Construction and Immunogenicity Comparison of Three Virus-Like Particles Carrying Different Combinations of Structural Proteins of Avian Coronavirus Infectious Bronchitis Virus

Abstract

Infectious bronchitis virus (IBV) poses massive economic losses in the global poultry industry. Here, we firstly report the construction and immunogenicity comparison of virus-like particles (VLPs) carrying the S, M and E proteins (SME-VLPs); VLPs carrying the S and M proteins (SM-VLPs); and VLPs carrying the M and E proteins (ME-VLPs) from the dominant serotype representative strain GX-YL5 in China. The neutralizing antibody response induced by the SME-VLPs was similar to that induced by the inactivated oil vaccine (OEV) of GX-YL5, and higher than those induced by the SM-VLPs, ME-VLPs and commercial live vaccine H120. More importantly, the SME-VLPs elicited higher percentages of CD4⁺ and CD8⁺ T lymphocytes than the SM-VLPs, ME-VLPs and OEV of GX-YL5. Compared with the OEV of GX-YL5, higher levels of IL-4 and IFN-γ were also induced by the SME-VLPs. Moreover, the mucosal immune response (sIgA) induced by the SME-VLPs in the tear and oral swabs was comparable to that induced by the H120 vaccine and higher than that induced by the OEV of GX-YL5. In the challenge experiment, the SME-VLPs resulted in significantly lower viral RNA levels in the trachea and higher protection scores than the OEV of GX-YL5 and H120 vaccines, and induced comparable viral RNA levels in the kidneys, and tear and oral swabs to the OEV of GX-YL5. In summary, among the three VLPs, the SME-VLPs carrying the S, M and E proteins of IBV could stimulate the strongest humoral, cellular and mucosal immune responses and provide effective protection, indicating that it would be an attractive vaccine candidate for IB.

Keywords: construction; immunogenicity; infectious bronchitis virus; virus-like particles.

Figure 1

Immunofluorescent staining analysis of M, S and E protein expression in the recombinant baculovirus-infected Sf9 cells at 60 h post-infection (200×). (A) Sf9 cells infected with recombinant baculovirus rHBM-S. (B) Sf9 cells infected with recombinant baculovirus rHBM-M. (C) Sf9 cells infected with recombinant baculovirus rHBM-E. (D) Sf9 cells infected with wild-type baculovirus. (E) Sf9 cells. FITC was antibody conjugated (green); DAPI was used to stain cell nuclei (blue), and merging signified that FITC merged with DAPI.

Figure 2

Identification of the recombinant proteins’ expression using Western blotting. (A) The S protein expressed by recombinant baculovirus rHBM-S. (B) The M protein expressed by recombinant baculovirus rHBM-M. (C) The E protein expressed by recombinant baculovirus rHBM-E. Lane M, protein molecular weight marker; Lane 1, culture supernatant; Lane 2, cell lysate; Lane 3, wild-type baculovirus; Lane 4, Sf9 cells.

Figure 3

Western blot analysis of virus-like particles (VLPs). (A) Western blot analysis of SME-VLPs purified from culture supernatants of cells co-infected with rHBM-S, rHBM-M and rHBM-E. (B) Western blot analysis of SM-VLPs purified from culture supernatants of cells co-infected with rHBM-S and rHBM-M. (C) Western blot analysis of ME-VLPs purified from culture supernatants of cells co-infected with rHBM-M and rHBM-E. Lane M, protein molecular weight marker; Lane 1, purified VLPs containing S, M and E proteins; Lane 4, purified VLPs containing S and M proteins; Lane 5, purified VLPs containing M and E proteins; Lanes 2, 3 and 6, wild-type baculovirus.

Figure 4

The formation of infectious bronchitis virus (IBV) VLPs identified by TEM. (A) Purified SME-VLPs. (B) Purified SM-VLPs. (C) Purified ME-VLPs. Bar = 100 nm.

