Natural killer T cell immunotherapy combined with IL-15-expressing oncolytic virotherapy and PD-1 blockade mediates pancreatic tumor regression.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death, with a 5-year -year survival rate of less than 10%. This results from late detection, high rates of metastasis, and resistance to standard chemotherapies. Furthermore, chemotherapy and radiation are associated with significant morbidity, underscoring the need for novel therapies. Recent clinical studies have shown that immunotherapies can provide durable outcomes in cancer patients, but successes in pancreatic cancer have been limited. It is likely that novel and combined therapies will be needed to achieve clinical benefits. METHODS: Using experimental mouse models of pancreatic ductal adenocarcinoma, we examined natural killer T (NKT) cell activation therapy in combination with a recombinant oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express the cytokine IL-15 (VSV-IL-15). Panc02 pancreatic ductal carcinoma cells were implanted subcutaneously or orthotopically into syngeneic C57BL/6 mice. Mice were then treated with VSV expressing green fluorescent protein (VSV-GFP) or VSV-IL-15 and/or NKT cell activation therapy via delivery of α-GalCer-loaded DCs. We further assessed whether the addition of PD-1 blockade could increase the therapeutic benefit of our combination treatment. Three days after NKT cell activation, some groups of mice were treated with anti-PD-1 antibodies weekly for 3 weeks. RESULTS: VSV-GFP and VSV-IL-15 mediated equal killing of human and mouse pancreatic cancer lines in vitro. In vivo, VSV-IL-15 combined with NKT cell activation therapy to enhance tumor regression and increase survival time over individual treatments, and was also superior to NKT cell therapy combined with VSV-GFP. Enhanced tumor control was associated with increased immune cell infiltration and anti-tumor effector functions (cytotoxicity and cytokine production). While ineffective as a monotherapy, the addition of blocking PD-1 antibodies to the combined protocol sustained immune cell activation and effector functions, resulting in prolonged tumor regression and complete tumor clearance in 20% of mice. Mice who cleared the initial tumor challenge exhibited reduced tumor growth uponon rechallenge, consistent with the formation of immune memory. CONCLUSION: TThese results demonstrate that NKT cell immunotherapy combined with oncolytic VSV-IL-15 virotherapy and PD-1 blockade enhances tumor control and presents a promising treatment strategy for targeting pancreatic cancer.

Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

Aerosol delivery, but not intramuscular injection, of adenovirus-vectored tuberculosis vaccine induces respiratory-mucosal immunity in humans.

BackgroundAdenovirus-vectored (Ad-vectored) vaccines are typically administered via i.m. injection to humans and are incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized, and its ability to induce mucosal immunity in humans is unknown. This phase Ib trial evaluated the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or i.m. injection.MethodsThirty-one healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb Solo Nebulizer or by i.m. injection. The study consisted of the low-dose (LD) aerosol, high-dose (HD) aerosol, and i.m. groups. The adverse events were assessed at various times after vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline, as well as at select time points after vaccination.ResultsThe nebulized aerosol droplets were < 5.39 µm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and i.m. injection were safe and well tolerated. Both aerosol doses, particularly LD, but not i.m., vaccination markedly induced airway tissue-resident memory CD4+ and CD8+ T cells of polyfunctionality. While as expected, i.m. vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages.ConclusionInhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens, including TB and COVID-19.Trial registrationClinicalTrial.gov, NCT02337270.FundingThe Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada funded this work.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

CXCR6 by increasing retention of memory CD8+ T cells in the ovarian tumor microenvironment promotes immunosurveillance and control of ovarian cancer.

