Design of a peptide-based vaccine from late stage specific immunogenic cross-reactive antigens of PE/PPE proteins of Mycobacterium tuberculosis

Since decades now, Tuberculosis (TB) is among the leading cause of death globally. Innovative and extensive research strategies are necessary to lower TB incidence and achieve the End TB Strategy milestone. Epitope-based vaccine designing and development provides a promising solution with high efficacy and effectiveness. Mining of less studied genes of Mycobacterium tuberculosis (Mtb) is crucial for recognizing potential antigenic peptide epitopes which can mount protective immune response in host. Many proteins from ESX associated Proline-Glutamate (PE)/ Proline-Proline-Glutamate (PPE) family are virulence factors and alter host mediated immune response against the pathogen. In the present study, we have targeted 34 late stage expressing (being expressed at 90 days) PE/PPE proteins of Mtb for prediction and identification of promiscuous, immunogenic and cross-reactive CD4+ T cell specific epitopes. We found a total of 149 promiscuous and cross-reactive epitopes out of which 42 were antigenic as well. Further, we shortlisted top 10 Promiscuous, Cross-reactive CD4+ T cell specific, Antigenic Peptide Epitopes (PCAPEs) which were characterized to be non-allergenic and pro-inflammatory cytokine inducing in nature. These epitopes also showed strong binding affinity for CD8+ T cell restricted Major Histo-compatibility Complex (MHC) class I alleles. Additionally, these PCAPEs showed wide population coverage of 99.6% globally for both MHC class I and class II alleles. Molecular docking studies were conducted to confirm the affinity of these shortlisted peptides for widely occurring MHC alleles. Additionally, we performed codon adaptation and in silico cloning of the recombinant vaccine construct incorporating EsxA (ESAT-6) as an adjuvant and the 10 selected PCAPEs joined by linkers. The recombinant vaccine construct showed strong affinity for Toll-like receptor2 (TLR2) immune receptor in docking studies. In silico prediction based study using C-ImmSim server shows significant population of Th1 type immune cells with memory cells lasting for months in response to our vaccine administration. Since, majority of TB vaccines under clinical trials are antigens expressed at early stages; a combinatorial approach inclusive of peptide epitopes derived from proteins being expressed at all stages could be a promising strategy to design and develop effective TB vaccine. Synthesis and experimental validation of this multi-epitopic recombinant TB vaccine construct may result in an effective vaccine to confer protection against Mtb.