Pf332-C231-reactive antibodies affect growth and development of intra-erythrocytic Plasmodium falciparum parasites

The Plasmodium falciparum antigen 332 (Pf332), is a megadalton parasite protein expressed at the surface of infected red cells during later stages of the parasite's developmental cycle. Antibodies to different parts of this antigen have been shown to inhibit parasite growth and adherence to host cells with or without ancillary cells. However, the mechanisms involved in these inhibitions remain largely unknown. We further analysed the activities of specific antibodies with regard to their specific mechanisms of action. For these analyses, affinity purified human antibodies against epitopes in the C-terminal fragment of Pf332 (Pf332-C231) were employed. All purified antibodies recognized Pf332-C231 both by immunofluorescence and ELISA. IgG was the main antibody isotype detected, although all sera investigated had varying proportions of IgG and IgM content. All the antibodies showed a capacity to inhibit parasite growth in P. falciparum cultures to different extents, mainly by acting on the more mature parasite stages. Morphological analysis revealed the antibody effects to be characterized by the presence of a high proportion of abnormal schizonts (15-30%) and pyknotic parasites. There was also an apparent antibody effect on the red cell integrity, as many developing parasites (up to 10% of trophozoites and schizonts) were extracellular. In some cases, the infected red cells appeared to be disintegrating/fading, staining paler than surrounding infected and uninfected cells. Antigen reversal of inhibition confirmed that these inhibitions were antigen specific. Furthermore, the growth of parasites after 22-42 h exposure to antibodies was investigated. Following the removal of antibody pressure, a decreased growth rate of these parasites was seen compared to that of control parasites. The present study confirms the potential of Pf332 as a target antigen for parasite neutralizing antibodies, and further indicates that epitopes within the C231 region of Pf332 should constitute important tools in the dissection of the role of Pf332 in the biology of the malaria parasite, as well as in the design of a malaria vaccine.     

Parasite cultivation in relation to research on the chemotherapy of malaria.

Attempts to develop techniques for the continuous in vitro cultivation of the malaria parasite have not yet proved successful. It has not been possible to obtain the complete sporogonic development of the parasite in vitro although some progress was made with Plasmodium relictum and P. berghei. Exoerythrocytic stages of P. gallinaceum have been successfully cultivated in vitro in tissue explants and those of P. fallax have been grown in turkey primary embryo tissue cells. With the recent development of mammalian liver cell lines, prospects for the in vitro cultivation of exoerythrocytic stages of mammalian plasmodia are greatly improved. While it is still not possible to cultivate erythrocytic stages of plasmodia serially in vitro some species have been successfully grown through one asexual cycle. This progress has led to a number of applications of parasite cultivation to chemotherapeutic studies, to the testing of new antimalarial drugs, and especially to the testing of the susceptibility of P. falciparum to chloroquine. Cultivation technique is greatly improved by an appropriate choice of culture media. The addition of fresh red cells to the subculture system permits relatively high rates of invasion and multiplication of the parasite to be obtained. As well as its application in the screening and evaluation of antimalarial compounds, the in vitro cultivation technique is also very suitable for studying the entry mechanism of the parasite into red blood cells.     

Studies on serum requirements for the cultivation of Plasmodium falciparum. 2. Medium enrichment

Previous experiments using RPMI 1640 medium have indicated that the dialysis of human serum removes components of low relative molecular mass (6000-8000 RMM) that are essential for continuous cultivation of Plasmodium falciparum. To determine which low-RMM components are important for parasite development, we compared growth in normal serum to that in dialysed serum using a number of other commercially available media, which we considered to be richer than RPMI 1640. Through these comparisons, we determined that hypoxanthine was the major dialysable nutrient required for parasite development. High quality bovine serum requires 3 - 12 × 10^-5 mol/litre of hypoxanthine as a supplement to support continuous cultures of P. falciparum. Thus far we have been unable to attain parasite growth in medium containing supplemented bovine serum that is as good as growth in medium containing human serum.     

