In vitro and in vivo activities of T-705 and oseltamivir against influenza virus

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has a potent and selective inhibitory activity against influenza virus. We studied the effects of an infectious dose on the anti-influenza virus activities of T-705 and oseltamivir, a commercially available neuraminidase inhibitor, both in vitro and in vivo. Plaque formation of influenza A/PR/8/34 virus was completely inhibited by 10 microg/ml of T-705 after 72 h incubation, whereas visible plaque formation was detected in the plate treated with GS 4071, the active form of oseltamivir (10 microg/ml). The antiviral activity of T-705 was not influenced by an increase in multiplicity of infection (MOI) from 0.0001 to 1, but that of GS 4071 was influenced in a yield reduction assay. No increase in viral yield was seen in either culture supernatant or cells after removal of T-705 (10 microg/ml) but, in contrast, productive infection recurred in culture supernatant and in cells after removal of GS 4071. In mice infected with a high challenge dose of influenza A/PR/8/34 virus, orally administered T-705 (200 and 400 mg/kg/day) completely prevented the death of mice and the survival rates of mice were significantly higher than those in mice treated with oseltamivir (P<0.01). When the treatment was delayed at 1, 13 and 25 h post infection, oral administration of 200 mg/kg of T-705 significantly prevented the death of mice (P<0.01), and the survival rates of mice treated with T-705 were comparable to those of mice treated with oseltamivir. These results suggest that T-705 has the potential to be a potent inhibitor of human influenza virus infections.     

Sialic Acid-Binding Protein Sp2CBMTD Protects Mice against Lethal Challenge with Emerging Influenza A (H7N9) Virus

Compounds that target the cellular factors essential for influenza virus replication represent an innovative approach to antiviral therapy. Sp2CBMTD is a genetically engineered multivalent protein that masks sialic acid-containing cellular receptors on the respiratory epithelium, which are recognized by influenza viruses. Here, we evaluated the antiviral potential of Sp2CBMTD against lethal infection in mice with an emerging A/Anhui/1/2013 (H7N9) influenza virus and addressed the mechanistic basis of its activity in vivo. Sp2CBMTD was administered to mice intranasally as a single or repeated dose (0.1, 1, 10, or 100 μg) before (day −7, −3, and/or −1) or after (6 or 24 h) H7N9 virus inoculation. A single Sp2CBMTD dose (10 or 100 μg) protected 80% to 100% of the mice when administered 7 days before the H7N9 lethal challenge. Repeated Sp2CBMTD administration conferred the highest protection, resulting in 100% survival of the mice even at the lowest dose tested (0.1 μg). When treatment began 24 h after exposure to the H7N9 virus, a single administration of 100 μg of Sp2CBMTD protected 40% of the mice from death. The administration of Sp2CBMTD induced the pulmonary expression of proinflammatory mediators (interleukin-6 [IL-6], IL-1β, RANTES, monocyte chemotactic protein-1 [MCP-1], macrophage inflammatory protein-1α [MIP-1α], and inducible protein [IP-10]) and recruited neutrophils to the respiratory tract before H7N9 virus infection, which resulted in less pronounced inflammation and rapid virus clearance from mouse lungs. Sp2CBMTD administration did not affect the virus-specific adaptive immune response, which was sufficient to protect against reinfection with a higher dose of homologous H7N9 virus or heterologous H5N1 virus. Thus, Sp2CBMTD was effective in preventing H7N9 infections in a lethal mouse model and holds promise as a prophylaxis option against zoonotic influenza viruses.     

Treatment of murine pulmonary cryptococcosis with a combination of fluconazole and interleukin-12

Interleukin-12 (IL-12) is known to protect the host from various pathogens by inducing cell-mediated immunity. In a murine model of pulmonary infection withCryptococcus neoformans, the daily administration of 0.1 μg/day of IL-12 for 7 day failed to protect mice from infection when treatment commenced 7 days after intratracheal instillation of the pathogen. However, IL-12 administered prophylactically or on the day of infection was effective in combating the infection. Because the number of yeast cells increase by 10-fold in the lungs within a short period of time, and in an attempt to control the growth of the fungus, a combination therapy of an antifungal agent, fluconazole (FCZ), and IL-12 was initiated 7 days after infection. Treatment with FCZ significantly prolonged survival time, and when combined with IL-12, there was a further prolongation of survival time. In addition, administration of both agents significantly reduced the number of live microorganisms isolated from the lung tissue. Our results suggest that the combined use of FCZ and IL-12 may represent a new therapeutic strategy for cryptococcal infections in patients with impaired cell-mediated immunity.     

