Decreased neutralizing antigenicity in IBV S1 protein expressed from mammalian cells

Abstract

We evaluated the antigenicity of recombinant infectious bronchitis virus (IBV) S1 protein expressed in mammalian cells. Recombinant S1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using Ni Sepharose. The purified protein was analyzed by Western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. Six weeks after immunization, anti-IBV neutralizing titer and anti-S1 ELISA titer were determined; immunized chickens then were inoculated with IBV via the trachea and ciliary activity was observed. Results showed that the recombinant S1 protein was highly glycosylated, and the neutralizing antigenicity of recombinant S1 protein was lower than that of inactivated virus. However, anti-S1 ELISA indicated that the recombinant S1 protein induced antibodies against S1. These results suggest that the recombinant S1 may retain non-neutralizing epitopes but have unnatural glycosylation pattern and conformation, resulting in lacking neutralizing conformational epitopes. In conclusion, the neutralizing antigenicity of recombinant S1 protein expressed from mammalian cells was decreased, and was not sufficient to induce neutralizing antibodies.

Keywords: Conformational epitope; Infectious bronchitis virus; Neutralizing antibody; S1; Spike protein.

Fig. 1

Schematics of the IBV S1 segment expressed as a secreted protein. To construct the secreted S1 expression plasmid, the nucleotide sequence encoding the S1 domain lacking the cleavage sequence (RRFRR) was amplified from the IBV TM86 genome. To increase expression levels of the recombinant S1 protein, the nucleotide sequence encoding the signal sequence was replaced with a nucleotide sequence encoding MDV gA. To facilitate purification and multimerization, the resulting sequence was cloned upstream and in-phase with nucleotide sequences permitting expression as C-terminal fusions to a 6× His-tag peptide sequence with or without a T4 phage fibritin coiled-coil trimerization motif.

Fig. 2

Western blotting analysis of the recombinant S1 protein with (+) and without (−) fibritin motif expressed in 293T and 293 GnTI⁻ cells. IBV derived from chicken embryonated eggs was included as a positive control. SDS-PAGE was carried out under denaturing conditions.

Fig. 3

Western blotting analysis of the recombinant S1 protein with fibritin motif expressed in 293T (lane 1), 293 GnTI⁻ (lane 2), and CEF cells (lane 3). IBV derived from chicken embryonated eggs was included as a positive control (lane 4). SDS-PAGE was carried out under denaturing conditions.

Fig. 4

Western blotting analysis of the recombinant S1 protein derived from 293T cells with (+) and without (−) fibritin motif. Recombinant proteins were purified using Ni Sepharose and SDS-PAGE was carried out under non-denaturing conditions.

Fig. 5

Anti-IBV viral neutralization (VN) titer of immunized chickens. Sera were collected at 6 weeks post-immunization. Sera were serially diluted two-fold with EMEM in microplates, then mixed with 200 TCID₅₀ of IBV. VN titer was defined as the reciprocal of the highest dilution showing no cytopathic effect (CPE). The asterisk indicates statistical significance (P < 0.05) and the error bar indicates standard deviation. Comparison between two groups was performed using t test.

Fig. 6

Indirect sandwich ELISA using recombinant S1 protein (a) and the lysate from IBV-infected cells (b). Primary sera were collected from immunized chickens and diluted 1:100. In recombinant S1 ELISA, primary sera were pre-adsorbed with purified recombinant His-tagged protein to remove the antibodies against the His-tag peptide sequence. Reactivities are shown as the S/P ratio of absorbance at 450 nm and 650 nm. Comparisons between three groups were performed using steel-Dwass test and n.s. indicated not significant.