Abstract
Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins including antibodies, antigens and hormones. Here, we report expression of a full-length nucleoprotein gene of rabies virus in transgenic tomato plants. The nucleoprotein was also transiently expressed in Nicotiana benthamiana plants by agroinfiltration. In both cases, the nucleoprotein was expressed at high levels, 1–5% of total soluble protein in tomato and 45% in N. benthamiana. Previously, only epitopes of the nucleoprotein had been expressed in plants. The presence and expression of the transgene was verified by PCR, Southern, northern and western blots. Mice were immunized both intraperitoneally (i.p.) and orally with tomato protein extracts containing the N protein induced the production of antibodies. The antibody titer of mice immunized i.p., was at least four times higher than that of mice immunized orally. These results were reflected in the challenge experiments where i.p.-immunized mice were partially protected against a peripheral virus challenge whereas orally immunized mice were not. This protection was comparable to that obtained in previous experiments employing different expression systems. Work is in progress to express both G and N proteins in transgenic plants and evaluate protection in mice.
Similar content being viewed by others
References
Aldrich J, Cullis CA (1993) RAPD analysis in flax: optimization of yield and reproducibility using KlenTaq1 DNA polymerase, Chelex 100 and gel purification of genomic DNA. Plant Mol Biol Rep 11:128–141
Ashraf S, Singh PK, Yadav DK, Shahnawaz M, Mishra S, Sawant SV, Tuli R (2005) High level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice. J Biotechnol 119:1–14
Astoul E, Lafage M, Lafon M (1996) Rabies superantigen as a Vbeta T-dependent adjuvant. J Exp Med 183:1623–1631
Bendahmane M, Koo M, Karrer E, Beachy RN (1999) Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions. J Mol Biol 290:9–20
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Christou P (1996) Transformation technology. Trends Plants Sci 1:423–431
Cox JH (1982) The structural proteins of rabies virus. Comp Immunol Microbiol Infect Dis 5:21–25
da Cruz FW, McBride AJ, Conceicao FR, Dale JW, McFadden J, Dellagostin OA (2002) Expression of the B-cell and T-cell epitopes of the rabies virus nucleoprotein in Mycobacterium bovis BCG and induction of an humoral response in mice. Vaccine 20:731–736
Dietzschold B, Tollis M, Rupprecht CE, Celis E, Koprowski H (1987a) Antigenic variation in rabies and rabies-related viruses: cross-protection independent of glycoprotein-mediated virus-neutralizing antibody. J Infect Dis 156:815–822
Dietzschold B, Wang HH, Rupprecht CE, Celis E. Tollis M, Ertl H, Heber-Katz E, Koprowski H (1987b) Induction of protective immunity against rabies by immunization with rabies virus ribonucleoprotein. Proc Natl Acad Sci USA 84:9165–9169
Dietzschold B, Rupprecht CE, Tollis M, Lafon M, Mattei J, Wiktor TJ, Koprowski H (1988) Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemiology and control of rabies. Rev Infect Dis 10(Suppl 4):S785–S798
Drings A, Jallet C, Chambert B, Tordo N, Perrin P (1999) Is there an advantage to including the nucleoprotein in a rabies glycoprotein subunit vaccine? Vaccine 17:1549–1557
Dus Santos MJ, Wigdorovitz A (2005) Transgenic plants for the production of veterinary vaccines. Immunol Cell Biol 83:229–238
Ertl HC, Dietzschold B, Gore M, Otvos L Jr, Larson JK, Wunner WH, Koprowski H (1989) Induction of rabies virus-specific T-helper cells by synthetic peptides that carry dominant T-helper cell epitopes of the viral ribonucleoprotein. J Virol 63:2885–2892
Fekadu M, Sumner JW, Shaddock JH, Sanderlin DW, Baer GM (1992) Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein. J Virol 66:2601–2604
Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158
Flamand A, Wiktor TJ, Koprowski H (1980) Use of hybridoma monoclonal antibodies in the detection of antigenic differences between rabies and rabies-related virus proteins II. The glycoprotein. J Gen Virol 48:105–109
Fu ZF, Dietzschold B, Schumacher CL, Wunner WH, Ertl HC, Koprowski H (1991) Rabies virus nucleoprotein expressed in and purified from insect cells is efficacious as a vaccine. Proc Natl Acad Sci USA 88:2001–2005
Gil F, Brun A, Wigdorovitz A, Catala R, Martinez-Torrecuadrada JL, Casal I, Salinas J, Borca MV, Escribano JM (2001) High-yield expression of a viral peptide vaccine in transgenic plants. FEBS Lett 488:13–17
Gils M, Kandzia R, Marillonnet S, Klimyuk V, Gleba Y (2005) High-yield production of authentic human growth hormone using a plant virus-based expression system. Plant Biotech J 3:613–620
