Vaccination alters the balance between protective immunity, exhaustion, escape, and death in chronic infections

While T cell-based vaccines have the potential to provide protection against chronic virus infections, they also have the potential to generate immunopathology following subsequent virus infection. We develop a mathematical model to investigate the conditions under which T cells lead to protection versus adverse pathology. The model illustrates how the balance between virus clearance and immune exhaustion may be disrupted when vaccination generates intermediate numbers of specific CD8 T cells. Surprisingly, our model suggests that this adverse effect of vaccination is largely unaffected by the generation of mutant viruses that evade T cell recognition and cannot be avoided by simply increasing the quality (affinity) or diversity of the T cell response. These findings should be taken into account when developing vaccines against persistent infections.     

The antiviral CD8+ T cell response is differentially dependent on CD4+ T cell help over the course of persistent infection

Although many studies have investigated the requirement for CD4(+) T cell help for CD8(+) T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4(+) T cells in maintaining ongoing CD8(+) T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4(+) T cell help is required to maintain antiviral CD8(+) T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8(+) T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8(+) T cell population that was maintained long term. These unhelped PyV-specific CD8(+) T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8(+) T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4(+) T cells differentially affect CD8(+) T cell responses over the course of a persistent virus infection.     

Immunogenicity of cytopathic and noncytopathic viral vectors

The impact of cytolytic versus noncytolytic viral infections on host responses is not well understood, due to limitations of the systems that have been used to address this issue. Using paired cytopathic and noncytopathic rabies viruses that differ by only two amino acids, we investigated several fundamental aspects of the immune response to these viral vectors. Greater cytopathic capacity translated into a greater degree of cross-priming to CD8(+) T cells (T(CD8)(+)) and more-robust short-term humoral and cellular responses. However, long-term responses to the two viruses were similar, suggesting that direct priming drives the bulk of the T(CD8)(+) antirabies response and that enhanced acute responses associated with greater virally mediated cellular destruction were balanced by other factors, such as prolonged antigen expression associated with noncytopathic virus. Such compensatory mechanisms may be in place to ensure comparable immunologic memories to various pathogens.     

Comprehensive early and lasting loss of memory CD8 T cells and functional memory during acute and persistent viral infections

Viral infections have been shown to induce lymphopenias that lower memory CD8 T cell frequencies, and they also have been shown to cause a permanent loss of memory cells specific to previously encountered pathogens. In this study, the patterns and significance of virus-induced memory CD8 T cell depletion were examined in mice immune to heterologous (Pichinde, vesicular stomatitis, vaccinia) viruses and subsequently challenged with acute or persistent lymphocytic choriomeningitis virus infections. Memory CD8 T cell loss was comprehensive and occurred in both lymphoid and peripheral tissues of the immune host. The impact of the loss of memory T cells was reflected by in vivo cytotoxicity assays, which showed decreased clearance of epitope-expressing targets. Memory CD8 T cell loss occurred very early (day 2) after infection, and was thereafter sustained, consistent more with an active deletion model than with a competition model. Cross-reactive T cells, in contrast, increased in number, but memory cells were reduced whether or not there was competition from cross-reactive T cells. Memory T cell loss was more profound during persistent infection than after acute infection. Adoptive transfer studies showed that, unlike the resolved acute infection, in which the reduced memory frequencies became stable, memory T cell loss was a continuously ongoing process during persistent infection. This study therefore links an early virus-induced lymphopenia to a subsequent long-term loss of CD8 T cell memory and offers a new mechanism for immune deficiency during persistent viral infections.     

