Intraperitoneal administration of a modified vaccinia virus Ankara confers single-chain interleukin-12 expression to the omentum and achieves immune-mediated efficacy against peritoneal carcinomatosis.

BACKGROUND: Peritoneal carcinomatosis is an advanced stage of cancer in which the disease has spread to the peritoneal cavity. In order to restore antitumor immunity subverted by tumor cells in this location, we evaluated intraperitoneal administrations of modified vaccinia virus Ankara (MVA) engineered to express single-chain interleukin 12 (scIL-12) to increase antitumor immune responses. METHODS: MVA encoding scIL-12 (MVA.scIL-12) was evaluated against peritoneal carcinomatosis models based on intraperitoneal engraftment of tumor cells. CD8-mediated immune responses, elucidated antitumor efficacy, and safety were evaluated following intravenous, intratumoral, or intraperitoneal administration of the viral vector. The immune response was measured by ELISpot (enzyme-linked immunosorbent spot), RNA sequencing, flow cytometry, intravital microscopy, and depletion of lymphocyte subsets with monoclonal antibodies. Safety was assessed by body-weight follow-up and blood testing. Tissue tropism on intravenous or intraperitoneal administration was assessed by bioluminescence analysis using a reporter MVA encoding luciferase. RESULTS: Intraperitoneal or locoregional administration, but not other routes of administration, resulted in a potent immune response characterized by increased levels of tumor-specific CD8+ T lymphocytes with the ability to produce both interferon-γ and tumor necrosis factor-α. The antitumor immune response was detectable not only in the peritoneal cavity but also systemically. As a result of intraperitoneal treatment, a single administration of MVA.scIL-12 encoding scIL-12 completely eradicated MC38 tumors implanted in the peritoneal cavity and also protected cured mice from subsequent subcutaneous rechallenges. Bioluminescence imaging using an MVA encoding luciferase revealed that intraperitoneal administration targets transgene to the omentum. The omentum is considered a key tissue in immune protection of the peritoneal cavity. The safety profile of intraperitoneal administration was also better than that following intravenous administration since no weight loss or hematological toxicity was observed when the vector was locally delivered into the peritoneal cavity. CONCLUSION: Intraperitoneal administration of MVA vectors encoding scIL-12 targets the omentum, which is the tissue where peritoneal carcinomatosis usually begins. MVA.scIL-12 induces a potent tumor-specific immune response that often leads to the eradication of experimental tumors disseminated to the peritoneal cavity.

Armored modified vaccinia Ankara in cancer immunotherapy.

Cancer immunotherapy relies on unleashing the patient´s immune system against tumor cells. Cancer vaccines aim to stimulate both the innate and adaptive arms of immunity to achieve durable clinical responses. Some roadblocks for a successful cancer vaccine in the clinic include the tumor antigen of choice, the adjuvants employed to strengthen antitumor-specific immune responses, and the risks associated with enhancing immune-related adverse effects in patients. Modified vaccinia Ankara (MVA) belongs to the family of poxviruses and is a versatile vaccine platform that combines several attributes crucial for cancer therapy. First, MVA is an excellent inducer of innate immune responses leading to type I interferon secretion and induction of T helper cell type 1 (Th1) immune responses. Second, it elicits robust and durable humoral and cellular immunity against vector-encoded heterologous antigens. Third, MVA has enormous genomic flexibility, which allows for the expression of multiple antigenic and costimulatory entities. And fourth, its replication deficit in human cells ensures a excellent safety profile. In this review, we summarize the current understanding of how MVA induces innate and adaptive immune responses. Furthermore, we will give an overview of the tumor-associated antigens and immunomodulatory molecules that have been used to armor MVA and describe their clinical use. Finally, the route of MVA immunization and its impact on therapeutic efficacy depending on the immunomodulatory molecules expressed will be discussed.

Preclinical development of a first-in-class vaccine encoding HER2, Brachyury and CD40L for antibody enhanced tumor eradication.

The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8+ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.

