Tarlan MAMEDOV, Gülshan MAMMADOVA, Nedim MUTLU, Nilüfer GUN, İrem GÜRBÜZASLAN, Damla YÜKSEL, Gülnara HASANOVA

Engineering, production, and immunogenicity studies of a truncated form of rabies virus glycoprotein produced in Nicotiana benthamiana plant

Rabies is a viral disease caused by the rabies virus (Lyssavirus) that affects the central nervous system, ultimately leading to death and brain disease. Rabies usually transmitted through a bite among domestic dogs and wild carnivorous animals. The rabies virus (RABV) infects the central nervous system causing disease in the brain and death. Rabies is 100% fatal if untreated and ultimately causing more than 70,000 deaths annually worldwide, especially among children in developing countries. Rabies can be prevented by vaccination and can be cured immediately after infection. Although several human and animal vaccines against rabies are available, they are expensive, have relatively low immunogenicity and also difficult to produce. Therefore, less expensive, more immunogenic and safe rabies vaccines that can be produced in the large quantities are urgently needed. The transient plant expression system has become a more attractive and promising platform for the expression of a wide range of recombinant proteins such as vaccines, antibodies, therapeutic proteins, including mammalian complex proteins. In this study, we engineered and produced a truncated form of glycoprotein (G protein) of rabies virus in Nicotiana benthamiana (N. benthamiana) plant for the first time, using transient expression system. Plant produced RG2 protein was purified through Ni-NTA column chromatography. The purification yield of RG2 protein was ~32 mg/ kg plant leaf. The results of immunogenicity studies showed that the plant produced G-protein elicited significantly high antibody titers in mice. Plant-produced G-protein could be a cost-effective, safe and highly immunogenic rabies vaccine candidate.