Expansion of protective CD8+ T-cell responses driven by recombinant cytomegaloviruses

CD8(+) T cells are critical for the control of many persistent viral infections, such as human immunodeficiency virus, hepatitis C virus, Epstein-Barr virus, and cytomegalovirus (CMV). In most infections, large CD8(+)-T-cell populations are induced early but then contract and are maintained thereafter at lower levels. In contrast, CD8(+) T cells specific for murine CMV (MCMV) have been shown to gradually accumulate after resolution of primary infection. This unique behavior is restricted to certain epitopes, including an immunodominant epitope derived from the immediate-early 1 (IE1) gene product. To explore the mechanism behind this further, we measured CD8(+)-T-cell-mediated immunity induced by recombinant MCMV-expressing epitopes derived from influenza A virus or lymphocytic choriomeningitis virus placed under the control of an IE promoter. We observed that virus-specific CD8(+)-T-cell populations were induced and that these expanded gradually over time. Importantly, these CD8(+) T cells provided long-term protection against challenge without boosting. These results demonstrate a unique pattern of accumulating T cells, which provide long-lasting immune protection, that is independent of the initial immunodominance of the epitope and indicates the potential of T-cell-inducing vaccines based on persistent vectors.     

Control of memory CD8+ T cell differentiation by CD80/CD86-CD28 costimulation and restoration by IL-2 during the recall response

Memory CD8+ T cell responses have been considered to be independent of CD80/CD86-CD28 costimulation. However, recall responses are often severely blunted in CD28-/- mice. Whether this impairment represents a requirement for CD28 costimulation for proper memory CD8+ T cell development or a requirement during the recall response is unknown. Furthermore, how CD28 costimulation affects the phenotype and function of memory CD8+ T cells has not been characterized in detail. In this study, we investigate these questions by studying the role of the CD28 costimulatory pathway in memory CD8+ T cell responses to acute and persistent DNA virus infections. Memory CD8+ T cells against vaccinia virus (VV) infection which develop without CD28 costimulation exhibit lower expression of differentiation markers CD27 and CD122 (IL-15Rbeta). These memory CD8+ T cells also fail to produce IL-2. Our data indicate that for an optimal recall response, CD28 costimulation is required both for T cell priming and also during the recall response. Similar requirements were observed for memory CD8+ T cell responses during persistent infection with murine gammaherpesvirus 68 (MHV-68) infection, indicating CD28 may play the same role in both acute and persistent infections. Finally, we show deficits in the recall response are restored by IL-2 signaling during recall, but not during priming. The data presented show that CD28 costimulation not only controls the magnitude of the primary response but also affects development of memory CD8+ T cells and is required during the recall response in addition to initial T cell priming.     

Antibodies and CD8+ T cells are complementary and essential for natural resistance to a highly lethal cytopathic virus

It is believed that CD8+ T lymphocytes or Abs can independently clear many primary viral infections, including those caused by Orthopoxviruses (OPV), a genus that includes the human pathogens variola and monkeypox and the vaccine species vaccinia virus. However, most experiments addressing the role of Abs and CD8+ T cells in protection have used viruses that are not specific for the host. In the present study, we used the mouse-specific OPV ectromelia virus and mice deficient in CD40, B cells, or CD8+ T cells and adoptive transfers of CD8+ T or B lymphocytes to show that the protection afforded by CD8+ T cells is incomplete. Despite sustained CD8+ T cell responses, in the absence of Ab responses ectromelia virus persists. This results in delayed disease and inexorably leads to death. Therefore, CD8+ T lymphocytes and Abs are not redundant but complementary and essential to survive infections with a highly pathogenic viruses in the natural host.     

Late priming and variability of epitope-specific CD8+ T cell responses during a persistent virus infection

