Skip to main content
PMC home page
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 2003;51(4):459–469. doi: 10.1023/A:1022354322226

Neutralizing immunogenicity of transgenic carrot (Daucus carota L.)-derived measles virus hemagglutinin

E Marquet-Blouin 1, FB Bouche 2, A Steinmetz 1, CP Muller 2,✉
PMCID: PMC7088612  PMID: 12650613

Abstract

Although edible vaccines seem to be feasible, antigens of human pathogens have mostly been expressed in plants that are not attractive for human consumption (such as potatoes) unless they are cooked. Boiling may reduce the immunogenicity of many antigens. More recently, the technology to transform fruit and vegetable plants have become perfected. We transformed carrot plants with Agrobacterium tumefaciens to generate plants (which can be eaten raw) transgenic for an immunodominant antigen of the measles virus, a major pathogen in man. The hemagglutinin (H) glycoprotein is the principle target of neutralizing and protective antibodies against measles. Copy numbers of the H transgene were verified by Southern blot and specific transcription was confirmed by RT-PCR. The H protein was detected by western blot in the membrane fraction of transformed carrot plants. The recombinant protein seemed to have a 8% lower molecular weight than the viral protein. Although this suggests a different glycosylation pattern, proper folding of the transgenic protein was confirmed by conformational-dependent monoclonal antibodies. Immunization of mice with leaf or root extracts induced high titres of IgG1 and IgG2a antibodies that cross-reacted strongly with the measles virus and neutralized the virus in vitro. These results demonstrate that transgenic carrot plants can be used as an efficient expression system to produce highly immunogenic viral antigens. Our study may pave the way towards an edible vaccine against measles which could be complementary to the current live-attenuated vaccine.

Keywords: carrots, Daucus carota, hemagglutinin protein, measles virus, neutralization, vaccine

