ABSTRACTRespiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-α/β) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-α/β receptor.

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, infect > 90 % of the population worldwide, and latent infection is associated with numerous malignancies. Rational vaccination and therapeutic strategies require an understanding of virus-host interactions during the initial asymptomatic infection. Primary EBV infection is associated with virus replication at epithelial sites and entry into the circulating B lymphocyte pool. The virus exploits the life cycle of the B cell and latency is maintained long term in resting memory B cells. In this study, using a murine gamma-herpesvirus model, we demonstrate an early dominance of latent virus at the site of infection, with lung B cells harboring virus almost immediately after infection. These data reinforce the central role of the B cell not only in the later phase of infection, but early in the initial infection. Early inhibition of lytic replication does not impact the progression of the latent infection, and latency is established in lymphoid tissues following infection with a replication-deficient mutant virus. These data demonstrate that lytic viral replication is not a requirement for gamma-herpesvirus latency in vivo and suggest that viral latency can be disseminated by cellular proliferation. These observations emphasize that prophylactic vaccination strategies must target latent gamma-herpesvirus at the site of infection.

The murine gamma-herpesvirus-68 (gammaHV68) establishes viral latency in dendritic cells (DCs). In the present study, we examined the specific consequences of DC infection by gammaHV68, both in vivo and in vitro. Ex vivo analysis of infected mice showed that the virus colonizes respiratory DCs very early after infection and that all subsets of splenic DCs analyzed are viral targets. We have developed and characterized an in vitro model of gammaHV68 infection of DCs. Using this model, we demonstrated that viral infection neither induces full DC maturation nor interferes with exogenous activation, which is assessed by cell surface phenotypic changes. However, whereas gammaHV68 infection alone failed to elicit cytokine secretion, IL-10 secretion of exogenously activated DCs was enhanced. Furthermore, gammaHV68-infected DCs efficiently stimulated virus-specific T cell hybridomas but failed to induce alloreactive stimulation of normal T cells. These data indicate that viral infection doesn't interfere with Ag processing and presentation but does interfere with the ability of DCs to activate T cells. The inhibition of T cell activation was partially reversed by blocking IL-10. Analysis of infected mice shows elevated levels of IL-10 expression in DCs and that lack of endogenous IL-10 is associated with decreased gammaHV68 long-term latency. Taken together, these observations indicate that gamma2-herpesvirus infection of DCs is a mechanism of viral immune evasion, partially mediated by IL-10.

The murine γ-herpesvirus-68 (γHV68) establishes viral latency in dendritic cells (DCs). In the present study, we examined the specific consequences of DC infection by γHV68, both in vivo and in vitro. Ex vivo analysis of infected mice showed that the virus colonizes respiratory DCs very early after infection and that all subsets of splenic DCs analyzed are viral targets. We have developed and characterized an in vitro model of γHV68 infection of DCs. Using this model, we demonstrated that viral infection neither induces full DC maturation nor interferes with exogenous activation, which is assessed by cell surface phenotypic changes. However, whereas γHV68 infection alone failed to elicit cytokine secretion, IL-10 secretion of exogenously activated DCs was enhanced. Furthermore, γHV68-infected DCs efficiently stimulated virus-specific T cell hybridomas but failed to induce alloreactive stimulation of normal T cells. These data indicate that viral infection doesn’t interfere with Ag processing and presentation but does interfere with the ability of DCs to activate T cells. The inhibition of T cell activation was partially reversed by blocking IL-10. Analysis of infected mice shows elevated levels of IL-10 expression in DCs and that lack of endogenous IL-10 is associated with decreased γHV68 long-term latency. Taken together, these observations indicate that γ2-herpesvirus infection of DCs is a mechanism of viral immune evasion, partially mediated by IL-10.

Intranasal infection of mice with murine gammaherpesvirus 68 causes a dramatic increase in numbers of activated CD8+ T cells in the blood, analogous in many respects to EBV-induced infectious mononucleosis in humans. In the mouse model, this lymphocytosis has two distinct components: an early, conventional virus-specific CD8+ T cell response, and a later response characterized by a dramatic increase among CD8+ T cells that bear Vβ4+ TCRs. We previously demonstrated that Vβ4+CD8+ T cells recognize an uncharacterized ligand expressed on latently infected B cells in an MHC-independent manner. The frequency of Vβ4+CD8+ T cells increases dramatically following the peak of viral latency in the spleen. In the current studies, we show that elevated Vβ4+CD8+ T cell levels are sustained long-term in persistently infected mice, apparently a consequence of continued ligand expression. In addition, we show that Vβ4+CD8+ T cells can acquire effector functions, including cytotoxicity and the capacity to secrete IFN-γ, although they have an atypical activation profile compared with well-characterized CD8+ T cells specific for conventional viral epitopes. The characteristics of Vβ4+CD8+ T cells (potential effector function, stimulation by latently infected B cells, and kinetics of expansion) suggested that this dominant T cell response plays a key role in the immune control of latent virus. However, Ab depletion and adoptive transfer studies show that Vβ4+CD8+ T cells are not essential for this function. This murine model of infection may provide insight into the role of unusual populations of activated T cells associated with persistent viral infections.