Mannosylated liposomes formulated with whole parasite P. falciparum blood-stage antigens are highly immunogenic in mice

The development of a blood-stage malaria vaccine has largely focused on the subunit approach. However, the limited success of this strategy, mainly due to antigenic polymorphism and the failure to maintain potent parasite-specific immune responses, indicates that other approaches must be considered. Whole parasite (WP) vaccines offer many advantages over sub-units; they represent every antigen on the organism, thus limiting the effects of antigenic polymorphism, and similarly they compensate for individual Immune-Response (Ir) gene-regulated non-responsiveness to any particular antigen. From a development perspective, they negate the need to identify and compare the relative efficacies of individual candidate antigens. WP vaccines induce protective immunity that is largely cell-mediated.However, WP blood-stage vaccines present a number of challenges for the development pathway. Key issues are cryopreservation and storage and the possible induction of antibodies against red blood cell surface antigens, even if the parasites are grown in blood group O, Rh negative blood. Here, we used a novel adaptation of an immunomagnetic method from STEMCELL™ Technologies to remove the red cell membranes from human red blood cells parasitized with P. falciparum. We then used these antigens to construct liposomes which were modified to present mannose on their membrane to target the liposome to antigen presenting cells. We then compared the immunogenicity of freshly prepared and lyophilized liposome vaccines. Following vaccination of mice, liposomes induced significantly lower antibody responses to human red cells but potent strain- and species-transcending cell-mediated immune responses to parasite antigens. These data support transitioning the P. falciparum liposomal vaccine into clinical studies.     

Decreased growth of Plasmodium falciparum in red cells containing haemoglobin E, a role for oxidative stress, and a sero-epidemiological correlation

The in vitro growth of Plasmodium falciparum in red cells containing haemoglobin E (HbE) was studied at oxygen concentration of 5 to 20%, with and without antioxidants. Under all conditions, parasite growth decreased as the concentration of HbE increased as compared with growth in red cells containing only HbA. The decreases were proportionately greatest at the highest oxygen concentration. The antioxidant vitamin C partially reversed the decreases in growth observed in HbE-containing cells at 20% oxygen. South-east Asian refugees with HbAE or HbEE had high antimalarial IFA titres, indicative of exposure to malaria more frequently than did refugees with HbAA. The decreased growth of P. falciparum in HbE-containing red cells may reduce the severity of malaria infections, conferring a survival advantage and thus increasing the numbers of individuals with HbE in local areas of South-east Asia with high incidences of malaria.     

Mini-Column for Cytoadherence: A New Method for Measuring the Relative Size of Binding Subpopulations in Plasmodium falciparum Isolates

Background

Cytoadherence of Plasmodium falciparum- infected red blood cells to endothelial cells is an important mechanism for parasite survival and a major trigger for diseases pathology. Here, we describe a new adhesion assay in which different cell types (CHO, CHO/CD36 and CHO/ICAM-1) are attached to Cytodex beads in a mini-column format to measure the relative sizes of various binding subpopulations as a percentage of the total population.

Methods

Relative size of CD36 and ICAM-1-binding subpopulations of erythrocytes infected with P. falciparum were measured by amount of parasitemia before and after passing the infected erythrocytes through a particular column.

Results

The mini-column adhesion assay was a suitable method as parasitemia always reduced after passing through a particular column in independent experiments. For example, in a typical experiment using P. falciparum ITG line, 75% of the parasites are retained on a CHO/ICAM-1 while 0% of clone 3D7 is retained.

Conclusion

This work introduced and validated a method for measuring the relative size of parasite binding subpopulations and the selection of them. Also, the mini-column method is of value for assessments of cytoadherence and can be used as tool for different applications.     

Diagnosis of Plasmodium falciparum infection using a solid-phase radioimmunoassay for the detection of malaria antigens

A serodiagnostic test has been developed for the detection of Plasmodium falciparum in infected blood. Using parasite antigens and infected red blood cells from in vitro cultures of P. falciparum and malaria antibody from high-titre Gambian sera, parasites were detected in a solid-phase radioimmunoassay (RIA) that measured antibody-binding inhibition. Lysed RBC were incubated with labelled IgG purified from immune sera and were then placed in antigen-coated microtubes and incubated. The degree of inhibition of antibody binding in the tubes correlated with the level of parasitaemia in the test RBC and, using dilutions of RBC from in vitro cultures of P. falciparum, the test detected infection at a level of 8 parasites/10⁶ red blood cells. The test was applied to RBC from 100 healthy European blood donors and to samples of RBC from 500 Gambians from the up-country villages of Keneba and Manduar. More samples were positive by RIA than by microscopy and there was a highly significant degree of correlation between the RIA and microscopy results.     