References

  1. Arakawa T., Chong D.K.X., Langridge W.H.R. Efficacy of a food plant based cholera toxin B subunit vaccine. Nature Biotechnol. 1998;16:292–297. doi: 10.1038/nbt0398-292. [DOI] [PubMed] [Google Scholar]
  2. Arakawa T., Jie Y., Chong D.K.X., Langridge W.H.R. A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nature Biotechnol. 1998;16:934–938. doi: 10.1038/nbt1098-934. [DOI] [PubMed] [Google Scholar]
  3. Bardor M., Faye L., Lerouge P. Analysis of the N-glycosylation of recombinant glycoproteins produced in transgenic plants. Trends Plant Sci. 1999;4:376–380. doi: 10.1016/s1360-1385(99)01461-2. [DOI] [PubMed] [Google Scholar]
  4. Bevan M. Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 1984;12:8711–8721. doi: 10.1093/nar/12.22.8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bouche F., Ammerlaan W., Berthet F., Houard S., Schneider F., Muller C.P. Immunosorbent assay based on recombinant hemagglutinin protein produced in a high-efficiency mammalian expression system for surveillance of measles immunity. J. Clin. Microbiol. 1998;36:721–726. doi: 10.1128/jcm.36.3.721-726.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bouche F., Ammerlaan W., Fournier P., Schneider F., Muller C.P. A simplified immunoassay based on measles virus recombinant hemagglutinin protein for testing the immune status of vaccinees. J. Virol. Meth. 1998;74:77–87. doi: 10.1016/s0166-0934(98)00073-1. [DOI] [PubMed] [Google Scholar]
  7. Brodzik R., Gadanidze D., Hennig J., Muszynska G., Koprowski H., Sirko A. Transgenic plants as a potential source of an oral vaccine against Helicobacter pylori. In: Bielecki S., Tramper J., Polak J., editors. Food Biotechnology. Amsterdam: Elsevier Science; 2000. pp. 000–000. [Google Scholar]
  8. Cardineau, G.A and Curtiss, R. III. 1990. Oral immunization by transgenic plants. Patent WO9002484.
  9. Carrillo C., Wigdorovitz A., Oliveros J.C., Zamorano P.I., Sadir A.M., Gomez N., Salinas J., Escribano J.M., Borca M.V. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. J. Virol. 1998;72:1688–1690. doi: 10.1128/jvi.72.2.1688-1690.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. de Swart R.L., Vos H.W., UytdeHaag F.G., Osterhaus A.D., Binnendijk R.S. Measles virus fusion protein-and hemagglutinin-transfected cell lines are sensitive tool for the detection of specific antibodies by FACS-measured immunofluorescence assay. J. Virol. Meth. 1997;2:123–126. doi: 10.1016/s0166-0934(97)00188-2. [DOI] [PubMed] [Google Scholar]
  11. Deroo S., El Kasmi K.C, Fournier P., Theisen D., Brons N.H.C., Hermann M., Desmet J., Muller C.P. Enhanced antigenicity of a four-contact-residue epitope of the measles virus hemagglutinin protein by phage display libraries: evidence of a helical structure in the putative active site. Mol. Immunol. 1998;35:435–443. doi: 10.1016/s0161-5890(98)00057-1. [DOI] [PubMed] [Google Scholar]
  12. Faye L., Fitchette-Laine A.C., Gomord V., Chekkati A., Delaunay A.M., Driouich A. Detection, biosynthesis and some functions of glycans N-linked to plant secreted proteins. Soc. Exp. Biol. Semin. Ser. 1993;53:213–242. [Google Scholar]
  13. Feinberg A.P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 1983;132:6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Fournier P., Brons N.H.C., Berbers G.A.M., Wiesmüller K.H., Fleckenstein B.T., Schneider F., Jung G., Muller C.P. Antibodies to a new linear site at the topographical or functional interface between the haemagglutinin and fusion proteins protect against measles encephalitis. J. Gen. Virol. 1997;78:1295–1302. doi: 10.1099/0022-1317-78-6-1295. [DOI] [PubMed] [Google Scholar]
  15. Giraudon P., Wild F. Correlation between epitopes on hemagglutinin of measles virus and biological activities: passive protection by monoclonal antibodies is related to their hemagglutination inhibiting activity. Virology. 1985;144:46–58. doi: 10.1016/0042-6822(85)90303-4. [DOI] [PubMed] [Google Scholar]