PURPOSE: Resident memory CD8 T cells, owing to their ability to reside and persist in peripheral tissues, impart adaptive sentinel activity and amplify local immune response, and have beneficial implications for tumor surveillance and control. The current study aimed to clarify the less known chemotactic mechanisms that govern the localization, retention, and residency of memory CD8 T cells in the ovarian tumor microenvironment. EXPERIMENTAL DESIGN: RNA and protein expressions of chemokine receptors in CD8+ resident memory T cells in human ovarian tumor-infiltrating CD8+ T cells and their association with survival were analyzed. The role of CXCR6 on antitumor T cells was investigated using prophylactic vaccine models in murine ovarian cancer. RESULTS: Chemokine receptor profiling of CD8+CD103+ resident memory tumor-infiltrating lymphocytes in patients with ovarian cancer revealed high expression of CXCR6. Analysis of The Cancer Genome Atlas (TCGA) (ovarian cancer database revealed CXCR6 to be associated with CD103 and increased patient survival. Functional studies in mouse models of ovarian cancer revealed that CXCR6 is a marker of resident, but not circulatory, tumor-specific memory CD8+ T cells. CXCR6-deficient tumor-specific CD8+ T cells showed reduced retention in tumor tissues, leading to diminished resident memory responses and poor control of ovarian cancer. CONCLUSIONS: CXCR6, by promoting retention in tumor tissues, serves a critical role in resident memory T cell-mediated immunosurveillance and control of ovarian cancer. Future studies warrant exploiting CXCR6 to promote resident memory responses in cancers.

Synergistic anti-tumor efficacy of oncolytic influenza viruses and B7-H3 immune- checkpoint inhibitors against IC-resistant lung cancers.

Non-small cell lung cancers (NSCLCs) establish a highly immunosuppressive tumor microenvironment supporting cancer growth. To interfere with cancer-mediated immunosuppression, selective immune-checkpoint inhibitors (ICIs) have been approved as a standard-of-care treatment for NSCLCs. However, the majority of patients poorly respond to ICI-based immunotherapies. Oncolytic viruses are amongst the many promising immunomodulatory treatments tested as standalone therapy or in combination with ICIs to improve therapeutic outcome. Previously, we demonstrated the oncolytic and immunomodulatory efficacy of low-pathogenic influenza Aviruses (IAVs) against NSCLCs in immunocompetent transgenic mice with alung-specific overexpression of active Raf kinase (Raf-BxB). IAV infection not only resulted in significant primary virus-induced oncolysis, but also caused afunctional reversion of tumor-associated macrophages (TAMs) comprising additional anti-cancer activity. Here we show that NSCLCs as well as TAMs and cytotoxic immune cells overexpress IC molecules of the PD-L2/PD-1 and B7-H3 signaling axes. Thus, we aimed to combine oncolytic IAV-infection with ICIs to exploit the benefits of both anti-cancer approaches. Strikingly, IAV infection combined with the novel B7-H3 ICI led to increased levels of M1-polarized alveolar macrophages and increased lung infiltration by cytotoxic Tlymphocytes, which finally resulted in significantly improved oncolysis of about 80% of existing tumors. In contrast, application of clinically approved α-PD-1 IC antibodies alone or in combination with oncolytic IAV did not provide additional oncolytic or immunomodulatory efficacy. Thus, individualized therapy with synergistically acting oncolytic IAV and B7-H3 ICI might be an innovative future approach to target NSCLCs that are resistant to approved ICIs in patients.

RNA editing enzyme APOBEC3A promotes pro-inflammatory M1 macrophage polarization.

Pro-inflammatory M1 macrophage polarization is associated with microbicidal and antitumor responses. We recently described APOBEC3A-mediated cytosine-to-uracil (C > U) RNA editing during M1 polarization. However, the functional significance of this editing is unknown. Here we find that APOBEC3A-mediated cellular RNA editing can also be induced by influenza or Maraba virus infections in normal human macrophages, and by interferons in tumor-associated macrophages. Gene knockdown and RNA_Seq analyses show that APOBEC3A mediates C>U RNA editing of 209 exonic/UTR sites in 203 genes during M1 polarization. The highest level of nonsynonymous RNA editing alters a highly-conserved amino acid in THOC5, which encodes a nuclear mRNA export protein implicated in M-CSF-driven macrophage differentiation. Knockdown of APOBEC3A reduces IL6, IL23A and IL12B gene expression, CD86 surface protein expression, and TNF-α, IL-1ß and IL-6 cytokine secretion, and increases glycolysis. These results show a key role of APOBEC3A cytidine deaminase in transcriptomic and functional polarization of M1 macrophages.

Immunological considerations for COVID-19 vaccine strategies.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most formidable challenge to humanity in a century. It is widely believed that prepandemic normalcy will never return until a safe and effective vaccine strategy becomes available and a global vaccination programme is implemented successfully. Here, we discuss the immunological principles that need to be taken into consideration in the development of COVID-19 vaccine strategies. On the basis of these principles, we examine the current COVID-19 vaccine candidates, their strengths and potential shortfalls, and make inferences about their chances of success. Finally, we discuss the scientific and practical challenges that will be faced in the process of developing a successful vaccine and the ways in which COVID-19 vaccine strategies may evolve over the next few years.

Enhanced immunotherapeutic profile of oncolytic virus-based cancer vaccination using cyclophosphamide preconditioning.

Despite a sizeable body of research, the efficacy of therapeutic cancer vaccines remains limited when applied as sole agents. By using a prime:boost approach involving two viral cancer vaccines, we were able to generate large tumor-specific CD8+ T-cell responses in a murine model of disseminated pulmonary melanoma. Significant increases in the number and quality of circulating effector T-cells were documented when low-dose cyclophosphamide (CTX) was administered pre-vaccination to tumor-bearing but not tumor-free hosts. Interestingly, tumor-bearing mice receiving CTX and co-primed with a melanoma differentiation antigen together with an irrelevant control antigen exhibited significantly enhanced immunity against the tumor, but not the control antigen, in secondary lymphoid organs. This result highlighted an increased cancer-specific reactivity of vaccine-induced T-cell responses following CTX preconditioning. Additionally, an acute reduction of the frequency of peripheral regulatory T-cells (Tregs) was noticeable, particularly in the proliferating, presumably tumour-reactive, subset. Enhanced infiltration of lungs with multifunctional T-cells resulted in overt reduction in metastatic burden in mice pretreated with CTX. Despite doubling the median survival in comparison to untreated controls, most vaccinated mice ultimately succumbed to cancer progression. However, preconditioning of the virus-based vaccination with CTX resulted in a remarkable improvement of the therapeutic activity leading to complete remission in the majority of the animals. Collectively, these data reveal how CTX can potentiate specific cellular immunity in an antigen-restricted manner that is only observed in vaccinated tumor-bearing hosts while depleting replicating Tregs. A single low dose of CTX enhances antitumor immunity and the efficacy of this potent prime:boost platform by modulating the kinetics of the vaccine-specific responses. Clinical assessment of CTX combined with next-generation cancer vaccines is indicated.

Spray dried VSV-vectored vaccine is thermally stable and immunologically active in vivo.

Effective vaccine delivery and coverage to rural and resource-poor countries is hindered by the dependence on cold chain storage. As such, developments of cold chain-free technologies are highly sought. Although spray dried adenoviral vectors have shown long term stability at ambient temperatures and relatively low humidity, it remains to be determined whether similar excipient formulations are applicable to other viral vectors. To address this, we have spray dried vesicular stomatitis virus (VSV)-vectors with a panel of well-characterized sugar excipients to determine the optimal formulation for vector stabilization. Upon reconstitution, we show that trehalose conferred superior stability of VSV both in vitro and in vivo. Importantly, following cold chain-free storage at elevated temperatures at 37 °C for 15 days, we show that a VSV-vectored vaccine retains its in vivo immunogenicity, whereas a liquid control completely lost its immune-stimulating ability. Our results provide foundational evidence that spray drying with properly tested excipients can stabilize viral vectors such as VSV, allowing them to be stored long-term at elevated temperatures without dependency on cold chain conditions.

Measles Vaccines Designed for Enhanced CD8+ T Cell Activation.

