Lethal Lymphocytic Choriomeningitis Research Articles

ID: 51: Functional exhaustion of the immune response protects IRF9-deficient mice from lethal lymphocytic choriomeningitis

The interferon regulatory factor 9 (IRF9) is a key component of type I interferon signalling and critical for the host anti-viral response. While wild-type mice die following intracranial infection with lymphocytic choriomeningitis virus (LCMV), IRF9-deficient mice survive and develop a persistent infection and chronic encephalitis. Here we studied whether functional exhaustion of the T-cell response contributes to survival and virus persistence in IRF9-deficient mice. We found that CD8+ T-cells from LCMV-infected IRF9-deficient mice show upregulation of PD-1, indicative of functional exhaustion. In contrast to IRF9-deficient mice, mice lacking both IRF9 and PD-L1, the primary ligand for PD-1, developed a lethal wasting disease following LCMV infection. Surprisingly, expression of key proinflammatory cytokines and T-cell infiltrates in the CNS and peripheral organs were more pronounced in infected IRF9-deficient mice than in the double-deficient mice. Further, neutralisation of PD-L1 during the subacute and chronic stages of LCMV infection in IRF9-deficient mice increased cytokine serum levels and decreased viral RNA levels. Importantly, neutralisation of PD-L1 had no impact on the clinical symptoms or body weight. These findings suggest that PD-L1 dependent T-cell exhaustion is crucial for the survival of LCMV-infected IRF9-deficient mice. Furthermore neutralisation of PD-L1 results in a ‘controlled’ reactivation of the immune response.

80: Loss of IRF9 prevents lethal lymphocytic choriomeningitis virus infection in mice at the cost of viral persistence and chronic inflammation in the CNS

80: Loss of IRF9 prevents lethal lymphocytic choriomeningitis virus infection in mice at the cost of viral persistence and chronic inflammation in the CNS

PS1-113 Loss of IRF9 prevents lethal lymphocytic choriomeningitis virus infection in mice at the cost of viral persistence and chronic inflammation in the CNS

Functional type I interferon (IFN-I) signalling is critical for the host response to viruses. Cellular responses IFN-Is depend largely on STAT1, STAT2 and IRF9. Here we studied the effects of IRF9-deficiency on the host response to a viral infection in the CNS. Wild-type (WT) mice and mice lacking IRF9 (IRF9 KO) were infected intracranially with lymphocytic choriomeningitis virus (LCMV). In WT mice, lethal disease occurred by day 7 with characteristic cerebral seizures and LCMV being largely confined to the CNS. In contrast, LCMV-infection of IRF9 KO mice caused a transient non-fatal disease and virus spread to peripheral organs. Viral RNA levels decreased slowly over time and became undetectable in some peripheral organs such as liver and spleen but remained detectable in the CNS for more than 150 days. In the CNS, sites of viral infection were associated with foci of activated microglia/ macrophages and moderate T-cell infiltrates. The persistent infection in the CNS and peripheral organs was paralleled by significantly increased levels of various cytokine mRNAs, including IFN-g and TNF, as well as of the co-inhibitory molecule B7-H1 (or PD-L1). In conclusion, these findings indicate that the absence of IRF9 prevents lethal LCM but results in persistent infection and chronic inflammation in the CNS. The presence of T cells and the slow decrease in viral RNA levels in the CNS and peripheral organs argue for a retarded rather than an exhausted or incapacitated anti-viral response.

A highly optimized DNA vaccine confers complete protective immunity against high-dose lethal lymphocytic choriomeningitis virus challenge

Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while ‘first-generation’ DNA vaccines were poorly immunogenic, new genetic ‘optimization’ strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency. We developed a highly optimized plasmid DNA vaccine that expresses the lymphocytic choriomeningitis virus (LCMV) nucleocapsid protein (NP) and evaluated it using the LCMV challenge model, a gold standard for studying infection and immunity. When administered intramuscularly with EP, robust NP-specific cellular and humoral immune responses were elicited, the magnitudes of which approached those following acute LCMV infection. Furthermore, these responses were capable of providing 100% protection against a high-dose, normally lethal virus challenge. This is the first non-infectious vaccine conferring complete protective immunity up to 8 weeks after vaccination and demonstrates the potential of ‘next-generation’ DNA vaccines.

