Host protection from infection relies on the recognition of pathogens by innate pattern-recognition receptors such as Toll-like receptors (TLRs). Here, we show that the orphan receptor TLR13 in mice recognizes a conserved 23S ribosomal RNA (rRNA) sequence that is the binding site of macrolide, lincosamide, and streptogramin group (MLS) antibiotics (including erythromycin) in bacteria. Notably, 23S rRNA from clinical isolates of erythromycin-resistant Staphylococcus aureus and synthetic oligoribonucleotides carrying methylated adenosine or a guanosine mimicking a MLS resistance-causing modification failed to stimulate TLR13. Thus, our results reveal both a natural TLR13 ligand and specific mechanisms of antibiotic resistance as potent bacterial immune evasion strategy, avoiding recognition via TLR13.

In humans and mice, CD8α+ conventional dendritic cells are the primary source of interferon-λ released in response to the adjuvant and Toll-like receptor 3 agonist poly IC.

Modified vaccinia Ankara (MVA) is a safe and promising viral vaccine vector that is currently investigated in several clinical and pre-clinical trials. In contrast to inactivated or sub-unit vaccines, MVA is able to induce strong humoral as well as cellular immune responses. In order to further improve its CD8 T cell inducing capacity, we genetically adjuvanted MVA with the coding sequence of murine CD40L, a member of the tumor necrosis factor superfamily. Immunization of mice with this new vector led to strongly enhanced primary and memory CD8 T cell responses. Concordant with the enhanced CD8 T cell response, we could detect stronger activation of dendritic cells and higher systemic levels of innate cytokines (including IL-12p70) early after immunization. Interestingly, acquisition of memory characteristics (i.e., IL-7R expression) was accelerated after immunization with MVA-CD40L in comparison to non-adjuvanted MVA. Furthermore, the generated cytotoxic T-lymphocytes (CTLs) also showed improved functionality as demonstrated by intracellular cytokine staining and in vivo killing activity. Importantly, the superior CTL response after a single MVA-CD40L immunization was able to protect B cell deficient mice against a fatal infection with ectromelia virus. Taken together, we show that genetic adjuvantation of MVA can change strength, quality, and functionality of innate and adaptive immune responses. These data should facilitate a rational vaccine design with a focus on rapid induction of large numbers of CD8 T cells able to protect against specific diseases.

BackgroundDendritic cells (DCs) are specialized first-line sensors of foreign materials invading the organism. These sentinel cells rely on pattern recognition receptors such as Nod-like or Toll-like receptors (TLRs) to launch immune reactions against pathogens, but also to mediate tolerance to self-antigens and, in the intestinal milieu, to nutrients and commensals. Since inappropriate DC activation contributes to inflammatory diseases and immunopathologies, a key question in the evaluation of orally ingested nanomaterials is whether their contact with DCs in the intestinal mucosa disrupts this delicate homeostatic balance between pathogen defense and tolerance. Here, we generated steady-state DCs by incubating hematopoietic progenitors with feline McDonough sarcoma-like tyrosine kinase 3 ligand (Flt3L) and used the resulting immature DCs to test potential biological responses against food-grade synthetic amorphous silica (SAS) representing a common nanomaterial generally thought to be safe.ResultsInteraction of immature and unprimed DCs with food-grade SAS particles and their internalization by endocytic uptake fails to elicit cytotoxicity and the release of interleukin (IL)-1α or tumor necrosis factor-α, which were identified as master regulators of acute inflammation in lung-related studies. However, the display of maturation markers on the cell surface shows that SAS particles activate completely immature DCs. Also, the endocytic uptake of SAS particles into these steady-state DCs leads to induction of the pro-IL-1β precursor, subsequently cleaved by the inflammasome to secrete mature IL-1β. In contrast, neither pro-IL-1β induction nor mature IL-1β secretion occurs upon internalization of TiO2 or FePO4 nanoparticles. The pro-IL-1β induction is suppressed by pharmacologic inhibitors of endosomal TLR activation or by genetic ablation of MyD88, a downstream adapter of TLR pathways, indicating that endosomal pattern recognition is responsible for the observed cytokine response to food-grade SAS particles.ConclusionsOur results unexpectedly show that food-grade SAS particles are able to directly initiate the endosomal MyD88-dependent pathogen pattern recognition and signaling pathway in steady-state DCs. The ensuing activation of immature DCs with de novo induction of pro-IL-1β implies that the currently massive use of SAS particles as food additive should be reconsidered.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-017-0202-8) contains supplementary material, which is available to authorized users.

