Secreted Antibody Is Required for Immunity to Plasmodium berghei

Infection with Plasmodium berghei is lethal to mice, causing high levels of parasitemia, severe anemia, and death. However, when mice are treated with antimalarial drugs during acute infection, they have enhanced immunity to subsequent infections. With this infection and cure model of immunity, we systematically examined the basis of adaptive immunity to infection using immunodeficient mice. In order to induce adaptive immunity, mice were infected with blood-stage parasites. When the mice developed 2 to 3% parasitemia, they were treated with chloroquine to cure the infection. These convalescent mice were then challenged with homologous blood-stage parasites. Immunized wild-type mice were able to control the level of infection. In contrast, mice lacking mature B cells and T cells were unable to control a challenge infection, indicating the critical role of lymphocytes in immunity to P. berghei. Furthermore, mice lacking secreted antibody were unable to control the level of parasitemia following a challenge infection. Our results indicate that secreted antibody is a requirement for immunity to P. berghei.Each year there are approximately 500 million cases of malaria worldwide, resulting in 2 to 3 million deaths, primarily in children in sub-Saharan Africa (42). Malaria is caused by infection with one of four protozoan Plasmodium species: P. falciparum, P. vivax, P. malariae, and P. ovale. P. falciparum is responsible for the majority of severe disease, which can manifest itself in anemia, cerebral malaria, organ failure, and death. Repeated infection and treatment of individuals in areas of malaria endemicity eventually induce a level of immunity that limits morbidity and results in chronic infection with low levels of parasitemia (41). A fully effective vaccine that reduces parasite burden and severe disease has not been developed.Murine models of malaria have long been used to examine the immune response to Plasmodium parasites and to understand the host factors required for the development of immunity. P. berghei infection in mice is lethal, causing high levels of parasitemia, severe anemia, and body weight loss. However, mice can become resistant to subsequent infections by treatment with antimalarial drugs during acute infection (27). This is known as the infection and cure model, and mice that develop this immunity mimic the human experience of disease in that they are reinfected but experience low-level patent parasitemia and survive. However, it takes years to establish this level of immunity in humans (36), while in mice it is accomplished by only one infection and drug cure, which provides long-lasting protection (12, 13, 48). An understanding of the basis of rodent immunity to blood-stage infection will help to direct future vaccine approaches.The fact that immunity induced by infection and cure is long-lasting suggests that the adaptive immune system is required for immunity. Evidence from previous work indicates a role for B and T cells in immunity. Mice lacking both mature B and T cells (SCID mice) (6), as well as mice deficient in mature B cells (μ-MT mice) (23), were unable to eliminate primary and secondary infection, suggesting that B cells are required for adaptive immunity to Plasmodium species (30, 47). Immunity to P. yoelii, another rodent parasite, can also be induced by infection and cure. In the P. yoelii system, it has been shown that immunity can be passively transferred to naïve recipient mice (17, 21, 34). Hyperimmune serum (from mice infected and challenged multiple times) is most effective; it allowed mice with an active infection to clear blood-stage parasites within 48 h (17). The results from these studies suggest that B cells and antibody are required for immunity, but the requirement for secreted antibody has not been well defined. While all of the studies thus far have relied on mice lacking mature B cells, in this study we were able to examine immunity to P. berghei in a mouse with intact B cells, which express surface immunoglobulin M (IgM) but are unable to secrete antibody (25).Here we use the infection and cure model to examine the requirement for secreted antibody in immunity to P. berghei in the murine host. We establish a model of infection and cure with P. berghei and demonstrate the pivotal requirement for secreted antibody in adaptive immunity to P. berghei.     

TNF and Plasmodium berghei ANKA-induced cerebral malaria

The cerebral pathology observed in Plasmodium berghei ANKA-infected CBA mice has been attributed to overproduction of TNF, the mice in which this syndrome is seen being those with the highest serum TNF levels. To investigate this further, we injected recombinant human TNF into malaria-primed mice to see if we could reproduce the cerebral changes observed in P. berghei ANKA infections. A range of doses, administered as a single or repeated injections, or via osmotic pumps, failed to reproduce these changes, but did induce hypoglycaemia, midzonal liver necrosis and neutrophil adhesion in pulmonary vessels. This pathology is seen in terminal Plasmodium vinckei infections, but absent in terminal P. berghei ANKA. In addition, the permeability of the blood-brain barrier to Evan's blue, which is present in P. berghei ANKA but not in normal or P. vinckei-infected mice, was not induced by exogenous TNF. Serum levels of TNF were measured in an ELISA assay, and found to be consistently higher in P. vinckei rather than P. berghei ANKA terminal infections. This is consistent with the pathological changes we could reproduce by injecting TNF. For these reasons we suggest that the cerebral pathology seen in mice infected with P. berghei ANKA may be governed by TNF produced locally by monocytes sequestered within the cerebral blood vessels, not simply by systemic levels of this cytokine.     

