Mucosal immunization with Salmonella typhimurium expressing Lassa virus nucleocapsid protein cross-protects mice from lethal challenge with lymphocytic choriomeningitis virus.

OBJECTIVES: Lassa fever virus (LAS) is transmitted to man by rodent carriers and is fatal in a third of untreated cases. Our goal is to provide immune protection from Lassa fever by mucosal vaccination. STUDY DESIGN/METHODS: Mice were vaccinated intragastrically with control vectors or with vectors (vaccinia or Salmonella) expressing LAS nucleocapsid protein (NP). Mice were challenged intracranially with a lethal dose of the related arenavirus, lymphocytic choriomeningitis virus (LCMV), as a measure of the vaccine's ability to elicit cross-protection. RESULTS: Salmonella and vaccinia vectors expressing LAS NP each protected a third of the mice from lethal challenge with LCMV. All mice vaccinated with a vector expressing LCMV NP were protected as expected. CONCLUSIONS: The LAS recombinant Salmonella vector is comparable to the LAS recombinant vaccinia vector in its ability to cross-protect mice from lethal challenge. Nucleocapsid protein is an inadequate immunogen on its own, but provides sufficient cross-protection to make it a useful component of a broadly reactive arenavirus vaccine.     

Analyses of the cytotoxic T lymphocyte responses to glycoprotein and nucleoprotein components of lymphocytic choriomeningitis virus

The outcome of infection by lymphocytic choriomeningitis virus (LCMV) in the natural murine host is determined in large part by the cytotoxic T lymphocyte response (CTL) mounted by the host. In order to define the specificities of CTL induced by LCMV infection, we have cloned and expressed the full-length nucleoprotein (NP) gene and 75% of the glycoprotein (GP) gene of LCMV in vaccinia virus vectors and have used these recombinant viruses to sensitize syngeneic target cells to lysis by anti-LCMV CTL. We have studied the anti-LCMV CTL responses induced on three different mouse H2 (major histocompatibility complex) backgrounds. First, we find that the relative recognition of the two LCMV proteins differs markedly on different H2 haplotypes; both proteins are seen on the H2bb background, while only NP is recognized on two other haplotypes (H2dd and H2qq). Second, we show that on the H2bb background the anti-GP CTL response comprises a major component of the overall CTL response, in marked contrast to several other viruses, e.g., influenza virus, vesicular stomatitis virus, and respiratory syncytial virus where anti-GP responses, if present, comprise only a minor portion of the whole. Third, LCMV GP can be a major target antigen for CTL induced by a serotypically distinct strain of LCMV, again in contrast to the above virus systems in which CTL cross-reactivity among different serotypes is dependent largely on the recognition of "internal" proteins.     

An acquired immune suppression in mice caused by infection with lymphocytic choriomeningitis virus

A murine model of virally induced acquired immunodeficiency was analyzed in mice. The effect of systemic infection with various isolates of lymphocytic choriomeningitis virus (LCMV) on the capacity of mice to mount a T cell-independent IgM and a T cell-dependent IgG neutralizing antibody response against a subsequent infection with vesicular stomatitis virus (VSV) was analyzed. DBA/2 mice infected with the LCMV-WE isolate were impaired in their IgM and IgG responses to VSV. Immune suppression was not caused by interferons inhibiting proper VSV antigen expression, since responses to inactivated VSV were also suppressed. The higher the dose of the LCMV and the lower the dose of the challenging VSV infection the more drastic was the apparent lack of immune responsiveness and the longer it lasted. Kinetics of induction of suppression of the T cell-independent IgM responses closely followed that of a normal cytotoxic T cell response to LCMV-WE, starting on day 6 and reaching maximal levels by day 8 to 10. The T cell-dependent IgG response to VSV was suppressed with a kinetics that was shifted by about 6 days when compared with suppression of IgM responses, i.e. LCMV infection on the same day or before (but not after) VSV infection led to suppression of IgG responses that are usually first detected by day 6-7 after initiation of the VSV infection. Severity and duration of immunosuppressiveness depended upon the LCMV isolate and the mouse strain used: LCMV-WE and LCMV-Docile were most, whereas LCMV-Armstrong was in general least immunosuppressive. Antibody responses to VSV-NJ seemed to be more subject to LCMV-induced immune suppression than VSV-IND-specific responses. Mouse strains differed considerably with respect to extent of suppression, dependent upon both major histocompatibility genes (MHC) and non-MHC genes. DBA and Swiss type mice were generally more susceptible than C57BL and CBA mice, and H-2q and H-2k seemed to be more susceptible than H-2b or H-2d mice. Mice infected with LCMV-WE showed signs of acquired immunodeficiency diseases since they were more susceptible to superinfection with VSV and developed paralytic disease and tended to die from VSV infection. Since LCMV is basically a noncytopathic virus, this murine model of virally induced immune suppression may serve to analyze immune pathogenesis of virus-induced acquired immunodeficiency.     

