Plasmodium berghei: antibody response and protective immunity induced by vaccination

The authors have studied the behaviour of Swiss mice and of 5 inbred strains of mice in order to investigate: the protective effect, in the homologous infection test, of six vaccine inoculations of irradiated parasites belonging to two strains of Plasmodium berghei: ISTISAN and K173; the capacity to produce humoral antibodies after vaccine treatments and during infection; the probable correlation between the high antibody titre and the protection against infection. The results of the present study show that the antibody response plays a precise role in the immunity induced by vaccination. There is a certain degree of correlation, which is more evident for K173 vaccine, between the level of antibody response during infection and the protective efficacy of vaccination.     

Plasmodium berghei XAT: protective 155/160 kDa antigens are located in parasitophorous vacuoles of schizont-stage parasite

Effective blood-stage malaria vaccine candidates have been mainly developed from the proteins in exposed locations on the parasite such as the surface of free merozoites or infected red blood cells. In the present study, we identified and localized novel protective antigens derived from the blood-stage of Plasmodium berghei XAT after establishment of hybridomas producing protective monoclonal antibodies (mAbs) against the parasites. The protective antigens were expressed in schizonts but not in trophozoites, and located in the parasitophorous vacuoles in the infected erythrocyte cytoplasm. The antigens, with molecular weight of 155/160 kDa, were not identical to any merozoite/schizont antigens that have been reported as target molecules recognized by mAbs developed to rodent malaria parasites. The characterization of new malarial antigenic targets of potentially protective antibody responses following infection would give us new insights for the selection of candidate antigens for malaria vaccine.     

Characterization of the antigenic determinant on Fusarium oxysporum recognized by a genus-specific monoclonal antibody

The antigenic determinant of a monoclonal antibody (MAb) (API9-2) having specific reactivity with the fungi grouped into the genus Fusarium was analyzed. The culture supernatant of the fungi showed antigenicity against MAb API9-2, proving that the antigen exists as an exoantigen. The heat-resistant, proteinase K-resistant and periodate oxidation-labile features of the antigenic determinant indicated its carbohydrate nature. Also, lectin affinity tests and thin-layer chromatography analysis suggested that the monosaccharide making up the antigenic determinant was mainly mannose. Considering previous reports that the antigen exists on the surface of mycelia (by immunofluorescence assay) and is a - 55 kDa molecule (by Western blotting analysis), it was concluded that the antigenic determinant of MAb API9-2 on F. oxysporum is a mannan component existing on the surface of mycelia.     

HLA-DR antigens and mitogenic factors released by PWM-stimulated T lymphocytes share the framework determinant recognized by the monoclonal antibody Q5/13

Human T lymphocytes release factors which enhance the mitogenic response of B lymphocytes to PWM. These mitogenic factors share with HLA-DR antigens the framework determinant recognized by the monoclonal antibody Q5/13 (MoAb Q5/13) since adsorption of T-cell medium with an excess of insolubilized MoAb Q5/13 significantly reduces the enhancing activity of the T-cell supernatant on the proliferative response of B cells to PWM. On the other hand, incubation of the T-cell supernatant with an excess of insolubilized anti-HLA-A,B MoAb Q1/28 did not effect the activity of the T-cell supernatant in the proliferative assay.     

Use of a DNA probe to measure the neutralization of Plasmodium berghei sporozoites by a monoclonal antibody

A specific DNA probe has been used to quantify the neutralizing effects of monoclonal antibodies (3D11) against the circumsporozoite protein of Plasmodium berghei sporozoites. The amount of parasite DNA was measured in the livers of Norway Brown rats at the peak of proliferation of the exoerythrocytic forms (EEF). In vitro treatment of 1.5 X 10(5) sporozoites with 0.36 microgram/0.5 ml of whole 3D11 IgG neutralized about 90% of the sporozoite infectivity. When the dose was 3.6 micrograms no signal was detected, indicating that less than ten sporozoites developed into EEF in the liver. In contrast, 3.6 micrograms of Fab obtained from 3D11 neutralized sporozoite infectivity by only 60%. Although the neutralizing effect of 3D11 was very marked, the infected rats developed parasitemias after a prolonged delay in patency, suggesting that a small proportion of sporozoites was resistant to the effects of 3D11. The sporozoites were subjected to four cycles of 3D11-mediated selection, each one involving treatment of sporozoites with the antibodies, injection of the mixture into rats, infection of hamsters with blood stage parasites obtained from the rats, feeding of Anopheles stephensi on these hamsters, and obtaining sporozoites from the salivary glands of the infected mosquitoes. After four cycles of selection, the susceptibility of the resulting sporozoites to different concentrations of 3D11 was compared with that of nonselected sporozoites. No differences were detected, indicating that the capacity of a few sporozoites to escape the neutralizing effect of 3D11 antibodies is not inherited.     

