A persistent virus vector confers superior anti-tumor immunity, compared with a non-persistent vector.

Active vaccination strategies using viral vectors often give disappointing protection from tumor development, and usually require multiple immunizations. These approaches normally use viruses that cause acute infections, as they provoke potent CD8 T cell responses. Persistent virus vectors have not been used in this setting due to the perception that exhaustion of the T cell response occurs and would lead to poor anti-tumor protection. However, such exhaustion generally only occurs in high-load virus infections, whereas T cell function is intact in lower-load persistent infections. In fact, CD8 T cell responses in these infections, which are adapted for long-term immune surveillance, have properties that may make them more desirable for long-term anti-tumor immunity. In this report, we show that a persistent gammaherpesvirus vector provides superior protection against melanoma, relative to a non-persistent mutant of the same virus. These data suggest that vaccine vectors derived from persistent viruses may perform better than those from acute viruses at mediating anti-tumor protection.

Vaccination against murine gamma-herpesvirus infection.

The gamma-herpesviruses establish life-long latency in the host and are important human pathogens. T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T-cell response to gamma-herpesvirus infection and the requirements for effective vaccination are poorly understood. The recent development of a murine gamma-herpesvirus (murine herpesvirus-68 [MHV-68]) has made it possible to analyze T-cell responses and test vaccination strategies in a small animal model. Intranasal infection with MHV-68 induces an acute infection in the lung and the subsequent establishment of long-term latency, which is associated with splenomegaly and an infectious mononucleosis-like syndrome. Here we review the T-cell response to different phases of the infection and the impact of vaccination against either lytic-cycle, or latency-associated T-cell epitopes.

Latent antigen vaccination in a model gammaherpesvirus infection.

Vaccines that can reduce the load of latent gammaherpesvirus infections are eagerly sought. One attractive strategy is vaccination against latency-associated proteins, which may increase the efficiency with which T cells recognize and eliminate latently infected cells. However, due to the lack of tractable animal model systems, the effect of latent-antigen vaccination on gammaherpesvirus latency is not known. Here we use the murine gammaherpesvirus model to investigate the impact of vaccination with the latency-associated M2 antigen. As expected, vaccination had no effect on the acute lung infection. However, there was a significant reduction in the load of latently infected cells in the initial stages of the latent infection, when M2 is expressed. These data show for the first time that latent-antigen vaccination can reduce the level of latency in vivo and suggest that vaccination strategies involving other latent antigens may ultimately be successfully used to reduce the long-term latent infection.

Protection from respiratory virus infections can be mediated by antigen-specific CD4(+) T cells that persist in the lungs.

Although CD4(+) T cells have been shown to mediate protective cellular immunity against respiratory virus infections, the underlying mechanisms are poorly understood. For example, although phenotypically distinct populations of memory CD4(+) T cells have been identified in different secondary lymphoid tissues, it is not known which subpopulations mediate protective cellular immunity. In this report, we demonstrate that virus-specific CD4(+) T cells persist in the lung tissues and airways for several months after Sendai virus infection of C57BL/6 mice. A large proportion of these cells possess a highly activated phenotype (CD44(hi), CD62L(lo), CD43(hi), and CD25(hi)) and express immediate effector function as indicated by the production of interferon gamma after a 5-h restimulation in vitro. Furthermore, intratracheal adoptive transfer of lung memory cells into beta2m-deficient mice demonstrated that lung-resident virus-specific CD4(+) T cells mediated a substantial degree of protection against secondary virus infection. Taken together, these data demonstrate that activated memory CD4(+) T cells persisting at mucosal sites play a critical role in mediating protective cellular immunity.

Regression of a murine gammaherpesvirus 68-positive b-cell lymphoma mediated by CD4 T lymphocytes.

Murine gammaherpesvirus 68-infected S11 cells were injected subcutaneously into nude mice. Adoptively transferred restimulated lymphocytes consistently elicited the regression of S11 tumors. CD4 T lymphocytes were most effective in preventing tumor formation, and immunohistochemistry highlighted populations of CD4 T cells in regressing tumors.

