ABSTRACT
Although vaccines have been used for a long time and different types of vaccines have been developed, as yet no fully synthetic vaccines have been produced. The production of fully synthetic vaccines has probably not been realized so far due to the structural limitations of linear synthetic peptides to mimic the native shape of protein fragments which is often needed to induce protective antibodies. In this report we used the Bordetella pertussis protein pertactin as a model and show that a novel synthetic scaffold can be used to mimic structurally defined epitopes by confined presentation of several different peptide arms. Guided by modelling a construct was synthesized that induced protective antibodies directed towards a discontinuous epitope. This approach opens up the possibility to the design of new and fully synthetic vaccines that can induce protective antibodies.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Epitopes/chemistry , Epitopes/immunology , Molecular Mimicry/immunology , Virulence Factors, Bordetella/chemistry , Virulence Factors, Bordetella/immunology , Animals , Antibodies/immunology , Bacterial Outer Membrane Proteins/chemical synthesis , Bacterial Outer Membrane Proteins/genetics , Bordetella pertussis/immunology , Crystallography, X-Ray , Mice , Models, Molecular , Molecular Structure , Mutation/genetics , Virulence Factors, Bordetella/chemical synthesis , Virulence Factors, Bordetella/geneticsABSTRACT
A synthetic tridecapeptide, corresponding to the 30-42 fragment of the S1 subunit of pertussis toxin, has been structurally characterised by using NMR spectroscopy. The molecule corresponds to a T-cell epitope of the bacterial toxin which has been extensively analysed with the alanine scanning approach to check the relevance of each residue for the biological activity of the peptide. Five of these Ala-substituted analogs have also been spectroscopically studied. In the experimental conditions used, different extents of helicity were found for the six peptides in a way which cannot be related to their capabilities of of binding to major histocompatibility complex (MHC) class II and inducing T-cell proliferation. Backbone flexibility around helical transient conformations seems to constitute the structural intermediate step between the structure of the corresponding sequence within the parental protein and in the MHC class II complex. A model of the latter complex, which accounts for the different biological activities of the analogs, is proposed.
Subject(s)
Epitopes, T-Lymphocyte/chemistry , Pertussis Toxin , Virulence Factors, Bordetella/chemistry , Virulence Factors, Bordetella/immunology , Amino Acid Sequence , Drug Design , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Histocompatibility Antigens Class II/chemistry , Humans , Macromolecular Substances , Magnetic Resonance Spectroscopy , Models, Molecular , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/immunology , Protein Conformation , Protein Structure, Secondary , Structure-Activity Relationship , Vaccines, Synthetic/chemistry , Virulence Factors, Bordetella/chemical synthesisABSTRACT
A manually operated apparatus for parallel multiple column soli-phase peptide synthesis is described. It employs Fmoc-amino acid-O-Dhbt or -Pfp esters in the continuous flow version of the polyamide method on small packed columns of kieselguhr supported resin in a reaction block of Teflon. The solvents and deprotecting reagents are dispensed from two washers in a parallel fashion and reagent consumption is low. Activated and protected amino acids are transferred from a dispenser tray as solutions, eight at a time. The use of the method is demonstrated by the synthesis of overlapping peptides from a protein structure and of analogous protease substrates. The products have been characterized by HPLC, FAB mass spectroscopy and amino acid analysis.
