A novel chimpanzee adenovirus vector vaccine for protection against infectious bronchitis and Newcastle disease in chickens

Abstract

The development of effective poultry vaccines is crucial for maintaining flock health and productivity. In this study, we developed and evaluated a recombinant chimpanzee adenovirus vaccine (PAD-S1-HN) that simultaneously expresses the infectious bronchitis virus (IBV) spike subunit protein S1 and Newcastle disease virus (NDV) hemagglutinin-neuraminidase HN protein. The recombinant virus was successfully rescued in HEK293 cells, and transmission electron microscopy confirmed its typical adenoviral morphology. The expression of the IBV S1 and NDV HN proteins was validated by indirect immunofluorescence assay and western blotting. The vaccine demonstrated genetic stability over multiple passages and exhibited growth kinetics similar to those of the empty chimpanzee adenovirus vector. In animal trials, PAD-S1-HN effectively induced IBV- and NDV-specific antibodies, increased key cytokine levels, and stimulated mucosal immune responses, resulting in reduced viral loads, and alleviated clinical symptoms in vaccinated chickens. These findings indicate that the PAD-S1-HN vaccine provides strong immunogenicity and protective efficacy against IBV and NDV infections. Therefore, it presents a promising alternative to conventional vaccines, offering a novel approach for improving poultry disease management.

Keywords: IBV S1 protein; NDV HN protein; bivalent vaccine; nonreplicating chimpanzee adenovirus; poultry immunization.

Figure 1

Rescue and characterization of the recombinant chimpanzee adenovirus PAD-S1-HN. A Schematic diagram of the construction of the recombinant chimpanzee adenovirus vector. B IFA identification of the expression of the target protein PAD-S1-HN from the recombinant chimpanzee adenovirus. C Western blot analysis of target protein expression of the recombinant chimpanzee adenovirus PAD-S1-HN, PAD-S1-HN (Lane 1), the control recombinant adenovirus PAD-S from a previous study (Lane 2), the empty adenovirus PAD (Lane 3), and the normal cell control (Lane 4). D Transmission electron microscopy of the recombinant adenovirus (150 000 ×).

Figure 2

Growth kinetics and preliminary immunogenicity investigation of the recombinant chimpanzee adenovirus PAD-S1-HN in chickens. A One-step growth curve of the recombinant adenovirus PAD-S1-HN, with the empty adenovirus PAD used as a control. B Growth trends of chicken body weight after immunization in different groups. C IBV-specific IgG antibody levels in chicken serum. D NDV-specific IgG antibody levels in chicken serum.

Figure 3

Measurement of major cytokine levels in chickens post-immunization. A Measurement of IL-2 levels in chickens at 14 and 28 days after the first immunization (dpv: days post-vaccination). B Measurement of IL-4 levels in chickens at 14 and 28 days after the first immunization. C Measurement of IL-6 levels in chickens at 14 and 28 days after the first immunization. D Measurement of IFN-γ levels in chickens at 14 and 28 days after the first immunization.

Figure 4

Detection of specific mucosal IgA antibodies and total mucosal IgA antibodies. A IBV-specific IgA results in chicken trachea at 14 and 28 days after the first immunization. B IBV-specific IgA results in chicken intestines at 14 and 28 days after the first immunization. C NDV-specific IgA results in chicken trachea at 14 and 28 days after the first immunization. D NDV-specific IgA results in chicken intestines at 14 and 28 days after the first immunization. E Total mucosal IgA results in chicken trachea at 14 and 28 days after the first immunization. F Total mucosal IgA results in chicken intestines at 14 and 28 days after the first immunization.

Figure 5

Viral challenge in chickens. A Schematic diagram of the animal experimental protocol. B Survival curve after IBV and NDV challenge (n = 24). C Detection of IBV shedding in throat swabs on days 2, 5, and 8 post-challenge. D Detection of IBV shedding in cloacal swabs on days 2, 5, and 8 post-challenge. E Detection of NDV shedding in throat swabs on days 2, 5, and 8 post-challenge. F Detection of NDV shedding in cloacal swabs on days 2, 5, and 8 post-challenge.

Figure 6

Measurement of the viral load in organs and pathological analysis post-challenge. A Viral load of IBV in different organs post-challenge. B Viral load of NDV in different organs post-challenge. C Pathological changes in major organs and tissues post-challenge.