Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules.

BACKGROUND: Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS), the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii. METHODS AND FINDINGS: We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS) are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species. CONCLUSION: This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that efficient production of edible vaccines can be genetically produced in Chlamydomonas.

Enzymatic degradation of hydroxypropyltrimethylammonium wheat starches.

The enzymatic degradation of hydroxypropyltrimethylammonium modified starches synthesised by dry process was compared with that of hydroxypropyltrimethylammonium modified starches synthesised in glycerol-water plasticised molten medium. The enzymatic degradation rate of products from both origins decreased as the degree of substitution increased. However, two distinct enzymatic degradation profiles were obtained. Dry process products displayed a regular decrease pattern as DS increased. Molten medium synthesised cationic starches displayed a constant degradation level on a wide DS range with alpha,beta-amylase and amyloglucosidase, whereas isoamylase degradation rapidly reached its degradation limit at DSs 0.05. The various plasticising conditions used to synthesise cationic starch in molten medium show no influence on the enzymatic degradation. By measuring the affinity of alpha-amylase, beta-amylase and isoamylase for native, extruded non-modified and hydroxypropyltrimethylammonium-modified starches. It was evident that the enzymes' affinity for the substrate diminishes with increasing chemical modification, particularly in the case of alpha-amylase, suggesting that the location of cationic groups impairs the enzyme's recognition of the substrate. Structural elements of limit dextrins were analysed by (1)H NMR.

The endopolysaccharide metabolism of the hyperthermophilic archeon Thermococcus hydrothermalis: polymer structure and biosynthesis.

The endopolysaccharide accumulated by Thermococcus hydrothermalis was extracted and purified from a 4 h culture. It presented an "amylopectin-like" structure with an average chain length of 14 and a ramification degree of 7.5%. The glucosyltransferase was isolated, partially purified and characterized. The molecular mass was 42 kDa by SDS PAGE and 85 +/- 5 kDa by gel filtration. This enzyme was able to use both Uridine-5'-DiPhosphoGlucose (UDPG) and Adenosine-5'-DiPhosphoGlucose (ADPG) as substrates. Optimal pH and temperature for the enzyme were 5.5 and 80 degrees C, respectively. In the presence of 3.2 mM ADPG, the half life of the protein was 6 min at 110 degrees C. The apparent Km value with the two substrates was 0.9 mM, but the Vmax was 9.7 fold higher for ADPG. A branching activity was also detected at high temperature, up to 80 degrees C by different methods: phosphorylase stimulation, iodine, and branching linkage assays.