Control of persistently infecting viruses requires that antiviral CD8(+) T cells sustain their numbers and effector function. In this study, we monitored epitope-specific CD8(+) T cells during acute and persistent phases of infection by polyoma virus, a mouse pathogen that is capable of potent oncogenicity. We identified several novel polyoma-specific CD8(+) T cell epitopes in C57BL/6 mice, a mouse strain highly resistant to polyoma virus-induced tumors. Each of these epitopes is derived from the viral T proteins, nonstructural proteins produced by both productively and nonproductively (and potentially transformed) infected cells. In contrast to CD8(+) T cell responses described in other microbial infection mouse models, we found substantial variability between epitope-specific CD8(+) T cell responses in their kinetics of expansion and contraction during acute infection, maintenance during persistent infection, as well as their expression of cytokine receptors and cytokine profiles. This epitope-dependent variability also extended to differences in maturation of functional avidity from acute to persistent infection, despite a narrowing in TCR repertoire across all three specificities. Using a novel minimal myeloablation-bone marrow chimera approach, we visualized priming of epitope-specific CD8(+) T cells during persistent virus infection. Interestingly, epitope-specific CD8(+) T cells differed in CD62L-selectin expression profiles when primed in acute or persistent phases of infection, indicating that the context of priming affects CD8(+) T cell heterogeneity. In summary, persistent polyoma virus infection both quantitatively and qualitatively shapes the antiviral CD8(+) T cell response.     

CD8 T cells recruited early in mouse polyomavirus infection undergo exhaustion

Repetitive Ag encounter, coupled with dynamic changes in Ag density and inflammation, imparts phenotypic and functional heterogeneity to memory virus-specific CD8 T cells in persistently infected hosts. For herpesvirus infections, which cycle between latency and reactivation, recent studies demonstrate that virus-specific T cell memory is predominantly derived from naive precursors recruited during acute infection. Whether functional memory T cells to viruses that persist in a nonlatent, low-level infectious state (smoldering infection) originate from acute infection-recruited naive T cells is not known. Using mouse polyomavirus (MPyV) infection, we previously showed that virus-specific CD8 T cells in persistently infected mice are stably maintained and functionally competent; however, a sizeable fraction of these memory T cells are short-lived. Further, we found that naive anti-MPyV CD8 T cells are primed de novo during persistent infection and contribute to maintenance of the virus-specific CD8 T cell population and its phenotypic heterogeneity. Using a new MPyV-specific TCR-transgenic system, we now demonstrate that virus-specific CD8 T cells recruited during persistent infection possess multicytokine effector function, have strong replication potential, express a phenotype profile indicative of authentic memory capability, and are stably maintained. In contrast, CD8 T cells recruited early in MPyV infection express phenotypic and functional attributes of clonal exhaustion, including attrition from the memory pool. These findings indicate that naive virus-specific CD8 T cells recruited during persistent infection contribute to preservation of functional memory against a smoldering viral infection.     

Critical Role for CD4+ T Cells in Controlling Retrovirus Replication and Spread in Persistently Infected Mice

Reactivations of persistent viral infections pose a significant medical problem in immunocompromised cancer, transplant, and AIDS patients, yet little is known about how persistent viral infections are immunologically controlled. Here we describe a mouse model for investigating the role of the immune response in controlling a persistent retroviral infection. We demonstrate that, following recovery from acute Friend virus infection, a small number of B cells evade immunological destruction and harbor persistent virus. In vivo depletions of T-cell subsets in persistently infected mice revealed a critical role for CD4⁺ T cells in controlling virus replication, spread to the erythroid lineage, and induction of erythroleukemia. The CD4⁺ T-cell effect was independent of CD8⁺ T cells and in some cases was also independent of virus-neutralizing antibody responses. Thus, the CD4⁺ T cells may have had a direct antiviral effect. These results may have relevance for human immunodeficiency virus (HIV) infections where loss of CD4⁺ T cells is associated with an increase in HIV replication, reactivation of persistent viruses, and a high incidence of virus-associated cancers.     