Figure 5

Analysis of the presence of S protein in the IBV VLPs by immunoelectron microscopy (IEM). (A) Purified SME-VLPs. (B) Purified SM-VLPs. Bar = 100 nm.

Figure 6

IBV-specific antibody levels in vaccinated chicken sera (0, 7, 14, 21 and 28 days post-vaccination (dpv)) detected by indirect ELISA. Letters (a–c) indicate significant differences among vaccinated groups (p < 0.05). ** and * indicate p < 0.01 and p < 0.05 between vaccinated and nonvaccinated groups, respectively. (n = 10 chickens/group.)

Figure 7

IBV-neutralizing antibodies measured in sera (0, 14 and 28 dpv) by trachea organ ring (TOC) neutralization test. Letters (a,b) indicate significant differences among vaccinated groups (p < 0.05). * indicates p < 0.05 between vaccinated and nonvaccinated groups. (n = 10 chickens/group.)

Figure 8

Percentages of CD3⁺, CD4⁺ and CD8⁺ T lymphocytes in peripheral blood of vaccinated chickens. Peripheral blood lymphocytes were isolated from vaccinated chickens at 0, 7, 14, 21 and 28 dpv and analyzed by flow cytometry. (A) Percentages of CD3⁺ T lymphocytes. (B) Percentages of CD4⁺ T lymphocytes. (C) Percentages of CD8⁺ T lymphocytes. Letters (a–c) indicate significant differences among vaccinated groups (p < 0.05). ** indicates p < 0.01 between vaccinated and nonvaccinated groups. (n = 10 chickens/group.)

Figure 8

Percentages of CD3⁺, CD4⁺ and CD8⁺ T lymphocytes in peripheral blood of vaccinated chickens. Peripheral blood lymphocytes were isolated from vaccinated chickens at 0, 7, 14, 21 and 28 dpv and analyzed by flow cytometry. (A) Percentages of CD3⁺ T lymphocytes. (B) Percentages of CD4⁺ T lymphocytes. (C) Percentages of CD8⁺ T lymphocytes. Letters (a–c) indicate significant differences among vaccinated groups (p < 0.05). ** indicates p < 0.01 between vaccinated and nonvaccinated groups. (n = 10 chickens/group.)

Figure 9

IL-4 and IFN-γ concentrations in the sera of vaccinated chickens at 0, 14 and 28 dpv. (A) IL-4 concentrations. (B) IFN-γ concentrations. Letters (a,b) indicate significant differences among vaccinated groups (p < 0.05). ** indicates p < 0.01 between vaccinated and nonvaccinated groups. (n = 10 chickens/group.)

Figure 10

sIgA concentrations in tear and oral swabs at 0, 7, 14, 21 and 28 dpv. (A) sIgA concentrations in tear swabs. (B) sIgA concentrations in oral swabs. Letters (a,b) indicate significant differences among vaccinated groups (p < 0.05). ** indicates p < 0.01 between vaccinated and nonvaccinated groups. (n = 10 chickens/group.)

Figure 11

Viral loads in tracheas and kidneys. IBV RNA levels in tracheas and kidneys were evaluated at 5 days post-challenge (dpc). (A) Viral loads in tracheas. (B) Viral loads in kidneys. Letters (a–c) indicate significant differences among vaccinated groups (p < 0.05). ** indicates p < 0.01 between vaccinated and nonvaccinated groups. (n = 10 chickens/group.)

Figure 12

Viral loads in tear and oral swabs. IBV RNA levels in tear and oral swabs were evaluated at 2, 4, 6, 8, 10, 12 and 14 dpc. (A) Viral loads in tear swabs. (B) Viral loads in oral swabs. Letters (a,b) indicate significant differences among vaccinated groups (p < 0.05). ** and * indicate p < 0.01 and p < 0.05 between vaccinated and nonvaccinated groups, respectively. (n = 10 chickens/group.)

Figure 13

Ciliostasis protection scores for tracheas in challenged chickens at 5 dpc (n = 10 chickens/group).