Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus

Lv, Lishan; Li, Xiaoming; Liu, Genmei; Li, Ran; Liu, Qiliang; Shen, Huifang; Wang, Wei; Xue, Chunyi; Cao, Yongchang

The fusion protein NA/S1 was composed of S1 protein (521 amino acids without signal peptide) of H120 S protein and the CT and TM domain (40 amino acids) of the NA Fig. 1 . Fusion protein NA/S1 and protein expression in sf9 cells. (A) The construct of fusion protein NA/S1. S1 protein (521 amino acids) was fused to the CT and TM domain of the NA protein of H5N1. (B) Western blot analysis of the expression of fusion protein NA/S1 in sf9 cells. Sf9 cell were infected with rBV-N/S1 at a multiplicity of infection (MOI) of 5, after which the cell lysates were separated by SDS-PAGE and probed with S1-specific antibodies. One clear band was observed with a molecular mass of 100 KD, which corresponds to mass estimated according to its amino acid composition. (C) Western blot analysis of the expression of M1 protein in sf9 cells. Sf9 cells were infected by rBV-M1 at a MOI of 5, after which the cell lysates were separated by SDS-PAGE and probed with M1-specific antibodies. One band of 25 KD was shown to correspond to M1 molecular weight. NC: negative control. Fig. 2 . Incorporation of fusion protein NA/S1 into influenza VLPs. Chimeric. (A) Indirect immunofluorescence staining of NA/S1 and M1. Sf9 cells were co-infected with both rBV-NA/S1 and rBV-M1 at an MOI of 5; the control cells were infected by wt BV at an MOI of 5; the infected cells were fixed and stained by antibodies as described in the Materials and Methods. NA/S1 was stained by FITC (green); M1 protein by CY3 (red); and the nucleus by DAPI (blue). Merge indicates the combination of the three pictures. Magnifications of 1,000× were used. (B) NA/S1 and M1 proteins in the purified chimeric VLPs. Sf9 cells were co-infected with rBV-NA/S1 and rBV-M1 using different combinations of MOIs to give ratios of 1 : 1, 1 : 5, 1 : 10, 5 : 5, 5 : 1 and 10 : 1. Infection with wt BV was used as NC. The supernatants were then collected and purified by discontinuous sucrose step density gradient centrifugation. The 40∼50% fraction was collected and analyzed by western blot for detection of NA/S1 and M1. (C) Negative staining electron microscopy of chimeric VLPs composed of NA/S1 fusion and M1 proteins. Scale Bar = 100 nm. Fig. 3 . Serum IgG elicited by chimeric VLPs and inactivated H120 in mice. Six-week-old BALB/c mice were vaccinated via intramuscular injection at weeks 0, 2 and 4 with chimeric VLPs, H120 or PBS. Serum for each group was tested for IgG antibody by indirect ELISA. Pre-immune sera from mice were diluted 400 times and analyzed, and no detectable specific anti-H120 antibodies were observed. Optical densities were read at 450 nm. Bars represent the arithmetic mean antibody titers ± standard errors (SE). Fig. 4 . ELISPOT assays of IL-4 and IFN-γ cytokine-secreting splenocytes in mice. Splenocytes were isolated from immunized mice two weeks after the final immunization and re-stimulated by inactivated IBV H120 viruses, while cytokine-secreting cells were determined by ELISPOT assays. The spots for cytokine-producing cells from the spleen were counted and expressed based on 5 × 10 5 cells per well.