Priming and activation of CD8+ T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics systems allow for insertion of additional genes, including heterologous antigens. Here, we designed recombinant measles vaccine vectors for priming and activation of antigen-specific CD8+ T cells. For proof-of-concept, we used cytotoxic T lymphocyte (CTL) lines specific for the melanoma-associated differentiation antigen tyrosinase-related protein-2 (TRP-2), or the model antigen chicken ovalbumin (OVA), respectively. We generated recombinant measles vaccine vectors with TRP-2 and OVA epitope cassette variants for expression of the full-length antigen or the respective immunodominant CD8+ epitope, with additional variants mediating secretion or proteasomal degradation of the epitope. We show that these recombinant measles virus vectors mediate varying levels of MHC class I (MHC-I)-restricted epitope presentation, leading to activation of cognate CTLs, as indicated by secretion of interferon-gamma (IFNγ) in vitro. Importantly, the recombinant OVA vaccines also mediate priming of naïve OT-I CD8+ T cells by dendritic cells. While all vaccine variants can prime and activate cognate T cells, IFNγ release was enhanced using a secreted epitope variant and a variant with epitope strings targeted to the proteasome. The principles presented in this study will facilitate the design of recombinant vaccines to elicit CD8+ responses against pathogens and tumor antigens.

Detection of Tumor Antigen-Specific T-Cell Responses After Oncolytic Vaccination.

Oncolytic vaccines, which consist of recombinant oncolytic viruses (OV) encoding tumor-associated antigens (TAAs), have demonstrated potent antitumor efficacy in preclinical models and are currently evaluated in phase I/II clinical trials. On one hand, oncolysis of OV-infected malignant entities reinstates cancer immunosurveillance. On the other hand, overexpression of TAAs in infected cells further stimulates the adaptive arm of antitumor immunity. Particularly, the presence of tumor-specific CD8+ T lymphocytes within the tumor microenvironment, as well as in the periphery, has demonstrated prognostic value for cancer treatments. These effector CD8+ T cells can be detected through their production of the prototypical Tc1 cytokine: IFN-γ. The quantitative and qualitative assessment of this immune cell subset remains critical in the development process of efficient cancer vaccines, including oncolytic vaccines. The present chapter will describe a single-cell immunological assay, namely the intracellular cytokine staining (ICS), that allows the enumeration of IFN-γ-producing TAA-specific CD8+ T cells in various tissues (tumor, blood, lymphoid organs) following oncolytic vaccination.

Endogenous T cells prevent tumor immune escape following adoptive T cell therapy.

While the outcome of adoptive T cell therapy (ACT) is typically correlated with the functionality of the inoculated T cells, the role of the endogenous T cells is unknown. The success of checkpoint blockade therapy has demonstrated the potentially curative value of preexisting tumor-primed T cells in cancer treatment. Given the results from checkpoint blockade therapy, we hypothesized that endogenous T cells contribute to long-term survival following ACT. Here, we describe a therapeutic approach combining ACT with an oncolytic vaccine that allows simultaneous analysis of antitumor immunity mediated by transferred and endogenous T cells. We found that, in addition to promoting the expansion and tumor infiltration of the transferred T cells, oncolytic vaccines boosted tumor-primed host T cells. We determined that transferred T cells contributed to rapid destruction of large tumor masses while endogenous T cells concurrently prevented the emergence of antigen-loss variants. Moreover, while transferred T cells disappeared shortly after tumor regression, endogenous T cells secured long-term memory with a broad repertoire of antigen specificity. Our findings suggest that this combination strategy may exploit the full potential of ACT and tumor-primed host T cells to eliminate the primary tumor, prevent immune escape, and provide long-term protective memory.

Oncolytic Maraba virus armed with tumor antigen boosts vaccine priming and reveals diverse therapeutic response patterns when combined with checkpoint blockade in ovarian cancer.

