Designing a multiepitope construct as a therapeutic vaccine for HPV using E1, E5, and E6 proteins
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Mobina Madihi,1 Mehrdad Ameri,2,*
1. Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran / Research Center for Clinical Virology, Tehran University of Medical Sciences, Tehran, Iran
2. Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
- Introduction: Introduction:
Cervical cancer is the fourth most frequent cancer in women worldwide. Human papillomavirus (HPV) is the most important etiological factor for this cancer. Prophylactic vaccines have no therapeutic effect against infection; therefore, it is important to use new approaches such as therapeutic vaccines. E5 and E6 oncoproteins cause proliferation. However, E5 downregulates the expression of HLA-class 1 then disrupts CD8+ cytotoxic T lymphocytes responses. Thereby, causing immune evasion. E6 suppress P53 and is responsible for transforming infected cell. E1 is essential for initiating DNA replication and is a helicase protein.
It is the preferable strategy to induce a broad CTL response directed against several CTL epitopes for treatment. Using reverse vaccinology and bioinformatic tools we can predict potential T cell binding epitopes. This study aims to design a therapeutic multi-epitope vaccine construct using E1, E5, and E6 Tcells epitopes.
- Methods: The reference sequence of E1 (P03114), E5 (P06927), and E6 (P03126) proteins were obtained from UniPort. To predict CTL and HTL epitopes we used netCTL 1.2 and netMHCII servers respectively. netCTL predict 9mer epitopes for 12 MHC class I supertypes (A1, A2, A3, A24, A26, B7, B8, B27, B39, B44, B58, and B62). For epitope identification in this server, 0.75 was used as the threshold. Using netMHCII we predicted 15mer HTL epitopes for HLA-DR, HLA-DQ, HLA-DP alleles. Predicted epitopes were submitted to the VaxiJen v2.0 server for evaluation of antigenicity, AllerTOP v. 2.0 server for allergenicity assessment, and ToxinPred to select non-toxic epitopes. Additionally, HTL epitopes were used for their inducibility of interferon-γ (IFN-γ), and interleukin-4 (IL-4) using IFNepitope and IL4pred respectively. After selecting the final epitopes for each protein, the vaccine construct has been constructed. Ultimately, the final construct was evaluated for antigenicity (using ANTIGENpro), allergenicity (using AllerTOP v. 2.0), toxicity (using ToxinPred), and physicochemical properties (using ProtParam).
- Results: Primarily, we selected epitopes that cover more MHC alleles in each protein. At least each epitope in two alleles must have the ability of MHC binding. In the next step, selected epitopes were assessed for antigenicity using VaxiJen. Epitopes with a threshold > 0.4 were considered Probable antigens. Moreover, non-allergen and non-toxic epitopes were selected for further analysis. HTL epitopes with the ability to induce IFN-γ and IL-4 were selected as potential epitopes. Acceptable epitopes with mentioned filters from each protein were used in the final construct. EAAAK, AAY, and GPGPG linkers were used for linking selected epitopes. CTL epitopes were linked using AAY and HTL epitopes were linked using GPGPG linkers. Moreover, 50S ribosomal protein L7/L12 (Locus RL7_MYCTU) as an Adjuvant was linked to the construct in N-terminal using the EAAAK linker.
- Conclusion: In this study, we designed a multiepitope construct with the potential to be considered a therapeutic vaccine for HPV. Epitopes were selected from E1, E5, and E6 proteins. Using proper linkers and adjuvant, we constructed a multiepitope construct that has proper physicochemical properties, it is antigenic and has no allergenicity. However, further analysis is needed to confirm the functionality of this structure.
- Keywords: HPV - Human papillomavirus - multiepitope - vaccine- bioinformatic
