Protracted course of lymphocytic choriomeningitis virus WE infection in early life: induction but limited expansion of CD8+ effector T cells and absence of memory CD8+ T cells

Viral infections in human infants frequently follow a protracted course, with higher viral loads and delayed viral clearance compared to viral infections in older children. To identify the mechanisms responsible for this protracted pattern of infection, we developed an infant infection murine model using the well-characterized lymphocytic choriomeningitis virus (LCMV) WE strain in 2-week-old BALB/c mice. In contrast to adult mice, in which viral clearance occurred as expected 8 days after infection, LCMV titers persisted for several weeks after infection of infant mice. LCMV-specific effector CD8(+) T cells were elicited in infant mice and fully functional on day 7 but rapidly waned and could not be recovered from day 12 onwards. We show here that this results from the failure of LCMV-specific CD8(+) T cells to expand and the absence of protective LCMV-specific memory CD8(+) T cells. Under these early life conditions, viral control and clearance are eventually achieved only through LCMV-specific B cells that contribute to protect infant mice from early death or chronic infection.     

MyD88 plays a critical T cell-intrinsic role in supporting CD8 T cell expansion during acute lymphocytic choriomeningitis virus infection

During acute lymphocytic choriomeningitis virus (LCMV) infection, CD8 T cells rapidly expand and differentiate into effectors that are required for viral clearance. The accumulation of activated T cells is greatly reduced in mice lacking the adaptor molecule MyD88. Although MyD88 has generally been considered to indirectly regulate adaptive immune responses by controlling inflammatory cytokine production and Ag presentation in innate immune cells, in this study, we identify an unappreciated cell-intrinsic role for MyD88 in LCMV-specific CD8 T cells. Using reciprocal adoptive transfer models and bone marrow chimeras, we show that Myd88(-/-) CD8 T cells are defective in their clonal expansion in response to LCMV infection, independent of their environment. Furthermore, we show that while MyD88 is dispensable for initial activation and division of LCMV-specific CD8 T cells during the early stages of viral infection, MyD88-dependent signals are critical for supporting their survival and sustained accumulation.     

Abrogation of tolerance to a chronic viral infection

This study documents failure of peripheral tolerance mechanisms in a chronic viral infection and shows that T cell tolerance to a viral Ag seen as self from fetal life can be broken despite the presence of this Ag in extrathymic tissues. Congenital infection of mice with lymphocytic choriomeningitis virus (LCMV) results in T cell tolerance to the virus. Such mice become carriers for life harboring virus in many tissues including the thymus and exhibit no LCMV-specific CTL responses. Our previous studies have documented the curing of this congenitally acquired chronic infection after adoptive transfer of CD8+ T cells from LCMV-immune mice and the presence of host-derived, LCMV-specific CTL in these "cured" carriers. In this study we have examined the mechanism by which these carriers acquired T cell competence and show that these CTL differentiated from the bone marrow after elimination of viral Ag from the thymus. These results demonstrate that even when a chronic infection has been established in utero, the adult thymus retains the ability to restore immunocompetence to the host and to provide protection against reinfection. Surprisingly, these LCMV specific CTL were acquired at a time when infectious virus and intracellular viral Ag, although cleared from the thymus, were readily detectable in organs such as the kidney, testes, and brain. In fact, active viral replication in peripheral tissues was ongoing when these mice acquired new virus-specific T cells. These results show that clearance of virus form the thymus was sufficient to abrogate tolerance to a congenitally acquired chronic infection and that Ag in peripheral tissues did not tolerize newly developing T cells. These findings suggest that mechanisms that operate on immature cells within the thymus to silence self-reactive T cells are effective in induction of tolerance to viruses, but mechanisms of tolerizing mature T cells are likely to breakdown. This has implications for virus-induced autoimmunity and for treatment of chronic infections.     

