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Summary The immunological outcome of infections and vaccinations is largely determined during the initial first days in which antigen‐presenting cells instruct T cells to expand and differentiate into effector and memory cells. Besides the essential stimulation of the T‐cell receptor complex a plethora of co‐stimulatory signals not only ensures a proper T‐cell activation but also instils phenotypic and functional characteristics in the T cells appropriate to fight off the invading pathogen. The tumour necrosis factor receptor/ligand pair CD27/CD70 gained a lot of attention because of its key role in regulating T‐cell activation, survival, differentiation and maintenance, especially in the course of viral infections and cancer. We sought to investigate the role of CD70 co‐stimulation for immune responses induced by the vaccine vector modified vaccinia virus Ankara–Bavarian Nordic® (MVA‐BN ®). Short‐term blockade of CD70 diminished systemic CD8 T‐cell effector and memory responses in mice. The dependence on CD70 became even more apparent in the lungs of MHC class II‐deficient mice. Importantly, genetically encoded CD70 in MVA‐BN ® not only increased CD8 T‐cell responses in wild‐type mice but also substituted for CD4 T‐cell help. MHC class II‐deficient mice that were immunized with recombinant MVA‐CD70 were fully protected against a lethal virus infection, whereas MVA‐BN ®‐immunized mice failed to control the virus. These data are in line with CD70 playing an important role for vaccine‐induced CD8 T‐cell responses and prove the potency of integrating co‐stimulatory molecules into the MVA‐BN ® backbone.

Bacterial flagellin enhances innate and adaptive immune responses and is considered a promising adjuvant for the development of vaccines against infectious diseases and cancer. Antigen-presenting cells recognize flagellin with the extracellular TLR5 and the intracellular NLRC4 inflammasome-mediated pathway. The detailed cooperation of these innate pathways in the induction of the adaptive immune response following intranasal (i.n.) administration of a recombinant modified vaccinia virus Ankara (rMVA) vaccine encoding flagellin (rMVA-flagellin) is not known. rMVA-flagellin induced enhanced secretion of mucosal IL-1β and TNF-α resulting in elevated CTL and IgG2c antibody responses. Importantly, mucosal IgA responses were also significantly enhanced in both bronchoalveolar (BAL) and intestinal lavages accompanied by the increased migration of CD8+ T cells to the mesenteric lymph nodes (MLN). Nlrc4−/− rMVA-flagellin-immunized mice failed to enhance pulmonary CTL responses, IgG2c was lower, and IgA levels in the BAL or intestinal lavages were similar as those of control mice. Our results show the favorable adjuvant effect of rMVA-flagellin in the lung as well as the intestinal mucosa following i.n. administration with NLRC4 as the essential driver of this promising mucosal vaccine concept.

Virus-based vaccines and appropriate costimulation potently enhance antigen-specific T cell immunity against cancer. In the present study, we exploit both innate and adaptive immune responses triggered by recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against large, established tumors in different combinatory settings. Therapeutic treatment with rMVA-CD40L resulted in the control or eradication of solid tumors in several unrelated tumor models. The expansion of non-exhausted, tumor-specific cytotoxic CD8+ T cells was essential for the therapeutic activity and was partially dependent on CD8α+ cross-presenting dendritic cells. Combination of rMVA-CD40L with PD-1 checkpoint blockade further enhanced the therapeutic activity of MVA virotherapy in colorectal carcinoma. In addition, rMVA-CD40L combined with antibodies targeting Tumor-Associated Antigens (TAA) resulted in increased therapeutic antitumor efficacy against two unrelated tumor models. We describe a translationally relevant therapeutic synergy between viral vaccination and CD40L costimulation. We show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combining immunotherapeutic regimes, such as checkpoint blockade and TAA targeting antibodies. This novel immunotherapeutic approach could translate into clinical cancer therapies where ADCC competent TAA targeting antibodies PD-1 checkpoint blockade are employed. Citation Format: Jose Medina-Echeverz, Maria Hinterberger, Raphael Giessel, Barbara Bathke, Ronny Kassub, Giovanna Fiore, Paul Chaplin, Hubertus Hochrein, Henning Lauterbach. Synergistic cancer immunotherapy combination of MVA-CD40L with tumor targeting antibodies or checkpoint blockade to achieve strong antitumor immune responses against large, established tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1468.

Plasmacytoid dendritic cells (pDCs) play a critical role in antiviral immunity through their ability to produce large amounts of type I IFNs. Activation of pDCs upon viral infection has been shown to be dependent on MyD88 and mediated by Toll-like receptors (TLR) 7 and 9, which sense viral ssRNA and CpG DNA, respectively. In this study, we showed that murine pDC recognition of vaccinia virus (VV), a dsDNA virus, was MyD88-dependent but TLR9-independent. Using HEK293 cells transfected with murine TLR7 or TLR8 and a NF-kappaB luciferase reporter, we demonstrated that stimulation of TLR8-, but not TLR7-, transfected cells with either VV or VV DNA resulted in substantial NF-kappaB activation, and that siRNA-mediated knockdown of TLR8 expression in pDCs led to a complete ablation of VV-induced type I IFN production. We further identified that the VV genome was rich in poly(A)/T sequences, and synthetic poly(A) and poly T oligodeoxynucleotides were capable of activating pDCs in a TLR8-dependent manner. In vivo, TLR8-MyD88-dependent pDC activation played a critical role in innate immune control of VV infection. Collectively, our data are unique in demonstrating that TLR8 is required for sensing poly(A)/T-rich DNA in pDCs, and that murine TLR8 is functional in the context of a viral infection.

Current prophylactic vaccines work via the induction of B and T cell mediated memory that effectively control further replication of the pathogen after entry. In the case of therapeutic or post-exposure vaccinations the situation is far more complex, because the pathogen has time to establish itself in the host, start producing immune-inhibitory molecules and spread into distant organs. So far it is unclear which immune parameters have to be activated in order to thwart an existing lethal infection. Using the mousepox model, we investigated the immunological mechanisms responsible for a successful post-exposure immunization with modified vaccinia Ankara (MVA). In contrast to intranasal application of MVA, we found that intravenous immunization fully protected mice infected with ectromelia virus (ECTV) when applied three days after infection. Intravenous MVA immunization induced strong innate and adaptive immune responses in lethally infected mice. By using various gene-targeted and transgenic mouse strains we show that NK cells, CD4 T cells, CD8 T cells and antibodies are essential for the clearance of ECTV after post-exposure immunization. Post-exposure immunization with MVA is an effective measure in a murine model of human smallpox. MVA activates innate and adaptive immune parameters and only a combination thereof is able to purge ECTV from its host. These data not only provide a basis for therapeutic vaccinations in the case of the deliberate release of pathogenic poxviruses but possibly also for the treatment of chronic infections and cancer.