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Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology

  • Laura A. Palomares
  • Indresh K. Srivastava
  • Octavio T. Ramírez
  • Manon M. J. Cox
Chapter
Part of the Advances in Biochemical Engineering/Biotechnology book series

Abstract

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed.

Graphical Abstract

Keywords

Baculovirus Cell engineering Glycobiotechnology Glycosylation Insect cells Recombinant protein 

Abbreviations

BEST

Baculovirus expression system technology

CHO

Chinese hamster ovary

CHST2

Carbohydrate sulfotransferase 2

CSAS

Sialic acid synthetase

DOT

Dissolved oxygen tension

eLH/CG

Recombinant equine luteinizing hormone/chorionic gonadotropin

ER

Endoplasmic reticulum

FBS

Fetal bovine serum

FDL

Fused lobes protein

Fuc

Fucose

FucT

Fucosyltransferase

FucT3

α-1,3 Fucosyltransferase

GalT

β-(1➔4)-Galactosyltransferase

Gal

Galactose

Gal3ST2

Gal-3-O-sulfotransferase 2

GalNAc

N-Acetylgalactosamine

Glc

Glucose

GlcNAc

N-Acetylglucosamine

GlcNAcase

β-N-Acetylglucosaminidase

GlcNAcT

GlcNAc transferase

hpi

Hours postinfection

IgG

Immunoglobulin

Man

Mannose

ManNAc

N-Acetylmannosamine

MP

Baculovirus basic protein promoter

ND

Not detected

Neu5Ac

N-Acetylneuraminic acid

NR

Not reported

PAP

Rat purple acid phosphatase

rHA

Recombinant influenza hemagglutinin

rLRE

Recombinant lutropin receptor ectodomain

RMD

Guanosine-5′-diphospho (GDP)4-dehydro-6-deoxy-d-mannose reductase

SAS

Sialic acid 9-phosphate synthase

SeAP

Secreted human alkaline phosphatase

SialT

Sialyltransferase

TPA

Tissue plasminogen activator

βhCG

β subunit of human chorionic gonadotropin

Notes

Acknowledgements

Research performed thanks to the support by UNAM-DGAPA-PAPIIT IT-200315. Technical assistance from Luis Alberto Diaz is acknowledged.

