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.

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.

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.

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.

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.

Genetic Adjuvantation of Recombinant MVA with CD40L Potentiates CD8 T Cell Mediated Immunity.

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.

TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification.

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.

Mouse CD8alpha+ DCs and human BDCA3+ DCs are major producers of IFN-lambda in response to poly IC.

Polyinosinic:polycytidylic acid (poly IC), a double-stranded RNA, is an effective adjuvant in vivo. IFN-λs (also termed IL-28/29) are potent immunomodulatory and antiviral cytokines. We demonstrate that poly IC injection in vivo induces large amounts of IFN-λ, which depended on hematopoietic cells and the presence of TLR3 (Toll-like receptor 3), IRF3 (IFN regulatory factor 3), IRF7, IFN-I receptor, Fms-related tyrosine kinase 3 ligand (FL), and IRF8 but not on MyD88 (myeloid differentiation factor 88), Rig-like helicases, or lymphocytes. Upon poly IC injection in vivo, the IFN-λ production by splenocytes segregated with cells phenotypically resembling CD8α(+) conventional dendritic cells (DCs [cDCs]). In vitro experiments revealed that CD8α(+) cDCs were the major producers of IFN-λ in response to poly IC, whereas both CD8α(+) cDCs and plasmacytoid DCs produced large amounts of IFN-λ in response to HSV-1 or parapoxvirus. The nature of the stimulus and the cytokine milieu determined whether CD8α(+) cDCs produced IFN-λ or IL-12p70. Human DCs expressing BDCA3 (CD141), which is considered to be the human counterpart of murine CD8α(+) DCs, also produced large amounts of IFN-λ upon poly IC stimulation. Thus, IFN-λ production in response to poly IC is a novel function of mouse CD8α(+) cDCs and their human equivalents.