Prime-Boost Vaccination With a Novel Hemagglutinin Protein Produced in Bacteria Induces Neutralizing Antibody Responses Against H5-Subtype Influenza Viruses in Commercial Chickens

Abstract

The highly pathogenic (HP) avian influenza virus (AIV), H5N1 and reassortant H5-subtype HPAIVs, H5N2, H5N6, and H5N8, cause high mortality in domestic birds, resulting in economic losses in the poultry industry. H5N1 and H5N6 also pose significant public health risks and H5N1 viruses are a permanent pandemic threat. To control HPAIVs, eukaryotic expression systems have traditionally been exploited to produce vaccines based on hemagglutinin (HA), a protective viral antigen. In contrast, we used a bacterial expression system to produce vaccine targeting the HA protein. A fragment of the HA ectodomain from H5N1, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. The resulting antigen, rH5-E. coli, was validated in terms of conformational integrity and oligomerization status. Previously, the protective efficacy of rH5-E. coli adjuvanted with aluminum hydroxide, has been positively verified by challenging the specific pathogen-free layer chickens with homologous and heterologous H5N1 HPAIVs. Protection was provided primarily by the H5 subtype-specific antibodies, as detected in the FluAC H5 test. The present studies were conducted to assess the performance of alum-adjuvanted rH5-E. coli in commercial birds. Broiler chickens were vaccinated twice with 25 μg of rH5-E. coli at 2- and 4-week intervals, while the layer chickens were vaccinated with 5- to 25-μg antigen doses at 4- and 6-week intervals. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition (HI) tests. Prime-boost immunizations with rH5-E. coli elicited H5 HA-specific antibodies in all the chickens tested. Two antigen doses administered at 4- or 6-week intervals were sufficient to induce neutralizing antibodies against H5-subtype HAs; however, they were ineffective when applied with a 2-week delay. In the layers, 80% to 100% of individuals developed antibodies that were active in the FluAC H5 and/or HI tests. A dose-sparing effect was seen when using the longer prime-boost interval. In the broiler chickens, 62.5% positivity was achieved in the FluAC H5 and/or HI tests. The trials confirmed the vaccine potential of rH5-E. coli and provided indications for anti-influenza vaccination with respect to the chicken type and immunization scheme.

Keywords: H5-subtype influenza viruses; H5N1; chicken; hemagglutinin; influenza; poultry; recombinant vaccine; vaccination and immunization.

Figure 1

Serum antibody responses of broiler chickens to vaccination with alum-adjuvanted rH5-E. coli or rH5-BEVS. Four groups of 1-week-old broiler chickens (8 or 7 per group) in Exp 1 were vaccinated twice at 2- and 4-week intervals, with 25 μg of rH5-E. coli (B-25/2 and B-25/4) or rH5-BEVS (B-25_ref/2 and B-25_ref/4) and aluminum hydroxide (alum) adjuvant. Sera collected post-prime (pp) and/or post-boost (pb) were analyzed for the presence of anti-H5 HA antibodies using the indirect and competitive ELISAs, H5 iELISA and FluAC H5, respectively, and a hemagglutination inhibition (HI) assay with H5N2 low-pathogenic (LP) AIV. Tests were performed and interpreted as described in the Materials and Methods section. In (A), the results for all of the test vaccine groups are presented. In (B,C), only the results for chickens vaccinated at a 4-week interval are shown. The chickens vaccinated at a 2-week interval were seronegative in relevant tests (Table 3). (A) Endpoint titers of anti-H5 HA IgY antibodies in post-boost sera. Each bar represents the result for the pooled sera collected from one group of chickens at the indicated time point. (B) Titers of HI antibodies against H5N2 LPAIV in post-prime and post-boost sera. Each symbol represents the result for one chicken at the specified time point. A value of 1.0 indicates that the HI antibody titer was below the detection limit (1:8). The dashed line indicates the cut-off value in the HI test (1:16). (C) Pattern of immune responses to the prime-boost vaccination. Considering the results from all of the post-vaccination sera, chickens were classified as positive (+) or negative (–) in H5 iELISAs (iELISA) or HI and FluAC H5 tests (HI/FluAC). Each bar represents a percentage of the specified category of chickens in each group.

Figure 2

Serum antibody responses of layer chickens to vaccination with alum-adjuvanted rH5-E. coli. Eight groups of 3-week-old layer chickens (10 per group) in Exp 2 were vaccinated twice at 4-week (A,C,E) or 6-week (B,D,F) intervals with 25, 15, 10, or 5 μg of rH5-E. coli and aluminum hydroxide (alum) adjuvant. Sera collected post-prime (pp) and/or post-boost (pb) were analyzed for the presence of anti-H5 HA antibodies using the indirect and competitive ELISAs, H5 iELISA and FluAC H5, respectively, and a hemagglutination inhibition (HI) assay with H5N2 low-pathogenic (LP) AIV. Tests were performed and interpreted as described in the Materials and Methods section. (A,B) Endpoint titers of anti-H5 HA IgY antibodies in post-boost sera from chickens vaccinated at (A) 4-week and (B) 6-week intervals. Each bar represents the result for pooled sera collected from one group of chickens at the indicated time point. (C,D) Titers of HI antibodies against H5N2 LPAIV in post-prime and post-boost sera from chickens vaccinated at (C) 4-week and (D) 6-week intervals. Each symbol represents the result for one chicken at the specified time point. A value of 1.0 indicates that the HI antibody titer was below the detection limit (1:8). The horizontal lines are the geometric mean titers (GMT) of HI-positive sera (≥1:16), detected 1 or 2 weeks post-boost in particular groups of chickens. The dashed line indicates the cut-off value in the HI test (1:16). (E,F) Pattern of immune responses to the prime-boost vaccination at (E) 4-week and (F) 6-week intervals. Considering the results from all of the post-vaccination sera, chickens were classified as positive (+) or negative (−) in H5 iELISA (iELISA) or HI and FluAC H5 tests (HI/FluAC). Each bar represents a percentage of the specified category of chickens in each group.