Non-specific immunity to Plasmodium falciparum: in vitro studies

Peripheral blood mononuclear cells (PBM) were cultured with Plasmodium falciparum malaria for several cycles of parasite growth. Non-immune PBM inhibited P. falciparum more frequently than immune PBM. The inhibiting factor appears to be released during the first 24 hours of culture and is a non-dialysable factor, probably originating from monocytes. The relevance of these findings to in vivo immunity is discussed.     

5. Variation of Plasmodium falciparum msp1 block 2 and msp2 allele prevalence and of infection complexity in two neighbouring Senegalese villages with different transmission conditions

To investigate the impact of transmission on the development of immunity to malaria and on parasite diversity, longitudinal surveys have been conducted for several years in Dielmo and Ndiop, 2 neighbouring Senegalese villages with holo- and mesoendemic transmission conditions, respectively. We analysed Plasmodium falciparum msp1 block 2 and msp2 genotypes of isolates collected from 58% of the Dielmo villagers during the same week as those studied recently from Ndiop. Allele frequencies differed in both villages, indicating considerable microgeographical heterogeneity of parasite populations. The complexity of the infections, estimated using individual or combined msp1 and msp2 genotyping, in Dielmo was more than double that in Ndiop and it was age-dependent in Dielmo but not in Ndiop. Thus, this study confirmed the influence of age on the complexity of asymptomatic malaria infections in a holoendemic area. The age distribution of complexity in Dielmo substantiates the interpretation that the number of parasite types per isolate reflects acquired antiparasite immunity. This cross-sectional survey also confirms that the sickle cell trait has no impact on complexity but influences the distribution of P. falciparum genotypes.     

Diagnosis of Plasmodium falciparum infection by spot hybridization assay: specificity, sensitivity, and field applicability

The spot hybridization assay for the detection of Plasmodium falciparum reported here uses as probe a repetitive DNA sequence from this species and exhibits a high degree of species specificity. Isolates from African, Asian, and South American patients were positive in the assay and gametocytes could be detected at the same level of parasitaemia as asexual parasites. An RNA probe containing the same repetitive sequence as the DNA probe has a detection limit of 1 parasite per 10⁶ red blood cells. Comparison of the results of the assay with those obtained by microscopic examination of blood films indicated that the assay was more sensitive than microscopy if the blood films were examined for only 10 minutes; however, 40 minutes'' examination by microscopy was slightly more sensitive than the assay.     

Plasmodium falciparum polypeptides released during in vitro cultivation

Synchronous cultures of Plasmodium falciparum were successively labelled with (³⁵S)-methionine and both the supernatants and the pellets of infected red blood cells were collected. The release of TCA-precipitable material in the culture supernatants was low during the development of ring forms and trophozoites, increased during schizogony, and was maximum at the time of schizont rupture and merozoite reinvasion. Analysis of the supernatants by SDS — PAGE and autoradiography showed that both polypeptides common to the various developmental stages of the parasite and schizont/merozoite-specific polypeptides were released. Polypeptides of relative molecular mass 140 000, 82 000 and, to a lower degree, 41 000 were present in high amounts in the culture supernatants. These polypeptides have been shown to be the target of monoclonal antibodies that are able to inhibit the growth of P. falciparum cultures, and may be involved in protective immunity. The released polypeptides may also be used as target antigens in immunodiagnostic tests aiming at the detection of malaria infection.     

Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response

Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. Like most living organisms, it is susceptible to oxidative stress, and must adapt to distinct environments. Hence, DNA repair is essential for its survival and the persistence of infection. Therefore, we studied whether T. cruzi has a homolog counterpart of the MutY enzyme (TcMYH), important in the DNA Base Excision Repair (BER) mechanism. Analysis of T. cruzi genome database showed that this parasite has a putative MutY DNA glycosylase sequence. We performed heterologous complementation assays using this genomic sequence. TcMYH complemented the Escherichia coli MutY- strain, reducing the mutation rate to a level similar to wild type. In in vitro assays, TcMYH was able to remove an adenine that was opposite to 8-oxoguanine. We have also constructed a T. cruzi lineage that overexpresses MYH. Although in standard conditions this lineage has similar growth to control cells, the overexpressor is more sensitive to hydrogen peroxide and glucose oxidase than the control, probably due to accumulation of AP sites in its DNA. Localization experiments with GFP-fused TcMYH showed this enzyme is present in both nucleus and mitochondrion. QPCR and MtOX results reinforce the presence and function of TcMYH in these two organelles. Our data suggest T. cruzi has a functional MYH DNA glycosylase, which participates in nuclear and mitochondrial DNA Base Excision Repair.     

