Synthesis, Conformational Properties, and Immunogenicity of a Cyclic Template-Bound Peptide Mimetic Containing an NPNA Motif from the Circumsporozoite Protein of Plasmodium falciparum

The immunodominant central portion of the circumsporozoite (CS) surface protein of the malaria parasite Plasmodium falciparum contains a tetrapeptide motif, Asn-Pro-Asn-Ala (NPNA), tandemly repeated almost 40 times. The three-dimensional structure of the CS protein, including the central repeat region, is presently unknown. We have investigated an approach to stabilize beta -turns in a single NPNA motif, by its incorporation into a template-bound cyclic peptide comprising the sequence ANPNAA. The template was designed to stabilize beta -turns in the peptide loop and to allow its conjugation to T-cell epitopes in a multiple-antigen-peptide. NMR studies and MD simulations with time-averaged NOE-derived upper distance restraints support the formation of a stable beta -I turn conformation in the NPNA motif of this template-bound antigen. Balb/c mice immunized with a multiple-antigen-peptide containing four copies of the template-bound loop conjugated to a single universal T-cell epitope produced antibodies that bound P. falciparum sporozoites in immunofluorescence assays. These results provide further support for the immunological relevance of a type-I beta -turn conformation based on the NPNA cadence in the repeat region of the CS protein and illustrate the use of a novel template for the evaluation of conformationally constrained peptide immunogens.