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High-Throughput Production of Influenza Virus-Like Particle (VLP) Array by Using VLP-factory, a MultiBac Baculoviral Genome Customized for Enveloped VLP Expression

Part of the Methods in Molecular Biology book series (MIMB,volume 2025)

Abstract

Baculovirus-based expression of proteins in insect cell cultures has emerged as a powerful technology to produce complex protein biologics for many applications ranging from multiprotein complex structural biology to manufacturing of therapeutic proteins including virus-like particles (VLPs). VLPs are protein assemblies that mimic live viruses but typically do not contain any genetic material, and therefore are safe and attractive alternatives to life attenuated or inactivated viruses for vaccination purposes. MultiBac is an advanced baculovirus expression vector system (BEVS) which consists of an engineered viral genome that can be customized for tailored applications. Here we describe the creation of a MultiBac-based VLP-factory, based on the M1 capsid protein from influenza, and its application to produce in a parallelized fashion an array of influenza-derived VLPs containing functional mutations in influenza hemagglutinin (HA) thought to modulate the immune response elicited by the VLP.

Key words

  • Baculovirus expression vector system (BEVS)
  • Small-scale production
  • Virus-like particle (VLP)
  • MultiBac
  • Cre recombinase
  • Cre-LoxP fusion
  • Influenza
  • Hemagglutinin (HA)

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References

  1. Cox MM, Hollister JR (2000) FluBlok: a next generation influenza vaccine manufactured in insect cells. Biologicals 37(3):182–189

    CrossRef  Google Scholar 

  2. Schiller JT, Lowy DR (2006) Prospects for cervical cancer prevention by human papillomavirus vaccination. Cancer Res 66(21):10229–10232

    CAS  CrossRef  Google Scholar 

  3. Temchura V, Überla K (2017) Intrastructural help: improving the HIV-1 envelope antibody response induced by virus-like particle vaccines. Curr Opin HIV AIDS 12(3):272–277

    CAS  CrossRef  Google Scholar 

  4. Charlton Hume HK, Lua LH (2017) Platform technologies for modern vaccine manufacturing. Vaccine 35(35 Pt A):4480–4485

    CAS  CrossRef  Google Scholar 

  5. Jeong H, Seong BL (2017) Exploiting virus-like particles as innovative vaccines against emerging viral infections. J Microbiol 55(3):220–230

    CAS  CrossRef  Google Scholar 

  6. Pouyanfard S, Müller M (2017) Human papillomavirus first and second generation vaccines – current status and future directions. Biol Chem 398(8):871–889. https://doi.org/10.1515/hsz-2017-0105

    CAS  CrossRef  PubMed  Google Scholar 

  7. Zaykov AN, Mayer JP, DiMarchi RD (2016) Pursuit of a perfect insulin. Nat Rev Drug Discov 15(6):425–439

    CAS  CrossRef  Google Scholar 

  8. Jia B, Jeon CO (2016) High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biol 6(8). pii: 160196

    CrossRef  Google Scholar 

  9. Angius F, Ilioaia O, Uzan M, Miroux B (2016) Membrane protein production in Escherichia coli: protocols and rules. Methods Mol Biol 1432:37–52

    CAS  CrossRef  Google Scholar 

  10. Vincentelli R, Romier C (2016) Complex reconstitution and characterization by combining co-expression techniques in Escherichia coli with high-throughput. Adv Exp Med Biol 896:43–58

    CAS  CrossRef  Google Scholar 

  11. Vincentelli R, Romier C (2013) Expression in Escherichia coli: becoming faster and more complex. Curr Opin Struct Biol 23(3):326–334

    CAS  CrossRef  Google Scholar 

  12. Nettleship JE, Assenberg R, Diprose JM, Rahman-Huq N, Owens RJ (2010) Recent advances in the production of proteins in insect and mammalian cells for structural biology. J Struct Biol 172:55–65

    CAS  CrossRef  Google Scholar 

  13. Nettleship JE, Watson PJ, Rahman-Huq N, Fairall L, Posner MG, Upadhyay A, Reddivari Y, Chamberlain JM, Kolstoe SE, Bagby S, Schwabe JW, Owens RJ (2015) Transient expression in HEK 293 cells: an alternative to E. coli for the production of secreted and intracellular mammalian proteins. Methods Mol Biol 1258:209–222

    CAS  CrossRef  Google Scholar 

  14. Nie Y, Viola C, Bieniossek C, Trowitzsch S, Vijay-achandran LS, Chaillet M, Garzoni F, Berger I (2009) Getting a grip on complexes. Curr Genomics 10:558–572

    CAS  CrossRef  Google Scholar 

  15. Vijayachandran LS, Viola C, Garzoni F, Trowitzsch S, Bieniossek C, Chaillet M, Schaffitzel C, Busso D, Romier C, Poterszman A et al (2011) Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. J Struct Biol 175:198–208

    CAS  CrossRef  Google Scholar 

  16. Nie Y, Chaillet M, Becke C, Haffke M, Pelosse M, Fitzgerald D, Collinson I, Schaffitzel C, Berger I (2016) ACEMBL tool-kits for high-throughput multigene delivery and expression in prokaryotic and eukaryotic hosts. Adv Exp Med Biol 896:27–42

    CAS  CrossRef  Google Scholar 

  17. Berger I, Fitzgerald DJ, Richmond TJ (2004) Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 22(12):1583–1587

    CAS  CrossRef  Google Scholar 

  18. Fitzgerald DJ, Berger P, Schaffitzel C, Yamada K, Richmond TJ, Berger I (2006) Protein complex expression by using multigene baculoviral vectors. Nat Methods 3(12):1021–1032