Identification of immunodominant Trypanosoma musculi antigens recognized by monoclonal antibody and curative immunoglobulin G2a antibody.

Trypanosoma musculi obtained from normal or irradiated (900 rad) hosts or from in vitro cultures were lysed and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar protein banding patterns with a molecular weight (mol. wt) range from 34 to 68 kDa were observed between the two bloodstream forms. In comparison, lysates of cultured parasites showed a unique banding pattern of antigens within the same mol. wt range. Western blot of bloodstream form lysates, probed with immune plasma (IP), revealed a wide range of parasite proteins. However, when probed with the IgG2a-enriched fraction of IP, a major band of approximately 66 kDa was detected on the blot. Several bands of higher mol. wt were also observed. When anti-T. musculi monoclonal antibodies were used to probe the blot, the 66 kDa protein was again recognized. Using indirect fluorescence, live bloodstream form parasites were analysed by flow cytometry and the p66 protein was determined to be a surface molecule. Finally, lysates of 35S-methionine-labelled trypanosomes were immunoprecipitated with Sepharose linked anti-T. musculi monoclonal antibodies and the eluted ligand analysed by SDS-PAGE and autoradiographed. The 66 kDa band was identified, therefore confirming that this protein was of parasite origin.     

Plasmodium berghei: characterization of antigens and their role in inducing immunity to infection

In vitro, Plasmodium berghei infected erythrocytes incorporated 35S-methionine into 31 polypeptides with molecular weights from 21 kd to 300 kd. Hemoglobin and additional smaller molecular weight polypeptides were labelled with 35S-methionine by a population of uninfected, reticulocyte-rich rat erythrocytes. 3H-glucosamine was incorporated into at least 3 components by Plasmodium berghei infected erythrocytes. Uninfected, reticulocyte-rich rat erythrocytes did not incorporate 3H-glucosamine. Rabbit antisera against small, free plasmodia formed complexes which contained between 12 and 22 of the 31 labelled polypeptides in the 35S-methionine labelled antigen preparation. Rabbit antisera against soluble antigens washed from small, free plasmodia formed complexes containing many of the same labelled plasmodial polypeptides, however the reactions were particularly strong with those components which yielded polypeptides with molecular weights of 25 kd and 31 kd. Rabbit origin antisera against the 2 preparations did not form detectable complexes with the 3H-glucosamine labelled plasmodial components. Sera from rats undergoing progressive P. berghei infection formed complexes containing an increasing number of 35S-methionine labelled plasmodial polypeptides. Hyperimmune rat serum, the only serum protective upon passive transfer into mice, formed complexes containing 7 polypeptides with molecular weights of 35 kd, 75 kd, 80 kd, 92 kd, 100 kd, 150 kd and 190 kd. Antigens containing 1 or more of these polypeptides may be important in the induction of a protective antibody response against the parasite.     

Plasmodium berghei: inhibition of the sporogonous cycle by alpha-difluoromethylornithine

alpha-Difluoromethylornithine (DFMO), an enzyme inhibitor of ornithine decarboxylase, inhibits the sporogonous cycle of the malaria parasite Plasmodium berghei in the mosquito vector Anopheles stephensi. DFMO was administered to the mosquitoes dissolved either in the sugar solution at their disposal in the cages or through blood meals taken from treated mice. The mice subsequently bitten by mosquitoes treated with DFMO by both routes of administration did not contract malaria.     

