BY265株减毒子孢子免疫能否诱导小鼠产生完全保护性免疫及其效应分子。方法确定减毒子孢子合适辐照剂量并免疫小鼠后,间接免疫荧光和ELISPOT分别检测抗环子孢子蛋白(CSP)抗体滴度和CSP特异的CD8+T细胞分泌IFN-γ及其抵御子孢子的攻击感染情况。结果免疫小鼠的外周血能检测到子孢子CSP特异的抗体(1∶400)和产生IFN-γ的CSP特异CD8+T细胞;与对照小鼠相比,1000个子孢子尾静脉注射攻击减毒子孢子免疫小鼠后,直到14d也检测不到红内期疟原虫。结论经辐照减毒子孢子免疫后的小鼠对野生株子孢子产生了完全保护性免疫,为红外期疫苗的研究提供基础与理论依据。     

Host-parasite interactions with peritoneal macrophages of mice and rats in vitro and in vivo.

This paper deals with the intracellular multiplication of mycobacteria in peritoneal macrophages from mice and rats immunized with tubercle bacilli or pretreated with Triton WR 1339. If unstimulated macrophages were used, almost unrestricted multiplication of mycobacteria was observed in macrophages from both vaccinated and pretreated hosts after infection of the cells in vitro. Only when the infection of the cells was perfored in the peritoneal cavity of vaccinated hosts did the macrophages display a high degree of inhibition. This striking difference in the behavior of macrophages infected in vitro and in vivo is explained by the local inflammation caused by the intraperitoneal infection, which leads to an influx of T-cell mediators. When macrophages from hosts pretreated with Triton WR 1339 were used, inhibition of the multiplication of mycobacteria within cells infected in vitro or in vivo was very slight, though this compound displayed a marked protective effect in the host. Addition of streptomycin to the culture medium caused a strong inhibition of intracellular mycobacteria even in small concentrations; there was no difference between normal and "immune" macrophages. When rats were infected with virulent tubercle bacilli, they were initially fully susceptible to the infection but showed rapid onset of a strong immune response.     

Vaccination with irradiated Listeria induces protective T cell immunity

We evaluated gamma-irradiated Listeria monocytogenes as a killed bacterial vaccine, testing the hypothesis that irradiation preserves antigenic and adjuvant structures destroyed by traditional heat or chemical inactivation. Irradiated Listeria monocytogenes (LM), unlike heat-killed LM, efficiently activated dendritic cells via Toll-like receptors and induced protective T cell responses in mice. Like live LM, irradiated LM induced Toll-like-receptor-independent T cell priming. Cross-presentation of irradiated listerial antigens to CD8(+) T cells involved TAP- and proteasome-dependent cytosolic antigen processing. These results establish that killed LM can induce protective T cell responses, previously thought to require live infection. gamma-irradiation may be potentially applied to numerous bacterial vaccine candidates, and irradiated bacteria could serve as a vaccine platform for recombinant antigens derived from other pathogens, allergens, or tumors.     

Immunoglobulin E and eosinophil-dependent protective immunity to larval Onchocerca volvulus in mice immunized with irradiated larvae

Mice immunized with irradiated Onchocerca volvulus third-stage larvae developed protective immunity. Eosinophil levels were elevated in the parasite microenvironment at the time of larval killing, and measurements of total serum antibody levels revealed an increase in the immunoglobulin E (IgE) level in immunized mice. The goal of the present study was to identify the role of granulocytes and antibodies in the protective immune response to the larval stages of O. volvulus in mice immunized with irradiated larvae. Immunity did not develop in mice if granulocytes, including both neutrophils and eosinophils, were eliminated, nor did it develop if only eosinophils were eliminated. Moreover, larvae were killed in na?ve interleukin-5 transgenic mice, and the killing coincided with an increase in the number of eosinophils and the eosinophil peroxidase (EPO) level in the animals. To determine if EPO was required for protective immunity, mice that were genetically deficient in EPO were immunized, and there were no differences in the rates of parasite recovery in EPO-deficient mice and wild-type mice. Two mouse strains were used to study B-cell function; micro MT mice lacked all mature B cells, and Xid mice had deficiencies in the B-1 cell population. Immunity did not develop in the micro MT mice but did develop in the Xid mice. Finally, protective immunity was abolished in mice treated to eliminate IgE from the blood. We therefore concluded that IgE and eosinophils are required for adaptive protective immunity to larval O. volvulus in mice.     