The Quest for a Respiratory Syncytial Virus Vaccine for Older Adults: Thinking beyond the F Protein.

Respiratory syncytial virus (RSV) is a common cause of paediatric respiratory tract infection and causes a significant health burden in older adults. Natural immunity to RSV is incomplete, permitting recurrent symptomatic infection over an individual's lifespan. When combined with immunosenescence, this increases older adults' susceptibility to more severe disease symptoms. As RSV prophylaxis is currently limited to infants, older adults represent an important target population for RSV vaccine development. The relationship between RSV and our immune systems is complex, and these interactions require deeper understanding to tailor an effective vaccine candidate towards older adults. To date, vaccine candidates targeting RSV antigens, including pre-F, F, G (A), G (B), M2-1, and N, have shown efficacy against RSV infection in older adults in clinical trial settings. Although vaccine candidates have demonstrated robust neutralising IgG and cellular responses, it is important that research continues to investigate the RSV immune response in order to further understand how the choice of antigenic target site may impact vaccine effectiveness. In this article, we discuss the Phase 3 vaccine candidates being tested in older adults and review the hurdles that must be overcome to achieve effective protection against RSV.

A Recombinant MVA-Based RSV Vaccine Induces T-Cell and Antibody Responses That Cooperate in the Protection Against RSV Infection.

Respiratory syncytial virus (RSV) causes a respiratory disease with a potentially fatal outcome especially in infants and elderly individuals. Several vaccines failed in pivotal clinical trials, and to date, no vaccine against RSV has been licensed. We have developed an RSV vaccine based on the recombinant Modified Vaccinia Virus Ankara-BN® (MVA-RSV), containing five RSV-specific antigens that induced antibody and T-cell responses, which is currently tested in clinical trials. Here, the immunological mechanisms of protection were evaluated to determine viral loads in lungs upon vaccination of mice with MVA-RSV followed by intranasal RSV challenge. Depletion of CD4 or CD8 T cells, serum transfer, and the use of genetically engineered mice lacking the ability to generate either RSV-specific antibodies (T11µMT), the IgA isotype (IgA knockout), or CD8 T cells (ß2M knockout) revealed that complete protection from RSV challenge is dependent on CD4 and CD8 T cells as well as antibodies, including IgA. Thus, MVA-RSV vaccination optimally protects against RSV infection by employing multiple arms of the adaptive immune system.

Synergistic antitumor response with recombinant modified virus Ankara armed with CD40L and CD137L against peritoneal carcinomatosis.

Recombinant-modified vaccinia virus Ankara (rMVA) is known to elicit potent antitumor immune responses in preclinical models due to its inherent ability to activate the innate immune system and the activation of adaptive responses mediated by the expression of tumor antigens and costimulus-providing molecules, such as CD40L and CD137L. Here, we evaluated different rMVA vectors in preclinical peritoneal carcinomatosis models (ID8.OVA-Vegf/GFP and MC38). We compared rMVA vectors expressing a tumor antigen (OVA or gp70) either alone or co-expressed with CD40L or/and CD137L. In tumor-free mice, the vector coding for the triple combination was only slightly superior, whereas, in tumor-bearing animals, we observed a synergistic induction of T lymphocytes specific against vector-encoded and non-encoded tumor-associated antigens. The enhanced activation of the immune response was associated with improved survival in mice with peritoneal carcinomatosis treated with a rMVA vector encoding both CD40L and CD137L. Thus, the triple transgene combination in vaccinia viral vectors represents a promising strategy for the treatment of peritoneal carcinomatosis.