Essential Role for TLR9 in Prime but Not Prime-Boost Plasmid DNA Vaccination To Activate Dendritic Cells and Protect from Lethal Viral Infection

One of the requirements for efficient vaccination against infection is to achieve the best combination of an adequate adjuvant with the antigenic information to deliver. Although plasmid DNA is a promising tool bearing the unique potential to activate humoral and cellular immunity, an actual challenge is to increase plasmid immunogenicity in human vaccination protocols in which efficacy has proven rather limited. Previous work showed that the bacterial DNA backbone of the plasmid has potent adjuvant properties because it contains CpG motifs that are particular activating nucleotidic sequences. Among TLRs, which are key sensors of microbial products, TLR9 can detect CpG motifs and confer activation of APCs, such as dendritic cells. However, whether the immunogenic properties of plasmid DNA involve TLR9 signaling has not been clearly established. In the current study, we demonstrate that TLR9 determines the effectiveness of vaccination against lethal lymphocytic choriomeningitis virus infection using plasmid DNA in a prime, but not prime-boost, vaccination regimen. Furthermore, we provide evidence that the presence of TLR9 in dendritic cells is necessary for effective and functional priming of virus-specific CD8+ T cells upon plasmid exposure in vitro or single-dose vaccination in vivo. Therefore, at single or low vaccine doses that are often used in human-vaccination protocols, CpG/TLR9 interactions participate in the immunogenicity of plasmid DNA. These results suggest that the TLR9 signaling pathway is involved in the efficacy of plasmid vaccination; therefore, it should remain a focus in the development or amelioration of vaccines to treat infections in humans.

55 Lethal Lymphocytic Choriomeningitis Virus (LCMV) Infection in STAT1 KO Mice is Associated with Dysregulated PD1/B7-H1 and A Phenotypic Switch in the T-cell Response

55 Lethal Lymphocytic Choriomeningitis Virus (LCMV) Infection in STAT1 KO Mice is Associated with Dysregulated PD1/B7-H1 and A Phenotypic Switch in the T-cell Response

HLA-A2-restricted protection against lethal lymphocytic choriomeningitis.

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8(+) T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides-nucleoprotein NP(69-77), glycoprotein precursor GPC(10-18), GPC(447-455), and zinc-binding protein Z(49-58)-that displayed high-affinity binding (< or =275 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC(447-455)), after peptide immunization of HLA-A*0201 mice, induced CD8(+) T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.

Induction of protective cytotoxic T cell responses in the presence of high titers of virus-neutralizing antibodies: implications for passive and active immunization.

The effect of preexistent virus-neutralizing antibodies on the active induction of antiviral T cell responses was studied in two model infections in mice. Against the noncytopathic lymphocytic choriomeningitis virus (LCMV), pretreatment with neutralizing antibodies conferred immediate protection against systemic virus spread and controlled the virus below detectable levels. However, presence of protective antibody serum titers did not impair induction of antiviral cytotoxic T lymphocyte (CTL) responses after infection with 10(2) PFU of LCMV. These CTLs efficiently protected mice independent of antibodies against challenge with LCMV-glycoprotein recombinant vaccinia virus; they also protected against otherwise lethal lymphocytic choriomeningitis caused by intracerebral challenge with LCMV-WE, whereas transfused antibodies alone did not protect, and in some cases even enhanced, lethal lymphocytic choriomeningitis. Against the cytopathic vesicular stomatitis virus (VSV), specific CTLs and Th cells were induced in the presence of high titers of VSV-neutralizing antibodies after infection with 10(6) PFU of VSV, but not at lower virus doses. Taken together, preexistent protective antibody titers controlled infection but did not impair induction of protective T cell immunity. This is particularly relevant for noncytopathic virus infections since both virus-neutralizing antibodies and CTLs are essential for continuous virus control. Therefore, to vaccinate against such viruses parallel or sequential passive and active immunization may be a suitable vaccination strategy to combine advantages of both virus-neutralizing antibodies and CTLs.