Investigation of antigenic expression of Bacteroides fragilis by immunogold labelling and immunoblotting with a monoclonal antibody

Abstract Electron microscopy and immunogold labelling with monoclonal antibody (McAb) Bfl identified an antigen expressed on some in vitro and in vivo grown Bacteroides fragilis NCTC9343 cells.
Immunoprecipitation with this McAb was used to enrich for B. fragilis NCTC9343 cells expressing the Bfl antigen. The McAb Bfl bound to an epitope close to the surface of the outer membrane, but the fibrous capsular network radiating from the bacterial surface was not labelled. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting identified 3 high- M _r bands which resisted heating and protease digestion but were partially sensitive to sodium periodate treatment.     

Localization of protective 143/140 kDa antigens of Plasmodium knowlesi by the use of antibodies and ultracryomicrotomy

Immune sera from mice immunized with the 143/140 kDa protein have been shown to partially block erythrocyte invasion by P. knowlesi merozoites. Therefore, immunoelectron microscopy utilizing ultracryomicrotomy, antibody to 143/140 kDa protein, and protein A gold particles were used to determine the precise localization of this protein in malarial parasites. Gold particles were not seen associated with young trophozoites but appeared in the parasite cytoplasm as the parasites grew to multi-nucleate schizonts. In presegmenter-schizonts, gold particles were associated with the well-developed endoplasmic reticulum, the parasite plasma membrane, and the parasitophorous vacuole membrane. The surface of merozoites was covered with gold particles. Maurer's clefts, which appeared in Plasmodium infected erythrocytes, were also associated with gold particles. These observations suggest that 143/140 kDa protective malarial proteins may be synthesized in the endoplasmic reticulum of P. knowlesi schizonts before being transported to the surface of the schizonts and merozoites. Shedding of the merozoite surface coat may be responsible for the presence of the 143/140 kDa proteins in the parasitophorous vacuole and Maurer's clefts.     

Plasmodium berghei: inhibition by splenectomy of a paralyzing syndrome in infected rats

In recent attempts to isolate the factor causing paralysis in rats infected with a mousepassed KBG 173 strain of Plasmodium berghei Splenectomy was employed. The effects of Splenectomy on the paralyzing syndrome are discussed in the present report. Paralysis was inhibited in rats splenectomized prior to inoculation. Serial bloodpassaging of the strain in the splenectomized host however, apparently enhanced its virulence. Spleen-intact rats used as controls exhibited a marked increase in incidence of paralysis. Rats splenectomized a day before paralysis became evident were paralyzed significantly more frequently than those splenectomized a day earlier, indicating the apparent requirement of an incubation period for the expression of the paralytic effect. The enhanced virulence did not appear to be related to the level of parasitemia of the splenectomized rats used as donors. The spleen appears to provide the optimal conditions required for the elaboration of the paralyzing factor.     

Lysozyme expression in the rat parotid gland: light and electron microscopic immunogold studies

 Lysozyme (muramidase) is capable of direct bacteriolytic action by hydrolyzing glycosidic bonds in bacterial cell walls. Although it is broadly distributed in vertebrate tissues and secretions, the cellular and subcellular localizations of the enzyme are still not well known. The present study examines the distribution of lysozyme expression in the various cell types of LR gold-embedded rat parotid gland, applying a postembedding immunogold-silver staining technique for light microscopy. Simultaneously, a postembedding immunogold method for electron microscopy was used to determine the cellular compartments engaged in the biosynthesis and exocytosis of lysozyme. Silver-amplified immunogold staining for lysozyme demonstrated identical localization in both paraffin and semithin LR-gold sections: in the supranuclear parts of acinar and intercalated duct cells. Staining intensity varied even between adjacent cells. In the electron microscope, immunogold labeling was detected over the cell compartments associated with protein synthesis and exocytosis in acinar and intercalated duct cells. Lysozyme antigenic sites were visible over endoplasmic reticulum and throughout the Golgi apparatus, being intense over the trans-Golgi network, but even stronger in the condensing vacuoles and most prominent over secretory granules in both cell types. The findings provide the first immunocytochemical evidence of the synthesis and secretion of lysozyme in parotid acinar and intercalated duct cells. Accepted: 3 December 1996     

