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.     

Cross-protection against lymphocytic choriomeningitis virus mediated by a CD4+ T-cell clone specific for an envelope glycoprotein epitope of Lassa virus.

Recombinant vaccinia virus expressing the Lassa virus (LV) envelope glycoprotein precursor, V-LSGPC, was used to study the basis of LV-induced cross-protective immunity against the closely related arenavirus lymphocytic choriomeningitis virus (LCMV). C3H/HeJ mice primed with V-LSGPC developed neither circulating antibodies nor CD8+ cytotoxic T cells specific for LCMV, yet they resisted a normally lethal LCMV challenge. Spleen cells from such mice gave a proliferative response to LCMV in vitro that was inhibitable by anti-CD4 antibody. Synthetic peptides corresponding to predicted T-cell sites common to the envelope glycoprotein precursor (GP-C) of LV and that of LCMV were used to map the specificity of the proliferative response to an epitope located between amino acids 403 and 417 of LV GP-C. Several CD4+ T-cell clones specific for the 403-417 peptide were isolated and found to produce gamma interferon in response to both the peptide and LCMV. One of these clones, C9, was selected for further study. C9 lysed I-AK-bearing target cells, and when adoptively transferred to C3H/HeJ mice, it was capable of mediating both a peptide-specific delayed hypersensitivity reaction and resistance to lethal LCMV challenge. These collective findings demonstrate, for the first time, that CD4+ T cells can play a major role in arenavirus-specific cross-protective immunity.     

Direct ex vivo kinetic and phenotypic analyses of CD8(+) T-cell responses induced by DNA immunization

CD8(+) T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8(+) T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8(+) T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8(+) T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8(+) T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8(+) T-cell response.     

Theiler's virus and Mengo virus induce cross-reactive cytotoxic T lymphocytes restricted to the same immunodominant VP2 epitope in C57BL/6 mice.

C57BL/6 mice develop a virus-specific cytotoxic T-lymphocyte (CTL) response after intraperitoneal inoculation with either the DA strain of Theiler's virus or Mengo virus, two members of the Cardiovirus genus. These CTLs contribute to viral clearance in the case of Theiler's virus but do not protect the mice from the fatal encephalomyelitis caused by Mengo virus. In this study we show that DA and Mengo virus-induced CTLs are cross-reactive. The cross-reactivity is due to a conserved, H-2Db-restricted epitope located between amino acid residues 122 and 130 of the VP2 capsid protein (VP2(122-130)). This epitope is immunodominant in C57BL/6 mice infected with Theiler's virus. The VP2(122-130) epitope, initially identified for Mengo virus, is the first CTL epitope described for Theiler's virus.     

Further studies on the relative antiviral efficacies of interferons induced by poly I:C and Mengo virus.

Mouse serum interferons induced by polyI:C, vesicular stomatitis virus (VSV), reovirus, and Mengo virus were assayed in monolayers of mouse L-929 cells by the plaque-reduction method using both VSV and Mengo as challenge viruses. Titers obtained with Mengo virus as challenge were all lower than with VSV. With the interferons induced by VSV, reovirus, and ployI:C, the reductions were of the order of two- to three-fold. With Mengo virus-induced interferon the reduction was much greater (about 17-fold). This offers an explanation for the observation that, unit for unit (measured by the plaque reduction of VSV), Mengo virus-induced interferon is only about 1/10 as effective as polyI:C-induced interferon in protecting mice against lethal infection with Mengo virus. The data are consistent with the hypothesis that an interferon antagonist is produced in the serum of mice infected with Mengo virus. This antagonist, which is not produced in mice inoculated with polyI:C, or reovirus, effectively blocks the antiviral action of interferon during Mengo virus infections, both in vivo and in vitro.     

Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4.