Toll-like receptor 7 is required for effective adaptive immune responses that prevent persistent virus infection

TLR7 is an innate signaling receptor that recognizes single-stranded viral RNA and is activated by viruses that cause persistent infections. We show that TLR7 signaling dictates either clearance or establishment of life-long chronic infection by lymphocytic choriomeningitis virus (LCMV) Cl 13 but does not affect clearance of the acute LCMV Armstrong 53b strain. TLR7(-/-) mice infected with LCMV Cl 13 remained viremic throughout life from defects in the adaptive antiviral immune response-notably, diminished T?cell function, exacerbated T?cell exhaustion, decreased plasma cell maturation, and negligible antiviral antibody production. Adoptive transfer of TLR7(+/+) LCMV immune memory cells that enhanced clearance of persistent LCMV Cl 13 infection in TLR7(+/+) mice failed to purge LCMV Cl 13 infection in TLR7(-/-) mice, demonstrating that a TLR7-deficient environment renders antiviral responses ineffective. Therefore, methods that promote TLR7 signaling are promising treatment strategies for chronic viral infections.     

CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.     

Enhanced establishment of a virus carrier state in adult CD4+ T-cell-deficient mice.

CD4+ T cells play an important role in regulating the immune response; their contribution to virus clearance is variable. Mice that lack CD4+ T cells (CD4-/- mice) and are therefore unable to produce neutralizing antibodies cleared viscero-lymphotropic lymphocytic choriomeningitis virus (LCMV) strain WE when infected intravenously with a low dose (2 x 10(2) PFU) because of an effective CD8+ cytotoxic T-cell (CTL) response. In contrast, infection with a high dose (2 x 10(6) PFU) of LCMV strain WE led to expansion of antiviral CTL, which disappeared in CD4-/- mice; in contrast, CD4+ T-cell-competent mice developed antiviral memory CTL. This exhaustion of specific CTL caused viral persistence in CD4-/- mice, whereas CD4+ T-cell-competent mice eliminated the virus. After infection of CD4-/- mice with the faster-replicating LCMV strain DOCILE, abrogation of CTL response and establishment of viral persistence developed after infection with a low dose (5 x 10(2) PFU), i.e., an about 100-fold lower dose than in CD(4+)-competent control mice. These results show that absence of T help enhances establishment of an LCMV carrier state in selected situations.     

DNA vaccination against persistent viral infection.

This study shows that DNA vaccination can confer protection against a persistent viral infection by priming CD8+ cytotoxic T lymphocytes (CTL). Adult BALB/c (H-2d) mice were injected intramuscularly with a plasmid expressing the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) under the control of the cytomegalovirus promoter. The LCMV NP contains the immunodominant CTL epitope (amino acids 118 to 126) recognized by mice of the H-2d haplotype. After three injections with 200 micrograms of NP DNA, the vaccinated mice were challenged with LCMV variants (clones 13 and 28b) that establish persistent infection in naive adult mice. Fifty percent of the DNA-vaccinated mice were protected, as evidenced by decreased levels of infectious virus in the blood and tissues, eventual clearance of viral antigen from all organs tested, the presence of an enhanced LCMV-specific CD8+ CTL response, and maintenance of memory CTL after clearance of virus infection. However, it should be noted that protection was seen in only half of the vaccinated mice, and we were unable to directly measure virus-specific immune responses in any of the DNA-vaccinated mice prior to LCMV challenge. Thus, at least in the system that we have used, gene immunization was a suboptimal method of inducing protective immunity and was several orders of magnitude less efficient than vaccination with live virus. In conclusion, our results show that DNA immunization works against a persistent viral infection but that efforts should be directed towards improving this novel method of vaccination.     