Treatment of mannan-enhanced influenza B virus infections in mice with oseltamivir, ribavirin and viramidine

Mannan, a polysaccharide preparation from Saccharomyces cerevisiae, has previously been shown to enhance influenza virus replication in mice by inhibiting host defense collectins. The use of mannan in infections may serve to broaden the types of influenza viruses that can be studied in rodent infection models. When mannan was co-administered with influenza B/Sichuan/379/99 virus to mice, the animals died from the infection, whereas mice infected with only virus survived. Three types of influenza A (H1N1) and another influenza B (Hong Kong/330/01) virus infection were also enhanced by mannan, but not four types of influenza A (H3N2) viruses. Mannan was used at 0.16 or 0.5 mg/mouse for optimal disease-enhancing activity using influenza B/Sichuan/379/99 virus. Using this model, influenza B/Sichuan/379/99 infections were treated with oseltamivir, ribavirin or viramidine (the carboxamidine derivative of ribavirin). When oral gavage treatments started 4 h before virus and mannan challenge, oseltamivir was effective at 2.5, 5 and 10 mg/kg/day. Ribavirin was active at 20, 40 and 80 mg/kg/day. Viramidine was effective at 80 and 160 mg/kg/day but not at 40 mg/kg/day. Active drug doses improved lung consolidation scores and lung weights, with decreases in lung virus titres also noted. Arterial oxygen saturation values in treated groups were significantly better than those of the placebo group on days 7-11 of the infection. Oseltamivir (5 mg/kg/day) and ribavirin (40 mg/kg/day) were used alone and in combination to determine how late after infection they could be beneficially administered. Ribavirin alone was very effective (90-100% survival of mice) when treatments started as late as 3 days after infection. Forty percent survival was evident even when treatments started 4 days post-infection. Oseltamivir was active starting treatments 1 day after virus exposure, but lost considerable efficacy when treatments began after that time. The combination of ribavirin and oseltamivir appeared to be no better than ribavirin alone, due to the stronger beneficial effect of ribavirin in this model. The overall results demonstrate that mannan can be used to enhance certain non-lethal influenza virus infections sufficiently to allow antiviral studies.     

Effect of oral gavage treatment with ZnAL42 and other metallo-ion formulations on influenza A H5N1 and H1N1 virus infections in mice

Avian influenza H5N1 infections can cause severe, lethal human infections. Whether influenza A virus treatments effectively ameliorate avian influenza H5N1 human infections is uncertain. The research objective was to evaluate the efficacy of novel zinc and other metallo-ion formulations in two influenza A mouse models. Mice infected with influenza A/Duck/MN/1525/81 (H5N1) virus were treated orally 48 h before virus exposure and then twice daily for 13 days with ZnAL42. The optimal dosing regimen for ZnAL42 was achieved at 17.28 mg/kg 48 h prior to virus exposure, twice daily for 7 days. The survival rate was 80% compared with 10% in the untreated control group and a 100% survival rate with ribavirin (75 mg/kg/day, twice a day for 5 days, beginning 4 h before virus exposure). ZnAL42 treatment significantly lessened the decline in arterial oxygen saturation (SaO2; P < 0.001). This regimen was also well tolerated by the mice. Manganese and selenium formulations were not inhibitory to virus replication when given therapeutically. Mice were also infected with influenza A/NWS/33 (H1N1) virus and were treated 48 h before virus exposure with three dosages of ZnAL42 (8.64, 1.46 or 0.24 mg/kg/day). Treatment was by oral gavage twice daily for 13 days. The highest dose of ZnAL42 was significantly inhibitory to the virus infection as seen by prevention of deaths and lessening of decline in SaO2. The data suggest that the prophylactic use of ZnAL42 is effective against avian influenza H5N1 or H1N1 virus infection in mice and should be further explored as an option for treating human influenza virus infections.     

Genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12

Despite the efficacy of IL-12 in cancer experimental models, clinical trials with systemic recombinant IL-12 showed unacceptable toxicity related to endogenous IFNgamma production. We report that systemic administration of a recombinant adenovirus encoding IL-12 (AdCMVmIL-12) has a dramatically different survival outcome in a number of mouse pure strains over a wide range of doses. For instance at 2.5 x 10(9) p.f.u., systemic AdCMVmIL-12 killed all C57BL/6 mice but spared all BALB/c mice. Much higher IFNgamma concentrations in serum samples of C57BL/6 than in those from identically treated BALB/c were found. Causes for heterogeneous toxicity can be traced to differences among murine strains in the levels of gene transduction achieved in the liver, as assessed with adenovirus coding for reporter genes. In accordance, IL-12 serum concentrations are higher in susceptible mice. In addition, sera from C57BL/6 mice treated with AdCMVmIL-12 showed higher levels of IL-18, a well-known IFNgamma inducer. Interestingly, lethal toxicity in C57BL/6 mice was abolished by administration of blocking anti-IFNgamma mAbs and also by simultaneous depletion of T cells, NK cells, and macrophages. These observations together with the great dispersion of IFNgamma produced by human PBMCs upon in vitro stimulation with IL-12, or infection with recombinant adenovirus encoding IL-12, suggest that patients might also show heterogeneous degrees of toxicity in response to IL-12 gene transfer.     

Effect of intramuscularly injected polyprenols on influenza virus infection in mice

This study demonstrates the possibility of achieving a prophylactic effect by intramuscular injection of Abies sibirica polyprenols for the control of influenza virus infection in mice. One of the five polyprenol preparations tested, preparation N1, which had the lowest hydrophilic-lipophilic balance (8.6), produced a significant protective effect when injected in a dose of 2000 microg/mouse 2 days before aerosol infection of mice with influenza virus. A moderate protective effect was also observed using a second preparation, designated N2. One day after aerosol infection, animals pre-treated with 2000 microg doses of the polyprenol preparations or Hanks' solution showed no difference in the level of interferon accumulation in the lungs. Three days after injection of preparation N2 and N1, a significant decrease in spleen and thymus weights was, observed in the mice. One day after injection of these preparations, the number of lymphocytes in the bronchoalveolar tract of the mice exceeded almost twice that seen in mice treated with placebo. After 3 days, relative and absolute numbers of macrophages decreased, whereas those of lymphocytes increased significantly. Three days after the administration of preparations N1 and N2, macrophages became approximately twice as active in absorbing zymozan granules. Preparation N1 affected the system of superoxide radical anion production to a greater extent than preparation N2. The production of radical anions by the macrophages of the bronchoalveolar tract in the mice, 1 day after intramuscular injection of preparation N1, was significantly higher than that seen on day 3 and that induced by preparation N2 1 and 3 days after injection. These data indicate that emulsions of polyprenols that have relatively low hydrophilic-lipophilic balance, inhibit influenza virus infection in mice through a modulation of the host immune response.     

Utilization of alpha-1-acid glycoprotein levels in the serum as a parameter for in vivo assay of influenza virus inhibitors

Alpha-1-acid glycoprotein (AGP), an acute phase protein in serum assayed by single radial immunodiffusion using a commercially available kit, was found to significantly increase in mice infected with influenza A and B viruses. Experiments were run to determine the rate of increase of serum AGP and its relation to other influenza disease parameters, including lung consolidation, development of lung virus titres, decline in arterial oxygen saturation (SaO2), histopathological changes in the lung, and death of the animal. Maximal AGP levels occurred by day 3 in the animals, at about the same time lung virus titres reached their peak and inflammatory effects were evident in the lung. Serum levels of AGP were then compared with other disease parameters in the evaluation of the anti-influenza A and B virus efficacy of oseltamivir and ribavirin in mice. Treatment was by oral gavage twice daily for 5 days, beginning 4 h before virus exposure using doses of 100, 10, and 1 mg/kg per day of oseltamivir and 75 mg/kg per day of ribavirin. Against the influenza A infection, significant inhibition of death, SaO2 decline, and lung consolidation was seen at all doses of each compound; day-6 AGP levels were reduced in a dose-responsive manner. Lung virus titres were lessened at this time, but to a significant degree only at the high dose of oseltamivir and by ribavirin. The influenza B virus infection, which appeared more severe than the influenza A infection, was also significantly inhibited by both compounds, but to a lesser extent. The serum AGP levels were again lessened by therapy with both compounds. The influence of challenge dose of influenza A virus on AGP level and on the antiviral activity of 20 mg/kg per day of oseltamivir, administered by oral gavage, was determined in mice. The AGP level was in proportion to the viral challenge dose; oseltamivir significantly inhibited AGP levels and all other disease parameters regardless of size of viral inoculum. These data indicate murine AGP levels to be markedly stimulated by infection with influenza A and B viruses, and the level of the protein to be an additional measure of antiviral efficacy.     

