Immunomodulation and Vaccination with RHDV VLP

Abstract

Colorectal cancer (CRC) is an insidious disease, responsible for almost 700,000 deaths annually worldwide. The incidence of CRC in New Zealand is more than double the world average, at 48.1 and 36.7 cases per 100,000 annually for men and women respectively, with the highest rates of distant metastases on presentation around the world at 24% of cases. The predominance of this late stage presentation may be attributed to poor public awareness, exacerbated by the current absence of the nationwide screening program that is due to begin in 2017. Combined with limited long-term treatment options, the prognosis for patients diagnosed with CRC in New Zealand is relatively poor. The availability of alternative treatment options, such as immunotherapy, may help to improve patient outcomes, promoting remission and recovery while limiting the development of adverse side effects. Immunotherapy involves the engagement and manipulation of the patients own immune system, re-educating the effector cells of the immune system to recognise, respond and eliminate tumour cells. One of the most promising types of immunotherapy is vaccination, capable of reinvigorating the immune system to recognise and target molecules associated with CRC.

This study used virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) as a vaccine construct for the treatment of CRC. VLP are a form of subunit vaccine composed of components derived from a virus, but without the ability to infect or replicate. These inert shells can be modified to incorporate heterologous antigens, such as those derived from tumour cells, as well as additional molecules that can manipulate and modify the immune responses induced. In previous work undertaken with RHDV VLP, these molecules have included adjuvants such as α-galactosylceramide and deoxyribonucleic acid (DNA) oligonucleotides containing unmethylated CpG islands (CpGs), as well as compounds that promote uptake, such as mannose and surface conjugated CpGs. During this study, it was found that RHDV VLP have a natural affinity for CpGs, facilitating the co-delivery of this adjuvant into antigen-presenting cells (APCs) that uptake VLP. This property was identified based on the protection of CpG oligonucleotides from DNase digestion in the presence of RHDV VLP, and the further retention of CpGs following dialysis. The functionality of CpGs associated with RHDV VLP was investigated using the RAW-Blue reporter cell line, and confirmed in assays evaluating the activation of murine bone marrow-derived dendritic cells (BMDCs). CpGs delivered in association with RHDV VLP were found to induce enhanced activation of BMDCs in comparison to an equivalent quantity of CpGs administered alone. This enhancement could not be explained by the direct effects of RHDV VLP on these cells, as VLP alone was found to be slightly suppressive relative to untreated controls. RHDV VLP were further found to have a natural affinity for other short nucleic acids, such as short interfering RNA (siRNA). This affinity was identified following dialysis of siRNA laced RHDV VLP. The efficacy of siRNA delivery was investigated using signal transducer and activator of transduction (STAT) 3 siRNA, based upon its ability to knockdown STAT3 protein levels in a model cell line following administration in combination with RHDV VLP. The functionality of STAT3 siRNA delivery by RHDV VLP was further investigated based on the hypothesised ability to interfere with interleukin-6 (IL-6) secretion in activated murine macrophages. Although RHDV VLP was found to be capable of delivering STAT3 siRNA to murine macrophages, inducing a significant knockdown in STAT3 protein levels, this effect did not functionally translate into significant alterations in IL-6 secretion. RHDV VLP-based vaccines developed for the treatment of cancer have previously included the incorporation of model antigens, limiting direct relevance of these vaccines to targeting true tumour-associated antigens. More recently, a study involving the incorporation of the melanoma-associated antigen gp100 proved that RHDV VLP-based vaccines can induce responses against tumour-associated antigens. However, this study also showed that targeting gp100 alone was insufficient for inducing responses that could induce complete remission of murine melanoma tumours, indicating that escape mechanisms remained intact. This may be overcome by inducing simultaneous immune responses against multiple tumour-associated antigens, potentially limited the ability of tumours to adapt and escape. In order to test this hypothesis, RHDV VLP were produced that contained an epitope derived from murine topoisomerase IIα (T.VP60), an epitope derived from murine survivin (S.VP60) or both in combination (TS.VP60). The identity of these chimaeric VLP was confirmed through sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), electron microscopy and mass spectrometry. Their ability to induce targeted immune responses against their respective epitopes was assessed with in vivo cytotoxicity assays. The efficacy of these chimaeric VLP constructs to combat subcutaneously engrafted MC38-OVA murine CRC tumours in combination with CpGs as an adjuvant was evaluated in therapeutic tumour trials, with RHDV VLP containing the model chicken ovalbumin epitope SIINFEKL (SIIN.VP60) used as a model control. Both T.VP60 and S.VP60 delayed the growth of MC38-OVA tumours with comparable efficacy to SIIN.VP60, attributable to 60% overall survival amongst mice combined across all trials. TS.VP60 exhibited further delays in the growth of MC38-OVA tumours, attributable to 67% overall survival amongst mice combined across all trials. Although this trend for enhanced efficacy with vaccination targeting multiple tumour-epitopes was repeatable between trials, the difference between mono- and multi-target therapies was not statistically significant. All mice that survived following vaccination were rechallenged with a subcutaneous MC38-OVA tumour on the opposing flank at day 100, and by day 200 none of these mice had grown tumours. Although these results are promising, combinations of greater than two target antigens may be required to provide significant benefits with a multi-target vaccine. Finally, RHDV VLP was assessed for its ability to be expressed by the oncolytic virus Vesicular stomatitis virus (VSV). The purpose of this investigation was to facilitate the development of a combination vaccine capable of utilising the targeted tumour cell-specific lytic infection of an oncolytic virus, and the therapeutic vaccination properties of RHDV VLP. To this end, recombinant VSV expressing RHDV VP60 (VSV-VP60), SIIN.VP60 (VSV-SIIN.VP60) and TS.VP60 (VSV-TS.VP60) were produced. The viability of recombinant VSV was assessed by plaque assay, the incorporation of each VLP construct was confirmed through sequencing, and the production of each VLP in baby hamster kidney (BHK) cells was identified by Western blot and electron microscopy. In MC38-OVA tumour trials, none of the VLP expressing VSV constructs provided significant benefits in either growth rate or survival. The absence of the usual oncolytic properties of VSV may be due to impaired or altered activity in the MC38-OVA murine CRC model system, or through impairment in combination with RHDV VLP expression. The apparent inefficacy of the VLP vaccines expressed by each VSV construct may be due to inadequate dosage, with in vivo expression from the administered quantity of recombinant VSV possibly incapable of recapitulating levels known to be required for successful RHDV VLP vaccination. These results indicate that although VSV is able to express RHDV VLP, further work is required to facilitate the development of this potential combination therapy.