Characterization of the vaccinia virus M1 protein on caspase inhibition and the MVA/5.2KB virus on immunogenicity

Vaccinia virus (VACV) is a master manipulator of the immune response, and is also a prime candidate for vaccine vectors. This thesis explored both properties through (i) identification and characterization of the VACV M1 protein as a caspase inhibitor and (ii) investigating a previously unexplored method to alter the immunogenicity of the attenuated VACV MVA. One anti-viral host response inhibited by VACV during infection is apoptosis. The attenuated MVA strain of VACV, however, stimulates apoptosis in several immune cell types due to loss of inhibitors. This thesis demonstrates that reinsertion of the M1L gene into MVA results in a virus that inhibits apoptosis. Furthermore, M1 can specifically inhibit intrinsic apoptosis. Inhibition was identified to be via M1-apoptosome (Apaf-1 and caspase-9) interactions, resulting in caspase-9 inactivation. This is the first VACV protein shown to inhibit after formation of the apoptosome, making M1 a novel inhibitor of apoptosis. Because M1 inhibited caspase-9 activation, the inhibition of other caspases by M1 was also examined. Via expression of M1 in mammalian cells or yeast, M1 inhibited caspases-1, -2, -3, -5, -7 and -8 in addition to its inhibition of caspase-9. This suggests M1 is a broad-spectrum caspase inhibitor. Further studies determining the mechanism of M1 inhibition of caspases could yield valuable information about poxviral-host interactions. Additionally, M1 features 14 predicted ANK repeat domains, a domain important for protein-protein interactions. Novel designed ANK repeat proteins (DARPins), are being created to specifically bind proteins, such as caspases, with high affinity. Therefore, information from the study of M1 could potentially aid in DARPin development. Due to the truncation or deletion of multiple viral proteins, MVA is a highly attenuated virus that is replication-restricted. These qualities make MVA a safe vaccine vector. However, higher doses or multiple boosts of MVA are necessary to elicit an immune response similar to wild-type VACV. Multiple strategies have been used to create modified MVA viruses that remain safe, but have increased immunogenicity, such as deletion of remaining VACV immune regulatory proteins. This thesis investigated the opposite and previously unexamined strategy; re-inserting three VACV immunomodulatory proteins (M1, K1, and M2) into MVA (MVA/5.2kb). The addition of one apoptosis inhibitor (M1) and two inhibitors of NF-κB (K1 and M2) was hypothesized to increase immunogenicity by delaying viral clearance and increasing the potential for antigen presentation. MVA/5.2kb-infection of antigen presenting cells (APCs) in vitro showed similar APC maturation and cytokine production compared to MVA-infection. However, this did not translate to increased immunogenicity as VACV-specific T-cells were dampened in vivo after infection with MVA/5.2kb. While a more immunogenic virus was not created, a previously unexplored method for altering MVA immunogenicity was tested. The studies in this thesis reveal novel information that is pertinent to the poxvirus field. First, a function was identified for the previously uncharacterized VACV M1 protein. Secondly, a previously unexplored method to alter MVA immunogenicity was explored. The information here expands the knowledge of poxviral-host interactions and could aid in the modification of MVA in the future.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste