Development of diagnostic tools for IBDV detection using plants as bioreactors

Abstract

Infectious bursal disease virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds, thus causing important economic losses in the poultry industry. Multimeric particles with different architectures based on the capsid protein VP2 have been widely produced for different purposes. We hereby show the production and easy recovery of IBDV subviral particles (SVP) from transiently transformed Nicotiana benthamiana. The SVP, which were observed by electronic microscopy, proved to be antigenically and immunogenically similar to the virion. Indeed, anti-IBDV antibodies from samples of infected birds recognized these SVP and, when injected intramuscularly, these subviral particles also evoked a humoral immune response in chickens. We developed an in-house ELISA using SVP as coating reagent that demonstrated to be highly accurate and in good agreement with a commercial ELISA. This study demonstrates that the recombinant antigen generated and the technology used to produce it are suitable for developing a diagnostic tool against Infectious bursal disease.

The expression in plants of an engineered VP2 protein of Infectious Bursal Disease Virus induces formation of structurally heterogeneous particles that protect from a very virulent viral strain

“…Although the assembly of the VP2 in capsid-like particles (mainly T = 1, T = 4 and T = 13 symmetries) or tubular structures in eukaryotic expression systems (yeast and insect cells) has been described [ 16 – 18 ], the formation of SVP in plants was only recently described and these structures used to set up a diagnostic assay [ 23 ].…”

“…The plant-produced monomeric VP2 was able to elicit protective IBDV-specific humoral responses in chickens [ 20 – 22 ]. Recently a paper was published showing also the assembly in plants of the VP2 in SVP and the use of these structures to develop a diagnostic tool [ 23 ]. Nonetheless, the ability of plant-produced VP2-based structures to induce protection against IBDV infection has never been demonstrated [ 20 , 24 – 26 ].…”

Oral Immunization With Plant-Based Vaccine Induces a Protective Response Against Infectious Bursal Disease

“…This suggests that, although the antigen is not bioencapsulated by plant cell wall, since our formulation consists of extracted proteins, VP2 is able to resist degradation in the digestive tract of chicken until it is taken by M cells which allows it to reach immunocompetent cells in the gut-associated lymphoid tissue (Taghavian et al, 2013). This resistance in the GIT might be explained by the fact that, as recently demonstrated by us and other authors, VP2 is able to correctly self-assemble into IBD-SVP in plant cells (Gómez et al, 2020;Marusic et al, 2021) that are very stable under harsh conditions (Taghavian et al, 2012;Gómez et al, 2020).…”

“…Transient expression was performed by infiltrating 5- to 6-week-old greenhouse-grown Nicotiana benthamiana leaves with a suspension of recombinant Agrobacterium tumefaciens strain, GV3101 harboring pEAQ-VP2 vector as previously described ( Lucero et al, 2019 ; Gómez et al, 2020 ). Briefly, the recombinant bacteria were cultured in Luria–Bertani medium containing 100 μg/mL Kanamycin, 100 μg/mL of Rifampicin, and 50 μg/mL of Gentamicin for 32 h at 28°C, pelleted and resuspended in an infiltration solution [10 mM of morpholinoethanesulfonic acid (MES), pH 5.5; 10 mM MgSO 4 , and 100 μM acetosyringone] to an OD 600 of 0.8–1.…”

“…Sera were tested for specific anti-VP2 antibodies using an indirect in-house ELISA based on IBDV SVP (Gómez et al, 2020). Briefly, 96-well MaxiSorp TM Nunc TM flat-bottom plates (Thermo Fischer Scientific, MA, United States) were coated with 95 ng of SVP per well in 0.1 M carbonate-bicarbonate buffer, with pH of 9.6, overnight at 4 • C. After blocking with 4% skimmed milk in PBS-T (0.05% Tween 20), the plates were subsequently incubated with a 1:400 dilution of sample sera, washed and incubated again with a 1:4000 dilution of goat antichicken IgG antibodies coupled to horseradish peroxidase (Bethyl Laboratories, United States).…”