Plant Cell Reports

, Volume 34, Issue 6, pp 969–980 | Cite as

Expression of H3N2 nucleoprotein in maize seeds and immunogenicity in mice

  • Hartinio N. Nahampun
  • Brad Bosworth
  • Joan Cunnick
  • Mark Mogler
  • Kan Wang
Original Paper

Abstract

Key message

Oral administration of maize-expressed H3N2 nucleoprotein induced antibody responses in mice showing the immunogenicity of plant-derived antigen and its potential to be utilized as a universal flu vaccine.

Abstract

Influenza A viruses cause influenza epidemics that are devastating to humans and livestock. The vaccine for influenza needs to be reformulated every year to match the circulating strains due to virus mutation. Influenza virus nucleoprotein (NP) is a multifunctional RNA-binding protein that is highly conserved among strains, making it a potential candidate for a universal vaccine. In this study, the NP gene of H3N2 swine origin influenza virus was expressed in maize endosperm. Twelve transgenic maize lines were generated and analyzed for recombinant NP (rNP) expression. Transcript analysis showed the main accumulation of rNP in seed. Protein level of rNP in T1 transgenic maize seeds ranged from 8.0 to 35 µg of NP/g of corn seed. The level increased up to 70 µg of NP/g in T3 seeds. A mouse study was performed to test the immunogenicity of one line of maize-derived rNP (MNP). Mice were immunized with MNP in a prime-boost design. Oral gavage administration showed that a humoral immune response was elicited in the mice treated with MNP indicating the immunogenicity of MNP. NP-specific antibody responses in the MNP group showed comparable antibody titer with the groups receiving positive controls such as Vero cell-derived NP (VNP) or alphavirus replicon particle-derived NP (ANP). Cytokine analysis showed antigen-specific stimulation of IL-4 cytokine elicited in splenocytes from mice treated with MNP further confirming a TH2 humoral immune response induced by MNP administration.

Keywords

Nucleoprotein H3N2 Plant-based vaccine Antigens Transgenic maize 

Notes

Acknowledgments

HN and KW thank Meaghan Nelson and Pam Whitson for their technical assistance in animal experiment, Dr. Ryan Vander Veen for providing H3N2 NP gene cassette and technical assistance in the experiment, and Dr. Hank Harris for his expertise in vaccines and initial scientific discussion. This work was supported in part by the U.S. Department of Agriculture National Institute of Food and Agriculture (Hatch Project No. IOW05162), the Plant Sciences Institute of Iowa State University and Charoen Pokphand Indonesia.

Conflict of interest

HN, BB, JC and KW declare that they have no conflict of interest. MM is an employee of Harrisvaccines, Inc., which provided materials and expertise for the work. However, this does not alter the author’s adherence to all the Plant Cell Reports policies on sharing data and materials.

Supplementary material

299_2015_1758_MOESM1_ESM.docx (39 kb)
Supplementary material 1 (DOCX 38 kb)