Recombinant parvovirus-like particles as an antigen carrier: a novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells.

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8(+) T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8(+) class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells.

Fas-dependent CD4+ cytotoxic T-cell-mediated pathogenesis during virus infection.

beta 2-Microglobulin-deficient (beta 2m-) mice generate a CD4+ major histocompatibility complex class II-restricted cytotoxic T-lymphocyte (CTL) response following infection with lymphocytic choriomeningitis (LCM) virus (LCMV). We have determined the cytotoxic mechanism used by these CD4+ CTLs and have examined the role of this cytotoxic activity in pathogenesis of LCM disease in beta 2m- mice. Lysis of LCMV-infected target cells by CTLs from beta 2m- mice is inhibited by addition of soluble Fas-Ig fusion proteins or by pretreatment of the CTLs with the protein synthesis inhibitor emetine. In addition, LCMV-infected cell lines that are resistant to anti-Fas-induced apoptosis are refractory to lysis by these virus-specific CD4+ CTLs. These data indicate that LCMV-specific CD4+ CTLs from beta 2m- mice use a Fas-dependent lytic mechanism. Intracranial (i.c.) infection of beta 2m- mice with LCMV results in loss of body weight. Fas-deficient beta 2m- Jpr mice develop a similar wasting disease following i.c. infection. This suggests that Fas-dependent cytotoxicity is not required for LCMV-induced weight loss. A potential mediator of this chronic wasting disease is tumor necrosis factor (TNF)-alpha, which is produced by LCMV-specific CD4+ CTLs. In contrast to LCMV-induced weight loss, lethal LCM disease in beta 2m- mice is dependent on Fas-mediated cytotoxicity. Transfer of immune splenocytes from LCMV-infected beta 2m- mice into irradiated infected beta 2m- mice results in death of recipient animals. In contrast, transfer of these splenocytes into irradiated infected beta 2m- Jpr mice does not cause death. Thus a role for CD4+ T-cell-mediated cytotoxicity in virus-induced immunopathology has now been demonstrated.

Immunity to viruses in B cell-deficient mice: influence of antibodies on virus persistence and on T cell memory.

Mice rendered B cell deficient by targeted disruption of the immunoglobulin mu chain gene (IgM-/- mice) were used to analyze the role of antibodies and B cells in viral infections; homozygous IgM-/- mice were bred in a way to avoid transmission of maternal antibodies. After infection with vesicular stomatitis virus (VSV), IgM-/- mice developed paralytic disease and subsequently died, whereas C57BL/6 control mice or IgM-/- mice passively protected with VSV-neutralizing antibodies survived. Furthermore, IgM-/- mice showed increased natural killer (NK) activity upon exposure to either lymphocytic choriomeningitis virus (LCMV) or to poly(I).poly(C), while NK activity in untreated IgM-/- mice was within normal ranges. Cytotoxic T cell responses were comparable in IgM-/- and control mice infected either with VSV or with vaccinia virus or with low doses of LCMV (10(2) infectious focus-forming units [ifu]). After intracerebral infection with LCMV-Armstrong, CD8+ T cell-mediated lethal lymphocytic choriomeningitis developed independently of the presence of B cells and antibodies. After infection with high doses (2 x 10(6) - 5 x 10(6) ifu) of LCMV-WE or LCMV-Docile, IgM-/- mice exhibited a reduced capacity to control these primary infections and had elevated virus titers for prolonged times (> 60 days). Nevertheless, the cytotoxic T cell response against LCMV in the early phase of infection was comparable in IgM-/- and control mice, but disappeared in those IgM-/- mice which had a persistent viral infection. Cytotoxic T cell memory was apparently unimpaired in low-dose-primed IgM-/- mice, which were able to control the primary virus infection; both IgM-/- and control mice cleared a high intravenous dose of virus within 2 days after challenge infection. This indicates that an efficient T cell memory against LCMV was established in the absence of B cells.

Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus.

Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA immunization confers protection against lethal lymphocytic choriomeningitis virus infection

DNA vaccination has been evaluated with the lymphocytic choriomeningitis virus (LCMV) model system. Plasmid DNA encoding the LCMV nucleoprotein, when injected intramuscularly, induces both antiviral antibodies and cytotoxic T lymphocytes. Injection of DNA encoding the nucleoprotein or the viral glycoprotein confers protection against normally lethal LCMV challenge in a major histocompatibility complex-dependent manner. The protection conferred is incomplete, but it is most probably mediated by the induced cytotoxic T lymphocytes.

Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes.

The reverse genetics system has made it possible to modify the influenza virus genome. By this method, we were able to assess influenza virus as a vaccine vector for protecting BALB/c mice against otherwise lethal lymphocytic choriomeningitis virus (LCMV) infection. A single dose of influenza virus [A/WSN/33 (H1N1)] bearing a cytotoxic T-lymphocyte-specific epitope of the LCMV nucleoprotein (residues 116 to 127) in the neuraminidase stalk protected mice against LCMV challenge for at least 4 months. The immunity was mediated by cytotoxic T lymphocytes and was haplotype specific, indicating that the observed protective response was solely a consequence of prior priming with the H-2d LCMV nucleoprotein epitope expressed in the recombinant influenza virus. We also found that as many as 58 amino acids could be inserted into the neuraminidase stalk without loss of viral function. These findings demonstrate the potential of influenza virus as a vaccine vector, with the neuraminidase stalk as a repository for foreign epitopes.

Production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of interleukin-6 (IL-6) but not IL-4, IL-5, and gamma interferon

The activities of cytokines were determined in cerebrospinal fluid (CSF) and serum of mice persistently or intracerebrally acutely infected with lymphocytic choriomeningitis (LCM) virus (LCMV). In contrast to CBA/J (LCMV carrier) mice that responded with low levels of LCMV-specific antibody, high-responder NMRI (carrier) mice showed antibody production by B cells outside of lymphoid organs. The B cells that had infiltrated the brains of LCMV carrier mice exhibited no preferential immunoglobulin isotype or subtype virus-specific antibody production. Phenotypic analysis of the brain infiltrates in virus carrier mice revealed dominance of CD4+ T cells in contrast to virtual absence of CD4+ and dominance of CD8+ in mice with acute LCM. In NMRI but not in CBA/J carrier mice, significant concentrations of interleukin-6 (IL-6) were detected in CSF and serum; IL-2, IL-4, IL-5, granulocyte-macrophage CSF (GM-CSF), and gamma interferon (IFN-gamma) were not elevated. In contrast, during acute, lethal LCM, IL-6 and IFN-gamma were found at high concentrations, and IL-4, IL-5, and GM-CSF were detectable in CSF and serum, but virus-specific antibody-producing cells were not (yet) detectable in the brain. Thus, distinct cytokine patterns are found in acute versus chronic LCMV infection of the brain: in LCM carrier mice, local random-class immunoglobulin production correlated with the absence of IL-2, IL-4, IL-5, and IFN-gamma but active secretion of IL-6.

