Vilela, Julianne and Munir, Muhammad (2024) Development of CRISPR/Cas9-Based Novel Vaccines Against Poultry Viruses. PhD thesis, Lancaster University.
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Abstract
The poultry industry significantly depends on the use of vaccinations as a primary strategy for disease control. Consequently, there exists a critical need for further research and development of novel vaccines to effectively address the challenges posed by the emergence and re-emergence of novel virus strains. The utilisation of recombinant viral vaccine vectors has played a crucial role in the advancement of novel vaccines, presenting several advantages in comparison to traditional approaches. These advantages include enhanced precision and efficacy in cell targeting, as well as the capacity to elicit robust immune responses and augment cellular immunity. Recent developments in the fields of pathogen biology and recombinant DNA technologies have opened up new avenues for addressing challenges associated with traditional vaccination creation. In recent times, the application of CRISPR/Cas9 genome editing technology has facilitated the creation of innovative recombinant viral vectors, thereby facilitating progress in the field of vaccine development. The primary objective of this PhD thesis is to create recombinant viral vectors derived from herpesvirus, employing the CRISPR/Cas9 genome editing approach. These vectors will be specifically designed to combat economically significant poultry viruses, including NDV, IBV, and AIV. The findings of this study successfully facilitated the identification of prospective antigenic proteins and epitopes that may be utilised in the development of vaccines. Additionally, the genotypic profiling of the primary antigens, namely F and HN for NDV, S for IBV, and HA for AIV H9N2, was conducted on circulating field strains in the Philippines. The findings of this study demonstrate the efficacy of the CRISPR/Cas9 genome editing method in accurately targeting sections of the ILTV genome and inducing the necessary cleavage, hence enabling the successful integration of foreign gene cassettes into the ILTV genome. As a consequence, a total of ten recombinant viral vectors were generated, specifically referred to as ILTV-NDV-F (-UL0, -UL50, and -US4), ILTV-NDV-HN, ILTV-NDVFHN, ILTV-NDVF-Variants (-Fd, -Fc, and -FcTail), ILTV-IBV-S, and ILTV-HAH9N2. Following molecular and protein investigations, it was shown that these recombinant ILTV strains demonstrate stability and comparable functionality to the wild-type ILTV. The results presented in this study offer evidence to support the concept that ILTV might potentially serve as a tool for developing recombinant viral vectors, particularly for the creation of a multivalent viral vector. Moreover, it underscores the efficacy of CRISPR/Cas9 genome editing technology as the preferred technique for the advancement of novel vaccines.