Induction or prevention of immunopathological disease by cloned cytotoxic T cell lines specific for lymphocytic choriomeningitis virus

Cloned lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T lymphocyte (CTL) lines were prepared from spleens of 129/J (H-2^b) mice immunized 7-9 months earlier with LCMV (UBC strain), or of C57BL/10J (H-2^b) mice immunized 4 to 5 weeks earlier with LCMV (Armstrong strain). One uncloned and 3 cloned cytotoxic T cell lines were assessed for their respective abilities to produce, or protect against, fatal disease upon transfer to appropriate recipients or to induce specific footpadswelling reaction. The effects of all lines were essentially identical. In recipient mice acutely infected with LCMV and immunosuppressed either by irradiation (750-990 rds) or treatment with cyclophosphamide, cloned T cells administered intracerebrally (i.c.) caused a convulsive disease and death within 1-4 days. No disease was produced when the same CTL were transferred to uninfected recipients or when they had been frozen and thawed prior to transfer to infected recipients. When admixed with 500 plaque-forming units of LCMV and transferred i.c. to immunocompetent H-2^b mice, the T cell clones prevented overt disease. Allogeneic (H-2^k) recipients of this same admixture all developed typical LCM disease as did H-2^b recipients of the admixture after T cells had been frozen and thawed. Inoculation of cloned CTL into preinfected footpads induced a specific footpad-swelling reaction, which reached maximum levels after about 36 h. Irradiated and infected recipients of cloned LCMV-specific T cells showed the footpad-swelling reaction only when they had been reconstituted with bone marrow cells. In contrast, cloned T cells induced LCM disease in i.c. infected and irradiated mice independent of bone marrow reconstitution. These findings indicate that both fatal LCMV-induced neurologic disease and protection against it are mediated directly by virus-specific CTL.     

Chronic production of interferon in carrier mice congenitally infected with lymphocytic choriomeningitis virus

Congenital infection of mice with lymphocytic choriomeningitis virus leads to a lifelong virus carrier state. Here we provide evidence for the presence and action of interferon in such mice. The level of circulating interferon in adult carrier mice is 8–16 NIH units/ml of plasma. This interferon is acid stable and is capable of inducing 2–5A synthetase in mouse L 929 cells but not in human HeLa cells. Control, noinfected mice show less than 1 NIH unit/ml of plasma. In accord with these results, adult carrier mice have higher levels of the interferon-mediated pppA(2′p5′A)_n synthetase (2–5A synthetase) in their liver and spleen than normal mice. Congenitally infected newborn mice also have higher levels of 2–5A synthetase in their liver in contrast to newborn control mice. These results in congenitally infected newborn and adult mice suggest that interferon may play a role, at least in part, in the pathogenesis of infection.     

Control of lymphocytic choriomeningitis virus infection in granzyme B deficient mice

We have investigated whether granzyme B (GzmB) is required for effective cytotoxic T lymphocyte (CTL) mediated control of lymphocytic choriomeningitis virus (LCMV) infection. Clearance of LCMV from tissues of GzmB-deficient (GzmB-) mice following intraperitoneal infection with LCMV was impaired compared with control mice; however, the virus was ultimately eliminated. The impaired clearance of LCMV in GzmB- mice was not due to a deficiency in the generation of LCMV-specific T cells. In addition, CTL from LCMV-infected GzmB- mice efficiently lysed virus-infected cells in vitro, but were deficient in their ability to induce rapid DNA fragmentation in target cells. We examined whether the development of protective immunity against intracranial (i.c.) rechallenge with LCMV was compromised in GzmB- mice. We found that clearance of LCMV from the brain following secondary i.c. infection also was slower in the absence of GzmB; however, the virus was ultimately eliminated and the mice survived. Our data indicate that clearance of LCMV is delayed in the absence of GzmB expression, but that other CTL effector molecules can compensate for the absence of this granule constituent in vivo.     