  16. Gomez N., Carrillo C., Salinas J., Parra F., Borca M.V., Escribano J.M. Expression of immunogenic glycoprotein S polypeptides from transmissible gastroenteritis coronavirus in transgenic plants. Virology. 1998;249:352–358. doi: 10.1006/viro.1998.9315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haq T.A., Mason H.S., Clements J.D., Arntzen C.J. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science. 1995;268:714–716. doi: 10.1126/science.7732379. [DOI] [PubMed] [Google Scholar]
  18. Hardegger M., Sturm A. Transformation and regeneration of carrot (Daucus carota L.) Mol. Breed. 1998;4:119–127. [Google Scholar]
  19. Herrera-Estrella L., Simpson J. Foreign gene expression in plants. In: Shaw C.H., editor. Plant Molecular Biology: A Practical Approach. Oxford: IRL Press; 1988. pp. 131–159. [Google Scholar]
  20. Hu A., Norrby E. Role of individual cysteine residues in the processing and antigenicity of measles virus hemagglutinin protein. Arch. Virol. 1995;136:239–253. [Google Scholar]
  21. Hu A., Cattaneo R., Schwartz S., Norrby E. Role of N-linked oligosaccharide chains in the processing and the antigenicity of the measles virus haemagglutinin protein. J. Gen. Virol. 1994;75:2173–2181. doi: 10.1099/0022-1317-75-5-1043. [DOI] [PubMed] [Google Scholar]
  22. Huang Z., Dry I., Webster D., Strugnell R., Wesseling S. Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice. Vaccine. 2001;19:2163–2171. doi: 10.1016/s0264-410x(00)00390-x. [DOI] [PubMed] [Google Scholar]
  23. Hughes D.W., Galau G. Preparation of RNA from cotton leaves and pollen. Plant Mol. Biol. Rep. 1988;6:253–257. [Google Scholar]
  24. Kapusta J., Modelska A., Figlerowicz M., Pniewski T., Letellier M., Lisowa O., Yusibov V., Koprowski H., Plucienniczak A., Legocki B. A plant-derived edible vaccine against hepatitis B virus. FASEB J. 1999;13:1796–1799. doi: 10.1096/fasebj.13.13.1796. [DOI] [PubMed] [Google Scholar]
  25. Kong Q., Richter L., Yang Y.F., Arntzen C.J, Mason H.S., Thanavala J. Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc. Natl. Acad. Sci. USA. 2001;98:11539–11544. doi: 10.1073/pnas.191617598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mantis N.J., Kozlowski P.A., Mielcarz D.W., Weissenhorn W., Neutra M.R. Immunization of mice with recombinant gp41 in a systemic prime/mucosal boost protocol induces HIV-1-specific serum IgG and secretary IgA antibodies. Vaccine. 2001;19:3990–4001. doi: 10.1016/s0264-410x(01)00115-3. [DOI] [PubMed] [Google Scholar]
  27. Mason H.S., Lam D.M.-K., Arntzen C.J. Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA. 1992;89:11745–11749. doi: 10.1073/pnas.89.24.11745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mason H.S., Ball J.M., Shi J.J., Jiang X., Estes M.K., Arntzen C.J. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc. Natl. Acad. Sci. USA. 1996;93:5335–5340. doi: 10.1073/pnas.93.11.5335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McFarlin D.E., Bellini W.J., Mingioli E.S., Behar T.N., Trudgett A. Monospecific antibody to the haemagglutinin of measles virus. J. Gen. Virol. 1980;48:425–429. doi: 10.1099/0022-1317-48-2-425. [DOI] [PubMed] [Google Scholar]
  30. McGarvey P.B., Hammond J., Dienelt M., Hooper D.C., Fu Z.F., Dietzschold B., Koprowski H., Michaels F.H. Expression of the rabies virus glycoprotein in transgenic tomatoes. Bio/technology. 1995;13:1484–1487. doi: 10.1038/nbt1295-1484. [DOI] [PubMed] [Google Scholar]
  31. Miller E., Hill A., Morgan-Capner P., Forsey P., Rush M. Antibodies to measles, mumps, and rubella in UK children 4 years after vaccination with different MMR vaccines. Vaccine. 1995;13:799–802. doi: 10.1016/0264-410x(94)00086-3. [DOI] [PubMed] [Google Scholar]
  32. Modelska A., Dietzschold B., Sleysh N., Fu Z.F., Steplewski K., Hooper D.C., Koprowski H., Yusibov V. Immunization against rabies with plant-derived antigen. Proc. Natl. Acad. Sci. USA. 1998;95:2481–2485. doi: 10.1073/pnas.95.5.2481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mossong J., Nokes D.J., Edmunds W.J., Cox M.J., Ratnam S., Muller C.P. Modeling the impact of subclinical measles transmission in vaccinated populations with waning immunity. Am. J. Epidemiol. 1999;150:1238–1249. doi: 10.1093/oxfordjournals.aje.a009951. [DOI] [PubMed] [Google Scholar]
