Rhoptry-associated protein 1-binding monoclonal antibody raised against a heterologous peptide sequence inhibits Plasmodium falciparum growth in vitro.

Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1(23-711)) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-1(23-711) MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.

Preventive effect of artemether in experimental animals infected with Schistosoma mansoni.

The effect of artemether, an antimalarial drug developed from the plant Artemisia annua, has been tested against the larval stages of Schistosoma mansoni covering the time from skin penetration to the early adult liver-stage. The results show that the experimental animals used (hamster and mice) do not develop schistosomiasis mansoni if treated with artemether during the first month after infection. The parasite was found to be especially susceptible between the 3rd and 4th week after infection, resulting in worm reductions of 75.3-82.0% compared to non-treated controls. This level was boosted to 97.2-100% when the animals were subjected to various schedules of repeated treatment. Almost complete protection was also reached in parallel experiments with repeated infections carried out to mirror more closely the real situation of trickle infection.

Antibodies against Plasmodium falciparum vaccine candidates in infants in an area of intense and perennial transmission: relationships with clinical malaria and with entomological inoculation rates.

Serum immunoglobulin (Ig)G1, IgG3 and total IgG were assessed by immunoabsorbent assay in 198 infants from a Tanzanian village highly endemic for Plasmodium falciparum. Antibodies were measured against epitopes of the circumsporozoite protein (the repetitive epitope (NANP)50 and a construct of the flanking regions (CS27IC)), the malaria vaccine SPf66, and two constructs of the merozoite surface protein-1 (MSP-1), a 19-kDa fragment from the C-terminal domain (MSP-119) and an N-terminal fragment spanning blocks 1-6 (H6-p190 M-1/6-H6). IgG1 and total IgG titres showed similar age profiles, all decreasing for the first 2 months of life. Anti-(NANP)50 titres remained very low throughout the first year of life, while anti-CS27IC antibody appeared to peak around 7 months of age. Only a slight tendency to increase with age was observed for levels of the other antibodies studied. IgG3 titres except for H6-p190(1/6), were very low initially and remained very low throughout the first year of life. Clinical malaria incidence at the village dispensary was analysed prospectively in relation to antibody. No IgG1 or total IgG titre showed protective effects, but low IgG3 against p190(1/6) appeared to be a risk factor in some age groups. Given the large number of antibodies tested, this single indication of possible protection could merely be chance. There were no strong associations between antibody titres and entomologically assessed sporozoite exposure suggesting that transmission-reducing interventions may have little effect on antibody levels in such children.

Role of ICAM-1 (CD54) in the development of murine cerebral malaria.

In susceptible mouse strains, infection of mice with Plasmodium berghei ANKA (PbA) results in a lethal complication, cerebral malaria. Cerebral malaria is due to the immune response induced by the parasite, which results in an increased production of TNF, known to increase the expression of adhesion molecules on the endothelia. To investigate the role of the adhesion molecule ICAM-1 (CD54), we infected wild-type (+/+) and ICAM-1-deficient (-/-) mice with PbA. While +/+ mice died 6-8 days after infection, -/- mice survived > 15 days. Parasitaemia was similar in +/+ and -/- mice. Serum TNF concentration was increased by the infection and was significantly higher in infected +/+ than in -/- mice. However, TNF mRNA levels in spleen, lungs, and brain were elevated in both infected +/+ and -/- mice. For IFN-gamma, serum levels were similar in both groups. A breakdown of the blood-brain barrier was evident in infected +/+ mice only. Interestingly, thrombocytopenia was profound in infected +/+, but practically absent in -/- mice. Moreover, macrophage sequestration was evident in brain venules and lung capillaries of +/+ mice and was significantly less important in the alveolar capillaries of infected -/- mice. In contrast, neutrophil sequestration in the lung was similar in both +/+ and -/- mice. Sequestration of parasitized red blood cells was significantly greater in the alveolar capillaries from +/+ than -/- mice. These results indicate that while the immune response is similar in both +/+ and ICAM-1(-/-) mice, the absence of mortality in ICAM(-/-) mice correlates with a decrease of macrophage and parasitized RBC trapping and a less severe thrombocytopenia.

Immune responses to Plasmodium falciparum antigens during a malaria vaccine trial in Tanzanian children.