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.     

Transgenic Plasmodium berghei sporozoites expressing beta-galactosidase for quantification of sporozoite transmission

Malaria transmission occurs during a blood-meal of an infected Anopheles mosquito. Visualization and quantification of sporozoites along the journey from the mosquito midgut, where they develop, to the vertebrate liver, their final target organ, is important for understanding many aspects of sporozoite biology. Here we describe the generation of Plasmodium berghei parasites that express the reporter gene lacZ as a stable transgene, under the control of the sporozoite-specific CSP promoter. Transgenic sporozoites expressing beta-galactosidase can be simply visualized and quantified in an enzymatic assay. In addition, these sporozoites can be used to quantify sporozoites deposited in subcutaneous tissue during natural infection.     

Plasmodium berghei resists killing by reactive oxygen species

Reactive oxygen species (ROS) are widely believed to kill malarial parasites. C57BL/6 mice injected with P. berghei inocula incubated with supraphysiological doses of NO (< or =150 microM) or with peroxynitrite (220 microM), however, exhibited parasitemia similar to that seen with those given control inocula, and there was no difference in disease development. Only treatment of inocula with NO doses nearing saturation (> or =1.2 mM) resulted in no detectable parasitemia in the recipients; flow cytometric analysis with a vital dye (hydroethidine) indicated that 1.5 mM NO lysed the erythrocytes rather than killing the parasites. The hemoglobin level in the inocula was about 8 muM; the hemoglobin was mainly oxyhemoglobin (oxyHb) (96%), which was converted to methemoglobin (>95%) after treatment with 150 microM NO. The concentrations of 150 microM of NO and 220 microM of peroxynitrite were far in excess of the hemoglobin concentration (approximately 8 microM), and yet no parasite killing was detected. We therefore conclude that hemoglobin protects Plasmodium parasites from ROS, but the parasite likely possesses intrinsic defense mechanisms against ROS.     

Interaction between Host Complement and Mosquito-Midgut-Stage Plasmodium berghei

After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement on Plasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules.     

Variant-Specific Immunity to Plasmodium berghei in Pregnant Mice

We have investigated the immunological basis of pregnancy-related Plasmodium berghei recrudescence in immune mice with substantial preexisting immunity. Specifically, we examined the relevance of this experimental model to the study of pregnancy-associated malaria (PAM) caused by P. falciparum in women with substantial preexisting protective immunity. We used mice with immunity induced prior to pregnancy and employed flow cytometry to assess their levels of immunoglobulin G (IgG) recognizing antigens on the surfaces of infected erythrocytes (IEs) in plasma. After immunization, the mice did not possess IgG specific for antigens on IEs obtained during pregnancy-related recrudescence but they acquired recrudescence-specific IgG over the course of several pregnancies and recrudescences. In contrast, levels of antibodies recognizing IEs from nonpregnant mice did not increase with increasing parity. Furthermore, maternal hemoglobin levels increased and pregnancy-related parasitemia decreased with increasing parity. Finally, parasitemic mice produced smaller litters and pups with lower weights than nonparasitemic mice. Taken together, these observations suggest that levels of antibodies specific for recrudescence-type IEs are related to the protection of pregnant mice from maternal anemia, low birth weight, and decreased litter size. We conclude that the model replicates many of the key parasitological and immunological features of PAM, although the P. berghei genome does not encode proteins homologous to the P. falciparum erythrocyte membrane protein 1 adhesins, which are of key importance in P. falciparum malaria. The study of P. berghei malaria in pregnant, immune mice can be used to gain significant new insights regarding malaria pathogenesis and immunity in general and regarding PAM in particular.Pregnancy-associated malaria (PAM) is a major cause of mother-offspring morbidity and mortality in areas with stable transmission of Plasmodium falciparum parasites, despite protective immunity to P. falciparum malaria acquired by the mother prior to the first pregnancy (7, 29). Susceptibility to PAM declines with increasing parity due to the acquisition of protective immunoglobulin G (IgG) with specificity for parasite-encoded, clonally variant surface antigens (VSA) that are selectively expressed on infected erythrocytes (IEs) that become sequestered in the placenta (14, 34). The PAM-specific VSA (VSA_PAM) are functionally and antigenically distinct from the VSA expressed by P. falciparum parasites infecting nonpregnant hosts, and the lack of VSA_PAM-specific IgG appears to be the main reason for the high susceptibility to PAM in primigravidae possessing substantial preexisting protective immunity (4, 13, 28). The best-studied VSA are the high-molecular-weight P. falciparum erythrocyte membrane protein 1 (PfEMP1) molecules encoded by the var gene family, with about 60 members per haploid parasite genome (17, 35). VAR2CSA appears to be the only PfEMP1 molecule involved in the pathogenesis of, and protective immunity to, PAM (30, 31). Studies of VSA-specific immunity to P. falciparum malaria have been frustrated by the lack of convenient and relevant animal models. Although rodent malaria parasites lack var gene orthologs, antigenic variation and IE sequestration occur with several Plasmodium species (1, 19, 23, 41), and these species possess multigene families that appear to encode IE surface-expressed VSA (6, 16). This fact notwithstanding, only limited information regarding the roles of the products of these gene families in pathogenesis and immunity is available (21, 22).In a series of papers published in the ''80s, Van Zon and coworkers developed a mouse model to study the impact of pregnancy on immunity to P. berghei infection. Importantly, they used the model to demonstrate pregnancy-related recrudescences accompanied by severe clinical symptoms in mice with preexisting acquired protective immunity (38). Furthermore, they found that susceptibility to recrudescence appeared to decrease with increasing parity (39, 40). In these aspects, their model resembles PAM caused by P. falciparum in areas where malaria is endemic, where women generally develop substantial clinical immunity to malaria before reproductive age. In the present study, we reevaluated the model developed by Van Zon et al. in view of the recent evidence pointing to the clinical importance of VSA-specific antibody responses in PAM. We show that the apparent breakdown of preexisting protective immunity to P. berghei K173 infection during pregnancy is in fact the consequence of the emergence of parasites expressing pregnancy-specific VSA to which the animals do not possess antibodies if they have never been pregnant before. Furthermore, antibodies to these pregnancy-specific VSA are acquired in a parity-dependent manner and appear to be related to protection from pregnancy-related recrudescence, maternal anemia, low birth weight, and reduced litter size.     

