Vaccines 2022, 10, 1850. https://doi.org/10.3390/vaccines10111850 www.mdpi.com/journal/vaccines Article Designing of Peptide Based Multi-Epitope Vaccine Construct against Gallbladder Cancer Using Immunoinformatics and Computational Approaches Mukhtar Ahmad Dar 1 , Pawan Kumar 2 , Prakash Kumar 2 , Ashish Shrivastava 3 , Muneer Ahmad Dar 4 , Richa Chauhan 5 , Vinita Trivedi 5 , Ashutosh Singh 3 , Eshan Khan 6 , Ravichandiran Velayutham 7 and Sameer Dhingra 1, * 1 Department of Pharmacy Practice, National Institute of Pharmaceutical Education and Research (NIPER), Hajipur 844102, Bihar, India 2 Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Hajipur 844102, Bihar, India 3 Translational Bioinformatics and Computational Genomics Research Lab, Department of Life Sciences, Shiv Nadar University, G.B. Nagar 201314, Uttar Pradesh, India 4 Division of Biotechnology, Sher-e-Kashmir University of Agricultural Sciences and Technology Kashmir (SKUAST-K), Shuhama 191201, India 5 Department of Radiotherapy, Mahavir Cancer Sansthan and Research Centre (MCSRC), Phulwarisharif Patna 801505, Bihar, India 6 Department of Cancer Biology and Genetics, The Ohio State University, 460 West 12th Avenue, Columbus, OH 43210, USA 7 Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Kolkata 700054, India * Correspondence: sameerdhingra78@gmail.com Abstract: Gallbladder cancer (GBC) is an aggressive and difficult to treat biliary tract carcinoma with a poor survival rate. The aim of this study was to design a peptide-based multi-epitope vaccine construct against GBC using immunoinformatics approaches. Three proteins implicated in the progression of GBC were selected for B and T cell epitope prediction and the designing of the potential vaccine construct. Seven CTL, four HTL and six Bcell epitopes along with a suitable adjuvant were selected and connected using linkers for designing the vaccine construct. The secondary and tertiary models of the designed vaccine were generated and satisfactorily validated. A Ramachandran plot of the final 3D model showed more than 90% of the residues in allowed regions and only 0.4% in disallowed regions. The binding affinity of a vaccine construct with TLR 2, 3 and 4 receptors was assessed through molecular docking and simulation. The average numbers of hydrogen bonds for vaccine-TLR 2, 3 and 4 complexes in the simulation were 15.36, 16.45, and 11.98, respectively, and remained consistent over a 100ns simulation period, which is critical for their function. The results of this study provide a strong basis for further evaluation through in vitro/in vivo experimental validation of the safety and efficacy of the designed vaccine construct. Keywords: immunoinformatics; vaccine; epitope; antigenicity; TLR; GBC 1. Introduction The global burden of cancer is increasing rapidly with more than 19.3 million new incident cancers and 9.9 million deaths estimated in 2020 [1,2]. Worldwide, the incidence and mortality of GBC in 2020 was reported to be 115,949 and 84,695, respectively [1]. GBC is the most frequently diagnosed biliary tract cancer with poor prognosis and a high fatality rate [3]. The median survival of GBC is less than one year with an overall five-year survival rate ranging from 5–20% [4–6]. The poor survival of GBC is mostly associated Citation: Dar, M.A.; Kumar, P.; Kumar, P.; Shrivastava, A.; Dar, M.A.; Chauhan, R.; Trivedi, V.; Singh, A.; Khan, E.; Velayutham, R.; Dhingra, S. Designing of Peptide Based Multi-Epitope Vaccine Construct against Gallbladder Cancer Using Immunoinformatics and Computational Approaches. Vaccines 2022, 10, 1850. https:// doi.org/10.3390/vaccines10111850 Academic Editor: Takumi Kumai Received: 25 September 2022 Accepted: 26 October 2022 Published: 31 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/).