Natural immunity to CMV dominates the CD4 and CD8 memory compartments of the CMV-seropositive host. This property has been recently exploited for experimental CMV-based vaccine vector strategies, and it has shown promise in animal models of AIDS and Ebola disease. Although it is generally agreed that CMV-based vaccine vectors may induce highly protective and persistent memory T cells, the influence of the gene expression context on Ag-specific T cell memory responses and immune protection induced by CMV vectors is not known. Using murine CMV (MCMV) recombinants expressing a single CD8 T cell epitope from HSV-1 fused to different MCMV genes, we show that magnitude and kinetics of T cell responses induced by CMV are dependent on the gene expression of CMV Ags. Interestingly, the kinetics of the immune response to the HSV-1 epitope was paralleled by a reciprocal depression of immune responses to endogenous MCMV Ags. Infection with a recombinant MCMV inducing a vigorous initial immune response to the recombinant peptide resulted in a depressed early response to endogenous MCMV Ag. Another recombinant virus, which induced a slowly developing “inflationary” T cell response to the HSV-1 peptide, induced weaker long-term responses to endogenous CMV Ags. Importantly, both mutants were able to protect mice from a challenge with HSV-1, mediating strong sterilizing immunity. Our data suggest that the context of gene expression markedly influences the T cell immunodominance hierarchy of CMV Ags, but the immune protection against HSV-1 does not require inflationary CD8 responses against the recombinant CMV-expressed epitope.

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy.

Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. Experimental studies of immunity against CMV in animal models of infection, such as the infection of mice with mouse CMV (MCMV), have relied mainly on parenteral infection protocols, although the virus naturally transmits by mucosal routes via body fluids. To characterize the biology of infections by mucosal routes, we compared the kinetics of virus replication, latent viral load and CD8 T-cell responses in lymphoid organs upon experimental intranasal (targeting the respiratory tract) and intragastric (targeting the digestive tract) infection with systemic intraperitoneal infection of two unrelated mouse strains. We observed that intranasal infection induced robust and long-term virus replication in the lungs and salivary glands but limited replication in the spleen. CD8 T-cell responses were somewhat weaker than upon intraperitoneal infection but showed similar kinetic profiles and phenotypes of antigen-specific cells. In contrast, intragastric infection resulted in abortive or poor virus replication in all tested organs and poor T-cell responses to the virus, especially at late times after infection. Consistent with the T-cell kinetics, the MCMV latent load was high in the lungs but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we showed that intranasal but not intragastric infection of mice with MCMV represents a robust model to study the short-and long-term biology of CMV infection by a mucosal route.

Experimental CMV-based vaccine vectors expressing a single MHC class I-restricted high-avidity epitope provided strong, T cell-dependent protection against viruses or tumors. In this study we tested the low-avidity epitope KCSRNRQYL, and show that a mouse CMV (MCMV) vector provides complete immune control of recombinant vaccinia virus expressing the same epitope if KCSRNRQYL is expressed within the immediate-early MCMV gene The same epitope expressed within the early gene provided no protection, although MCMV vectors expressing the high-avidity epitope SSIEFARL induced protective immunity irrespective of gene expression context. Immune protection was matched by Ag-induced, long-term expansion of effector memory CD8 T cells, regardless of epitope avidity. We explained this pattern by observing regularities in Ag competition, where responses to high-avidity epitopes outcompeted weaker ones expressed later in the replicative cycle of the virus. Conversely, robust and early expression of a low-avidity epitope compensated its weak intrinsic antigenicity, resulting in strong and sustained immunity and immune protection.

