Organ-Specific Expression of IL-1 Receptor Results in Severe Liver Injury in Type I Interferon Receptor Deficient Mice

Upon treatment with polyinosinic:polycytidylic acid [poly(I:C)], an artificial double-stranded RNA, type I interferon receptor-deficient (IFNAR−/−) mice develop severe liver injury seen by enhanced alanine aminotransferase (ALT) activity in the serum that is not observed in their wildtype (WT) counterparts. Recently, we showed that liver injury is mediated by an imbalanced expression of interleukin (IL)-1β and its receptor antagonist (IL1-RA) in the absence of type I IFN. Here we show that despite comparable expression levels of IL-1β in livers and spleens, spleens of poly(I:C)-treated IFNAR−/− mice show no signs of injury. In vitro analyses of hepatocytes and splenocytes revealed that poly(I:C) had no direct toxic effect on hepatocytes. Furthermore, expression levels of cytokines involved in other models for liver damage or protection such as interferon (IFN)-γ, transforming growth factor (TGF)-β, IL-6, IL-10, IL-17, and IL-22 were comparable for both organs in WT and IFNAR−/− mice upon treatment. Moreover, flow cytometric analyses showed that the composition of different immune cells in livers and spleens were not altered upon injection of poly(I:C). Finally, we demonstrated that the receptor binding IL-1β, IL1R1, is specifically expressed in livers but not spleens of WT and IFNAR−/− mice. Accordingly, mice double-deficient for IFNAR and IL1R1 developed no liver injury upon poly(I:C) treatment and showed ALT activities comparable to those of WT mice. Collectively, liver injury is mediated by the organ-specific expression of IL1R1 in the liver

Loss of hCD45⁺ or hCD3⁺ cells from the peripheral blood upon TGN1412 or OKT3 treatment.

<p>Humanized mice were injected i.v. with 20 μg OKT3 or 20 μg TGN1412 per 10 grams body weight. <b>(A)</b> Before (black bars) and 2–6 hours post OKT3 (n = 11–12) or TGN1412 (n = 18–20) application (time point of sacrifice; gray bars), percentage and composition of hCD45⁺ cells in peripheral blood of reconstituted mice were analyzed by flow cytometric analysis. PBS-treated mice (n = 12) were used as control. Each line represents an individual mouse. *** p < 0.001; n.s., not significant (paired t-test for difference after-before). <b>(B)</b> Before and 4 hours post OKT3 application (n = 3) CD3 expression on hCD4⁺ or hCD8⁺ T cells in peripheral blood of humanized mice was investigated by flow cytometric analysis. Data from one individual animal, being representative for the indicated groups, are shown. The relative number of hCD4⁺ or hCD8⁺ T cells 4 hours after OKT3 application (gray bars) is given as percentages of total hCD4⁺ or hCD8⁺ T cells, normalized to “before”. *** p < 0.001 (paired t-test for difference after-before). <b>(C)</b> CD3 expression on human CD4⁺/CD8⁺ T cells in peripheral blood of humanized mice was investigated by flow cytometric analysis after OKT3 in vivo application (n = 3; upper panel). CD3 expression on CD4⁺/CD8⁺ T cells was investigated by flow cytometry before and after in vitro stimulation of purified human T cells with coated OKT3 (n = 3; lower panel). As control, primary human T cells were left unstained (light gray-shaded curve). Data from one individual animal or independent T cell donor, being representative for the indicated groups, are shown. Data shown in (A) are taken from 5–8 independent experiments. Data shown in (B) are representative for 2, in (C) for 3 independent experiments.</p

OKT3 application selectively downmodulates CD3 on T cells in humanized mice.

<p>Humanized mice were injected i.v. with 20 μg OKT3 or 20 μg TGN1412 per 10 grams body weight. <b>(A)</b> 2–6 hours (time point of sacrifice) post OKT3 (gray bars; n = 6–16) or TGN1412 (white bars; n = 6–16) application, percentage and composition of hCD45⁺ cells in spleen, PEC, LN, and thymus of humanized mice were analyzed by flow cytometry. PBS-treated mice (black bars; n = 6–16) were used as control. *** p < 0.001; n.s., not significant (t-test, for comparison of hCD45⁺ cells adjusted according Dunnett for multiple comparisons). <b>(B)</b> 4 hours post OKT3 (n = 3) application, CD3 expression on hCD4⁺ or hCD8⁺ T cells in spleen, PEC, LN, and thymus of humanized mice was analyzed by flow cytometry. PBS-treated mice (n = 3) were used as control to adjust quadrants for definition of positive and negative cells. Data from one individual animal, being representative for the indicated groups, are shown. <b>(C)</b> 4 hours post OKT3 (n = 3) or TGN1412 (n = 3) application, mAbs bound by their target CD3 or CD28 on human CD4⁺/CD8⁺ T cells in spleen, PEC, LN, and thymus of humanized mice were analyzed by flow cytometry (black curves). In vivo receptor occupancy of CD3 or CD28 was analyzed by exogenously adding OKT3 or TGN1412 to cells isolated from spleen, PEC, LN, and thymus of in vivo mAb-treated humanized mice (dotted curve). PBS-treated mice (n = 3) were used as control (gray-shaded curves). Data from one individual animal, being representative for the indicated groups, are shown. Data shown in (A) are taken from 2–6 independent experiments. Data shown in (B) and (C) are representative for 2 independent experiments.</p

A Highly Immunogenic and Protective Middle East Respiratory Syndrome Coronavirus Vaccine Based on a Recombinant Measles Virus Vaccine Platform.

In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR(-/-))-CD46Ge mice with 2 × 10(5) 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice

TGN1412 Induces Lymphopenia and Human Cytokine Release in a Humanized Mouse Model.

Therapeutic monoclonal antibodies (mAbs) such as the superagonistic, CD28-specific antibody TGN1412, or OKT3, an anti-CD3 mAb, can cause severe adverse events including cytokine release syndrome. A predictive model for mAb-mediated adverse effects, for which no previous knowledge on severe adverse events to be expected or on molecular mechanisms underlying is prerequisite, is not available yet. We used a humanized mouse model of human peripheral blood mononuclear cell-reconstituted NOD-RAG1-/-Aβ-/-HLADQ(tg+ or tg-)IL-2Rγc-/- mice to evaluate its predictive value for preclinical testing of mAbs. 2-6 hours after TGN1412 treatment, mice showed a loss of human CD45+ cells from the peripheral blood and loss of only human T cells after OKT3 injection, reminiscent of effects observed in mAb-treated humans. Moreover, upon OKT3 injection we detected selective CD3 downmodulation on T cells, a typical effect of OKT3. Importantly, we detected release of human cytokines in humanized mice upon both OKT3 and TGN1412 application. Finally, humanized mice showed severe signs of illness, a rapid drop of body temperature, and succumbed to antibody application 2-6 hours after administration. Hence, the humanized mouse model used here reproduces several effects and adverse events induced in humans upon application of the therapeutic mAbs OKT3 and TGN1412