Antiviral action of carminomycin and some of its derivatives]

Carminomycin was shown to inhibit the development of both the DNA-containing variolovaccine virus and the RNA-containing grippe virus in chick embryos. Comparison of the effects of rubomycin, carminomycin, 14-oxy-carminomycin and carminomycin complex with bovine serum albumin in experiments with chick embryos showed that the inhibitory effect of carminomycin and its derivatives on the development of the grippe virus was much higher than that of rubomycin. The carminomycin derivatives proved to be much more active in this respect than the initial antibiotic. Carminomycin and its derivatives had a therapeutic effect on mice with experimental grippe pneumonia also on their oral use.     

Antiviral action of carbonyl-conjugated pentaene macrolides

Clear antiviral activity of carbonyl-conjugated pentaene macrolides, such as flavofungin, mycothicin, brunefungin and flavopentin was shown on models with infectious and oncogenic viruses. The antibiotics were active against influenza A and B virus. The effect was most pronounced in the in vitro and in ovo systems. On a model of experimental influenza infection of mice with the lethal outcome, antiinfluenzal activity of flavofungin was comparable to that of remantadin. However, unlike the latter one flavofungin and brunefungin inhibited the growth of influenza B virus. The drugs had a pronounced inhibitory effect on variolavaccine virus and prevented formation of foci of cell neoplastic transformation infected with various strains of Rous sarcoma virus.     

The role of natural killer cells and interferon in resistance to acute infection of mice with herpes simplex virus type 1

The relative roles of interferon (IFN) and natural killer (NK) cells in herpes simplex virus type 1 (HSV-1) infection of mice were examined. Adoptive transfer of adult mouse leukocytes into 4- to 6-day-old suckling mice protected the recipients from HSV-1 infection, as judged by viral titers in the spleen 2 days postinfection. Protection was mediated by several classes of leukocytes, including those depleted of NK cell activity by antibody to asialo GM1 and those depleted of macrophages by size separation. Mice receiving these leukocytes produced significantly higher levels of IFN 6 hr postinfection (early IFN) than did HSV-1-infected mice not receiving donor leukocytes. Antibody to IFN, under conditions that blocked early but not late IFN synthesis, greatly enhanced HSV-1 synthesis in mice receiving leukocytes and completely removed the protective effect mediated by leukocytes. High doses of anti-asialo GM1 blocked both NK cell activity and early IFN production and resulted in high titers of HSV-1. This effect on virus synthesis was not seen if mice were given antibody 1 day postinfection. Lower doses of anti-asialo GM1, which still depleted NK cell activity but had no effect on early IFN production, did not enhance HSV-1 synthesis. Depletion of NK cell activity with a low dose of antibody had no effect on the reduced HSV-1 synthesis resulting from prophylactic IFN treatment or on the enhanced HSV-1 synthesis resulting from antibody to IFN treatment. Thus, resistance to acute HSV-1 infection in mice correlates with early IFN production but not with NK cell activity, suggesting that NK cells are not major mediators of natural resistance in this model and that the antiviral effect of IFN is not mediated by NK cells.     

Anti-influenza effect of bacterial RNAse and the pharmacokinetic basis of its administration in experimental studies

Anti-influenzal action of bacterial and pancreatic RNAases was studied. It was shown in ovo that the RNAases had distinct virus inhibiting activity with respect to various strains of the grippe A virus and did not practically differ by their activity from remantadin but unlike it had inhibitory action on the grippe B virus. The anti-influenzal activity of bacterial RNAase in contrast to pancreatic one was detected not only in experiments with developing chick embryos but also in albino mice with lethal influenzal infection. The index of the animal protection by the preparation amounted to 54-90 per cent depending on the virus infecting dose and RNAase administration route, the lifespan of the animals being increased by 2.4 to 3.8 days. It was shown that the anti-influenzal effect of bacterial RNAase correlated with high levels of the exogenic enzyme in blood of the animals after the preparation intravenous administration. Elimination of RNAase was observed already within the first 4 hours after the experiment start. Intranasal administration allowed to increase the residence time of RNAase in blood up to 8 hours at the account of its gradual absorption from the administration site and the preparation availability increased more than 2-fold. The results provided the basis for recommending the intranasal route of bacterial RNAase administration for use in further investigation of RNAase antiviral activity.     

