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.

Immunogenicity of propagation-restricted vesicular stomatitis virus encoding Ebola virus glycoprotein in guinea pigs.

Vesicular stomatitis virus (VSV) expressing the Ebola virus (EBOV) glycoprotein (GP) in place of the VSV glycoprotein G (VSV/EBOV-GP) is a promising EBOV vaccine candidate which has already entered clinical phase 3 studies. Although this chimeric virus was tolerated overall by volunteers, it still caused viremia and adverse effects such as fever and arthritis, suggesting that it might not be sufficiently attenuated. In this study, the VSV/EBOV-GP vector was further modified in order to achieve attenuation while maintaining immunogenicity. All recombinant VSV constructs were propagated on VSV G protein expressing helper cells and used to immunize guinea pigs via the intramuscular route. The humoral immune response was analysed by EBOV-GP-specific fluorescence-linked immunosorbent assay, plaque reduction neutralization test and in vitro virus-spreading inhibition test that employed recombinant VSV/EBOV-GP expressing either green fluorescent protein or secreted Nano luciferase. Most modified vector constructs induced lower levels of protective antibodies than the parental VSV/EBOV-GP or a recombinant modified vaccinia virus Ankara vector encoding full-length EBOV-GP. However, the VSV/EBOV-GP(F88A) mutant was at least as immunogenic as the parental vaccine virus although it was highly propagation-restricted. This finding suggests that VSV-vectored vaccines need not be propagation-competent to induce a robust humoral immune response. However, VSV/EBOV-GP(F88A) rapidly reverted to a fully propagation-competent virus indicating that a single-point mutation is not sufficient to maintain the propagation-restricted phenotype.

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.

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.

The mycobacterial cord factor adjuvant analogue trehalose-6,6'-dibehenate (TDB) activates the Nlrp3 inflammasome.

The success of a vaccine consists in the induction of an innate immune response and subsequent activation of the adaptive immune system. Because antigens are usually not immunogenic, the addition of adjuvants that activate innate immunity is required. The mycobacterial cord factor trehalose-6,6'-dimycolate (TDM) and its synthetic adjuvant analogue trehalose-6,6'-dibehenate (TDB) rely on the C-type lectin Mincle and the signaling molecules Syk and Card9 to trigger innate immunity. In this study, we show that stimulation of bone marrow-derived dendritic cells (BMDCs) with TDB induces Nlrp3 inflammasome-dependent IL-1ß secretion. While Card9 is required for NF-κB activation by TDB, it is dispensable for TDB-induced activation of the Nlrp3 inflammasome. Additionally, efflux of intracellular potassium, lysosomal rupture, and oxygen radical (ROS) production are crucial for caspase-1 processing and IL-1ß secretion by TDB. In an in vivo inflammation model, we demonstrate that the recruitment of neutrophils by TDB is significantly reduced in the Nlrp3-deficient mice compared to the wild-type mice, while the production of chemokines in vitro is not influenced by the absence of Nlrp3. These results identify the Nlrp3 inflammasome as an essential mediator for the induction of an innate immune response triggered by TDB.

The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence.

Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo.

Immediate-early expression of a recombinant antigen by modified vaccinia virus ankara breaks the immunodominance of strong vector-specific B8R antigen in acute and memory CD8 T-cell responses.

Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediate-early protein, and remained higher over the first 6 h of infection than p7.5- or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5- and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigen-specific CD8 T-cell responses.

The fusion kinase ITK-SYK mimics a T cell receptor signal and drives oncogenesis in conditional mouse models of peripheral T cell lymphoma.

Peripheral T cell lymphomas (PTCLs) are highly aggressive malignancies with poor prognosis. Their molecular pathogenesis is not well understood and small animal models for the disease are lacking. Recently, the chromosomal translocation t(5;9)(q33;q22) generating the interleukin-2 (IL-2)-inducible T cell kinase (ITK)-spleen tyrosine kinase (SYK) fusion tyrosine kinase was identified as a recurrent event in PTCL. We show that ITK-SYK associates constitutively with lipid rafts in T cells and triggers antigen-independent phosphorylation of T cell receptor (TCR)-proximal proteins. These events lead to activation of downstream pathways and acute cellular outcomes that correspond to regular TCR ligation, including up-regulation of CD69 or production of IL-2 in vitro or deletion of thymocytes and activation of peripheral T cells in vivo. Ultimately, conditional expression of patient-derived ITK-SYK in mice induces highly malignant PTCLs with 100% penetrance that resemble the human disease. Our work demonstrates that constitutively enforced antigen receptor signaling can, in principle, act as a powerful oncogenic driver. Moreover, we establish a robust clinically relevant and genetically tractable model of human PTCL.

