Malaria in the post-genomics era: light at the end of the tunnel or just another train?

Malaria remains the third leading cause of death attributable to an infectious disease worldwide, with an estimated death toll of over 2 million per year, predominately in sub-Saharan Africa. The first serious attempt to eradicate this disease was unsuccessful, and 50 years later in 1998 a second programme coined "roll back malaria" was started. While this programme is at present unlikely to reach its stated aims, the completion of the genome sequencing projects on the human host, the mosquito vector, and the malaria parasite offers new hope. It is probable that the burden of disease caused by the most malignant form of the parasite Plasmodium falciparum can be, if not eliminated, then effectively suppressed within a generation through new and novel treatments aimed at all three arms of malaria control.

Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA.

We have previously shown by targeted gene disruption that the clag9 gene of Plasmodium falciparum is essential for cytoadherence to CD36. Here we report inhibition of the function of clag9 by the use of an antisense RNA vector as an alternative to targeted gene disruption. We transfected an antisense construct of clag9 into the P. falciparum clone 3D7 and when the resulting line was cultured in the presence of pyrimethamine it showed 15-fold lower cytoadherence to C32 melanoma cells than the control. Reversion to wildtype upon removal of the introduced plasmid provides direct evidence that the event responsible for the phenotypic change is not at an unrelated site and this approach provides a valuable new tool in malaria transfection technology.

clag9: A cytoadherence gene in Plasmodium falciparum essential for binding of parasitized erythrocytes to CD36.

The propensity of isolates of the malaria parasite Plasmodium falciparum to delete a segment of chromosome 9 has provided positional information that has allowed us to identify a gene necessary for cytoadherence. It has been termed the cytoadherence-linked asexual gene (clag9). clag9 encodes at least nine exons and is expressed in blood stages. The hydrophobicity profile of the predicted CLAG9 protein identifies up to four transmembrane domains. We show here that targeted gene disruption of clag9 ablated cytoadherence to C32 melanoma cells and purified CD36. DNA-induced antibodies to the clag9 gene product reacted with a polypeptide of 220 kDa in the parental malaria clone but not in clones with a disrupted clag9 gene.

The cytoadherence linked asexual gene family of Plasmodium falciparum: are there roles other than cytoadherence?

The binding of erythrocytes infected with Plasmodium falciparum to the endothelium lining the small blood vessels of the brain and other organs can mediate severe pathology. A region at the right end of chromosome 9 has been implicated in the binding of parasitised erythrocytes to the endothelial receptor CD36. A gene expressed in asexual erythrocytic stage parasites has been identified in this region and termed the cytoadherence linked asexual gene (clag). Antisense RNA production and targeted gene disruption of clag resulted in greatly reduced binding to CD36. Hybridisation to 3D7 chromosomes showed clag to be a part of a gene family of at least nine members. All members analysed so far have a conserved gene structure of at least nine exons, as well as putative transmembrane domains. The possible functions of the gene family are discussed.

Aggregation and distribution of strains in microparasites.

Recent research has shown that many parasite populations are made up of a number of epidemiologically distinct strains or genotypes. The implications of strain structure or genetic diversity for parasite population dynamics are still uncertain, partly because there is no coherent framework for the interpretation of field data. Here, we present an analysis of four published data sets for vector-borne microparasite infections where strains or genotypes have been distinguished: serotypes of African horse sickness (AHS) in zebra; types of Nannomonas trypanosomes in tsetse flies; parasite-induced erythrocyte surface antigen (PIESA) based isolates of Plasmodium falciparum malaria in humans, and the merozoite surface protein 2 gene (MSP-2) alleles of P. falciparum in humans and in anopheline mosquitoes. For each data set we consider the distribution of strains or types among hosts and any pairwise associations between strains or types. Where host age data are available we also compare age-prevalence relationships and estimates of the force of infection. Multiple infections of hosts are common and for most data sets infections have an aggregated distribution among hosts with a tendency towards positive associations between certain strains or types. These patterns could result from interactions (facilitation) between strains or types, or they could reflect patterns of contact between hosts and vectors. We use a mathematical model to illustrate the impact of host-vector contact patterns, finding that even if contact is random there may still be significant aggregation in parasite distributions. This effect is enhanced if there is non-random contact or other heterogeneities between hosts, vectors or parasites. In practice, different strains or types also have different forces of infection. We anticipate that aggregated distributions and positive associations between microparasite strains or types will be extremely common.

Diversity in repeat-containing surface proteins of Leishmania major.