Immunoprotection in mice susceptible to waning memory against the pre-erythrocytic stages of malaria after validated immunisation with irradiated sporozoites ofPlasmodium berghei

The induction of immunity by irradiated sporozoites has been a bench-mark of immunological protection against the malaria parasite. Herein we confirm that different mouse strains exhibit different susceptibilities to sporozoite-induced infection ofPlasmodium berghei. We note, however, that after hepatic schizogony,early parasite growth in the blood demonstrates no strain preference between C57BL/6 and BALB/c mice. Sporozoite-susceptible C57BL/6 mice, although initially protected by irradiated sporozoite immunisation against a challenge of 10³ live sporozoites, progressively lose this protection; a challenge with fewer sporozoites 2 months later elicits a blood infection. BALB/c mice treated in parallel remain protected. Analysis of the kinetics of blood parasitaemia (a measure of hepatic schizont burden) with waning protection shows clearly that immunocompetence remains, as indicated by a reduction in the effective exo-erythrocytic schizont load. This immunocompetence can be shown to be absolutely protective, given an appropriately low dose of viable infective sporozoites. We discuss the testable proposition that this elicitation of protective memory is a consequence either of unsaturated threshold levels of recirculating immunoeffector CD8⁺ cells or of CD4 cell activation by nonviable sporozoites.This work was supported by a grant from the Medical Research Council     

The role of intrahepatic lymphocytes in mediating protective immunity induced by attenuated Plasmodium berghei sporozoites

Summary: Exposure to irradiated Plasmodium sporozoites (g‐spz) results in protection against malaria. Like infectious spz, g‐spz colonize hepatocytes to undergo maturation. Disruption of liver stage development prevents the generation of protection, which appears, therefore, to depend on liver stage antigens. Although some mechanisms of protection have been identified, they do not include a role for intrahepatic mononuclear cells (IHMC). We demonstrated that P. berghei g‐spz‐immune murine IHMC adoptively transfer protection to naive recipients. Characterization of intrahepatic CD4+ T cells revealed an immediate, albeit transient, response to g‐spz, while the response of CD8+ T cells is delayed until acquisition of protection. It is presumed that activated CD8+ T cells home to the liver to die; g‐spz‐induced CD8+CD45RBloCD44hi T cells, however, persist in the liver, but not the spleen, during protracted protection. The association between CD8+CD45RBloCD44hi T cells and protection has been verified using MHC class I and CD1 knockout mice and mice with disrupted liver stage parasites. Based on kinetic studies, we propose that interferon‐g, presumably released by intrahepatic effector CD8+ T cells, mediates protection; the persistence of CD8+ T cells is, in turn, linked to Plasmodium antigen depots and cytokines released by CD4+ T cells and/or NK T cells.     

Recruitment of lymphocytes to the lung through vaccination enhances the immunity of mice exposed to irradiated schistosomes.

The effector mechanism, which operates against challenge parasites in the lungs of C57BL/6 mice vaccinated once with irradiated cercariae of Schistosoma mansoni, is mediated by CD4+ T helper lymphocytes. However, adoptive transfer of immunity from vaccinated donors to naive recipients by using sensitized T cells has not proved successful. One explanation may be that the recruitment of sensitized T lymphocytes to the lungs by vaccinating parasites to arm that organ is not reproduced by transfer protocols. We have used the technique of parabiosis, as a means of adoptive transfer, to demonstrate the relevance of pulmonary T cells to protection. Sensitized and naive partners were joined surgically for a 28-day period, coincident with priming of the immune system. A vascular union rapidly developed, and sensitized T cells were detected in the spleens of the naive partners. When parabionts were challenged percutaneously 10 days after separation, the level of immunity transferred to the naive partners was approximately two-thirds that of their vaccinated counterparts. The naive partners, unlike the vaccinated animals, did not recruit lymphocytes to the lungs during the priming period. In contrast, after percutaneous challenge, schistosome-specific lymphocytes were recruited to the lungs of both separated parabionts. The importance of lymphocytes recruited to the lungs during the primary response was revealed by an intravenous challenge with lung schistosomula; this eliminates the opportunity for secondary immune responses prior to parasite arrival in the lungs. In this situation, the vaccinated partners showed 47% immunity while the naive partners were not protected. We conclude that the presence of specific T cells in the lungs at the time of challenge confers a significant advantage, permitting a more effective recall response than in animals lacking such resident cells.