Analysis of the species barrier of human cells against infection with canine distemper Virus (CDV)

Die geplante Ausrottung der Masern bis 2020 und die damit eventuell einhergehende Beendigung der Masernimpfung könnten die Voraussetzungen dafür schaffen, dass andere Morbilliviren, wie beispielsweise das Hundestaupevirus (CDV), einen Wirtswechsel zum Menschen vollbringen könnten. CDV ist ein hoch ansteckendes Pathogen und besitzt einen weiten Wirtstropismus, der sich aktuell immer weiter ausbreitet. Im Gegensatz dazu kann das Masernvirus (MV) nahezu ausschließlich Menschen und nur sehr bedingt wenige Affenarten infizieren. In dieser Doktorarbeit konnte gezeigt werden, dass eine Adaptierung des rekombinanten wildtypischen CDV-Stammes CDV-75/17red an den humanen Rezeptor SLAM (signaling lymphocytic activation molecule, CD150) reproduzierbar und innerhalb weniger Passagen erfolgt. Bei der Adaptierung an das humane SLAM ist dabei nur eine Mutation in dem Gen für das virale Hämagglutinin notwendig. Diese Mutation an Position 8697 von A zu G im viralen Genom (Aminosäure D540G im Hämagglutinin) konnte reproduzierbar detektiert werden, obwohl veröffentlicht wurde, dass unterschiedliche Mutationen im Hämagglutinin verschiedener CDV-Stämme eine SLAM-Adaptierung ermöglichen. Die Mutation D540G im Hämagglutinin des humanen SLAM-adaptierten CDV-A75/17red kompensiert eine negative Ladung der Aminosäure 71E, die speziesspezifisch im humanen SLAM vorhanden ist. Durch Wachstumskinetiken konnte belegt werden, dass das an humanes SLAM-adaptierte CDV-A75/17red auch weiterhin das canine SLAM effizient verwendet. Ein weiterer Eintrittsrezeptor, humanes Nectin4, konnte mit demselben CDV-Stamm ohne adaptive Mutation in den viralen Hüllproteingenen benutzt werden. Wachstumskurven auf verschiedenen humanen B-Lymphozyten Zelllinien zeigen allerdings, dass eine alleinige Adaptierung an die humanen Wirtszellrezeptoren, für eine effiziente Virusreplikation, nicht ausreicht. Damit das CDV die Speziesbarriere durchbrechen kann, muss offenbar ein weiterer Adaptierungsprozess an die humanen Wirtszellen erfolgen, der voraussichtlich mit umfangreicheren Mutationen des viralen Genoms einhergehen würde. Diese Ergebnisse unterstreichen, dass intrinsische Faktoren und das angeborene Immunsystem eine wichtige Barriere bilden und den Menschen vor einer CDV-Infektion schützen. Allerdings würde eine Fortführung der MV-Impfung auch nach Ausrottung der Masern, aufgrund der Kreuzreaktivität gegen andere Morbilliviren, den Schutz vor einer möglichen Adaptierung eines Morbillivirus, wie CDV, an den Menschen deutlich verstärken.Analysis of the species barrier of human cells against infection with canine distemper Virus (CDV

Comparison of single step growth curves of parental and adapted CDV-A75/17^red using canine and human CD150-expressing target cells.

<p>Vero-cSLAM cells (open circles) and Vero-hSLAM cells (closed circles) were infected with parental CDV-A75/17^red, and Vero-cSLAM cells (blue triangles) and Vero-hSLAM cells (red squares) were infected with human CD150-adapted CDV-A75/17^red (CDV-A75-ad) at a MOI of 0.01 and incubated for indicated times before harvesting cell bound plus released virus. The viruses were then titrated using the optimal target cells for each virus (n = 3, using 3 independently adapted viruses).</p

Infection of human epithelial cells H358 with CDV-A75/17^red.

<p>H358 cells were infected with recombinant wild-type CDV-A75/17^red at a MOI of 1.0 (<b>A</b>), incubated for the indicated times and pictures of the red autofluorescence taken under UV light. At 8 days post-infection (dpi) virus was prepared and titrated using Vero-cSLAM cells, and H358 cells infected with this virus at a MOI of 0.01 (<b>B</b>). After 8 dpi virus was again prepared and titrated using Vero-cSLAM cells, and H358 cells were infected with this virus at a MOI of 0.01 for indicated times (<b>C</b>). Photomicrographs of the autofluorescent virus-encoded tD tomato red were taken under UV light (bar = 100 µm).</p

Comparison of the CD150 binding sites of Morbillivirus H proteins.

