Activity 3.2: Development of Novel Nanoparticle-Base Vaccines for Infectious Bronchitis Virus
PI: Mazhar Khan and Peter Burkhard
Infectious bronchitis virus (IBV) is one of the primary causes of respiratory disease in domestic fowl. It affects the respiratory tract but also the gut, reproductive systems and kidney of chickens. The effectiveness of currently available attenuated vaccines and inactivated vaccines is reduced by poor cross-protection. Live attenuated vaccine has a strong probability of spreading of vaccine virus and help evolving variants. IBV genome encodes four major structural proteins, known as spike (S) protein, nucleocapsid (N) protein, membrane (M) protein and envelope (E) protein. The S protein is post-translationally cleaved into the outer S1 and the membrane bound S2 proteins. S1 protein comprises major antigenic determinants that induce neutralizing antibodies which make it a major target of vaccine design and immune therapy. S2 protein is conserved and comprises epitopes inducing cross-reactive Abs and cell –mediated immune responses. N protein is largely conserved and contains epitopes which induce cytotoxic T lymphocyte (CTL) responses and help in protection as well as activating B cell. To generate a potent vaccine the best conserved and protective B and T cell epitopes should be combined into a highly immunogenic epitope delivery and presenting system. The S protein of IBV contains two coiled-coil sequences. We will engineer this coiled-coil sequence onto the trimeric coiled-coil of several versions of our current SAPNs. Thus, these nanoparticles will present this epitope in a conformation-specific manner to the immune system, hence inducing conformation-specific antibodies with the potential to neutralize the virus in a viral infectivity assay.
Year 1: Bio-production of SAPN constructs and Structural and biophysical analyses.
DNA oligos coding for the epitope will be ligated into the plasmid. SAPN constructs will be designed and the nucleotide sequence of each SAPN will be synthesized.. Transformation, expression, purification, refolding and biophysical analyses will be performed. The criteria for the selection of the constructs will be their aggregation behavior their ability to form nicely shaped and sized nanoparticles as well as their ease of expression in E. coli. Thus, these nanoparticles will present this epitope in a conformation-specific manner to the immune system, hence inducing conformation-specific antibodies with the potential to neutralize the virus in a viral infectivity assay.
Year 2: To test immunogenicity of the SAPN-vaccine constructs
SPF-chickens will be injected with SAPN vaccine constructs. Elicit immune responses in chickens will be tested at various time intervals. For this purpose, ELISA, virus neutralization, T-cell lymphocyte proliferation and flow cytometry analyses .will be used.
Year 3: Protective efficacy against challenge with a pathogenic infectious bronchitis virus strain.
The efficacy of the nanoparticle vaccines will be tested using an established challenge of immunity model. The model will: 1) evaluate clinical disease 2) evaluate pre- and post-challenge serum antibody assess challenge virus shedding and 3) evaluate IBV gross and microscopic lesions in tissues after challenge.
Years 4 & 5: Application of Nanoparticle base IBV vaccine under Field condition.
Nanoparticle based IBV vaccine will be compared for its protection with the commercial live vaccine on the poultry farm.
Expected Results and Criteria or Means to Evaluate Your Research Success:
Direct outcomes of this project will include: evaluation of a peptide platform that will deliver antigenic epitopes of IBV virus to the host immune system and induce protective immune response. The virus peptide will be used to generate B and T-cell immune responses to mimicking those of attenuated vaccination but without risks of using live IBV virus.