PDF

___

1. Dietzschold B, Schnell M, Koprowski H. Pathogenesis of rabies. Curr Top Microbiol Immunol. 2005;292:45-56.
2. Knobel DL, Cleaveland S, Coleman PG, et al. Re-evaluating the burden of rabies in Africa and Asia. Bull World Health Organ. 2005;83:360-8.
3. Dhingra V, Li X, Liu Y, Fu ZF. Proteomic profiling reveals that rabies virus infection results in differential expression of host proteins involved in ion homeostasis and synaptic physiology in the central nervous system. J Neurovirol. 2007;13:107-17.
4. Gillet JP, Derer P, Tsiang H. Axonal transport of rabies virus in the central nervous system of the rat. J Neuropathol Exp Neurol. 1986;45:619-34.
5. Davis BM, Rall GF, Schnell MJ. Everything You Always Wanted to Know About Rabies Virus (But Were Afraid to Ask). Annu Rev Virol. 2015;2:451-71.
6. Fletcher MA , Hessel L, Plotkin SA. Human diploid cell strains (HDCS) viral vaccines. Developments in Biological Standardization. 1998;93:97-107.
7. Dhankhar P, Vaidya SA, Fishbien DB, Meltzer MI. Cost effectiveness of rabies post exposure prophylaxis in the United States. Vaccine. 2008;26: 4251–5.
8. Freuling C, Selhorst T, Batza HJ, Muller T. The financial challenge of keeping a large region rabies-free– the EU example. Dev Biol. 2008;131:273–282.
9. Schnell MJ, McGettigan JP, Wirblich C, Papaneri A. The cell biology of rabies virus: using stealth to reach the brain. Nat Rev Microbiol. 2010;8:51-61.
10. Foley HD, McGettigan JP, Siler CA, Dietzschold B, Schnell MJ. A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection. Proc Natl Acad Sci USA. 2000;97:14680-5.
11. Macfarlan RI, Dietzschold B, Koprowski H. Stimulation of cytotoxic T-lymphocyte responses by rabies virus glycoprotein and identification of an immunodominant domain. Mol Immunol. 1986;23:733-41.
12. Gaudin Y, Ruigrok RW, Tuffereau C, Knossow M, Flamand A. Rabies virus glycoprotein is a trimer. Virology. 1992; 187:627–32.
13. Prehaud C, Takehara K, Flamand A, Bishop DH. Immunogenic and protective properties of rabies virus glycoprotein expressed by baculovirus vectors. Virology. 1989;173:390-9.
14. Ashraf S, Singh PK, Yadav DK, et al. High level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice. J Biotechnol. 2005;119:1-14.
15. Yokomizo AY, Jorge SA, Astray RM, et al. Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co-transfected cell line. Biotechnol J. 2007;2:102-9.
16. Huang H, Xiao S, Qin J, et al. Construction and immunogenicity of a recombinant pseudotype baculovirus expressing the glycoprotein of rabies virus in mice. Arch Virol. 2011;156:753-8.
17. Ramya R, Mohana Subramanian B, Sivakumar V, et al. Expression and solubilization of insect cell-based rabies virus glycoprotein and assessment of its immunogenicity and protective efficacy in mice. Clin Vaccine Immunol. 2011;18:1673-9.
18. Koraka P, Bosch BJ, Cox M, et al. A recombinant rabies vaccine expressing the trimeric form of the glycoprotein confers enhanced immunogenicity and protection in outbred mice. Vaccine. 2014;32:4644-50.
19. Yang DK, Nakagawa K, Ito N, et al. A single immunization with recombinant rabies virus (ERAG3G) confers complete protection against rabies in mice, Clin Exp Vaccine Res. 2014;3:176–184.
20. McGettigan JP, David F, Figueiredo MD, et al. Safety and serological response to a matrix gene-deleted rabies virus-based vaccine vector in dogs. Vaccine. 2014;32:1716-9.
21. Huang F, Ahmad W, Duan M, et al. Efficiency of live attenuated and inactivated rabies viruses in prophylactic and post exposure vaccination against the street virus strain. Acta Virologica. 2015;59:117–24.
22. Klimyuk V, Pogue G, Herz S, e at. Production of recombinant antigens and antibodies in Nicotiana benthamiana using 'magnifection' technology: GMP-compliant facilities for small- and large-scale manufacturing. Curr Top Microbiol Immunol. 2014;375:127-54.
23. Mamedov T, Musayeva I, Acsora R, et al. Engineering, and production of functionally active human Furin in N. benthamiana plant: In vivo posttranslational processing of target proteins by Furin in plants. PLoS One. 2019;14:e0213438.
24. Mamedov T, Cicek K, Miura K, et al. Plant produced in vivo deglycosylated full-length Pfs48/45 as a transmission-blocking vaccine candidate against malaria. Sci. Rep. 2019;9-9868.
25. Mamedov T, Yuksel D, Ilgin M et al. Plant-Produced Glycosylated and In vivo Deglycosylated Receptor Binding Domain Proteins of SARS-CoV-2 Induce Potent Neutralizing Responses in Mice. Viruses. 2021;13:1595.
26. Mamedov T, Cicek K, Gulec B, et al. In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus. PLoS One. 2017;12:e0183589.
27. Mamedov T, Ghosh A, Jones RM, Mett V et al. Production of nonglycosylated recombinant proteins in Nicotiana benthamiana plants by coexpressing bacterial PNGase F. Plant Biotechnol. J. 2012;10:773-82.
28. McGarvey PB, Hammond J, Dienelt MM, et al. Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnology. 1995;13:1484-7.
29. Loza-Rubio E, Rojas-Anaya E, López J, et al. Induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein. Vaccine. 2012;30:5551-6.
30. Sainsbury F, Thuenemann EC, Lomonossoff GP. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J. 2009;7;682–93.
31. Starodubova ES, Kuzmenko YV, Pankova EO, et al. Rabies Virus Glycoprotein with a Consensus Amino Acid Sequence and a Lysosome Targeting Signal Causes Effective Production of Antibodies in DNAImmunized Mice. Mol Biol. 2018;52:314-317.
32. Rupprecht CE, Nagarajan T, Ert H. Current Status and Development of Vaccines and Other Biologics for Human Rabies Prevention. Expert Rev Vaccines. 2016;15:731-49.
33. Margolin E, Oh YJ, Verbeek M, et al. Co-expression of human calreticulin significantly improves the production of HIV gp140 and other viral glycoproteins in plants. Plant Biotechnol J. 2020;18;2109–117.
34. Siriwattananon K, Manopwisedjaroen S, Shanmugaraj B, et al. PlantProduced Receptor-Binding Domain of SARS-CoV-2 Elicits Potent Neutralizing Responses in Mice and Non-human Primates. Front Plant Sci. 2021;13:682953.
35. Fleysh N, Deka D, Drath M, et al. Pathogenesis of Alfalfa mosaic virus in soybean (Glycine max) and expression of chimeric rabies peptide in virusinfected soybean plants. Phytopathology. 2001;91:941–7.
36. Roy S, Tyagi A, Tiwari S, et al. Rabies glycoprotein fused with B subunit of cholera toxin expressed in tobacco plants folds into biologically active pentameric protein. Protein Expr Purif. 2010;70:184-90.
37. Singh A, Srivastava S, Chouksey A, et al. Expression of rabies glycoprotein and ricin toxin B chain (RGP-RTB) fusion protein in tomato hairy roots: a step towards oral vaccination rabies. Mol Biotechnol. 2015;57:359–70.
38. Messeguer J. Gene flow assessment in transgenic plants. PCTOC. 2003;73:201–12.
39. Park Y, Kang H, Min K, et al. Rabies virus glycoprotein produced in Nicotiana benthamiana is an immunogenic antigen in mice. Czech J. Genet. Plant Breed. 2021;57:26–35.
40. Stronge VS, Saito Y, Ihara Y, Williams DB. Relationship between calnexin and BiP in suppressing aggregation and promoting refolding of protein and glycoprotein substrates. J Biol Chem. 2001;276:39779–87.