Among Tanzanian children living in an area of intense and perennial malaria transmission, prevalence of naturally acquired IgG antibodies that recognize SPf66, NANP, p190 and a 19 kDa fragment of the merozoite surface protein-1 (MSP-1) is high and increases with age. This possibly reflects the high level of natural exposure of the children to P. falciparum. The prevalences of IgG antibodies that recognize the three putative merozoite derived sequences contained in the malaria vaccine SPf66 (83.1, 55.1 and 35.1) is low but also show some age dependence. Three doses of the SPf66 vaccine induce a strong IgG antibody response against both the SPf66 construct, NANP and the three individual peptides. Vaccination with SPf66 did not result in an increase of anti19 kDa fragment antibodies. This reflects the specificity of the humoral immune response induced by the SPf66 construct. Among vaccinated children, antibody titres against SPf66 decreased over time following the third dose. However, 18 months after the third dose, SPf66 recipients still had significantly higher IgG titres and stimulation indices of peripheral blood mononuclear cells (PBMC) than placebo recipients. Within the vaccine group, there is a trend for increasing anti-SPf66 IgG titre to be associated with decreasing risk of clinical malaria but this was not statistically significant. Results also show the difficulties of establishing whether antibody responses are related to protection in field trials in endemic areas.

In vivo sequestration of Plasmodium falciparum-infected human erythrocytes: a severe combined immunodeficiency mouse model for cerebral malaria.

Cerebral malaria is a fatal complication of infection by Plasmodium falciparum in man. The neurological symptoms that characterize this form of malarial disease are accompanied by the adhesion of infected erythrocytes to the vasculature of the brain. To study this phenomenon in vivo, an acute phase severe combined immunodeficiency (SCID) mouse model was developed in which sequestration of P. falciparum-infected human erythrocytes took place. During acute cerebral malaria in humans, the expression of intercellular adhesion molecule-1 (ICAM-1) is induced in vascular endothelium by inflammatory reactions. Acute phase ICAM-1 expression can also be obtained in SCID mice. The endothelium of the midbrain region was the most responsive to such inflammatory stimulus. It is noteworthy that the reticular formation in the midbrain controls the level of consciousness, and loss of consciousness is a symptom of cerebral malaria. We found that infected human erythrocytes were retained 24 times more than normal erythrocytes in ICAM-1-positive mouse brain. Sequestration to the brain was reduced by anti-ICAM-1 antibodies. These in vivo results were confirmed by the binding of P. falciparum-infected erythrocytes to the ICAM-1-positive endothelium in tissue sections of mouse brain. We conclude that the SCID mouse serves as a versatile in vivo model that allows the study of P. falciparum-infected erythrocyte adhesion as it occurs in human cerebral malaria. Upregulation of ICAM-1 expression in the region of the midbrain correlates with increased retention of malaria-infected erythrocytes and with the symptoms of cerebral malaria.

Evaluation of C-reactive protein and haptoglobin as malaria episode markers in an area of high transmission in Africa.

Field studies of malaria in endemic areas frequently use the presence or levels of parasitaemia, together with the measurement of fever, as the primary criteria with which to identify cases. However, since malaria cases do not always present with measurable fever, and since asymptomatic parasitaemia occurs, additional episode markers might be useful epidemiological tools. We have measured the C-reactive protein and haptoglobin levels in paediatric patients presenting to a village health post in the Kilombero District in Tanzania and in convalescent sera from the same patients, in order to evaluate these acute-phase reactants as alternative markers of Plasmodium falciparum episodes. Among afebrile patients, C-reactive protein levels were highly correlated with parasite density. High C-reactive protein levels are therefore probably indicative of recent clinical malaria episodes in currently afebrile individuals with high parasite densities. An appropriate case definition for malaria in epidemiological studies in endemic areas might therefore be hyperparasitaemia accompanied by either, or both, measurable fever and raised C-reactive protein levels. This would give less biased estimates of the overall burden of malaria morbidity than does a definition which requires measurable fever. Levels of haptoglobin were highly negatively correlated with parasitaemia, but did not appear to be useful episode markers because this correlation was probably not related to acute morbidity. However, haptoglobin can be useful to assess at community level the impact of interventions on parasitaemia.