References

  1. 1.
    Palomares LA, Realpe M, Ramírez OT (2015) An overview of cell culture engineering for the insect cell-baculovirus expression vector system (BEVS). In: Al-Rubeai M (ed) Animal cell culture. Cell engineering, vol 9. Cham, Springer International, pp 501–519Google Scholar
  2. 2.
    Cox MMJ, Hashimoto Y (2011) A fast track influenza virus vaccine produced in insect cells. J Invert Pathol 107:s31–s41Google Scholar
  3. 3.
    Orlova OV, Drutsa VL, Spirin PV, Popenko VI, Prasolov VS, Rubtsov PM, Kochetkov SN, Belzhelarskaya SN (2013) Role of N-linked glycans of HCV glycoprotein E1 in folding of structural proteins and formation of viral particles. Mol Biol 47:131–139Google Scholar
  4. 4.
    Wang Y, Norgård M, Andersson G (2005) N-Glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase. Arch Biochem Biophys 435:147–156Google Scholar
  5. 5.
    Cox MMJ (2012) Recombinant protein vaccines produced in insect cells. Vaccine 30:1759–1766Google Scholar
  6. 6.
    Palomares LA, Ramírez OT (2009) Challenges for the production of virus-like particles in insect cells: the case of rotavirus-like particles. Biochem Eng J 45:158–167Google Scholar
  7. 7.
    Palomares LA, Joosten CE, Hughes PR, Granados RR, Shuler ML (2003) Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins. Biotechnol Prog 19:185–192Google Scholar
  8. 8.
    Lipscomb ML, Palomares LA, Hernández V, Ramírez OT, Kompala DS (2005) Effect of production method and gene amplification on the glycosylation pattern of a secreted reporter protein in CHO cells. Biotechnol Prog 21:40–49Google Scholar
  9. 9.
    Ailor E, Takahashi N, Tsukamoto Y, Masuda K, Rahman BA, Jarvis DL, Lee YC, Betenbaugh MJ (2000) N-Glycan pattern of human transferrin produced in Trichoplusia ni insect cells: effects of mammalian galactosyltransferase. Glicobiology 10:837–847Google Scholar
  10. 10.
    Hsu TA, Takahashi N, Tsukamoto Y, Kato K, Shimada I, Masuda K, Whiteley E, Fan JQ, Lee YC, Betenbaugh MJ (1997) Differential N-glycan patterns of secreted and intracellular IgG produced by Trichoplusia ni cells. J Biol Chem 272:9062–9070Google Scholar
  11. 11.
    Davidson DJ, Castellino FJ (1991) Asparagine-linked oligosaccharide processing in Lepidopteran insect cells. Temporal dependence of the nature of the oligosaccharides assembled on asparagine-289 of recombinant human plasminogen produced in baculovirus vector infected Spodoptera frugiperda (IPLB-SF-21AE) cells. Biochemistry 30:6167–6174Google Scholar
  12. 12.
    Joshi L, Davis TR, Mattu TS, Rudd PM, Dwek RA, Shuler ML, Wood HA (2000) Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein. Biotechnol Prog 7:9–14Google Scholar
  13. 13.
    Joosten CE, Park TH, Shuler ML (2003) Effect of silkworm hemolymph on N-linked glycosylation in two Trichoplusia ni insect cell lines. Biotechnol Prog 83:695–705Google Scholar
  14. 14.
    Abdul-Rahman B, Ailor E, Jarvis D, Betenbaugh M, Lee YC (2002) β-(1→4)-Galactosyltransferase activity in native and engineered insect cells measured with time-resolved europium fluorescence. Carbohydr Res 337:2181–2186Google Scholar
  15. 15.
    van Die I, van Tetering A, Bakker H, van den Eijnden DH, Joziasse DH (1996) Glycosylation of lepidopteran insect cells: identification of a β1→4-N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains. Glycobiology 6:157–164Google Scholar
  16. 16.
    An Y, Rininger JA, Jarvis DL, Jing X, Ye Z, Aumiller JJ, Eichelberger M, Cipollo JF (2013) Comparative glycomics analysis of influenza hemagglutinin (H5N1) produced in vaccine relevant cell platforms. J Proteome Res 12:3707–3720Google Scholar
  17. 17.