Independent regulation of lymphocytic choriomeningitis virus-specific T cell memory pools: relative stability of CD4 memory under conditions of CD8 memory T cell loss

Infection of mice with a series of heterologous viruses causes a reduction of memory CD8(+) T cells specific to viruses from earlier infections, but the fate of the virus-specific memory CD4(+) T cell pool following multiple virus infections has been unknown. We have previously reported that the virus-specific CD4(+) Th precursor (Thp) frequency remains stable into long-term immunity following lymphocytic choriomeningitis virus (LCMV) infection. In this study, we questioned whether heterologous virus infections or injection with soluble protein CD4 Ags would impact this stable LCMV-specific CD4(+) Thp memory pool. Limiting dilution analyses for IL-2-producing cells and intracellular cytokine staining for IFN-gamma revealed that the LCMV-specific CD4(+) Thp frequency remains relatively stable following multiple heterologous virus infections or protein Ag immunizations, even under conditions that dramatically reduce the LCMV-specific CD8(+) CTL precursor frequency. These data indicate that the CD4(+) and CD8(+) memory T cell pools are regulated independently and that the loss in CD8(+) T cell memory following heterologous virus infections is not a consequence of a parallel loss in the memory CD4(+) T cell population.     

MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis

An optimal vaccine against leishmaniasis should elicit parasite specific CD4+ and cytotoxic CD8+ T cells. In this investigation, we described a prime/boost immunization approach based on DNA and on poxvirus vectors (Western Reserve, WR, and the highly attenuated modified vaccinia virus Ankara, MVA), both expressing the LACK antigen of Leishmania infantum, that triggers different levels of specific CD8+ T cell responses and protection (reduction in lesion size and parasitemia) against L. major infection in mice. A prime/boost vaccination with DNA-LACK/MVA-LACK elicits higher CD8+ T cell responses than a similar protocol with the replication competent VV-LACK. Both CD4+ and CD8+ T cells were induced by DNA-LACK/MVA-LACK immunization. The levels of IFN-gamma and TNF-alpha secreting CD8+ T cells were higher in splenocytes from DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals. Moreover, protection against L. major was significantly higher in DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals when boosted with the same virus dose, and correlated with high levels of IFN-gamma and TNF-alpha secreting CD8+ T cells. In DNA-LACK/MVA-LACK vaccinated animals, the extent of lesion size reduction ranged from 65 to 92% and this protection was maintained for at least 17 weeks after challenge with the parasite. These findings demonstrate that in heterologous prime/boost immunization approaches, the protocol DNA-LACK/MVA-LACK is superior to DNA-LACK/VV-LACK in triggering specific CD8+ T cell immune responses and in conferring protection against cutaneous leishmaniasis. Thus, MVA-LACK is a safe and efficient vector for vaccination against leishmaniasis.     

Extralymphatic virus sanctuaries as a consequence of potent T-cell activation

T helper cells can support the functions of CD8(+) T cells against persistently infecting viruses such as murine lymphocytic choriomeningitis virus (LCMV), cytomegalovirus, hepatitis C virus and HIV. These viruses often resist complete elimination and remain detectable at sanctuary sites, such as the kidneys and other extralymphatic organs. The mechanisms underlying this persistence are not well understood. Here we show that mice with potent virus-specific T-cell responses have reduced levels and delayed formation of neutralizing antibodies, and these mice fail to clear LCMV from extralymphatic epithelia. Transfer of virus-specific B cells but not virus-specific T cells augmented virus clearance from persistent sites. Virus elimination from the kidneys was associated with the formation of IgG deposits in the interstitial space, presumably from kidney-infiltrating B cells. CD8(+) T cells in the kidneys of mice that did not clear virus from this site were activated but showed evidence of exhaustion. Thus, we conclude that in this model of infection, site-specific virus persistence develops as a consequence of potent immune activation coupled with reductions in virus-specific neutralizing antibodies. Our results suggest that sanctuary-site formation depends both on organ anatomy and on the induction of different adaptive immune effector mechanisms. Boosting T-cell responses alone may not reduce virus persistence.     