Antiviral immune responses of mice lacking interleukin-2 (IL-2) or IL-4 or both IL-2 and IL-4 (IL-2/4) were compared by using different viruses. Primary cytotoxic T-lymphocyte (CTL) responses against lymphocytic choriomeningitis virus (LCMV) were only moderately reduced in mice lacking IL-2 and were normal in mice lacking IL-4. Mice deficient in both interleukins exhibited variable and more strongly reduced but nevertheless in vivo protective LCMV-specific CTL responses. Similar results were obtained with vaccinia virus. Upon virus-specific restimulation in vitro, spleen cells from IL-2- and IL-2/4-deficient mice failed to generate CTL responses against virus-infected target cells, whereas the response of mice deficient in only IL-4 was comparable to that of control mice. The addition of IL-2 during in vitro restimulation completely restored the responses of both IL-2 and IL-2/4-deficient mice. T-helper-cell-independent immunoglobulin M and T-helper-cell-dependent immunoglobulin G antibody responses against vesicular stomatitis virus glycoprotein were within normal ranges for the various mutant mice. After LCMV infection, specific antibody responses against LCMV nucleoprotein were reduced four- to eightfold. These results show that mice lacking IL-2/4 have an overall tendency to exhibit more severely reduced CTL responses than IL-2- or IL-4-deficient mice. Nevertheless, and surprisingly, in vivo protective immune responses were mounted in the absence of IL-2/4, suggesting that besides a minor contribution from IL-4, other interleukins compensate in vivo for the lack of IL-2 in IL-2-deficient mice.     

Functional analysis of T lymphocyte subsets in antiviral host defense

The role of different T cell subsets in antiviral host defense was investigated by treating thymectomized C57BL/6 and CBA/J mice with monoclonal rat anti-Lyt-2 or anti-L3/T4 IgG 2b antibodies 14 and 10 days before infection. This treatment depleted the respective T cell subsets to undetectable levels in peripheral blood when assayed by immunofluorescence. In mice treated with anti-Lyt-2, induction of cytotoxic T cells was reduced to less than 1 to 2% after intravenous infection with Armstrong strain of lymphocytic choriomeningitis virus (LCMV). In addition, no primary swelling of the footpad could be detected following local inoculation of the virus. In animals treated with anti-L3/T4, antiviral cytotoxic T lymphocyte responses were reduced by a factor of 10. These L3/T4+ cell-depleted mice showed delayed footpad swelling after local injection of LCMV Armstrong. After intracerebral infection with LCMV, anti-Lyt-2-treated mice were resistant and those injected with anti-L3/T4 were totally susceptible to LCMV Armstrong-triggered immunopathologic disease. Virus could be detected in the blood of antibody-treated mice 7 days after inoculation; however, no virus could be measured in the blood of surviving anti-Lyt-2-treated animals 15 days after intracerebral infection. Serum titers of interferon-alpha,beta induced by viral infection remained unaffected by depletion of T cell subsets. Anti-L3/T4 antibody-treated C57BL/6 mice failed to generate IgG antibodies against the New Jersey strain of vesicular stomatitis virus, whereas Lyt-2+ cell-depleted mice had normal antivesicular stomatitis virus (New Jersey strain) IgG antibody titers.     

The S RNA segment of lymphocytic choriomeningitis virus codes for the nucleoprotein and glycoproteins 1 and 2.

The lymphocytic choriomeningitis virus (LCMV) genome consists of a large RNA segment and a small RNA segment. The three major structural proteins of this virus are an internal nucleoprotein and two surface glycoproteins. Intertypic reassortants between the Armstrong and WE strains of LCMV were made to map proteins encoded by the LCMV genome segments. Using monoclonal antibodies specific for the nucleoprotein and the glycoproteins of WE and Armstrong, we showed that the small RNA segment of LCMV codes for the three major structural polypeptides.     

Vaccination with a synthetic peptide modulates lymphocytic choriomeningitis virus-mediated immunopathology.

