Type I Interferon α/ß Receptor-Mediated Signaling Negatively Regulates Antiviral Cytokine Responses in Murine Bone-Marrow-Derived Mast Cells and Protects the Cells from Virus-Induced Cell Death.

Mast cells (MCs) are critical for initiating inflammatory responses to pathogens including viruses. Type I interferons (IFNs) that exert their antiviral functions by interacting with the type I IFN receptor (IFNAR) play a central role in host cellular responses to viruses. Given that virus-induced excessive toxic inflammatory responses are associated with aberrant IFNAR signaling and considering MCs are an early source of inflammatory cytokines during viral infections, we sought to determine whether IFNAR signaling plays a role in antiviral cytokine responses of MCs. IFNAR-intact, IFNAR-blocked, and IFNAR-knockout (IFNAR-/-) bone-marrow-derived MCs (BMMCs) were treated in vitro with a recombinant vesicular stomatitis virus (rVSVΔm51) to assess cytokine production by these cells. All groups of MCs produced the cytokines interleukin-6 and tumor necrosis factor-α in response to rVSVΔm51. However, production of the cytokines was lowest in IFNAR-intact cells as compared with IFNAR-/- or IFNAR-blocked cells at 20 h post-stimulation. Surprisingly, rVSVΔm51 was capable of infecting BMMCs, but functional IFNAR signaling was able to protect these cells from virus-induced death. This study showed that BMMCs produced pro-inflammatory cytokines in response to rVSVΔm51 and that IFNAR signaling was required to down-modulate these responses and protect the cells from dying from viral infection.

Recombinant Newcastle disease viruses expressing immunological checkpoint inhibitors induce a pro-inflammatory state and enhance tumor-specific immune responses in two murine models of cancer.

Introduction: Tumor microenvironments are immunosuppressive due to progressive accumulation of mutations in cancer cells that can drive expression of a range of inhibitory ligands and cytokines, and recruitment of immunomodulatory cells, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages, and regulatory T cells (Tregs). Methods: To reverse this immunosuppression, we engineered mesogenic Newcastle disease virus (NDV) to express immunological checkpoint inhibitors anti-cytotoxic T lymphocyte antigen-4 and soluble programmed death protein-1. Results: Intratumoral administration of recombinant NDV (rNDV) to mice bearing intradermal B16-F10 melanomas or subcutaneous CT26LacZ colon carcinomas led to significant changes in the tumor-infiltrating lymphocyte profiles. Vectorizing immunological checkpoint inhibitors in NDV increased activation of intratumoral natural killer cells and cytotoxic T cells and decreased Tregs and MDSCs, suggesting induction of a pro-inflammatory state with greater infiltration of activated CD8+ T cells. These notable changes translated to higher ratios of activated effector/suppressor tumor-infiltrating lymphocytes in both cancer models, which is a promising prognostic marker. Whereas all rNDV-treated groups showed evidence of tumor regression and increased survival in the CT26LacZ and B16-F10, only treatment with NDV expressing immunological checkpoint blockades led to complete responses compared to tumors treated with NDV only. Discussion: These data demonstrated that NDV expressing immunological checkpoint inhibitors could reverse the immunosuppressive state of tumor microenvironments and enhance tumor-specific T cell responses.

Mechanisms that allow vaccination against an oncolytic vesicular stomatitis virus-encoded transgene to enhance safety without abrogating oncolysis.

Vaccination can prevent viral infections via virus-specific T cells, among other mechanisms. A goal of oncolytic virotherapy is replication of oncolytic viruses (OVs) in tumors, so pre-existing T cell immunity against an OV-encoded transgene would seem counterproductive. We developed a treatment for melanomas by pre-vaccinating against an oncolytic vesicular stomatitis virus (VSV)-encoded tumor antigen. Surprisingly, when the VSV-vectored booster vaccine was administered at the peak of the primary effector T cell response, oncolysis was not abrogated. We sought to determine how oncolysis was retained during a robust T cell response against the VSV-encoded transgene product. A murine melanoma model was used to identify two mechanisms that enable this phenomenon. First, tumor-infiltrating T cells had reduced cytopathic potential due to immunosuppression. Second, virus-induced lymphopenia acutely removed virus-specific T cells from tumors. These mechanisms provide a window of opportunity for replication of oncolytic VSV and rationale for a paradigm change in oncolytic virotherapy, whereby immune responses could be intentionally induced against a VSV-encoded melanoma-associated antigen to improve safety without abrogating oncolysis.