Activated antigen-specific CD8+ T cells persist in the lungs following recovery from respiratory virus infections.

The poor correlation between cellular immunity to respiratory virus infections and the numbers of memory CD8(+) T cells in the secondary lymphoid organs suggests that there may be additional reservoirs of T cell memory to this class of infection. Here we identify a substantial population of Ag-specific T cells in the lung that persist for several months after recovery from an influenza or Sendai virus infection. These cells are present in high numbers in both the airways and lung parenchyma and can be distinguished from memory cell populations in the spleen and peripheral lymph nodes in terms of the relative frequencies among CD8(+) T cells, activation status, and kinetics of persistence. In addition, these cells are functional in terms of their ability to proliferate, express cytolytic activity, and secrete cytokines, although they do not express constitutive cytolytic activity. Adoptive transfer experiments demonstrated that the long-term establishment of activated T cells in the lung did not require infection in the lung by a pathogen carrying the inducing Ag. The kinetics of persistence of Ag-specific CD8(+) T cells in the lung suggests that they play a key role in protective cellular immunity to respiratory virus infections.

Antigen expression during murine gamma-herpesvirus infection.

Gammaherpesviruses (gammaHV) establish a life-long latency in the host and are associated with a number of malignant human diseases. It is generally believed that T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T cell response to gamma-herpesvirus infections is poorly understood. In the current report we took advantage of a mouse model of gammaHV infection (murine herpesvirus-68, MHV-68) to investigate the T cell response to different phases of the infection. Intranasal infection with MHV-68 induces an acute infection in lung epithelial cells and long-term latency in B cells. The kinetics of the CD8+ T cell response to different lytic cycle and latency-associated antigens was highly complex and distinct patterns of response could be identified. These responses were regulated by multiple factors including differences in temporal expression of the relevant antigens, differences in the presentation of antigen in different organs, and differential expression of antigen in different types of antigen presenting cells. For example, some antigens were expressed at distinct phases of the infection and in specific organs or subsets of antigen presenting cells. In addition, recent data suggest that in addition to B cells, both macrophages and dendritic cells harbor latent MHV-68 infection, adding further complexity to their role in controlling the T cell response to this infection.

Control of gammaherpesvirus latency by latent antigen-specific CD8(+) T cells.

The contribution of the latent antigen-specific CD8(+) T cell response to the control of gammaherpesvirus latency is currently obscure. Some latent antigens induce potent T cell responses, but little is known about their induction or the role they play during the establishment of latency. Here we used the murine gammaherpesvirus system to examine the expression of the latency-associated M2 gene during latency and the induction of the CD8(+) T cell response to this protein. M2, in contrast to the M3 latency-associated antigen, was expressed at day 14 after infection but was undetectable during long-term latency. The induction of the M2(91-99)/K(d) CD8(+) T cell response was B cell dependent, transient, and apparently induced by the rapid increase in latently infected cells around day 14 after intranasal infection. These kinetics were consistent with a role in controlling the initial "burst" of latently infected cells. In support of this hypothesis, adoptive transfer of an M2-specific CD8(+) T cell line reduced the initial load of latently infected cells, although not the long-term load. These data represent the first description of a latent antigen-specific immune response in this model, and suggest that vaccination with latent antigens such as M2 may be capable of modulating latent gammaherpesvirus infection.

Rta of murine gammaherpesvirus 68 reactivates the complete lytic cycle from latency.

