Systemic and local infection routes govern different cellular dissemination pathways during gammaherpesvirus infection in vivo.

Human gammaherpesviruses cause morbidity and mortality associated with infection and transformation of lymphoid and endothelial cells. Knowledge of cell types involved in virus dissemination from primary virus entry to virus latency is fundamental for the understanding of gammaherpesvirus pathogenesis. However, the inability to directly trace cell types with respect to virus dissemination pathways has prevented definitive conclusions regarding the relative contribution of individual cell types. Here, we describe that the route of infection affects gammaherpesvirus dissemination pathways. We constructed a recombinant murine gammaherpesvirus 68 (MHV-68) variant harboring a cassette which switches fluorescent markers in a Cre-dependent manner. Since the recombinant virus which was constructed on the wild-type background was attenuated, in this study we used an M1-deleted version, which infected mice with normal kinetics. Infection of Cre-transgenic mice with this convertible virus was used to estimate the quantitative contribution of defined cell types to virus productivity and dissemination during the acute phase of MHV-68 infection. In systemic infection, we found splenic vascular endothelial cells (EC) among the first and main cells to produce virus. After local infection, the contribution of EC to splenic virus production did not represent such early kinetics. However, at later time points, B cell-derived viruses dominated splenic productivity independently of systemic or local infection. Systemic versus local infection also governed the cell types involved in loading peritoneal exudate cells, leading to latency in F4/80- and CD11b-positive target cells. Systemic infection supported EC-driven dissemination, whereas local infection supported B cell-driven dissemination.

The role of cell types in cytomegalovirus infection in vivo.

Human cytomegalovirus (HCMV) is the major viral cause of morbidity in immune compromised patients and of pre- and perinatal pathology in newborns. The clinical manifestations are highly variable and the principles which govern these differences cannot be understood without detailed knowledge on tissue specific aspects of HCMV infection. For decades the role of individual cell types during cytomegalovirus infection and disease has been discussed. The pathogenesis of mouse cytomegalovirus (MCMV) mirrors the human infection in many aspects. Although only MCMV infection is studied extensively at the level of organs, the relative contribution of specific cell types to viral pathogenesis in vivo has remained enigmatic. Here we discuss new approaches based on the cre/loxP-system to label nascent virus progeny or to lift a replication block. The salient aspect of this technique is the change of viral genome properties selectively in cells that express cre during infection in vivo. This allowed us to study the role of endothelial cells and hepatocytes for virus dissemination and will permit detailed studies on innate and adaptive immune responses to CMV.

Shedding light on the elusive role of endothelial cells in cytomegalovirus dissemination.

Cytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelial cells (EC) are suited for bidirectional virus spread from and to the transplant. We applied Cre/loxP-mediated green-fluorescence-tagging of EC-derived murine CMV (MCMV) to quantify the role of infected EC in transplantation-associated CMV dissemination in the mouse model. Both EC- and non-EC-derived virus originating from infected Tie2-cre(+) heart and kidney transplants were readily transmitted to MCMV-naïve recipients by primary viremia. In contrast, when a Tie2-cre(+) transplant was infected by primary viremia in an infected recipient, the recombined EC-derived virus poorly spread to recipient tissues. Similarly, in reverse direction, EC-derived virus from infected Tie2-cre(+) recipient tissues poorly spread to the transplant. These data contradict any privileged role of EC in CMV dissemination and challenge an indiscriminate applicability of the primary and secondary viremia concept of virus dissemination.

A spread-deficient cytomegalovirus for assessment of first-target cells in vaccination.

Human cytomegalovirus (HCMV) is a human pathogen that causes severe disease primarily in the immunocompromised or immunologically immature individual. To date, no vaccine is available. We describe use of a spread-deficient murine CMV (MCMV) as a novel approach for betaherpesvirus vaccination. To generate a spread-deficient MCMV, the conserved, essential gene M94 was deleted. Immunization with MCMV-DeltaM94 is apathogenic and protective against wild-type challenge even in highly susceptible IFNalphabetaR(-/-) mice. MCMV-DeltaM94 was able to induce a robust CD4(+) and CD8(+) T-cell response as well as a neutralizing antibody response comparable to that induced by wild-type infection. Endothelial cells were identified as activators of CD8(+) T cells in vivo. Thus, the vaccination with a spread-deficient betaherpesvirus is a safe and protective strategy and allows the linkage between cell tropism and immunogenicity. Furthermore, genomes of MCMV-DeltaM94 were present in lungs 12 months after infection, revealing first-target cells as sites of genome maintenance.