The gene B protein (GBP) is one of the products of the LmcDNA16 gene family, a cluster of related but non-identical genes that are differentially-expressed during the Leishmania life cycle. This protein, which is found on the surface of infective stage parasites, contains an extensive region of proline-rich amino acid repeats, constituting 45% of the total protein. The structure and stability of these repeats have been investigated in a number of L. major strains by polymerase chain reaction (PCR) amplification and Southern blotting. Data reported in this paper demonstrate variability between strains with respect to the number of repeats encoded by GBP, although those strains isolated within adjacent geographical regions have conserved repeat structures. The data also reveal that some parasite lines have additional repeat sequences within a second, related gene in the LmcDNA16 array. Western blotting experiments have established that these sequences are expressed in vivo, indicating that L. major strains are heterogeneous in their surface complement of gene B repeat-containing proteins.

Molecular cloning of a myoD-like gene from the parasitic nematode, Trichinella spiralis.

The infective larval stage of the nematode Trichinella spiralis is an intracellular parasite of skeletal muscle cells. Infection with T. spiralis results in dedifferentiation of the host cell and the formation of a host/parasite complex. A gene encoding a T. spiralis Helix-Loop-helix (HLH) protein with homology to the myogenic transcription factor, MyoD, and to the Caenorhabditis elegans protein, CeMyoD, has been identified and partially characterized. The tsmyd-1 gene is expressed constitutively during the muscle-larval and adult stages. A purified recombinant Tsmyd-1 protein, expressed in Escherichia coli, binds to a high affinity mouse MyoD DNA binding site in vitro. The present study describes the first HLH gene to be identified in Trichinella.

Heterogeneity of IgG antibody responses to cloned Onchocerca volvulus antigens in microfiladermia positive individuals from Esmeraldas Province, Ecuador.

The prevalence of IgG antibodies to three recombinant O. volvulus antigens, OvMBP/10, OvMBP/11 and OvMBP/29 was determined in a group of 94 microfilaria positive (mf+) individuals resident in the hyperendemic onchocercal area of Esmeraldas Province, Ecuador. Clone OvMBP/11 was the antigen most frequently recognized by patients sera followed by OvMBP/10 and OvMBP/29. When a cocktail of the three recombinant antigens was used the proportion of positive sera increased to 100%. Antibody responses to the fusion partner maltose binding protein (MBP) were low in comparison with those to the cloned antigens and no correlation of responses between individual antigens was observed. The relative level of antibody response to each of the clones in the cocktail varied between individuals. The distribution of IgG responses to OvMBP/11 was bimodal and those to OvMBP/29 and OvMBP/10 were positively and negatively skewed, respectively. When the three recombinant antigens were used in combination this variation was minimized and the pattern of responses showed a normal distribution as was also seen to crude O. volvulus antigen. The cocktail of recombinants thus offers excellent diagnostic sensitivity in combination with the parasite specificity demonstrated previously.

Antigenic diversity and the transmission dynamics of Plasmodium falciparum.

The average age of humans at their first infection with Plasmodium falciparum is typically less than 1 year in most endemic areas. This has been interpreted as evidence of the high transmissibility of the parasite, with the implication that control of malaria will require high levels of coverage with a potential vaccine. This interpretation is challenged by mathematical models that demonstrate that the long period required to develop immunity to malaria permits a high risk (or low average age) of infection even when parasite transmissibility is low. Patterns of seroconversion to five antigenically distinct isolates of P. falciparum in a highly malarious area of Papua New Guinea indicate that each is only mildly transmissible and that malaria, as a construct of several such independently transmitted strains, has a basic reproductive rate (or transmissibility) that is an order of magnitude lower than other estimates.

Resistance of Plasmodium falciparum to amodiaquine in Papua New Guinea.

The sensitivity of Plasmodium falciparum to amodiaquine was assessed in children in the Madang region of Papua New Guinea. Fifty-four tests in vivo were carried out and 19 (35%) of these showed resistance at the RII or RIII level. Twenty-two of the 43 isolates tested showed some degree of resistance to amodiaquine in vitro. A negative correlation between the age of the patient and apparent resistance of the parasite in vivo was observed. There was no correlation between resistance in vivo and in vitro.

The effects of vector control on the antibody response to antigens of Onchocerca volvulus.

