Birnaviruses as Candidates for Oncolytic Virotherapy
Restricted (Penn State Only)
- Author:
- Ravichandran, Abirami
- Graduate Program:
- Integrative and Biomedical Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 14, 2024
- Committee Members:
- Prashant Nighot, Outside Unit Member
Anthony Schmitt, Major Field Member
Vishal Singh, Outside Field Member
Robert Paulson, Major Field Member
Marco Archetti, Chair & Dissertation Advisor
Gregory Shearer, Program Head/Chair - Keywords:
- oncolytic virus
immunotherapy
cancer treatment
infectious bursal disease virus
infectious pancreatic necrosis virus
viruses
immune response
breast cancer - Abstract:
- Oncolytic virotherapy is a type of cancer therapy that involves the use of live, replication-competent viruses as therapeutic agents. These viruses have the potential to selectively replicate in cancer cells while causing minimal harm to normal, non-cancerous cells. Due to their cancer-selective nature, they cause minimal side effects when employed as cancer therapeutics and are emerging as a promising immunotherapeutic treatment option. A number of oncolytic viruses of different viral families are currently undergoing preclinical as well as clinical testing for their oncolytic properties. From the work done so far in the field, it has been recognized that a single oncolytic virus may not be able to target all types of cancer and that the development of different oncolytic virus platforms will be necessary to target diverse cancer types. With the goal of identifying novel candidates for oncolytic virotherapy, studies using two viruses from the Birnaviridae family, namely Infectious Pancreatic Necrosis Virus (IPNV) and Infectious Bursal Disease Virus (IBDV), were undertaken in this dissertation. In vitro testing, one of the initial steps undertaken in the development of any novel cancer therapeutic, was the focus of the first study. Here, we assessed the presence of oncolytic properties in Birnaviruses, IPNV and IBDV. Two main properties oncolytic viruses contain are cancer-selective replication and lysis. These two properties were explored for IPNV and IBDV using a range of cancer and non-cancer cells of different cell lineages. When the cancer cells were treated with IPNV, a decline in viability was observed in breast, liver, and renal cancer cells that were tested. With IBDV, a decrease in viability was observed in breast, renal, liver, colorectal cancer, and leukemic cells. The non-cancerous normal cells did not undergo lysis on treatment with IPNV or IBDV. The viruses were able to replicate productively in the susceptible cancer cells but not in the non-cancer normal cells. The levels of caspases 3 and 7, involved in executing apoptosis, were elevated in all the susceptible cancer cells. This indicates that apoptosis, the predominant cell death pathway activated by many viruses, may also be involved in Birnavirus-induced oncolytic cell death. On further exploration of innate immune cell pathways in the infected cells, upregulation of Toll-like receptor 3 (TLR3) pathway components was observed, implying that this could be a major pathway activated by Birnaviruses post infection. Overall, the first study findings indicated the presence of oncolytic properties in both IPNV and IBDV, with a greater range of oncolytic action observed while using IBDV. Subsequently, we explored the potential for engineering IBDV with the goal of attaining better oncolytic properties. In this study, the first experiment was aimed at standardizing and establishing a reverse genetics system for the virus rescue of IBDV. For the viral rescue experiments, a plasmid-based reverse genetics system with bidirectional promoters and viral cDNA sequences was designed. When experimentally tested, this plasmid-based system successfully produced the IBDV wildtype virus with good efficiency. The rescued WT virus is referred to as IBDV rWT. After the successful rescue of the wildtype IBDV, the same reverse genetics methodology was employed to attempt the rescue of recombinant IBDV. The first recombinant IBDV design that was tested had a deletion of the accessory viral protein, VP5 (dVP5). Since it was known that VP5 assists in the non-lytic egress of the virus, it was hypothesized that the deletion of VP5 might result in enhanced oncolytic properties. The IBDV with deleted VP5 (IBDV dVP5) was successfully recovered using the reverse genetics system. Following the recovery of the IBDV dVP5, experiments were conducted to test if IBDV dVP5 had better oncolytic properties compared to the wildtype virus. It was observed that IBDV dVP5 had oncolytic properties; however, the oncolytic efficacy was not increased compared to the rescued IBDV rWT. Another recombinant IBDV was designed with the addition of csf gene as a payload to enhance oncolytic action. The payload csf gene encodes for the cytokine, Granulocyte-macrophage colony-stimulating factor (GM-CSF). However, when experiments were conducted, the recombinant virus with the csf gene insert could not be successfully rescued. In summary, a bidirectional promoter-based reverse genetics system was established in this study, which opens avenues for further engineering of recombinant viruses using this reverse genetics system. Further, recombinant IBDV dVP5 was rescued, and its oncolytic activity was tested in comparison with IBDV WT. Although the reasons for the decreased oncolytic activity of IBDV dVP5, as well as the unsuccessful rescue of recombinant IBDV csf virus, are not fully understood, these are interesting areas to explore in future studies. Finally, in vivo studies were performed to assess the oncolytic effects of IBDV and IPNV in a mouse breast cancer model. Model characterization experiments were initially performed to identify a suitable murine tumor model for preclinical validation studies. From these experiments, a cell-derived xenograft model using HCC1187 breast cancer cells was chosen for studying in vivo oncolytic activity. HCC1187 tumor cells were injected into the immunocompromised NSG mice (NOD-scid IL2Rgammanull) mice to generate mammary tumors, and the viruses were injected subsequently to test for their oncolytic activity. The viral treatments did not significantly reduce the tumor growth; however, there was a marginal difference in survival outcomes among the groups. Further, the viral treatments were found to be well tolerated by the mice. In summary, an in vivo model system for preclinical validation studies of Birnavirus oncolysis was established, and one of the first studies was conducted to evaluate the in vivo oncolytic activity of IPNV and IBDV. Although this initial study did not find significant differences in tumor outcomes, further studies can be undertaken using this tumor model to optimize various treatment parameters, including the dose, schedule, and mode of administration, with the goal of enhancing treatment efficacy. In conclusion, this is one of the first studies to extensively characterize the oncolytic properties of Birnaviruses, IPNV and IBDV. The viruses were found to possess innate tumor tropic, replicative, and lytic abilities against different cancer cell types in vitro. A high-efficiency reverse genetics system was established in the study to facilitate genetic modification of IBDV, and this system can be used in designing recombinant IBDV for further studies of varied applications, including oncolytic virotherapy, vaccine studies, or studies regarding virus biology. Furthermore, initial in vivo studies were conducted to test the oncolytic properties of IPNV and IBDV, and this murine tumor model can be used in further optimization studies of the viral treatments for better efficacy. Additionally, previous evidence from the literature suggests that these Birnaviruses may be safer for human use due to their non-pathogenic profile and may be strongly immunostimulatory in vivo due to their dsRNA genome. Collectively, these findings indicate that Birnaviruses, IPNV and IBDV, possess oncolytic properties with important advantages and therefore, can be good candidates for oncolytic virotherapy.