References

  1. Ashraf S, Kong W, Wang S, Yang J, Curtiss R 3rd (2011) Protective cellular responses elicited by vaccination with influenza nucleoprotein delivered by a live recombinant attenuated Salmonella vaccine. Vaccine 29(23):3990–4002CrossRefPubMedCentralPubMedGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  3. Brett IC, Johansson BE (2005) Immunization against influenza A virus: comparison of conventional inactivated, live-attenuated and recombinant baculovirus produced purified hemagglutinin and neuraminidase vaccines in a murine model system. Virology 339(2):273–280CrossRefPubMedGoogle Scholar
  4. Carragher DM, Kaminski DA, Moquin A, Hartson L, Randall TD (2008) A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus. Journal of immunology 181(6):4168–4176CrossRefGoogle Scholar
  5. Chen Z, Kadowaki S, Hagiwara Y, Yoshikawa T, Matsuo K, Kurata T, Tamura S (2000) Cross-protection against a lethal influenza virus infection by DNA vaccine to neuraminidase. Vaccine 18(28):3214–3222CrossRefPubMedGoogle Scholar
  6. Chen Q, Kuang H, Wang H, Fang F, Yang Z, Zhang Z, Zhang X, Chen Z (2009) Comparing the ability of a series of viral protein-expressing plasmid DNAs to protect against H5N1 influenza virus. Virus Genes 38(1):30–38CrossRefPubMedGoogle Scholar
  7. Chikwamba R, Cunnick J, Hathaway D, McMurray J, Mason H, Wang K (2002) A functional antigen in a practical crop: LT-B producing maize protects mice against Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT). Transgenic Res 11(5):479–493CrossRefPubMedGoogle Scholar
  8. Ciacci-Zanella JR, Vincent AL, Prickett JR, Zimmerman SM, Zimmerman JJ (2010) Detection of anti-influenza A nucleoprotein antibodies in pigs using a commercial influenza epitope-blocking enzyme-linked immunosorbent assay developed for avian species. J Vet Diagn Invest 22(1):3–9CrossRefPubMedGoogle Scholar
  9. Cohen AD, Boyer JD, Weiner DB (1998) Modulating the immune response to genetic immunization. FASEB J 12(15):1611–1626PubMedGoogle Scholar
  10. Crawford J, Wilkinson B, Vosnesensky A, Smith G, Garcia M, Stone H, Perdue ML (1999) Baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian H5 and H7 subtypes. Vaccine 17(18):2265–2274CrossRefPubMedGoogle Scholar
  11. Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6(5):219–226CrossRefPubMedGoogle Scholar
  12. Daniell H, Singh ND, Mason H, Streatfield SJ (2009) Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci 14(12):669–679CrossRefPubMedCentralPubMedGoogle Scholar
  13. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  14. Du L, Zhou Y, Jiang S (2010) Research and development of universal influenza vaccines. Microbes Infect 12(4):280–286CrossRefPubMedGoogle Scholar
  15. Eliasson DG, El Bakkouri K, Schon K, Ramne A, Festjens E, Lowenadler B, Fiers W, Saelens X, Lycke N (2008) CTA1-M2e-DD: a novel mucosal adjuvant targeted influenza vaccine. Vaccine 26(9):1243–1252CrossRefPubMedGoogle Scholar
  16. Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4(3):181–191CrossRefPubMedGoogle Scholar
  17. Epstein SL, Tumpey TM, Misplon JA, Lo CY, Cooper LA, Subbarao K, Renshaw M, Sambhara S, Katz JM (2002) DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice. Emerg Infect Dis 8(8):796–801CrossRefPubMedCentralPubMedGoogle Scholar
  18. Epstein SL, Kong W-P, Misplon JA, Lo C-Y, Tumpey TM, Xu L, Nabel GJ (2005) Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine 23(46–47):5404–5410CrossRefPubMedGoogle Scholar
  19. Flavell RB (1994) Inactivation of gene expression in plants as a consequence of specific sequence duplication. Proc Natl Acad Sci USA 91(9):3490–3496CrossRefPubMedCentralPubMedGoogle Scholar
  20. Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129(1):13–22CrossRefPubMedCentralPubMedGoogle Scholar