Using a Prime-Boost Vaccination Strategy That Proved Effective for High Resolution Epitope Mapping to Characterize the Elusive Immunogenicity of Survivin.

Survivin is a member of the inhibitor of apoptosis family of proteins and has been reported to be highly expressed in a variety of cancer types, making it a high priority target for cancer vaccination. We previously described a heterologous prime-boost strategy using a replication-deficient adenovirus, followed by an oncolytic rhabdovirus that generates unprecedented antigen-specific T cell responses. We engineered each vector to express a mutated version of full-length murine survivin. We first sought to uncover the complete epitope map for survivin-specific T cell responses in C57BL/6 and BALB/c mice by flow cytometry. However, no T cell responses were detected by intracellular cytokine staining after re-stimulation of T cells. Survivin has been found to be expressed by activated T cells, which could theoretically cause T cell-mediated killing of activated T cells, known as fratricide. We were unable to recapitulate this phenomenon in experiments. Interestingly, the inactivated survivin construct has been previously shown to directly kill tumor cells in vitro. However, there was no evidence in our models of induction of death in antigen-presenting cells due to treatment with a survivin-expressing vector. Using the same recombinant virus-vectored prime-boost strategy targeting the poorly immunogenic enhanced green fluorescent protein proved to be a highly sensitive method for mapping T cell epitopes, particularly in the context of identifying novel epitopes recognized by CD4+ T cells. Overall, these results suggested there may be unusually robust tolerance to survivin in commonly used mouse strains that cannot be broken, even when using a particularly potent vaccination platform. However, the vaccination method shows great promise as a strategy for identifying novel and subdominant T cell epitopes.

Intranasal vaccination with a Newcastle disease virus-vectored vaccine protects hamsters from SARS-CoV-2 infection and disease.

The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Worldwide efforts are being made to develop vaccines to mitigate this pandemic. We engineered two recombinant Newcastle disease virus (NDV) vectors expressing either the full-length SARS-CoV-2 spike protein (NDV-FLS) or a version with a 19 amino acid deletion at the carboxy terminus (NDV-Δ19S). Hamsters receiving two doses (prime-boost) of NDV-FLS developed a robust SARS-CoV-2-neutralizing antibody response, with elimination of infectious virus in the lungs and minimal lung pathology at five days post-challenge. Single-dose vaccination with NDV-FLS significantly reduced SARS-CoV-2 replication in the lungs but only mildly decreased lung inflammation. NDV-Δ19S-treated hamsters had a moderate decrease in SARS-CoV-2 titers in lungs and presented with severe microscopic lesions, suggesting that truncation of the spike protein was a less effective strategy. In summary, NDV-vectored vaccines represent a viable option for protection against COVID-19.

Tumour vasculature: Friend or foe of oncolytic viruses?

In the past two decades there have been substantial advances in understanding the anti-cancer mechanisms of oncolytic viruses (OVs). OVs can mediate their effects directly, by preferentially infecting and killing tumour cells. Additionally, OVs can indirectly generate anti-tumour immune responses. These differing mechanisms have led to a paradoxical divergence in strategies employed to further increase the potency of oncolytic virotherapies. On one hand, the tumour neovasculature is seen as a vital lifeline to the survival of the tumour, leading some to use OVs to target the tumour vasculature in hopes to starve cancers. Therapeutics causing vascular collapse can potentiate tumour hypoxia, nutrient restriction and pro-inflammatory cytokine release, which has shown promise in oncological studies. On the other hand, the same vasculature plays an important role for the dissemination of OVs, trafficking of effector cells and other therapeutics, which has prompted researchers to find ways of normalizing the vasculature to enhance infiltration of leukocytes and delivery of therapeutic agents. This article describes the recent developments of therapies aimed to shut down versus normalize tumour vasculature in order to inform researchers striving to optimize OV-based therapies.

Myeloid Cells during Viral Infections and Inflammation.