Preapoptotic phenotype of viral epitope-specific CD8 T cells precludes memory development and is an intrinsic property of the epitope

Virus-specific CD8 T cells after clearance of infection reduce their number in lymphoid organs by apoptotic death and by migration into peripheral tissues. During and after infection, many lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells in lymphoid but not peripheral tissues are in a preapoptotic state, as detected by the early apoptosis marker annexin V. In this report, we investigated the significance of this preapoptotic state and how it may be influenced by viral epitope specificity. Stimulation with anti-CD3 or IL-2 in vitro postponed DNA fragmentation in annexin V+ cells, but adoptive transfer studies in vivo showed that this preapoptotic phenotype precluded the development of functional memory. CD8 T cells specific to LCMV epitopes NP396 and gp33 differed in their preapoptotic state, with NP396-specific T cells binding more annexin V than gp33-specific T cells. These epitope- and tissue-dependent differences were seen in primary, memory, and secondary responses and in mice receiving different displays of Ag by infection with LCMV strains of different tropisms or by infection with vaccinia virus recombinants expressing LCMV proteins. Thus, the epitope-dependent differences in apoptosis were independent of virus tropisms, duration of Ag exposure, and competition within APCs, and were an intrinsic property of the epitope. The tissue-dependent and epitope-dependent preapoptotic state correlated with reduced expression of IL-7Ralpha.     

Critical role for alpha/beta and gamma interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic T cells

Under conditions of high antigenic load during infection with invasive lymphocytic choriomeningitis virus (LCMV) strains, virus can persist by selective clonal exhaustion of antigen-specific CD8(+) T cells. In this work we studied the down-regulation of the virus-specific CD8(+)-T-cell response during a persistent infection of adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense. Studies were conducted by infecting mice deficient in receptors for type I (alpha/beta interferon [IFN-alpha/beta]), type II (IFN-gamma), and both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-cell exhaustion. The main conclusions of this study are as follows. (i) IFNs play a critical role in LCMV infection by reducing viral loads in the initial stages of infection and thus modifying both the extent of CD8(+)-T-cell exhaustion and the course of infection. The importance of IFNs in this context varies with the biological properties of the LCMV strain. (ii) An inverse correlation exists between antigen persistence and responsiveness of virus-specific CD8(+) T cells. This results in distinct programs of activation or tolerance (functional unresponsiveness and/or physical elimination of antigen-specific cells) during acute and chronic virus infections, respectively. (iii) A successful immune response associated with definitive viral clearance requires an appropriate balance between cellular and humoral components of the immune system. We discuss the role of IFNs in influencing virus-specific T cells that determine the outcome of persistent infections.     

Dissection of antiviral and immune regulatory functions of tumor necrosis factor receptors in a chronic lymphocytic choriomeningitis virus infection

The effector function of CD8 T cells is mediated via cell-mediated cytotoxicity and production of cytokines like gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). While the roles of perforin-dependent cytotoxicity, IFN-gamma, and TNF-alpha in controlling acute viral infections are well studied, their relative importance in defense against chronic viral infections is not well understood. Using mice deficient for TNF receptor (TNFR) I and/or II, we show that TNF-TNFR interactions have a dual role in mediating viral clearance and downregulating CD8 and CD4 T-cell responses during a chronic lymphocytic choriomeningitis virus (LCMV) infection. While wild-type (+/+) and TNFR II-deficient (p75(-/-)) mice cleared LCMV from the liver and lung, mice deficient in TNFR I (p55(-/-)) or both TNFR I and TNFR II (double knockout [DKO]) exhibited impaired viral clearance. The inability of p55(-/-) and DKO mice to clear LCMV was not a sequel to either suboptimal activation of virus-specific CD8 or CD4 T cells or impairment in trafficking of LCMV-specific CD8 T cells to the liver and lung. In fact, the expansion of LCMV-specific CD8 and CD4 T cells was significantly higher in DKO mice compared to that in +/+, p55(-/-), and p75(-/-) mice. TNFR deficiency did not preclude the physical deletion of CD8 T cells specific for nucleoprotein 396 to 404 but delayed the contraction of CD8 T-cell responses to the epitopes GP33-41 and GP276-285 in the viral glycoprotein. The antibody response to LCMV was not significantly altered by TNFR deficiency. Taken together, these findings have implications in development of immunotherapy in chronic viral infections of humans.     

