Attenuated immunogenic parasites are essential in the transfer of immunity to virulent Plasmodium berghei.

A less virulent parasite of Plasmodium berghei K173 was isolated that induced immunity against the more virulent parasite. Immunity to this parasite but not to the virulent one, could be transferred by immune spleen cells but not by immune lymph node cells. However, the immune spleen cells did transfer immunity to the virulent strain if accompanied by infection with viable parasites of the less virulent strain, but only if they were allowed to proliferate for a period of 1 week before challenge with the virulent strain. Immune spleen cells could survive two cycles of mouse to mouse transfer. The induction of immunity by transfer of immune spleen cells was associated with the production of anti-parasitic antibody.     

Murine model for assessment of Plasmodium falciparum transmission-blocking vaccine using transgenic Plasmodium berghei parasites expressing the target antigen Pfs25

Currently, there is no animal model for Plasmodium falciparum challenge to evaluate malaria transmission-blocking vaccines based on the well-established Pfs25 target antigen. The biological activity of transmission-blocking antibodies is typically assessed using an assay known as the membrane feeding assay (MFA). It is an in vitro method that involves mixing antibodies with cultured P. falciparum gametocytes and feeding them to mosquitoes through an artificial membrane followed by assessment of infection in the mosquitoes. We genetically modified Plasmodium berghei to express Pfs25 and demonstrated that the transgenic parasites (TrPfs25Pb) are susceptible to anti-Pfs25 antibodies during mosquito-stage development. The asexual growth kinetics and mosquito infectivity of TrPfs25Pb were comparable to those of wild-type parasites, and TrPfs25Pb displayed Pfs25 on the surface of ookinetes. Immune sera from nonhuman primates immunized with a Pfs25-based vaccine when passively transferred to mice blocked transmission of TrPfs25Pb to Anopheles stephensi. Furthermore, mice immunized with Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with wild-type plasmid. These studies describe development of an animal malaria model alternative to the in vitro MFA and show that the model can facilitate P. falciparum transmission-blocking vaccine evaluation based on the target antigen Pfs25. We believe that an animal model to test transmission-blocking vaccines would be superior to the MFA, since there may be additional immune factors that synergize the transmission-blocking activity of antibodies in vivo.     

Complementation of Plasmodium berghei TRAP knockout parasites using human dihydrofolate reductase gene as a selectable marker

Previously we have used the Plasmodium dihydrofolate reductase thymidylate synthase (DHFR-TS) selectable marker to generate Plasmodium berghei TRAP null mutant parasites. These TRAP null mutants do not glide and they showed a great reduction in their ability to infect mosquito salivary glands and the hepatocytes of the vertebrate host. Thus far, complementation of these knockout parasites was not possible due to the lack of additional selectable markers. Recently, a new selectable marker, based on the human dihydrofolate reductase (hDHFR) gene, has been developed which confers resistance to the antifolate drug WR99210. This drug has been found to be highly active against pyrimethamine-sensitive and -resistant strains of P. berghei. In this study, we have used the hDHFR gene as a second selectable marker for the complementation of P. berghei TRAP null mutant parasites. Restoration of the TRAP null mutant parasites to the wild-type phenotype was achieved in this study via autonomously replicating episomes bearing a wild-type copy of the TRAP gene. This is the first report of complementation of a mutant phenotype in malaria parasites.     

Differential gene expression in the ookinete stage of the malaria parasite Plasmodium berghei

Plasmodium, the malaria parasite, undergoes a complex developmental program in its mosquito vector. The ookinete is the parasite form which invades the mosquito midgut and is an important stage for genetic mixing. To identify genes expressed during ookinete development and mosquito midgut invasion, purified zygotes and ookinetes of the rodent parasite Plasmodium berghei were used to construct a suppression subtractive hybridization cDNA library, enriched in sequences expressed in the ookinete stage. In addition to four genes coding for previously described major ookinete-secreted proteins, we isolated ookinete-expressed sequences representing 18 predicted genes. Their gene products include proteins involved in signal transduction and regulatory processes. For six of these genes our analysis provides the first evidence for expression in the ookinete stage. A majority of the genes are not expressed in the zygote, the preceding developmental stage. Furthermore, four of the genes are also transcribed in sporozoites, and one of these in merozoites, suggesting that they code for proteins with a function common to Plasmodium invasive stages.     

Stage-specific expression of aldolase isoenzymes in the rodent malaria parasite Plasmodium berghei.