  21. Garten RJ, Davis CT, Russell CA et al (2009) Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325(5937):197–201CrossRefPubMedCentralPubMedGoogle Scholar
  22. Geisbert TW (2014) Medical research: ebola therapy protects severely ill monkeys. Nature 514(7520):41–43CrossRefPubMedGoogle Scholar
  23. Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotech 18(11):1151–1155CrossRefGoogle Scholar
  24. Gupta R, Jung E, Brunak S (2004) Prediction of N-glycosylation sites in human proteins (in preparation)Google Scholar
  25. Hayden CA, Egelkrout EM, Moscoso AM, Enrique C, Keener TK, Jimenez-Flores R, Wong JC, Howard JA (2012) Production of highly concentrated, heat-stable hepatitis B surface antigen in maize. Plant Biotechnol J 10(8):979–984CrossRefPubMedCentralPubMedGoogle Scholar
  26. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168(3):1291–1301PubMedCentralPubMedGoogle Scholar
  27. Hood EE, Bailey MR, Beifuss K, Magallanes-Lundback M, Horn ME, Callaway E, Drees C, Delaney DE, Clough R, Howard JA (2003) Criteria for high-level expression of a fungal laccase gene in transgenic maize. Plant Biotechnol J 1(2):129–140CrossRefPubMedGoogle Scholar
  28. Huang B, Wang W, Li R, Wang X, Jiang T, Qi X, Gao Y, Tan W, Ruan L (2012) Influenza A virus nucleoprotein derived from Escherichia coli or recombinant vaccinia (Tiantan) virus elicits robust cross-protection in mice. Virol J 9(1):322CrossRefPubMedCentralPubMedGoogle Scholar
  29. Hutchinson EC, von Kirchbach JC, Gog JR, Digard P (2010) Genome packaging in influenza A virus. J Gen Virol 91(2):313–328CrossRefPubMedGoogle Scholar
  30. Jimenez GS, Planchon R, Wei Q, Rusalov D, Geall A, Enas J, Lalor P, Leamy V, Vahle R, Luke CJ, Rolland A, Kaslow DC, Smith LR (2007) Vaxfectin-formulated influenza DNA vaccines encoding NP and M2 viral proteins protect mice against lethal viral challenge. Hum Vaccines 3(5):157–164CrossRefGoogle Scholar
  31. Kim S-H, Kim JY, Choi Y, Nguyen HH, Song MK, Chang J (2013) Mucosal vaccination with recombinant adenovirus encoding nucleoprotein provides potent protection against influenza virus infection. PLoS ONE 8(9):e75460CrossRefPubMedCentralPubMedGoogle Scholar
  32. Kodihalli S, Goto H, Kobasa DL, Krauss S, Kawaoka Y, Webster RG (1999) DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice. J Virol 73(3):2094–2098PubMedCentralPubMedGoogle Scholar
  33. Kreijtz JHCM, de Mutsert G, van Baalen CA, Fouchier RAM, Osterhaus ADME, Rimmelzwaan GF (2008) Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus. J Virol 82(11):5161–5166CrossRefPubMedCentralPubMedGoogle Scholar
  34. Kuiken T, Holmes EC, McCauley J, Rimmelzwaan GF, Williams CS, Grenfell BT (2006) Host species barriers to influenza virus infections. Science 312(5772):394–397CrossRefPubMedGoogle Scholar
  35. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  36. Lambert LC, Fauci AS (2010) Influenza vaccines for the future. N Engl J Med 363(21):2036–2044CrossRefPubMedGoogle Scholar
  37. LaMere MW, Lam HT, Moquin A, Haynes L, Lund FE, Randall TD, Kaminski DA (2011a) Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus. J Immunol 186(7):4331–4339CrossRefPubMedCentralPubMedGoogle Scholar
  38. LaMere MW, Moquin A, Lee FE, Misra RS, Blair PJ, Haynes L, Randall TD, Lund FE, Kaminski DA (2011b) Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance. J Virol 85(10):5027–5035CrossRefPubMedCentralPubMedGoogle Scholar
  39. Lee G, Na YJ, Yang B-G et al (2014) Oral immunization of haemaggulutinin H5 expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza A virus infection. Plant Biotechnol J 13(1):62–72CrossRefPubMedGoogle Scholar
