J Vet Sci.  2014 Jun;15(2):209-216. 10.4142/jvs.2014.15.2.209.

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

• Lv L
• Li X
• Liu G
• Li R
• Liu Q
• Shen H
• Wang W
• Xue C
• Cao Y

• KMID: 1784641
• DOI: http://doi.org/10.4142/jvs.2014.15.2.209

Abstract

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

Keyword

chimeric virus-like particle; immune response; infectious bronchitis virus

MeSH Terms

Animals
Antibodies, Viral/blood
*Chickens
Chimera/genetics/immunology
Coronavirus Infections/prevention & control/*veterinary/virology
Female
*Immunity, Innate
Infectious bronchitis virus/genetics/*immunology
Influenza A Virus, H5N1 Subtype/genetics/immunology
Injections, Intramuscular/veterinary
Mice
Mice, Inbred BALB C
Neuraminidase/genetics
Poultry Diseases/*prevention & control/virology
Recombinant Fusion Proteins/genetics/immunology
Spike Glycoprotein, Coronavirus/genetics/*immunology
Vaccines, Synthetic/administration & dosage/genetics/immunology
Vaccines, Virus-Like Particle/administration & dosage/genetics/*immunology
Viral Proteins/genetics
Antibodies, Viral
Recombinant Fusion Proteins
Neuraminidase
Spike Glycoprotein, Coronavirus
Vaccines, Synthetic
Vaccines, Virus-Like Particle
Viral Proteins

Figure

• 

  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 × 105 cells per well.

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  Fig. 5 IBV-specific neutralization antibody titers in immunized mice. Serum samples were collected on the 14th day after the third immunization to determine neutralization antibody titers. Data represent the mean ± SD of six mice.

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  Fig. 6 Serum S1-specific IgG antibodies. Ten-day-old SPF chickens were intramuscularly immunized twice at 2-week intervals with chimeric VLPs, H120 or PBS. Sera were tested for S1-specific IgG antibodies by ELISA. Pre-immune sera from SPF chickens had no detectable S1-specific antibodies. Optical densities were read at 450 nm. Bars represent the arithmetic mean antibody titers ± SE.

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  Fig. 7 IBV-specific neutralization antibody titers in immunized SPF chickens. Serum samples from SPF chickens were collected on the 28th day post immunization. 100× TCID50 IBV H120 was mixed with serial diluted sera, after which the mixtures were inoculated into 9-day-old SPF embryonated chicken eggs. The neutralization titer was then expressed as the reciprocal of the highest serum dilution that exhibited neutralization in 50% of the eggs.

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