title: Recombinant replication-defective human adenovirus serotype 5 vaccine vectors expressing proteins of interest induce ubiquitination modification: a potential T cell vaccine
Creators: name: Tospitakkul, Pawana
Publisher: name: The University of Edinburgh
Year: 2021
Subjects: subject: inactivated foot-and-mouth disease (FMD) vaccines
subject: cell-mediated immunity
subject: infectious bovine rhinotracheitis
subject: bovine herpesvirus 1
subject: ubiquitin-proteasome system
subject: major histocompatibility complex
subject: live replicationdefective human adenovirus recombinant vector
subject: Lys-48
subject: AdP1-2A
subject: AdP1-2A3C
subject: AdBHV-1 gB
subject: AdBHV-1 tgB
subject: Madin-Darby bovine kidney (MDBK) cell cultures

description: Commercially available inactivated foot-and-mouth disease (FMD) vaccines provide a robust protective immune response, often attributed to neutralising antibodies. However, animals with vaccine-induced, high titres of the virus-specific antibody are often not protected and protection has also been demonstrated in animals that didn’t have any detectable humoral immune response suggesting a contribution of other immune system components including cell-mediated immunity (CMI). Similarly, antibody responses to virus pathogens in infectious bovine rhinotracheitis (IBR), called bovine herpesvirus 1 (BHV-1), are induced by vaccination; however, the role of CMI in these important livestock diseases is not well understood. The post-translational modification of proteins by ubiquitin regulates a variety of biological processes and initiates the CMI to pathogens via a pathway called the ubiquitin-proteasome system (UPS). The UPS yields antigenic peptides that bind to the major histocompatibility complex (MHC) class I and are presented to stimulate a cytotoxic T cells response. The aim of this study was to express proteins of interest using a live replication-defective human adenovirus recombinant vector (Ad) and evaluate the induction of post-translation modification by ubiquitin especially for the lysine residue at position 48 (Lys-48) linkage which mediates the ubiquitin-conjugated protein to the proteasome degradation. Our hypothesis was that an engineered cytosolic synthesised protein had a higher ubiquitination level, specifically in Lys-48 chains, hence become targeted for proteasome degradation resulting in peptides available for MHC class I binding and stimulate cytotoxic T cell response. In order to investigate this hypothesis, we constructed an Ad recombinant vector to express native and modified forms of protein by insertion of cDNA encoding the proteins of interest; AdP1-2A (structural protein) and AdP1-2A3C (structural protein plus proteolytic enzyme) of FMDV as well as AdBHV-1 gB (mature protein) and AdBHV-1 tgB (truncated protein of BHV-1 glycoprotein B). Recombinant viruses were propagated and purified to high titres of &gt;10^8 tissue culture infectious dose (TCID50) per mL with the proteins of interest shown to be successfully expressed in vitro in Madin-Darby bovine kidney (MDBK) cell cultures. The expressed proteins and Lys-48 linkage conjugated proteins in the immunoprecipitated sample were analysed by western blot and mass spectrometry assays. The results demonstrated that the cytoplasm accumulated proteins, expressed by the vector, indeed served as a target for Lys-48 modification and therefore mediated the proteasome recognition.