Efficacy of orally administered T-705 on lethal avian influenza A (H5N1) virus infections in mice

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was inhibitory to four strains of avian H5N1 influenza virus in MDCK cells, with the 90% effective concentrations ranging from 1.3 to 7.7 microM, as determined by a virus yield reduction assay. The efficacy was less than that exerted by oseltamivir carboxylate or zanamivir but was greater than that exerted by ribavirin. Experiments with mice lethally infected with influenza A/Duck/MN/1525/81 (H5N1) virus showed that T-705 administered per os once, twice, or four times daily for 5 days beginning 1 h after virus exposure was highly inhibitory to the infection. Dosages from 30 to 300 mg/kg of body weight/day were well tolerated; each prevented death, lessened the decline of arterial oxygen saturation (SaO(2)), and inhibited lung consolidation and lung virus titers. Dosages from 30 to 300 mg/kg/day administered once or twice daily also significantly prevented the death of the mice. Oseltamivir (20 mg/kg/day), administered per os twice daily for 5 days, was tested in parallel in two experiments; it was only weakly effective against the infection. The four-times-daily T-705 treatments at 300 mg/kg/day could be delayed until 96 h after virus exposure and still significantly inhibit the infection. Single T-705 treatments administered up to 60 h after virus exposure also prevented death and the decline of SaO(2). Characterization of the pathogenesis of the duck influenza H5N1 virus used in these studies was undertaken; although the virus was highly pathogenic to mice, it was less neurotropic than has been described for clinical isolates of the H5N1 virus. These data indicate that T-705 may be useful for the treatment of avian influenza virus infections.     

Activities of oseltamivir and ribavirin used alone and in combination against infections in mice with recent isolates of influenza A (H1N1) and B viruses

Mouse models have been widely used for evaluating potential influenza virus inhibitors. However, the viral strains traditionally used in these models are fairly old and do not represent currently circulating viruses in nature. We developed two new lethal infection models in mice using mouse-adapted influenza A/New Caledonia/20/99 (H1N1) and influenza B/Sichuan/379/99 viruses. Both virus infections were used to study oral treatment with oseltamivir and ribavirin, both alone and in combination. Oral treatments were given twice daily for 5 days starting 4 h before infection in initial studies. Against influenza A, oseltamivir was active at 10, 20, and 40 mg/kg/day, protected 80-100% of mice from death and reduced lung consolidation - ribavirin was similarly effective at 20, 40, and 80 mg/kg/day. When treatments were initiated after virus challenge, delaying treatment with oseltamivir even 1 day caused it to be ineffective. Ribavirin prevented mortality by 50-80% when treatments were delayed 1-4 days after infection. The combination of the two drugs (oseltamivir at 20 mg/kg/day and ribavirin at 40 mg/kg/day) was no better than ribavirin alone. In contrast to what we observed with influenza A virus infections, oseltamivir and ribavirin showed similar dose-related antiviral activities against influenza B virus infections. The compounds both significantly increased survival when treatments started up to 4 days after infection, but ribavirin was more active than oseltamivir (50-80% survival compared to 30-40% survival, respectively, when starting treatments on days 2-4 after infection). By varying the doses of each drug that were used in combination (oseltamivir at 1.25, 2.5 and 5 mg/kg/day; ribavirin at 5, 10 and 20 mg/kg/day) certain dosage combinations were superior to either compound used alone as assessed by decreased mortality, lung virus titre, lung score and lung weight parameters. These activities differed from published results with older, more established virus strains as oseltamivir was less effective and ribavirin was more active than previously reported.     