Interleukin-12 exhibits potent antiviral activity in experimental herpesvirus infections.

The prophylactic and therapeutic efficacy of interleukin-12 was studied by using murine models of herpes simplex virus infection. Prophylactic administration consisted of two intraperitoneal doses of interleukin-12 given 48 and 24 h prior to infection. Therapeutic intraperitoneal administration of interleukin-12 commenced 6 h after the mice were infected with herpes simplex virus and was continued daily for a total of 5 days. Interleukin-12 therapy improved the survival rates of mice with systemic herpes simplex virus infection compared with those of placebo-treated infected mice. Subcutaneous administration of interleukin-12 also improved the rate of survival of mice after systemic herpes simplex virus infection, although higher doses were required to give comparable effects. Combined prophylactic and therapeutic administration of interleukin-12 produced the greatest effect on survival after an otherwise lethal systemic infection. Intraperitoneal administration of interleukin-12 for 2 days before and 3 days after systemic infection with herpes simplex virus resulted in survival of 80% of the mice. These surviving mice were resistant to subsequent reinfection with herpes simplex virus. Such resistance was apparently specific for herpes simplex virus infection, since a second group of survivors succumbed to a lethal infection with murine cytomegalovirus. Infectious virus was recovered from lumbar ganglia explants dissected from survivors of prophylactic interleukin-12 therapy and cultured for 5 days in vitro, suggesting that interleukin-12 treatment did not prevent the establishment of latent herpes simplex virus infection. One action of interleukin-12 may be to enhance natural killer cell-mediated clearance of the virus. However, interleukin-12 therapy was also effective in mice carrying the beige mutation, which reduces natural killer cell lytic activity, suggesting that interleukin-12 has additional activities in vivo.     

Impaired anti-viral T cell responses due to expression of the Ly49A inhibitory receptor.

Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.     

The role of NK cell activity in age-dependent resistance of mice to murine cytomegalovirus infection

The resistance of mice to cell culture passaged murine cytomegalovirus (CC-MCMV) infection developed with age. In parallel with this finding, augmentation of the splenic NK cell activity in older mice was always higher than that of younger mice. The splenic NK cell activity reached the maximum level at 6 day post infection (PI) in 2-4-week-old mice while in 6-8-week-old mice it peaked at 4 days PI. When the dose of CC-MCMV was increased, the NK cell activity was potentiated accordingly. However, it was decreased on the infection with increased doses of the salivary gland passaged MCMV (SG-MCMV). NK cells augmented by MCMV infection actually inhibited in vitro replication of MCMV when they were added to mouse embryonic fibroblast (MEF) monolayers infected with CC-MCMV. Splenic and peritoneal macrophages inhibited in vitro replication of MCMV, but their activities were less potent than those of NK cells.     

In vivo significance of NK cell on resistance against virus (HSV-1) infections in mice

Susceptibility to infection with herpes simplex virus type 1 (HSV-1) was examined in euthymic as well as athymic nude mice which were continuously depleted of natural killer (NK) cell activity by i.v. injection of anti-asialo GM1. In those NK cell activity-depleted mice, the mortality rate of infection with HSV-1 and the virus titers in the brain, liver, and spleen were notably higher than in the control mice. The enhanced susceptibility was demonstrated only in the mice receiving anti-asialo GM1 and HSV-1 simultaneously, but not in the mice in which NK cell deletion was postponed by injecting the antisera 5 days after the virus inoculation. Interferon (IFN) production of peritoneal exudate cells was also reduced in the anti-asialo GM1-injected mice. The decline of resistance against HSV-1 infection proved to be primarily due to deletion of NK cells, but not due to the inability to produce IFN, because repeated injections of IFN increased the NK cell activity and prolonged the life of HSV-1-infected mice with an intact NK cell activity. In the NK cell activity-depleted mice, however, neither the NK cell activity nor the life span was improved by the administration of IFN.     