    Walski T, De Schutter K, Van Damme EJM, Smagghe G (2017) Diversity and function of protein glycosylation in insects. Insect Biochem Mol Biol 83:21–34Google Scholar
  18. 18.
    Geisler C, Jarvis DL (2012) Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway. J Biol Chem 287:7084–7097Google Scholar
  19. 19.
    Geisler C, Jarvis DL (2012) Innovative use if a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells. Metab Eng 14:642–652Google Scholar
  20. 20.
    Schachter H (2009) The functions of high mannose N-glycans in Caenorhabditis elegans. Trends Glycosci. Glycotechnol 119:131–148Google Scholar
  21. 21.
    Licari PJ, Jarvis DL, Bailey JE (1993) Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity. Biotechnol Prog 9:146–152Google Scholar
  22. 22.
    Watanabe S, Kokuho T, Takakashi H, Takakashi M, Kubota T, Inumaru S (2002) Sialylation of N-glycans on the recombinant proteins expressed by a baculovirus-insect cell system under β-N-acetylglucosaminidase inhibition. J Biol Chem 277:5090–5093Google Scholar
  23. 23.
    Léonard R, Rendic D, Rabouille C, Wilson IBH, Préat T, Altmann F (2006) The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing. J Biol Chem 281:4867–4875Google Scholar
  24. 24.
    Altmann F, Schwihla H, Staudacher E, Glössl J, März L (1995) Insect cells contain an unusual, membrane bound β-N-acetylglucosaminidase probably involved in the processing of protein N-glycans. J Biol Chem 270:17344–17349Google Scholar
  25. 25.
    Geisler C, Aumiller JJ, Jarvis DL (2008) A fused lobes gene encodes the processing β-N-acetylglucosaminidase in Sf9 cells. J Biol Chem 283:11330–11339Google Scholar
  26. 26.
    Dragosits M, Yan S, Razzazi-Fazeli E, Wilson IBH, Rendic D (2015) Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases. Glycobiology 25:448–464Google Scholar
  27. 27.
    Tomiya N, Narang S, Park J, Abdul_Rahman B, Choi O, Singh S, Hiratake J, Sakata K, Betenbaugh MJ, Palter KB, Lee YC (2006) Purification, characterization and cloning of a Spodoptera frugiperda Sf9 β-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core. J Biol Chem 281:19545–19560Google Scholar
  28. 28.
    Hollister J, Grabenhorst E, Nimtz M, Donradt H, Jarvis DL (2002) Engineering the protein N-glycosylation pathway in insect cells for production of biantennary, complex N-glycans. Biochemistry 41:15093–15104Google Scholar
  29. 29.
    Kim K, Lawrence SM, Park J, Pitts L, Vann WF, Betenbaugh MJ, Palter KB (2002) Expression of a functional Drosophila melanogaster N-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects. Glycobiology 12:73–83Google Scholar
  30. 30.
    Koles K, Irvine KD, Panin VM (2004) Functional characterization of Drosophila sialyltransferase. J Biol Chem 279:4346–4357Google Scholar
  31. 31.
    Viswanathan K, Tomiya N, Park J, Singh S, Lee YC, Palter K, Betenbaugh MJ (2006) Expression of a functional Drosophila melanogaster CMP-sialic acid synthetase. J Biol Chem 281:15929–15940Google Scholar
  32. 32.
    Schauer R (2001) The occurrence and significance of sialic acids in insects. Trends Glycosci Glycotechnol 13:507–517Google Scholar
  33. 33.
    Kajiura H, Hamaguchi Y, Mizushima H, Misaki R, Fujiyama K (2015) Sialylation potentials of the silk worm, Bombyx mori; B. mori possesses an active α2,6-sialyltransferase. Glycobiology 25:1441–1453Google Scholar
  34. 34.
    Davidson DJ, Castellino FJ (1991) Structure of the asparagine-289-linked oligosaccharides assembled on recombinant human plasminogen expressed in a Mamestra brassicae cell line (IZD-MBO503). Biochemistry 30:6689–6696Google Scholar
  35. 35.