In vitro and in vivo protection against the highly pathogenic H5N1 influenza virus by an antisense phosphorothioate oligonucleotide

BACKGROUND: Current vaccination strategies and antiviral drugs only provide limited protection against influenza virus infection. In this study, we investigated the use of a novel antisense oligonucleotide (named IV-AS), which is specific for the 5'-terminal conserved sequence found in all eight viral RNA segments of influenza A virus. METHODS: The activity of IV-AS was monitored both in vitro, in Madin-Darby canine kidney (MDCK) cells, and in vivo using a mouse model. IV-AS was given intranasally to H5N1-infected mice once daily for 6 days starting 6 h after infection. A three-base mismatch of IV-AS was used as a control. RESULTS: IV-AS inhibited influenza virus A induced cytopathic effects in MDCK cells with the 50% effective concentration (EC50) ranging from 2.2 to 4.4 microM. IV-AS was effective against H5N1 virus in preventing death, lessening weight reduction, inhibiting lung consolidation and reducing lung virus titres. Dosages of 40 and 60 mg/kg/day provided 40% and 60% survival rates and prolonged mean survival days in comparison with the infected control group (P<0.05). The lung index in mice treated with IV-AS, at a dose of 20, 40 or 60 mg/kg/day, had been inhibited on day 4 or 6 (P<0.05 or P<0.01); virus titres in lung had declined to 2.42, 1.51 and 1.54 log10 TCID50/g of lung, respectively, whereas the yields in the infected control mice were 6.00 log10 TCID50/g of lung. CONCLUSIONS: Our results suggest that the 5'-terminal conserved region of influenza A virus RNA segments can be targeted using antisense technology; therefore, IV-AS is a potential drug for prophylaxis and control of influenza virus infections.     

Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.     

Efficacy of Repeated Intravenous Administration of Peramivir against Highly Pathogenic Avian Influenza A (H5N1) Virus in Cynomolgus Macaques

Highly pathogenic avian influenza A (H5N1) viruses cause severe and often fatal disease in humans. We evaluated the efficacy of repeated intravenous dosing of the neuraminidase inhibitor peramivir against highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) infection in cynomolgus macaques. Repeated dosing of peramivir (30 mg/kg/day once a day for 5 days) starting immediately after infection significantly reduced viral titers in the upper respiratory tract, body weight loss, and cytokine production and resulted in a significant body temperature reduction in infected macaques compared with that of macaques administered a vehicle (P < 0.05). Repeated administration of peramivir starting at 24 h after infection also resulted in a reduction in viral titers and a reduction in the period of virus detection in the upper respiratory tract, although the body temperature change was not statistically significant. The macaque model used in the present study demonstrated that inhibition of viral replication at an early time point after infection by repeated intravenous treatment with peramivir is critical for reduction of the production of cytokines, i.e., interleukin-6 (IL-6), tumor necrosis factor α, gamma interferon, monocyte chemotactic protein 1, and IL-12p40, resulting in amelioration of symptoms caused by highly pathogenic avian influenza virus infection.     

Treatment with interleukin 12 in combination with atovaquone or clindamycin significantly increases survival of mice with acute toxoplasmosis.

The capacity of interleukin 12 (IL-12) to potentiate drugs in the treatment of murine toxoplasmosis was examined. IL-12 (100 ng/injection), atovaquone (10 mg/kg of body weight/day), or clindamycin (5 mg/kg/day) administered alone caused delayed time to death or minimal survival rates. In contrast, significant survival rates resulted when the same dose of IL-12 was used in combination the same doses of atovaquone (P=0.01) or clindamycin (P=0.001). Infected mice treated with IL-12 plus drug produced significantly higher levels of gamma interferon than controls. Although IL-12 was effective only when administered before infection, these results suggest that this cytokine may be a useful adjunct in the therapy of human toxoplasmosis in situations when cysts reactivate and tachyzoites start multiplying in immunocompromised patients.     

The stimulatory effects of interleukin (IL)-12 on hematopoiesis are antagonized by IL-12-induced interferon gamma in vivo

Interleukin (IL)-12 synergizes with other cytokines to stimulate the proliferation and differentiation of early hematopoietic progenitors in vitro. However, in vivo administration of IL-12 decreases peripheral blood counts and bone marrow hematopoiesis. Here, we used interferon (IFN) gamma receptor-deficient (IFN gamma R-/-) mice to investigate whether the in vivo inhibition of hematopoiesis by IL-12 is indirectly mediated by IL-12-induced IFN-gamma. IL-12 administered for 4 d (1 microgram/mouse per day) resulted in lower peripheral blood counts and a 2-fold decrease in bone marrow cellularity in wild-type mice, but not in IFN gamma R-/- mice. Bone marrow hematopoietic progenitors were decreased after IL-12 treatment in wild-type mice, but rather increased in IFN gamma R-/- mice. Splenic cellularity was 2.3-fold higher after IL-12 administration in wild-type mice, largely due to natural killer (NK) cell and macrophage infiltration together with some extramedullary hematopoiesis. In IFN gamma R-/- mice, spleen cellularity was less increased, there were fewer infiltrating NK cells, but a strong extramedullary hematopoiesis. Thus, alterations mediated by IL-12- induced IFN-gamma include reduction in bone marrow cellularity and hematopoietic progenitors, as well as pronounced splenomegaly, largely caused by NK cell infiltration. In the absence of IFN-gamma signaling, IL-12 promotes hematopoiesis, consistent with its in vitro activities.     

