Despite decades of intensive research efforts there is currently no vaccine available that provides sterile immunity against malaria. Various proteins from different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates but none of them have fulfilled the expectations. Therefore, combinations of key antigens from a single life cycle stage or multi-stage vaccine cocktails comprising promising antigens selected from different life cycle stages of the Plasmodium falciparum parasites may be essential for the development of efficacious malaria vaccines. Additionally, vaccines targeting poverty related diseases, and especially malaria, have to be produced at a low cost as well as in... More
Despite decades of intensive research efforts there is currently no vaccine available that provides sterile immunity against malaria. Various proteins from different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates but none of them have fulfilled the expectations. Therefore, combinations of key antigens from a single life cycle stage or multi-stage vaccine cocktails comprising promising antigens selected from different life cycle stages of the Plasmodium falciparum parasites may be essential for the development of efficacious malaria vaccines. Additionally, vaccines targeting poverty related diseases, and especially malaria, have to be produced at a low cost as well as in large quantities for mass immunization campaigns. The Agrobacterium tumefaciens-based transient expression in plants has emerged as a fast and low-cost protein production system that fulfil these requirements in terms of flexibility and scalability. Both crucial aspects of malaria vaccine development (novel vaccine cocktails and low cost vaccine production in large quantities) have been addressed in the present PhD thesis and the detailed characterization of a plant-derived multi-stage multi-component malaria vaccine cocktail is described. In the first part of the PhD thesis, single stage malaria vaccine candidates either a fusion protein (CCT comprising PfCelTos, the TSR domains of PfCSP and PfTRAP, a pre-erythrocytic stage malaria vaccine candidate) or variants of a leading blood stage vaccine candidate (PfAMA1) were produced at high levels up to 1.5 mg/g FLW in N. benthamiana and their vaccine potential was evaluated in stage-specific in vitro assays after mice (CCT) or rabbit (PfAMA1) immunization studies. Inhibition in the pre-erythrocytic stage of 35% were determined for antibodies induced by CCT immunization and antibodies against PfAMA1 reduced the growth of the blood stage parasites by 90%. In the second part, a multi-stage multi-component malaria vaccine cocktail (PlasmoMix) was generated by combining both promising vaccine candidates (CCT and PfAMA1) and two additional fusion proteins E3 (blood stage) and F0 (sexual stage). The fusion protein E3 comprises EGF-like domains from the blood stage antigens PfMSP1, PfMSP4 and PfMSP8 as well as a fragment of PfMSP3 and the F0 sexual stage vaccine candidate comprises the well characterized antigens Pfs25 and the C0 fragment of Pfs230. After rabbit immunization studies, the immune response against the multi-stage multi-component malaria vaccine cocktail as well as against the components (CCT, PfAMA1, E3 and F0) and all single antigens/domains were analyzed. Furthermore, the component-specific antibodies in the purified total antibody preparations were quantified by calibration-free concentration analysis (CFCA) using surface plasmon resonance (SPR) spectroscopy and correlated with the observed stage-specific in vitro inhibition. Maximum inhibition values of 80% (pre-erythrocytic stage), 90% (blood stage) and 100% (sexual stage) were detected with noticeable differences in the IC50-values. While the IC50-values for the pre-erythrocytic stage (17-25 μg/ml) and the blood stage (33-45 μg/ml) are comparable the IC50-values for the sexual stage is more than ten times lower (1.75 μg/ml). The results from the detailed analysis of the PlasmoMix vaccine cocktail provided valuable insights in the development of next-generation malaria vaccine cocktails (VaxDrIME) which should especially circumvent the hurdles of blood stage antigen polymorphisms and the study indicated that the different components should not be mixed in equal amounts but in optimal ratios to generate what may be called “balanced efficacy” by taken into account the different stage-specific IC50-requirements.
