H9 subtype influenza vaccine in MDCK single‐cell suspension culture with stable expression of TMPRSS2: Generation and efficacy evaluation

Abstract

Vaccination is the most effective way to protect chickens from avian influenza pandemics. Mammalian cells, especially MDCK cells, are being investigated as a good alternative to the embryonated chicken eggs for avian influenza vaccine production. In this work, we developed a new MDCK cell line, which has two main features: single‐cell suspension growth and stable expression of human TMPRSS2 protein (transmembrane protease serine S1 member 2) anchored on cells membrane, named MDCK‐Sus‐TMPRSS2. Reverse‐transcription polymerase chain reaction, western blot analysis and indirect immunofluorescence assay were employed to detect the expression of TMPRSS2 in the MDCK‐Sus‐TMPRSS2 cells. Then, H9 subtype avian influenza virus, NJ02/01 strain, was adapted in MDCK‐Sus‐TMPRSS2 cells with serial blind passages and propagated in stirred bioreactor with 9.5 log2/25 μL hemagglutination titers for inactivated avian influenza vaccine production without addition of exogenous trypsin. The MDCK‐Sus‐TMPRSS2 cell‐derived H9 subtype avian influenza vaccine could induce high titers of hemagglutiniton inhibition antibodies in specific pathogen‐free chickens and protect chickens against heterologous H9 subtype influenza virus challenge, showing lower virus shedding rate and sickness rate than in the egg‐derived vaccines group. High titers of H9 subtype‐specific antibodies above 6 log2 were maintained for 6 months in commercial chickens vaccinated by the MDCK‐Sus‐TMPRSS2 cells derived H9 subtype avian influenza vaccine and there were no negative effects on body weight increase in these chickens. Taken together, the results of this work revealed that MDCK‐Sus‐TMPRSS2 cell line could be a promising and safe substitute for embryonated chicken eggs as a host cell line for H9 subtype avian influenza virus propagation.

Expression of recombinant classical swine fever virus E2 glycoprotein by endogenous Txnip promoter in stable transgenic CHO cells

“…CHO cells with monolayer were transfected with the pCMV‐rE2 and different pTxnip‐rE2 vectors respectively using branched 25 kDa polyethylenimine (PEI, Sigma–Aldrich, 764604). The transfection procedure based on PEI was performed as previously described [14,15]. One day after the transfection, the different transfected cell groups were digested with trypsin and split into 10‐cm plates respectively in growth media with dialyzed FBS and without H/T addition for transgenic cells forming, followed with two rounds drug selection of 800 and 1200 μg/mL G418 (Roche, 04727878001).…”

Animal Cell Lines as Expression Platforms in Viral Vaccine Production: A Post Covid-19 Perspective

“…In this respect, it is a frequently preferred expression platform in influenza virus studies, vaccine development processes and vaccine production. ,, However, in order to improve the production process, a protease enzyme must be added to the production medium to convert hemagglutinin to its active form during the production process of influenza viruses . To eliminate this, there are studies conducted with MDCK cells modified to express TMPRSS2 (transmembrane protease, serine S1 family member 2) and HAT (human airway trypsin like protease) proteases, which altering hemagglutinin to the active form in natural influenza infection …”