Development of a cross-strain effective vaccine against H. influenzae strains using In-silico and In-vivo Approaches

Abstract

Haemophilus influenzae is a Gram-negative, nonmotile, facultative anaerobic coccobacillus that commonly causes a variety of invasive and non-invasive infections. It colonizes the respiratory tract and is associated with life-threatening invasive infections. The recent rise in its global prevalence, even in the presence of multiple vaccines, indicates an urgent need to develop effective cross-strain vaccine strategies. First objective of current work focused on identifying the universally conserved antigenic regions of H. influenzae that can be used to develop new vaccines. A variety of bioinformatics tools (subcellular localization, essentiality, virulence, and non-host homology, 3D protein structures determinations, B and T cell epitope mapping, molecular docking and comparative genome analysis) were applied for the comprehensive geno-proteomic analysis of H. influenzae type ‘a’ strain, as reference serotype, through which the candidature of the identified regions was established. Based on the established vaccinomics criteria, five target proteins were predicted as novel vaccine candidates. Among these, nine epitopic regions that could regulate lymphocyte activity through strong protein–protein interactions were identified. Comparative genomic analysis revealed that the identified regions were highly conserved among the different strains of H. influenzae. Based on multiple immunogenic factors, five prioritized proteins and their predicted epitopes were identified as ideal common putative vaccine candidates against typeable strains. The second objective aimed to validate the immune potential of highly conserved synthetic tbp1 (transferrin-binding protein 1) peptide-based vaccine candidates (tbp1-E1 and tbp1-E2) predicted using In-silico approaches. The candidacy of the epitopes was confirmed by finding their Cytokine induction ability, immune simulations, and molecular dynamics (MD) simulations of docked complexes. BALB/c mouse were injected with vaccine formulations of peptides: adjuvants (BGs; Bacterial Ghosts) and CFA/IFA (complete/incomplete Freund’s adjuvant), in three booster shots at two-week intervals. Endpoint antibody titers was determined using the Student’s t-distribution method following an indirect ELISA. The results revealed that combining peptides (tbp1-E1 and tbp1-E2) with adjuvants produced better results. These findings suggest that the tbp1 peptide-based vaccine candidates could potentially be used to develop a cross-strain vaccine against H. influenzae in the future, as Abstract xvi they are highly conserved. The goal of the third objective was to generate an edible vaccine against the H. influenzae OMP P6 gene in a model plant, Nicotiana tabacum, using Agrobacterium-mediated plant transformation. The synthetic OMP P6 gene was cloned in a Binary vector pGreen0029 and transformed into Agrobacterium tumefaciens strain LBA4404. The N. tabacum leaf discs were co-cultured with Agrobacterium and transferred to selective media, for regeneration following standard optimized protocols and media/antibiotics compositions. The callogenesis started in leaf discs transformed with the construct, vector control, and healthy leaf discs while the leaf disc turned yellow in the selection media. The Calli was transferred in a shoot-inducing medium and then in a root inducing medium for 15 days each. The plantlets were moved to sterile soil pots, planted in covered trays, and placed in a glasshouse with a 12-hour day and 8-hour night cycle. Protocols have been optimized for the expression of the gene of interest for the development of an edible vaccine. This was demonstrated by the successful development of calluses and shoot regeneration over the selection marker in the binary construct, in contrast to the control where the presence of antibiotic markers resulted in the death of the plants. Thus, the study design—using bioinformatics to predict target vaccines and validate methods has the potential to drive future vaccine research and development. This could lead to creating vaccines that are effective, affordable, and safe for many diseases.