Molekulare Quantifizierung der mCMV-Immunevasion

Die Kontrolle der murinen Cytomegalovirus (mCMV)-Infektion wird vom Organismus der Maus primär durch antivirale CD8 T-Zellen vermittelt. Diese erkennen auf MHC-Klasse-I-Molekülen präsentierte virale Peptide und lysieren die infizierte Zelle. Um der Immun¬kontrolle zu entkommen, entwickelte mCMV im Laufe der Evolution der Virus-Wirtbezie¬hung Strategien der Immunevasion, die direkt die Expression der Peptid-MHC-Klasse-I Komplexe an der Zelloberfläche beeinflussen. Die hierfür verantwortlichen Early (E)-Glykoproteine m06/gp48 und m152/gp40 werden auf Grund ihres regulatorischen Einflusses auf die Antigenpräsentation als vRAPs (viral regulators of antigen presentation) bezeichnet. Die vRAPs interferieren mit dem Transport Peptid-beladener MHC-Klasse-I-Moleküle und reduzieren in ihrer kooperativen Wirkung die Präsentation viraler Peptide an der Zelloberfläche, mit der Folge, dass die Erkennung infizierter Zellen inhibiert wird. Bisherige funktionelle Untersuchungen zeigten die qualitative Verringerung der MHC-Klasse-I-Expression auf der Zelloberfläche nach Expression der vRAPs und wiesen auf eine differentielle Wirkung der vRAPs auf die Gesamtpopulation der MHC-Klasse-I-Moleküle im Vergleich zu den Peptid-MHC-Klasse-I Komplexen hin. In der vorliegenden Arbeit sollte die Effizienz und Spezifität der von mCMV vermittelten CD8 T-Zell-Immunevasion näher untersucht werden. Hierfür war es notwendig ein Modell zu etablieren, das es ermöglichte, selektiv die Präsentation eines viral kodierten Peptids auf der Zelloberfläche zu quantifizieren. Ziel war es, zwischen der Gesamtheit an MHC-Klasse-I-Molekülen eines bestimmten Allels und denjenigen, die ein definiertes virales Peptid präsentieren, unterscheiden zu können. Um dieses zu gewährleisten, wurde in dieser Arbeit der monoklonale Antikörper T-AG25-DL1.16 eingesetzt, der selektiv den Präsentationskomplex aus dem MHC-Klasse-I-Molekül Kb und dem gut charakterisierten Modell-Peptid SIINFEKL (OVA257-264) aus dem Ovalbumin (OVA) nachweist. Um SIINFEKL im System der mCMV-Infektion nutzbar zu machen, wurden mittels BAC-Mutagenese die mCMV-Rekombinanten mCMV-vRAP-SIINFEKL und mCMV-ΔvRAP-SIINFEKL generiert. In diesen Viren wurde mittels „orthotopen Peptidaustauschs“ im E-Protein m164/gp36,5 das immundominante virale Peptid m164150-158 gegen SIINFEKL ausgetauscht. Wie gezeigt werden konnte wird SIINFEKL nach Infektion von Fibroblasten prozessiert und von den vRAPs wie ein mCMV-Peptid kontrolliert. Die Infektion von C57BL/6-Mäusen mit beiden Rekombinanten führt zum SIINFEKL-spezifischen CD8 T-Zell Priming in der Akut- und Memory-Phase der Infektion. In einer Analyse der CD8 T-Zell-Frequenzen ordnet sich SIINFEKL in einer intermediären Position des Spektrums der authentischen mCMV-Peptide ein. Die erfolgreiche Integration von SIINFEKL in das virale Immunom erlaubte zum ersten Mal die absolute Quantifizierung der Effektivität der viralen Immunevasion. Dabei ergab sich, dass die vRAPs die Präsentation von Kb-SIINFEKL um einen Faktor von > 100 reduzieren, während die Expression aller Kb-Moleküle an der Zelloberfläche nur um einen Faktor von ~4 reduziert wird. Dies belegt den starken Einfluss der vRAPs auf die Präsentation der endogen mit viralen Peptiden beladenen MHC-Klasse-I Moleküle im Vergleich zur Gesamtpopulation der Klasse-I-Moleküle an der Zelloberfläche. In weiteren Experimenten der Arbeit konnte gezeigt werden, dass eine endogene Beladung der MHC-Klasse-I-Moleküle mit SIINFEKL im ER deutlich effizienter ist als ihre exogene Beladung mit synthetischem Peptid an der Zelloberfläche

Reverse Genetics Modification of Cytomegalovirus Antigenicity and Immunogenicity by CD8 T-Cell Epitope Deletion and Insertion

The advent of cloning herpesviral genomes as bacterial artificial chromosomes (BACs) has made herpesviruses accessible to bacterial genetics and has thus revolutionised their mutagenesis. This opened all possibilities of reverse genetics to ask scientific questions by introducing precisely accurate mutations into the viral genome for testing their influence on the phenotype under study or to create phenotypes of interest. Here, we report on our experience with using BAC technology for a designed modulation of viral antigenicity and immunogenicity with focus on the CD8 T-cell response. One approach is replacing an intrinsic antigenic peptide in a viral carrier protein with a foreign antigenic sequence, a strategy that we have termed “orthotopic peptide swap”. Another approach is the functional deletion of an antigenic peptide by point mutation of its C-terminal MHC class-I anchor residue. We discuss the concepts and summarize recently published major scientific results obtained with immunological mutants of murine cytomegalovirus

