Viral Delivery of IL-7 Is a Potent Immunotherapy Stimulating Innate and Adaptive Immunity and Confers Survival in Sepsis Models.

Persistence of an immunosuppressive state plays a role in septic patient morbidity and late mortality. Both innate and adaptive pathways are impaired, pointing toward the need for immune interventions targeting both arms of the immune system. We developed a virotherapy using the nonpropagative modified vaccinia virus Ankara (MVA), which harbors the intrinsic capacity to stimulate innate immunity, to deliver IL-7, a potent activator of adaptive immunity. The rMVA-human IL-7 (hIL-7)-Fc encoding the hIL-7 fused to the human IgG2-Fc was engineered and shown to express a dimeric, glycosylated, and biologically active cytokine. Following a single i.v. injection in naive mice, the MVA-hIL-7-Fc increased the number of total and activated B, T, and NK cells but also myeloid subpopulations (Ly6Chigh, Ly6Cint, and Ly6Cneg cells) in both lung and spleen. It triggered differentiation of T cells in central memory, effector memory, and acute effector phenotypes and enhanced polyfunctionality of T cells, notably the number of IFN-γ-producing cells. The MVA vector contributed significantly to immune cell activation, particularly of NK cells. The MVA-hIL-7-Fc conferred a significant survival advantage in the cecal ligation and puncture (CLP) and Candida albicans sepsis models. It significantly increased cell numbers and activation in both spleen and lung of CLP mice. Comparatively, in naive and CLP mice, the rhIL-7-Fc soluble counterpart overall induced less vigorous, shorter lasting, and narrower immune activities than did the MVA-hIL-7-Fc and favored TNF-α-producing cells. The MVA-hIL-7-Fc represents a novel class of immunotherapeutic with clinical potential for treatment of septic patients.

Impaired NFAT transcriptional activity in antigen-stimulated CD8 T cells linked to defective phosphorylation of NFAT transactivation domain.

NFAT transcription factors play critical roles in CD4 T cell activation and differentiation. Their function in CD8 T cell is, however, unknown. We show in this study that, in contrast to CD4 T cells, Ag-stimulated CD8 T cells do not demonstrate NFAT transcriptional activity despite normal regulation of NFAT nuclear shuttling. Further analysis of the signaling defect shows that phosphorylation of the (53)SSPS(56) motif of the NFAT transactivation domain is essential for NFAT-mediated transcription in primary T cells. Although Ag stimulation induces in CD4 T cells extensive phosphorylation of this motif, it does so only minimally in CD8 T cells. Although Ag stimulation triggers only modest activation of the p38 MAPK in CD8 T cells as opposed to CD4 T cells, p38 MAPK is not the upstream kinase that directly or indirectly phosphorylates the NFAT (53)SSPS(56) motif. These findings reveal an unsuspected difference between CD4 and CD8 T cells in the TCR downstream signaling pathway. Therefore, whereas in CD4 T cells TCR/CD28 engagement activates a yet unknown kinase that can phosphorylate the NFAT (53)SSPS(56) motif, this pathway is only minimally triggered in CD8 T cells, thus limiting NFAT transcriptional activity.

Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

[Th17 cells, a novel proinflammatory effector CD4 T cell population]. / Les cellules Th17: Une nouvelle population de cellules T CD4 effectrices pro-inflammatoires.

After more than 20 years of hegemony, the Th1-Th2 paradigm was recently shaken by the discovery of a novel population of CD4 effector T cells, the Th17 cells. Th17 effector cells produce IL-17 and IL-22 and thus have pro-inflammatory properties notably favoring neutrophils recruitment and thus control of extracellular bacteria mainly at the epithelium surface. Th17 cells appear also as the major inducer of organ specific autoimmune pathologies such as EAE or rheumatoid arthritis, a function previously attributed to Th1 effector cells. The discovery of Th17 cells further supports the notion that effector CD4 T cells responses are diverse in vivo and that fine tuning of these different effector cells is critical to maintain tissue integrity.

A multi-antigenic MVA vaccine increases efficacy of combination chemotherapy against Mycobacterium tuberculosis.

Despite the existence of the prophylactic Bacille Calmette-Guérin (BCG) vaccine, infection by Mycobacterium tuberculosis (Mtb) remains a major public health issue causing up to 1.8 million annual deaths worldwide. Increasing prevalence of Mtb strains resistant to antibiotics represents an urgent threat for global health that has prompted a search for alternative treatment regimens not subject to development of resistance. Immunotherapy constitutes a promising approach to improving current antibiotic treatments through engagement of the host's immune system. We designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara (MVA) virus, denoted MVATG18598, which expresses ten antigens classically described as representative of each of different phases of Mtb infection. In vitro analysis coupled with multiple-passage evaluation demonstrated that this vaccine is genetically stable, i.e. fit for manufacturing. Using different mouse strains, we show that MVATG18598 vaccination results in both Th1-associated T-cell responses and cytolytic activity, targeting all 10 vaccine-expressed Mtb antigens. In chronic post-exposure mouse models, MVATG18598 vaccination in combination with an antibiotic regimen decreases the bacterial burden in the lungs of infected mice, compared with chemotherapy alone, and is associated with long-lasting antigen-specific Th1-type T cell and antibody responses. In one model, co-treatment with MVATG18598 prevented relapse of the disease after treatment completion, an important clinical goal. Overall, results demonstrate the capacity of the therapeutic MVATG18598 vaccine to improve efficacy of chemotherapy against TB. These data support further development of this novel immunotherapeutic in the treatment of Mtb infections.