Herpesviruses are characterized as having two distinct life cycle phases: lytic replication and latency. The mechanisms of latency establishment and maintenance, as well as the switch from latency to lytic replication, are poorly understood. Human gammaherpesviruses, including Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), are associated with lymphoproliferative diseases and several human tumors. Unfortunately, the lack of cell lines to support efficient de novo productive infection and restricted host ranges of EBV and HHV-8 make it difficult to explore certain important biological questions. Murine gammaherpesvirus 68 (MHV-68, or gammaHV68) can establish de novo lytic infection in a variety of cell lines and is also able to infect laboratory mice, offering an ideal model with which to study various aspects of gammaherpesvirus infection. Here we describe in vitro studies of the mechanisms of the switch from latency to lytic replication of MHV-68. An MHV-68 gene, rta (replication and transcription activator), encoded primarily by open reading frame 50 (ORF50), is homologous to the rta genes of other gammaherpesviruses, including HHV-8 and EBV. HHV-8 and EBV Rta have been shown to play central roles in viral reactivation from latency. We first studied the kinetics of MHV-68 rta gene transcription during de novo lytic infection. MHV-68 rta was predominantly expressed as a 2-kb immediate-early transcript. Sequence analysis of MHV-68 rta cDNA revealed that an 866-nucleotide intron 5' of ORF50 was removed to create the Rta ORF of 583 amino acids. To test the functions of MHV-68 Rta in reactivation, a plasmid expressing Rta was transfected into a latently infected cell line, S11E, which was established from a B-cell lymphoma in an MHV-68-infected mouse. Rta induced expression of viral early and late genes, lytic replication of viral DNA, and production of infectious viral particles. We conclude that Rta alone is able to disrupt latency, activate viral lytic replication, and drive the lytic cycle to completion. This study indicates that MHV-68 provides a valuable model for investigating regulation of the balance between latency and lytic replication in vitro and in vivo.

T-cell vaccination alters the course of murine herpesvirus 68 infection and the establishment of viral latency in mice.

Diseases caused by gammaherpesviruses such as Epstein-Barr virus are a major health concern, and there is significant interest in developing vaccines against this class of viral infections. However, the requirements for effective control of gammaherpesvirus infection are only poorly understood. The recent development of the murine herpesvirus MHV-68 model provides an experimental tool to dissect the immune response to gammaherpesvirus infections. In this study, we investigated the impact of priming T cells specific for class I- and class II-restricted epitopes on the acute phase of the infection and the subsequent establishment of latency and infectious mononucleosis. The data show that vaccination with either major histocompatibility complex class I- or class II-restricted T-cell epitopes derived from lytic cycle proteins significantly reduced lung viral titers during the acute infection. Moreover, the peak level of latently infected spleen cells was significantly reduced following vaccination with immunodominant CD8(+) T-cell epitopes. However, this vaccination approach did not prevent the long-term establishment of latency or the development of the infectious mononucleosis-like syndrome in infected mice. Thus, the virus is able to establish latency efficiently despite strong immunological control of the lytic infection.

Apoptotic cells are generated at every division of in vitro cultured T cell lines.

Long- and short-term T cell lines form the backbone of many assays for T cell function and also represent important tools for use in human immunotherapy. Despite much study concerning the requirements for T cell activation and growth in culture there is relatively little information about the kinetics of proliferation and cell death in such cultures. Here we studied these parameters in a long-term CD8(+) T cell line using a tetrameric MHC reagent and the fluorescent dye CFSE. We observed proliferation of the T cells within 24 h of restimulation with antigen and IL-2 and the cells continued to divide once every 12 h on average. Interestingly, a proportion of cells entered apoptosis with each cell division, showing that a degree of programmed cell death occurred constantly in vitro, not merely at the end of the culture period when antigen or the necessary growth factors became limiting. This information should assist in the design of more efficient protocols for generating large numbers of specific T cells for clinical use.

Functionally heterogeneous CD8(+) T-cell memory is induced by Sendai virus infection of mice.

It has recently been established that memory CD8(+) T cells induced by viral infection are maintained at unexpectedly high frequencies in the spleen. While it has been established that these memory cells are phenotypically heterogeneous, relatively little is known about the functional status of these cells. Here we investigated the proliferative potential of CD8(+) memory T cells induced by Sendai virus infection. High frequencies of CD8(+) T cells specific for both dominant and subdominant Sendai virus epitopes persisted for many weeks after primary infection, and these cells were heterogeneous with respect to CD62L expression (approximately 20% CD62L(hi) and 80% CD62L(lo)). Reactivation of these cells with the antigenic peptide in vitro induced strong proliferation of antigen-specific CD8(+) T cells. However, approximately 20% of the cells failed to proliferate in vitro in response to a cognate peptide but nevertheless differentiated into effector cells and acquired full cytotoxic potential. These cells also expressed high levels of CD62L (in marked contrast to the CD62L(lo) status of the proliferating cells in the culture). Direct isolation of CD62L(hi) and CD62L(lo) CD8(+) T cells from memory mice confirmed the correlation of this marker with proliferative potential. Taken together, these data demonstrate that Sendai virus infection induces high frequencies of memory CD8(+) T cells that are highly heterogeneous in terms of both their phenotype and their proliferative potential.

