Protective efficacy of crude virus-like particle vaccine against HPAI H5N1 in chickens and its application on DIVA strategy

Abstract

Background: Currently, Asian lineage highly pathogenic avian influenza (HPAI) H5N1 has become widespread across continents. These viruses are persistently circulating among poultry populations in endemic regions, causing huge economic losses, and raising concerns about an H5N1 pandemic. To control HPAI H5N1, effective vaccines for poultry are urgently needed.

Objective: In this study, we developed HPAI virus-like particle (VLP) vaccine as a candidate poultry vaccine and evaluated its protective efficacy and possible application for differentiating infected from vaccinated animals (DIVA).

Methods: Specific pathogen-free chickens received a single injection of HPAI H5N1 VLP vaccine generated using baculovirus expression vector system. Immunogenicity of VLP vaccines was determined using hemagglutination inhibition (HI), neuraminidase inhibition (NI), and ELISA test. Challenge study was performed to evaluate efficacy of VLP vaccines.

Results and conclusions: A single immunization with HPAI H5N1 VLP vaccine induced high levels of HI and NI antibodies and protected chickens from a lethal challenge of wild-type HPAI H5N1 virus. Viral excretion from the vaccinated and challenged group was strongly reduced compared with a mock-vaccinated control group. Furthermore, we were able to differentiate VLP-vaccinated chickens from vaccinated and then infected chickens with a commercial ELISA test kit, which offers a promising strategy for the application of DIVA concept.

Figure 1

Identification of proteins in A/chicken/Korea/ES/2003(H5N1) VLPs. (A) Coomassie‐stained SDS‐PAGE analysis of VLPs expressing HA, NA, and M1. M, a standard molecular size marker (in kilodaltons); Lane 1, 2¹⁰ HAU of VLP protein; Lane 2, 2⁹ HAU of VLP protein; Lane 3, 2⁸ HAU of VLP protein. (B) Analysis of VLPs by Western blot using mouse anti‐H5 monoclonal antibody and rabbit polyclonal antibodies for N1 or M1. Molecular weights of expressed HA, NA, and M1 are indicated on the left.

Figure 2

Mean serum hemagglutination inhibition (HI) titers (log2) induced in specific pathogen‐free (SPF) chickens after a single dose of virus‐like particle (VLP) vaccine with ISA70 adjuvant. A total of 32 five‐week‐old SPF chickens (8 per group) were intramuscularly immunized with HPAI H5N1 VLP vaccines. HI titers against the homologous antigen (A) A/chicken/Korea/ES/2003 (clade 2·5) and heterologous antigen (B) A/Indonesia/5/2005 (clade 2·1) and (C) A/Vietnam/1194/2004 (clade 1) were determined 3 weeks after vaccination. Mock – emulsified solution of conditioned SF900III medium with ISA70. *P < 0·05 and ***P < 0·001 by anova with Tukey–Kramer post‐test compared with other groups.

Figure 3

Mean serum neuraminidase inhibition (NI) titers (log2) induced in specific pathogen‐free (SPF) chickens after a single dose of virus‐like particle (VLP) vaccine with ISA70 adjuvant. A total of 32 five‐week‐old SPF chickens (8 per group) were intramuscularly immunized with HPAI H5N1 VLP vaccines. NI titers against the homologous antigen were determined 3 weeks after vaccination. Mock – emulsified solution of conditioned SF900III medium with ISA70. ***P < 0·001 by anova with Tukey–Kramer post‐test compared with other groups.

Figure 4

Differentiation of vaccinated chickens from vaccinated and then infected chickens and changes in hemagglutination inhibition (HI) titers following infection. Serum samples were taken 3 weeks post‐vaccination from 2⁸ and 2¹⁰ VLP‐vaccinated chickens. 10 days post‐challenge (dpc) infection, serum samples were taken from 2⁸ and 2¹⁰ VLP‐vaccinated and then infected chickens. (A) NP antibody levels from each serum samples were tested with commercially available NP‐cELISA Kit. Each dot represents the NP‐specific antibody value of each chicken. (B) Hemagglutination inhibition (HI) titers (log2) against homologous antigen at 10 dpc were compared with pre‐challenge sera. Data shown are the meant titers of each group ± standard deviation.