The effects of exposure to infective larvae on the antibody response to a cocktail of specific recombinant antigens of Onchocerca volvulus and to a worm extract were evaluated by comparing the responses of individuals from a vector controlled area with those from an area of continuing transmission by ELISA. Individuals from the vector controlled areas were found to have reduced responses to both antigen preparations. A microfilerdermic (mf-) individuals from the area of vector control exhibited significantly lower total and subclass IgG responses to the worm extract. In contrast, the responses to the cocktail of specific recombinants were significantly reduced in individuals from the area of vector control who were still microfilerdermia positive (mf+). The distribution of IgG subclass specific responses was similar to both antigen preparations, both dominated by the IgG4 and IgG1 subclasses. IgG1 responses to the worm extract remained elevated in the vector controlled individuals but IgG4 was significantly reduced in the mf- individuals. Both subclasses reflected the total IgG response to the cocktail of recombinants and were significantly reduced in individuals from the vector controlled area, when compared to individuals from the hyperendemic area. IgG1 responses to the cocktail of recombinants are significantly lower than IgG4 in all individuals and virtually absent in individuals from the vector-controlled area. Measuring total IgG and IgG4 is more sensitive than IgG1 in detecting infection, 100 or 97% respectively, but they remain elevated in the individuals from the vector controlled areas even after 8-10 years interruption of transmission. These results have important implications for the serological monitoring of control programmes in individuals who have previously been infected.

A sensitive serodiagnostic test for onchocerciasis using a cocktail of recombinant antigens.

A specific serodiagnostic test for onchocerciasis has been a priority objective of the World Health Organization. Fragments of cDNA encoding Onchocerca volvulus antigens selected on the basis of their specificity for this parasite were subcloned into a protein purification and expression system. No individual recombinant antigen showed a high sensitivity in an enzyme-linked immunosorbent assay because of heterogeneity in the response of O. volvulus-infected individuals. However, a cocktail of three recombinant proteins showed 96% sensitivity with 100% specificity, compared with 99% sensitivity and only 59% specificity against a crude O. volvulus extract. The sensitivity of detection of individual antigens varied between sera taken from individuals from different geographic areas infected with O. volvulus, but when used as a cocktail, all but one of the microfilaria-positive individuals from all the geographic areas studied were detected. Recombinant probes provide a practical basis for specific diagnostic tests for helminth infections.

Chemoprophylaxis against malaria in Papua New Guinea: a trial of amodiaquine and a combination of dapsone and pyrimethamine.

A placebo-controlled chemoprophylaxis trial was carried out in 1980 in 318 semi-immune school children in the Madang area of Papua New Guinea, where there was a high prevalence of strains of Plasmodium falciparum resistant to 4-aminoquinolines. Since prophylaxis with amodiaquine at 5 mg/kg weekly had failed, amodiaquine at a dose of 10mg/kg weekly and Maloprim (half a tablet or one tablet depending on body weight, which gave ranges of dapsone of 1.7-3.3mg/kg and pyrimethamine 0.2-0.4 mg/kg) weekly were tried. Neither regimen was completely successful in preventing parasitaemia, though after 13 weeks of prophylaxis the slide positivity rate was 16% for the amodiaquine group and 2% for the Maloprim group, which was in each case significantly lower than the normal baseline rate in the controls of 42%. Amodiaquine was completely successful in suppressing Plasmodium vivax infections. Breakthrough parasitaemia occurred, with either P. falciparum or P. vivax, in 5% of subjects on Maloprim at some time during the 13-week period of prophylaxis. Significantly more children in both the amodiaquine and Maloprim groups than in the placebo group showed a reduction in spleen size. All groups showed an unexplained fall in haemoglobin level over the study period but the fall was significantly less in both the prophylaxis groups. There was no adverse effect on white cell counts by either drug regimen. Chemoprophylaxis as a component of an integrated malaria control program should not be overlooked, provided that compliance can be maintained. However, in this particular case the principal purpose of the study had been to evaluate the proposed chemoprophylactic regimens in school children before embarking on an intervention study in young children. As a result of this study it was decided not to go ahead with the chemoprophylactic intervention in young children but to adopt an approach based on early presumptive treatment.

The effect of a fibroblast feeder layer, L-cysteine and 2-mercaptoethanol on the growth of Plasmodium falciparum in vitro.

A fibroblast-like cell line derived from the mountain vole Microtus montanus was effective in promoting an increase in the multiplication rate of new and established isolates of Plasmodium falciparum. An increase in multiplication rate was also achieved in the absence of M. montanus cells when cultures of P. falciparum were grown in medium which was supplemented with L-cysteine at 12 h intervals, and to a lesser extent when medium was supplemented with 2-mercaptoethanol instead of L-cysteine.

The use of murine feeder cells in the cultivation of Plasmodium falciparum asexual blood stages.

Increased multiplication rates were observed in asexual erythrocytic stages of Plasmodium falciparum grown in the presence of a feeder-cell layer of mouse peritoneal wash cells (PWCs) using the candle-jar method of Trager and Jensen (1977). This held true for both new and established isolates of the parasite. When the PWC population was separated into adherent and non-adherent fractions, the adherent PWC population promoted an increase in parasite growth but the non-adherent population did not. An increase in parasite multiplication was not promoted by PWC-conditioned medium.