Etablierung von Zellkultur und Tiermodellen zum Studium neu identifizierter Atemwegspathogene (hMPV, hCoV-NL63 und SARS-CoV)

Im Rahmen der vorliegenden Dissertation wurden das humane Metapneumovirus (hMPV) und das respiratorische Synzitial Virus (hRSV) in vitro und in vivo untersucht. Beide Viren entstammen der Familie der Paramyxoviridae, verursachen milde bis schwerwiegende Atemwegsinfektionen und sind anhand der Krankheitssymptome nicht voneinander unterscheidbar. Davon betroffen sind vor allem Kleinkinder, immunsupprimierte und ältere Menschen. Es wurde eine humane Zelllinie neu für die Replikation beider Viren identifiziert, die im Vergleich zu bisher verwendeten, nicht-humanen Zelllinien sowohl eine hochtitrige Replikation beider Viren erlaubt, als auch eine in vitro Charakterisierung molekularer Mechanismen vor humanem Hintergrund ermöglicht. Dabei zeigte sich, dass eine hMPV-Infektion andere zellmorphologische Strukturen induziert sowie unterschiedliche Zellsignalwege aktiviert als eine hRSV-Infektion. An einem optimierten BALB/c-Mausmodell wurde weiterhin gezeigt, dass hMPV und hRSV als Einzel- und als Doppelinfektion auch in vivo Unterschiede auf zellulärer Ebene hervorrufen. Da bekannt ist, dass auch ältere Individuen trotz der Anwesenheit neutralisierender Antikörper schwerwiegend an hMPV und hRSV erkranken, wurden ebenfalls altersbedingte Unterschiede untersucht. Es zeigten sich gegensätzliche immunologische Reaktionen auf zellulärer Ebene abhängig vom Alter des Tieres und dem Virus, der zur Infektion verwendet wurde: beispielsweise war der NF-kB-Spiegel in hMPV- und doppelinfizierten jungen Tieren deutlich erhöht, verblieb jedoch niedrig bei hRSV-infizierten Tieren, während bei alten Tieren das gegensätzliche Resultat beobachtet wurde. Bei keiner der infizierten Tiergruppen wurde eine Veränderung der Aktivität, des Gewichts und der Futteraufnahme im Vergleich zu Mock- oder Negativkontrolltiergruppen beobachtet, obwohl eine Replikation des jeweilig eingesetzten Virus zweifelsfrei nachgewiesen wurde. Die produktive Infektion in Zusammenhang mit den unterschiedlichen zellulären Reaktionen führt zu der Annahme, dass junge und alte Individuen jeweils unterschiedliche Mechanismen zur Bekämpfung viraler Pathogene anwenden. Der letzte Teil dieser Arbeit beschäftigt sich mit der Implementierung einer Tierversuchsplattform für das Severe acute respiratory syndrome- assoziierte Coronavirus (SARS-CoV) und das humane Coronavirus NL63 (hCoV-NL63). Untersucht wurde die Suszeptibilität syrischer Goldhamster und alter C57BL/6-Mäuse für hCoV-NL63 und verschiedene wt-Stämme von SARS-CoV

Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo

During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells)

The SARS-coronavirus-host interactome

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

Impact of everolimus plus calcineurin inhibitor on formation of non-HLA antibodies and graft outcomes in kidney transplant recipients: 12-month results from the ATHENA substudy

BackgroundNon-human leukocyte antigen (non-HLA) antibodies including antibodies targeting Angiotensin II type 1 (AT1R) and Endothelin-1 type A (ETAR) receptors represent a topic of interest in kidney transplantation (KTx). This exploratory substudy evaluated the impact of everolimus (EVR) or mycophenolic acid (MPA) in combination with tacrolimus (TAC) or cyclosporine A (CsA) in patients with preformed non-HLA antibodies, potentially associated rejections and/or their impact on renal function over 1 year.MethodsAll eligible patients were randomized (1:1:1) before transplantation to receive either EVR/TAC, EVR/CsA, or MPA/TAC regimen. The effect of these regimens on the formation of non-HLA antibodies within one year post de novo KTx and the association with clinical events was evaluated descriptively in randomized (n = 268) population.ResultsAt Month 12, in EVR/TAC group, higher incidence of patients negative for AT1R- and ETAR-antibodies (82.2% and 76.7%, respectively) was noted, whereas the incidence of AT1R- and ETAR-antibodies positivity (28.1% and 34.7%, respectively) was higher in the MPA/TAC group. Non-HLA antibodies had no influence on clinical outcomes in any treatment group and no graft loss or death was reported.ConclusionsThe studied combinations of immunosuppressants were safe with no influence on clinical outcomes and suggested minimal exposure of calcineurin inhibitors for better patient management.Clinical Trial Registrationhttps://clinicaltrials.gov/ (NCT01843348; EudraCT number: 2011-005238-21)