    Hollister J, Conradt H, Jarvis DL (2003) Evidence for a sialic acid salvaging pathway in lepidopteran insect cells. Glycobiology 13:487–495Google Scholar
  36. 36.
    Rendic D, Wilson IBH, Paschinger K (2008) The glycosylation capacity of insect cells. Croatica Chem Acta 81:7–21Google Scholar
  37. 37.
    Hillar A, Jarvis DL (2010) Re-visiting the endogenous capacity for recombinant glycoprotein sialylation by baculovirus-infected Tn-4h and DpN1 cells. Glycobiol 20:1323–1330Google Scholar
  38. 38.
    Palmberger D, Ashjaei K, Strell S, Hoffmann-Sommergruber K, Grabherr R (2014) Minimizing fucosylation in insect cell-derived glycoproteins reduces binding to IgE antibodies from sera of patients with allergy. Biotechnol J 9:1206–1214Google Scholar
  39. 39.
    Paschinger K, Staudacher E, Stemmer U, Fabini G, Wilson IBH (2005) Fucosyltransferase substrate specificity and the order of fucosylation in vertebrates. Glycobiology 15:463–474Google Scholar
  40. 40.
    Seismann H, Blank S, Braren I, Greunke K, Cifuentes L, Grunwald T, Bredehorst R, Ollert M, Spillner E (2010) Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of α-1,3-core fucosylation. Mol Immunol 47:799–808Google Scholar
  41. 41.
    Minagawa S, Sekiguchi S, Nakaso Y, Tomita M, Takahisa M, Yasuda H (2015) Identification of core alpha 1,3-fucosyltransferase gene from silkworm: An insect popularly used to express mammalian proteins. J Insect Sci 15:110Google Scholar
  42. 42.
    Stanton R, Hykollary A, Eckmair B, Malzl D, Dragostis M, Palmberg D, Wang P, Wilson IBH, Paschinger K (2017) The underestimated N-glycomes of lepidopteran species. Biochim. Biophys Acta 1861:699–714Google Scholar
  43. 43.
    Vadaie N, Jarvis DL (2004) Molecular cloning and functional characterization of a lepidopteran insect β4-N-acetylgalactosaminyltransferase with broad substrate specificity, a functional role in glycoprotein biosynthesis and a potential functional role in glycolipid biosynthesis. J Biol Chem 279:33501–33518Google Scholar
  44. 44.
    Thomsen DR, Post LE, Elhammer AP (1990) Structure of O-glycosidically linked oligoencephalitis virus glycoprotein saccharides synthesized by the insect cell line Sf9. J Cell Biochem 43:67–79Google Scholar
  45. 45.
    Lopez M, Tetaert D, Juliant S, Gazon M, Cerutti M, Verbert A, Delannoy P (1999) O-Glycosylation potential of lepidopteran insect cell lines. Biochim Biophys Acta 1427:49–61Google Scholar
  46. 46.
    Gaunitz S, Jin C, Nilsson A, Liu J, Karlsson NG, Holgersson J (2013) Mucin-type proteins produced in the Trichoplusia ni and Spodoptera frugiperda insect cell lines carry novel O-glycans with phosphocholine and sulfate substitutions. Glycobiology 23:778–796Google Scholar
  47. 47.
    Spearman M, Butler M (2015) Glycosylation in cell culture. In: Al-Rubeai M (ed) Animal cell culture, cell engineering 9. Springer International Publishing, Cham, pp 237–258Google Scholar
  48. 48.
    Ooi BG, Miller LK (1988) Regulation of host RNA levels during baculovirus infection. Virology 166:515–523Google Scholar
  49. 49.
    Palomares LA, Estrada-Mondaca S, Ramírez OT (2006) Principles and applications of the insect-cell baculovirus expression vector system. In: Ozturk SS, Hu WS (eds) Cell culture technology for pharmaceutical and cell based therapies. Taylor and Francis, Boca Raton, pp 627–692Google Scholar
  50. 50.
    Palomares LA, López S, Ramírez OT (2004) Utilization of oxygen uptake rate to assess the role of glucose and glutamine in the metabolism of infected insect cell cultures. Biochem Eng J 19:87–93Google Scholar
  51. 51.