Virus-based vaccines and appropriate costimulation potently enhance antigen-specific T cell immunity against cancer. Here we report the use of recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against solid tumors. Therapeutic treatment with rMVA-CD40L-expressing tumor-associated antigens results in the control of established tumors. The expansion of tumor-specific cytotoxic CD8+ T cells is essential for the therapeutic antitumor effects. Strikingly, rMVA-CD40L also induces strong natural killer (NK) cell activation and expansion. Moreover, the combination of rMVA-CD40L and tumor-targeting antibodies results in increased therapeutic antitumor efficacy relying on the presence of Fc receptor and NK cells. We describe a translationally relevant therapeutic synergy between systemic viral vaccination and CD40L costimulation. We show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combination with tumor-targeting antibodies. This immunotherapeutic approach could translate into clinical cancer therapies where tumor-targeting antibodies are employed.

The findings of Martinez et al.(1) that vaccinia virus and its DNA are potent inducers of plasmacytoid dendritic cell (pDC)-derived IFN-α in a Toll-like receptor (TLR)9-independent, exclusively TLR8-dependent way came to us as a great surprise (2). The data (1) contradict our results and those of others.Murine TLR8 gene-transfected cells show a strong induction of an NF-κB luciferease reporter construct after stimulation with CL075 (1). In contrast, others have shown that sole CL075 (also known as 3M-002) was able to trigger murine TLR7 but not murine TLR8-dependent reporter activity (3). This is consistent with our data showing stimulation of human but not murine TLR8 transfectants.The A-type CpG oligodeoxynucleotides (CpG-ODN) used (1) was fully phosphorothioated, which we found to be biologically inactive. Furthermore, the used concentration of 1 nM is below the range of activity for A-type ODN.Martinez et al. (1) showed that in vitro flt3 ligand-generated pDC (FL-pDC) produces large amounts of IFN-α in response to CL075, CL087, or CpG-ODN, but the response to CL075 was lost in TLR8-knockdown pDC. In contrast, we found that CL087 and CL075 induced only very little IFN-α in sorted wildtype (WT) FL-pDCs compared with large amounts induced by CpG-ODN or Sendai virus. Furthermore, TLR7-deficient FL-pDCs still produced large amounts of IFN-α, IFN-λ, IL-6, TNF-α, CC chemokine ligand (CCL)3, CCL4, and CCL5 on stimulation with CpG-ODN, but no cytokines were produced in response to Sendai virus, CL075, or CL087.Data (1) indicate that poly(A) and poly T ODNs were highly stimulatory for IFN-α production in WT but not in TLR8-knockdown pDCs. In our hands, poly A10 and two different poly T ODNs did not induce IFN-α or other cytokines in sorted FLpDCs or ex vivo-isolated pDCs.High levels of TLR7, TLR8, and TLR9 transcripts were detected in murine pDCs (1). Our analyses show high-level transcripts for TLR7 and TLR9 but not for TLR8 in murine pDC. In addition, our quantitative proteomic analysis of isolated splenic pDC (4) or sorted FL-pDC revealed the presence of TLR7 and TLR9 but not of TLR8 protein.A central part of the paper (1) is the observation that the i.v. injection of 10 7 pfu of vaccinia virus strain Western Reserve (VV-WR) induced large amounts of IFN-α in WT but not MyD88-deficient mice 48 h after infection. We and others (5) found that VV-WR is highly inhibitory for the production of IFN-α in vitro and in vivo, and we could not detect any IFN-α in the sera in time kinetics up to 48 h. In summary, our experiments are consistent with previously published work in the field of vaccinia infection and its recognition, CpG-ODN use, and the role of TLR7, TLR8, and TLR9 for the response of murine pDCs (2-5). However, they largely contradict the results of Martinez et al. (1), which leads us to question the conclusions drawn that VV and VV-DNA are major agonists for murine pDC-expressed TLR8.

Background Human cancers are extraordinarily heterogeneous in terms of tumor antigen expression, immune infiltration and composition. A common feature, however, is the host′s inability to mount potent immune responses that prevent tumor growth effectively. Often, naturally primed CD8+ T cells against solid tumors lack adequate stimulation and efficient tumor tissue penetration due to an immune hostile tumor microenvironment.Methods To address these shortcomings, we cloned tumor-associated antigens (TAA) and the immune-stimulatory ligand 4-1BBL into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy.Results Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Intratumoral injection of MVA localized mainly to the tumor with minimal leakage to the tumor-draining lymph node. In situ infection by MVA-TAA-4-1BBL triggered profound changes in the tumor microenvironment, including the induction of multiple proinflammatory molecules and immunogenic cell death. These changes led to the reactivation and expansion of antigen-experienced, tumor-specific cytotoxic CD8+ T cells that were essential for the therapeutic antitumor effect. Strikingly, we report the induction of a systemic antitumor immune response including tumor antigen spread by local MVA-TAA-4-1BBL treatment which controlled tumor growth at distant, untreated lesions and protected against local and systemic tumor rechallenge. In all cases, 4-1BBL adjuvanted MVA was superior to MVA.Conclusion Intratumoral 4-1BBL-armed MVA immunotherapy induced a profound reactivation and expansion of potent tumor-specific CD8+ T cells as well as favorable proinflammatory changes in the tumor microenvironment, leading to elimination of tumors and protective immunological memory.

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.