Virally infected mouse liver endothelial cells trigger CD8+ T-cell immunity.

BACKGROUND & AIMS: Dendritic cell activation through ligation of pattern recognition receptors leading to full functional maturation causes induction of CD8(+) T-cell immunity through increased delivery of costimulatory signals instead of tolerance. Here we investigate whether organ-resident antigen-presenting cells, such as liver sinusoidal endothelial cells (LSECs), also switch from tolerogenic to immunogenic CD8(+) T-cell activation upon such stimulation. METHODS: Murine LSECs were isolated by immunomagnetic separation and analyzed for functional maturation upon triggering pattern recognition receptors or viral infection employing gene expression analysis and T cell coculture assays. In vivo relevance of the findings was confirmed with bone-marrow chimeric animals. RESULTS: LSECs expressed numerous pattern recognition receptors that allowed for sentinel function, but ligand-induced activation of these receptors was not sufficient to overcome tolerance induction of CD8(+) T cells. Importantly, viral infection with murine cytomegalovirus caused functional maturation of antigen-presenting LSECs and was sufficient to promote antigen-specific differentiation into effector CD8(+) T cells in the absence of dendritic cells and independent of CD80/86. CONCLUSIONS: These results shed new light on the generation of organ-specific immunity and may contribute to overcoming tolerance in relevant situations, such as cancer.

Tumor agonist peptides break tolerance and elicit effective CTL responses in an inducible mouse model of hepatocellular carcinoma.

Tumors often induce tolerance in the immune system, which may contribute to the limited success of clinical vaccination against tumors. In order to develop strategies for overcoming tumor tolerance we have developed an inducible mouse model of autochthonus hepatocellular carcinoma growth, which relates more closely to the clinical situation than transplantation tumors. These so-called AST mice harbour a construct consisting of the hepatocyte-specific albumin promoter, a loxP flanked stop-cassette, and the oncogene SV40 large T antigen (Tag). By intravenous application of an adenovirus encoding Cre recombinase the stop cassette was excised, thereby inducing Tag expression and formation of hepatoma nodules in a dose-dependent fashion in about 3 months. Non-induced AST mice showed tumor tolerance, as demonstrated by the failure to reject Tag-positive transplantation tumors and the inability to mount CTL following Tag immunization. Dendritic cell-based immunization with an agonist Tag peptide was able to overcome tolerance and resulted in marked CTL activity against naturally occurring Tag epitopes. Importantly, vaccination with the agonist peptide prevented growth of the autochthonous liver tumors and significantly prolonged survival of the animals. Our findings demonstrate that agonist peptides can be used in immunization protocols for breaking of tolerance and induction of CTL that mediate effective anti-tumor responses. In addition, the inducible hepatoma model described here can be used for the design of therapeutic strategies against hepatocellular carcinoma.

The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host.

The course of systemic viral infections is determined by the virus productivity of infected cell types and the efficiency of virus dissemination throughout the host. Here, we used a cell-type-specific virus labeling system to quantitatively track virus progeny during murine cytomegalovirus infection. We infected mice that expressed Cre recombinase selectively in vascular endothelial cells or hepatocytes with a murine cytomegalovirus for which Cre-mediated recombination would generate a fluorescently labeled virus. We showed that endothelial cells and hepatocytes produced virus after direct infection. However, in the liver, the main contributor to viral load in the mouse, most viruses were produced by directly infected hepatocytes. Remarkably, although virus produced in hepatocytes spread to hepatic endothelial cells (and vice versa), there was no significant spread from the liver to other organs. Thus, the cell type producing the most viruses was not necessarily the one responsible for virus dissemination within the host.

Conditional gene expression systems to study herpesvirus biology in vivo.