Cytomegalovirus (CMV) is a ubiquitous beta-herpesvirus whose reactivation from latency is a major cause of morbidity and mortality in immunocompromised hosts. Mouse CMV (MCMV) is a well-established model virus to study virus-host interactions. We showed in this study that the CD8-independent antiviral function of myeloid dendritic cells (mDC) is biologically relevant for the inhibition of MCMV replication in vivo and in vitro. In vivo ablation of CD11c ؉ DC resulted in higher viral titers and increased susceptibility to MCMV infection in the first 3 days postinfection. We developed in vitro coculture systems in which we cocultivated MCMV-infected endothelial cells or fibroblasts with T cell subsets and/or dendritic cells. H uman cytomegalovirus (HCMV) is a betaherpesvirus which establishes a lifelong latent infection in immunocompetenthosts. Latent HCMV is present in the majority of people worldwide, but the primary infection is usually asymptomatic. The primary infection is well contained by the immune cells, such as natural killer (NK) cells and T cells, which also prevent viral reactivation from latency (1, 2).Their activation depends on cross talk with dendritic cells (DC) (3, 4), and this interaction plays an important role in CMV control (5-7). The direct effect of DC on viral replication remains, however, unclear.In immunocompromised hosts, like AIDS patients or people undergoing transplantation, the virus cannot be contained, and its reactivation from latency has been associated with severe disease (8). Therefore, to develop new therapeutic approaches against CMV disease, it is exceedingly important to understand the immune mechanisms that drive the virus into latency.Murine cytomegalovirus (MCMV) is a natural pathogen of the mouse. It shows numerous analogies in latency and reactivation to the human virus, and its genome displays substantial similarity to the HCMV one (9). Therefore, MCMV is a widely used model for CMV infection and immunity (10)(11)(12).During primary infection, MCMV infects various different cell types, such as macrophages and DC but also nonhematopoietic cells, including endothelial and epithelial cells (13). On the other hand, the establishment of latency appears to be restricted to certain cell types. Latent HCMV was found in blood monocytes and in progenitor cells of the myeloid lineage (14-19), whereas liver sinusoidal endothelial cells (LSEC) were shown to be a site of MCMV latency and reactivation (20, 21), although myeloid cells might also present a latent reservoir in the mouse (22, 23).

f Latent herpesvirus infections alter immune homeostasis. To understand if this results in aging-related loss of immune protection against emerging infections, we challenged old mice carrying latent mouse cytomegalovirus (CMV), herpes simplex virus 1 (HSV-1), and/or murine gammaherpesvirus 68 (MHV-68) with influenza virus, West Nile virus (WNV), or vesicular stomatitis virus (VSV). We observed no increase in mortality or weight loss compared to results seen with herpesvirus-negative counterparts and a relative but not absolute reduction in CD8 responses to acute infections. Therefore, the presence of herpesviruses does not appear to increase susceptibility to emerging infections in aging patients.

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Cytomegalovirus (CMV) is the most common congenital infection in the United States. The major target of congenital CMV is the brain, with clinical manifestations including mental retardation, vision impairment and sensorineural hearing loss. Previous reports have shown that CD8+ T cells are required to control viral replication and significant numbers of CMV-specific CD8+ T cells persist in the brain even after the initial infection has been cleared. However, the dynamics of CD8+ T cells in the brain during latency remains largely undefined. In this report, we used T cell receptor (TCR) sequencing to track the development and maintenance of neonatal clonotypes in the brain and spleen of mice during chronic infection. Given the discontinuous nature of tissue resident memory CD8+ T cells, we hypothesized that neonatal TCR clonotypes would be ‘locked-in’ the brain and persist into adulthood. Surprisingly, we found that the antigen-specific T cell repertoire in neonatal-infected mice diversified during persistent infection in both the brain and spleen, while maintaining substantial similarity between the CD8+ T cell populations in the brain and spleen in both early and late infection. However, despite the diversification of, and potential interchange between, the spleen and brain antigen-specific T cell repertoires, we observed that germline-encoded TCR clonotypes, characteristic of neonatal infection, persisted in the brain, albeit sometimes in low abundance. These results provide valuable insights into the evolution of CD8+ T cell repertoires following neonatal CMV infection and thus have important implications for the development of therapeutic strategies to control CMV in early life.