Suberoylanilide hydroxamic acid reactivates HIV from latently infected cells.

Human immunodeficiency virus (HIV) persists in a latent form in infected individuals treated effectively with highly active antiretroviral therapy (HAART). In part, these latent proviruses account for the rebound in viral replication observed after treatment interruption. A major therapeutic challenge is to purge this reservoir. In this study, we demonstrate that suberoylanilide hydroxamic acid (SAHA) reactivates HIV from latency in chronically infected cell lines and primary cells. Indeed, P-TEFb, a critical transcription cofactor for HIV, is released and then recruited to the viral promoter upon stimulation with SAHA. The phosphatidylinositol 3-kinase/Akt pathway is involved in the initiation of these events. Using flow cytometry-based single cell analysis of protein phosphorylation, we demonstrate that SAHA activates this pathway in several subpopulations of T cells, including memory T cells that are the major viral reservoir in peripheral blood. Importantly, SAHA activates HIV replication in peripheral blood mononuclear cells from individuals treated effectively with HAART. Thus SAHA, which is a Food and Drug Administration-approved drug, might be considered to accelerate the decay of the latent reservoir in HAART-treated infected humans.

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection.

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1-infected individuals to a mean of 49.4 +/- SD 12.9% of CD8(+) T cells expressing Tim-3 in HIV-1-infected chronic progressors versus 28.5 +/- 6.8% in HIV-1-uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1-infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4(+) T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1-specific CD8(+) T cells. Tim-3-expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1-specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1-associated T cell dysfunction.

HIV-induced changes in T cell signaling pathways.

Infection with HIV usually results in chronic activation of the immune system, with profound quantitative and qualitative changes in the T cell compartment. To better understand the mechanistic basis for T cell dysfunction and to discern whether such mechanisms are reversed after effective antiviral treatment, we analyzed changes in signaling pathways of human CD4(+) and CD8(+) T cells from 57 HIV-infected subjects in varying stages of disease progression and treatment, including long-term nonprogressors, progressors, and chronically infected subjects provided effective antiretroviral therapy (responders). A previously described PhosFlow method was adapted and optimized so that protein phosphorylation could be visualized in phenotypically defined subpopulations of CD4(+) and CD8(+) T cells (naive, memory, and effector) by flow cytometry. T cell signaling induced by TCR cross-linking, IL-2, or PMA/ionomycin was found to be blunted within all T cell subpopulations in those with progressive HIV disease compared with long-term nonprogressors and responders. Although alterations in cellular signaling correlated with levels of basal phosphorylation, viral load, and/or expression of programmed death-1, it was the level of basal phosphorylation that appeared to be the factor most dominantly associated with impaired signaling. Notably, provision of effective antiretroviral therapy was associated with a normalization of both basal phosphorylation levels and T cell signaling. These data, in aggregate, suggest that generalized dysfunction of the T cell compartment during progressive HIV disease may be in part dependent upon an increased basal level of phosphorylation, which itself may be due to the heightened state of immune activation found in advanced disease.

WD-repeat-propeller-FYVE protein, ProF, binds VAMP2 and protein kinase Czeta.

We have recently identified a protein, consisting of seven WD repeats, presumably forming a beta-propeller, and a domain identified in Fab1p, YOTB, VAC1p, and EEA1 (FYVE) domain, ProF. The FYVE domain targets the protein to vesicular membranes, while the WD repeats allow binding of the activated kinases Akt and protein kinase (PK)Czeta. Here, we describe the vesicle-associated membrane protein 2 (VAMP2) as interaction partner of ProF. The interaction is demonstrated with overexpressed and endogenous proteins in mammalian cells. ProF and VAMP2 partially colocalize on vesicular structures with PKCzeta and the proteins form a ternary complex. VAMP2 can be phosphorylated by activated PKCzeta in vitro and the presence of ProF increases the PKCzeta-dependent phosphorylation of VAMP2 in vitro. ProF is an adaptor protein that brings together a kinase with its substrate. VAMP2 is known to regulate docking and fusion of vesicles and to play a role in targeting vesicles to the plasma membrane. The complex may be involved in vesicle cycling in various secretory pathways.

A WD-FYVE protein binds to the kinases Akt and PKCzeta/lambda.

WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein-protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCzeta/lambda (protein kinase Czeta/lambda) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.