Cytomegalovirus (CMV), a prototypic beta-herpesvirus, is an important human pathogen causing protean clinical manifestations in immature and immunocompromised patients. Mechanisms of infection can be studied in a mouse model. Mouse cytomegalovirus (MCMV) resembles in pathogenesis its human counterpart in many ways. Although MCMV infection is studied extensively on the level of organs, the contribution of specific cell types to viral replication in vivo is still elusive. Here we describe our approach based on the the Cre/loxP-system to investigate MCMV infection at the level of cell types in vivo. Using bacterial artificial chromosome (BAC)-technology, we created an MCMV virus containing an enhanced green fluorescent protein (egfp) reporter-gene which is not expressed due to a 'Stop' cassette flanked by two loxP-sites between promoter and coding sequence. Infection of cre-transgenic mice with this reporter virus results in the deletion of the 'Stop' cassette and expression of EGFP in a cell type-specific manner. Using this conditional gene expression system we are able to quantify viral productivity in specific cell types and to determine their contribution to viral dissemination in vivo. Furthermore, the deletion of viral genes can be used to screen for cell type-specificity of viral gene functions. Hence, conditional MCMV mutants allow the study of herpesvirus biology on the level of cell types in vivo.

Engineering of cytomegalovirus genomes for recombinant live herpesvirus vaccines.

The advances of sequence knowledge and genetic engineering hold a great promise for a rational approach to vaccine development. Herpesviruses are important pathogens of all vertebrates. They cause acute and chronic infections and persist in their hosts for life. In man there are eight herpesviruses known and most of them can be linked to diseases. To date only one licensed vaccine against a human herpesvirus exists and there is no proven successful concept on rational design for herpesvirus vaccines available. Here, we use new reverse genetic systems, based on the 230-kb mouse cytomegalovirus genome to explore new methods of vaccine delivery and of virus attenuation. With regard to virus delivery, we show that the bacterial transfer of the infectious DNA in vivo is theoretically possible but not yet a practical option. With regard to a rational approach of virus attenuation, we consider a selective deletion of viral genes that modulate the immune response of the host.

Natural killer cells promote early CD8 T cell responses against cytomegalovirus.

Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-alpha/beta production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-alpha administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity.

Conditional cytomegalovirus replication in vitro and in vivo.

We have established a conditional gene expression system for cytomegalovirus which allows regulation of genes independently from the viral replication program. Due to the combination of all elements required for regulated expression in the same viral genome, conditional viruses can be studied in different cell lines in vitro and in the natural host in vivo. The combination of a self-sufficient tetracycline-regulated expression cassette and Flp recombinase-mediated insertion into the viral genome allowed fast construction of recombinant murine cytomegaloviruses carrying different conditional genes. The regulation of two reporter genes, the essential viral M50 gene and a dominant-negative mutant gene (m48.2) encoding the small capsid protein, was analyzed in more detail. In vitro, viral growth was regulated by the conditional expression of M50 by 3 orders of magnitude and up to a millionfold when the dominant-negative small capsid protein mutant was used. In vivo, viral growth of the dominant-negative mutant was reduced to detection limits in response to the presence of doxycycline in the organs of mice. We believe that this conditional expression system is applicable to genetic studies of large DNA viruses in general.

CpG-ODN-induced inflammation is sufficient to cause T-cell-mediated autoaggression against hepatocytes.

Autoimmune diseases are often associated with microbial infections. Molecular mimicry between microbial antigens and self-epitopes has been suggested as a mechanism for breaking self-tolerance and induction of autoimmunity. Since infections also cause inflammatory responses we explored the role of local inflammation in organ-specific autoimmunity. For this purpose, transgenic mice were used expressing the MHC class I molecule Kb exclusively on hepatocytes. These mice exhibit Kb-specific tolerance as exemplified by the acceptance of Kb+ grafts. Inflammatory reactions were induced by injection of immunostimulatory cytosine-phosphorothioate-guanine (CpG)-rich oligodeoxynucleotides (ODN). Application of CpG-ODN is sufficient to break tolerance in vivo, and to cause activation of Kb-specific CD8+ T cells and subsequent autoaggression against hepatocytes. The CpG-ODN-induced inflammation appears to have two major effects. First, it causes infiltration of T cells into the liver parenchyma. Second, adhesion and costimulatory molecules are up-regulated on hepatocytes so that the infiltrating CD8+ T cells encounter Kb on hepatocytes, which display an APC-like phenotype, resulting in activation and tissue damage. Autoimmune hepatitis can be maintained for at least eight weeks by repeated application of CpG-ODN but subsides after termination of the inflammatory stimulus, suggesting the requirement of additional factors for a self-perpetuation of autoimmunity. These observations describe an additional pathway for the induction of autoimmunity, i.e. in the absence of microbial antigens inflammatory reactions alone can lead to infiltration of T cells into organs, resulting in breaking of tolerance and autoaggression. Moreover, the results provide evidence that T cell activation can take place not only in draining lymph nodes but also directly on parenchymal cells.