Vaccination with a nucleopeptide (NP 118; amino acids 118 to 132) representing a cytotoxic T-cell epitope of lymphocytic choriomeningitis virus (LCMV) can modulate immunopathology. Immunization with NP 118 protected H-2d mice against intracerebral infection with the LCMV-ARMSTRONG isolate. However, when NP 118-primed H-2d mice were challenged intracerebrally with an intermediate dose (5 x 10(4) PFU) of the LCMV-DOCILE strain, all mice primed with NP 118 emulsified in incomplete Freund's adjuvant died, whereas unprimed mice survived. Correspondingly, peptide vaccination enhanced specifically the cytotoxic T-cell response, influencing the critical balance between T-cell response and virus spread.     

CD8(+) T cells secreting type 2 lymphokines are defective in protection against viral infection

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8(+) T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-gamma secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules.     

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.     

Inhibition of Mengo virus by interferon

Gauntt, Charles J. (The University of Texas, Austin), and Royce Z. Lockart, Jr. Inhibition of Mengo virus by interferon. J. Bacteriol. 91:176-182. 1966.-The inhibition of Mengo virus replication in L cells resulting from interferon was studied quantitatively. Interferon was titrated on L cells with Western equine encephalomyelitis (WEE) virus as the challenge virus. One protective unit (PU) of interferon is the least amount of interferon which prevents cytopathic effects when a large multiplicity of WEE virus is added subsequent to overnight incubation with interferon. Ten PU of interferon reduced the yields of Mengo virus by about 90%. Larger doses of interferon, up to 220 PU, caused no further reduction in the amount of virus produced. Plaque formation by Mengo virus was also reduced in number by about 85 to 90%, but could not be further reduced. The plaques which formed on interferon-treated cells were reduced in size. We were unable to obtain a virus population with increased resistance to interferon action by use of five successive growth cycles in interferon-treated cultures. Analysis of the cell population for the proportion of cells able to act as infectious centers revealed that incubation of cells with 10 PU of interferon decreased the proportion of virus-yielding cells by 80%. The yield of virus per virus-producing cell was decreased by 40 to 60%. Despite the reduction in yields, plaques, and infectious centers resulting from interferon, all doses of interferon failed to prevent the complete destruction of the cells. Experiments with puromycin indicated that the cytopathic effects observed in L cells infected with Mengo virus required that a virus-directed protein be synthesized between 4 and 5 hr postinfection. The evidence suggested, therefore, that the Mengo virus genome was able to code for new protein synthesis in the absence of the production of infectious 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.     

CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge.

Although primary antiviral CD8+ cytotoxic T lymphocytes (CTL) can be induced in mice depleted of CD4+ T cells, the role of CD4+ T lymphocytes in the generation and maintenance of antiviral memory CTL is uncertain. This question, and the consequences upon vaccine-mediated protection, were investigated in transgenic CD4 knockout (CD4ko) mice, which lack CD4+ T lymphocytes. Infection of immunocompetent C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV), or with recombinant vaccinia viruses bearing appropriate LCMV sequences, induces long-lasting protective immunity, mediated mainly by antiviral CD8+ CTL. Here we report two important findings. First, LCMV-specific CD8+ memory CTL are maintained at considerably lower levels in CD4ko mice than in normal C57BL/6J mice; we demonstrate a reduction in precursor CTL evident as soon as 30 days postimmunization and declining, by day 120, to levels 1 to 2 log units below those in normal mice. Thus, CD4+ T cells appear to be important to the generation and maintenance of their CD8+ counterparts. Second, this reduction has an important biological consequence; compared with immunocompetent mice, CD4ko mice immunized with vaccinia virus recombinants expressing nucleoprotein or glycoprotein of LCMV are less effectively protected from subsequent LCMV challenge. Thus, this study underscores the potential importance of CD4+ T lymphocytes in generation of appropriate levels of CD(8+)-cell-mediated immunoprotective memory and has implications for vaccine efficacy in individuals with immune defects in which CD4 levels may be reduced, such as AIDS.     

