Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.

Emerging viral infections are difficult to control because heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) successively emerged, causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. We show that a nucleotide prodrug, GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems, including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat CoVs, prepandemic bat CoVs, and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory function. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and, possibly most importantly, emerging CoV of the future.

Bruton's Tyrosine Kinase Synergizes with Notch2 To Govern Marginal Zone B Cells in Nonobese Diabetic Mice.

Expansion of autoimmune-prone marginal zone (MZ) B cells has been implicated in type 1 diabetes. To test disease contributions of MZ B cells in NOD mice, Notch2 haploinsufficiency (Notch2(+/-)) was introduced but failed to eliminate the MZ, as it does in C57BL/6 mice. Notch2(+/-)/NOD have MZ B cell numbers similar to those of wild-type C57BL/6, yet still develop diabetes. To test whether BCR signaling supports Notch2(+/-)/NOD MZ B cells, Bruton's tyrosine kinase (Btk) deficiency was introduced. Surprisingly, MZ B cells failed to develop in Btk-deficient Notch2(+/-)/NOD mice. Expression of Notch2 and its transcriptional target, Hes5, was increased in NOD MZ B cells compared with C57BL/6 MZ B cells. Btk deficiency reduced Notch2(+/-) signaling exclusively in NOD B cells, suggesting that BCR signaling enhances Notch2 signaling in this autoimmune model. The role of BCR signaling was further investigated using an anti-insulin transgenic (Tg) BCR (125Tg). Anti-insulin B cells in 125Tg/Notch2(+/-)/NOD mice populate an enlarged MZ, suggesting that low-level BCR signaling overcomes reliance on Notch2. Tracking clonotypes of anti-insulin B cells in H chain-only VH125Tg/NOD mice showed that BTK-dependent selection into the MZ depends on strength of antigenic binding, whereas Notch2-mediated selection does not. Importantly, anti-insulin B cell numbers were reduced by Btk deficiency, but not Notch2 haploinsufficiency. These studies show that 1) Notch2 haploinsufficiency limits NOD MZ B cell expansion without preventing type 1 diabetes, 2) BTK supports the Notch2 pathway in NOD MZ B cells, and 3) autoreactive NOD B cell survival relies on BTK more than Notch2, regardless of MZ location, which may have important implications for disease-intervention strategies.

Tolerant anti-insulin B cells are effective APCs.

Autoreactive B lymphocytes that are not culled by central tolerance in the bone marrow frequently enter the peripheral repertoire in a state of functional impairment, termed anergy. These cells are recognized as a liability for autoimmunity, but their contribution to disease is not well understood. Insulin-specific 125Tg B cells support T cell-mediated type 1 diabetes in NOD mice, despite being anergic to B cell mitogens and T cell-dependent immunization. Using this model, the potential of anergic, autoreactive B cells to present Ag and activate T cells was investigated. The data show that 1) insulin is captured and rapidly internalized by 125Tg BCRs, 2) these Ag-exposed B cells are competent to activate both experienced and naive CD4(+) T cells, 3) anergic 125Tg B cells are more efficient than naive B cells at activating T cells when Ag is limiting, and 4) 125Tg B cells are competent to generate low-affinity insulin B chain epitopes necessary for activation of diabetogenic anti-insulin BDC12-4.1 T cells, indicating the pathological relevance of anergic B cells in type 1 diabetes. Thus, phenotypically tolerant B cells that are retained in the repertoire may promote autoimmunity by driving activation and expansion of autoaggressive T cells via Ag presentation.