Skip to main content

Expression of H3N2 nucleoprotein in maize seeds and immunogenicity in mice

Plant Cell Reports volume 34, pages 969–980 (2015)Cite this article

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.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  • 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):3990–4002

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • 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):273–280

    Article  CAS  PubMed  Google Scholar 

  • 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):4168–4176

    Article  CAS  Google Scholar 

  • 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):3214–3222

    Article  CAS  PubMed  Google Scholar 

  • 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):30–38

    Article  CAS  PubMed  Google Scholar 

  • 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):479–493

    Article  CAS  PubMed  Google Scholar 

  • 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):3–9

    Article  PubMed  Google Scholar 

  • Cohen AD, Boyer JD, Weiner DB (1998) Modulating the immune response to genetic immunization. FASEB J 12(15):1611–1626

    CAS  PubMed  Google Scholar 

  • 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):2265–2274

    Article  CAS  PubMed  Google Scholar 

  • Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6(5):219–226

    Article  CAS  PubMed  Google Scholar 

  • Daniell H, Singh ND, Mason H, Streatfield SJ (2009) Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci 14(12):669–679

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Du L, Zhou Y, Jiang S (2010) Research and development of universal influenza vaccines. Microbes Infect 12(4):280–286

    Article  CAS  PubMed  Google Scholar 

  • 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):1243–1252

    Article  CAS  PubMed  Google Scholar 

  • Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4(3):181–191

    Article  CAS  PubMed  Google Scholar 

  • 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):796–801

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):5404–5410

    Article  CAS  PubMed  Google Scholar 

  • Flavell RB (1994) Inactivation of gene expression in plants as a consequence of specific sequence duplication. Proc Natl Acad Sci USA 91(9):3490–3496

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):13–22

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):197–201

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Geisbert TW (2014) Medical research: ebola therapy protects severely ill monkeys. Nature 514(7520):41–43

    Article  CAS  PubMed  Google Scholar 

  • Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotech 18(11):1151–1155

    Article  CAS  Google Scholar 

  • Gupta R, Jung E, Brunak S (2004) Prediction of N-glycosylation sites in human proteins (in preparation)

  • 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):979–984

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):1291–1301

    PubMed Central  CAS  PubMed  Google Scholar 

  • 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):129–140

    Article  CAS  PubMed  Google Scholar 

  • 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):322

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hutchinson EC, von Kirchbach JC, Gog JR, Digard P (2010) Genome packaging in influenza A virus. J Gen Virol 91(2):313–328

    Article  CAS  PubMed  Google Scholar 

  • 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):157–164

    Article  CAS  Google Scholar 

  • 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):e75460

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):2094–2098

    PubMed Central  CAS  PubMed  Google Scholar 

  • 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):5161–5166

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kuiken T, Holmes EC, McCauley J, Rimmelzwaan GF, Williams CS, Grenfell BT (2006) Host species barriers to influenza virus infections. Science 312(5772):394–397

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lambert LC, Fauci AS (2010) Influenza vaccines for the future. N Engl J Med 363(21):2036–2044

    Article  CAS  PubMed  Google Scholar 

  • 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):4331–4339

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):5027–5035

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):62–72

    Article  PubMed  Google Scholar 

  • 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):286

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ma JKC, Drake PMW, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4(10):794–805

    Article  CAS  PubMed  Google Scholar 

  • 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):223–230

    Article  CAS  PubMed  Google Scholar 

  • Marks MD, Lindell JS, Larkins BA (1985) Quantitative analysis of the accumulation of Zein mRNA during maize endosperm development. J Biol Chem 260(30):16445–16450

    CAS  PubMed  Google Scholar 

  • Mason HS, DeWald DB, Mullet JE (1993) Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter. Plant Cell 5(3):241–251

    PubMed Central  CAS  PubMed  Google Scholar 

  • Medina RA, García-Sastre A (2011) Influenza A viruses: new research developments. Nat Rev Micro 9(8):590–603

    Article  CAS  Google Scholar 

  • 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):3337–3352

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):1647–1657

    Article  CAS  PubMed  Google Scholar 

  • Munro S, Pelham HRB (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48(5):899–907

