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

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 Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tailanchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction

Direct Evidence for Viral Antigen Presentation during Latent Cytomegalovirus Infection

Murine models of cytomegalovirus (CMV) infection have revealed an immunological phenomenon known as “memory inflation” (MI). After a peak of a primary CD8+ T-cell response, the pool of epitope-specific cells contracts in parallel to the resolution of productive infection and the establishment of a latent infection, referred to as “latency.” CMV latency is associated with an increase in the number of cells specific for certain viral epitopes over time. The inflationary subset was identified as effector-memory T cells (iTEM) characterized by the cell surface phenotype KLRG1+CD127−CD62L−. As we have shown recently, latent viral genomes are not transcriptionally silent. Rather, viral genes are sporadically desilenced in a stochastic fashion. The current hypothesis proposes MI to be driven by presented viral antigenic peptides encoded by the corresponding, stochastically expressed viral genes. Although this mechanism suggests itself, independent evidence for antigen presentation during viral latency is pending. Here we fill this gap by showing that T cell-receptor transgenic OT-I cells that are specific for peptide SIINFEKL proliferate upon adoptive cell transfer in C57BL/6 recipients latently infected with murine CMV encoding SIINFEKL (mCMV-SIINFEKL), but not in those latently infected with mCMV-SIINFEKA, in which antigenicity is lost by mutation L8A of the C-terminal amino acid residue

Mouse Model of Cytomegalovirus Disease and Immunotherapy in the Immunocompromised Host: Predictions for Medical Translation that Survived the “Test of Time”

Human Cytomegalovirus (hCMV), which is the prototype member of the β-subfamily of the herpesvirus family, is a pathogen of high clinical relevance in recipients of hematopoietic cell transplantation (HCT). hCMV causes multiple-organ disease and interstitial pneumonia in particular upon infection during the immunocompromised period before hematopoietic reconstitution restores antiviral immunity. Clinical investigation of pathomechanisms and of strategies for an immune intervention aimed at restoring antiviral immunity earlier than by hematopoietic reconstitution are limited in patients to observational studies mainly because of ethical issues including the imperative medical indication for chemotherapy with antivirals. Aimed experimental studies into mechanisms, thus, require animal models that match the human disease as close as possible. Any model for hCMV disease is, however, constrained by the strict host-species specificity of CMVs that prevents the study of hCMV in any animal model including non-human primates. During eons of co-speciation, CMVs each have evolved a set of “private genes„ in adaptation to their specific mammalian host including genes that have no homolog in the CMV virus species of any other host species. With a focus on the mouse model of CD8 T cell-based immunotherapy of CMV disease after experimental HCT and infection with murine CMV (mCMV), we review data in support of the phenomenon of “biological convergence„ in virus-host adaptation. This includes shared fundamental principles of immune control and immune evasion, which allows us to at least make reasoned predictions from the animal model as an experimental “proof of concept.„ The aim of a model primarily is to define questions to be addressed by clinical investigation for verification, falsification, or modification and the results can then give feedback to refine the experimental model for research from “bedside to bench„

Mast Cells Meet Cytomegalovirus: A New Example of Protective Mast Cell Involvement in an Infectious Disease

Cytomegaloviruses (CMVs) belong to the β-subfamily of herpesviruses. Their host-to-host transmission involves the airways. As primary infection of an immunocompetent host causes only mild feverish symptoms, human CMV (hCMV) is usually not considered in routine differential diagnostics of common airway infections. Medical relevance results from unrestricted tissue infection in an immunocompromised host. One risk group of concern are patients who receive hematopoietic cell transplantation (HCT) for immune reconstitution following hematoablative therapy of hematopoietic malignancies. In HCT patients, interstitial pneumonia is a frequent cause of death from hCMV strains that have developed resistance against antiviral drugs. Prevention of CMV pneumonia requires efficient reconstitution of antiviral CD8 T cells that infiltrate lung tissue. A role for mast cells (MC) in the immune control of lung infection by a CMV was discovered only recently in a mouse model. MC were shown to be susceptible for productive infection and to secrete the chemokine CCL-5, which recruits antiviral CD8 T cells to the lungs and thereby improves the immune control of pulmonary infection. Here, we review recent data on the mechanism of MC-CMV interaction, a field of science that is new for CMV virologists as well as for immunologists who have specialized in MC

Consequence of Histoincompatibility beyond GvH-Reaction in Cytomegalovirus Disease Associated with Allogeneic Hematopoietic Cell Transplantation: Change of Paradigm

