Role of NS1 and TLR3 in Pathogenesis and Immunity of WNV.

West Nile Virus (WNV) is a mosquito-transmitted flavivirus which causes encephalitis especially in elderly and immunocompromised individuals. Previous studies have suggested the protective role of the Toll-like receptor 3 (TLR3) pathway against WNV entry into the brain, while the WNV non-structural protein 1 (NS1) interferes with the TLR3 signaling pathway, besides being a component of viral genome replication machinery. In this study, we investigated whether immunization with NS1 could protect against WNV neuroinvasion in the context of TLR3 deficiency. We immunized mice with either an intact or deleted TLR3 system (TLR3KO) with WNV envelope glycoprotein (gE) protein, NS1, or a combination of gE and NS1. Immunization with gE or gE/NS1, but not with NS1 alone, induced WNV neutralizing antibodies and protected against WNV brain invasion and inflammation. The presence of intact TLR3 signaling had no apparent effect on WNV brain invasion. However, mock-immunized TLR3KO mice had higher inflammatory cell invasion upon WNV brain infection than NS1-immunized TLR3KO mice and wild type mice. Thus, immunization against NS1 may reduce brain inflammation in a context of TLR3 signaling deficiency.

Corrigendum: Pramel7 mediates ground-state pluripotency through proteasomal-epigenetic combined pathways.

This corrects the article DOI: 10.1038/ncb3554.

Pramel7 mediates ground-state pluripotency through proteasomal-epigenetic combined pathways.

Naive pluripotency is established in preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of naive pluripotency. 2i culture has optimized this state, leading to a gene signature and DNA hypomethylation closely comparable to preimplantation epiblast, the developmental ground state. Here we show that Pramel7 (PRAME-like 7), a protein highly expressed in the inner cell mass (ICM) but expressed at low levels in ESCs, targets for proteasomal degradation UHRF1, a key factor for DNA methylation maintenance. Increasing Pramel7 expression in serum-cultured ESCs promotes a preimplantation epiblast-like gene signature, reduces UHRF1 levels and causes global DNA hypomethylation. Pramel7 is required for blastocyst formation and its forced expression locks ESCs in pluripotency. Pramel7/UHRF1 expression is mutually exclusive in ICMs whereas Pramel7-knockout embryos express high levels of UHRF1. Our data reveal an as-yet-unappreciated dynamic nature of DNA methylation through proteasome pathways and offer insights that might help to improve ESC culture to reproduce in vitro the in vivo ground-state pluripotency.

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice.

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 µg compared to those immunized with 3 or 10 µg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn(®) WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.

Pramel7 mediates LIF/STAT3-dependent self-renewal in embryonic stem cells.

A unique and complex signaling network allows ESCs to undergo extended proliferation in vitro, while maintaining their capacity for multilineage differentiation. Genuine ESC identity can only be maintained when both self-renewal and suppression of differentiation are active and balanced. Here, we identify Pramel7 (preferentially expressed antigen in melanoma-like 7) as a novel factor crucial for maintenance of pluripotency and leukemia inhibitory factor (LIF)-mediated self-renewal in ESCs. In vivo, Pramel7 expression was exclusively found in the pluripotent pools of cells, namely, the central part of the morula and the inner cell mass of the blastocyst. Ablation of Pramel7 induced ESC differentiation, whereas its overexpression was sufficient to support long-term self-renewal in the absence of exogenous LIF. Furthermore, Pramel7 overexpression suppressed differentiation in ESCs in vitro and in vivo. This process was reversible, as on transgene excision cells reverted to a LIF-dependent state and regained their capacity to participate in the formation of chimeric mice. Molecularly, LIF directly controls Pramel7 expression, involving both STAT3-dependent transcriptional regulation and PI3K-dependent phosphorylation of glycogen synthase kinase 3ß. Pramel7 expression in turn confers constitutive self-renewal and prevents differentiation through inactivation of extracellular signal-regulated kinase phosphorylation. Accordingly, knockdown of Pramel7 promotes ESC differentiation in presence of LIF and even on forced STAT3-activation. Thus, Pramel7 represents a central and essential factor in the signaling network regulating pluripotency and self-renewal in ESCs.