Cytokine treatment of central nervous system infection: efficacy of interleukin-12 alone and synergy with conventional antifungal therapy in experimental cryptococcosis.

Cell-mediated immune responses appear to be critical in the outcome of cryptococcosis. Interleukin-12 (IL-12) was studied for its potential use as a therapeutic agent because of its stimulation of natural killer cells and gamma interferon production by stimulated T cells and natural killer cells. Gamma interferon-activated macrophages are important in host resistance against cryptococcosis. In two separate studies, male BALB/c mice were infected intravenously with Cryptococcus neoformans. In the first study, mice received either no treatment, 5.0 mg of fluconazole alone per kg of body weight per day (by gavage twice daily), or IL-12 subcutaneously at 0.01, 0.1, or 1.0 microgram/day once daily (low-dose study) alone or in combination with 5.0 mg of fluconazole per kg/day. In a second study (high dose), the dosages of IL-12 used were 1.0, 2.5, or 5.0 micrograms/day. Therapy was given for 10 consecutive days, and the number of CFU of C. neoformans remaining in various organs was quantitated 1 or 2 days after administration of the last dose. In the low-dose study, IL-12 at 0.1 or 1.0 microgram reduced the level of brain infection by approximately 10-fold (P < 0.05) and IL-12 at 1.0 or 0.1 microgram/day enhanced the efficacy of fluconazole. In liver, both the efficacy of IL-12 alone (0.01 or 0.1 microgram; P < 0.05) and enhancement of the efficacy of fluconazole (P < 0.05) were seen. No efficacy of IL-12 was seen in spleens or lungs, although spleen weights increased fourfold in mice given 1.0 microgram of IL-12 per day. In the high-dose study, all IL-12 doses alone again reduced the levels of brain infection (5- to 8-fold; P < 0.05) when the two were given in combination. No overt toxicities were observed at any dose, and overall, 1.0 microgram of IL-12 per day was found to be the optimal dosage for reducing infection in the brain. To our knowledge, this is the first demonstration of the efficacy of cytokine therapy in systemic and particularly brain infections with C. neoformans. The stimulation of cell-mediated immunity represents a new approach to therapy and can enhance suboptimal antimicrobial chemotherapy. IL-12 should be considered for further study and for clinical trials. These studies suggest that other opportunistic central nervous system pathogens should also be investigated.     

Evaluation of the anti-influenza virus activities of 1,3,4-thiadiazol-2-ylcyanamide (LY217896) and its sodium salt.

1,3,4-Thiadiazol-2-ylcyanamide (LY217896) and its sodium salt were shown to be effective against influenza A and B viruses in vitro and in the mouse model. In nondividing confluent MDCK cells, the 50% inhibitory concentration of LY217896 ranged from 0.37 to 1.19 micrograms/ml against various strains of influenza A virus and from 0.75 to 1.54 micrograms/ml against various strains of influenza B virus, with no apparent cytotoxicity. However, at a concentration of 0.31 microgram/ml, LY217896 inhibited the replication of dividing MDCK cells. LY217896 (9 mg/m2 of body surface area per day) administered in the diet, in the drinking water, by oral gavage, by intraperitoneal injection, or by aerosolization was well tolerated and protected CD-1 mice infected with a lethal dose of influenza A or B virus. Effective administration of the compound could be delayed for up to 96 h postinfection. Virus titer was reduced by 1 to 2 log10 units in lungs of mice given LY217896 in the drinking water. Mice treated initially with protective levels of LY217896 were resistant to a subsequent challenge of influenza virus in the absence of the compound, indicating that the animals were able to develop immunity to the initial infection. Administration of LY217896 to uninfected mice did not induce interferon-like activity or interfere with natural killer cell function. In the ferret, LY217896 was effective in preventing fever induced by influenza virus.     

Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells.