Murine gamma-herpesvirus 68 (MHV-68) provides an important experimental model for analyzing gamma-herpesvirus latent infection. After intranasal infection with MHV-68, we analyzed the distribution of the virus in different anatomical locations and purified populations of cells. Our data show that long-term latency is maintained in a variety of anatomical locations and cell populations with different frequencies. Importantly, we demonstrate that although latency in the lung is established in a variety of cell subsets, long-term latency in the lung is only maintained in B cells. In contrast, splenic latency is maintained in macrophages and dendritic cells, as well as in B cells. In blood, isotype-switched B cells constitute the major viral reservoir. These results show that the cell subsets in which latency is established vary within different anatomical sites. Finally, we demonstrate that long-term latency is accompanied by a low level of infectious virus in lung and spleen. These data have important implications for understanding the establishment and maintenance of latency by gamma(2)-herpesviruses.

It has been proposed that the γ-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40+ and CD40− B cells from CD40+CD40− mixed bone marrow chimera mice after infection with a murine γ-herpesvirus, MHV-68. CD40+ B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40− B cells and preferentially maintained in the long-lived, isotype-switched CD40+ B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.

It has been proposed that the gamma-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40(+) and CD40(-) B cells from CD40(+)CD40(-) mixed bone marrow chimera mice after infection with a murine gamma-herpesvirus, MHV-68. CD40(+) B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40(-) B cells and preferentially maintained in the long-lived, isotype-switched CD40(+) B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.

ABSTRACTThis work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68)M2gene. A frameshift mutation was made in theM2open reading frame that caused premature termination of translation of M2 after amino acid residue 90. TheM2mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vβ4 T-cell-receptor-positive CD8+T-cell mononucleosis, and establishment of latency were unaffected. However, theM2mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression ofM2is restricted to B cells in the spleen and thatM2encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.

The γ-herpesviruses are oncogenic B cell lymphotrophic viruses that establish life-long latency in the host. Murine γ-herpesvirus 68 (MHV-68) infection of mice represents a unique system for analyzing γ-herpesvirus latency in splenic B cells at different stages of infection. After intranasal infection with MHV-68 we analyzed the establishment of latency 14 days after infection, and the maintenance of latency 3 months after infection in different purified subpopulations of B cells in the spleen. The data show that MHV-68 latency is mainly established in germinal center B cells and that long-term latency is preferentially maintained in two different subsets of isotype-switched B cells, germinal center and memory B cells. Cell cycle analysis indicates that MHV-68 is located in both cycling and resting isotype-switched B cells. Analysis of viral gene expression showed that both lytic and latent viral transcripts were differentially expressed in germinal center and memory B cells during long-term latency. Together, these observations suggested that γ-herpesviruses exploit the B cell life cycle in the spleen.

Intranasal infection of mice with murine gamma-herpesvirus 68 (MHV-68) elicits a striking CD8(+) T-cell lymphocytosis following the establishment of latency, which includes a marked increased frequency of Vbeta4(+) CD8(+) T cells. The Vbeta4(+) CD8(+) T cells do not recognize a conventional viral peptide, but are stimulated by an uncharacterized ligand expressed on latently infected, activated B cells. The selective expansion of Vbeta4(+) CD8(+) T cells after MHV-68 infection is observed in all mouse strains examined, although the fold-increase varies widely, ranging from less than twofold to greater than 10-fold. The factors controlling the variation are currently undefined. In the current study, CD8(+) T cell activation and Vbeta4(+) CD8(+) T-cell frequencies were analyzed in 18 inbred strains of mice. The data show that the magnitude of the Vbeta4(+) CD8(+) T-cell response correlates with the degree of CD8(+) T cell-activation, and that both major histocompatibility complex (MHC) and non-MHC genes contribute to the magnitude of the activation. Furthermore, the magnitude of the response does not reflect major differences in susceptibility to viral infection and/or corresponding differences in the acute response. Rather the degree of Vbeta4(+) CD8(+) T cell activation may be determined by differences in levels of expression of the stimulatory ligand at the peak of latency.

Major histocompatibility complex class II-mediated antigen presentation after intranasal infection with murine gammaherpesvirus 68 differs in mediastinal lymph nodes and spleen. Evidence that virus-specific CD4(+) T cells were being stimulated was found as late as 6 to 8 months after infection, and cells specific for the viral gp150(67-83) and ORF11(168-180) peptides were maintained as a fairly stable proportion of the total response.

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.

