Iranian Biomedical Journal

en Packaging, Purification, and Titration of Replication-Deficient Semliki Forest Virus-Derived Particles as a Self-Amplifying mRNA Vaccine Vector Molecular Microbiology Molecular Microbiology مقاله کامل Full Length/Original Article <div style="text-align: justify;">Background: Self-amplifying mRNA is the next-generation vaccine platform with the potential advantages in efficacy and speed of development against infectious diseases and cancer. The main aim was to present optimized and rapid methods for Semliki Forest virus (SFV)-PD self-amplifying mRNA (SAM) preparation, its packaging, and titer determination. These protocols are provided for producing and harvesting the high yields of virus replicon particle (VRP)-packaged SAM for vaccine studies. Methods: pSFV-PD-EGFP plasmid was linearized and subjected to in vitro transcription. Different concentrations of SFV-PD SAM were first transfected into human embryonic kidney 293 cells (HEK-293) and baby hamster kidney cell line 21 (BHK-21) cell lines, and EGFP expression at different time points was evaluated by fluorescent microscopy. Replicon particle packaging was achieved by co-transfection of SFV-PD SAM and pSFV-Helper2 RNA into BHK-21 cells. The VRPs were concentrated using ultrafiltration with 100 kDa cut-off. The titers of replicon particles were determined by reverse transcription quantitative real-time PCR (RT-qPCR). Results: In vitro transcribed SAM encoding EGFP was successfully transfected and expressed in HEK-293 and BHK-21 cell lines. Higher levels of EGFP expression was observed in BHK-21 compared to HEK-293 cells showing more stable protein overexpression and VRP packaging. Using ultrafiltration, the high yields of purified SFV-PD-EGFP particles were rapidly obtained with only minor loss of replicon particles. Accurate and rapid titer determination of replication-deficient particles was achieved by RT-qPCR. Conclusion: Using optimized methods for SAM transfection, VRP packaging, and concentration, high yields of SFV-PD VRPs could be produced and purified. The RT-qPCR demonstrated to be an accurate and rapid method for titer determination of replication deficient VRPs.</div> mRNA vaccines, Semliki Forest virus, Vaccines 269 278 http://ibj.pasteur.ac.ir/browse.php?a_code=A-10-4670-1&slc_lang=en&sid=1 Nastaran Sadat Savar nstrn.savar@gmail.com 0000-0003-2509-2127 No Immunology Department, Pasteur Institute of Iran, Tehran, Iran Thomas Vallet thomas.vallet@pasteur.fr 0000-0001-7775-7448 No Institut Pasteur, Viral Populations and Pathogenesis Unit, Centre National de la Recherche Scientifique UMR 3569, Paris, France Arash Arashkia arash.arashkia@gmail.com 0000-0002-3117-3217 No Virology Department, Pasteur Institute of Iran, Tehran, Iran; 4PanTherapeutics, CH 1095 Lutry, Switzerland Kenneth Lundstrom lundstromkenneth@gmail.com 0000-0002-0580-5209 No PanTherapeutics, CH 1095 Lutry, Switzerland Marco Vignuzzi* marco.vignuzzi@pasteur.fr 0000-0002-4400-771X No Institut Pasteur, Viral Populations and Pathogenesis Unit, Centre National de la Recherche Scientifique UMR 3569, Paris, France Hamid Mahmoudzadeh Niknam hamid.m.niknam@gmail.com 0000-0001-9338-4266 Yes Immunology Department, Pasteur Institute of Iran, Tehran, Iran