Involvement of cholecystokinin 2 receptor in food intake regulation: hyperphagia and increased fat deposition in cholecystokinin 2 receptor-deficient mice.

The role of cholecystokinin (CCK) as a satiety factor has been extensively documented. Although most work implies that CCK1 receptor mediates the control of food intake, a contributing role for CCK2 receptor (CCK2R) in the CCK-induced satiety cannot be totally excluded. The hypothesis that CCK2R invalidation disrupts regulatory pathways with impact on feeding behavior was examined in CCK2R(-/-) mice. CCK2R(-/-) mice developed obesity that was associated with hyperphagia. Obesity was related with increased fat deposition resulting from adipocyte hypertrophy. Expression of several adipokines was dysregulated consistently with obesity. Moreover, obesity was associated with disturbed glucose homeostasis as revealed by increased fasting glycemia and insulinemia, impaired glucose tolerance, and hepatic insulin resistance in CCK2R(-/-) mice. In vitro analysis of isolated adipocytes metabolism was consistent with increased storage but preserved insulin sensitivity. Suppression of feeding and concomitant increased expression of hypothalamic proopiomelanocortin after intracerebroventricular injection of gastrin into control mice demonstrates that hypothalamic CCK2 receptors mediate inhibition of food intake. Comparative analysis of hypothalamic mediator gene expression in fed knockout and control mice demonstrated overexpression of ghrelin receptors in CCK2R(-/-) mice, indicating up-regulation of orexigenic pathways. This effect was also observed after body weight normalization, indicating a causative role in the development of hyperphagia and obesity of CCK2R(-/-) mice. Our results give evidence that CCK2 receptor activity plays a contributing regulatory role in the control of food intake.

GTL001 and bivalent CyaA-based therapeutic vaccine strategies against human papillomavirus and other tumor-associated antigens induce effector and memory T-cell responses that inhibit tumor growth.

GTL001 is a bivalent therapeutic vaccine containing human papillomavirus (HPV) 16 and HPV18 E7 proteins inserted in the Bordetella pertussis adenylate cyclase (CyaA) vector intended to prevent cervical cancer in HPV-infected women with normal cervical cytology or mild abnormalities. To be effective, therapeutic cervical cancer vaccines should induce both a T cell-mediated effector response against HPV-infected cells and a robust CD8+ T-cell memory response to prevent potential later infection. We examined the ability of GTL001 and related bivalent CyaA-based vaccines to induce, in parallel, effector and memory CD8+ T-cell responses to both vaccine antigens. Intradermal vaccination of C57BL/6 mice with GTL001 adjuvanted with a TLR3 agonist (polyinosinic-polycytidylic acid) or a TLR7 agonist (topical 5% imiquimod cream) induced strong HPV16 E7-specific T-cell responses capable of eradicating HPV16 E7-expressing tumors. Tumor-free mice also had antigen-specific memory T-cell responses that protected them against a subsequent challenge with HPV18 E7-expressing tumor cells. In addition, vaccination with bivalent vaccines containing CyaA-HPV16 E7 and CyaA fused to a tumor-associated antigen (melanoma-specific antigen A3, MAGEA3) or to a non-viral, non-tumor antigen (ovalbumin) eradicated HPV16 E7-expressing tumors and protected against a later challenge with MAGEA3- and ovalbumin-expressing tumor cells, respectively. These results show that CyaA-based bivalent vaccines such as GTL001 can induce both therapeutic and prophylactic anti-tumor T-cell responses. The CyaA platform can be adapted to different antigens and adjuvants, and therefore may be useful for developing other therapeutic vaccines.

A Novel MVA-Based Multiphasic Vaccine for Prevention or Treatment of Tuberculosis Induces Broad and Multifunctional Cell-Mediated Immunity in Mice and Primates.

Bacille Calmette-Guérin (BCG) vaccination of new born babies can protect children against tuberculosis (TB), but fails to protect adults consistently against pulmonary TB underlying the urgent need to develop novel TB vaccines. Majority of first generation TB vaccine candidates have relied on a very limited number of antigens typically belonging to the active phase of infection. We have designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara virus (MVA). Up to fourteen antigens representative of the three phases of TB infection (active, latent and resuscitation) were inserted into MVA. Using three different strains of mouse (BALB/c, C57BL/6 and C3H/HeN), we show that a single vaccination results in induction of both CD4 and CD8 T cells, displaying capacity to produce multiple cytokines together with cytolytic activity targeting a large array of epitopes. As expected, dominance of responses was linked to the mouse haplotype although for a given haplotype, responses specific of at least one antigen per phase could always be detected. Vaccination of non-human primates with the 14 antigens MVA-TB candidate resulted in broad and potent cellular-based immunogenicity. The remarkable plasticity of MVA opens the road to development of a novel class of highly complex recombinant TB vaccines to be evaluated in both prophylactic and therapeutic settings.