Herpesviruses are double-stranded DNA viruses that establish life-long latency in the host and are important human pathogens1. They can be classified into three major groups, largely determined by the growth characteristics of the virus and the tissue types in which viral latency is established. The a-herpesviruses include herpes simplex virus, the b-herpesviruses include cytomegalovirus and the lymphotropic g-herpesviruses include Epstein‐Barr virus (EBV) and Kaposi’s sarcoma associated herpesvirus (KSHV or human herpesvirus-8). EBV is ubiquitous in the human population. The virus establishes a lytic infection in the oropharynx and life-long latency, predominantly in B cells. The latent stage is benign in individuals with a competent immune system but is associated with the development of a range of malignancies in immunocompromised individuals. Infectious mononucleosis is a common sequela of primary EBV infection. The syndrome is characterized by lymphadenopathy, splenomegaly, heterophilic antibodies and peripheral blood lymphocytosis with atypical lymphocytes, and usually resolves spontaneously. Interestingly, infectious mononucleosis occurs frequently in developed countries where initial EBV infection is delayed until adolescence or adulthood, but is not observed in developing countries where EBV infection occurs early in life. In many cases, infectious mononucleosis is the first clinical indication of EBV infection and the precise relationship of the syndrome with the lytic infection has not been determined.

Intranasal infection of mice with the murine gamma-herpesvirus MHV-68 results in an acute lytic infection in the lung, followed by the establishment of lifelong latency. Development of an infectious mononucleosis-like syndrome correlates with the establishment of latency and is characterized by splenomegaly and the appearance of activated CD8+ T cells in the peripheral blood. Interestingly, a large population of activated CD8+ T cells in the peripheral blood expresses the V beta 4+ element in their TCR. In this report we show that MHV-68 latency in the spleen after intranasal infection is harbored in three APC types: B cells, macrophages, and dendritic cells. Surprisingly, since latency has not previously been described in dendritic cells, these cells harbored the highest frequency of latent virus. Among B cells, latency was preferentially associated with activated B cells expressing the phenotype of germinal center B cells, thus formally linking the previously reported association of latency gene expression and germinal centers to germinal center B cells. Germinal center formation, however, was not required for the establishment of latency. Significantly, although three cell types were latently infected, the ability to stimulate V beta 4+CD8+ T cell hybridomas was limited to latently infected, activated B cells.

Like EBV-infected humans with infectious mononucleosis, mice infected with the rodent gammaherpesvirus MHV-68 develop a profound increase in the number of CD8+ T cells in the circulation. In the mouse model, this lymphocytosis consists of highly activated CD8+ T cells strikingly biased toward V beta 4 TCR expression. Moreover, this expansion of V beta 4+CD8+ T cells does not depend on the MHC haplotype of the infected animal. Using a panel of lacZ-inducible T cell hybridomas, we have detected V beta 4-specific T cell stimulatory activity in the spleens of MHV-68-infected mice. We show that the appearance and quantity of this activity correlate with the establishment and magnitude of latent viral infection. Furthermore, on the basis of Ab blocking studies as well as experiments with MHC class II, beta2-microglobulin (beta2m) and TAP1 knockout mice, the V beta 4-specific T cell stimulatory activity does not appear to depend on conventional presentation by classical MHC class I or class II molecules. Taken together, the data indicate that during latent infection, MHV-68 may express a T cell ligand that differs fundamentally from both conventional peptide Ags and classical viral superantigens.

A CD8(+) T cell lymphocytosis in the peripheral blood is associated with the establishment of latency following intranasal infection with murine gammaherpesvirus-68. Remarkably, a large percentage of the activated CD8(+) T cells of mice expressing different MHC haplotypes express V beta 4(+) TCR, Identification of the ligand driving the V beta 4(+)CD8(+) T cell activation remains elusive, but there is a general correlation between V beta 4(+)CD8(+) T cell stimulatory activity and establishment of latency in the spleen. In the current study, the role of CD4(+) T cells in the V beta 4(+)CD8(+) T cell expansion has been addressed. The results show that CD4(+) T cells are essential for expansion of the V beta 4(+)CD8(+) subset, but not other V beta subsets, in the peripheral blood. CD4(+) T cells are required relatively late in the antiviral response, between 7 and 11 days after infection, and mediate their effect independently of IFN-gamma, Assessment of V beta 4(+)CD8(+) T cell stimulatory activity using murine gammaherpesvirus-68-specific T cell hybridomas generated from latently infected mice supports the idea that CD4(+) T cells control levels of the stimulatory ligand that drives the V beta 4(+)CD8(+) T cells. As V beta 4(+)CD8(+) T cell expansion also correlates with levels of activated B cells, these data raise the possibility that CD4(+) T cell-mediated B cell activation is required for optimal expression of the stimulatory ligand. In addition, in cases of low ligand expression, there may also be a direct role for CD4(+) T cell-mediated help for V beta 4(+)CD8(+) T cells.