This Is AuburnElectronic Theses and Dissertations

Novel Infectious Bronchitis Virus Vaccines and Immune Responses




Zegpi, Ramon

Type of Degree

PhD Dissertation


General Veterinary Medicine

Restriction Status


Restriction Type


Date Available



Infectious bronchitis virus (IBV) occurs as multiple serotypes and genotypes worldwide. In the United States IBV belonging to the Arkansas (Ark) Delmarva Poultry Industry (DPI) genotype is the most frequently isolated genotype despite extensive vaccination with ArkDPI-derived vaccines. ArkDPI vaccines are heterogeneous, and distinct viral subpopulations become predominant in vaccinated chickens. Vaccine heterogeneity and selection of viral subpopulations after vaccination are likely responsible for the continuous isolation of ArkDPI vaccine-like strains from outbreaks of disease. We previously demonstrated that adaptation of an embryo-attenuated IBV ArkDPI-derived vaccine to chicken embryo kidney cells (CEK) shifted the virus population towards homogeneity in spike (S) and non-structural protein (NSP) genes. Moreover, the typical Ark vaccine subpopulations emerging in chickens vaccinated with commercial Ark vaccines were not detected in chickens vaccinated with CEK-adapted vaccine virus. Using both conventional and next generation sequencing, we now demonstrate that the changes achieved during CEK adaptation remained after back passages in embryos. We further demonstrate that 1-day-old chickens vaccinated with the CEK-adapted virus are protected against Ark virulent challenge. Moreover, we compared protection conferred by the CEK-adapted virus with the protection conferred by an attenuated commercial ArkDPI-derived vaccine different from which the CEK-adapted virus originated. All vaccinated chicken groups showed a significant reduction of respiratory signs and viral load after Ark virulent challenge compared to unvaccinated-challenged controls. In CEK-adapted virus vaccinated chickens, viral subpopulations different from the challenge virus were detected after challenge in a marginal number (7-8%) of chickens. In contrast, IBV S1 sequences that differed from the predominant population in the challenge virus were detected after challenge in a large number (77%) of chickens vaccinated with the commercial Ark attenuated vaccine. The CEK-adapted IBV ArkDPI-derived vaccine is a stable and effective vaccine, which drastically reduces the emergence of Ark-like viruses both at vaccination and after challenge. To further understand the impact of population genetic structure on generation of immune responses and protection, we used comparison of responses to the homogeneous population of the CEK-adapted ArkDPI vaccine and the commercial ArkDPI from which the CEK adapted virus originated as a model. In a first experiment, vaccinated chickens were challenged with an IBV Ark99-type virulent strain (AL4614/98). Despite extensive sequence similarity between the vaccines, the more heterogeneous commercial ArkDPI was more efficient at reducing viral loads in challenged chickens, while respiratory signs and tracheal lesions were reduced similarly by either vaccine. A distinct subpopulation of the Ark challenge virus showing asparagine at S1 position 56 was consistently negatively selected by immune pressure originating from vaccination with either vaccine. Antibody levels and antibody avidity to Ark-type S1 protein were greater in CEK-ArkDPI-vaccinated chickens compared to chickens vaccinated with the more diverse commercial ArkDPI vaccine. Synchronous replication of a homogeneous virus population likely elicits clonal expansion and affinity maturation of a greater number of responding B cells compared to a diverse virus population continuously changing its proportion of phenotypes during replication. The results of a second experiment showed that during initial vaccine virus replication in chickens (24 and 48 hrs post-vaccination), the virus population showing increased diversity (commercial ArkDPI vaccine) achieved higher concentrations of IBV RNA in the trachea compared to the more homogenous CEK-adapted virus. mRNA expression in the trachea of genes associated with immune responses generally showed greater upregulation 48 hrs post-vaccination in chickens vaccinated with the heterogeneous commercial ArkDPI vaccine compared to the CEK-adapted virus. The greater upregulation of these genes is likely associated with higher virus replication achieved by the heterogeneous commercial vaccine. Thus, while the adaptive antibody response was favored by the more homogenous structure of the CEK-ArkDPI vaccine population (higher antibody levels and antibody avidity), the innate immune response was favored by the more diverse viral population