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BackgroundFunctional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry.MethodsBoth asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay.ResultsMitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of parasite counts through CMXRos staining and flow cytometry.ConclusionsA flow cytometry method based on CMXRos staining for detection of live parasite populations has been optimized. This is a rapid and sensitive method with high inter-assay reproducibility which can reliably determine the anti-parasite effect mediated by antibodies in functional in vitro assays such as ADCI assay.

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The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria transmission-blocking vaccine (TBV). Previously, we showed that a Pfs230–Pfs48/45 fusion protein elicits higher levels of functional antibodies than the individual antigens, but low yields hampered progression to clinical evaluation. Here we identified a modified construct (ProC6C) with a circumsporozoite protein (CSP) repeat-linker sequence that enhances expression. A scalable and reproducible process in the Lactococcus lactis expression system was developed and ProC6C was successfully transferred for manufacturing under current Good Manufacturing Practices (cGMP). In addition, a panel of analytical assays for release and stability were developed. Intact mass spectrometry analysis and multiangle light scattering showed that the protein contained correct disulfide bonds and was monomeric. Immunogenicity studies in mice showed that the ProC6C adsorbed to Alhydrogel®, with or without Matrix-MTM, elicited functional antibodies that reduced transmission to mosquitoes and sporozoite invasion of human hepatocytes. Altogether, our data support manufacture and clinical evaluation of ProC6C as a multistage malaria-vaccine candidate.

Water and nutrient deficiency are two major constraints that drastically affect rapeseed and mustard production under semi-arid regions of North-Western India. To cope with this problem, a study was undertaken to optimize irrigation, nitrogen and sulphur levels for productivity and quality of Ethiopian mustard(Brassica carinata).Field experiments were conducted during winter seasons of 2004-05 and 2005-06 at the Water Technology Centre (WTC), Indian Council of Agricultural Research (ICAR) -Indian Agricultural Research Institute (IARI), New Delhi, India. Irrigation, nitrogen and sulphur levels significantly (P<0.05) affected plant height, LAI, siliqua weight, seeds/siliqua, test weight, seed and biomass yield of Ethiopian mustard. Application of three irrigations (seedling, 50% flowering and pod development stage) to Ethiopian mustard increased seed yield by 27-28% compared to one irrigation (seedling stage). Nitrogen @ 90 kg ha -1 produced 49-54 % higher seed yield of Ethiopian mustard compared to no application. Similarly, sulphur application @ 40 kg ha -1 increased seed yield by 33-34 % compared to no application of sulphur. The oil content of Ethiopian mustard was significantly (P<0.05) affected by nitrogen and sulphur levels. Among the three treatments, irrigation treatment only significantly affected evapotranspiration of the studied crop. From the above study, it is suggested that application of 3 irrigations with 90 kg N ha -1 and 40 kg S ha -1 may be practiced for achieving higher seed yield, quality and water use efficiency of Ethiopian mustard in the semi-arid environment of Northern part of India.