Molecular mechanism of complex infection by bacteria and virus analyzed by a model using serratial protease and influenza virus in mice.

We examined the effect of a serratial exoprotease on the pathogenesis of influenza virus infection in mice as a model of complicated respiratory infection by bacteria and virus in humans. The 56-kilodalton (56-kDa) protease from Serratia marcescens was administrated intranasally to mice at a dose of 10, 20, or 40 micrograms from day 0 to day 3 after inoculation of the influenza virus. Administration of the protease resulted in remarkable enhancement of the lethal effect of the virus and enhancement of pathological changes in the lungs. Influenza virus replication, determined by plaque-forming assay, was accelerated by the protease. Namely, we found a 100-fold increase in virus yield by day 2. The 56-kDa protease caused generation of plasmin activity in the lungs. In vitro experiments showed that plasmin greatly enhanced the yield of influenza virus, although the effect of the 56-kDa protease by itself was much lower than that of plasmin. Furthermore, the 56-kDa protease could induce plasmin production indirectly via activation of plasminogen by the Hageman factor-dependent cascade in the in vitro system. We conclude that this major serratial exoprotease has a deleterious effect on mice infected with influenza virus and that this effect seems to result from enhancement of viral growth by indirect acceleration of plasmin generation induced by the protease.     

Rapid conversion of effector mechanisms from NK to T cells during virus-induced lysis of allogeneic implants in vivo

Viral infections can strongly stimulate both NK cell and allospecific CD8 T cell responses, and these same effector cells can lyse allogeneic cell lines in vitro. However, the impact of viral infections on the effector systems mediating rejection of allogeneic tissues in vivo has not been fully explored. Using in vivo cytotoxicity assays, we evaluated the effector systems mediating the rejection of CFSE-labeled allogeneic splenocytes after an infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus. Naive B6 mice predominantly used a NK cell-effector mechanism to reject allogeneic splenocytes because they rejected BALB/C (H2(d)) splenocytes but not CBA (H2(k)) splenocytes, and the rejection was prevented by immunodepletion of NK1.1(+) or Ly49D(+) NK cells. This rapid and efficient in vivo cytotoxicity assay recapitulated the specificity of NK cell-mediated rejection seen in longer duration in vivo assays. However, as early as 1 day after infection with lymphocytic choriomeningitis virus, a CD8 T cell-dependent mechanism participated in the rejection process and a broader range of tissue haplotypes (e.g., H2(k)) was susceptible. The CD8 T cell-mediated in vivo rejection process was vigorous at a time postinfection (day 3) when NK cell effector functions are peaking, indicating that the effector systems used in vivo differed from those observed with in vitro assays measuring the killing of allogeneic cells. This rapid generation of allospecific CTL activity during a viral infection preceded the peak of viral epitope-specific T cell responses, as detected by in vivo or in vitro cytotoxicity assays.     

Methodologic approaches to assessing cellular protection in influenza (the model: splenocyte versus splenocyte)

To study the cell-mediated link of immune response in mice in experimental influenza, both spleen cells obtained from intact mice and infected with the virus in vitro and spleen cells obtained from infected mice on day 6 after infection may equally be used with success as target cells. This opens the possibility of studying the role of virus-specific modifications of the cell membranes of immunocytes in the pathogenesis of influenza infection. The use of effector cells without their additional stimulation with homologous virus in vitro permits the simultaneous study of different mechanisms of specific (cytotoxic T lymphocytes and antibody-dependent cell-mediated cytotoxicity) and nonspecific (natural killer cells) cell-mediated immunity developing in influenza, as well as the study of the functional activity of spleen cells under the conditions similar to those existing in the body when the duration of the experiment is 5-7 days shorter.     

Infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro splenic T cell proliferation and cytokine production.

Earlier studies from this laboratory showed that infection of BALB/cByJ mice by a natural route with mouse hepatitis virus, strain JHM (MHV-JHM), results in functional splenic T cell suppression in vitro. This was evidenced by reduced concanavalin A-driven spleen cell proliferation and interleukin (IL)2 production measured after conventional intervals of cell culture (72 and 24 h, respectively). The purpose of the present work was to determine whether MHV-induced T cell dysfunction is kinetic or absolute and whether production of other T-cell derived cytokines is defective. Bioassays revealed that production of IL2, gamma interferon, and IL4 by spleen cells from acutely infected mice is suppressed and that some of the defects are kinetic as well as absolute. Proliferative responses of both CD4+ and CD8+ T cells were depressed, but neither cell type contained infectious virus. Cells that proliferated poorly in response to concanavalin A were fully capable of responding to specific virus stimulation. These results further emphasize the potential complications that MHV infection may pose to immunologic research using mice.     

Resistance to Pseudorabies Virus with Enhanced Interferon Production and Natural Killer Cell Activity in Mice Treated with Serum Thymic Factor

Susceptibility to pseudorabies virus (PRV) infection in mice, which were continuously depleted of natural killer (NK) cell activity by injection of anti-asialo GM1, was examined. Effects of serum thymic factor (FTS) on susceptibility of mice to PRV were also investigated. In mice with depleted NK cell activity, the mortality of PRV-infected mice was markedly high, whereas that of FTS-pretreated mice was significantly lower than the controls. Reduced susceptibility to PRV was demonstrated in mice treated with anti-asialo GM1 antisera before the PRV infection. Such a reduced susceptibility was not observed in mice inoculated with the antisera on day 1 post-infection (PI). To analyze the FTS-induced resistance to PRV infection, NK cell activity, macrophage activity, and interferon (IFN) productions were studied. Interferon production and NK cell activity were enhanced in the FTS-pretreated mice, suggesting that interferon may play an important role in this FTS-induced resistance to PRV infection.     

Effect of rabbit anti-asialo GM1 treatment in vivo or with anti-asialo GM1 plus complement in vitro on cytotoxic T cell activities

The susceptibility of cytotoxic effector lymphocytes and their induction to in vivo or in vitro treatment with rabbit anti-neutral glycolipid ganglio-N-tetraosylceramide (anti-ASGM1) antiserum was investigated. Intravenous injection of anti-ASGM1 antiserum eliminated measurable natural killer (NK) cell activity in spleen cells of mice infected for 5 days with Vaccinia virus, or for 8 days with lymphocytic choriomeningitis virus (LCMV) if injected 24 hr prior to testing. In addition, this treatment lowered measurable virus-specific cytotoxic T cell activity by 60 to 95%. Virus-specific cytotoxic T cell and NK cell activity generated during a primary infection in vivo was also sensitive to treatment in vitro with anti-ASGM1 antiserum (1/300 to 1/600 dilution) plus rabbit complement at a dilution of 1/15 (20 to 50% cell death, more than 30-fold decrease of cytotoxic activity); in vitro treatment with rabbit complement alone often enhanced NK and cytotoxic T cell activity slightly. In vivo treatment with anti-ASGM1 before primary immunization decreased generation of primary CTL only if high doses of anti-ASGM1 antiserum were injected twice. Antiviral T cells generated during secondary stimulation in vitro and alloreactive cytotoxic T cells from a mixed lymphocyte culture were resistant to treatment in vitro with anti-ASGM1 plus complement at the end of the culture period. Treatment in vitro of in vivo-primed responder spleen cells with anti-ASGM1 plus complement before their addition to a secondary restimulation culture resulted in complete inhibition of a secondary antiviral cytotoxic T cell response. In vivo treatment with anti-ASGM1 24 hr before their spleen cells were harvested and restimulated in vitro significantly reduced the virus-specific T cell activity of mice that had been immunized with virus several weeks previously. A cloned T cell line exclusively exerting NK-like activity was resistant, and two cloned virus-specific cytotoxic T cell lines were susceptible to treatment with anti-ASGM1 plus complement in vitro. These results caution the general use of rabbit anti-ASGM1 as a marker to distinguish NK from CTL cells; they indicate a possible relationship between NK and CTL cells and suggest that in vitro culture of lymphocytes may alter or select the cell surface expression or availability of the ASGM1 marker(s).     

