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

Abstract

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.

Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate

“…Several studies examined conformationally constrained NANP mimetics by NMR and CD (37), cyclic ANPNAA peptides by NMR (57), and Ac-ANPNA-NH 2 peptides by crystallography (36), all of which demonstrated the NPNA motif adopts a ␤-turn (58). Speculation by Nanzer et al (59) suggests that by affixing multiple NPNA repeats together, a stable "stemlike" structure may be formed.…”

Synthesis, Conformational Analysis, and Biological Activity of Proposed Vitronectin Antagonists

“…In addition to their utility as replacements for i+\ and i+2 amino acids of a ß-turn in acyclic peptides, bicyclic heterocycles have also been used to tie peptides into a loop [30]. In research directed toward the development of an antimalarial vaccine, Asn-Pro-Asn-Ala (NPNA, a repetitive sequence found in a protein believed to be involved in the transference of malaria) was predicted to exist in a ßl turn.…”

Preclinical profiling of the immunogenicity of a two-component subunit malaria vaccine candidate based on virosome technology

“…25 We have previously shown the development of synthetic peptides based on these two proteins for inclusion into a multivalent subunit malaria vaccine. [26][27][28][29][30] Peptide UK-39 is based on the NANP repeats of CSP 30 and peptide AMA49-C1 on the erythrocyte-binding loop 1 of domain III from AMA-1. 27 Both peptides were developed in a stepwise optimization process and the optimized peptides were able Presentation of synthetic peptides on immunopotentiating reconstituted influenza virosomes is a promising technology for subunit vaccine development.…”