Chairat Uthaipibull

Fine Specificity of Serum Antibodies to Plasmodium falciparum Merozoite Surface Protein, PfMSP-1 19 , Predicts Protection from Malaria Infection and High-Density Parasitemia†

ABSTRACT Antibodies to the C terminus of the Plasmodium falciparum merozoite surface protein, PfMSP-119, may inhibit merozoite invasion or block the effects of inhibitory antibodies. Here, using a competition enzyme-linked immunosorbent assay and antibody binding to wild-type and mutated recombinant proteins, we show that there are marked variations between individuals in the fine specificity of naturally acquired anti-MSP-119 antibodies. Furthermore, although neither the prevalence nor the concentration of total anti-MSP-119 antibodies was associated with resistance to malaria in African children, significant associations were observed between antibody fine specificity and subsequent risk of infection and high-density parasitemia during a follow-up period. Thus, the fine specificity of naturally acquired human anti-MSP-119 antibodies is crucial in determining their function. Future field studies, including the evaluation of PfMSP-1 vaccine trials, should include assays that explore antibody fine specificity as well as titer.

The Fine Specificity, but Not the Invasion Inhibitory Activity, of 19-Kilodalton Merozoite Surface Protein 1-Specific Antibodies Is Associated with Resistance to Malarial Parasitemia in a Cross-Sectional Survey in The Gambia

ABSTRACT In a cross-sectional survey of 187 Gambian children and adults, we have analyzed prevalence, fine specificity, and 19-kilodalton merozoite surface protein 1 (MSP-119)-specific erythrocyte invasion inhibitory activity of antibodies to MSP-119 but find no significant association between any of these parameters and prevalence or density of malarial parasitemia, except that, after correcting for total anti-MSP-119 antibody levels, individuals with anti-MSP-119 antibodies that compete with an invasion inhibitory monoclonal antibody (12.10) were significantly less likely to have malaria infections with densities of ≥1,000 parasites/μl than were individuals without such antibodies. This association persisted after correction for age and ethnic origin.

Inducible knockdown of Plasmodium gene expression using the glmS ribozyme.

Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection.

Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection

BackgroundMSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP119), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP119 had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP119 would affect critical T-cell responses to epitopes in this antigen.MethodsThe cellular responses to wild-type MSP119 and a panel of modified MSP119 antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults.ResultsInterestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP119. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins.ConclusionThis study suggests that specific MSP119 variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.

A nuclear targeting system in Plasmodium falciparum

BackgroundThe distinct differences in gene control mechanisms acting in the nucleus between Plasmodium falciparum and the human host could lead to new potential drug targets for anti-malarial development. New molecular toolkits are required for dissecting molecular machineries in the P. falciparum nucleus. One valuable tool commonly used in model organisms is protein targeting to specific sub-cellular locations. Targeting proteins to specified locations allows labeling of organelles for microscopy, or testing of how the protein of interest modulates organelle function. In recent years, this approach has been developed for various malaria organelles, such as the mitochondrion and the apicoplast. A tool for targeting a protein of choice to the P. falciparum nucleus using an exogenous nuclear localization sequence is reported here.MethodsTo develop a nuclear targeting system, a putative nuclear localization sequence was fused with green fluorescent protein (GFP). The nuclear localization sequence from the yeast transcription factor Gal4 was chosen because of its well-defined nuclear localization signal. A series of truncated Gal4 constructs was also created to narrow down the nuclear localization sequence necessary for P. falciparum nuclear import. Transfected parasites were analysed by fluorescent and laser-scanning confocal microscopy.ResultsThe nuclear localization sequence of Gal4 is functional in P. falciparum. It effectively transported GFP into the nucleus, and the first 74 amino acid residues were sufficient for nuclear localization.ConclusionsThe Gal4 fusion technique enables specific transport of a protein of choice into the P. falciparum nucleus, and thus provides a tool for labeling nuclei without using DNA-staining dyes. The finding also indicates similarities between the nuclear transport mechanisms of yeast and P. falciparum. Since the nuclear transport system has been thoroughly studied in yeast, this could give clues to research on the same mechanism in P. falciparum.

Effect of Aqueous Crude Extract of Tinospora Crispa on Plasmodium Berghei Induced Liver Damage in Mice

Malaria is still a serious problem with increasing of mortality in children annually. One of major causes of death in malaria, organ damage especially liver, has been observed. Hence, we aimed to investigate hepatoprotective effect of traditional plant, Tinospora crispa during malaria infection using Plasmodium berghei infected mice as in vivo model. Aqueous crude extract of T. crispa was freshly prepared. For in vivo test, groups of ICR mice were intraperitoneally injected with 6×106 parasitized erythrocytes of P. berghei ANKA, and given with the extract at doses 10, 50, and 500 mg/kg twice a day for 4-consecutive days. Aspartate aminotransferase and alanine aminotransferase are measured for liver damage while albumin measurement is for liver function. The results showed that liver damage was observed during malaria infection as indicating by significantly (p<0.05) increase of AST and ALT, and markedly decrease of albumin. Interestingly, T. crispa extract exerted hepatoprotective effect during malaria infection. The highest hepatoprotective activity of the extract was shown at dose of 500 mg/kg. Additionally, there were no any toxics to liver function in normal mice treated with this extract. It can be concluded that aqueous crude extract of T. crispa exerts hepatoprotective effect during P. berghei infection.