Protective effect of biological response modifiers on murine cytomegalovirus infection

Pretreatment with two biological response modifiers (BRM), OK-432 and PS-K, protected mice from lethal infection by murine cytomegalovirus (MCMV). This was evidenced by an increase in 50% lethal doses and a decrease in titers of infectious viruses replicated in the liver and spleen. Spleen cells from the BRM-treated mice augmented the natural killer (NK) cell activity and suppressed the replication of MCMV in vitro. During MCMV infection, the NK cell activity of the spleen cells was maintained at a high level in the BRM-treated mice, whereas it was severely impaired in untreated mice. The BRM-induced protection was nullified by concomitant administration of antiasialo GM1 antibody. Interferon was neither induced by BRM treatment nor enhanced in BRM-pretreated and MCMV-infected mice. Thus, the protective effect of OK-432 and PS-K seems to be based on activation of NK cells and prevention of MCMV-induced inhibition of the NK cell activity.     

Experimental effect of lysozyme on macrophage metabolism and resistance to infection

The peritoneal macrophages of mice treated with lysozyme were studied by cytochemical assay. In single and repeated doses of 0.5-5 mg/kg lysozyme induced an increase in macrophage metabolism. This was evident from an increased activity of succinate dehydrogenase, NADP X N-DH and the enzymes catalyzing glycolysis typical of these cells (lactate dehydrogenase and alpha-glycerophosphate). The changes in the activity of the enzymatic systems were most pronounced in minute and less mature macrophages after repeated administrations of the drugs. In a dose of 50 mg/kg lysozyme somewhat decreased the activity of a number of the enzymes. In the doses optimal for the macrophage activity lysozyme had a low effect on the infection resistance and slightly increased the cephotaxim efficiency in experimental staphylococcal infection. This may be mainly due to the immunomodulating effect of lysozyme and its low effect on the large macrophages having the bactericidal effect.     

X-irradiation enhances in vitro human immunodeficiency virus replication correlation with cellular levels of cAMP.

Total body x-irradiation has been utilized in the treatment of several human diseases, including leukemia, where it is followed by bone marrow transplantation, and in some autoimmune disorders. Recently, it was reported that total body irradiation appeared useful in the treatment of Friend leukemia virus infection in mice. In this report, the effect of x-irradiation on the replication of human immunodeficiency virus (HIV) in vitro in CD4+ cells was examined. MT-4 cells and HIV strain human T cell lymphotropic virus Type IIIB were used to conduct this study. Infected MT-4 cells were irradiated at the time of infection or following infection with x-ray doses of 25-300 cGy. Doses of 50, 150, and 300 cGy enhanced HIV replication by 1.6-, 2-, and 4.8-fold, respectively. Irradiating the cells prior to infection also resulted in similar enhancement of HIV replication. This phenomenon was also observed with wild-type HIV isolates grown in peripheral blood mononuclear and in HIV chronically infected cells. In addition, the enhancement was associated with a radiation-induced increase in intracellular levels of cAMP. The use of the cAMP-dependent protein kinase A inhibitor, H-8, inhibited HIV replication by 65%. These data suggest that in vitro exposure to low doses of x-ray enhances HIV replication partially via a cAMP-dependent pathway.     

