Palle Jakobsen

Primary structure and localization of a conserved immunogenicPlasmodium falciparum glutamate rich protein (GLURP) expressed in both the preerythrocytic and erythrocytic stages of the vertebrate life cycle

A gene coding for a 220-kDa glutamate rich protein (GLURP), an exoantigen of Plasmodium falciparum, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence contains 2 repeat regions. The sequence of one of these was shown to be conserved among geographically dispersed isolates, and a fusion protein containing that sequence was able to stimulate B- and T-cells. Antibodies against GLURP stained erythrocytic stages of the parasite as well as the hepatic stage as detected by electron microscopy.

Relationships between maternal malaria and malarial immune responses in mothers and neonates

Immune responses of 97 Gambian women and their neonates were studied. New methods distinguished between active and previous placental malaria, were used to examine relationships between maternal malaria and neonatal immune responses. Many placentas (61%) had active or previous malarial infection. Maternal and cord malarial IgG levels correlated (P < 0–001). Malarial IgG was raised in cord blood in active placental malaria; IgM was not detected. Mean lymphoproliferation and the proportion of responders to soluble P. falciparum antigens (F32) and conserved regions of p190 expressed on trophozoites and schizonts (190L and 190N) were higher in neonates than mothers. There was no clear relationship between maternal malaria and neonatal mean lymphoproliferation to malarial antigens, although fewer neonates responded when mothers were actively infected. Matched maternal and neonatal lymphoproliferation responses did not correlate. However, first born neonatal lymphoproliferation to PPD and malarial antigens appeared lower than other neonates, in agreement with lower lymphoproliferation in primigravidae compared with multigravidae. Also in common with mothers, autologous plasma suppressed neonatal lymphoproliferation to PPD and malarial antigens, suggesting common immunoregulation. Higher Cortisol or other circulating factors in first pregnancies may be implicated. The relevance of cell‐mediated malarial immune responses detected at birth remains to be established.

Malaria: toxins, cytokines and disease

In this review the old concept of severe malaria as a toxic disease is re‐examined in the light of recent discoveries in the field of cytokines. Animal studies suggest that the induction of TNF by parasite‐derived molecules may be partly responsible for cerebral malaria and anaemia, while hypoglycaemia may be due to direct effects of similar molecules on glucose metabolism. These molecules appear to be phospholipids and we suggest that when fully characterized they might form the basis of antitoxic therapy for malaria.,

Diversity of Plasmodium falciparum clones infecting children living in a holoendemic area in north-eastern Tanzania

The diversity of Plasmodium falciparum clones and their role in progression from asymptomatic to symptomatic condition in children have been investigated. Attempts to identify whether particular parasite genotypes were associated with the development of clinical symptoms have been made. A cohort of 34 initially asymptomatic parasitaemic children aged 1-5 years were followed daily for 31 days. Clinical examinations were made each day for signs and symptoms of clinical malaria, followed by parasitological investigation. Nineteen children developed symptoms suggestive of clinical malaria during this period. Daily blood parasite samples from 13 children who developed clinical malaria symptoms and 7 who remained asymptomatic were genotyped by PCR-amplification of the polymorphic regions of the merozoite surface proteins 1 and 2 (MSP1 and MSP2) and the glutamate rich protein (GLURP) genes. Infections were found to be highly complex in both groups of children. Every isolate examined from both groups had a mixture of parasite clones. Daily changes were observed in both parasite density and genotypic pattern. The mean number of genotypes per individual was estimated at 4.9 and 2.7 for asymptomatic and symptomatic groups of children, respectively. Analysis of allele frequency distributions showed that these differed significantly for the MSP1 locus only.

IgG reactivities against recombinant Rhoptry-Associated Protein-1 (rRAP-1) are associated with mixed Plasmodium infections and protection against disease in Tanzanian children

A cross-sectional sero-epidemiological study was performed in Magoda, Tanzania, an area where malaria is holoendemic. Blood samples were collected from children (1-4 years) and tested for IgG antibody reactivity against 2 recombinant protein fragments of Plasmodium falciparum Rhoptry-Associated Protein-1 (rRAP-1). The data were related to the prevalence of malarial disease and single P. falciparum or mixed Plasmodium infections. Fever (> or = 37.5 degrees C) in combination with parasite densities > 5000/microliter were used to distinguish between children with asymptomatic malaria infections and those with acute clinical disease. Furthermore, C-reactive protein (CRP) was applied as a surrogate marker of malaria morbidity. The prevalence of Plasmodium infections was 96.0%. Eleven children were defined as clinical malaria cases, all with single P. falciparum infections. The density of P. falciparum was significantly lower in children with mixed Plasmodium infections compared to those with single P. falciparum infections. Children with asymptomatic P. falciparum infections had higher IgG reactivities to rRAP-1, compared to IgG reactivities of children with malarial disease. Children with mixed Plasmodium infections generally showed elevated IgG reactivity to rRAP-1, when compared to children with single P. falciparum infections. The possible relationship between mixed species infections, clinical outcome of the disease and antibody responses to RAP-1 is discussed.

