Replication-incompetent influenza A viruses armed with IFN-γ effectively mediate immune modulation and tumor destruction in mice harboring lung cancer.

Low pathogenic influenza A viruses (IAVs) have shown promising oncolytic potential in lung cancer-bearing mice. However, as replication-competent pathogens, they may cause side effects in immunocompromised cancer patients. To circumvent this problem, we genetically engineered nonreplicating IAVs lacking the hemagglutinin (HA) gene (ΔHA IAVs), but reconstituted the viral envelope with recombinant HA proteins to allow a single infection cycle. To optimize the therapeutic potential and improve immunomodulatory properties, these replication-incompetent IAVs were complemented with a murine interferon-gamma (mIFN-γ) gene. After intratracheal administration to transgenic mice that develop non-small cell lung cancer (NSCLC), the ΔHA IAVs induced potent tumor destruction. However, ΔHA IAVs armed with mIFN-γ exhibited an even stronger and more sustained effect, achieving 85% tumor reduction at day 12 postinfection. In addition, ΔHA-mIFN-γ viruses were proven to be efficient in recruiting and activating natural killer cells and macrophages from the periphery and in inducing cytotoxic T lymphocytes. Most important, both viruses, and particularly IFN-γ-encoding viruses, activated tumor-associated alveolar macrophages toward a proinflammatory M1-like phenotype. Therefore, replication-incompetent ΔHA-mIFN-γ-IAVs are safe and efficient oncolytic viruses that additionally exhibit immune cell activating properties and thus represent a promising innovative therapeutic option in the fight against NSCLC.

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.

PD-1 IC Inhibition Synergistically Improves Influenza A Virus-Mediated Oncolysis of Metastatic Pulmonary Melanoma.

Recently, we showed that infection of primary lung tumor-bearing mice with oncolytic influenza A viruses (IAVs) led to strong virus-induced tumor cell lysis but also to restoration of immune competence of innate immune cells. Murine B16-F10 melanoma cells are known for their high lung tropism and progressive growth. As these cells are also highly permissive for IAVs, we analyzed their oncolytic and immunomodulatory efficiency against pulmonary B16-F10 lung metastases in vivo. IAV infection abrogated the melanoma-mediated immune suppression in the lung and induced a more than 50% cancer cell lysis. The oncolytic effect reached maximal efficacy 3 days post-infection, but it was not sustained over time. In order to maintain the virus-induced anti-tumor effect, mice with melanoma-derived lung cancers were treated in addition to influenza virus infection with an immune checkpoint inhibitor against programmed death-1 receptor (PD-1). The combined IAV and immune checkpoint inhibition (ICI) therapy resulted in a sustained anti-tumor efficacy, keeping the lung melanoma mass at day 12 of IAV infection still reduced by 50% over the control mice. In conclusion, ICI treatment strongly enhanced the oncolytic effect of influenza virus infection, suggesting that combined treatment is a promising approach against metastatic pulmonary melanoma.

The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model.

Pattern recognition receptors (PRRs) are key elements in the innate immune response. Formyl peptide receptor (FPR) 2 is a PRR that, in addition to proinflammatory, pathogen-derived compounds, also recognizes the anti-inflammatory endogenous ligand annexin A1 (AnxA1). Because the contribution of this signaling axis in viral infections is undefined, we investigated AnxA1-mediated FPR2 activation on influenza A virus (IAV) infection in the murine model. AnxA1-treated mice displayed significantly attenuated pathology upon a subsequent IAV infection with significantly improved survival, impaired viral replication in the respiratory tract, and less severe lung damage. The AnxA1-mediated protection against IAV infection was not caused by priming of the type I IFN response but was associated with an increase in the number of alveolar macrophages (AMs) and enhanced pulmonary expression of the AM-regulating cytokine granulocyte-M-CSF (GM-CSF). Both AnxA1-mediated increase in AM levels and GM-CSF production were abrogated when mouse (m)FPR2 signaling was antagonized but remained up-regulated in mice genetically deleted for mFPR1, an mFPR2 isoform also serving as AnxA1 receptor. Our results indicate a novel protective function of the AnxA1-FPR2 signaling axis in IAV pathology via GM-CSF-associated maintenance of AMs, expanding knowledge on the potential use of proresolving mediators in host defense against pathogens.-Schloer, S., Hübel, N., Masemann, D., Pajonczyk, D., Brunotte, L., Ehrhardt, C., Brandenburg, L.-O., Ludwig, S., Gerke, V., Rescher, U. The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model.

Oncolytic influenza virus infection restores immunocompetence of lung tumor-associated alveolar macrophages.