  40. Luo J, Zheng D, Zhang W et al (2012) Induction of cross-protection against influenza A virus by DNA prime-intranasal protein boost strategy based on nucleoprotein. Virol J 9(1):286CrossRefPubMedCentralPubMedGoogle Scholar
  41. Ma JKC, Drake PMW, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4(10):794–805CrossRefPubMedGoogle Scholar
  42. Markine-Goriaynoff D, van der Logt JT, Truyens C et al (2000) IFN-gamma-independent IgG2a production in mice infected with viruses and parasites. Int Immunol 12(2):223–230CrossRefPubMedGoogle Scholar
  43. Marks MD, Lindell JS, Larkins BA (1985) Quantitative analysis of the accumulation of Zein mRNA during maize endosperm development. J Biol Chem 260(30):16445–16450PubMedGoogle Scholar
  44. Mason HS, DeWald DB, Mullet JE (1993) Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter. Plant Cell 5(3):241–251PubMedCentralPubMedGoogle Scholar
  45. Medina RA, García-Sastre A (2011) Influenza A viruses: new research developments. Nat Rev Micro 9(8):590–603CrossRefGoogle Scholar
  46. Moeller L, Gan Q, Wang K (2009) A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway. J Exp Bot 60(12):3337–3352CrossRefPubMedCentralPubMedGoogle Scholar
  47. Moravec T, Schmidt MA, Herman EM, Woodford-Thomas T (2007) Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine. Vaccine 25(9):1647–1657CrossRefPubMedGoogle Scholar
  48. Munro S, Pelham HRB (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48(5):899–907CrossRefPubMedGoogle Scholar
  49. Orellana-Escobedo L, Korban SS, Rosales-Mendoza S (2014) Seed-Based Expression Strategies. In: Rosales-Mendoza S (ed) Genetically engineered plants as a source of vaccines against wide spread diseases. Springer, New York, pp 79–93CrossRefGoogle Scholar
  50. Ou J, Guo Z, Shi J, Wang X, Liu J, Shi B, Guo F, Zhang C, Yang D (2014) Transgenic rice endosperm as a bioreactor for molecular pharming. Plant Cell Rep 33(4):585–594CrossRefPubMedGoogle Scholar
  51. Paz M, Shou H, Guo Z, Zhang Z, Banerjee A, Wang K (2004) Assessment of conditions affecting Agrobacterium -mediated soybean transformation using the cotyledonary node explant. Euphytica 136(2):167–179CrossRefGoogle Scholar
  52. Perea Arango I, Loza Rubio E, Rojas Anaya E et al (2008) Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice. Plant Cell Rep 27(4):677–685CrossRefPubMedGoogle Scholar
  53. Pertmer TM, Roberts TR, Haynes JR (1996) Influenza virus nucleoprotein-specific immunoglobulin G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery. J Virol 70(9):6119–6125PubMedCentralPubMedGoogle Scholar
  54. Portela AN, Digard P (2002) The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83(4):723–734PubMedGoogle Scholar
  55. Qiu X, Wong G, Audet J et al (2014) Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 514(7520):47–53CrossRefPubMedCentralPubMedGoogle Scholar
  56. Rosales-Mendoza S, Salazar-Gonzalez JA (2014) Immunological aspects of using plant cells as delivery vehicles for oral vaccines. Expert Rev Vaccines 13(6):737–749CrossRefPubMedGoogle Scholar
  57. Rose MA, Zielen S, Baumann U (2012) Mucosal immunity and nasal influenza vaccination. Expert Rev Vaccines 11(5):595–607CrossRefPubMedGoogle Scholar
  58. Russell D, Fromm M (1997) Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice. Transgenic Res 6(2):157–168CrossRefPubMedGoogle Scholar
  59. Rybicki EP (2010) Plant-made vaccines for humans and animals. Plant Biotechnol J 8(5):620–637CrossRefPubMedGoogle Scholar
  60. Sanchez MV, Ebensen T, Schulze K et al (2014) Intranasal delivery of influenza rNP adjuvanted with c-di-AMP induces strong humoral and cellular immune responses and provides protection against virus challenge. PLoS ONE 9(8):e104824CrossRefPubMedCentralPubMedGoogle Scholar