of the commercial ArkDPI. We confirmed previous results that distinct subpopulations in the Ark challenge virus become selected by immune pressure originating from vaccination and concluded that the population structure of IBV vaccines impacts innate immune response, antibody avidity, and protection. We also explored expression of an immunologically important IBV protein from a vector as a possibly better vaccine alternative to protect chicken flocks against Ark-type virus. A recombinant Newcastle disease virus (NDV) LaSota (LS) expressing secreted trimeric spike (S)-ectodomain (Se) of IBV (rLS/IBV.Se) was developed and evaluated for protection conferred against IBV challenge. The IBV S-ectodomain protein, which is S excluding the transmembrane anchor and short cytoplasmic domain of S2, expressed from recombinant LS corresponds to an Ark-type IBV. In a first experiment, chickens were primed at 1-day of age or primed at 1 day-old and boosted at 14 days-old with 104 EID50/bird of rLS/IBV.Se and challenged with a virulent Ark strain. While single vaccination proved completely ineffective at protecting chickens against challenge, priming and boosting reduced clinical signs and tracheal lesions but did not reduce viral load in lachrymal fluids. In experiment 2, the vaccine dose was increased to 107 EID50/bird and a different virulent Ark strain was used for challenge. In addition, chickens were singly immunized on either day 1 or day 10 after hatch. NDV antibody levels detected in vaccinated chickens were moderate, with hemagglutination inhibition titers varying between 4 and 5 log2. Slightly higher antibody levels to NDV were observed in chickens vaccinated on day 10 versus day 1 but without the difference achieving statistical significance. In contrast, antibody responses measured using recombinant IBV S1 protein-coated ELISA plates were significantly greater in chickens vaccinated on day 10 compared to day 1. The use of a higher rLS/IBV.Se dose proved to enhance the success of single vaccination substantially compared to experiment 1. Signs and tracheal lesions were reduced more effectively in chickens vaccinated at day 10 after hatch. However, as in experiment 1, vaccination did not reduce the viral loads in tear fluids of challenged chickens. Similar results, in which no reduction in viral load in the trachea was apparent following rLS/IBV.S vaccination, have been obtained by others. Further work is needed to understand the immune responses induced by this recombinant virus that seems to provide some protection against the disease but does not reduce viral loads in the upper respiratory tract. Finally, we explored the possibility that failure of vaccination might be due to the fact that chickens are vaccinated immediately after hatch. Previous work in our laboratory had shown that antibody responses were reduced in chickens vaccinated at 1 day of age compared to chickens vaccinated beyond 1-day of age. In the present work, we examined cross-protection and immune responses elicited by IBV vaccination on day 1 of age or at later time points. Chickens were vaccinated with a Massachusetts (Mass)-type vaccine and heterologous challenge performed with an Ark-type virulent strain. In experiment 1, chickens vaccinated on day 1 or 10 of age were challenged 21 days after vaccination. Analysis of tracheal histopathology and viral load demonstrated enhanced cross-protection when vaccination was postponed beyond day 1 of age. In experiment 2, chickens were vaccinated on day 1 or 14 days of age. A somewhat stronger systemic antibody response to IBV was detected in chickens vaccinated at 14 days of age. In addition, avidity of antibodies to Ark-type S1 protein elicited by vaccination at 14 days of age was greater. Few differences were noted between chickens vaccinated at 1 or 14 days of age in immune cell populations in the Harderian gland (HG) at the time of sampling 35 days following vaccination. In birds 7 days after challenge both non-vaccinated/challenged groups showed significantly higher (P<0.05) proportions of B cells and CD8+ T cells than age-matched vaccinated/challenged groups or age-matched non-vaccinated/non-challenged control groups. These results indicate infiltration and/or expansion of B cells and CD8+ cells in HGs following challenge of non-vaccinated chickens. A fortuitous finding was that the more immature immune system of 1-day-old chickens was less effective at clearing vaccine virus after vaccination. Collectively, the current results indicate that IBV vaccination at least ten days after hatch induces more effective cross-protection than vaccination on day of hatch. Greater antibody affinity maturation likely contributes to increased cross-protection.