Non-small-cell lung cancer (NSCLC) is the most frequent type of lung cancer and demonstrates high resistance to radiation and chemotherapy. These tumors evade immune system detection by promoting an immunosuppressive tumor microenvironment. Genetic analysis has revealed oncogenic activation of the Ras/Raf/MEK/ERK signaling pathway to be a hallmark of NSCLCs, which promotes influenza A virus (IAV) infection and replication in these cells. Thus, we aimed to unravel the oncolytic properties of IAV infection against NSCLCs in an immunocompetent model in vivo. Using Raf-BxB transgenic mice that spontaneously develop NSCLCs, we demonstrated that infection with low-pathogenic IAV leads to rapid and efficient oncolysis, eliminating 70% of the initial tumor mass. Interestingly, IAV infection of Raf-BxB mice caused a functional reversion of immunosuppressed tumor-associated lung macrophages into a M1-like pro-inflammatory active phenotype that additionally supported virus-induced oncolysis of cancer cells. Altogether, our data demonstrate for the first time in an immunocompetent in vivo model that oncolytic IAV infection is capable of restoring and redirecting immune cell functions within the tumor microenvironment of NSCLCs.

Employing RNA viruses to fight cancer: novel insights into oncolytic virotherapy.

Within recent decades, viruses that specifically target tumor cells have emerged as novel therapeutic agents against cancer. These viruses do not only act via their cell-lytic properties, but also harbor immunostimulatory features to re-direct the tumor microenvironment and stimulate tumor-directed immune responses. Furthermore, oncolytic viruses are considered to be superior to classical cancer therapies due to higher selectivity towards tumor cell destruction and, consequently, less collateral damage of non-transformed healthy tissue. In particular, the field of oncolytic RNA viruses is rapidly developing since these agents possess alternative tumor-targeting strategies compared to established oncolytic DNA viruses. Thus, oncolytic RNA viruses have broadened the field of virotherapy facilitating new strategies to fight cancer. In addition to several naturally occurring oncolytic viruses, genetically modified RNA viruses that are armed to express foreign factors such as immunostimulatory molecules have been successfully tested in early clinical trials showing promising efficacy. This review aims to provide an overview of the most promising RNA viruses in clinical development, to summarize the current knowledge of clinical trials using these viral agents, and to discuss the main issues as well as future perspectives of clinical approaches using oncolytic RNA viruses.

Macrophage-mediated psoriasis can be suppressed by regulatory T lymphocytes.

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis-like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro-inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis-like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF-mediated psoriasis, ihTNFtg mice were crossbred into RAG1KO mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1KO or wild-type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild-type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3-positive cells into RAG1KO or wild-type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro-inflammatory activity of macrophages, which are the major immune effector cells in hTNF-mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In vitro and in vivo antitumor activity of a novel semisynthetic derivative of cucurbitacin B.

Lung cancer is the most deadly type of cancer in humans, with non-small-cell lung cancer (NSCLC) being the most frequent and aggressive type of lung cancer showing high resistance to radiation and chemotherapy. Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task. Here we describe a new semisynthetic derivative of cucurbitacin B (DACE) as a potent inhibitor of NSCLC cell proliferation. DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3. Consistent with our in vitro studies, intraperitoneal application of DACE significantly suppressed the growth of mouse NSCLC that arises from type II alveolar pneumocytes due to constitutive expression of a human oncogenic c-RAF kinase (c-RAF-1-BxB) transgene in these cells. Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug.

MAPKAP kinase 3 suppresses Ifng gene expression and attenuates NK cell cytotoxicity and Th1 CD4 T-cell development upon influenza A virus infection.

MK2 and MK3 are downstream targets of p38 and ERK1/2. They control the mRNA stability of several inflammatory cytokines, including TNF-α and IL-10. Whereas MK2 is expressed ubiquitously, the expression of MK3 is restricted to muscle, liver, and heart tissues and T and NK cells. Using Mk-deficient and wild-type (WT) mice, we demonstrated an inhibitory effect of MK3, but not of MK2, on interferon (IFN)-γ expression in T and NK lymphocytes. The results provided evidence that the inhibitory effect of MK3 is based on negative feedback phosphorylation of p38 and ERK1/2, which causes decreased binding of Stat4 to the IFN-γ promoter and reduced expression of IFN-γ mRNA and protein. Consequently, all Mk3(-/-) mice challenged with the Th1-inducing influenza A virus (IAV) survived the WT LD50 virus dose. The reduced disease severity in the Mk3(-/-) mice was accompanied by a >10-fold reduction in viral lung titer and an increase in the number of activated NK cells and enhanced Th1 activation of CD4 T cells. Thus, our data describe the protein kinase MK3 as a novel regulator of the innate and adaptive immune responses.-Köther, K., Nordhoff, C., Masemann, D., Varga, G., Bream, J. H., Gaestel, M., Wixler, V., Ludwig, S. MAPKAP kinase 3 suppresses Ifng gene expression and attenuates NK cell cytotoxicity and Th1 CD4 T-cell development upon influenza A virus infection.