<p>The receptor (CD150) binding site of the Morbillivirus H-proteins comprises approximately amino acid 500 to 550. CDV-H position 540 (corresponding to MV-H position 544) is located nearby the cognate receptor interacting residues 525, 526, and 529 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057488#pone.0057488-Zipperle1" target="_blank">[25]</a>. CDV-A75-ad = human CD150-adapted CDV-A75/17^red.</p

Comparison of the canine and human CD150 sequences around the Morbillivirus H binding site.

<p>The MV-H binding site of CD150 was mapped between amino acid 61 and 131 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057488#pone.0057488-Hashiguchi2" target="_blank">[33]</a>. Amino acid 71 is an uncharged G in dog CD150, and a negatively charged E in human CD150 (NM_001003084 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057488#pone.0057488-Zipperle1" target="_blank">[25]</a>).</p

Infection of Vero-hSLAM cells with CDV-A75/17^red.

<p>Human CD150-expressing Vero cells (Vero-hSLAM) were infected with recombinant wild-type CDV-A75/17^red at a MOI of 1.0 (<b>A</b>), incubated for the indicated times and pictures of the red autofluorescence taken under UV light. At 4 days post-infection (dpi) virus was prepared and titrated, and Vero-hSLAM cells infected at a MOI of 0.01 (<b>B</b>). After 4 dpi virus was again prepared and titrated, and Vero-hSLAM cells were infected with this virus at a MOI of 0.01 for indicated times (<b>C</b>). Titres of the virus preparations were determined using Vero-cSLAM cells and are given in (<b>D</b>). Photomicrographs of the autofluorescent virus-encoded tD tomato red were taken under UV light (bar = 100 µm).</p

Antigen receptor-mediated depletion of FOXP3 in induced regulatory T-lymphocytes via PTPN2 and FOXO1

Regulatory T-cells induced via IL-2 and TGF in vitro (iTreg) suppress immune cells and are potential therapeutics during autoimmunity. However, several reports described their re-differentiation into pathogenic cells in vivo and loss of their key functional transcription factor (TF) FOXP3 after T-cell antigen receptor (TCR)-signalling in vitro. Here, we show that TCR-activation antagonizes two necessary TFs for foxp3 gene transcription, which are themselves regulated by phosphorylation. Although the tyrosine phosphatase PTPN2 is induced to restrain IL-2-mediated phosphorylation of the TF STAT5, expression of the TF FOXO1 is downregulated and miR-182, a suppressor of FOXO1 expression, is upregulated. TGF counteracts the FOXP3-depleting TCR-signal by reassuring FOXO1 expression and by re-licensing STAT5 phosphorylation. Overexpressed phosphorylation-independent active versions of FOXO1 and STAT5 or knockdown of PTPN2 restores FOXP3 expression despite TCR-signal and absence of TGF. This study suggests novel targets for stabilisation and less dangerous application of iTreg during devastating inflammation

Antigen receptor-mediated depletion of FOXP3 in induced regulatory T-lymphocytes via PTPN2 and FOXO1

Regulatory T-cells induced via IL-2 and TGFβ in vitro (iTreg) suppress immune cells and are potential therapeutics during autoimmunity. However, several reports described their re-differentiation into pathogenic cells in vivo and loss of their key functional transcription factor (TF) FOXP3 after T-cell antigen receptor (TCR)-signalling in vitro. Here, we show that TCR-activation antagonizes two necessary TFs for foxp3 gene transcription, which are themselves regulated by phosphorylation. Although the tyrosine phosphatase PTPN2 is induced to restrain IL-2-mediated phosphorylation of the TF STAT5, expression of the TF FOXO1 is downregulated and miR-182, a suppressor of FOXO1 expression, is upregulated. TGFβ counteracts the FOXP3-depleting TCR-signal by reassuring FOXO1 expression and by re-licensing STAT5 phosphorylation. Overexpressed phosphorylation-independent active versions of FOXO1 and STAT5 or knockdown of PTPN2 restores FOXP3 expression despite TCR-signal and absence of TGFβ. This study suggests novel targets for stabilisation and less dangerous application of iTreg during devastating inflammation