Over the past several years, we have been interested in variant surface antigens and, in particular, the erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of red blood cells infected (iRBC) with Plasmodium falciparum, the causative agent of malaria. Our objectives are to identify PfEMP1 proteins involved in the pathogenesis of malaria and to use them in the development of a malaria vaccine to protect children against severe malaria.
P. falciparum malaria remains one of the greatest disease burdens in developing countries. An estimated 1 to 2 million children under the age of five years die from malaria each year, and the parasite causes enormous suffering from nonfatal disease, particularly in young children and pregnant women. Parasites resistant to available and affordable drugs are spreading, and replacement drugs tend to be expensive and associated with side effects. In the long term, vaccination appears to be the most sustainable and cost-effective solution. However, despite decades of research, no clinically applicable vaccine exists.
Several independent lines of evidence reported during the last few years indicate that it is highly likely that naturally acquired immunity relies on the accumulation of IgG with specificity for variant surface antigens such as PfEMP1. PfEMP1 is expressed on the surface of iRBC, thus making it an attractive target for vaccination against malaria.
P. falciparum causes severe syndromes by adhering to endothelium and becoming sequestered in deep vascular beds, where it elicits an inflammatory response. Naturally exposed children develop immunity that prevents severe malaria relatively quickly, suggesting that the parasites that are targeted by protective immunity have limited diversity.
On the basis of our previous studies and those of others, we hypothesized that distinct parasites cause severe malaria and that individuals become resistant to severe malaria as they acquire antibodies against the relevant parasite isolate. It appears that severe and life-threatening P. falciparum malaria is associated with parasites that express a restricted and antigenically conserved PfEMP1 subset (PfEMP1SM). PfEMP1SM expression appears to confer a selective advantage to parasites in non-immune individuals, perhaps by allowing particularly efficacious iRBC sequestration and high growth rates. As PfEMP1SM-specific immunity is acquired, this advantage gradually declines. Survival rates of parasites expressing less virulent and more diverse PfEMP1s (PfEMP1UM) eventually surpass those of PfEMP1SM-expressing parasites, causing PfEMP1UM-expressing parasites to dominate infections in semi-immune individuals. Thus, theoretically, it should be possible to protect non-immune children against severe and complicated malaria by accelerating the acquisition of PfEMP1SM-specific immunity through vaccination.
We recently showed that, in vitro, sera from children who are resistant to symptomatic malaria will cull 3D7 parasite strains that express distinct variants of PfEMP1. PfEMP1 variants are encoded by approximately 60 var genes per haploid genome of P. falciparum. Most var genes are subtelomeric and display extensive variation. Diversity of var genes may be generated by recombination between telomeres that cluster during mitosis and by independent chromosomal segregation during meiosis. Despite the enormous diversity of var gene sequences, PfEMP1 molecules that are highly conserved or semi-conserved among isolates from around the world have been identified. Some of them, such as the semi-conserved VAR4, have been implicated as parasite proteins presenting at the surface of the iRBC and causing severe malaria.
VAR4 is composed of an intracellular domain and various extracellular domains: NTS, DBL1α, CIDR1α, DBL2β, C2, DBL3β, DBL4γ, DBL5δ, and CIDR2β. Homologues with a conserved domain structure similar to that of the 3D7 var4 gene have been found in P. falciparum field isolates from Ghana; we are using these data to look at sequence diversity, recombination hot spots, and so forth.
To assess a possible protective effect of VAR4 antibodies, we measured CIDR1α antibody levels in the plasma of more than 500 individuals living in Tanzanian villages with either high (Mkokola) or moderate (Kwamasimba) transmission and correlated the levels with protection against malaria. The risk of anemia was reduced in VAR4-CIDR1α responders in both villages and that of malaria fever was reduced among individuals with a measurable VAR4-CIDR1α response from Mkokola village. Antibody levels to three other malaria antigens were not associated with protection against morbidity.
The 60 PfEMP1 variants of 3D7 can be put into groups A and B/A (category A), B, B/C, and C (category non-A), expression of category A molecules is associated with severe malaria and category non-A expression is associated with uncomplicated malaria and asymptomatic infection. By using competition ELISA and 60 recombinant 3D7 PfEMP1 domains, we were able to show that naturally acquired antibodies are largely directed toward epitopes varying between different domains with a few, mainly category A, domains sharing cross-reactive antibody epitopes. Given that identification of groups of serological cross-reacting molecules is pivotal for the development of vaccines based on PfEMP1, we are continuing and extending this work to look at cross-reactivity among VAR4 variants and other PfEMP1 molecules encoded by different P. falciparum genomes.
We previously suggested that antibodies to category A molecules are acquired earlier in life than antibodies to PfEMP1 belonging to category non-A. In a recent study, we compared the acquisition of IgG to 20 PfEMP1 domains derived from 3D7 in individuals living under markedly different malaria transmission intensity; we were unable to find differences in the antibody acquisition rate to PfEMP1 of different groupings or domain type. Antibodies were acquired early in life in individuals living in the high-transmission village and, by the age of two to four years, most individuals had antibodies against most constructs. This level of reactivity was found at the age of 10–20 years in the medium-transmission village and was never reached by individuals living under low-transmission conditions. Nevertheless, the sequence in which individuals acquired antibodies to particular constructs was largely the same in the three villages, indicating that the pattern of PfEMP1 expression by parasites transmitted at the different sites was similar and suggesting that PfEMP1 expression is nonrandom and shaped by host-parasite relationship factors operating at all transmission intensities. We believe that, as more extensive analysis including full genome sequences become available, we might be able detect differences between antibody recognition of category A and non-A molecules and are still pursuing such an analysis.
We are also exploring a novel expression platform, Tetrahymena thermophila, or the expression of heterologous, pure, and correctly folded P. falciparum antigen, which can be used for structural studies and for vaccine development.
Last updated August 2009