Increased plasma levels of soluble IL-2R are associated with severe Plasmodium falciparum malaria.

Plasma samples from children with mild and severe Plasmodium falciparum malaria and from children with unrelated diseases were collected to investigate whether the clinical outcome of infection was associated with plasma factors which reflected the activity of different cells of the immune system. Children with severe P. falciparum malaria had significantly higher plasma levels of soluble IL‐2R than children with mild malaria. Plasma levels of 1L‐2R and levels of parasitaemia were significantly correlated. Neither parasitaemia nor plasma levels of tumour necrosis factor‐alpha (TNF ‐a). IL‐6. lymphotoxin (LT). interferon‐gamma (IFN‐γ). IL‐4, soluble IL‐4R or soluble CD8 differed significantly between the two groups of children with malaria. High plasma levels of soluble CD8 were associated with failure of lymphocytes to produce I FN‐γin vitro following stimulation with P. falciparum antigen. We conclude that soluble IL‐2R is a useful marker of disease severity independently of the association with levels of parasitaemia, and that functional regulation of different lymphocyte subsets occurs during acute malaria episodes.

Tumour necrosis factor and interleukin-6 production induced by components associated with merozoite proteins of Plasmodium falciparum.

P. falciparum merozoite antigens, merozoite surface protein‐l (MSP‐1) and rhoptry associated protein‐1 (RAP‐1), were shown to be liberated into the supernatant of in vitro parasite cultures and to be included in the endotoxin‐like exoantigen complex, previously designated Ag7. Material affinity purified from culture supernatants, using immobilized monoclonal antibodies specific for RAP‐1 or MSP‐1, stimulated normal human mononuclear cells to produce TNF and IL‐6 in vitro. However, stimulation of TNF was absent, and that of IL‐6 was reduced, when the antigens were purified from detergent extracts of infected erythro‐cytes. These results indicate that the RAP‐1 and MSP‐1 proteins themselves do not stimulate the production of TNF. Instead, other components associating with these exoantigens may be responsible for the TNF production. Mouse antisera blocking TNF production stimulated by P. yoelii exoantigens also blocked TNF production stimulated by material affinity purified from P. falciparum culture supernatants using RAP‐1 specific monoclonal antibody, indicating the conserved structure of the TNF inducing component.

Reduced cellular immune reactivity in healthy individuals during the malaria transmission season.

Antigen-induced cellular immune responses are suppressed during acute malaria. The present study engages the possibility that malaria-induced alterations in cellular immune reactivity extend beyond the clinical disease. Thus, lymphoproliferative responses of healthy individuals were diminished during the malaria transmission period in individuals living in an area of highly seasonal, unstable malaria transmission. This finding may have important implications for the design of studies of stimulatory properties of antigens using lymphocytes of endemic origin.

Antibody responses to Rhoptry-Associated Protein-1 (RAP-1) of Plasmodium falciparum parasites in humans from areas of different malaria endemicity

Plasma IgM and IgG antibody reactivities against the recombinant Plasmodium falciparum protein, Rhoptry Associated Protein‐1 (rRAP‐1) were measured by ELISA in individuals from Sudan, Indonesia, Kenya and The Gambia living in areas of different malaria endemicity. IgG and IgM reactivities to rRAP‐1 increased with malaria endemicity. IgG reactivities were associated with spleen rates in Indonesia with high malaria endemicity while IgM reactivities were associated with spleen rates in Kenya with low malaria endemicity. IgG and IgM reactivities to rRAP‐1 increased during acute episodes of P. falciparum malaria in Sudanese adults and IgG reactivities remained high one month after treatment in all adults tested. Antibody reactivities to rRAP‐1 in Gambian children in the dry season were higher in children with parasitaemia than in children without detectable parasitaemia. Antibody reactivities were not associated with protection against clinical episodes in the following rainy season but higher antibody reactivities were detectable at the end of the rainy season. There was no difference in antibody reactivity to rRAP‐1 between Gambian children with mild or severe malaria.

Antibody reactivity to conserved linear epitopes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of protein antigens are involved in adhesion of P. falciparum infected erythrocytes to the capillary endothelium of the host. Antibodies to variable regions of these proteins, measured by agglutination, correlates with clinical protection against falciparum malaria. In this study we investigated the occurrence of antibodies to conserved sequences of these very variable proteins in a population living in an area endemic for falciparum malaria. Using the ELISA method, we were able to measure an antibody response to three synthetic peptides derived from conserved regions of PfEMP1. The antibody responses to these peptides increased with age and were higher in individuals with asymptomatic P. falciparum infection compared to individuals presenting with fever attributable to falciparum malaria. This indicates that antibodies recognising the conserved regions of PfEMP1 arise upon exposure to the parasite, and that these may be involved in the development of protection against malaria. Antibodies to the Pfalhesin peptide of the human aniontransporter, band3, were measured by the same method. The magnitude of this antibody response did not correlate with neither age nor clinical protection.