The putative gene for the first enzyme of glutathione biosynthesis in Plasmodium berghei and Plasmodium falciparum

The putative gene for gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, has been characterized both in Plasmodium berghei and Plasmodium falciparum. Protein sequence comparison between these two species reveals large conserved regions sharing more than 80% similarity, separated by less conserved portions. When the comparison is extended to known gamma-glutamylcysteine synthetases from other eukaryotes, a number of high similarity blocks are observed which may help in identifying sequence essential for protein function.     

Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors

Experimental reverse genetic approaches have proven powerful in the study of the biology of the malaria parasite. The murine malaria model parasite Plasmodium berghei is the genetically most amendable Plasmodium species and allows full access to the entire life cycle in vivo. Here, we describe a next-generation, highly versatile transfection vector set that facilitates advancing experimental genetic strategies towards a genome-wide scale. Through 36 consecutive cloning and 17 subcloning steps an optimized vector set was generated from the standard transfection plasmid. These targeting vectors, collectively referred to as the Berghei Adaptable Transfection (pBAT) plasmids, contain key elements that permit recycling of the drug-selectable cassette, robust green fluorescent labelling of recombinant parasites, carboxy-terminal tagging of target proteins with a red fluorescent-epitope tag fusion, and expression of heterologous genes. The vectors were further optimized for small size, versatile restriction endonuclease recognition sites and potential exchange of individual vector elements. We show that stable integration into a transgene expression site, an intergenic locus at a synteny breakpoint on P. berghei chromosome 6, is phenotypically silent and generated a bright green fluorescent parasite line for imaging applications. We provide an example, P. berghei actin 2, for targeted gene deletion and illustrate that the positive selection marker can be recycled, thereby permitting multiple rounds of genetic manipulations. We propose that the vectors described herein will greatly facilitate functional assignment to predicted and orphan Plasmodium gene models by multiple experimental genetics approaches.     

Anti-thymocyte serum effects on Plasmodium berghei infection in rats

Rabbit anti-rat thymocyte serum (ATS) virtually abolished the innate resistance mechanism(s) of rats of different ages to primary infection with Plasmodium berghei. ATS apparently produced its immunosuppressant effect without arresting humoral antibody production as judged by agar-gel double diffusion tests. These results, when considered together with those of other workers, suggest that cell-mediated immunity is substantially involved in resistance to P. berghei.     

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.     

Small-scale in vitro culture and purification of Plasmodium berghei for transfection experiment

The standard protocol for genetic modification of the rodent malaria parasite Plasmodium berghei requires infected blood from one or more laboratory mice, followed by large-scale in vitro parasite culture and purification of mature schizonts. Here, protocols are described for small-scale in vitro culture from 20 μL of mouse tail blood and purification of mature P. berghei schizonts sufficient for a single transfection experiment. All procedures are performed in 1.5-mL microcentrifuge tubes. We confirmed that transgenic parasites could be obtained using schizonts prepared by this protocol. This small-scale protocol provides significant advantages, namely reduction of parasite sample, laboratory consumables and mice for transfection experiments.     

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.     

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.