  61. Schubert D, Lechtenberg B, Forsbach A, Gils M, Bahadur S, Schmidt R (2004) Silencing in arabidopsis T-DNA transformants: the predominant role of a gene-specific RNA sensing mechanism versus position effects. Plant Cell 16(10):2561–2572CrossRefPubMedCentralPubMedGoogle Scholar
  62. Stam M, Mol JNM, Kooter JM (1997) Review article: the silence of genes in transgenic plants. Ann Bot 79(1):3–12CrossRefGoogle Scholar
  63. Steel J, Lowen AC, Wang TT, Yondola M, Gao Q, Haye K, García-Sastre A, Palese P (2010) Influenza virus vaccine based on the conserved hemagglutinin stalk domain. MBio 1(1):e00018–10CrossRefPubMedCentralPubMedGoogle Scholar
  64. Stoger E, Fischer R, Moloney M, Ma JK-C (2014) Plant molecular pharming for the treatment of chronic and infectious diseases. Annu Rev Plant Biol 65(1):743–768CrossRefPubMedGoogle Scholar
  65. Townsend ARM, McMichael AJ, Carter NP, Huddleston JA, Brownlee GG (1984) Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells. Cell 39(1):13–25CrossRefPubMedGoogle Scholar
  66. Townsend ARM, Gotch FM, Davey J (1985) Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell 42(2):457–467CrossRefPubMedGoogle Scholar
  67. Turrell L, Lyall JW, Tiley LS, Fodor E, Vreede FT (2013) The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes. Nat Commun 4:1591CrossRefPubMedCentralPubMedGoogle Scholar
  68. Twyman R, Schillberg S, Fischer R (2012) The production of vaccines and therapeutic antibodies in plants. In: Wang A, Ma S (eds) Molecular farming in plants: recent advances and future prospects. Springer, Netherlands, pp 145–159CrossRefGoogle Scholar
  69. Vander Veen RL, Mogler MA, Russell BJ, Loynachan AT, Harris DL, Kamrud KI (2013) Haemagglutinin and nucleoprotein replicon particle vaccination of swine protects against the pandemic H1N1 2009 virus. Vet Rec 173(14):344CrossRefPubMedGoogle Scholar
  70. Wang W, Huang B, Jiang T, Wang X, Qi X, Tan W, Ruan L (2014) Maximal immune response and cross protection by influenza virus nucleoprotein derived from E. coli using an optimized formulation. Virology 468–470C:265–273CrossRefGoogle Scholar
  71. Wraith DC, Vessey AE, Askonas BA (1987) Purified influenza virus nucleoprotein protects mice from lethal infection. J Gen Virol 68(2):433–440CrossRefPubMedGoogle Scholar
  72. Wu J, Yu L, Li L, Hu J, Zhou J, Zhou X (2007) Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens. Plant Biotechnol J 5(5):570–578CrossRefPubMedGoogle Scholar
  73. Yewdell JW, Bennink JR, Smith GL, Moss B (1985) Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes. Proc Natl Acad Sci 82(6):1785–1789CrossRefPubMedCentralPubMedGoogle Scholar
  74. Zhai Z, Liu Y, Wu L, Senchina DS, Wurtele ES, Murphy PA, Kohut ML, Cunnick JE (2007) Enhancement of innate and adaptive immune functions by multiple Echinacea species. J Med Food 10(3):423–434CrossRefPubMedCentralPubMedGoogle Scholar
  75. Zheng M, Luo J, Chen Z (2014) Development of universal influenza vaccines based on influenza virus M and NP genes. Infection 42(2):251–262CrossRefPubMedGoogle Scholar
  76. Zhou D, Wu TL, Lasaro MO et al (2010) A universal influenza A vaccine based on adenovirus expressing matrix-2 ectodomain and nucleoprotein protects mice from lethal challenge. Mol Ther 18(12):2182–2189CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Hartinio N. Nahampun
    • 1
    • 2
  • Brad Bosworth
    • 3
  • Joan Cunnick
    • 3
  • Mark Mogler
    • 4
  • Kan Wang
    • 2
  1. 1.Interdepartmental Plant Biology MajorIowa State UniversityAmesUSA
  2. 2.Department of AgronomyIowa State UniversityAmesUSA
  3. 3.Department of Animal ScienceIowa State UniversityAmesUSA
  4. 4.Harrisvaccines, IncAmesUSA

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