Persistent infection of mice with the virus of lymphocytic choriomeningitis: virus-specific immunological tolerance

Lymphocytic choriomeningitis (LCM) virus-infected culture cells as targets were markedly reduced in numbers when incubated in vitro with spleen cells from LCM virus-immune mice, even if the cells were taken months after a subcutaneous immunizing infection of the donor animal. Spleen cells from mice persistently infected with LCM virus had no effect on target cells. Also, mitomycin C-blocked LCM virus-infected culture cells stimulated the incorporation of radiolabeled thymidine into spleen cells from LCM virus-immune mice. No stimulation was observed with spleen cells from LCM virus carrier mice. It was concluded that cell-mediated immunity directed against LCM virion surface antigen(s) is absent in LCM virus carrier mice.     

Immunity to lymphocytic choriomeningitis virus in B cell-depleted mice: evidence for B cell and antibody-independent protection by memory T cells

Immunity against lymphocytic choriomeningitis virus (LCMV) in anti-IgM-treated B cell-depleted mice was evaluated. We found that the following immune phenomena were independent of antibodies: the generation of virus-specific cytotoxic T cells; the footpad swelling response against locally injected LCMV; natural killer cell activity basic levels or after LCMV or poly(I) X poly(C) stimulation; immunopathologically mediated LCM after primary intracerebral inoculation; immunological memory in LCMV-immune mice assessed by immune protection against LCM after intracerebrally injected virus or as resistance against the local footpad swelling response to LCMV. This study demonstrates that humoral immunity plays no crucial role in immune protection and immunopathology in murine LCMV infection and suggests that protective memory T cell function is B cell and antibody independent.     

Beneficial effect of cyclosporin A on the lymphocytic choriomeningitis virus infection in mice

The effect of cyclosporin A on the course of lymphocytic choriomeningitis virus infection in mice was investigated. Evidence is presented that administration of this immunosuppressive drug spares a majority of lethally infected mice. This beneficial effect is different from the one obtained with other treatments leading to the abolition of T cell functions. Surviving animals rapidly eliminate the virus and produce high titers of neutralizing IgG antibodies.     

Stage of primary infection with lymphocytic choriomeningitis virus determines predisposition or resistance of mice to secondary bacterial infections

We investigated the effect of a primary non-lethal infection with lymphocytic choriomeningitis virus (LCMV) on the course and outcome of a secondary infection with the Gram-negative Salmonella enterica serovar Typhimurium or the Gram-positive Listeria monocytogenes in mice. We found that at each stage of the viral infection the susceptibility of mice to bacterial super-infections changes dramatically and depends also on whether the secondary infection is a Gram-positive or Gram-negative one. The study shows that the outcome of the secondary infection is determined by a delicate balance between the overproduction of and the hypersensitivity to inflammatory cytokines (TNF-α and IFN-γ), as well as by the changes in blood leukocytes occurring in mice in the course of viral infection.     

Protection against local Shigella sonnei infection in mice by parenteral immunization with a nucleoprotein subcellular vaccine.

Nucleoprotein subcellular (NPS) vaccine, consisting of ribosome-bound O polysaccharide, was prepared from avirulent Shigella sonnei. NPS vaccine was tested for safety and protective activity in the mouse intranasal challenge model of Shigella infection. The vaccine was nontoxic when injected in doses up to 10,000 micrograms, and a single subcutaneous injection of as little as 0.1 micrograms gave significant protection against a lethal intranasal challenge with S. sonnei. These data demonstrate the induction of local protective immunity by parenteral immunization, support the concept of the ribosome as a potent vaccine vector, and give additional evidence for the protective activity of the NPS vaccine against Shigella infection.     

Selective resistance to togaviral superinfection in mice with tolerant lymphocytic choriomeningitis virus infection.

Mice infected neonatally with lymphocytic choriomeningitis virus (LCMV) developed partial and complete resitance to cerebral superinfection with tick-borne encephalitis virus (TEV) in 10 and 20 days after birth, respectively. This resistance lasted at least till the age of 40 days. LCMV tolerant mice neither succumbed to TEV infection, nor circulated TEV in their blood. Moderate, gradually decreasing TEV titres were detected in the brains and TEV-induced brain interferon was lower than in control mice of the same age. TEV superinfection caused a significant depression of the blood titre of tolerated LCMV while the titres in the brains remained equal to those in tolerant but not superinfected mice. LCMV tolerant mice showed a similar resistance to another togavirus (chikungunya) but not to encephalitogenic picorna-, herpes- and rhabdoviruses.