Recent progress in the development of malaria vaccines: Memorandum from a WHO Meeting

The sixth meeting of the Scientific Working Group on the Immunology of Malaria reviewed studies on the identification and analysis of malarial antigens of asexual blood stages and sexual stages (gametes, zygotes, ookinetes) that may be exploited as targets for vaccination. Several proteins have been identified on the surface of mature schizonts and free merozoites, some of which can be recognized by antibodies which block in vitro parasite growth. Immunization of rodents and monkeys with purified antigens from the parasite surface membrane has conferred substantial immunity against subsequent challenge. A new class of malarial antigens has been identified which bind specifically to glycophorin, the major erythrocyte glycoprotein; these antigens are on the merozoite surface and it is possible that they mediate attachment to erythrocytes. Antibodies against these proteins also block parasite growth in vitro. The Plasmodium falciparum S-antigens have been characterized biochemically and the genes for two of these proteins sequenced. Several antigens have been localized in the invasion process, and monoclonal antibodies against these proteins block in vitro growth. Malarial antigens on the surface of P. falciparum trophozoite and schizont-infected erythrocytes may be involved in the cytoadherence of infected erythrocytes to endothelial cells. Surface antigens on gametes and zygotes of P. gallinaceum and P. falciparum have been shown to be the targets of transmission-blocking immunity. Monoclonal antibodies specific for these antigens block fertilization in the mosquito midgut. Transmission of P. gallinaceum can also be blocked by an antibody that blocks development of zygotes into ookinetes. Studies on the transmission of P. yoelii have identified a gamete protein that immunizes mice against transmission to mosquitos.     

A foreign invader or a reclusive native? DNA bar coding reveals a distinct European lineage of the zoonotic parasite Schistosoma turkestanicum (syn. Orientobilharzia turkestanicum ())

Natural foci of Schistosoma turkestanicum (syn. Orientobilharzia turkestanicum) has been identified in the Gemenc Forest regions of Hungary utilising red deer as the definitive host. In order to identify the origins of this parasite in Europe standard DNA bar coding techniques were employed to sequence fragments of the cytochrome oxidase 1 (cox1) and the nuclear ribosomal internal transcribed region (ITS) from 10 individual adult male worms. Phylogenetic reconstruction using maximum likelihood phylogenetic reconstruction and haplotype networks of the cox1 showed all the worms to be of a distinct unique Hungarian lineage although some ITS haplotypes were shared with worms from populations in China and Iran. Molecular clock analysis suggests an early divergence event around 270,000 years before present (YBP) between all S. turkestanicum populations giving rise to the Chinese, Iranian and Hungarian lineages. However, divergence of the sequences within the Hungarian population appears to have occurred approximately 63,000 YBP suggesting a long established population of S. turkestanicum in Europe. This suggests that the Hungarian population of S. turkestanicum has been native since the Ice Age and probably established itself during the last interglacial period as red deer moved into Europe from North Africa and the Middle East. This may also indicate that the parasite may have unknown populations established in several other countries in Eastern, Central and Southern Europe.     

Use of human plasma for continuous in vitro cultivation of Plasmodium falciparum

Plasma from units of human blood collected in CPDA-1 preservative were compared with human serum in cultures of Plasmodium falciparum. No significant differences in parasite growth and multiplication were seen between cultures containing serum or plasma. The wider availability of plasma makes it an attractive alternative to serum, especially in large scale cultures.     

Erythrocyte miRNA regulators and malarial pathophysiology

Pathophysiology of Plasmodium falciparum and Plasmodium vivax in malaria vis a vis host and the parasite genome interactions has been deciphered recently to present the biology of cerebral malaria, severe anaemia and placental malaria. Small non-coding RNAs have exhibited their potential to be considered as indicators and regulators of diseases. The malarial pathologies and their associated mechanisms mediated by miRNAs and their role in haematopoiesis and red cell-related disorders are elucidated. Evidence of miRNA carrying exosome-like vesicles released during infection, delivering signals to endothelial cells enhancing gene expression, resulting in parasite sequestration and complications leading to pathologies of cerebral malaria are important breakthroughs. Pregnancy malaria showed Plasmodium surface antigen promoted erythrocyte sequestration in the placental intervillous space, provoking disease development and assorted complications. Syncytiotrophoblast-derived microparticles during pregnancy and fetus development may predict pathophysiological progression on account of their altered miRNA cargoes in malaria.     

