Design, production and characterization of synthetic immunogens : application on Plasmodium falciparum malaria antigens

Abstract: The development of synthetic subunit vaccines is presently one of the major strategies to construct vaccines against infectious diseases. Such vaccines are based on isolated and characterized immunogens and should include appropriate B- and T-cell epitopes. The construction of synthetic vaccines is dependent on the accessibility of techniques for efficient production of the immunogens and the possibility to combine several immunogens and safe and potent adjuvant/ delivery systems in the same vaccine formulation. The present thesis describes methods for the design, production and immunological characterization of immunogens based on the Plasmodium falciparum malaria blood stage antigens Pf 155/RES A and Clustered-Asparagine- Rich-Protein (CARP).Genetic fusion systems based on the IgG-binding domains of staphylococcal protein A and the serum albumin binding region of streptococcal protein G were developed for the polymerization, assembly and expression of genes encoding epitope-carrying peptides and the subsequent affinity purification of the produced fusion proteins by a one-step procedure. Immunogens produced in this way were immunogenic in various animal models after administration in Freund's adjuvant and induced antibodies which reacted with the malaria sequence used for immunization as well as with the corresponding intact protein. Furthermore, these antibodies were efficient inhibitors of merozoite invasion of erythrocytes in vitro. The antibody responses were sustained over long time periods, could be efficiently boosted and were specific for Pfl55/RESA as well as the sequence used for immunization.Since Freund's adjuvant is not suitable for use in humans, the immunopotentiating capacity of several other adjuvant/delivery systems was investigated. Of these, only immunostimulating complexes (ISCOMs) induced antibody levels comparable to those obtained with Freund's adjuvant. ISCOMs are spherical particles composed by the glycoside adjuvant Quil A, cholesterol and phospholipids in which antigens are presented in a multimeric form. ISCOM-based immunogens were prepared by direct incorporation or by conjugation of fusion proteins to pre-formed influenza vims ISCOMs. Immunization with ISCOMs prepared in this way resulted in strong antibody responses to all polypeptide components of the particles and these antibodies recognized the corresponding intact proteins. The antibody responses induced by the ISCOMs were efficiently boosted, indicating the induction of immunological memory including memory specific for the malariaderived sequences. The efficient boosting of the antibody responses to the malaria sequences suggests that possible competitive or suppressive effects of the additional components of the ISCOM preparations in general were weak or absent. Taken together, these results imply that incorporation or conjugation of fusion proteins to ISCOMs may form a suitable basis for the construction of multivalent synthetic subunit vaccines.The immunogenicity in inbred mice of synthetic immunogens has been shown to be influenced by genetic restriction. Strong B- and T-cell responses in mice to fusion proteins containing large numbers of Pf 155/RES A repeat sequences were linked to expression of the MHC class II I-Ak allele. While a certain cross-reactivity between the different Pf 155/RES A repeat sequences was demonstrated for the T-cells that were stimulated by the fusion proteins, the corresponding antibody responses were specific for the repeat sequences used for immunization. These results indicate that the T-cells which were induced by immunization with the different fusion proteins and the corresponding B-cells had separate major specificities.