Myeloid cells represent a diverse range of innate leukocytes that are crucial for mounting successful immune responses against viruses. These cells are responsible for detecting pathogen-associated molecular patterns, thereby initiating a signaling cascade that results in the production of cytokines such as interferons to mitigate infections. The aim of this review is to outline recent advances in our knowledge of the roles that neutrophils and inflammatory monocytes play in initiating and coordinating host responses against viral infections. A focus is placed on myeloid cell development, trafficking and antiviral mechanisms. Although known for promoting inflammation, there is a growing body of literature which demonstrates that myeloid cells can also play critical regulatory or immunosuppressive roles, especially following the elimination of viruses. Additionally, the ability of myeloid cells to control other innate and adaptive leukocytes during viral infections situates these cells as key, yet under-appreciated mediators of pathogenic inflammation that can sometimes trigger cytokine storms. The information presented here should assist researchers in integrating myeloid cell biology into the design of novel and more effective virus-targeted therapies.

Use of Precision-Cut Lung Slices as an Ex Vivo Tool for Evaluating Viruses and Viral Vectors for Gene and Oncolytic Therapy.

Organotypic slice cultures recapitulate many features of an intact organ, including cellular architecture, microenvironment, and polarity, making them an ideal tool for the ex vivo study of viruses and viral vectors. Here, we describe a procedure for generating precision-cut ovine and murine tissue slices from agarose-perfused normal and murine melanoma tumor-bearing lungs. Furthermore, we demonstrate that these precision-cut lung slices can be maintained up to 1 month and can be used for a range of applications, which include characterizing the tissue tropism of viruses that cannot be propagated in cell monolayers, evaluating the transducing properties of gene therapy vectors, and, finally, investigating the tumor specificity of oncolytic viruses. Our results suggest that ex vivo lung slices are an ideal platform for studying the tissue specificity and cancer cell selectivity of gene therapy vectors and oncolytic viruses prior to in vivo studies, providing justification for pre-clinical work.

Immune responses in the thyroid cancer microenvironment: making immunotherapy a possible mission.

The incidence of thyroid cancers has been steadily increasing worldwide over the past few decades. Although five-year survival rates for differentiated thyroid cancers are upwards of 90%, clinical outcomes for patients with undifferentiated, recurrent and/or metastatic disease are often dismal despite conventional interventions. As such, there is a demand for novel treatment options. Cancer immunotherapy represents the ultimate form of personalized medicine by leveraging the specificity and potency of a patient's immune system to kill their tumor. The thyroid cancer microenvironment is rich in immunological cells, making it a reasonable candidate for immunotherapy. This review maps out the immunological features of thyroid cancers and how these can be modulated. There are surprising immunological consequences of conventional therapies that demand attention. Also, hormonal modulation of the immune system is highlighted as a unique and confounding feature of thyroid cancers. A variety of cutting-edge immune-based therapies are discussed, with an emphasis placed on how these can be integrated with the current standard of care. Several high priority areas in need of research are also highlighted.

Enhancing Immune Responses to Cancer Vaccines Using Multi-Site Injections.

For a vaccine to be effective it must induce a sufficiently robust and specific immune response. Multi-site injection protocols can increase the titers of rabies virus-neutralizing antibodies. Hypothetically, spreading a vaccine dose across multiple lymphatic drainage regions could also potentiate T cell responses. We used a replication-deficient adenovirus serotype 5-vectored cancer vaccine targeting the melanoma-associated antigen dopachrome tautomerase. Clinically, high numbers of tumor-infiltrating CD8+ T cells are a positive prognostic indicator. As such, there is interest in maximizing tumor-specific T cell responses. Our findings confirm a positive correlation between the number of tumor-specific T cells and survival. More importantly, we show for the first time that using multiple injection sites could increase the number of vaccine-induced CD8+ T cells specific for a self-tumor antigen. Further, the number of tumor antigen-specific antibodies, as well CD8+ T cells specific for a foreign antigen could also be enhanced. Our results show that multi-site vaccination induces higher magnitude immune responses than a single-bolus injection. This provides a very simple and almost cost-free strategy to potentially improve the efficacy of any current and future vaccine. Broader clinical adoption of multi-site vaccination protocols for the treatment of cancers and infectious diseases should be given serious consideration.