We have cloned two gene (aldo-1 and aldo-2) encoding the glycolytic enzyme aldolase of the rodent malaria parasite Plasmodium berghei. The amino acid sequence of one gene product, ALDO-1, is virtually identical to P. falciparum aldolase whereas ALDO-2, the second gene product, is different and has 13% sequence diversity to ALDO-1. We expressed ALDO-2 as an active enzyme in Escherichia coli and compared the biochemical and kinetic properties to that of P. falciparum recombinant aldolase (ALDO-1 type). Based on the Km and Vmax constants for FMP and FBP, neither ALDO-1 nor ALDO-2 can be clearly assigned to any of the known mammalian isoenzyme classes. We demonstrate that expression of the two isoenzymes is developmentally regulated: specific antibody probes detect ALDO-1 in sporozoite stages of P. berghei and ALDO-2 is found in blood stage parasites.     

Demonstration of the role of cytophilic antibody in resistance to malaria parasites (Plasmodium berghei) in rats.

This paper reports the results of a study of the nature of the immune response against Plasmodium berghei parasites by inbred rats. A macrophage-cytophilic antibody specific for malarial antigens was identified and characterized. Detection of the antibody on the macrophage surface was accomplished by the parasite adherence tests and by the indirect fluorescent antibody technique. Isolation and purification of the macrophage-cytophilic and opsonic antibodies from hyperimmune rat serum was accomplished by QAE-Sephadez A-50 elution chromatography, and of the macrophage-cytophilic antibody by adsorption with and elution from syngeneic macrophages as well. Characterization of the cytophilic antibody as immunoglobulin G1 was done by immunoelectrophoresis and by Ouchterlony-type double diffusion in gel. Passive protection tests in weanling inbred rats have demonstrated that the opsonizing antibody conferred some protection against P. berghei. The macrophage-cytophilic antibody, on the other hand, was not protective alone but acted synergistically with the opsonizing antibody.     

A marker epitope of attenuated Plasmodium berghei

Plasmodium berghei XAT is an irradiation-induced, permanent attenuated derivative from high-virulence P. berghei NK65. Monoclonal antibodies against XAT were developed. By immunofluorescence screening, one monoclonal antibody was identified that was reactive with XAT at the schizont stage but not with NK65 nor with any other stage of intra-erythrocytic development of either parasite. The monoclonal antibody precipitated a 240-kD molecule from metabolically labeled XAT antigens. This molecule was thought to be a marker epitope of the attenuated parasite.     

Molecular karyotyping of the rodent malarias Plasmodium chabaudi, Plasmodium berghei and Plasmodium vinckei

The molecular karyotypes of four isolates of Plasmodium chabaudi, three of the subspecies P. chabaudi adami and one P. chabaudi chabaudi, as well as P. berghei and P. vinckei were studied by means of pulsed field gradient (PFG) gel electrophoresis. Each species appears to have 14 chromosomes, ranging in size from approximately 730 kb to greater than 2000 kb. The three P. chabaudi adami isolates did not appear any more similar to each other than to the P. c. chabaudi isolate. The chromosome locations of genes for a heat shock protein (hsp) 70, ribosomal RNA (rRNA), the precursor to the major merozoite surface proteins, dihydrofolate reductase and P. falciparum antigen 352 as well as four cloned DNA markers and a telomere probe were determined. However, a number of probes representing cloned P. falciparum antigens failed to hybridize to P. chabaudi DNA. Hence genes for malaria antigens appear to be much more divergent than genes for housekeeping functions.     

Host defenses in murine malaria: failure of vaccination with formolized blood parasites to protect athymic mice from Plasmodium berghei

The thymus dependency of immunity to erythrocytic Plasmodium berghei (NYU-2) infection generated in response to injections of Formalin-killed mixed blood parasites was shown by the demonstration that the vaccine protected immunologically intact nu/+ mice, but not their athymic nu/nu littermates.     

