Abstract In the light of the tremendous progress that has been made in raising the transition temperature of the copper oxide superconductors. At present, the highest reported values of Tc for non-copper-oxide bulk superconductivity are 33 K in CsxRbyC60, and 30 K in Ba1-xKxBiO3. The recent discovery of superconductivity at 39 K in the sample binary ceramic compound magnesium diboride, MgB2(ref. 1), was therefore surprising. Indeed, this material has been known and structurally characterized since the mid 1950s, and is readily available from chemical suppliers. Here we try to addition of electrons to MgB2, through partial substitution of Al for Mg and a structural transition in Mg1-xAlxB2

In the present study, we characterized the ability of a novel oncolytic rhabdovirus - Maraba MG1 to boost Natural Killer (NK) cell activity. In tandem, we addressed the ability of this enhanced NK cell functionality to reduce the incidence of post-cancer surgery micrometastases. Due to the potential safety barriers associated with the use of a live virus immediately prior to surgery in cancer patients, we generated a single cycle replication virus (MG1-Gless) and UV-inactivated MG1 to stimulate NK cell function and reduce post-operative metastases. Our in vivo data demonstrate that significant NK cell activation and a similar level of reduction in postoperative tumor metastases was achieved with live MG1, MG1-Gless and UV-inactivated MG1, concluding that viral replication is important, but not necessary for NK cell activation. Mechanistically, we observed that dendritic cells (DCs) are necessary intermediates for MG1-induced NK cell activation. Finally, we characterized and compared a panel of UV-inactivated MG1 (2mins to 2hrs) to better understand the requirements for NK cell activation. Our results suggest that intact viral particle and cellular recognition and association are essential for NK cell mediated anti-tumor responses. These findings provide the preclinical rationale to develop safe and viable virotherapy-based interventional protocols that might reduce the risk of metastatic disease after cancer surgery.

In the present study, we characterized the ability of a novel oncolytic rhabdovirus - Maraba MG1 to boost Natural Killer (NK) cell activity. In tandem, we addressed the ability of this enhanced NK cell functionality to reduce the incidence of post-cancer surgery micrometastases. Due to the potential safety barriers associated with the use of a live virus immediately prior to surgery in cancer patients, we generated a single cycle replication virus (MG1-Gless) and UV-inactivated MG1 to stimulate NK cell function and reduce post-operative metastases. Our in vivo data demonstrate that significant NK cell activation and a similar level of reduction in postoperative tumor metastases was achieved with live MG1, MG1-Gless and UV-inactivated MG1, concluding that viral replication is important, but not necessary for NK cell activation. Mechanistically, we observed that dendritic cells (DCs) are necessary intermediates for MG1-induced NK cell activation. Finally, we characterized and compared a panel of UV-inactivated MG1 (2mins to 2hrs) to better understand the requirements for NK cell activation. Our results suggest that intact viral particle and cellular recognition and association are essential for NK cell mediated anti-tumor responses. These findings provide the preclinical rationale to develop safe and viable virotherapy-based interventional protocols that might reduce the risk of metastatic disease after cancer surgery.

HPV-associated cancer and carcinoma of the prostate are responsible for significant worldwide morbidity and mortality. The viral transforming proteins E6 and E7 make human papilloma virus positive (HPV+) malignancies an attractive target for cancer immunotherapy however, therapeutic vaccination exerts limited efficacy in the setting of advanced disease. In prostatic carcinoma therapeutic vaccination shows some therapeutic activity but is infrequently curative. A strategy to induce substantial specific immune responses against multiple epitopes of E6 and E7 proteins based on an attenuated transgene from HPV serotypes 16 and 18, that is incorporated into MG1-Maraba virotherapy (MG1-E6E7), was designed. MG1-E6E7 is able to boost specific immunity following priming with either an adenoviral vector (Ad-E6E7) or customised synthetic peptide vaccines resulting in multifunctional CD8+ T cell responses of an enormous magnitude. MG1-E6E7 vaccination in the HPV+ murine model TC1 is curative against large tumours in a CD8+ dependent manner and results in durable immunologic memory. Using the same adenoviral prime and MG1 boosting strategy targeting the prostatic antigen, STEAP, immunity against multiple CD8+ STEAP epitopes was induced. In a murine prostate cancer model, STEAP specific oncolytic virotherapy significantly improved the survival of mice bearing advanced TRAMP-C2 tumours. One significant obstacle to therapeutic cancer vaccination is an immunosuppressive tumour microenvironment. MG1 Maraba is able to lethally infect HPV-associated and prostate cancer cells, increase the immunologic activity within the tumour microenvironment in vivo and exploit molecular hallmarks of HPV-positive cancer and prostatic carcinoma enabling infection of bulky tumours. Pre-clinical data generated within this thesis has been instrumental in securing funding for future clinical trials assessing the safety and activity of MG1 Maraba virotherapy for HPV-associated cancer and prostatic carcinoma. This promising approach has the potential to be directly translatable to human clinical oncology to tackle these two highly prevalent and frequently lethal groups of epithelial neoplasia. / Thesis / Doctor of Philosophy (PhD) / Carcinoma (epithelial cancer) is the most common form of human cancer and two frequently encountered types, namely HPV-associated and prostatic carcinoma are responsible for a substantial worldwide cancer burden. Current therapeutic options show limited clinical benefit and/ or significant long-term side effects for advanced carcinomas, therefore new treatments are urgently required. Oncolytic viruses represent an exciting new form of anti-tumour immunotherapy capable of infecting and killing cancerous cells; here we present a virus called MG1 Maraba that is able to exploit molecular characteristics of these cancers. When MG1 Maraba is engineered to target proteins from HPV-associated cancer and prostatic carcinoma, specific immune attack against these tumours occur in mouse cancer models. MG1 Maraba offers a novel, selective, safe and highly promising therapeutic approach against advanced carcinomas. Based on the information within this thesis human clinical trials assessing MG1 Maraba are due to take place for both HPV-associated and prostate cancer.

