Clin Exp Vaccine Res.  2019 Jul;8(2):124-131. 10.7774/cevr.2019.8.2.124.

Transient expression of hemagglutinin antigen from canine influenza virus H3N2 in Nicotiana benthamiana and Lactuca sativa

• Maharjan P
• Choe S

• KMID: 2455084
• DOI: http://doi.org/10.7774/cevr.2019.8.2.124

Abstract

PURPOSE
Canine influenza virus (CIV), H3N2, carries potentiality for zoonotic transmission and genetic assortment which raises a concern on possible epidemics, and human threats in future. To manage possible threats, the development of rapid and effective methods of CIV vaccine production is required. The plant provides economical, safe, and robust production platform. We investigated whether hemagglutinin (HA) antigen from Korea-originated CIV could be produced in Nicotiana benthamiana and lettuce, Lactuca sativa by a DNA viral vector system.
MATERIALS AND METHODS
We used DNA sequences of the HA gene from Korean CIV strain influenza A/canine/Korea/S3001/2015 (H3N2) for cloning into a geminiviral expression vectors to express recombinant HA (rHA) antigen in the plant. Agrobacterium-mediated infiltration was performed to introduce HA-carrying vector into host plants cells. Laboratory-grown N. benthamiana, and grocery-purchased or hydroponically-grown lettuce plant leaves were used as host plants.
RESULTS
CIV rHA antigen was successfully expressed in host plant species both N. benthamiana and L. sativa by geminiviral vector. Both complex-glycosylated and basal-glycosylated form of rHA were produced in lettuce, depending on presence of endoplasmic reticulum (ER) retention signal. In terms of rHA expression level, canine HA (H3N2) showed preference to the native signal peptide than ER retention signal peptide in the tested geminiviral vector system.
CONCLUSION
Grocery-purchased lettuce leaves could serve as an instant host system for the transient expression of influenza antigen at the time of emergency. The geminiviral vector was able to induce expression of complex-glycosylated and basal-glycosylated rHA in lettuce and tobacco.

Keyword

Canine influenza H3N2; Lactuca sativa; Nicotiana benthamiana; Plant-made vaccine; Viral vector

MeSH Terms

Base Sequence
Clone Cells
Cloning, Organism
DNA
Emergencies
Endoplasmic Reticulum
Hemagglutinins*
Humans
Influenza, Human*
Lettuce*
Orthomyxoviridae*
Plant Leaves
Plants
Protein Sorting Signals
Tobacco*
DNA
Hemagglutinins
Protein Sorting Signals

Figure

• 

  Fig. 1 Expression of GFP and rHA in tobacco and lettuce. (A, B) Visualization of GFP expression by geminiviral vector system in tobacco (A), red lettuce and green lettuce (B) using ultra violet light. (C) Immunodetection of rHA in tobacco and lettuce leaves being agroinfiltrated with HA in geminiviral vector. (D) Quantification of the rHA yield in tobacco. Western blot analyses were performed using polyclonal anti-H3N2 HA antibody. ‘Housekeeping protein’ Rubisco, detected by Coomassie staining analysis, is shown as a loading control. N, Nicotiana benthamiana leaves; L, Lactuca sativa leaves; rHA, recombinant hemagglutinin; TSP, total soluble protein; cHA, commercial hemagglutinin (H3N2) of partial segment; GFP, green fluorescent protein.

• 

  Fig. 2 Expression of complex-glycosylated and basal-glycosylated recombinant hemagglutinin in lettuce. (A) Schematic representation of portion of expression cassette of pBYR11ek2Md flanking the cloned canine HA (H3N2) with N-SP at N-terminal (upper) or KEDL at C-terminal (lower). (B, C) Immunodetection of complex-glycosylated and basal-glycosylated rHA in leaves agroinfiltrated with HA in geminiviral vector into tobacco (B) and lettuce (C). (D) Predicted N-glycosylation site (N-Glyc) in HA sequence. N-Glyc results column output “+” represents potential > 0.5 and output “++” represents the potential > 0.5 and the jury agreement (9/9) where the potential score is the average of the output of the nine neural network and default threshold is 0.5. Western blot analyses were performed using polyclonal anti-H3N2 HA antibody. ‘Housekeeping protein’ RuBisCo, detected by Coomassie staining analysis, is shown as a loading control. N-SP, native signal peptide; rHA, recombinant hemagglutinin; HA, hemagglutinin; UTR, untranslated region; LIR, long intergenic region; SIR, short intergenic region; 35S, cauliflower mosaic virus (CaMV) 35S promoter; 3′ MAR, 3′ elements of tobacco Rb7 chromatin matrix attachment region.

Reference

1. Crawford PC, Dubovi EJ, Castleman WL, et al. Transmission of equine influenza virus to dogs. Science. 2005; 310:482–485.

2. Song D, Kang B, Lee C, et al. Transmission of avian influenza virus (H3N2) to dogs. Emerg Infect Dis. 2008; 14:741–746.

3. Center for Disease Control and Prevention. Key facts about canine influenza (dog flu). Atlanta, GA: Center for Disease Control and Prevention;2019.

4. Song DS, An DJ, Moon HJ, et al. Interspecies transmission of the canine influenza H3N2 virus to domestic cats in South Korea, 2010. J Gen Virol. 2011; 92(Pt 10):2350–2355.

