Bioinformatic development of a recombinant trivalent synthetic protein construct using PTXa, Tox, and TetX toxins as...

Traditionally, diphtheria-tetanus-pertussis (DTwP or DTaP) as pediatric vaccines are produced from the corresponding inactivated toxins or whole -cell pathogenic bacteria of Corynebacterium diphtheria toxin (Tox), Clostridium tetani toxin (TetX) and Bordetella pertussis. There are major concerns in the classic or acellular DTP (DTaP) vaccine production processes from native live bacterial sources as it may raise concerns on adverse effects and safety issues, complexity of the purifications for each agent as well as cost. Here, we designated a recombinant multi-epitope vaccine candidates by vaccino-informatics study to address the mentioned issues and to develop a single trivalent fusion protein as a potent recombinant DTP vaccine. To achieve these goals, stages of immune-bioinformatics were retrieved using proteinaceous toxins sequences, predicting secondary/tertiary structure and transmembrane topology, energy minimization, and model validation. Then, conformational and linear Bcell epitope prediction by several servers, mapping of consensus linear/discontinuous immunogenic regions and construction synthetic fusion vaccine candidates in respect to optimal immunogenic, physicochemical properties and high expression in prokaryote host were achieved. Finally, reverse translation, codon optimization, addition of cloning tags for pet 28a vector and optimization of physicochemical properties of synthetic trivalent fusion protein were performed. Through various hybrid immuno-informatics and structural bioinformatics analysis of predicted and experimental epitopes finally, 12 new consensus highly immunogenic linear and discontinuous epitopes in Tox, TetX and PTXa proteins were selected. The peptide sequences of these immunogenic regions were as follows: PTXA (AA34-64, AA184-256 and AA98-116), Tox (AA47-76, AA117-159, AA515-557 and AA245-265) and TetX (AA226-249, AA819-844, AA923-967, AA1009-1067 and AA1225-1315). In addition, the characteristics of the recombinant trivalent fusion construct were; 546 residue length, soluble (Grand average of hydropathicity (GRAVY) was -0.475), estimated half-life was >10 hours in Escherichia coli, pI 5.94 (a little acidic), stable protein (The instability index (II) 35.58) as well as thermally stable (Aliphatic index (AI) 71.67). The putative antigenic epitopes from different organisms in a single protein, as in the current study, will possibly improve the protective efficacy as novel potent, safe, cheap and broad-spectrum vaccines for better prevention of diphtheria, tetanus and pertussis infections in the future.