The efficacy of biological response modifiers against murine cytomegalovirus infection in normal and immunodeficient mice

The host-mediated antiviral effect of two biological response modifiers (BRM), OK-432 and PS-K, against murine cytomegalovirus (MCMV) was evaluated in normal and immunologically deficient mice of the same litters. In normal littermate mice, BALB/c (nu/+) or C57BL/6 (bg/+), the BRM-induced resistance against MCMV infection was evidenced by increase in fifty percent lethal doses, decrease in titers of viruses replicated in the target organs and augmentation of natural killer (NK) cell activity of the spleen cells. In T cell-deficient, athymic nude mice, BALB/c (nu/nu), the protective effect was manifested by prolongation of the survival, decrease in the virus titers, and increase in the NK-cell activity, but without decrease in mortality. In NK cell-deficient, beige mutant mice, C57BL/6 (bg/bg), the BRM-induced protection was nullified or minimized, and there was little difference in those parameters between BRM-treated and untreated mice. However, with higher doses of OK-432, but not PS-K, or with sublethal doses of MCMV, the NK cell activity was slightly augmented in the beige mutant mice. Thus both NK cell and T cell activity are essential for mice to overcome acute MCMV infection and it is likely that the protective effect of BRM manifests itself fully, at least in immunologically intact mice.     

Antiviral activity of a thymic factor in experimental viral infections. I. Thymic hormonal effect on survival, interferon production and NK cell activity in Mengo virus-infected mice

A partially purified thymic factor, thymostimulin (TS), significantly increased the survival rate of adult, immune-intact mice infected with the neurotropic Mengo virus. TS treatment was begun after virus inoculation by daily i.p. injections. In untreated C57BL/6 mice, LD50 was reached with 1 X 10(4) PFU, but 10-fold more virus (i.e., 1 X 10(5) PFU) was needed to reach LD50 in TS-treated animals. TS effect on survival, though, could be observed with several virus doses (1 X 10(3) to 1 X 10(6) PFU) (p less than 0.001). A significant effect on survival was also observed with outbred ICR mice (p less than 0.005). Serum interferon (IFN) levels in the Mengo virus-infected mice were relatively low (average peak 300 U/ml), but were significantly increased (two- to ninefold) in the TS-treated mice. Peak serum levels were reached earlier in TS than in control animals (24 hr and 72 hr, respectively). Both acid-labile and acid-stable type I IFN production were augmented by TS in the Mengo virus-infected mice. Natural killer activity was also enhanced by TS, in particular on the second day after virus inoculation. In addition, MP-virus was used as a second, unrelated virus challenge. This virus caused a nonlethal infection, with relatively high levels of serum IFN (average peak 10,000 U/ml). TS increased IFN levels (two- to eight-fold) also in this challenge system. In conclusion, TS causes a nonspecific enhancement of endogenous production of IFN and has a significant effect on the survival of lethally infected mice. The data indicate a potential application of thymic factors for the treatment of viral infections.     

A mouse model of the pathogenesis and postexposure prophylaxis of rabies

A mouse model for the study of postexposure prophylaxis of rabies was established. Mice injected intramuscularly with a street strain of rabies virus were significantly protected from death by five daily 0.2-ml doses of inactivated rabies vaccine of chick embryo cell culture origin initiated immediately or 3 hr after infection. In these mice, a large amount of circulating interferon was induced as early as 1 hr after the first dose of vaccine and lasted until at least 12 hr but no such amount of interferon was induced by additional doses of vaccine. Serum antibody was first detected in the mice on day 6. It was noted that some of the surviving mice manifested an ataxia or paralysis of the legs. Increasing mortality rates were shown in mice treated with decreasing doses of the vaccine. Passive protection tests using concentrated IgG and IgM antibodies with equivalent neutralization titers showed that IgG antibody gave total protection when given 24 hr before the infection, while it was almost totally ineffective in reducing the mortality when given 2 days or more after infection. IgM antibody did not protect the mice even when given 24 hr before infection. These results suggest that interferon production is more important than antibody production in the initial stages of protection by postexposure vaccination. However, the mechanisms of postexposure prophylaxis in this model could not be explained only by the interferon produced by the vaccine and the possible contributions of additional mechanisms were suggested.