Strain-specific immunity induced by immunization with pre-erythrocytic stages of Plasmodium chabaudi

One of the most promising approaches in the efforts to produce a malaria vaccine involves the use of attenuated whole sporozoite immunizations. Attenuation may be achieved by the use of genetic modification, irradiation, chemical attenuation, or by the contemporaneous administration of antimalarial drugs that target only the erythrocytic stages of the parasite. Most research to date has focused on the efficacy of these approaches upon challenge with parasites homologous to those used for the initial immunizations. We, as have others, have previously shown that a component of the immunity achieved against the erythrocytic stages of the rodent malaria parasite Plasmodium chabaudi chabaudi is strain-specific, with a stronger immune response targeting the immunizing strain than genetically distinct strains. Here, we show that the immunity induced by infection with the pre-erythrocytic stages of these parasites, achieved via inoculation of sporozoites contemporaneously with mefloquine, also has a strain-specific component.     

The role of reactive nitrogen intermediates in modulation of gametocyte infectivity of rodent malaria parasites

Direct feeding of Anopheles stephensi mosquitoes on mice infected with Plasmodium vinckei petteri showed that, during the periods of schizogony in the blood, the infectivity of gametocytes was markedly reduced. This could be prevented by prior injection of the L-arginine analogue, Nw-nitro-L-arginine (NwNLA) showing that the altered infectivity was due to reactive nitrogen intermediates (RNI). Similar effects on transmission of P. yoelii nigeriensis were demonstrated in vitro by membrane feeding of the mosquitoes. The in vitro reduction in infectivity could be reversed by injecting the L-arginine analogue either into the infected mouse donor of serum, or into the membrane feeding chamber. Elevated levels of TNF and IL-6 were demonstrated during the course of infection but did not correlate well with nitrogen radical activity. Similarly, direct measurements of NO₂- and NO₃- did not reflect the nitrogen radical activity revealed by addition of the specific L-arginine analogue.     

Mature liver stages of cloned Plasmodium falciparum share epitopes with proteins from sporozoites and asexual blood stages

The liver merozoites of malaria parasites are of paramount importance, as they initiate the parasite invasion of red blood cells and start the cycle associated with the clinical features of malaria. Investigating liver merozoite antigen is difficult because of the lack of a rodent model of human malaria. In addition, only a low proportion of cells are obtained in vivo, the parasites from Cebus and Aotus monkeys are immature, and in-vitro experiments with liver cells are often confounded by contamination with the natural mosquito flora copurified with the sporozoites used for seeding the liver cultures. In our study, mature liver schizonts were shown to possess many of the antigenic determinants recognized by MoAbs and sera specific for defined sporozoite and blood-stage antigens. We employed an immunofluorescence procedure based on evaluating parasites in cryosections prepared from infected chimpanzee liver. Sufficient numbers of sectioned parasites were evaluated with each antibody to assure the reproducibility of the results, and the fixation procedure used was sufficiently non-destructive to parasite antigens so that clear differences between reactions of specific antibodies and negative controls were observed. Our evidence for sharing of epitopes by liver merozoites and sporozoites or by liver merozoites and asexual blood-stage parasites raises the possibility that immune responses elicited against sporozoites or asexual stage antigens being considered as vaccine candidates may also act against this important, little-studied stage of the parasite.     

Vaccination to prevent transmission of Plasmodium yoelii malaria

Summary It was possible to block the transmission of infection of the rodent malaria parasite Plasmodium yoelii nigeriensis to Anopheles stephensi mosquitoes by immunizing mice with a vaccine containing formalin-fixed gametes. Both intramuscular and intravenous routes were effective, immunity was achieved with a single dose and the immunity persisted for 6 months at least. Transmission-blocking immunity was found to reside in a serum factor, probably antibody, and to be directed against extracellular gametes, acting on them in the gut of the mosquito, while gametocytes in the circulation of the vertebrate host remained unaffected. The gamete vaccine afforded partial protection against the disease, but immunization with asexual parasites alone showed that this protection was due largely to the presence of asexual forms as contaminants and that anti-gamete immunity is stage specific.     