The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

Respiratory Infections by HMPV and RSV Are Clinically Indistinguishable but Induce Different Host Response in Aged Individuals

Background: Human metapneumovirus and respiratory syncytial virus can cause severe respiratory diseases, especially in infants, young children, and the elderly. So far it remains unclear why infections in the elderly become life threatening despite the presence of neutralizing antibodies in the serum, and to which extent double infections worsen the clinical course. Methods: Young and aged BALB/c-mice were infected with RSV or/and HMPV. Appearance of the mice was observed during course of infection. On day 5 p.i. animals were dispatched by cervical dislocation and levels of TNF-a and NF-kB were determined. Results: The observation of activity, weight and appearance of the different mice showed no differences among the tested groups. Despite this, the immunologic response depends on the animals ’ age and the virus they were infected with. In young animals, NF-kB levels were elevated if infected with HMPV and HMPV/RSV but remained low in RSV infections, whereas in aged animals the opposite was observed: solely RSV-infected animals showed elevated levels of NF-kB. TNF-a was slightly elevated in HMPV-infected young and old animals, but only in young animals this elevation was significant. Conclusions: Contrary to other studies, no weight loss or change in activity despite productive lung infection with the different viruses were observed. This may be due to the weaker anaesthesia or the lesser volume of virus solution used

Daily food consumption of infected animals.

<p>Daily food-consumption of 4–6 weeks (A) and 19 month (B) old BALB/c mice before and after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c mice were infected with 2×10⁷ geq RSV or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. The food consumption was recorded at the beginning of the experiment, on the day of inoculation and at the end of the experiment. Values were standardized referring to the values before infection (n = 3–5, values are shown as mean ± standard deviation). + significanty different to the corresponding value before infection (b.i.) (p<0.05).</p

Impact of de novo donor-specific HLA antibodies detected by Luminex solid-phase assay after transplantation in a group of 88 consecutive living-donor renal transplantations

De novo donor-specific HLA antibodies (DSA) after renal transplantation are known to be correlated with poor graft outcome and the development of acute and chronic rejection. Currently, data for the influence of de novo DSA in patient cohorts including only living-donor renal transplantations (LDRT) are limited. A consecutive cohort of 88 LDRT was tested for the occurrence of de novo DSA by utilizing the highly sensitive Luminex solid-phase assay for antibody detection. Data were analyzed for risk factors for de novo DSA development and correlated with acute rejection (AR) and graft function. Patients with de novo DSA [31 (35%)] showed a trend for inferior graft function [mean creatinine change (mg/dL/year) after the first year: 0.15 DSA (+) vs. 0.02 DSA (-) (P=0.10)] and a higher rate of AR episodes, especially in case of de novo DSA of both class I and II [6 (55%), (P=0.05)]. Antibody-mediated rejection (AMR) appeared in five patients and was significantly correlated with de novo DSA (P=0.05). Monitoring for de novo DSA after LDRT may help to identify patients at risk of declining renal function. Especially patients with simultaneous presence of de novo DSA class I and class II are at a high risk to suffer AR episodes

NF-κB expression in the lung after infection.

<p>Amount of NF-κB in the lungs of 4–6 weeks (A) und 19 months (B) old BALB/c mice after infection with RSV, HMPV or double infection: 4–6 weeks and 19 months old BALB/c-mice were infected with 2×10⁷ geq RSV, or HMPV, or co-infected with 1×10⁷ geq of each virus. Untreated animals, animals anesthetized and treated with cell culture supernatant or PBS or only anesthetized served as controls. Mice were killed by cervical dislocation and the lungs were removed, homogenised. The amount of NF-κB was determined by ELISA. Values were standardized referring to non treated controls (n = 3–5, values are shown as mean ± standard deviation). * significantly different to untreated animals (p<0.05). # signifcantly different to cell culture infected animals (p<0.05). + significantly different to hMPV and hMPV/RSV-infected animals (p<0.05).</p