Costimulation requirements for antiviral CD8+ T cells differ for acute and persistent phases of polyoma virus infection

The requirement for costimulation in antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections, but it is less defined for CD8+ T cell responses to persistent virus infection. Using mouse polyoma virus (PyV) as a model of low-level persistent virus infection, we asked whether blockade of the CD40 ligand (CD40L) and CD28 costimulatory pathways impacts the magnitude and function of the PyV-specific CD8+ T response, as well as the humoral response and viral control during acute and persistent phases of infection. Costimulation blockade or gene knockout of either CD28 or CD40L substantially dampened the magnitude of the acute CD8+ T cell response; simultaneous CD28 and CD40L blockade severely depressed the acute T cell response, altered the cell surface phenotype of PyV-specific CD8+ T cells, decreased PyV VP1-specific serum IgG titers, and resulted in an increase in viral DNA levels in multiple organs. CD28 and CD40L costimulation blockade during acute infection also diminished the memory PyV-specific CD8+ T cell response and serum IgG titer, but control of viral persistence varied between mouse strains and among organs. Interestingly, we found that CD28 and CD40L costimulation is dispensable for generating and/or maintaining PyV-specific CD8+ T cells during persistent infection; however, blockade of CD27 and CD28 costimulation in persistently infected mice caused a reduction in PyV-specific CD8+ T cells. Taken together, these data indicate that CD8+ T cells primed within the distinct microenvironments of acute vs persistent virus infection differ in their costimulation requirements.     

In vitro suppression of CD8+ T cell function by Friend virus-induced regulatory T cells

Regulatory T cell (Treg)-mediated suppression of CD8+ T cells has been implicated in the establishment and maintenance of chronic viral infections, but little is known about the mechanism of suppression. In this study an in vitro assay was developed to investigate the suppression of CD8+ T cells by Friend retrovirus (FV)-induced Tregs. CD4+CD25+ T cells isolated from mice chronically infected with the FV suppressed the development of effector function in naive CD8+ T cells without affecting their ability to proliferate or up-regulate activation markers. In vitro restimulation was not required for suppression by FV-induced Tregs, correlating with their high activation state in vivo. Suppression was mediated by direct T cell-T cell interactions and occurred in the absence of APCs. Furthermore, suppression occurred irrespective of the TCR specificity of the CD8+ T cells. Most interestingly, FV-induced Tregs were able to suppress the function of CD8+ effector T cells that had been physiologically activated during acute FV infection. The ability to suppress the effector function of activated CTLs is likely a requisite role for Tregs in limiting immunopathology by CD8+ T cells during antiviral immune responses. Such activity may also have adverse consequences by allowing viruses to establish and maintain chronic infections if suppression of antiviral immune responses occurs before virus eradication.     

Persistent infection with ebola virus under conditions of partial immunity

Ebola hemorrhagic fever in humans is associated with high mortality; however, some infected hosts clear the virus and recover. The mechanisms by which this occurs and the correlates of protective immunity are not well defined. Using a mouse model, we determined the role of the immune system in clearance of and protection against Ebola virus. All CD8 T-cell-deficient mice succumbed to subcutaneous infection and had high viral antigen titers in tissues, whereas mice deficient in B cells or CD4 T cells cleared infection and survived, suggesting that CD8 T cells, independent of CD4 T cells and antibodies, are critical to protection against subcutaneous Ebola virus infection. B-cell-deficient mice that survived the primary subcutaneous infection (vaccinated mice) transiently depleted or not depleted of CD4 T cells also survived lethal intraperitoneal rechallenge for >/==" BORDER="0">25 days. However, all vaccinated B-cell-deficient mice depleted of CD8 T cells had high viral antigen titers in tissues following intraperitoneal rechallenge and died within 6 days, suggesting that memory CD8 T cells by themselves can protect mice from early death. Surprisingly, vaccinated B-cell-deficient mice, after initially clearing the infection, were found to have viral antigens in tissues later (day 120 to 150 post-intraperitoneal infection). Furthermore, following intraperitoneal rechallenge, vaccinated B-cell-deficient mice that were transiently depleted of CD4 T cells had high levels of viral antigen in tissues earlier (days 50 to 70) than vaccinated undepleted mice. This demonstrates that under certain immunodeficiency conditions, Ebola virus can persist and that loss of primed CD4 T cells accelerates the course of persistent infections. These data show that CD8 T cells play an important role in protection against acute disease, while both CD4 T cells and antibodies are required for long-term protection, and they provide evidence of persistent infection by Ebola virus suggesting that under certain conditions of immunodeficiency a host can harbor virus for prolonged periods, potentially acting as a reservoir.     