BACKGROUND: Cancer immunotherapies are emerging as promising treatment strategies for ovarian cancer patients that experience disease relapse following first line therapy. As such, identifying strategies to bolster anti-tumor immunity and limit immune suppression, while recognizing diverse patterns of tumor response to immunotherapy is critical to selecting treatment combinations that lead to durable therapeutic benefit. METHODS: Using a pre-clinical mouse model, we evaluated a heterologous prime/boost vaccine in combination with checkpoint blockade to treat metastatic intraperitoneal ovarian cancer. Vaccine-elicited CD8+ T cell responses and changes in the tumor microenvironment following treatment were analyzed and compared to treatment outcome. Kinetics of intraperitoneal tumor growth were assessed using non-invasive magnetic resonance imaging (MRI). RESULTS: Vaccine priming followed by antigen-armed oncolytic Maraba virus boosting elicited robust tumor-specific CD8+ T cell responses that improved tumor control and led to unique immunological changes in the tumor, including a signature that correlated with improved clinical outcome of ovarian cancer patients. However, this treatment was not curative and T cells in the tumor microenvironment (TME) were functionally suppressed. Combination PD-1 blockade partially overcame the adaptive resistance in the tumor observed in response to prime/boost vaccination, restoring CD8+ T cell function in the TME and enhancing the therapeutic response. Non-invasive MRI of tumors during the course of combination treatment revealed heterogeneous radiologic response patterns following treatment, including pseudo-progression, which was associated with improved tumor control prior to relapse. CONCLUSIONS: Our findings point to a key hierarchical role for PD-1 signaling and adaptive immune resistance in the ovarian TME in determining the functional fate of tumor-specific CD8+ T cells, even in the context of robust therapy mediated anti-tumor immunity, as well as the ability of multiple unique patterns of therapeutic response to result in durable tumor control.

Excipient selection for thermally stable enveloped and non-enveloped viral vaccine platforms in dry powders.

Two enveloped viral vectors, vesicular stomatitis virus and influenza virus, and a non-enveloped viral vector, human adenovirus type 5, were encapsulated by spray drying to enhance thermal stability.Results with these candidates led to the hypothesis that stability performance of chosen excipients may be less virus-specific, as previously postulated in the literature, and more differentiated based on whether the virus has a lipid envelope. Spray dried samples were characterized for their thermal properties, RNA viability and in vitro viral activity after storage at 37 °C for up to 30 days or at 45 °C for up to 3 days. The enveloped viral vectors, as a group, were more thermally stable in trehalose while the non-enveloped viral vector showed higher activity with mannitol as the primary excipient in blends. Trehalose shows strong hydrogen bonds with the envelope's lipid membrane than the other carbohydrates, more effectively replacing water molecules while maintaining the fluidity of the membrane. Conversely, the small size of mannitol molecules was attributed to the more effective hydrogen bonding between water and the protein capsid of non-enveloped viral vectors. In all cases, a matrix with high glass transition temperature contributed to thermal stabilization through vitrification. This work suggests that carbohydrate stabilizer selection may be more dependent on the envelope rather than the specific viral vector, which, if universally true, will provide a guideline for future formulation development.

Preclinical evaluation of a MAGE-A3 vaccination utilizing the oncolytic Maraba virus currently in first-in-human trials.

Multiple immunotherapeutics have been approved for cancer patients, however advanced solid tumors are frequently refractory to treatment. We evaluated the safety and immunogenicity of a vaccination approach with multimodal oncolytic potential in non-human primates (NHP) (Macaca fascicularis). Primates received a replication-deficient adenoviral prime, boosted by the oncolytic Maraba MG1 rhabdovirus. Both vectors expressed the human MAGE-A3. No severe adverse events were observed. Boosting with MG1-MAGEA3 induced an expansion of hMAGE-A3-specific CD4+ and CD8+ T-cells with the latter peaking at remarkable levels and persisting for several months. T-cells reacting against epitopes fully conserved between simian and human MAGE-A3 were identified. Humoral immunity was demonstrated by the detection of circulating MAGE-A3 antibodies. These preclinical data establish the capacity for the Ad:MG1 vaccination to engage multiple effector immune cell populations without causing significant toxicity in outbred NHPs. Clinical investigations utilizing this program for the treatment of MAGE-A3-positive solid malignancies are underway (NCT02285816, NCT02879760).

Development and applications of oncolytic Maraba virus vaccines.

