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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
In-silico design of a multi-epitope vaccine targeting conserved transmembrane proteins of Gardnerella vaginalis using
Adeeba Ali1, Nouman Ali2, Amna Saleem1
1Department of Biotechnology, Faculty of Science and Technology, University of Central Punjab, Lahore, Punjab, Pakistan.
Background:
Gardnerella vaginalis is one of the major etiological agents of bacterial vaginosis, which form persistent biofilms and impair the efficacy of antimicrobial agents and trigger recurrent episodes. As no licensed vaccine is currently in existence, computation-aided vaccinology is an affordable method to discover new immunogenic targets. The present study was carried out to develop multi-epitope subunit vaccine against conserved transmembrane proteins of G. vaginalis, utilizing an immune-informatics pipeline.
Methods:
Transmembrane proteins were identified as conserved by ClustalW multiple sequence alignment: C5a peptidase, phosphatidate cytidylyltransferase, and ATP-dependent zinc metalloprotease. Predictive tests of antigenicity, allergenicity and toxicity were performed using established tests. Cytotoxic T-cell (CTL), helper T-cell (HTL), and B-cell epitopes were screened for antigenicity, population coverage, and non-allergenicity and were assembled by means of flexible linkers using v -defensin as adjuvant. The three-dimensional structure of the vaccine was designed, optimized and validated. Molecular docking with Toll-like receptors TLR2 and TLR4, and HLA-DRB1. Complex stability was examined by the molecular dynamics simulations (100 ns), and the potential for immunogenicity of the vaccine was predicted using an immune simulation.
Results:
The selected epitopes were of high antigenicity with wide range of HLA coverage. Docking studies showed high binding affinities in the order of -65 to -80 a.u and predicted stable complex formation. The stability was validated by molecular dynamics simulations. Immune simulations predicted strong immune responses with high IgG/IgM titers, high IFN- and IL-2 secretion, and activation of memory B and T cells. Codon optimization for E. coli K12 resulted in a codon adaptation index of 0.97 and a GC content of 53.2%, suggesting good expression properties.
Conclusions:
The multi-epitope vaccine built in this study has an excellent theoretical immunogenicity, structural stability, and expression potential, which confirms its potential for experimental validation with in vitro and in vivo studies.
