In silico Development of Multi Epitope Based Recombinant Vaccine to Prevent Cervical Cancer

Aims:.Cervical cancer is predominantly associated with persistent infection from high-risk human papillomavirus (HPV) strains, especially HPV16.This study focuses on the in silico design of a multiepitope- based recombinant vaccine targeting the E6 and E7 oncoproteins of HPV16. Through computational epitope prediction using IEDB tools, we selected CTL, HTL, and B-cell epitopes exhibiting high antigenicity, non-allergenicity, and non-toxicity. These epitopes were linked using specific peptide linkers, and an immunostimulatory adjuvant was added to enhance immunogenicity. The final vaccine construct underwent structural modeling using SWISS-MODEL and validation using Ramachandran plot analysis. Molecular docking with TLR4 receptor was conducted using ClusPro to predict immune interaction. Codon optimization and in silico cloning were performed using SnapGene for expression in E. coli. The computational analysis demonstrated high antigenicity, structural stability, and effective immune interaction, supporting the potential of this construct as a therapeutic HPV vaccine candidate. Study Design: Experimental in silico study. Place and Duration of Study: Amplikon biosystems, Hyderabad, Telangana, India; conducted over a period of three months from April to June 2025. Methodology: The protein sequences of HPV16 E6 and E7 were collected from the UniProt database and examined to find potential regions (epitopes) that could trigger an immune response. For this, different tools from the IEDB resource were used: NetMHCpan EL 4.1 for cytotoxic T-cell epitopes, the IEDB recommended method for helper T-cell epitopes, and BepiPred-2.0 for B-cell epitopes. The best candidates were chosen based on their strength of binding, ability to act as antigens, and sequence conservation. These selected epitopes were then joined together using short linkers (AAY, GPGPG, and KK), and an adjuvant protein (β-defensin or 50S ribosomal protein L7/L12) was added at the beginning of the construct through an EAAAK linker to boost immune activity. The designed vaccine was then checked for its properties: VaxiJen v2.0 was used to confirm its antigenicity, AllerTOP v2.1 to ensure it is non-allergenic, and ToxinPred to verify that it is non-toxic. ProtParam was used to calculate basic features such as molecular weight, isoelectric point, stability, and half-life. A three- dimensional structure of the construct was built using modeling servers, and its quality was tested through a Ramachandran plot. To study how the vaccine might interact with the immune system, molecular docking was performed with Toll-like receptor 4 (TLR4) using the ClusPro server. Finally, the vaccine gene sequence was optimized for E. coli expression and virtually inserted into the pET-28a (+) vector using SnapGene for cloning and expression purposes. Results: The multiepitope vaccine construct designed from HPV16 E6 and E7 proteins showed promising immunological properties. Epitope prediction identified strong CTL, HTL, and B-cell epitopes, which were linked with suitable linkers and an adjuvant to form the final construct. Antigenicity testing with VaxiJen confirmed the construct as highly antigenic, while AllerTOP and ToxinPred predicted it to be non-allergenic and non-toxic, ensuring safety. Physicochemical profiling revealed a stable protein with favorable molecular weight, isoelectric point, and stability index for expression in E. coli. The 3D structural model displayed good stereochemical quality in Ramachandran analysis. Docking studies with Toll-like receptor 4 (TLR4) showed strong binding interactions, indicating the potential to stimulate innate immune responses. Codon optimization and in-silico cloning into the pET-28a (+) vector further confirmed the suitability of the construct for bacterial expression. Conclusion: The computational analysis demonstrated that the designed multiepitope vaccine construct is antigenic, safe, and structurally stable, with strong binding potential to immune receptors. These findings suggest that the construct is a promising candidate for further development and experimental validation against HPV16.