Effects of promyelocytic leukemia protein on virus-host balance

The cellular promyelocytic leukemia protein (PML) associates with the proteins of several viruses and in some cases reduces viral propagation in cell culture. To examine the role of PML in vivo, we compared immune responses and virus loads of PML-deficient and control mice infected with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). PML(-/-) mice exhibited accelerated primary footpad swelling reactions to very-low-dose LCMV, higher swelling peaks upon high-dose inoculation, and higher viral loads in the early phase of systemic LCMV infection. T-cell-mediated hepatitis and consequent mortality upon infection with a hepatotropic LCMV strain required 10- to 100-times-lower inocula despite normal cytotoxic T-lymphocyte reactivity in PML(-/-) mice. Furthermore, PML deficiency rendered mice 10 times more susceptible to lethal immunopathology upon intracerebral LCMV inoculation. Accordingly, 10-times-lower VSV inocula elicited specific neutralizing-antibody responses, a replication-based effect not observed with inactivated virus or after immunization with recombinant VSV glycoprotein. These in vivo observations corroborated our results showing more virus production in PML(-/-) fibroblasts. Thus, PML is a contributor to innate immunity, defining host susceptibility to viral infections and to immunopathology.     

Use of single-cycle infectious lymphocytic choriomeningitis virus to study hemorrhagic fever arenaviruses

Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus in West Africa and Argentina, respectively, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. The investigation of antiviral strategies to combat HF arenaviruses is hampered by the requirement of biosafety level 4 (BSL-4) facilities to work with these viruses. These biosafety hurdles could be overcome by the use of recombinant single-cycle infectious arenaviruses. To explore this concept, we have developed a recombinant lymphocytic choriomeningitis virus (LCMV) (rLCMVΔGP/GFP) where we replaced the viral glycoprotein (GP) with the green fluorescent protein (GFP). We generated high titers of GP-pseudotyped rLCMVΔGP/GFP via genetic trans complementation using stable cell lines that constitutively express LCMV or LASV GPs. Replication of these GP-pseudotyped rLCMVΔGP/GFP viruses was restricted to GP-expressing cell lines. This system allowed us to rapidly and reliably characterize and quantify the neutralization activities of serum antibodies against LCMV and LASV within a BSL-2 facility. The sensitivity of the GFP-based microneutralization assay we developed was similar to that obtained with a conventionally used focus reduction neutralization (FRNT) assay. Using GP-pseudotyped rLCMVΔGP/GFP, we have also obtained evidence supporting the feasibility of this approach to identify and evaluate candidate antiviral drugs against HF arenaviruses without the need of BSL-4 laboratories.     

RNA interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.     

Suppression by cyclosporin A of murine T-cell-mediated immunity against viruses in vivo and in vitro

The immunosuppressive effect of Cyclosporin A on T-cell-mediated antiviral immune responses was examined. When administered intraperitoneally CS-A abrogated anti-vaccinia virus, anti-lymphocytic choriomeningitis virus (LCMV), and anti-vesicular stomatitis virus (VSV) T-cell responses in a dose-dependent fashion. Usually 50-60 mg/kg were efficient in suppressing primary T-cell responses completely. In contrast, 10-20 mg/kg often enhanced T-cell responses significantly when compared with controls. Suppression was observed if CS-A treatment was started before virus injection and up to 12 hr after infection; CS-A given 24 hr after the virus still suppressed T-cell activity partially. A 50 mg/kg dose of CS-A suppressed secondary anti-vaccinia virus or anti-VSV T-cell responses in vivo by a factor of about 10. This dose suppressed the primary T-cell-dependent footpad swelling induced by local LCMV infection and prevented T-cell-mediated immunopathological death due to LCM when LCMV was injected intracerebrally. In addition, clearance of LCMV was delayed drastically by CS-A treatment. When added to cultures of in vivo-primed antiviral T cells that were restimulated in vitro, CS-A inhibited both proliferation as well as generation of virus-specific cytotoxic T cells in a dose-dependent way. The results show that in CS-A-treated mice primary and secondary antiviral T-cell responses are strongly inhibited; acute viral infections with cytopathic viruses may therefore be more dramatic. In contrast immunopathological T-cell-mediated disease caused by noncytopathic viruses such as LCMV may be prevented or attenuated.