In Silico and In Vitro Analysis of a Multiepitope L1-E7 Fusion Construct for Vaccine Development Against Human Papillomaviruses

Title:In Silico and In Vitro Analysis of a Multiepitope L1-E7 Fusion Construct for Vaccine Development Against Human Papillomaviruses

Volume: 18 Issue: 11

Author(s): Elnaz Abbasifarid, Azam Bolhassani*, Shiva Irani and Fattah Sotoodehnejadnematalahi

Affiliation:

• Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran,Iran

Keywords: HPV, High risk, L1 capsid protein, E7 oncoprotein, immunoinformatics, in vitro studies.

Abstract:

Background: Human papillomavirus (HPV) infection is the major risk factor for cervical cancer. Current prophylactic HPV vaccines provide immunity against most genital and carcinogenic HPV types. However, these vaccines failed to produce immune responses against already established HPV infections.

Methods: To design a therapeutic vaccine candidate, we utilized immunoinformatics tools to design a potential multiepitope fusion construct based on L1 and E7 genes from different high- and low-risk HPV types. After determination of CD4+ and CD8+ T cell epitopes, the allergenicity, toxicity, immunogenicity, conservancy, and population coverage were analyzed for epitopes selection. Then, hemolytic probability of the selected epitopes, and molecular docking between major histocompatibility complex (MHC) and the chosen epitopes were performed by different web servers. Next, a multiepitope peptide construct consisting of 12 epitopes linked by AAY proteasomal sequence was designed. After that, physicochemical properties, solubility, secondary and tertiary structures of this construct were evaluated by bioinformatics tools. Finally, after amino acid reverse translation of the multiepitope peptide construct, expression of the L1-E7 DNA construct (pEGFP-L1-E7) was investigated in HEK-293T cells using fluorescent microscopy, flow cytometry, and western blotting.

Results: Considering various parameters, the immunodominant peptides such as L1(MHC-I)- DLDQFPLGRKFLLQ, L1(MHC-II)-NQLFVTVVDTTRSTN, E7-HPV16(MHC-I)-AEPDRAHYNI VTF, E7-HPV18(MHC-I)-HGPKATVQDIVLHL, E7-HPV31(MHC-I)-KPDTSNYNIVTF, E7-HPV33 (MHC-I)-RPDGQAQPATADYYI, E7-HPV45(MHC-I)- RTLQQLFLSFV, E7-HPV16(MHC -II)-TLH EYMLDLQPETTD, E7-HPV18(MHC-II)-LRAFQQLFLNTLSFV, E7-HPV31(MHC-II)-PTLQDYVL DLQPEAT, E7-HPV33(MHC-II)-LKEYVLDLYPEPTDL and E7-HPV45(MHC-II)-LQQLFLSTLSF VCPW were determined to design the vaccine construct. The results indicated efficient expression of the L1-E7 DNA construct (74 ± 2.19%) in vitro. Moreover, the polyepitope peptide generated in the cells was detected as a clear band of ~ 50 kDa in western blotting.

Conclusion: Regarding the favorable transfection efficiency of the designed L1-E7 multi-epitope construct, in vivo validation study on its therapeutic potential is underway.

Cite this article as:

Abbasifarid Elnaz , Bolhassani Azam *, Irani Shiva and Sotoodehnejadnematalahi Fattah , In Silico and In Vitro Analysis of a Multiepitope L1-E7 Fusion Construct for Vaccine Development Against Human Papillomaviruses, Letters in Drug Design & Discovery 2021; 18 (11) . https://dx.doi.org/10.2174/1570180818666210602160150