HIV remains an incurable viral infection and a significant global health concern. Despite the advent of antiretroviral therapy, there are 36.9 million recorded cases of HIV worldwide, with an additional 1.8 million new infections recorded in 2017 alone. An HIV cure is therefore one of several priorities within the field, and will require HIV “reservoir” cells—comprised of latently-HIV infected CD4+ T cells and productively-infected, tissue resident macrophages—to be selectively killed in vivo. HIV reservoir cells are rarely found within the peripheral circulation, residing instead within inaccessible tissue sanctuaries. Consequently, their characterization has been limited to in vitro laboratory models. To complicate matters further, a definitive cellular surface marker of HIV infected cells has yet to be identified. Impairment of the type I interferon (IFN1) response has been observed during HIV infection, however, making it a unique intracellular maker of HIV-infected cells. The recent development of oncolytic viruses (OV) designed to selectively kill IFN-defective cancer cells also suggests that these IFN1 defects possess therapeutic value. It was therefore hypothesized that the impairment of the IFN1 response in HIV-infected CD4+ cells and macrophages could serve as a target for oncolytic virus-mediated killing. The induction of several antiviral IFN-stimulated proteins, including PKR and ISG15, was inhibited in HIV-infected monocyte-derived macrophages (MDM) following stimulation with IFNα or a synthetic RNA. Consequently, HIV-infected MDM were more susceptible to infection and killing by the oncolytic Maraba virus, MG1. Importantly, MG1-mediated killing required the presence of replication-competent OV, and could not be potentiated by UV-inactivated MG1 or supernatants from MG1-infected cells. The ability of MG1 to target the HIV reservoir was further confirmed using alveolar macrophages collected from the lungs of cART-suppressed individuals living with HIV. These findings indicate that IFN1 defects are a feature of HIV infected cells, which can be exploited for selective killing by OV. This project is therefore unique in that it demonstrates that HIV reservoir cells can be eradicated in a targeted manner by exploiting an intracellular marker of HIV infection. As MG1-based cancer therapies are currently being explored in Phase I/II clinical trials, there is potential for this approach to be adapted for use within the HIV cure field.

Owing to their key role in mucosal functioning as surface barriers with biospecific interaction potentials, the mucins are interesting candidates for use as surface modifiers in biomaterials applications. In this work, “mild” fractionation procedures were used to prepare mucins of bovine (BSM), porcine (PGM), and human (MG1) origin. Biophysicochemical analysis showed the prepared mucins to differ in size, charge, conformation, and composition. In turn, these factors were shown to govern mucin adsorption on hydrophilic and hydrophobic model surfaces. To enable for detailed coating analysis, methods for the qualitative and quantitative analysis of mucin-based coatings were developed. Of particular interest, a method for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating was described. It was shown, using microscopy and activation assays, that mucin precoating effectively suppresses the neutrophil response towards a polymeric model biomaterial. Under optimal coating conditions, all mucins performed equally well, thus indicating them to be functionally similar. Coating analysis suggested that efficient mucin surface-shielding is critical for good mucin coating performance. Following a study on the complexation of albumin with preadsorbed mucin, we investigated the effect of mucin precoating on the conformation and neutrophil-activating properties of adsorbed host proteins. We found that mucin precoating greatly reduces the strong immune-response normally caused by adsorbed proinflammatory proteins (IgG and sIgA). Detailed coating analysis revealed that the fraction of surface-exposed protein in the mucin-protein composite influences the neutrophil response. Unexpectedly low neutrophil activation for composites containing near-monolayer concentrations of exposed IgG, suggested IgG to act synergistically with mucin on the surface. Conformational analysis supported this by showing that a preadsorbed mucin layer could stabilize adsorbed IgG through complexation. Our findings link well to the complex in vivo situation and suggest that functional mucosal mimics can be created in situ for improved biomaterials performance.