Nitrogen transfer properties in tantalum nitride based materials

Ta3-xMxNy (M = Re, Fe, Co; x = 0, 0.25, 0.5, 1) materials with different microstructural features (e.g. surface area) were successfully prepared using different synthesis techniques. The dependence of nitrogen transfer properties upon tantalum nitride microstructure and its chemical composition was evaluated using the ammonia synthesis with a H2/Ar feedstream (a reaction involving lattice nitrogen transfer). It was shown that nitrogen reactivity for tantalum nitride is more dominated by lattice nitrogen stability rather than microstructural properties. In the case of non-doped tantalum nitride, only a limited improvement of reactivity with enhanced surface area was observed which demonstrates the limited impact of microstructure upon reactivity. However, the nature of the transition metal dopant as well as its content was observed to play a key role in the nitrogen transfer properties of tantalum nitride and to impact strongly upon its reactivity. In fact, doping tantalum nitride with low levels of Co resulted in enhanced reactivity at lower temperature

The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo This effect is ameliorated in the absence of STING Interestingly, while m152 inhibits STING-mediated IRF signaling, it did not affect STING-mediated NF-κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF-κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, providing an advantage in the establishment of an infection

Non-redundant and Redundant Roles of Cytomegalovirus gH/gL Complexes in Host Organ Entry and Intra-tissue Spread

Author Summary The role of viral glycoprotein entry complexes in viral tropism in vivo is a question central to understanding virus pathogenesis and transmission for any virus. Studies were limited by the difficulty in distinguishing between viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. Employing the murine cytomegalovirus entry complex gH/gL/gO as a paradigm for a generally applicable strategy to dissect these two events experimentally, we used a gO-transcomplemented ΔgO mutant for providing the complex exclusively for the initial cell entry step. In immunocompromised mice as a model for recipients of hematopoietic cell transplantation, our studies revealed an irreplaceable role for gH/gL/gO in initiating infection in host organs relevant to pathogenesis, whereas subsequent spread within tissues and infection of the salivary glands, the site relevant to virus host-to-host transmission, are double-secured by the entry complexes gH/gL/gO and gH/gL/MCK-2. As an important consequence, interventional strategies targeting only gO might be efficient in preventing organ manifestations after a primary viremia, whereas both gH/gL complexes need to be targeted for preventing intra-tissue spread of virus reactivated from latency within tissues as well as for preventing the salivary gland route of host-to-host transmission

The murine cytomegalovirus M35 protein antagonizes type I IFN induction downstream of pattern recognition receptors by targeting NF-κB mediated transcription.

The herpesvirus cytomegalovirus can cause severe morbidity in immunosuppressed people and poses a much greater global problem in the context of congenital infections than the Zika virus. To establish infection, cytomegalovirus needs to modulate the antiviral immune response of its host. One of the first lines of defense against viral infections is the type I interferon response which is activated by cellular sensors called pattern recognition receptors. These receptors sense viral entry and rapidly induce the transcription of type I interferons, which are instrumental for the induction of an antiviral state in infected and surrounding cells. We have identified the first viral protein encoded by murine cytomegalovirus, the M35 protein, that counteracts type I interferon transcription downstream of multiple pattern recognition receptors. We found that this viral countermeasure occurs shortly after viral entry into the host cell, as M35 is delivered with the viral particle. M35 then localizes to the nucleus where it modulates NF-κB-mediated transcription. In vivo, murine cytomegalovirus deficient of the M35 protein replicates to lower levels in spleen and liver and cannot establish a productive infection in the salivary glands, which is a key site of viral transmission, highlighting the important role of M35 for the establishment of infection. Our study provides novel insights into the complex interaction between cytomegalovirus and the innate immune response of its host

Enhancerless Cytomegalovirus Is Capable of Establishing a Low-Level Maintenance Infection in Severely Immunodeficient Host Tissues but Fails in Exponential Growth

Major immediate-early transcriptional enhancers are genetic control elements that act, through docking with host transcription factors, as a decisive regulatory unit for efficient initiation of the productive virus cycle. Animal models are required for studying the function of enhancers paradigmatically in host organs. Here, we have sought to quantitatively assess the establishment, maintenance, and level of in vivo growth of enhancerless mutants of murine cytomegalovirus in comparison with those of an enhancer-bearing counterpart in models of the immunocompromised or immunologically immature host. Evidence is presented showing that enhancerless viruses are capable of forming restricted foci of infection but fail to grow exponentially

Cytomegalovirus immunoevasin reveals the physiological role of “missing self” recognition in natural killer cell dependent virus control in vivo

Natural killer cell recognition of “missing self” contributes meaningfully to control of mouse cytomegalovirus infection in vivo

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1

Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, theattenuatedgrowth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell– based vaccine