Lytic cycle T cell epitopes are expressed in two distinct phases during MHV-68 infection.

Murine herpesvirus-68 (MHV-68) is a type 2 gamma herpesvirus that productively infects alveolar epithelial cells during the acute infection and establishes long-term latency in B cells and lung epithelial cells. In C57BL/6 mice, T cells specific for lytic cycle MHV-68 epitope p56/Db dominate the acute phase of the infection, whereas T cells specific for another lytic cycle epitope, p79/Kb, dominate later phases of infection. To further understand this response, we analyzed the kinetics of Ag presentation in vivo using a panel of lacZ-inducible T cell hybridomas specific for several lytic cycle epitopes, including p56/Db and p79/Kb. Two distinct peaks of Ag presentation were observed. The first peak was at day 6 in the mediastinal lymph nodes and correlated with the initial pulmonary lytic infection. The second peak was at day 18 in both the mediastinal lymph nodes and spleen and correlated with the peak of latent infection. Interestingly, the p56 epitope was detected only in the mediastinal lymph nodes at day 6 after infection whereas the p79 epitope was predominantly presented in the spleen at day 18, suggesting that differential epitope presentation drives the switch in T cell responses to this virus. Phenotypic analysis of APCs at day 18 postinfection revealed that dendritic cells, macrophages, and B cells all presented lytic cycle epitopes. Taken together, the data suggest that there is a resurgence of lytic cycle Ags in the spleen, which explains the kinetics and specificity of the T cell response to MHV-68.

Murine gammaherpesvirus M2 gene is latency-associated and its protein a target for CD8(+) T lymphocytes.

Murine gammaherpesvirus 68 (MHV-68) infection of mice is a potential model with which to address fundamental aspects of the pathobiology and host control of gammaherpesvirus latency. Control of MHV-68 infection, like that of Epstein-Barr virus, is strongly dependent on the cellular immune system. However, the molecular biology of MHV-68 latency is largely undefined. A screen of the MHV-68 genome for potential latency-associated mRNAs revealed that the region encompassing and flanking the genomic terminal repeats is transcriptionally active in the latently infected murine B-cell tumor line S11. Transcription of one MHV-68 gene, that encoding the hypothetical M2 protein, was detected in virtually all latently infected S11 cells and in splenocytes of latently infected mice, but not in lytically infected fibroblasts. Furthermore, an epitope was identified in the predicted M2 protein that is recognized by CD8(+) T cells from infected mice and a cytotoxic T lymphocyte line that recognizes this epitope killed S11 cells, indicating that the M2 protein is expressed during latent infection and is a target for the host cytotoxic T lymphocyte response. This work therefore provides essential information for modeling MHV-68 latency and strategies of immunotherapy against gammaherpesvirus-related diseases in a highly tractable animal model.

Long-term CD8+ T cell memory to Sendai virus elicited by DNA vaccination.

The capacity of DNA vaccines to prime CD8+ T cells makes them excellent candidates for vaccines that are designed to emphasize cellular immunity. However, the long-term stability of CD8+ T cell memory induced by DNA vaccination is poorly characterized. Here, the quality of CD8+ T cell recall responses in mice was investigated more than 1 year after DNA vaccination with the Sendai virus nucleoprotein gene. Cytotoxic T lymphocyte (CTL) activity specific for both dominant and subdominant epitopes could be recalled readily 1 year after vaccination and the frequencies of CTL precursors specific for both of these epitopes were relatively high. These CTL responded strongly to subsequent Sendai virus infection in terms of their ability to migrate to the lung and to differentiate into effector cells. In addition, the recall response to virus infection, as determined by CTL activity in the lungs and IFN-gamma responses in the spleen, was both faster and greater in magnitude than that in control-immunized mice. Significantly, virus titres were reduced at least 100-fold in the lungs of mice that were immunized more than 1 year before infection, as compared with control mice. These data demonstrate that CD8+ T cell memory elicited by DNA vaccination is functionally relevant and persists for at least 1 year.

