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.

A Novel Epitope-Blocking ELISA for Specific and Sensitive Detection of Antibodies Against H5-Subtype Influenza Virus Hemagglutinin

“…Experimental antisera were from previously presented e cacy trials for the HA protein of the H5N1 HPAIV produced in Escherichia coli [10]. The vaccine antigen, referred to as rH5-E. coli, was refolded and chromatographically puri ed from inclusion bodies.…”

“…The anti-H5 HA negative sera were from control groups in the immunization studies with rH5-E. coli, including those reported previously [10]. The studies were performed under semi eld conditions, described above, using the commercial layers and broilers-namely, the Rossa 1 and Ross 308 lines, respectively.…”

“…2 Collected in (a) the test vaccine and (b) control chicken groups during immunization studies with H5 HA protein produced in bacteria (rH5-E. coli) at the Institute of Biotechnology and Antibiotics (IBA; Warsaw, Poland). The number of samples described previously with HI titers [10] are provided in brackets. Details of the vaccine antigen and experimental antisera against H5 HA are presented in Additional le 1: Tables S1 and S4, respectively.…”

“…The HI assay was performed according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [11] following the previously described procedure [10]. Brie y, the sera were analyzed with the heterologous A/turk/Italy/80(H5N2) LPAIV strain at an HI unit (HIU) of 1:8 using SPF chicken erythrocytes (DPD NVRI, Puławy, Poland).…”

“…On this basis, each serum sample was scored as positive or negative for H5 subtype-speci c antibodies. The results of the HI assay for 115 antisera collected in the layers immunized with rH5-E. coli and 96 sera from the nonimmunized layers and broilers were adapted from our previous paper [10].…”