Oncolytic activity of the MG1 strain of the Maraba vesiculovirus has proven efficacy in numerous preclinical cancer models, and relied not only on a direct cytotoxicity but also on the induction of both innate and adaptive antitumor immunity. To further expand tumor-specific T-cell effector and long-lasting memory compartments, we introduced the MG1 virus in a prime-boost cancer vaccine strategy. To this aim, a replication-incompetent adenoviral [Ad] vector together with the oncolytic MG1 have each been armed with a transgene expressing a same tumor antigen. Immune priming with the Ad vaccine subsequently boosted with the MG1 vaccine mounted tumor-specific responses of remarkable magnitude, which significantly prolonged survival in various murine cancer models. Based on these promising results, we validated the safety profile of the Ad:MG1 oncolytic vaccination strategy in nonhuman primates and initiated clinical investigations in cancer patients. Two clinical trials are currently under way (NCT02285816; NCT02879760). The present review will recapitulate the discoveries that led to the development of MG1 oncolytic vaccines from bench to bedside.

Transforming the prostatic tumor microenvironment with oncolytic virotherapy.

Prostate cancer (PCa) was estimated to have the second highest global incidence rate for male non-skin tumors and is the fifth most deadly in men thus mandating the need for novel treatment options. MG1-Maraba is a potent and versatile oncolytic virus capable of lethally infecting a variety of prostatic tumor cell lines alongside primary PCa biopsies and exerts direct oncolytic effects against large TRAMP-C2 tumors in vivo. An oncolytic immunotherapeutic strategy utilizing a priming vaccine and intravenously administered MG1-Maraba both expressing the human six-transmembrane antigen of the prostate (STEAP) protein generated specific CD8+ T-cell responses against multiple STEAP epitopes and resulted in functional breach of tolerance. Treatment of mice with bulky TRAMP-C2 tumors using oncolytic STEAP immunotherapy induced an overt delay in tumor progression, marked intratumoral lymphocytic infiltration with an active transcriptional profile and up-regulation of MHC class I. The preclinical data generated here offers clear rationale for clinically evaluating this approach for men with advanced PCa.

Preclinical development of peptide vaccination combined with oncolytic MG1-E6E7 for HPV-associated cancer.

Human papilloma virus (HPV)-associated cancer is a significant global health burden and despite the presence of viral transforming antigens within neoplastic cells, therapeutic vaccinations are ineffective for advanced disease. HPV positive TC1 cells are susceptible to viral oncolysis by MG1-E6E7, a custom designed oncolytic Maraba virus. Epitope mapping of mice vaccinated with MG1-E6E7 enabled the rational design of synthetic long peptide (SLP) vaccines against HPV16 and HPV18 antigens. SLPs were able to induce specific CD8+ immune responses and the magnitude of these responses significantly increased when boosted by MG1-E6E7. Logically designed vaccination induced multi-functional CD8+ T cells and provided complete sterilising immunity of mice challenged with TC1 cells. In mice bearing large HPV-positive tumours, SLP vaccination combined with MG1-E6E7 was able to clear tumours in 60% of mice and these mice were completely protected against a long term aggressive re-challenge with the TC1 tumour model. Combining conventional SLPs with the multi-functional oncolytic MG1-E6E7 represents a promising approach against advanced HPV positive neoplasia.

Consecutive Spray Drying to Produce Coated Dry Powder Vaccines Suitable for Oral Administration.

Current global vaccination programs are challenged by costs associated with vaccine cold chain storage and administration. A solid, thermally stable oral dosage form for vaccines would alleviate these costs but is difficult to produce due to general vaccine instability and the complication of bypassing the gastric barrier. We developed a novel consecutive spray drying method that is suitable for use with biologics and employs Eudragit L100 polymer as the enteric coating. More specifically, in step 1, recombinant replication deficient human type-5 adenovirus and vesicular stomatitis virus were encapsulated by spray drying with sugars from a water solution, and in step 2, the microparticles from step 1 were suspended in ethanol with Eudragit and spray dried again. Up to 25% of the starting material was fully encapsulated within the enteric coating, and encapsulation efficiency was largely dependent on spray gas flow rate and the solids concentration in the feed. After step 2, the coated vaccine-sugar particles maintained their thermostability and were slightly larger in size with a rugous surface morphology compared to the particles produced in step 1. The coated particles retained viral vector activity in vitro after 15 min incubation in 1 M HCl (simulating the stomach environment) and anhydrous ethanol (to dissolve the Eudragit outer shell). The production of dry, orally administered vaccine particles from consecutive spray drying offers the potential to remedy a number of vaccine storage, transportation, and administration limitations.