Enumeration of antigen-presenting cells in mice infected with Sendai virus.

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.

Murine gamma-herpesvirus 68 glycoprotein 150 protects against virus-induced mononucleosis: a model system for gamma-herpesvirus vaccination.

Murine gamma-herpesvirus 68 (MHV-68) is a model for the study of the pathogenesis of gamma-herpesviruses. Epstein-Barr virus (EBV) is a highly related gamma-herpesvirus that causes significant disease in humans. The major membrane antigen gp350 of EBV is a candidate vaccine antigen for protection against EBV-related disease. An MHV-68 glycoprotein, gp150, has significant homology to EBV gp350. We have therefore used the MHV-68 gp150 to model the potential efficacy of EBV gp350 in protecting from virus-associated disease. A recombinant vaccinia virus expressing MHV-68 gp150 was constructed. This recombinant vaccinia virus was used to infect mice via the subcutaneous route. This vaccination resulted in production of MHV-68-neutralising antibodies. Mice were then challenged intra-nasally with MHV-68. MHV-68-associated mononucleosis was virtually abrogated in immunised mice. However, mice did establish MHV-68 latency. The results suggest that gp350 may be effective as an immunogen to prevent EBV-associated infectious mononucleosis in humans that are EBV-seronegative.

Lung epithelial cells are a major site of murine gammaherpesvirus persistence.

It is currently believed that latently infected, resting B lymphocytes are central to gammaherpesvirus persistence, whereas mucosal epithelial cells are considered nonessential. We have readdressed the question of nonlymphoid persistence using murine gammaherpesvirus 68 (MHV-68). To dissect lymphoid from nonlymphoid persistence, we used microMT transgenic mice that are defective in B cells. MHV-68 DNA persisted in the lungs of intact and B cell-deficient mice. Both episomal and linear forms of the virus genome were present in lungs, implying the presence of both latency and productive replication. In situ hybridization for virus tRNA transcripts revealed latent MHV-68 in pulmonary epithelial cells. Infectious virus was recovered from the lungs of microMT mice after T cell depletion, showing that the persisting virus DNA was reactivatable. Finally, using adoptive transfer of B cells into B cell-deficient mice, it was shown that virus persisting in lungs seeded splenic B cells, and virus resident in the spleen seeded the lungs. These results show that mucosal epithelia can act as a nonlymphoid reservoir for gammaherpesvirus persistence, and that there is a two-way movement of virus between lymphoid and nonlymphoid compartments during persistence.

Immunological control of murine gammaherpesvirus infection is independent of perforin.

Perforin-mediated cytotoxic T cell killing has been suggested to be of importance in the control of noncytopathic virus infections, based on studies with lymphocytic choriomeningitis virus (LCMV). We examined the role of perforin in a mouse model of gammaherpesvirus infection using transgenic perforin-deficient mice. Previous work from this laboratory has shown that CD8 T cells are essential for the resolution of the acute lung infection and control of latently infected B cells in murine gamma-herpesvirus 68 infection. The absence of perforin did not significantly affect the kinetics of either the lytic lung infection or the latent spleen infection. Lymphocytes from both perforin-deficient and control mice secreted comparable levels of IFN-gamma, IL-10 and IL-6. In addition, lymphocytes from both strains had similar levels of CD3epsilon-dependent cytotoxic activity in the spleen, draining lymph nodes and bronchoalveolar lavage. These data indicate that the lack of perforin has little affect on the ability of mice to control an experimental gammaherpesvirus infection.