Pathophysiology of hypoxia in mice infected with Plasmodium berghei

Pathophysiological significance of hypoxia in malarial infection was investigated in mice infected with Plasmodium berghei NK65. Intraperitoneal inoculation of mice with 1×10⁷ parasitized red blood cells resulted in death of the hosts 6–7 days later. Anaemia of infected animals developed on day 4 after inoculation and oxygen affinity of whole blood, measured as P₅₀ act pH, increased simultaneously. This change may be a physiological adaptive response to a reduction in oxygen delivery to the tissues to day 5. However, the blood oxygen supply on day 6 appeared to be deteriorating and this is thought to be an important factor contributing to the death of the host. The value of adenylate energy charge in red cells during malarial infection, however, was comparatively well-maintained. Allopurinol stimulated the multiplication of malaria parasites and seems to have induced collapse in host-parasite balance more rapidly. Decrease in blood pH and in blood oxygen transport may be important factors for the pathogenesis of the allopurinol-treated hosts.     

Immunization efficacy of cryopreserved genetically attenuated Plasmodium berghei sporozoites

Parasitology Research - Malaria is transmitted through the injection of Plasmodium sporozoites into the skin by Anopheles mosquitoes. The parasites first replicate within the liver before infecting...     

Purification and characterization of DNA polymerases from Plasmodium berghei

DNA polymerases from the malaria parasite Plasmodium berghei were purified more than 50-fold. Several distinct enzymatic activities were isolated that could be distinguished by the use of various specific DNA polymerase inhibitors. In particular, subdivision into an aphidicolin-sensitive and an aphidicolin-resistant group was possible. Further analysis allowed a better comparison with host DNA polymerases and indicated that one aphidicolin-sensitive DNA polymerase resembled DNA polymerase alpha displaying processive DNA synthesis and using RNA primers, whereas another aphidicolin-sensitive DNA polymerase was distributive and only used DNA primers. Marked differences from the host enzymes do exist, however, such as insensitivity to BuPdGTP. Another P. berghei DNA polymerase was isolated that showed characteristics of a DNA polymerase beta-like enzyme, but which differed from host DNA polymerase beta in its insensitivity to dideoxynucleotides.     

Reduced sodium appetite and increased oxytocin gene expression in mutant mice lacking beta-endorphin

Central opioid and oxytocinergic systems have been involved in the regulatory control of sodium appetite. In addition, previous studies support the existence of a functional interaction between opioid peptides and oxytocinergic pathways, and suggest that beta-endorphin neurons would modulate the activity of central oxytocinergic pathways, its pituitary secretion and sodium appetite. To investigate the role of this opioid peptide in the control of oxytocin (OT) synthesis and sodium appetite regulation we used mice with gene dosage-dependent variations in brain beta-endorphin content, expressing either 100%, 50%, or 0% of normal beta-endorphin content. Our results show that beta-endorphin knockout (KO) and heterozygous (HT) mutant mice consume approximately a 50% less 2% NaCl solution compared with wild type mice (WT), after furosemide and low sodium diet treatment. These data suggest that beta-endorphin may facilitate induced sodium appetite, giving new evidence about the role of beta-endorphin on sodium appetite behavior. Our data also indicate that OT mRNA levels evaluated by in situ hybridization significantly increased within the hypothalamic paraventricular nucleus of WT animals after induced sodium ingestion, giving support to former evidence indicating an inhibitory role for central OT in the control of sodium appetite. Moreover, beta-endorphin mutated mice have similar higher levels of OT mRNA expression after the different conditions analyzed: basal, control or experimental, compared with WT mice. Both control HT and KO mice showed higher OT mRNA expression levels than control WT group and these levels did not change after induced sodium intake. Taken together, our data suggest that the reduced sodium ingestion observed in beta-endorphin deficient mice could be due to a higher expression of the OT gene. This conclusion would support the hypothesis that OT inhibits sodium intake and provides new evidence about beta-endorphin modulation of OT synthesis and sodium appetite.     

Immunosuppression in malaria: effect of hemozoin produced by Plasmodium berghei and Plasmodium falciparum

To a considerable degree, malaria-induced immunosuppression has been attributed to an inhibition of macrophage accessory cell function. In this study hemozoin, a plasmodium hemoglobin degradation product which readily accumulates in phagocytic cells and tissues during infection, was examined for its influence on immune responses. Hemozoin-laden liver and splenic macrophages from Plasmodium berghei-infected mice, displayed accessory cell dysfunction which was likely due to hemozoin loading by these phagocytic cells. This indicated by the observation that hemozoin obtained from livers and spleens of infected mice as well as from Plasmodium falciparum cultures greatly inhibited splenic plaque-forming cell responses to sheep red blood cells. The results of the present study strongly suggest that the inhibition of macrophage accessory cell activity is due, at least in part, to the uptake and accumulation of hemozoin in their cytoplasms.