5. Kim H, Song D, Moon H, et al. Inter- and intraspecies transmission of canine influenza virus (H3N2) in dogs, cats, and ferrets. Influenza Other Respir Viruses. 2013; 7:265–270.

6. Lyoo KS, Kim JK, Kang B, et al. Comparative analysis of virulence of a novel, avian-origin H3N2 canine influenza virus in various host species. Virus Res. 2015; 195:135–140.

7. Sohrab SS, Suhail M, Kamal MA, Husen A, Azhar EI. Recent development and future prospects of plant-based vaccines. Curr Drug Metab. 2017; 18:831–841.

8. Pillet S, Aubin E, Trepanier S, et al. A plant-derived quadrivalent virus like particle influenza vaccine induces cross-reactive antibody and T cell response in healthy adults. Clin Immunol. 2016; 168:72–87.

9. Takeyama N, Kiyono H, Yuki Y. Plant-based vaccines for animals and humans: recent advances in technology and clinical trials. Ther Adv Vaccines. 2015; 3:139–154.

10. Medicago. Pipeline. Quebec City: Medicago;2019.

11. Mappbio. Product development. San Diego, CA: Mapp Biopharmaceautical;2019.

12. Skehel JJ, Wiley DC. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem. 2000; 69:531–569.

13. Grgacic EV, Anderson DA. Virus-like particles: passport to immune recognition. Methods. 2006; 40:60–65.

14. D'Aoust MA, Lavoie PO, Couture MM, et al. Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice. Plant Biotechnol J. 2008; 6:930–940.

15. Cummings JF, Guerrero ML, Moon JE, et al. Safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza A (H1N1)pdm09 virus: a Phase 1 dose-escalation study in healthy adults. Vaccine. 2014; 32:2251–2259.

16. Chichester JA, Jones RM, Green BJ, et al. Safety and immunogenicity of a plant-produced recombinant hemagglutinin-based influenza vaccine (HAI-05) derived from A/Indonesia/05/2005 (H5N1) influenza virus: a phase 1 randomized, double-blind, placebo-controlled, dose-escalation study in healthy adults. Viruses. 2012; 4:3227–3244.

17. Shoji Y, Farrance CE, Bautista J, et al. A plant-based system for rapid production of influenza vaccine antigens. Influenza Other Respir Viruses. 2012; 6:204–210.

18. Landry N, Pillet S, Favre D, et al. Influenza virus-like particle vaccines made in Nicotiana benthamiana elicit durable, poly-functional and cross-reactive T cell responses to influenza HA antigens. Clin Immunol. 2014; 154:164–177.

19. Diamos AG, Rosenthal SH, Mason HS. 5′ and 3′ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves. Front Plant Sci. 2016; 7:200.

20. Huang Z, Chen Q, Hjelm B, Arntzen C, Mason H. A DNA replicon system for rapid high-level production of virus-like particles in plants. Biotechnol Bioeng. 2009; 103:706–714.

21. Huang Z, Phoolcharoen W, Lai H, et al. High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnol Bioeng. 2010; 106:9–17.

22. Kapusta J, Modelska A, Figlerowicz M, et al. A plant-derived edible vaccine against hepatitis B virus. FASEB J. 1999; 13:1796–1799.

23. Pniewski T, Kapusta J, Bociag P, et al. Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation. J Appl Genet. 2011; 52:125–136.

24. Govea-Alonso DO, Rubio-Infante N, Garcia-Hernandez AL, et al. Immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic HIV protein. Planta. 2013; 238:785–792.

25. Lai H, He J, Engle M, Diamond MS, Chen Q. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce. Plant Biotechnol J. 2012; 10:95–104.

26. Santi L, Batchelor L, Huang Z, et al. An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles. Vaccine. 2008; 26:1846–1854.

27. Zost SJ, Parkhouse K, Gumina ME, et al. Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains. Proc Natl Acad Sci U S A. 2017; 114:12578–12583.

28. An Y, Parsons LM, Jankowska E, Melnyk D, Joshi M, Cipollo JF. N-glycosylation of seasonal influenza vaccine hemagglutinins: implication for potency testing and immune processing. J Virol. 2019; 93:e01693–e01618.

29. Gupta R, Jung E, Brunak S. Prediction of N-glycosylation sites in human proteins. NetNGlyc 1.0 Server. Lyngby: DTU Bioinformatics;2004.

30. Kalthoff D, Giritch A, Geisler K, et al. Immunization with plant-expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus H5N1 challenge infection. J Virol. 2010; 84:12002–12010.

31. Phoolcharoen W, Dye JM, Kilbourne J, et al. A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge. Proc Natl Acad Sci U S A. 2011; 108:20695–20700.

32. Chen Q, He J, Phoolcharoen W, Mason HS. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum Vaccin. 2011; 7:331–338.

33. Kwon KC, Nityanandam R, New JS, Daniell H. Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells. Plant Biotechnol J. 2013; 11:77–86.

34. D'Aoust MA, Couture MM, Charland N, et al. The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza. Plant Biotechnol J. 2010; 8:607–619.

35. Future vaccine DARPA research. Arlingotn, VA: Defense Advanced Research Projects Agency;2012.