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

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 β-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 β-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 β-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 β-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.