    Donaldson M, Wood HA, Kulakosky PC, Shuler ML (1999) Glycosylation of recombinant protein in the Tn5B1-4 insect cell line: influence of ammonia, time of harvest, temperature and dissolved oxygen. Biotechnol Bioeng 65:255–262Google Scholar
  52. 52.
    Joshi L, Shuler ML, Wood AH (2001) Production of a sialylated N-linked glycoprotein in insect cells. Biotechnol Prog 17:822–827Google Scholar
  53. 53.
    Zhang F, Saarinen MA, Itle LJ, Lang SC, Murhammer DW, Linhardt RJ (2002) The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell- baculovirus expression system. Biotechnol Bioeng 77:219–224Google Scholar
  54. 54.
    Joosten CE, Shuler ML (2003) Production of sialylated N-linked glycoprotein in insect cells: role of glycosidases and effect of harvest time on glycosylation. Biotechnol Prog 19:193–201Google Scholar
  55. 55.
    Donaldson M, Wood HA, Kulakosky PC, Shuler ML (1999) Use of mannosamine supplementation for inducing the addition of outer arm N-acetylglucosamine onto N-linked oligosaccharides of recombinant proteins in insect cells. Biotechnol Prog 15:168–173Google Scholar
  56. 56.
    Estrada-Mondaca S, Delgado-Bustos LA, Ramírez OT (2005) Mannosamine supplementation extends the N-acetylglucosaminylation of recombinant human secreted alkaline phosphatase produced in Trichoplusia ni (cabbage looper) insect cell cultures. Biotechnol Appl Biochem 42:25–34Google Scholar
  57. 57.
    Sridhar P, Panda AK, Pal R, Talwar GP, Hasnain SE (1993) Temporal nature of the promoter and not relative strength determines the expression of an extensively processed protein in a baculovirus system. FEBS Lett 315:282–286Google Scholar
  58. 58.
    Pajot-Augy E, Bozon V, Remy JJ, Couture L, Salesse R (1999) Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus-infected insect cells. Eur J Biochem 260:635–648Google Scholar
  59. 59.
    Ahn WS, Jeon JJ, Jeong YR, Lee SJ, Yoon SW (2008) Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells. Biotechnol Bioeng 101:1234–1244Google Scholar
  60. 60.
    Aloi LA, Cherry RS (1994) Intracellular calcium response of Sf-9 insect cells exposed to intense fluid forces. J Biotechnol 33:21–31Google Scholar
  61. 61.
    Godoy-Silva R, Chalmers JJ, Casnocha SA, Bass LA, Ma N (2009) Physiological responses CHO cells to repetitive hydrodynamic stress. Biotechnol Bioeng 103:1103–1117Google Scholar
  62. 62.
    Ramirez OT, Mutharasan R (1992) Effect of serum on the plasma membrane fluidity of hybridomas: an insight into its shear protective mechanism. Biotechnol Prog 8:40–50Google Scholar
  63. 63.
    Tomiya N, Ailor E, Lawrence SM, Betenbaugh MJ, Lee YC (2001) Determination of nucleotides and sugar nucleotides involved in protein glycosylation by high-performance anion-exchange chromatography: sugar nucleotide contents in cultured insect cells and mammalian cells. Anal Biochem 293:129–137Google Scholar
  64. 64.
    Jarvis DL, Weinkauf C, Guarino LA (1996) Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells. Protein Expr Purif 8:191–203Google Scholar
  65. 65.
    Toth AM, Geisler C, Aumiller JJ, Jarvis DL (2011) Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system. Protein Expr Purif 80:274–282Google Scholar
  66. 66.
    Jarvis DL, Finn EE (1996) Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nat Biotechnol 14:1288–1292Google Scholar
  67. 67.
    Geisler C, Jarvis D (2009) Insect cell glycosylation patterns in the context of biopharmaceuticals. In: Walsh G (ed) Post-translational modification of protein biopharmaceuticals. Wiley-VCH Weinheim, Weinheim, pp 165–191Google Scholar
  68. 68.
    Viswanathan K, Lawrence S, Hinderlich S, Yarema KJ, Lee YC, Betenbaugh MJ (2003) Engineering sialic acid synthetic ability into insect cells: identifying metabolic bottlenecks and devising strategies to overcome them. Biochemistry 42:15215–15225Google Scholar
