Establishment of MDCK Stable Cell Lines Expressing TMPRSS2 and MSPL and Their Applications in Propagating Influenza Vaccine Viruses in Absence of Exogenous Trypsin.

Zhiyuan Wen,Jianzhong Shi,Chao Wu,Weiye Chen,Jinying Ge,Xianying Zeng,Zhigao Bu,Hualan Chen

doi:10.1155/2015/402628

Abstract

We established two Madin-Darby canine kidney (MDCK) cell lines stably expressing human airway transmembrane protease: transmembrane protease, serine 2 (TMPRSS2) and mosaic serine protease large form (MSPL) which support multicycle growth of two H5 highly pathogenic avian influenza viruses (HPAIV) recombinant vaccines (Re-5 and Re-6) and an H9 avian influenza virus (AIV) recombinant vaccine (Re-9) in the absence of trypsin. Data showed that the cell lines stably expressed TMPRSS2 and MSPL after 20 serial passages. Both MDCK-TMPRSS2 and MDCK-MSPL could proteolytically cleave the HA of Re-5, Re-6, and Re-9 and supported high-titer growth of the vaccine without exogenous trypsin. Re-5, Re-6, and Re-9 efficiently infected and replicated within MDCK-TMPRSS2 and MDCK-MSPL cells and viral titer were comparable to the virus grown in MDCK cells with TPCK-trypsin. Thus, our results indicate a potential application for these cell lines in cell-based influenza vaccine production and may serve as a useful tool for HA proteolytic cleavage-related studies.

Highlights

Influenza is a major zoonotic threat to public health, which is caused by 3 types (A, B, and C) of influenza viruses [1, 2]
Zmora and colleagues demonstrated that mosaic serine protease large form (MSPL)could activate HA protein of H1N1 and H3N2 influenza virus [20], while Okumura and colleagues confirmed that MSPL can cleave the HA protein of H5 highly pathogenic avian influenza viruses (HPAIV) and support their multicycle replication [21]
Human type II transmembrane serine proteases (TTSPs) have come under study and TTSPs such as human airway trypsinlike (HAT), TMPRSS2, TMPRSS4, and MSPL were found to be capable of cleaving different subtypes of influenza viruses

Summary

Introduction

Influenza is a major zoonotic threat to public health, which is caused by 3 types (A, B, and C) of influenza viruses [1, 2]. HA proteins of H5 highly pathogenic avian influenza viruses (HPAIV) have multibasic cleavage sites (R-X-R/K-R) which can be cleaved by ubiquitously expressed furin or PC5/6 protease to cause fatal systemic infections [15,16,17]. HA of most of the other mammalian and avian influenza viruses contains a single arginine (or lysine) at the cleavage site, so cleavage of these HAs is restricted to the respiratory tract in mammals and to the respiratory and intestinal tracts in avians and assumed to be processed extracellularly by trypsin-like proteases Of these proteases, some type II transmembrane serine proteases (TTSPs) family members such as human airway trypsinlike (HAT) protease, transmembrane protease, serine 2 (TMPRSS2), transmembrane protease, serine 4 (TMPRSS4), Biotechnology Research International and mosaic serine protease large form (MSPL) play important roles in influenza viral infection. Zmora and colleagues demonstrated that mosaic serine protease large form (MSPL)could activate HA protein of H1N1 and H3N2 influenza virus [20], while Okumura and colleagues confirmed that MSPL can cleave the HA protein of H5 HPAIV and support their multicycle replication [21]

Methods

Results

Conclusion