Low-dose IL-2 therapy invigorates CD8+ T cells for viral control in systemic lupus erythematosus

Autoimmune diseases are often treated by glucocorticoids and immunosuppressive drugs that could increase the risk for infection, which in turn deteriorate disease and cause mortality. Low-dose IL-2 (Ld-IL2) therapy emerges as a new treatment for a wide range of autoimmune diseases. To examine its influence on infection, we retrospectively studied 665 patients with systemic lupus erythematosus (SLE) including about one third receiving Ld-IL2 therapy, where Ld-IL2 therapy was found beneficial in reducing the incidence of infections. In line with this clinical observation, IL-2 treatment accelerated viral clearance in mice infected with influenza A virus or lymphocytic choriomeningitis virus (LCMV). Noticeably, despite enhancing anti-viral immunity in LCMV infection, IL-2 treatment exacerbated CD8⁺ T cell-mediated immunopathology. In summary, Ld-IL2 therapy reduced the risk of infections in SLE patients and enhanced the control of viral infection, but caution should be taken to avoid potential CD8⁺ T cell-mediated immunopathology.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

GITR Intrinsically Sustains Early Type 1 and Late Follicular Helper CD4 T Cell Accumulation to Control a Chronic Viral Infection

CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR^+/+ and GITR^-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ⁺IL-2⁺ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.     

Virus-lymphocyte interaction: T cells of the helper subset are infected with lymphocytic choriomeningitis virus during persistent infection in vivo.

The lifelong persistence of lymphocytic choriomeningitis virus (LCMV) in neonatally or congenitally infected mice is accompanied by a suppression of virus-specific T-cell responses. In this study, we identified the subset of T cells infected with LCMV during persistent infection in vivo. Using specific monoclonal antibodies to separate the different lymphocyte cell populations and employing both an infectious center assay and immunofluorescence to detect the virus, we found that infection is confined primarily to T cells of the helper subset (L3T4+ Lyt2-), with minimal involvement of cytotoxic T cells (Lyt2+ L3T4-) and mature B cells. About 0.54 to 1.1% of L3T4+ T cells were producing the virus, as determined by the infectious center assay. In contrast, 9.1 to 12.2% of these L3T4+ T cells contained viral antigen, as shown by immunofluorescence studies. This finding suggested that, at any given time, a substantial number of infected T cells were not producing infectious virus. This infection of T helper cells may be involved in the suppression of LCMV-specific T-cell responses observed in persistently infected mice.     

CD8 T cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for clonal expansion

The role of type I IFN signaling in CD8 T cells was analyzed in an adoptive transfer model using P14 TCR transgenic CD8 T cells specific for lymphocytic choriomeningitis virus (LCMV) but deficient in type I IFNR. In the present study, we demonstrate severe impairment in the capacity of P14 T cells lacking type I IFNR to expand in normal type I IFNR wild-type C57BL/6 hosts after LCMV infection. In contrast, following infection of recipient mice with recombinant vaccinia virus expressing LCMV glycoprotein, P14 T cell expansion was considerably less dependent on type I IFNR expression. Lack of type I IFNR expression by P14 T cells did not affect cell division after LCMV infection but interfered with clonal expansion. Thus, direct type I IFN signaling is essential for CD8 T cell survival in certain viral infections.     

