The aim of present study was the production of protease from local Bacillus subtilis through solid state fermentation. Response Surface Methodology (RSM) was used for the optimization of all the culture conditions. Casein (1% w/v) was used as a substrate in nutrient agar medium for the screening of enzyme production potential and showed maximum zone of clearance (4.6 cm). It was identified as Bacillus subtilis K-5 by genetic identification based on 16S rRNA and blast technology of NCBI. Among culture conditions, incubation temperature, incubation time, pH of the medium and moisture level of the substrate were optimized. Maximum protease production was observed at 37^oC, pH 9.0 with incubation time of 36 h and moisture to substrate ratio of 1: 0.75. Maximum protease production of 70.21 U/mL was recorded when wheat bran was used as an agro-industrial substrate. The activity of crude protease was observed 99.63 % at 60^oC and pH 10.0 with protein concentration 0.63 mg/mL and specific activity of 111.56 U/mg. Protein contents of 0.57 mg/mL (specific activity of 124.72 U/mg) and protein contents of 0.44 mg/mL (specific activity of 143.65 U/mg) were observed by 70% saturation with ammonium sulphate and gel chromatography, respectively. Line Weaver Burk plot was used to find its Vmax and Km, which were 344. 83 mg/mL/min and 100.04 mg/mL, respectively. The study concluded that Bacillus subtilis K-5 is thermophilic and alkaliphilic strain which produces active protease and can be used as potential microorganism for industries.

Haemophilus influenzae is a Gram-negative bacterium characterized as a small, nonmotile, facultative anaerobic coccobacillus. It is a common cause of a variety of invasive and non-invasive infections. Among six serotypes (a–f), H. influenzae type b (Hib) is the most familiar and predominant mostly in children and immunocompromised individuals. Following Hib vaccination, infections due to other serotypes have increased in number, and currently, there is no suitable effective vaccine to induce cross-strain protective antibody responses. The current study was aimed to validate the capability of two 20-mer highly conserved synthetic tbp1 (transferrin-binding protein 1) peptide-based vaccine candidates (tbp1-E₁ and tbp1-E₂) predicted using in silico approaches to induce immune responses against H. influenzae strains. Cytokine induction ability, immune simulations, and molecular dynamics (MD) simulations were performed to confirm the candidacy of epitopic docked complexes. Synthetic peptide vaccine formulations in combination with two different adjuvants, BGs (Bacterial Ghosts) and CFA/IFA (complete/incomplete Freund’s adjuvant), were used in BALB/c mouse groups in three booster shots at two-week intervals. An indirect ELISA was performed to determine endpoint antibody titers using the Student’s t-distribution method. The results revealed that the synergistic use of both peptides in combination with BG adjuvants produced better results. Significant differences in absorbance values were observed in comparison to the rest of the peptide–adjuvant combinations. The findings of this study indicate that these tbp1 peptide-based vaccine candidates may present a preliminary set of peptides for the development of an effective cross-strain vaccine against H. influenzae in the future due to their highly conserved nature.

Dengue fever, a rapidly spreading mosquito-borne viral disease, poses a major global health issue, particularly in tropical and subtropical regions. The absence of a universally effective vaccine against all four dengue virus serotypes necessitates continued exploration of novel vaccine strategies. This study evaluated a PrM-derived multiple antigenic peptide (MAP) vaccine candidate in BALB/c mice. Molecular docking analyses predicted favourable interactions of the selected PrM-derived B-cell epitope with both human (human BCR Fab region PDB ID: 5IFH) and mouse (mouse BCR PDB ID: 8EMA) B-cell receptors, supporting its antigenic potential. ChemSketch was used to develop the PrM–MAP vaccine construct, which was then commercially synthesized. Mice received single, double, and triple booster doses of the vaccine, while control groups received either PBS or adjuvant alone. Humoral immune responses were measured by ELISA, and safety was evaluated through in vitro cytotoxicity assays on HEK293 cells and the in vivo histopathological examination of major organs. Mice immunized with a single booster dose produced significant levels of PrM-specific antibodies compared to subsequent booster doses, suggesting variability in the humoral response elicited. Optimal coating concentrations in ELISA demonstrated the importance of antigen concentrations in the sensitivity of the assay. The safety evaluation indicated high cell viability, up to 150 µg/mL of PrM–MAP, in HEK293 cells, and an absence of significant histopathological damage in treated animals. The PrM–MAP vaccine construct elicited a detectable humoral immune response while demonstrating a high safety profile in vitro and in vivo. These findings indicate the potential of MAP-based vaccine platforms as promising candidates for further development against the dengue virus.