The role of immunogenic cell death in oncolytic herpes simplex virus-1 infection of cancer cells

Binks, Alexander William David (2018) The role of immunogenic cell death in oncolytic herpes simplex virus-1 infection of cancer cells. PhD thesis, University of Glasgow.

Abstract

Patients living with many cancers, including ovarian cancer (OC), often suffer from a lack of adequate treatment options. In the case of OC, primary debulking surgery followed by platinum and paclitaxel chemotherapy has led to a vast improvement in patient survival over the past few decades, however, rates of drug-resistant recurrence remain high. Research into new, experimental treatment options is therefore warranted for OC and other cancers.
Oncolytic viruses (OVs) are replication-competent viruses that can selectively infect and destroy cancerous cell types, while leaving healthy cells unharmed. OVs do this by exploiting differences between cancer and normal cell phenotypes. Herpes simplex virus (HSV)-1, strain 1716 is one example of this type of virus that has shown selectivity for cancer cells in previous preclinical studies, as well as high levels of safety in humans. One prominent area of current OV study seeks to investigate the ability of OVs to induce immunogenic cell death (ICD) – this term describes multiple modes of programmed death pathways that culminate in release of proimmunogenic factors, which facilitate a modification of the host immune system. Two of the most prominent of these pathways are necroptosis and immunogenic apoptosis (IA).
Here, I show that while many OV cell lines express the necessary components for necroptosis, they are unable to undergo classical necroptotic death (induced by TSZ). Despite this, HSV-1716 can infect and kill a range of OC lines successfully. I showed that HSV-1716-induced cell death displays two markers of IA yet does not seem to rely solely on apoptosis to kill cells. In addition, it appears not to rely on any components of the necrosome in order to kill cells, even in cells that are competent to typical necroptosis. However, when RIPK3 is overexpressed in HeLa cells, virus-induced cell death increases, as do markers of both necroptosis and IA.
To investigate the role of ICP6 in HSV-1716-induced ICD, viral and cell mutants were made possessing various forms of the protein. Full-length ICP6 protein expressed in cell lines had the effect of blocking cellular response to TSZ, but constructs lacking a region known as the RHIM did not. A functionally similar mutation was produced within the RHIM of live HSV-1716 using CRISPR/Cas9 technology, which was shown to have the effect of disrupting ICP6/RIPK3 binding – thought to be the determinant of necroptotic cell death. Despite this, no changes in cell death signalling could be determined between the viruses at all.
Interestingly, when cells were infected in combination with TNF-α, or TNF-α in addition to SMAC mimetic, the RHIM-modified virus produced significantly more death than HSV-1716. This suggests that while loss of RIPK3 inhibition is not sufficient to lead to increased necrosis alone, cells infected with this virus are more sensitive to further necrosis induction. This finding may prove to have great utility for producing the next generation of oncolytic viral therapeutics which can induce greater levels of proimmunogenic cell death.
From this we can conclude that HSV-1716 is capable of inducing IA in OC cells. Death is not dependent on necroptosis, however additional RIPK3 seems to sensitise cells to death by other means. Cellular binding of viral ICP6 and RIPK3 can be disrupted by modification of the RHIM, although this change has no bearing on ICD signalling alone but can sensitise cells to TNF-α-induced death.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: HSV-1, herpesvirus, cancer, oncolytic, virus, immunogenic cell death, necroptosis, necrosis, ovarian cancer.
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QR Microbiology > QR180 Immunology; Q Science > QR Microbiology > QR355 Virology; R Medicine > RM Therapeutics. Pharmacology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Funder's Name: Medical Research Council (MRC)
• Supervisor's Name: McNeish, Professor Iain and Tait, Dr. Stephen
• Date of Award: 2018
• Depositing User: Dr Alex Binks
• Unique ID: glathesis:2018-30718
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 08 Aug 2018 09:41
• Last Modified: 18 Sep 2018 10:34
• URI: https://theses.gla.ac.uk/id/eprint/30718