Depression of virus-specific cytotoxic T-cell responses during murine malaria

Mice with self-limiting P. yoelii or fatal P. berghei infections exhibited a markedly impaired ability to mount specific splenic cytotoxic T-lymphocyte responses to immunization with infectious ectromelia (EV), vaccinia (VAC), or lymphocytic choriomeningitis viruses (LCMV). Lymph node responsiveness, however, was not impaired. Primary CTL responses were depressed in mice immunized 7 days after P. berghei infection, while in P. yoelii-infected mice, depressed responses were detected only during the period corresponding with maximal parasitemia (days 9-12). Secondary VAC-specific CTL responses in vitro by spleen cells of mice previously immunized during P. yoelii infection were also depressed if UV-inactivated rather than infectious VAC was used for immunization. In addition, spleen cells of mice already immune to VAC failed to yield normal secondary CTL responses in vitro during the period of maximal P. yoelii parasitaemia. Collectively, these findings indicate that, during patent malaria infections, priming for and expression of virus-specific CTL responses may be inhibited.     

Monoclonal anti-gamete antibodies prevent transmission of murine malaria

Three monoclonal antibodies prepared using spleen cells from mice immunized with microgametes of Plasmodium yoelii nigeriensis were tested for their ability to block transmission of the infection. Two of them agglutinated microgametes and blocked transmission, this effect being antibody-dose dependent. The third monoclonal used alone was ineffective in both these assays although it stained gametocytes and microgametes by immunofluorescence in the same way as the protective monoclonals. However, when it was administered in combination with one of the protective monoclonals the transmission blocking immunity was enhanced significantly, indicating a synergistic effect of the two antibodies.     

Effects of irradiation on Plasmodium falciparum sporozoite hepatic development: implications for the design of pre-erythrocytic malaria vaccines

Immunization with irradiation-attenuated Plasmodium sporozoites confer protection against live sporozoite challenge. Protection relies primarily on cytotoxic lymphocyte activity against infected hepatocytes, and is suppressed when sporozoites are over-irradiated. Here, we demonstrate that over-irradiated (25-30 krad) Plasmodium falciparum sporozoites invade human hepatocytes and transform into uninucleate liver-trophozoites with the same efficiency as non-irradiated and irradiation-attenuated (12-15 krad) sporozoites. Since hepatocytes infected with over-irradiated non-protective sporozoites are likely to express sporozoite-derived peptide/major histocompatibility complex class I molecules on their surface, our results strongly suggest that sporozoite proteins are not the main immunogens involved in protection, and thus may not per se constitute proper malaria vaccine candidates.     

Detection of malaria sporozoites by standard ELISA and VecTestTM dipstick assay in field-collected anopheline mosquitoes from a malaria endemic site in Ghana

We compared the VecTestTM dipstick assay for detection of Plasmodium sporozoites in Anopheles vectors of malaria with standard circumsporozoite (CS) microplate ELISA for detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in Anopheles mosquitoes. Mosquitoes were collected from a malaria endemic site (Kassena Nankana district) in northern Ghana. Of 2620 randomly sampled mosquitoes tested, the standard CS-ELISA gave a sporozoite rate of 10.8% compared with 11.2% by VecTestTM, which was not statistically different (P = 0.66). Visual reading of the CS-ELISA results gave a sporozoite rate of 13.4%, which was higher than the other tests (P > 0.05). To allow a more objective evaluation of the sensitivity of the dipstick, an additional 136 known CS-ELISA-positive specimens were analysed. The prevalence of the test (including the additional samples) was 14.6% and 14.7% for CS-ELISA and dipstick, respectively (P > 0.05). The estimated prevalence by visual assessment of the CS-ELISA results was 17.5%. The relative specificity and sensitivity of the VecTestTM dipstick and visually read ELISA were estimated based on the CS-ELISA as a gold standard. The specificities of the dipstick and visual ELISA were high, 98.0% and 96.6%, respectively. However, the sensitivities of the two assays were 88.8% for VecTest and 100% for visual ELISA (P < 0.01). Concordance between VecTest and CS-ELISA was good (kappa = 0.86). Similarly, there was a good concordance between the dipstick and the visually read ELISA (kappa = 0.88). Extrapolating from PfCSP controls (titrated quantities of P. falciparum sporozoites), mean sporozoite loads of CS-ELISA-positive An. gambiae (286 +/- 28.05) and An. funestus (236 +/- 19.32) were determined (P = 0.146). The visual dipstick grades showed high correlation with sporozoite load. The more intense the dipstick colour, the higher the mean sporozoite load (+ = 108, ++ = 207, +++ = 290, r = 0.99, r2 = 1). The VecTest dipstick offers practical advantages for field workers needing rapid and accurate means of detection of sporozoites in mosquitoes.     