  69. 69.
    Aumiller JJ, Hollister JR, Jarvis DL (2003) A transgenic insect cell line engineered to produce CMP-sialic acid and sialylated glycoproteins. Glycobiology 13:497–507Google Scholar
  70. 70.
    Chang GD, Chen CJ, Lin CY, Chen HC, Chen H (2003) Improvement of glycosylation in insect cells by mammalian glycosyltransferases. J Biotechnol 102:61–71Google Scholar
  71. 71.
    Okada T, Ihara H, Ito R, Nakano M, Matsumoto K, Yamaguchi Y, Taniguchi N, Ikeda Y (2010) N-Glycosylation engineering of lepidopteran insect cells by the introduction of the β1,4-N-acetylglucosaminyltransferase III gene. Glycobiology 20:1147–1159Google Scholar
  72. 72.
    Aumiller JJ, Mabashi-Asazuma H, Hillar A, Shi X, Jarvis DL (2012) A new glycoengineered insect cell line with an inducibly mammalianized protein N-glycosylation pathway. Glycobiology 22:417–428Google Scholar
  73. 73.
    Palmberger D, Wilson IBH, Berger I, Grabherr R, Rendic D (2012) SweetBac: a new approach for the production of mammalianised glycoproteins in insect cells. PLoS One 7:e34226Google Scholar
  74. 74.
    Kim NY, Baek JY, Choi HS, Chung IS, Shin S, Lee JI, Choi JY, Yang JM (2012) Short-hairpin RNA-mediated gene expression interference in Trichoplusia ni cells. J Microbiol Biotechnol 22:190–198Google Scholar
  75. 75.
    Mabashi-Asazuma H, Kuo CW, Khoo KH, Jarvis DL (2014) A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells. Glycobiology 24:325–340Google Scholar
  76. 76.
    Kati T, Kako N, Kikuta K, Miyazaki T, Kondi S, Yagi H, Kato K, Park EY (2017) N-Glycan modification of a recombinant protein via coexpression of human glycosyltransferases in silkworm pupae. Sci Rep 7:1409Google Scholar
  77. 77.
    Legardinier S, Klett D, Poirier JC, Combarnous Y, Cahoreau C (2005) Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in mimic™ insect cells. Glycobiology 15:776–790Google Scholar
  78. 78.
    Lin SC, Jan JT, Dionne B, Butler M, Huang MS, Wu CY, Wong CH, Wu SC (2013) Different immunity elicited by recombinant H5N1 hemagglutinin proteins containing pauci-mannose, high-mannose, or complex type N-glycans. PLoS One 8:e66719Google Scholar
  79. 79.
    Geisler C, Mabashi-Asazuma H, Jarvis DL (2015) An overview and history of glycol-engineering in insect expression systems. In: Castilho A (ed) Glyco-engineering: methods and protocols. Methods in Molecular Biology, vol 1321, pp 131–152Google Scholar
  80. 80.
    Hancock K, Narang S, Pattabhi S, Yushak ML, Khan A, Lin S, Plemons R, Betenbaugh MJ, Tsang VCW (2008) False positive reactivity of recombinant, diagnostic, glycoproteins produced in high five™ insect cells: effect of glycosylation. J Immunol Meth 330:130–136Google Scholar
  81. 81.
    Bantleon F, Wolf S, Seismann H, Dam S, Lorentzen A, Miehe M, Jabs F, Jakob T, Plum M, Spillner E (2016) Human IgE is efficiently produced in glycosylated and biologically active form in lepidopteran cells. Mol Immunol 72:49–56Google Scholar
  82. 82.
    Dunkle LM, Izikson R, Patriarca P, Goldenthal KL, Muse D, Callahan J, Cox MMJ (2017) Efficacy of recombinant influenza vaccine in adults 50 years of age or older. New Engl J Med 376:2427–2436Google Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Laura A. Palomares
    • 1
  • Indresh K. Srivastava
    • 2
  • Octavio T. Ramírez
    • 1
  • Manon M. J. Cox
    • 2
  1. 1.Instituto de Biotecnología, Universidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Protein Sciences Corporation, A Sanofi CompanyMeridenUSA

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