Thymus-specific serine protease controls autoreactive CD4 T cell development and autoimmune diabetes in mice.

Type 1 diabetes is a chronic autoimmune disease in which genetic predispositions affect the immune system, leading to a loss of T cell tolerance to ß cells and consequent T cell-mediated destruction of insulin-producing islet cells. Genetic studies have suggested that PRSS16 is linked to a diabetes susceptibility locus of the extended HLA class I region in humans. PRSS16 encodes what we believe to be a novel protease, thymus-specific serine protease (TSSP), which shows predominant expression in thymic epithelial cells and is suspected to have a restricted role in the class II presentation pathway. Consistently, Tssp is necessary for the intrathymic selection of few class II-restricted T cell receptor specificities in B6 mice. To directly assess the role of Tssp in autoimmune diabetes, we generated Tssp-deficient (Tssp°) NOD mice. While remaining immunocompetent, Tssp° NOD mice were protected from diabetes and severe insulitis. Diabetes resistance of Tssp° NOD mice was a property of the CD4 T cell compartment that is acquired during thymic selection and correlated with an impaired selection of CD4 T cells specific for islet antigens. Hence, in the NOD mouse, Tssp is a critical regulator of diabetes development through the selection of the autoreactive CD4 T cell repertoire.

Essential interaction of Egr-1 at an islet-specific response element for basal and gastrin-dependent glucagon gene transactivation in pancreatic alpha-cells.

The peptide hormone gastrin is secreted from G cells of the gastric antrum and is the main inducer of gastric acid secretion via activation of its receptor the cholecystokinin 2 (CCK2) receptor. Both gastrin and CCK2 receptors are also transiently detected in the fetal pancreas and believed to exert growth/differentiation effects during endocrine pancreatic development. We demonstrated previously that whereas gastrin expression is extinguished in adult pancreas, CCK2 receptors are present in human glucagon-producing cells where their activation stimulates glucagon secretion. Based on these findings, we investigate in the present study whether gastrin regulates glucagon gene expression. To this aim, the CCK2 receptor was stably expressed into a glucagon-producing pancreatic islet cell line, and a glucagon-reporter fusion gene was transiently transfected in this new cellular model. We report that gastrin stimulates glucagon gene expression in glucagon-producing pancreatic cells. By using progressively 5'-increased sequences of the glucagon gene, gastrin responsiveness was located within the minimal promoter. Moreover, we clearly identified early growth response protein 1 (Egr-1) as an essential transcription factor interacting with the islet cell-specific G4 element. Egr-1 was shown to be essential for basal and gastrin-dependent glucagon gene transactivation. Furthermore, our results demonstrate that the MEK1/ERK1/2 pathway couples the CCK2 receptor to nuclearization and DNA binding of Egr-1. In conclusion, our data provide new information concerning the transcriptional regulation of the glucagon gene. Moreover they open new working hypothesis with reference to a potential role of gastrin in glucagon-producing pancreatic cells.

Expression of CCK2 receptors in the murine pancreas: proliferation, transdifferentiation of acinar cells, and neoplasia.

BACKGROUND & AIMS: To explore the pancreatic function of CCK2/gastrin receptor, we created ElasCCK2 transgenic mice expressing the human receptor in pancreatic exocrine cells. In previous studies, the transgenic CCK2/gastrin receptor was demonstrated to mediate enzyme release and protein synthesis. We now report results of phenotypic and long-term studies. METHODS: Pancreas was characterized using morphometry and immunohistochemistry. ElasCCK2 mice were crossed with INS-GAS mice expressing gastrin in pancreatic beta cells to achieve continuous stimulation of the CCK2/gastrin receptor. RESULTS: The pancreatic weight of ElasCCK2 mice was increased by 40% and correlated with an increase in the area of exocrine tissue. Alterations in pancreatic histology were apparent from postnatal day 50. Crossing the ElasCCK2 mice with INS-GAS mice resulted in development of morphologic changes in younger animals. Malignant transformation occurred in 3 of 20 homozygous ElasCCK2 mice. Although tumors had different phenotypes, they all developed through an acinar-ductal carcinoma sequence. CONCLUSIONS: Our data show that transgenic expression of a G protein-coupled receptor can lead to cancer. This study also supports a key role of the CCK2/gastrin receptor in the development of pre- and neoplastic lesions of the pancreas. ElasCCK2 mice provide a model for carcinogenesis by transformation and dedifferentiation of acinar cells.