Plasmodium chabaudi: a rodent malaria model for in-vivo and in-vitro cytoadherence of malaria parasites in the absence of knobs

The ability of Plasmodium chabaudi infected erythrocytes to bind to endothelial cells in vivo and to various murine cell lines in vitro is described. A procedure for the selective recovery of a sequestering subpopulation of schizont-infected erythrocytes from hepatic sinusoids of BALB/c mice, using a combination of body perfusion, trypsin treatment and Percoll density centrifugation was developed. The serial subinoculation of such a subpopulation was used to select for a clone of parasites (strain AJJ) with considerably better cytoadherent characteristics than the parent line (strain AJ). In contrast, it was demonstrated that another clone (PC7), showed no cytoadherence in vivo or in vitro. This study shows that parasite induced alterations occurred on the surface of erythrocytes infected with late developmental stages of P. chabaudi. The antigenicity of these molecules in the infected mouse was demonstrated using immune serum and affinity chromatography. Cytoadherence and surface antigenic changes in P. chabaudi schizont-infected erythrocytes were demonstrated in the absence of the submembranous 'knobs' associated with cytoadherence in P. falciparum.     

Polyspecific malaria antibodies present at the time of infection inhibit the development of immunity to malaria but antibodies specific for the malaria merozoite surface protein,MSP1, facilitate immunity

Serum taken from mice immune to malaria as a result of infection and drug cure, or from mice immunized with a recombinant form of the merozoite surface protein, MSP1, can provide passive protection of recipient mice against the lethal parasite, Plasmodium yoelii YM. However, recipients of MSP1-immune serum go on to develop long-term immunity, whereas recipients of serum from mice naturally immune to malaria rapidly lose their resistance to infection. We demonstrate that 'infection/cure' serum suppresses the development of both antibody and cell-mediated parasite-specific responses in recipients, whereas these develop in recipients of MSP1-specific antibodies. These data have profound implications for our understanding of the development of malaria immunity in babies who passively acquire antibodies from their mothers.     

A comparison of saponin with other adjuvants for the potentiation of protective immunity by a killed Plasmodium yoelii vaccine in the mouse

The protective immunity conferred by subcutaneous injection of outbred CD-1 mice with a killed Plasmodium yoelii (YM strain) vaccine was strongly potentiated by saponin. By adjusting the dose of antigen, the number of immunizations and the number of living parasites in the challenge infection, conditions were defined where antigen alone was non-protective but 100% protection was obtained by the addition of saponin. Inbred BALB/c, CBA/CA and C57 Bl mice were much less responsive than the CD-1 mice. The following adjuvants were compared with saponin: mineral oil emulsions (Freund's incomplete and complete adjuvants); Al(OH)₃(Alhydrogel); bacteria and synthetic bacterial derivatives ( Bordetella pertussis, Corynebacterium parvum and muramyl dipeptide); surface active materials (digitonin, vitamin A, Arquad 18, dimethyldioctadecyl ammonium bromide, and the polyene antibiotics, Nystatin and Amphotericin B). None of these adjuvants were as effective as saponin, although FCA, Al(OH)₃ and C. parvum augmented immunity considerably. The possible reasons for the efficacy of saponin as an adjuvant for protozoal vaccines are discussed. The P. yoelli /mouse system provides a sensitive and rapid screening assay for comparison of potential adjuvants suitable for use with a malaria vaccine.     

Batf3 deficiency proves the pivotal role of CD8α+ dendritic cells in protection induced by vaccination with attenuated Plasmodium sporozoites

Increasing evidence indicates that hepatic CD8α⁺ dendritic cells (DCs) are important antigen cross‐presenting cells (APC) involved in the priming of protective CD8⁺ T‐cell responses induced by live‐attenuated Plasmodium sporozoites. Experimental proof for a critical role of CD8α⁺ DCs in protective pre‐erythrocytic malaria immunizations has pivotal implications for vaccine development, including improved vectored subunit vaccines. Employing Batf3^?/? mice, which lack functional CD8α⁺ DCs, we demonstrate that deficiency of these particular APCs completely abolishes protection and corresponding signatures of vaccine‐induced immunity. We show that in wild‐type, but not in Batf3^?/?, mice CD8α⁺ DCs accumulate in the liver after immunization with live irradiation‐attenuated P. berghei sporozoites. IFN‐γ production by Plasmodium antigen‐specific CD8⁺ T cells is dependent on functional Batf3. In addition, our results demonstrate that the dysfunctional cDC‐CD8+ T‐cell axis correlates with MHC class II upregulation on splenic CD8α^? DCs. Collectively, these findings underscore the essential role of CD8α⁺ DCs in robust protection induced by experimental live‐attenuated malaria vaccines.     