Requirement for neutralizing antibodies to control bone marrow transplantation-associated persistent viral infection and to reduce immunopathology

Bone marrow transplantation (BMT) is commonly used in the treatment of leukemia, however its therapeutic application is partly limited by the high incidence of associated opportunistic infections. We modeled this clinical situation by infecting mice that underwent BMT with lymphocytic choriomeningitis virus (LCMV) and investigated the potential of immunotherapeutic strategies to counter such infections. All mice that received BMT survived LCMV infection and developed a virus carrier status. Immunotherapy by adoptive transfer of naive splenocytes protected against low (200 PFU), but not high (2 x 10(6) PFU), doses of LCMV. Attempts to control infection of high viral titers using strongly elevated frequencies of activated LCMV-specific T cells failed to control virus and resulted in immunopathology and death. In contrast, virus neutralizing Abs combined with naive splenocytes were able to efficiently control high-dose LCMV infection without associated side effects. Thus, cell transfer combined with neutralizing Abs represented the most effective means of controlling BMT-associated opportunistic viral infection in our in vivo model. These data underscore the in vivo efficacy and immunopathological "safety" of neutralizing antibodies.     

Long-Term CD4 Th1 and Th2 Memory following Acute Lymphocytic Choriomeningitis Virus Infection

CD4 T cells play a central role in viral immunity. They provide help for B cells and CD8 T cells and can act as effectors themselves. Despite their importance, relatively little is known about the magnitude and duration of virus-specific CD4 T-cell responses. In particular, it is not known whether both CD4 Th1 memory and CD4 Th2 memory can be induced by viral infections. To address these issues, we quantitated virus-specific CD4 Th1 (interleukin 2 [IL-2] and gamma-interferon) and Th2 (IL-4) responses in mice acutely infected with lymphocytic choriomeningitis virus (LCMV). Using two sensitive assays (enzyme-linked immunospot assay and intracellular stain) to measure cytokine production at the single-cell level, we found that both CD4 Th1 and Th2 responses were induced during primary LCMV infection. At the peak (day 8) of the response, the frequency of LCMV-specific CD4 Th1 cells was 1/35 to 1/160 CD4 T cells, and the frequency of Th2 cells was 1/400. After viral clearance, the numbers of virus-specific CD4 T cells dropped to 1/260 to 1/3,700 and then were maintained at this level indefinitely. Upon rechallenge with LCMV, both CD4 Th1 and Th2 memory cells made an anamnestic response in vivo. These results show that unlike some microbial infections in which only Th1 or Th2 responses are seen, an acute viral infection can induce a mixed CD4 T-cell response with long-term memory.     

Vaccination against persistent viral infection exacerbates CD4+ T-cell-mediated immunopathological disease.

Lymphocytic choriomeningitis virus (LCMV) infection of normal mice results in a fatal immunopathologic meningitis mediated by CD8+ cytotoxic T lymphocytes (CTL). We have previously shown that female beta2-microglobulin-deficient (beta2m-/-) mice, which are also deficient in CD8+ T cells, are susceptible to LCMV-induced immune-mediated meningitis, characterized by significant weight loss and mortality. This LCMV disease in beta2m-/- mice is mediated by CD4+ T lymphocytes. Our previous studies have also demonstrated that male beta2m-/- mice are less susceptible than female beta2m-/- mice to LCMV-induced, immune-mediated mortality and weight loss. In this report, we show that vaccination of male beta2m-/- mice enhances immunopathology following intracranial infection with LCMV. We observed increased production of gamma interferon (IFN-gamma), an increase in CD4+ CTL precursor frequency, and an increased frequency of IFN-gamma-producing cells from spleen cells of vaccinated male beta2m-/- mice. Vaccinated male beta2m-/- mice also had significantly increased inflammation in the cerebrospinal fluid (CSF), characterized by a large CD4+ T-cell infiltrate. CSF cells from vaccinated mice showed increased production of IFN-gamma on day 7 postchallenge. Neither vaccinated nor control beta2m-/- mice were able to clear virus, and the two groups had similarly high levels of virus early after infection. These results suggest that the magnitude of the early immune response is more important than the level of virus in the brain in determining the outcome of immunopathology in beta2m-/- mice. We show here that vaccination can increase CD4+ T-cell-dependent immunopathology to a persistent viral infection.