A recombinant avian paramyxovirus serotype 3 expressing the hemagglutinin protein protects chickens against H5N1 highly pathogenic avian influenza virus challenge

Abstract

Highly pathogenic avian influenza (HPAI) is a devastating disease of poultry and a serious threat to public health. Vaccination with inactivated virus vaccines has been applied for several years as one of the major policies to control highly pathogenic avian influenza virus (HPAIV) infections in chickens. Viral-vectored HA protein vaccines are a desirable alternative for inactivated vaccines. However, each viral vector possesses its own advantages and disadvantages for the development of a HA-based vaccine against HPAIV. Recombinant Newcastle disease virus (rNDV) strain LaSota expressing HA protein vaccine has shown promising results against HPAIV; however, its replication is restricted only to the respiratory tract. Therefore, we thought to evaluate avian paramyxovirus serotype 3 (APMV-3) strain Netherlands as a safe vaccine vector against HPAIV, which has high efficiency replication in a greater range of host organs. In this study, we generated rAPMV-3 expressing the HA protein of H5N1 HPAIV using reverse genetics and evaluated the induction of neutralizing antibodies and protection by rAPMV3 and rNDV expressing the HA protein against HPAIV challenge in chickens. Our results showed that immunization of chickens with rAPMV-3 or rNDV expressing HA protein provided complete protection against HPAIV challenge. However, immunization of chickens with rAPMV-3 expressing HA protein induced higher level of neutralizing antibodies compared to that of rNDV expressing HA protein. These results suggest that a rAPMV-3 expressing HA protein might be a better vaccine for mass-vaccination of commercial chickens in field conditions.

Figure 1

The schematic diagram for the construct of recombinant avian paramyxovirus serotype 3 (rAPMV-3) containing HA gene of HPAIV. The full-length antigenomic cDNA of APMV-3 strain Netherlands was constructed into a plasmid pBR322 previously. A transcription cassette containing ORF sequence of HA gene of H5N1 HPAIV was flanked between P and M genes of APMV-3. The HA gene transcription cassette contains sequences of following elements arranged in 3ʹ to 5ʹ order; SacII restriction enzyme site, GC nucleotides for rule of six, Kozak, HA gene ORF, GE of APMV-3 P gene that here it serves as GE for HA gene, IGS, GS of APMV-3 M gene and SacII restriction enzyme site. The polybasic cleavage site motifs (PQRERRRKKR’G) of HPAIV strain A/Vietnam/1203/2004 (H5N1) was modified to monobasic cleavage site motifs (PQRETR’G) of LPAIV strain A/Mexico/31381/94 (H5N2).

Figure 2

The expression of HA protein by rAPMV-3 or rNDV and incorporation of HA protein into rAPMV-3 or rNDV particles. The monolayer of DF1 cells were infected with rNDV, rNDV/HA or rAPMV-3/HA at 0.1 MOI. The cell lysates were collected 30 hours after infection. The expression of HA protein was detected by Western blot analysis using a polyclonal chicken anti H5N1 serum. (A) Lanes 1–4 represents; DF1 cells, rNDV, rAPMV-3 HA and rNDV/HA, respectively. The recombinant viruses were inoculated in 10-day-embryonated SPF chicken eggs, the infected allantoic fluids were collected three days post-inoculation and were partially purified. The incorporation of HA protein into rAPMV-3 or rNDV particles were detected by Western blot analysis using the mentioned serum. (B) The lanes 1–4 represent; rNDV, rNDV/HA, empty lane and rAPMV-3/HA, respectively. The full-length gels are presented in Supplementary Fig. S1.

Figure 3

Multicycle growth kinetics and plaque morphology of rNDV or rAPMV-3 expressing HA protein in DF1 cells. The monolayers of DF1 cells were infected with rNDV, rNDV/HA, rAPMV-3 and rAPMV-3/HA at 0.1 MOI. The supernatant media containing virus were removed after one hour adsorbtion. The cells were washed and DMEM containing 10% allantoic fluid was added to cells. The titer of viruses in equal volumes of supernatants collected from infected cells in 8 hours intervals were detected by TCID₅₀ using DF1 cells. (A,B) The plaque morphology of rNDV, rNDV/HA, rAPMV-3 and rAPMV-3/HA in presence of 10% allantoic fluid were evaluated in DF1 monolayers (C).