The activity and inhibition of the food vacuole plasmepsin from the rodent malaria parasite Plasmodium chabaudi

The rodent malaria parasite Plasmodium chabaudi encodes one food vacuole plasmepsin-the aspartic proteinases important in haemoglobin degradation. A recombinant form of this enzyme was found to cleave a variety of peptide substrates and was susceptible to a selection of naturally occurring and synthetic inhibitors, displaying an inhibition profile distinct from that of aspartic proteinases from other malaria parasites. In addition, inhibitors of HIV proteinase that kill P. chabaudi in vivo were also inhibitors of this new plasmepsin. P. chabaudi is a widely used model for human malaria species and, therefore, the characterisation of this plasmepsin is an important contribution towards understanding its biology.     

Products of tryptophan catabolism induce Ca2+ release and modulate the cell cycle of Plasmodium falciparum malaria parasites

Intraerythrocytic malaria parasites develop in a highly synchronous manner. We have previously shown that the host hormone melatonin regulates the circadian rhythm of the rodent malaria parasite, Plasmodium chabaudi, through a Ca2+-based mechanism. Here we show that melatonin and other molecules derived from tryptophan, i.e. N-acetylserotonin, serotonin and tryptamine, also modulate the cell cycle of human malaria parasite P. falciparum by inducing an increase in cytosolic free Ca2+. This occurs independently of the extracellular Ca2+ concentration, indicating that these molecules induce Ca2+ mobilization from intracellular stores in the trophozoite. This in turn leads to an increase in the proportion of schizonts. The effects of the indolamines in increasing cytosolic free Ca2+ and modulating the parasite cell cycle are both abrogated by an antagonist of the melatonin receptor, luzindole, and by the phospholipase inhibitor, U73122.     

The molecular epidemiology of malaria

This review discusses how the use of molecular genetic techniques such as the polymerase chain reaction are helping in the management and prevention of malaria.     

Genetic studies on a major merozoite surface antigen of the malaria parasite of rodents, Plasmodium chabaudi

Diversity in a major merozoite surface antigen (MSA-1) of Plasmodium chabaudi has been examined using a panel of monoclonal antibodies (MoAbs). The antigen was found to be different in each of twelve cloned isolates, as shown by its reactivity with the MoAbs in immunofluorescence tests. In genetic crossing experiments, the diverse forms of this antigen were shown to be determined by allelic variation of a single gene. Recombination occurred between the MSA-1 gene, a second blood stage antigen and enzyme markers.     

Competitive release and facilitation of drug-resistant parasites after therapeutic chemotherapy in a rodent malaria model

Malaria infections frequently consist of mixtures of drug-resistant and drug-sensitive parasites. If crowding occurs, where clonal population densities are suppressed by the presence of coinfecting clones, removal of susceptible clones by drug treatment could allow resistant clones to expand into the newly vacated niche space within a host. Theoretical models show that, if such competitive release occurs, it can be a potent contributor to the strength of selection, greatly accelerating the rate at which resistance spreads in a population. A variety of correlational field data suggest that competitive release could occur in human malaria populations, but direct evidence cannot be ethically obtained from human infections. Here we show competitive release after pyrimethamine curative chemotherapy of acute infections of the rodent malaria Plasmodium chabaudi in laboratory mice. The expansion of resistant parasite numbers after treatment resulted in enhanced transmission-stage densities. After the elimination or near-elimination of sensitive parasites, the number of resistant parasites increased beyond that achieved when a competitor had never been present. Thus, a substantial competitive release occurred, markedly elevating the fitness advantages of drug resistance above those arising from survival alone. This finding may explain the rapid spread of drug resistance and the subsequently brief useful lifespans of some antimalarial drugs. In a second experiment, where subcurative chemotherapy was administered, the resistant clone was only partly released from competitive suppression and experienced a restriction in the size of its expansion after treatment. This finding raises the prospect of harnessing in-host ecology to slow the spread of drug resistance.