Pesticide-containing diets augment anti-sheep red blood cell nonreaginic antibody responses in mice but may prolong murine infection with Giardia muris

The effects of oral ingestion of different pesticides (lindane, phosalone, carbaryl) and of a polychlorinated biphenyl (pyralene) on nonreaginic antibody production in $\text{BALB}\text{c}$ mice and also on the duration of Giardia muris infection have been studied using the animal model of this disease. Only lindane and carbaryl produced significant effects on systemic antibody production following oral immunization with sheep red blood cells (SRBC). Lindane ingestion led to a twofold increase in IgG2b antibody titer to SRBC (P < 0.05) and carbaryl significantly increased both IgG1 and IgG2b titers. No reduction was seen in the synthesis of any antibody class. The effect of feeding with lindane was to increase the duration of giardiasis significantly (P < 0.002), and these mice developed systemic anti-Giardia antibodies more frequently than Giardia-infected mice on normal diets. These results suggest that in the doses used these pesticides may increase systemic antibody responses to orally ingested antigens, and by inference that spontaneous elimination of giardiasis is independent of the systemic nonreaginic antibody response to the parasite.     

Angiostrongylus cantonensis in a Red Ruffed Lemur at a Zoo,Louisiana, USA

A red ruffed lemur (Varecia rubra) from a zoo in Louisiana, USA, was euthanized for worsening paresis. Brain and spinal cord histology identified eosinophilic meningoencephalomyelitis with intralesional adult Angiostrongylus sp. nematodes. PCR and sequencing confirmed A. cantonensis infection, indicating this parasite constitutes an emerging zoonosis in the southeastern United States.     

A step forward in the understanding of the presence and expansion of Echinococcus multilocularis in Eastern Europe using microsatellite EmsB genotyping in Poland

Alveolar echinococcosis is a severe zoonotic disease caused by the parasite Echinococcus multilocularis. In Europe, the lifecycle of this cestode is mainly sylvatic based on a prey-predator interaction between the red fox and small rodents as definitive and intermediate hosts, respectively. National surveillance of E. multilocularis in red foxes in Poland has reported a clear distinction between low endemic areas (from 2 to 5.7%) in the western half and high endemic areas (11.8 to 50.0%) in the eastern half of the country. A drastic increase of prevalence has been observed in the eastern half of Poland since the 2000's. Microsatellite EmsB genotyping was performed on 301 E. multilocularis worms from 87 foxes sampled throughout Poland, leading to identification of 29 EmsB profiles. The main profile, Pol19, was identified across the country and accounted for 44.9% of the worms collected. The conformity of 18 Polish profiles was established by comparison with previous profiles identified in Europe, but none corresponded to the most common European profiles. Poland was confirmed as a peripheral area of the main European focus, with more recent colonization by the parasite. The sharing of common profiles mainly by neighboring provinces was confirmed by a clustering analysis identifying four main groups. Expansion of the parasite in Poland in these four groups appears to be influenced by the situation in neighboring countries. Acquiring EmsB genotyping data from eastern European countries, for which very few data are reported, is necessary to understand the expansion of the parasite in the whole of Europe.     

Observations on the epidemiology of sickle cell disease

The four common genotypes of sickle cell disease in Jamaica are homozygous sickle cell (SS) disease, sickle cell-haemoglobin C (SC) disease, sickle cell-β⁺ thalassaemia, and sickle cell-β⁰ thalassaemia with respective incidence at birth of 3·2, 2· 0,0· 34, and 0· 16 per 1000 live births. Haematological indices, clinical features, and over-all prognosis vary between these genotypes and also between patients within individual genotypes. Although symptomatic selection has tended to emphasize more severely affected patients, this wide variation of clinical and haematological severity is especially apparent in SS disease. Factors contributing to this variability in SS disease include the persistence of foetal haemoglobin, the association with alpha thalassaemia, and the interaction with environmental factors of which socioeconomic status is the most obvious. Further elucidation of factors determining the severity of SS disease will increase understanding of the pathogenetic mechanisms in the disease and may also identify new possibilities for therapeutic intervention.