Treatment with anti-tumor necrosis factor α does not influence the immune pathological response against lymphocytic choriomeningitis virus

The role of tumor necrosis factor alpha (TNF alpha) in the immunopathological events induced by infection with lymphocytic choriomeningitis (LCM) virus (LCMV) was assessed by treatment of C57Bl/6 mice with a sheep antibody to murine TNF alpha antiserum to strongly interfere with anti-Listeria host defense. However, despite its effectiveness in Listeria infections in vivo, antibody to TNF alpha used at 6 x 10(4) neutralizing units per day subcutaneously had no detectable influence on the kinetics of maturation of antiviral cytotoxic T-cell activity, inflammatory processes, or clearance of virus. First, onset and severity of LCMV-induced hepatitis, as assessed by cytotoxic T-cell activity, viral titers in the liver, serum liver enzyme values, and histology, were not detectably affected by antibody to TNF alpha. Second, incidence of lethal LCM disease after intracerebral infection and the kinetics of the primary footpad swelling reaction observed after local foot inoculation were not altered by anti-TNF alpha antibody treatment. From the data presented we conclude that TNF alpha as assayed by in vivo therapy with a polyclonal anti-TNF alpha antibody plays no detectable role in the host reaction against LCMV.

Effects of treatment with IL-2 receptor specific monoclonal antibody in mice. Inhibition of cytotoxic T cell responses but not of T help.

Contribution of IL-2R-bearing activated lymphocytes to antiviral host defense was investigated in C57BL/6 mice by treatment in vivo with IL-2R-specific mAb PC61. When treated on days 0 and 1 with respect to infection with either vaccinia virus, lymphocytic choriomeningitis (LCM) virus (LCMV) or vesicular stomatitis virus, 6-day immune mice had low numbers of CD8+ T cells that were reduced to about 10% of the values found for infected but otherwise untreated controls. In contrast, the number of CD4+ T cells was within normal ranges. Correspondingly, induction of strictly T help-dependent antiviral neutralizing IgG antibody titers remained unaffected by the mAb treatment, whereas generation of antiviral cytotoxic T cell activity was abrogated. Anti-IL-2R treatment of thymectomized mice 14 and 15 days after infection prevented generation of secondary antiviral cytotoxic T cells in restimulation cultures in vitro initiated 24 days later. Treatment with IL-2R-specific mAb was comparable to treatment with CD8-specific mAb in preventing mice to eliminate virus. Because of the involvement of antiviral cytotoxic T cells in disease manifestations, treatment with IL-2R-specific mAb protected mice from lethal LCM after intracerebral infection with LCMV and inhibited the footpad swelling reaction caused by local infection with the same virus.

Enhanced virus replication and inhibition of lymphocytic choriomeningitis virus disease in anti-gamma interferon-treated mice

The role of gamma interferon (IFN-gamma) induced during a viral infection in the ability of the host to acquire antiviral immunity was studied in mice. They were injected subcutaneously daily with an ammonium sulfate-precipitated sheep anti-IFN-gamma antibody preparation able to neutralize 10(4) U of IFN-gamma. Specificity of the anti-IFN-gamma antiserum was demonstrated by absence of detectable activity against natural IFN-alpha and -beta. Controls were treated with a similarly prepared normal sheep serum. Treatment with the IFN-gamma-specific antibody preparation had no influence on the ability of mice to generate anti-vaccinia virus- or anti-vesicular stomatitis virus (VSV)-specific cytotoxic T-cell (CTL) responses or T helper-dependent immunoglobulin G responses to VSV. In contrast, treatment of mice with sheep anti-IFN-gamma impaired CTL responses against lymphocytic choriomeningitis (LCM) virus (LCMV, Aggressive isolate); in addition, under the experimental conditions used, it prevented lethal LCM. Cytotoxic T-cell activity measured in the spleens of anti-IFN-gamma-treated mice was comparable to that found in mice initially infected with a 100-fold-larger dose of LCMV. Evaluation of the effects of treatment on the kinetics of virus replication revealed that in both euthymic and athymic nude C57BL/6 mice, anti-IFN-gamma treatment led to an increase of virus titers up to 100-fold compared with control mice. Therefore, IFN-gamma may play a role in controlling viruses with tropism for lymphocytes and monocytes/macrophages, such as LCMV.