    CAS  CrossRef  Google Scholar 

  19. Bieniossek C, Richmond TJ, Berger I (2008) MultiBac: multigene baculovirus-based eukaryotic protein complex production. Curr Protoc Protein Sci Chapter 5:Unit 5.20

    PubMed  Google Scholar 

  20. Trowitzsch S, Bieniossek C, Nie Y, Garzoni F, Berger I (2010) New baculovirus expression tools for recombinant protein complex production. J Struct Biol 172(1):45–54

    CAS  CrossRef  Google Scholar 

  21. Bieniossek C, Imasaki T, Takagi Y, Berger I (2012) MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci 37(2):49–57

    CAS  CrossRef  Google Scholar 

  22. Trowitzsch S, Palmberger D, Fitzgerald DJ, Takagi Y, Berger I (2012) MultiBac complexomics. Expert Rev Proteomics 9(4):363–373

    CAS  CrossRef  Google Scholar 

  23. Barford D, Takagi Y, Schultz P, Berger I (2013) Baculovirus expression: tackling the complexity challenge. Curr Opin Struct Biol 23(3):357–364

    CAS  CrossRef  Google Scholar 

  24. Berger I, Garzoni F, Chaillet M, Haffke M, Gupta K, Aubert A (2013) The MultiBac protein complex production platform at the EMBL. J Vis Exp 11(77):e50159

    Google Scholar 

  25. Sari D, Gupta K, Thimiri Govinda Raj DB, Aubert A, Drncová P, Garzoni F, Fitzgerald DJ, Berger I (2016) The MultiBac baculovirus/insect cell expression vector system for producing complex protein biologics. Adv Exp Med Biol 896:199–215

    CAS  CrossRef  Google Scholar 

  26. Palmberger D, Rendic D (2015) SweetBac: applying MultiBac technology towards flexible modification of insect cell glycosylation. Methods Mol Biol 1321:153–169

    CrossRef  Google Scholar 

  27. Palmberger D, Klausberger M, Berger I, Grabherr R (2013) MultiBac turns sweet. Bioengineered 4(2):78–83

    CrossRef  Google Scholar 

  28. Palmberger D, Wilson IB, Berger I, Grabherr R, Rendic D (2012) SweetBac: a new approach for the production of mammalianised glycoproteins in insect cells. PLoS One 7(4):e34226

    CAS  CrossRef  Google Scholar 

  29. Fitzgerald DJ, Schaffitzel C, Berger P, Wellinger R, Bieniossek C, Richmond TJ, Berger I (2007) Multiprotein expression strategy for structural biology of eukaryotic complexes. Structure 15(3):275–279

    CAS  CrossRef  Google Scholar 

  30. Koehler C, Sauter PF, Wawryszyn M, Girona GE, Gupta K, Landry JJ, Fritz MH, Radic K, Hoffmann JE, Chen ZA et al (2016) Genetic code expansion for multiprotein complex engineering. Nat Methods 13(12):997–1000

    CAS  CrossRef  Google Scholar 

  31. Bahrami S, Laska MJ, Pedersen FS, Duch M (2016) Immune suppressive activity of the influenza fusion peptide. Virus Res 211:126–131

    CAS  CrossRef  Google Scholar 

  32. Holm CH, Jensen SB, Jakobsen MR, Cheshenko N, Horan KA, Moeller HB, Gonzalez-Dosal R, Rasmussen SB, Christensen MH, Yarovinsky TO et al (2012) Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING. Nat Immunol 13(8):737–743

    CAS  CrossRef  Google Scholar 

  33. Haffke M, Viola C, Nie Y, Berger I (2013) Tandem recombineering by SLIC cloning and Cre-LoxP fusion to generate multigene expression constructs for protein complex research. Methods Mol Biol 1073:131–140

    CAS  CrossRef  Google Scholar 

  34. Casini A, Storch M, Baldwin GS, Ellis T (2015) Bricks and blueprints: methods and standards for DNA assembly. Nat Rev Mol Cell Biol 16(9):568–576

    CAS  CrossRef  Google Scholar 

  35. Celie PH, Parret AH, Perrakis A (2016) Recombinant cloning strategies for protein expression. Curr Opin Struct Biol 38:145–154

    CAS  CrossRef  Google Scholar 

  36. Benoit RM, Ostermeier C, Geiser M, Li JS, Widmer H, Auer M (2016) Seamless insert-plasmid assembly at high efficiency and low cost. PLoS One 11(4):e0153158

    CrossRef  Google Scholar 

  37. Metcalf WW, Jiang W, Wanner BL (1994) Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers. Gene 138:1–7

    CAS  CrossRef  Google Scholar 

  38. Monteiro F, Bernal V, Chaillet M, Berger I, Alves PM (2016) Targeted supplementation design for improved production and quality of enveloped viral particles in insect cell-baculovirus expression system. J Biotechnol 233:34–41

    CAS  CrossRef  Google Scholar 

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Acknowledgements

We thank all members of the Berger and Schaffitzel laboratories for helpful discussions, as well as Rob Ruigrok, Thibaut Crepin, and Darren Hart for expert insight in influenza biology. We are grateful to Karin Huard for assistance with gradient preparation, and Yan Nie for introduction to negative-stain EM. This work was supported by the European Commission Framework Programme 7 projects ComplexINC (contract nr. 279039) and SynSignal (contract nr. 613879).

Competing Financial Interest Statement

The authors declare competing financial interest. Parts of the technology here described are subject of international patent EP2403940 and licensed exclusively to Geneva Biotech SARL.

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Correspondence to Frederic Garzoni or Imre Berger .

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Sari-Ak, D. et al. (2019). High-Throughput Production of Influenza Virus-Like Particle (VLP) Array by Using VLP-factory, a MultiBac Baculoviral Genome Customized for Enveloped VLP Expression. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_10

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  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_10

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