A Capsid Virus-Like Particle-Based SARS-CoV-2 Vaccine Induces High Levels of Antibodies and Protects Rhesus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic. Here, we present non-human primate immunogenicity and protective efficacy data generated with the capsid virus-like particle (cVLP)-based vaccine ABNCoV2 that has previously demonstrated immunogenicity in mice. In rhesus macaques, a single vaccination with either 15 or 100 µg ABNCoV2 induced binding and neutralizing antibodies in a dose-dependent manner, at levels comparable to those measured in human convalescents. A second vaccine administration led to a >50-fold increase in neutralizing antibodies, with 2-log higher mean levels in the 100-µg ABNCoV2 group compared with convalescent samples. Upon SARS-CoV-2 challenge, a significant reduction in viral load was observed for both vaccine groups relative to the challenge control group, with no evidence of enhanced disease. Remarkably, neutralizing antibody titers against an original SARS-CoV-2 isolate and against variants of concern were comparable, indicating a potential for broad protection afforded by ABNCoV2, which is currently in clinical testing.

Discordance in STING-Induced Activation and Cell Death Between Mouse and Human Dendritic Cell Populations.

Stimulator of Interferon Genes (STING) is a cytosolic sensor of cyclic dinucleotides (CDNs). The activation of dendritic cells (DC) via the STING pathway, and their subsequent production of type I interferon (IFN) is considered central to eradicating tumours in mouse models. However, this contribution of STING in preclinical murine studies has not translated into positive outcomes of STING agonists in phase I & II clinical trials. We therefore questioned whether a difference in human DC responses could be critical to the lack of STING agonist efficacy in human settings. This study sought to directly compare mouse and human plasmacytoid DCs and conventional DC subset responses upon STING activation. We found all mouse and human DC subsets were potently activated by STING stimulation. As expected, Type I IFNs were produced by both mouse and human plasmacytoid DCs. However, mouse and human plasmacytoid and conventional DCs all produced type III IFNs (i.e., IFN-λs) in response to STING activation. Of particular interest, all human DCs produced large amounts of IFN-λ1, not expressed in the mouse genome. Furthermore, we also found differential cell death responses upon STING activation, observing rapid ablation of mouse, but not human, plasmacytoid DCs. STING-induced cell death in murine plasmacytoid DCs occurred in a cell-intrinsic manner and involved intrinsic apoptosis. These data highlight discordance between STING IFN and cell death responses in mouse and human DCs and caution against extrapolating STING-mediated events in mouse models to equivalent human outcomes.

Intratumoral virotherapy with 4-1BBL armed modified vaccinia Ankara eradicates solid tumors and promotes protective immune memory.

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.

Human monkeypox - After 40 years, an unintended consequence of smallpox eradication.

Smallpox eradication, coordinated by the WHO and certified 40 years ago, led to the cessation of routine smallpox vaccination in most countries. It is estimated that over 70% of the world's population is no longer protected against smallpox, and through cross-immunity, to closely related orthopox viruses such as monkeypox. Monkeypox is now a re-emerging disease. Monkeypox is endemic in as yet unconfirmed animal reservoirs in sub-Saharan Africa, while its human epidemiology appears to be changing. Monkeypox in small animals imported from Ghana as exotic pets was at the origin of an outbreak of human monkeypox in the USA in 2003. Travellers infected in Nigeria were at the origin of monkeypox cases in the UK in 2018 and 2019, Israel in 2018 and Singapore in2019. Together with sporadic reports of human infections with other orthopox viruses, these facts invite speculation that emergent or re-emergent human monkeypox might fill the epidemiological niche vacated by smallpox. An ad-hoc and unofficial group of interested experts met to consider these issues at Chatham House, London in June 2019, in order to review available data and identify monkeypox-related research gaps. Gaps identified by the experts included:The experts further agreed on the need for a better understanding of the genomic evolution and changing epidemiology of orthopox viruses, the usefulness of in-field genomic diagnostics, and the best disease control strategies, including the possibility of vaccination with new generation non-replicating smallpox vaccines and treatment with recently developed antivirals.

Synergistic cancer immunotherapy combines MVA-CD40L induced innate and adaptive immunity with tumor targeting antibodies.

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.

PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing.

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

Antibiotic treatment-induced secondary IgA deficiency enhances susceptibility to Pseudomonas aeruginosa pneumonia.