JM4 is a four-transmembrane protein binding to the CCR5 receptor.

The CC chemokine receptor 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV) and CCR5 mutants lacking the carboxy (C)-terminus interfere with HIV infection. Therefore, we analysed the C-terminus of CCR5 and here describe Jena-Muenchen 4 (JM4), a novel CCR5-interacting protein. JM4 is membrane-associated, co-precipitates with CCR5, and is ubiquitously expressed. It shares about 62% sequence similarity with JWA and glutamate transporter-associated protein 3-18 (GTRAP3-18), a regulator of an amino acid transporter. JWA, like JM4, is a four-transmembrane protein, which binds to the CCR5 receptor. Furthermore, JM4, JWA, and GTRAP3-18 co-localise and heterodimerise indicating a functional relationship. JM4 co-localises with calnexin in the endoplasmic reticulum and with the mannose 6-phosphate receptor in the Golgi. JM4 and GTRAP3-18 harbor a Rab-acceptor motif, indicating a function in vesicle formation at the Golgi complex. In conclusion, we describe a CCR5-interacting protein, which is suggested to function in trafficking and membrane localisation of the receptor, possibly also other receptors or amino acid transporters.

The HIV-1 co-receptor CCR5 binds to alpha-catenin, a component of the cellular cytoskeleton.

The chemokine receptors CCR5 and CXCR4 belong to the family of seven transmembrane-spanning G protein-coupled receptors, which have diverse functions in host cell defense and are associated with numerous diseases. CCR5 and CXCR4 are known as co-receptors for entry of HIV-1. In this study the intracellular carboxy-terminus of CCR5, which is deleted in HIV-infected long-term non-progressors, was shown to interact with the carboxy-terminus of alpha-catenin, a component of the cytoskeleton, in a yeast two-hybrid screen. This interaction was verified in mammalian cells. Furthermore, the interaction of alpha-catenin with CCR5 and CXCR4 at endogenous protein levels was demonstrated in PM1 T-lymphocytes, a host cell line of HIV-1. Our results suggest that alpha-catenin links CCR5 and CXCR4 to the cytoskeleton and is involved in the organization of these receptors at the membrane, thereby possibly affecting HIV-1 infection.

A combination of plasmid DNAs encoding murine fetal liver kinase 1 extracellular domain, murine interleukin-12, and murine interferon-gamma inducible protein-10 leads to tumor regression and survival in melanoma-bearing mice.

The vascular endothelial growth factor (VEGF) and its interaction with the vascular endothelial growth factor receptor 2 [VEGFR2/murine fetal liver kinase 1 (Flk-1), human kinase domain receptor] are an important angiogenic pathway leading to tumor vascularization. A plasmid DNA encoding the complete extracellular domain (ECD) of murine Flk-1 including the endogenous signal sequence was designed as a possible competitor of the receptor to sequester VEGF. The plasmid DNA was used to treat B16F10 cell-induced subcutaneous melanomas in syngeneic mice. The Flk-1 ECD-encoding plasmid DNA injected intramuscularly did not lead to tumor reduction. However, intratumoral injection caused a dose-dependent reduction and significant retardation of tumor growth. Blood vessels analyzed by immunohistochemistry with anti-CD31 antibodies as indicators of vascularization appeared smaller in diameter after treatment. A combination of Flk-1 ECD and DNA encoding murine interleukin-12 or murine interferon-gamma inducible protein-10 improved the effect, leading to tumor regression and long-term survival of the mice.

Regulation of Raf-Akt Cross-talk.

We have recently shown that the Ras-Raf-MEK-ERK and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathways can cross-talk in the human breast cancer cell line MCF-7. High Raf activity induces growth arrest and differentiation in these cells, whereas high PI3K/Akt activity correlates with cell survival and proliferation. Here we show that the Raf-Akt cross-talk is regulated in a concentration- and ligand-dependent manner. High doses of insulin-like growth factor I (IGF-I) activate Akt quickly and strongly enough to suppress Raf kinase activity via phosphorylation of Ser-259, whereas low doses of IGF-I do not trigger this cross-talk but are still mitogenic. Phorbol 12-myristate 13-acetate, a differentiation-inducing stimulus, potently activates the Ras-Raf-MEK-ERK pathway but only weakly activates PI3K/Akt and does not trigger the cross-talk. Thus, the herein analyzed parameters such as ligand type, concentration, and time course may contribute to the cellular response of either proliferation or differentiation. This is highly relevant to understanding cellular transformation and may be of use in areas like tissue engineering.