Timing and magnitude of type I interferon responses by distinct sensors impact CD8 T cell exhaustion and chronic viral infection

Type I interferon (IFN-I) promotes antiviral CD8(+)T cell responses, but the contribution of different IFN-I sources and signaling pathways are ill defined. While plasmacytoid dendritic cells (pDCs) produce IFN-I upon TLR stimulation, IFN-I is induced in most cells by helicases like MDA5. Using acute and chronic lymphocytic choriomeningitis virus (LCMV) infection models, we determined that pDCs transiently produce IFN-I that minimally impacts CD8(+)T cell responses and viral persistence. Rather, MDA5 is the key sensor that induces IFN-I required for CD8(+)T cell responses. In the absence of MDA5, CD8(+)T cell responses to acute infection rely on CD4(+)T cell help, and loss of both CD4(+)T cells and MDA5 results in CD8(+)T cell exhaustion and persistent infection. Chronic LCMV infection rapidly attenuates IFN-I responses, but early administration of exogenous IFN-I rescues CD8(+)T cells, promoting viral clearance. Thus, effective antiviral CD8(+)T cell responses depend on the timing and magnitude of IFN-I production.     

Differential evolution and stability of epitope-specific CD8(+) T cell responses in EBV infection

Murine models of lymphocytic choriomeningitis virus infection suggest that the memory CD8(+) T cell repertoire is reflective of the CD8(+) T cell repertoire generated during acute infection. Less is known regarding the evolution of CD8(+) T cell repertoires during human viral infections. We therefore examined epitope-specific CD8(+) T cell responses in a large cohort of individuals with acute through latent Epstein-Barr virus infection. Using 16 of 20 published EBV epitopes restricted by HLA-A2, HLA-A3 or HLA-B7, we showed that lytic cycle-specific CD8(+) T cell responses predominated during acute EBV infection. However, whereas HLA-A2(+)-restricted BMLF-1-specific CD8(+) T cell responses were maintained through latency, HLA-A2(+)- and HLA-B7(+)-restricted BZLF-1, as well as HLA-A3(+)-restricted BRLF-1 CD8(+) T cell responses, were generated but not readily maintained. Analyses of CD8(+) T cell responses to EBV latent cycle Ags showed delayed detection and lower frequencies of latent epitope-specific CD8(+) T cell responses during acute EBV infection, with maintenance of these responses 1 yr post-EBV infection. Early BMLF-1 and EBNA-3A epitope-specific CD8(+) T cell frequencies did not correlate with their frequencies at 1 yr postinfection. Interestingly, populations of EBV-specific CD8(+) T cells were stable during 20 mo in our long term EBV-seropositive populations, suggesting homeostasis between virus and the host immune system. This study demonstrates that CD8(+) T cell repertoires generated during persistent viral infections are not simply reflective of the initial pool of CD8(+) T cells and provides evidence that the generation of CD8(+) T cell responses to a persistent infection is a dynamic process.     

The dynamics of the cellular immune response to HIV infection: implications for vaccination

Recent advances in measuring T-cell responses to viruses have led to new insights into how these T cells respond. In the acute infection there are massive CD8+ T-cell responses to both Epstein-Barr virus (EBV) and to human immunodeficiency virus (HIV). Many of these T cells are effector cells and only a minority appear to be capable of maintaining immunological memory. In persistent virus infections, high levels of antigen-specific effector cells persist. If virus does not persist, the effectors fade in number but memory is maintained and is primed to react rapidly to a new challenge. A vaccine that stimulates only T-cell responses may protect when these memory cells respond rapidly enough to generate high numbers of effectors before the infecting virus becomes established.