Figure 4

The induction of neutralizing antibodies and protective efficacy of rAPMV-3 or rNDV expressing HA protein against H5N1 HPAIV in SPF chickens (experiment 1). Day old SPF chicks were immunized with 10^6.5 TCID₅₀/bird of rNDV/HA or rAPMV-3/HA through oculonasal route. Three weeks after immunization, chickens were infected with 10⁵ EID₅₀ of H5N1 HPAIV. The infected birds were observed for 10 days post-challenge and the mortality rate for each group were recorded daily. (A) At day four post-challenge oropharyngeal swabs were taken from survived birds and each swab sample were inoculated in one 10-day-old embryonated eggs for re-isolation of challenge virus. The infected eggs were detected by HA assay. (B) Three weeks post-immunization the sera were collected from all chickens and the level of antibodies induced against a H5N2 LPAIV were detected by micro-virus neutralization assay (C) and HI assay (D) and the antibody HI titers against NDV and APMV-3 also were detected by HI assay. (E) Serum titers are expressed as reciprocals Log2 dilution. Statistical significance among groups was: P < 0.05 (*), p < 0.01(**) and p < 0.0001 (***).

Figure 5

The induction of neutralizing antibodies and protective efficacy of rAPMV-3 or rNDV expressing HA protein against H5N1 HPAIV in SPF chickens (experiment 2). Day old SPF chicks were immunized with 10⁶ PFU/bird of rNDV/HA or rAPMV-3/HA through oculonasal route. Three weeks after immunization, chickens were infected with 10⁶ EID₅₀ of H5N1 HPAIV. The infected birds were observed for 10 days post-challenge and the mortality rate for each group were recorded daily. (A) At day four post-challenge oropharyngeal swabs were taken from survived birds and each swab sample were inoculated in five 10-day-old embryonated eggs for re-isolation of challenge virus. The infected eggs were detected by HA assay. (B) Three weeks post-immunization the sera were collected from all chickens and the level of antibodies induced against the H5N2 LPAIV were detected by micro-virus neutralization assay (C) and HI assay. (D) The humoral antibody responses against NDV and APMV-3 also were detected by HI assay. (E) Serum titers are expressed as reciprocals Log2 dilution. Statistical significance among groups was: p < 0.01(**) and p < 0.0001 (***).

Figure 6

The protective efficacy of rAPMV-3 or rNDV expressing HA against H5N1 HPAIV in broilers. The groups of five broiler chickens were immunized with 10⁶ PFU/bird of rNDV/HA and/or rAPMV-3/HA based on regimens of vaccination for each group mentioned in Table 1 through oculonasal route. Chickens were infected with 10⁶ EID₅₀ of H5N1 HPAIV by the oculonasal route at week five of age. The infected birds were observed for 10 days post-challenge and the mortality rate for each group were recorded daily. (A) Three weeks after prime-immunization and two weeks after boost-immunization the sera were collected from all chickens and the antibodies induced against a H5N2 LPAIV were detected by HI assay. (B) Serum titers are expressed as reciprocals Log2 dilution. Statistical significance among groups was: P < 0.05 (*).

Figure 7

The antibody responses against APMV-3 and NDV by rAPMV-3 or rNDV expressing HA in broilers. The groups of broilers were immunized with 10⁶ TCID₅₀/bird of rNDV/HA or rAPMV-3/HA based on regimen of vaccination for each group mentioned in Table 1 through oculonasal route. Three weeks after prime-immunization and two weeks after boost-immunization the sera were collected from all chickens and the antibodies induced against rAPMV-3 strain Netherlands (A) and rNDV strain LaSota (B) were detected by HI assay. The level of maternally derived antibodies (MDAs) against rAPMV-3 strain Netherlands (C) and rNDV strain LaSota (D) in non-infected broilers also were detected by HI assay. Serum titers are expressed as reciprocals Log2 dilution.

Figure 8

The antibody responses and the protective efficacy rAPMV-3 or rNDV expressing HA against NDV strain GB Texas in immunized SPF chickens. Four-day-old SPF chickens were immunized with 10^6.5 TCID₅₀/ml of rAPMV-3/HA or rNDV/HA through oculonasal route. Three weeks after immunization, chickens were infected with 100 CLD₅₀ of NDV strain GB Texas. The infected birds were observed for 10 days post-challenge and the mortality rate clinical signs of NDV were recorded daily. (A) Three weeks post-immunization the sera were collected from all chickens and the level of antibodies induced against APMV-3 and NDV were detected by HI assay. (B) Serum titers are expressed as reciprocals Log2 dilution. Statistical significance among groups was: P < 0.05 (*) and p < 0.0001 (***).