Hematopoietic cell (HC) transplantation (HCT) is the last resort to cure hematopoietic malignancies that are refractory to standard therapies. Hematoablative treatment aims at wiping out tumor cells as completely as possible to avoid leukemia/lymphoma relapse. This treatment inevitably co-depletes cells of hematopoietic cell lineages, including differentiated cells that constitute the immune system. HCT reconstitutes hematopoiesis and thus, eventually, also antiviral effector cells. In cases of an unrelated donor, that is, in allogeneic HCT, HLA-matching is performed to minimize the risk of graft-versus-host reaction and disease (GvHR/D), but a mismatch in minor histocompatibility antigens (minor HAg) is unavoidable. The transient immunodeficiency in the period between hematoablative treatment and reconstitution by HCT gives latent cytomegalovirus (CMV) the chance to reactivate from latently infected donor HC or from latently infected organs of the recipient, or from both. Clinical experience shows that HLA and/or minor-HAg mismatches increase the risk of complications from CMV. Recent results challenge the widespread, though never proven, view of a mechanistic link between GvHR/D and CMV. Instead, new evidence suggests that histoincompatibility promotes CMV disease by inducing non-cognate transplantation tolerance that inhibits an efficient reconstitution of high-avidity CD8+ T cells capable of recognizing and resolving cytopathogenic tissue infection

Localization of Viral Epitope-Specific CD8 T Cells during Cytomegalovirus Latency in the Lungs and Recruitment to Lung Parenchyma by Airway Challenge Infection

Interstitial pneumonia is a life-threatening clinical manifestation of cytomegalovirus infection in recipients of hematopoietic cell transplantation (HCT). The mouse model of experimental HCT and infection with murine cytomegalovirus revealed that reconstitution of virus-specific CD8(+) T cells is critical for resolving productive lung infection. CD8(+) T-cell infiltrates persisted in the lungs after the establishment of latent infection. A subset defined by the phenotype KLRG1(+)CD62L(-) expanded over time, a phenomenon known as memory inflation (MI). Here we studied the localization of these inflationary T effector-memory cells (iTEM) by comparing their frequencies in the intravascular and transmigration compartments, the IVC and TMC, respectively, with their frequency in the extravascular compartment (EVC), the alveolar epithelium. Frequencies of viral epitope-specific iTEM were comparable in the IVC and TMC but were reduced in the EVC, corresponding to an increase in KLRG1(-)CD62L(-) conventional T effector-memory cells (cTEM) and a decrease in functional IFN gamma(+)CD8(+) T cells. As maintained expression of KLRG1 requires stimulation by antigen, we conclude that iTEM lose KLRG1 and convert to cTEM after transmigration into the EVC because pneumocytes are not latently infected and, therefore, do not express antigens. Accordingly, antigen re-expression upon airway challenge infection recruited virus-specific CD8(+) T cells to TMC and EVC

Host-Adapted Gene Families Involved in Murine Cytomegalovirus Immune Evasion

Cytomegaloviruses (CMVs) are host species-specific and have adapted to their respective mammalian hosts during co-evolution. Host-adaptation is reflected by “private genes” that have specialized in mediating virus-host interplay and have no sequence homologs in other CMV species, although biological convergence has led to analogous protein functions. They are mostly organized in gene families evolved by gene duplications and subsequent mutations. The host immune response to infection, both the innate and the adaptive immune response, is a driver of viral evolution, resulting in the acquisition of viral immune evasion proteins encoded by private gene families. As the analysis of the medically relevant human cytomegalovirus by clinical investigation in the infected human host cannot make use of designed virus and host mutagenesis, the mouse model based on murine cytomegalovirus (mCMV) has become a versatile animal model to study basic principles of in vivo virus-host interplay. Focusing on the immune evasion of the adaptive immune response by CD8+ T cells, we review here what is known about proteins of two private gene families of mCMV, the m02 and the m145 families, specifically the role of m04, m06, and m152 in viral antigen presentation during acute and latent infection

Memory CD8 T Cells Protect against Cytomegalovirus Disease by Formation of Nodular Inflammatory Foci Preventing Intra-Tissue Virus Spread

Cytomegaloviruses (CMVs) are controlled by innate and adaptive immune responses in an immunocompetent host while causing multiple organ diseases in an immunocompromised host. A risk group of high clinical relevance comprises transiently immunocompromised recipients of hematopoietic cell transplantation (HCT) in the “window of risk” between eradicative therapy of hematopoietic malignancies and complete reconstitution of the immune system. Cellular immunotherapy by adoptive transfer of CMV-specific CD8 T cells is an option to prevent CMV disease by controlling a primary or reactivated infection. While experimental models have revealed a viral epitope-specific antiviral function of cognate CD8 T cells, the site at which control is exerted remained unidentified. The observation that remarkably few transferred cells protect all organs may indicate an early blockade of virus dissemination from a primary site of productive infection to various target organs. Alternatively, it could indicate clonal expansion of a few transferred CD8 T cells for preventing intra-tissue virus spread after successful initial organ colonization. Our data in the mouse model of murine CMV infection provide evidence in support of the second hypothesis. We show that transferred cells vigorously proliferate to prevent virus spread, and thus viral histopathology, by confining and eventually resolving tissue infection within nodular inflammatory foci