Análisis del componente alimentario de la CFB

vivo. Attempts to modify the standard MSP-1 processing assay to improve its sensitivity, reproducibility and ease of use are under way. Preliminary experiments have shown that semi-automation of the assay, based on both a capture ELISA and BIAcore assay, is

feasible and can provide quantitative data. However, further validation of the MSP-1 processing assay should be carried out. For this purpose sera from humans vaccinated with recombinant based MSP-1 vaccines as well as from humans living in malaria-endemic areas will be assayed for processing-inhibitory activity and any correlation with clinical status will be established.

In the third part of this study, a mechanism by which P. falciparum parasites may avoid the host immune response was described. Within the panel of antibodies that react against MSP-119 some were defined and extensively studied in the previous chapter as

“processing-inhibitory antibodies”. Other mAbs which have no effect in either processing or erythrocyte invasion were denoted “blocking antibodies” in this section. Blocking antibodies - antibodies which possess neither processing-inhibitory nor erythrocyte invasion-inhibitory activity, but which interfere with the activity of mAbs 1 2 . 8 and 1 2 . 1 0 -

were found to act by competitively preventing the binding of 12.8 and 1 2 . 1 0 to MSP-1.

Blocking antibodies were found to be induced not only by epitopes within the C-terminal domain of MSP-1, but also by epitopes outside MSP-119. It was shown that naturally acquired human antibodies specific for epitopes within MSP-183 were capable of blocking

the inhibitory activity of mAb 12.10. The potential protection mediated by processing- inhibitory antibodies could be diminished by the presence or induction of blocking antibodies. Further work in the laboratory has focused on the identification of epitopes involved in the induction of blocking and inhibitory antibodies. Currently, a number of rM SP-li9 mutants, containing single and double point mutations, have been expressed in

E. colt; the mutations were designed to knock out epitopes involved in the induction of

In the fourth part of this study, structural analysis of recombinant and native MSP-119 were carried out in order to determine the presence of possible post-translational

modifications. It was shown that rMSP-lig appeared to have the same protease-resistance folding as the parasite-derived MSP-119 . It is possible that this “protective” folding may

interfere with the proteolytic processing necessary for MHC class II presentation of MSP- 119-derived peptides by antigen presenting cells. Data obtained from lectin binding assays

suggest the absence of #-linked or 0-linked giycosylation in P. falciparum MSP-11 9 . However, further studies will be necessary in order to rule out the presence of TV-linked or 0-linked carbohydrates in MSP-119 and to provide more conclusive information on post-

translational modification of the protein. Further lectin analysis should be carried out, use should be made of ConA, and a saturating amount of nitrocellulose-immobilised merozoite proteins will be used to guarantee the presence of a significant amount of glycoproteins. In addition analysis of purified, parasite-derived (native) MSP-119 by

HPLC/MS will provide information on any oligosaccharide modifications. Determination of the sites and extent of possible giycosylation may provide insights into the function of MSP-1.

Malaria has become one of the most difficult obstacles to overcome in the development of third world countries. Human malaria is responsible for the death of approximately 2 million children per year and affects one sixth of the worlds total population, potentially leading to the collapse of already limited and precarious health budgets. The growing and alarming appearance of parasite drug resistance, the human toxicity of certain antimalarials, as well as the increasing cost of effective chemo­ prophylaxis in malaria endemic areas, has placed enormous pressure on the scientific community to work towards the development of an effective malaria vaccine. To date MSP-1 is the most promising candidate for a vaccine against malaria. The physiological function of either MSP-1 or the proteolytic processing of MSP-1, and the detailed identity of the protease which mediates it, are unknown. However, the data presented in this work have increased our understanding and re-emphasise the importance of the processing step in the survival of P. falciparum within the human host, and the potential of the relevant enzyme as a novel target for development of site-directed anti-protease peptidiomimetics. This work has possibly provided a reliable laboratory assay capable of predicting protection against malaria in vivo. In conclusion, the data obtained in this study have important implications in the rational design and optimal use of a MSP-1-based vaccine against human P. falciparum malaria.

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