    Article  CAS  PubMed  Google Scholar 

  • 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 79–93

    Chapter  Google Scholar 

  • 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):585–594

    Article  CAS  PubMed  Google Scholar 

  • 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):167–179

    Article  CAS  Google Scholar 

  • 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):677–685

    Article  CAS  PubMed  Google Scholar 

  • 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):6119–6125

    PubMed Central  CAS  PubMed  Google Scholar 

  • Portela AN, Digard P (2002) The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83(4):723–734

    CAS  PubMed  Google Scholar 

  • Qiu X, Wong G, Audet J et al (2014) Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 514(7520):47–53

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rosales-Mendoza S, Salazar-Gonzalez JA (2014) Immunological aspects of using plant cells as delivery vehicles for oral vaccines. Expert Rev Vaccines 13(6):737–749

    Article  CAS  PubMed  Google Scholar 

  • Rose MA, Zielen S, Baumann U (2012) Mucosal immunity and nasal influenza vaccination. Expert Rev Vaccines 11(5):595–607

    Article  CAS  PubMed  Google Scholar 

  • Russell D, Fromm M (1997) Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice. Transgenic Res 6(2):157–168

    Article  CAS  PubMed  Google Scholar 

  • Rybicki EP (2010) Plant-made vaccines for humans and animals. Plant Biotechnol J 8(5):620–637

    Article  CAS  PubMed  Google Scholar 

  • 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):e104824

    Article  PubMed Central  PubMed  Google Scholar 

  • 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):2561–2572

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Stam M, Mol JNM, Kooter JM (1997) Review article: the silence of genes in transgenic plants. Ann Bot 79(1):3–12

    Article  CAS  Google Scholar 

  • 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):e00018–10

    Article  PubMed Central  PubMed  Google Scholar 

  • 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):743–768

    Article  CAS  PubMed  Google Scholar 

  • 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):13–25

    Article  CAS  PubMed  Google Scholar 

  • Townsend ARM, Gotch FM, Davey J (1985) Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell 42(2):457–467

    Article  CAS  PubMed  Google Scholar 

  • 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 4:1591

    Article  PubMed Central  PubMed  Google Scholar 

  • 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 145–159

    Chapter  Google Scholar 

  • 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):344

    Article  CAS  PubMed  Google Scholar 

  • 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:265–273

    Article  Google Scholar 

  • Wraith DC, Vessey AE, Askonas BA (1987) Purified influenza virus nucleoprotein protects mice from lethal infection. J Gen Virol 68(2):433–440

    Article  CAS  PubMed  Google Scholar 

  • 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):570–578

    Article  CAS  PubMed  Google Scholar 

  • 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):1785–1789

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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):423–434

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zheng M, Luo J, Chen Z (2014) Development of universal influenza vaccines based on influenza virus M and NP genes. Infection 42(2):251–262

    Article  CAS  PubMed  Google Scholar 

  • 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):2182–2189

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Interdepartmental Plant Biology Major, Iowa State University, Ames, IA, 50011-1010, USA

    Hartinio N. Nahampun

  2. Department of Agronomy, Iowa State University, Ames, IA, 50011-1010, USA

    Hartinio N. Nahampun & Kan Wang

  3. Department of Animal Science, Iowa State University, Ames, IA, 50011-1010, USA

    Brad Bosworth & Joan Cunnick

  4. Harrisvaccines, Inc, Ames, IA, 50010, USA

    Mark Mogler

Authors
  1. Hartinio N. Nahampun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brad Bosworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joan Cunnick
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mark Mogler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kan Wang.

Additional information

Communicated by P. Lakshmanan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 38 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nahampun, H.N., Bosworth, B., Cunnick, J. et al. Expression of H3N2 nucleoprotein in maize seeds and immunogenicity in mice. Plant Cell Rep 34, 969–980 (2015). https://doi.org/10.1007/s00299-015-1758-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-015-1758-0

Keywords

  • Nucleoprotein
  • H3N2
  • Plant-based vaccine
  • Antigens
  • Transgenic maize