Gutierrez-Ortega A, Sandoval-Montes C, de Olivera-Flores TJ, Santos-Argumedo L, Gomez-Lim MA (2005) Expression of functional interleukin-12 from mouse in transgenic tomato plants. Transgenic Res 14:877–885
Hooper DC, Pierard I, Modelska A, Otvos L Jr, Fu ZF, Koprowski H, Dietzschold B (1994) Rabies ribonucleocapsid as an oral immunogen and immunological enhancer. Proc Natl Acad Sci USA 91:10908–10912
Howard JA (2004) Commercialization of plant-based vaccines from research and development to manufacturing. Anim Health Res Rev 5:243–245
Huang Z, Santi L, LePore K, Kilbourne J, Arntzen CJ, Mason HS (2006) Rapid, high-level production of hepatitis B core antigen in plant leaf and its immunogenicity in mice. Vaccine 24:2506–2513
Kawaguchi R, Bailey-Serres J (2002) Regulation of translational initiation in plants. Curr Op Plant Biol 5:460–465
Ku MS, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, Miyao M, Matsuoka M (1999) High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotechnol 17:76–80
Lafon M, Wiktor TJ, Macfarlan RI (1983) Antigenic sites on the CVS rabies virus glycoprotein: analysis with monoclonal antibodies. J Gen Virol 64:843–851
Lafon M, Scott-Algara D, Marche PN, Cazenave PA, Jouvin-Marche E (1994) Neonatal deletion and selective expansion of mouse T cells by exposure to rabies virus nucleocapsid superantigen. J Exp Med 180:1207–1215
Lodmell DL, Sumner JW, Esposito JJ, Bellini WJ, Ewalt LC (1991) Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection. J Virol 65:3400–3405
Lodmell DL, Esposito JJ, Ewalt LC (1993) Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro. J Virol 67:6080–6086
Lodmell DL, Smith JS, Esposito JJ, Ewalt LC (1995) Cross-protection of mice against a global spectrum of rabies virus variants. J Virol 69:4957–4962
Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci USA 101:6852–6857
Marquet-Blouin E, Bouche FB, Steinmetz A, Muller CP (2003) Neutralizing immunogenicity of transgenic carrot (Daucus carota L.)-derived measles virus hemagglutinin. Plant Mol Biol 51:459–469
McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Dietzschold B, Koprowski H, Michaels FH (1995) Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnol 13:1484–1487
Modelska A, Dietzschold B, Sleysh N, Fu ZF, Steplewski K, Hooper DC, Koprowski H, Yusibov V (1998) Immunization against rabies with plant-derived antigen. Proc Natl Acad Sci USA 95:2481–2485
Morimoto K, McGettigan JP, Foley HD, Hooper DC, Dietzschold B, Schnell MJ (2001) Genetic engineering of live rabies vaccines. Vaccine 19:3543–3551
Nadin-Davis SA, Loza-Rubio E (2006) The molecular epidemiology of rabies associated with chiropteran hosts in Mexico. Virus Res 117:215–226
Nel LH, Niezgoda M, Hanlon CA, Morril PA, Yager PA, Rupprecht CE (2003) A comparison of DNA vaccines for the rabies-related virus, Mokola. Vaccine 21:2598–2606
Rupprecht CE, Hanlon CA, Hemachudha T (2002) Rabies re-examined. Lancet Infect Dis 2:327–343
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor, New York
Smith JS (1995) Rabies virus. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds) Manual of clinical microbiology. American Society for Microbiology, Washington, DC pp 997–1003
Streatfield SJ, Lane JR, Brooks CA, Barker DK, Poage ML, Mayor JM, Lamphear BJ, Drees CF, Jilka JM, Hood EE, Howard JA (2003) Corn as a production system for human and animal vaccines. Vaccine 21:812–815
Summer JW, Fekadu M, Shaddock JH, Esposito JJ, Bellini WJ (1991) Protection of mice with vaccinia virus recombinants that express the rabies nucleoprotein. Virology 183:703–710
Yusibov V, Modelska A, Steplewski K, Agadjanyan M, Weiner D, Hooper DC, Koprowski H (1997) Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc Natl Acad Sci USA 94:5784–5788
Yusibov V, Hooper DC, Spitsin SV, Fleysh N, Kean RB, Mikheeva T, Deka D, Karasev A, Cox S, Randall J, Koprowski H (2002) Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. Vaccine 20:3155–3164
Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT, Lane JR, Delaney DE, McComas-Wagner JM, Mallubhotla HD, Hood EE, Dangott LJ, Tichy SE, Howard JA (2003) Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. Biotechnol Appl Biochem 38:123–130
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by G.D. May.
Rights and permissions
About this article
Cite this article
Perea Arango, I., Loza Rubio, E., Rojas Anaya, E. et al. Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice. Plant Cell Rep 27, 677–685 (2008). https://doi.org/10.1007/s00299-007-0324-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-007-0324-9