Development and expression of cytoplasmic antigens in Purkinje cells recognized by monoclonal antibodies

Summary Five monoclonal antibodies reacting with intracellular constituents of Purkinje cells were investigated by means of indirect immunofluorescence on fresh-frozen sections of the cerebellum and retina from developing and adult normal and mutant mice. Antibodies PC1, PC2 and PC3, which recognize Purkinje cells, but no other cerebellar neuron type, label these cells from day 4 onward. PC4 antigen is expressed in addition to Purkinje cells also in granule cells and neurons of deep cerebellar nuclei and appears in Purkinje cells at day 4. M1 antigen (Lagenaur et al. 1980) is first detectable in Purkinje cell bodies by day 5; it is also detectable in deep cerebellar neurons. In the adult retina, only PC4 antigen is detectably expressed and is localized in the inner segments of photoreceptor cells.The neurological mutants weaver, reeler,jimpy and wobbler show detectable levels of these antigens in Purkinje cells. However, the mutants staggerer and Purkinje cell degeneration are abnormal in expression PC1, PC2, PC3, and M1 antigens. Staggerer never starts to express the antigens during development, whereas Purkinje cell degeneration first expresses the antigens, but then loses antigen expression after day 23. PC4 antigen is detectable in the remaining Purkinje cells in staggerer and Purkinje cell degeneration mice at all ages tested in this study. Deep cerebellar neurons are positive for both antigens, PC4 and M1, in all mutants and at all ages studied. In retinas of staggerer and Purkinje cell degeneration mutants, PC4 antigen is normally detectable in the inner segments of photoreceptor cells, even when these have started to degenerate in the case of Purkinje cell degeneration.     

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: functional heterogeneity in cell subpopulations recognized by a monoclonal antibody

A hemocyte surface membrane marker (BGH₁) has been identified using hemocyte-specific monoclonal antibodies (mABs) generated by somatic cell fusion methods. The BGH₁ epitope was expressed on a subpopulation of circulating, glass-adherent blood cells from two strains of the snail, Biomphalaria glabrata. Approximately 40% of the circulating hemocytes from the PR albino (M-line) B. glabrata strain were BGH₁^?, compared to a prevalence of 10% BGH₁⁺ cells in the 10-R2 snail strain. When hemocytes were firmly attached and spread on a glass surface, BGH₁⁺ cells were morphologically distinguishable from BGH₁^? cells by their ovoid shape and the presence of short, thin filopodial projections along the ectoplasmic border. In contrast, BGH₁^? hemocytes were more pleomorphic and possessed long, spike-like filopodia. Moreover, the BGH₁ epitope was trypsin-resistant and retained its antigenic reactivity with probe mABs following fixation with paraformaldehyde or paraformaldehyde/MeOH. Fixation with glutaraldehyde, however, significantly reduced mAB binding to the BGH₁ surface epitope. There was no apparent age-dependent expression of the BGH₁ determinant since circulating hemocyte populations in very young (1–2 mm) to adult (10–12 mm) snails were composed of both BGH₁⁺ and BGH₁^? subpopulations. Quantitative shifts in the prevalence of epitope-bearing hemocytes between the smallest snail size class (1–2 mm) and the larger snails (3–4 and 10–12 mm) are believed to be due to a differential production and/or release of BGH₁^? hemocytes within the blood circulation rather than a gradual age-related change in the expression of surface antigens on individual cells. Experiments designed to assess the in vitro phagocytic capability and lysosomal acid phosphatase (APase) activity of mAB-reactive hemocytes revealed that BGH₁⁺ cells, when compared to those lacking the surface marker, were significantly reduced in both their phagocytic and APase-producing activities. Since the PR albino strain of B. glabrata possesses a higher proportion of BGH₁^? hemocytes and a lower total concentration of circulating cells than do snails of the 10-R2 strain, PR albino snails are thus potentially reduced in their natural capacity to mount cellular reactions against foreign materials.     

Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study

In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalancho? species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.     

The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum,and their transmission by Anopheles stephensi,Anopheles gambiae and Aedes aegypti

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.