Characterization of novel malaria vaccine candidates representing alpha-helical coiled coil domains

The future vision in the battle against malaria goes beyond controlling the disease. Envisioned is the world-wide eradication of malaria. A substantial contribution to reach this goal is the development of an effective vaccine. Today’s most advanced and most effective malaria vaccine, RTS,S/AS01, showed efficacy of 30 to 66% against all clinical episodes. There is a great need to increase efficacy by the next generation malaria vaccines. A strategy for increasing RTS,S efficacy could be to combine it with an effective blood stage vaccine. The disappointing outcomes of clinical trials conducted for most current blood stage vaccines demands the identification of novel promising candidates.
Under persistent exposure individuals develop immunity that protects against clinical disease but not parasitemia. This natural acquired immunity develops slowly and is reached in adolescence. In contrast, immunity against severe disease develops already after few infections. The mechanisms that underlie naturally acquired immunity or severe disease immunity remain poorly understood. Antibodies were demonstrated to play a critical role for controlling blood stage infection. It remains unclear which proteins elicit the production of protective antibodies and through which antibody effector function protection is provided. The relevance of antibodies in blood stage protection has the consequence that the immunogen correctly mimic the three-dimensional structure of the native protein. This PhD thesis has its major focus on immunogens that adopt a stable tertiary structure in aqueous environment.
The availability of the P. falciparum genome sequences, transcriptome and proteome data has opened the avenue for the identification of novel targets for vaccine development. However, blood stage vaccine development has focused on only a few candidates. Previously our collaborators in this project have identified promising candidates by genome-wide screening for alpha-helical coiled coil domains in proteins expressed in the erythrocytic parasite stages. The segments with high probability score for coiled coil formation where selected. The 166 coiled coil segments derived from 131 proteins representing 4% of the blood stage proteome. 95 coiled coil fragments of a length of 30-40 amino acids were synthesized and analyzed systematically in a pre-clinical evaluation pathway.
The aim of this thesis was to fill the gaps in the preclinical evaluation pathway of novel synthetic peptide vaccine candidates.
The extensive polymorphism found in most parasite antigens represents a major obstacle for the development of efficacious blood stage vaccines. The genetic diversity of the identified coiled coil protein segments was studied in great detail. We found that coiled coil segments are well conserved, 82% of all selected 166 segments showed complete sequence conservation. Polymorphism was found predominantly in segments containing almost perfect tandem repeats. Based on these findings an optimized bioinformatic selection strategy was formulated proposing to exclude coiled coil segments consisting of almost perfect tandem repeats.
The availability of basic knowledge about vaccine candidates is a prerequisite for vaccine development and is essential to attract further funding for continued clinical development. A detailed cell biological characterization was undertaken for the most promising candidate, PFF0165c (newly termed Trophozoite exported protein 1 (Tex1)) Transcript and protein levels were analyzed throughout the intra-erytrocytic development cycle. Tex1 transcripts were found up-regulated in the early trophozoite stage. This was supported by Tex1 protein levels. Tex1 abundance persisted until parasite egress. Immunofluorescence experiments revealed that Tex1 is exported and associates to parasite-derived structures, termed Maurer’s clefts. Before parasite egress Tex1 resides in close proximity to the red blood cell membrane. In the search of sequence motifs responsible for Tex1 export we found that the actual translational start site is positioned 43 amino acids upstream of the start site previously predicted. The additional 43 amino acids function as signal peptide, directing the protein into the classical secretory pathway.
This thesis contributed to the immunological characterization of the intrinsically unstructured region (P27A) of Tex1. P27A was evaluated for vaccine potential and met the principal requirements to be downselected for a phase 1 trial. P27A was recognized by a majority of naturally exposed individuals, highly immunogenic, highly conserved and P27A-specific human and mouse sera were effective in in vitro parasite killing by Antibody-dependent cellular inhibition assay. High P27A-specifc antibody titers were found to positively correlate with protection. Clinical grade P27A peptide is currently produced.
In order to validate synthetic peptides as antigens the recognition by sera of adults from endemic region was compared to the recognition of the antigen recombinant expressed in E. coli. Comparable recognition of both types of antigens was observed.
This thesis provides evidence that the approach initiated by our collaborators is invaluable. This strategy, if proven successful in clinical trials, could be applied for vaccine development against many other pathogens from which genome data is available.