  34. Muller C.P., Beauverger P., Schneider F., Jung G., Brons N.H. Cholera toxin B stimulates systemic neutralizing antibodies after intranasal co-immunization with measles virus. J. Gen. Virol. 1995;76:1371–1380. doi: 10.1099/0022-1317-76-6-1371. [DOI] [PubMed] [Google Scholar]
  35. Naniche D., Varior-Krishnan D.G., Cervoni F., Wild T.F., Rossi B., Rabourdin-Combe C., Gerlier D. Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J. Virol. 1993;67:6025–6032. doi: 10.1128/jvi.67.10.6025-6032.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Odell J.T., Nagy F., Chua N.H. Identification of DNA sequences required for activity of a plant promoter: the CaMV 35S promoter. Nature. 1985;313:810–812. doi: 10.1038/313810a0. [DOI] [PubMed] [Google Scholar]
  37. Pietrzak M., Shillito R.D., Hohn T., Potrykus I. Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucl. Acids Res. 1986;14:5857–5868. doi: 10.1093/nar/14.14.5857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Restrepo M.A., Freed D.D., Carrington J.C. Nuclear transport of plant potyviral proteins. Plant Cell. 1990;2:987–998. doi: 10.1105/tpc.2.10.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Richter L.J., Thanavala Y., Arntzen C.J., Mason H.S. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nature Biotechnol. 2000;18:1167–1171. doi: 10.1038/81153. [DOI] [PubMed] [Google Scholar]
  40. Sandhu J.S., Krasnyanski S.F., Domier L.L, Korban S.S., Osadjan M.D., Buetow D.E. Oral immunization in mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response. Transgenic Res. 2000;9:127–135. doi: 10.1023/a:1008979525909. [DOI] [PubMed] [Google Scholar]
  41. Schenk P.M., Remans T., Sagi L., Elliott A.R., Dietzgen R.G., Swennen R., Ebert P.R., Grof C.P., Manners J.M. Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression inmonocot and dicot plants. Plant Mol. Biol. 2001;47:399–412. doi: 10.1023/a:1011680008868. [DOI] [PubMed] [Google Scholar]
  42. Tacket C.O., Mason H.S., Losonsky G., Clements J.D., Levine M.M., Arntzen C.J. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nature Med. 1998;4:607–609. doi: 10.1038/nm0598-607. [DOI] [PubMed] [Google Scholar]
  43. Tacket C.O., Mason H.S., Losonsky G., Estes M.K., Levine M.M., Arntzen C.J. Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes. J. Infect. Dis. 2000;128:302–305. doi: 10.1086/315653. [DOI] [PubMed] [Google Scholar]
  44. Thanavala Y., Yang Y.F., Lyons P., Mason H.S., Arntzen C. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc. Natl. Acad. Sci. USA. 1995;92:3358–3361. doi: 10.1073/pnas.92.8.3358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tokuji Y., Fukuda H. A rapid method for transformation of carrot (Daucus carota L.) by using direct somatic embryogenesis. Biosci. Biotechnol. Biochem. 1999;63:519–523. doi: 10.1271/bbb.63.519. [DOI] [PubMed] [Google Scholar]
  46. Vialard J., Lalumiere M., Vernet T., Briedis D., Alkhatib G., Henning D., Richardson C. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the ?-galactosidase gene. J. Virol. 1990;64:37–50. doi: 10.1128/jvi.64.1.37-50.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wigdorovitz A., Carrillo C., Dus Santos M.J., Trono K., Perelta A., Gomez M.C., Rios R.D., Franzone P.M., Sadir A.M., Escribano J.M., Borca M.V. Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parental immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology. 1999;255:347–353. doi: 10.1006/viro.1998.9590. [DOI] [PubMed] [Google Scholar]
  48. Wild T.F., Malvoisin E., Buckland R. Measles virus: both hemagglutinin and fusion glycoproteins are requiered for fusion. J. Gen. Virol. 1991;72:439–442. doi: 10.1099/0022-1317-72-2-439. [DOI] [PubMed] [Google Scholar]
  49. Ziegler D., Fournier P., Berbers G.A.H., Steuer H., Wiesmüller K.H., Fleckenstein B., Schneider F., Jung G., King C.C., Muller C.P. Protection against measles virus encephalitis by monoclonal antibodies binding to a cystein loop domain of the H protein mimicked by peptides which are not recognized by maternal antibodies. J. Gen. Virol. 1996;77:2479–2489. doi: 10.1099/0022-1317-77-10-2479. [DOI] [PubMed] [Google Scholar]

Articles from Plant Molecular Biology are provided here courtesy of Nature Publishing Group

RESOURCES