Broad-spectrum antibiotics are widely used with patients in intensive care units (ICUs), many of whom develop hospital-acquired infections with Pseudomonas aeruginosa. Although preceding antimicrobial therapy is known as a major risk factor for P. aeruginosa-induced pneumonia, the underlying mechanisms remain incompletely understood. Here we demonstrate that depletion of the resident microbiota by broad-spectrum antibiotic treatment inhibited TLR-dependent production of a proliferation-inducing ligand (APRIL), resulting in a secondary IgA deficiency in the lung in mice and human ICU patients. Microbiota-dependent local IgA contributed to early antibacterial defense against P. aeruginosa. Consequently, P. aeruginosa-binding IgA purified from lamina propria culture or IgA hybridomas enhanced resistance of antibiotic-treated mice to P. aeruginosa infection after transnasal substitute. Our study provides a mechanistic explanation for the well-documented risk of P. aeruginosa infection following antimicrobial therapy, and we propose local administration of IgA as a novel prophylactic strategy.

CD70 encoded by modified vaccinia virus Ankara enhances CD8 T-cell-dependent protective immunity in MHC class II-deficient mice.

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.

MyD88-dependent pro-interleukin-1ß induction in dendritic cells exposed to food-grade synthetic amorphous silica.

BACKGROUND: Dendritic 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. RESULTS: Interaction 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-1ß precursor, subsequently cleaved by the inflammasome to secrete mature IL-1ß. In contrast, neither pro-IL-1ß induction nor mature IL-1ß secretion occurs upon internalization of TiO2 or FePO4 nanoparticles. The pro-IL-1ß 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. CONCLUSIONS: Our 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-1ß implies that the currently massive use of SAS particles as food additive should be reconsidered.

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles.

There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant.IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce noninfectious EBOV VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV VLPs, thus producing a highly authentic immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g., by including antigens from various filoviral species to develop multivalent VLP-producing MVA-based filovirus vaccines.

NLRC4 Inflammasome-Driven Immunogenicity of a Recombinant MVA Mucosal Vaccine Encoding Flagellin.

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-1ß 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.

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA.

Toll-like receptor (TLR) 13 and TLR2 are the major sensors of Gram-positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA-derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A-treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single-stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence-derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88-dependent manner. These ORNs, as well as S. aureus-, Escherichia coli-, and mt-RNA, also activate differentiated human monocytoid THP-1 cells, provided they express TLR8. Moreover, Unc93b1(-/-)- and Tlr8(-/-)-THP-1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif-dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria-driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.

Selective Expression of the MAPK Phosphatase Dusp9/MKP-4 in Mouse Plasmacytoid Dendritic Cells and Regulation of IFN-ß Production.

Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220(-) bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2 MAPK compared with cDCs. Enforced retroviral expression of Dusp9 in mouse GM-CSF-induced cDCs increased the expression of TLR9-induced IL-12p40 and IFN-ß, but not of IL-10. Conditional deletion of Dusp9 in pDCs was effectively achieved in Dusp9(flox/flox); CD11c-Cre mice at the mRNA and protein levels. However, the lack of Dusp9 in pDC did not restore ERK1/2 activation after TLR9 stimulation and only weakly affected IFN-ß and IL-12p40 production. Taken together, our results suggest that expression of Dusp9 is sufficient to impair ERK1/2 activation and enhance IFN-ß expression. However, despite selective expression in pDCs, Dusp9 is not essential for high-level IFN-ß production by these cells.

Recombinant modified vaccinia virus Ankara generating excess early double-stranded RNA transiently activates protein kinase R and triggers enhanced innate immune responses.

UNLABELLED: Double-stranded RNA (dsRNA) is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by dsRNA and triggers major cellular defenses against viral infection, including protein synthesis shutdown, apoptosis, and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-ß in murine and human cells in the presence of an intact E3L gene. IFN-ß induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR dependent. Excess early dsRNA produced by MVA activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-α, IFN-γ, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo. IMPORTANCE: Efficient cellular sensing of pathogen-specific components, including double-stranded RNA (dsRNA), is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a by-product of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector.