Volume 26, Issue 4 (7-2022)                   IBJ 2022, 26(4): 269-278 | Back to browse issues page

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Savar N S, Vallet T, Arashkia A, Lundstrom K, Vignuzzi* M, Mahmoudzadeh Niknam H. Packaging, Purification, and Titration of Replication-Deficient Semliki Forest Virus-Derived Particles as a Self-Amplifying mRNA Vaccine Vector. IBJ 2022; 26 (4) :269-278
URL: http://ibj.pasteur.ac.ir/article-1-3535-en.html
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.
Type of Study: Full Length | Subject: Molecular Microbiology

1. Blakney AK, Ip S, Geall AJ. An update on self-amplifying mRNA vaccine development. Vaccines (Basel) 2021; 9(2): 97. [DOI:10.3390/vaccines9020097]
2. Lundstrom K. RNA viruses as tools in gene therapy and vaccine development. Genes 2019; 10(3): 189. [DOI:10.3390/genes10030189]
3. Brito LA, Kommareddy S, Maione D, Uematsu Y, Giovani C, Berlanda Scorza F, Otten GR, Yu D, Mandl 1, Peter W Mason CW, Dormitzer PR, Ulmer JB, Geall AJ. Self-amplifying mRNA vaccines. Advances in genetics 2015; 89: 179-233. [DOI:10.1016/bs.adgen.2014.10.005]
4. Shin G, Yost SA, Miller MT, Elrod EJ, Grakoui A, Marcotrigiano J. Structural and functional insights into alphavirus polyprotein processing and pathogenesis. Proceedings of the national academy of sciences of the United States of America 2012; 109(41): 16534-16539. [DOI:10.1073/pnas.1210418109]
5. Pietila MK, Hellstrom K, Ahola T. Alphavirus polymerase and RNA replication. Virus research 2017; 234: 44-57. [DOI:10.1016/j.virusres.2017.01.007]
6. Geall AJ, Mandl CW, Ulmer JB. RNA: the new revolution in nucleic acid vaccines. Seminars in immunology 2013; 25(2): 152-159. [DOI:10.1016/j.smim.2013.05.001]
7. Lundstrom K. Alphavirus Vectors for Gene Therapy Applications. In: Hunt KK, Swisher SG, editor. Gene Therapy for Cancer Cancer Drug Discovery and Development: Humana Press; 2007.
8. Atkins GJ, Fleeton MN, Sheahan BJ. Therapeutic and prophylactic applications of alphavirus vectors. Expert reviews in molecular medicine 2008; 10: e33. [DOI:10.1017/S1462399408000859]
9. Rayner JO, Dryga SA, Kamrud KI. Alphavirus vectors and vaccination. Reviews in medical virology 2002; 12(5): 279-296. [DOI:10.1002/rmv.360]
10. Lundstrom K, Abenavoli A, Malgaroli A, Ehrengruber MU. Novel semliki forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression. Molecular therapy 2003; 7(2): 202-209. [DOI:10.1016/S1525-0016(02)00056-4]
11. Berglund P, Sjoberg M, Garoff H, Atkins GJ, Sheahan BJ, Liljestrom P. Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Biotechnology (NY) 1993; 11(8): 916-3-920. [DOI:10.1038/nbt0893-916]
12. Ballesteros-Briones MC, Silva-Pilipich N, Herrador-Canete G, Vanrell L, Smerdou C. A new generation of vaccines based on alphavirus self-amplifying RNA. Current opinion in virology 2020; 44: 145-153. [DOI:10.1016/j.coviro.2020.08.003]
13. Lundstrom K. Self-Amplifying RNA viruses as RNA vaccines. International journal of molecular sciences 2020; 21(14):5130. [DOI:10.3390/ijms21145130]
14. Komdeur FL, Singh A, van de Wall S, Meulenberg JJM, Boerma A, Hoogeboom BN, Paijens ST, Oyarce C, Bruyn Md, Schuuring E, Regts J, Marra R, Werner N, Sluis J, van der Zee AGJ, Wilschut JC, Allersma DP, van Zanten CJ, Kosterink JGW, Jorritsma-Smit A, Yigit R, Nijman HW, Daemen T. First-in-human Phase I clinical trial of an SFV-based RNA replicon cancer vaccine against HPV-Induced cancers. Molecular therapy 2021; 29(2): 611-625. [DOI:10.1016/j.ymthe.2020.11.002]
15. Lundstrom K. The potential of self-amplifying RNA vaccines for infectious diseases and COVID-19. Vaccine research 2020; 7(1): 25-37. [DOI:10.29252/vacres.7.1.25]
16. Vrba SM, Kirk NM, Brisse ME, Liang Y, Ly H. Development and applications of viral vectored vaccines to combat zoonotic and emerging public health threats. Vaccines (Basel) 2020; 8(4): 680. [DOI:10.3390/vaccines8040680]
17. Lundstrom K. Alphavirus-based vaccines. Methods in molecular biology 2016; 1404: 313-328. [DOI:10.1007/978-1-4939-3389-1_22]
18. Davis NL, Caley IJ, Brown KW, Betts MR, Irlbeck DM, McGrath KM, Connell MC, Montefiori DC, Frelinger JA, Swanstrom R, Johnson PR, Johnston RE. Vaccination of macaques against pathogenic simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles. Journal of virology 2000; 74(1): 371-378. [DOI:10.1128/JVI.74.1.371-378.2000]
19. Puglia AL, Rezende AG, Jorge SA, Wagner R, Pereira CA, Astray RM. Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus. Journal of virological methods 2013; 193(2): 647-652. [DOI:10.1016/j.jviromet.2013.07.058]
20. Pryor RJ, Wittwer CT. Real-time polymerase chain reaction and melting curve analysis. Methods in molecular biology 2006; 336: 19-32. [DOI:10.1385/1-59745-074-X:19]
21. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic acids research 2001; 29(9): e45. [DOI:10.1093/nar/29.9.e45]
22. Glover DJ. Artificial viruses: exploiting viral trafficking for therapeutics. Infectious disorders-drug targets 2012; 12(1): 68-80. [DOI:10.2174/187152612798995000]
23. Zeng C, Zhang C, Walker PG, Dong Y. Formulation and delivery technologies for mRNA vaccines. Current topics in microbiology and immunology 2020; 10. [DOI:10.1007/82_2020_217]
24. Demoulins T, Ruggli N, Gerber M, Thomann-Harwood LJ, Ebensen T, Schulze K, Guzmán CA, McCullough KC. Self-Amplifying pestivirus replicon RNA encoding influenza virus nucleoprotein and hemagglutinin promote humoral and cellular immune responses in pigs. Frontiers in immunology 2020; 11: 622385. [DOI:10.3389/fimmu.2020.622385]
25. Reap EA, Dryga SA, Morris J, Rivers B, Norberg PK, Olmsted RA, Chulay JD. Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins. Clinical and vaccine immunology 2007; 14(6): 748-755. [DOI:10.1128/CVI.00037-07]
26. Mori Y, Yoshida Y, Satoh A, Moriya H. Development of an experimental method of systematically estimating protein expression limits in HEK293 cells. Scientific reports 2020; 10(1): 4798. [DOI:10.1038/s41598-020-61646-3]
27. Fluet ME, Whitmore AC, Moshkoff DA, Fu K, Tang Y, Collier ML, West A, Moore DT, Swanstrom R, Johnston RE, Davis NL. Effects of rapid antigen degradation and VEE glycoprotein specificity on immune responses induced by a VEE replicon vaccine. Virology 2008; 370(1): 22-32. [DOI:10.1016/j.virol.2007.08.020]
28. Lundstrom K. Generation of recombinant alphaviral vectors. Cold spring harbor protocols 2012; 2012(7): 825-831. [DOI:10.1101/pdb.prot070151]
29. Pyke AT, Phillips DA, Chuan TF, Smith GA. Sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine. BMC microbiology 2004; 4: 3. [DOI:10.1186/1471-2180-4-3]
30. Sjöberg M, Garoff H. Chapter 51 - growth of semliki forest virus. Cell biology (Third edition) 2006; 1: 419-423. [DOI:10.1016/B978-012164730-8/50052-6]
31. Hutornojs A, Niedre-Otomere B, Kozlovska T, Zajakina A. Comparison of ultracentrifugation methods for concentration of recombinant alphaviruses: sucrose and iodixanol cushions. Environmental and experimental biology 2012; 10:117-123.
32. Reiser J. Production and concentration of pseudotyped HIV-1-based gene transfer vectors. Gene therapy 2000; 7(11): 910-913. [DOI:10.1038/sj.gt.3301188]
33. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nature protocols 2006; 1(3): 1559-1582. [DOI:10.1038/nprot.2006.236]
34. Ehrengruber MU, Lundstrom K, Schweitzer C, Heuss C, Schlesinger S, Gahwiler BH. Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proceedings of the National Academy of Sciences of the United States of America 1999; 96(12): 7041-7046. [DOI:10.1073/pnas.96.12.7041]

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