We investigated the role of antigen-presenting cells in early interferon (IFN)-gamma production in normal and recombinase activating gene 2-deficient (Rag-2(-/-)) mice in response to Listeria monocytogenes (LM) infection and interleukin (IL)-12 administration. Levels of serum IFN-gamma in Rag-2(-/-) mice were comparable to those of normal mice upon either LM infection or IL-12 injection. Depletion of natural killer (NK) cells by administration of anti-asialoGM1 antibodies had little effect on IFN-gamma levels in the sera of Rag-2(-/-) mice after LM infection or IL-12 injection. Incubation of splenocytes from NK cell-depleted Rag-2(-/-) mice with LM resulted in the production of IFN-gamma that was completely blocked by addition of anti-IL-12 antibodies. Both dendritic cells (DCs) and monocytes purified from splenocytes were capable of producing IFN-gamma when cultured in the presence of IL-12. Intracellular immunofluorescence analysis confirmed the IFN-gamma production from DCs. It was further shown that IFN-gamma was produced predominantly by CD8alpha+ lymphoid DCs rather than CD8alpha- myeloid DCs. Collectively, our data indicated that DCs are potent in producing IFN-gamma in response to IL-12 produced by bacterial infection and play an important role in innate immunity and subsequent T helper cell type 1 development in vivo.     

Mechanism of interleukin 12-mediated toxicities during experimental viral infections: role of tumor necrosis factor and glucocorticoids

Interleukin 12 (IL-12) doses in excess of 100 ng/d have been shown to induce profound immunotoxicities in mice infected with lymphocytic choriomeningitis virus (LCMV). These immunotoxicities are characterized by almost complete inhibition of virus-induced CD8+ T cell expansion and CTL activation, and up to 2 log increases in viral replication. They are accompanied by induction of serum tumor necrosis factor (TNF). The studies presented here were undertaken to characterize mechanisms for the IL-12-induced toxicities and to examine expression and function of TNF in this context. Several physiological changes were induced in IL-12-treated uninfected and dramatically elevated in IL-12-treated virus-infected mice. IL-12 induced (a) decreases in body weights, > 10% in uninfected and > 20% in LCMV-infected mice; (b) elevation of circulating glucocorticoid levels to > 10 micrograms/dl in uninfected and > 20 micrograms/dl in infected mice; and (c) decreases in thymic mass, > 30% in uninfected and up to 95% in infected mice. These changes are known to be associated with circulating TNF. Northern blot and in situ hybridization analyses demonstrated that IL-12 induced TNF-alpha expression and that LCMV infection synergized with IL-12 for induction of this factor. Antibodies neutralizing TNF reversed all of the IL-12- induced toxicities in LCMV-infected mice including the immunotoxicities against CD8+ T cells and anti-viral defenses. The TNF-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and CD8+ T cell subsets isolated from LCMV-infected mice were more sensitive to TNF- mediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. Experiments with the glucocorticoid type II receptor antagonist, RU486, demonstrated that endogenous glucocorticoids were secondary intermediaries in IL-12-induced thymic atrophy. Studies in IL-2-deficient mice showed that the synergism was dependent upon endogenous IL-2. The results delineate a unique mechanism of TNF-mediated toxicity. In addition, they have significant implications concerning potential detrimental consequences of in vivo TNF induction and of IL-12 administration for protective anti-viral responses.     

Direct Administration in the Respiratory Tract Improves Efficacy of Broadly Neutralizing Anti-Influenza Virus Monoclonal Antibodies

The emergence of influenza virus strains resistant to approved neuraminidase inhibitors and the time constrains after infection when these drugs can be effective constitute major drawbacks for this class of drugs. This highlights a critical need to discover new therapeutic agents that can be used for the treatment of influenza virus-infected patients. The use of broadly neutralizing anti-influenza monoclonal antibodies (MAbs) has been sought as an alternative immunotherapy against influenza infection. Here, we tested in mice previously characterized broadly neutralizing anti-hemagglutinin (HA) stalk MAbs prophylactically and therapeutically using different routes of administration. The efficacy of treatment against an influenza H1N1 pandemic virus challenge was compared between two systemic routes of administration, intraperitoneal (i.p.) and intravenous (i.v.), and two local routes, intranasal (i.n.) and aerosol (a.e.). The dose of MAb required for prophylactic protection was reduced by 10-fold in animals treated locally (i.n. or a.e.) compared with those treated systemically (i.p. or i.v.). Improved therapeutic protection was observed in animals treated i.n. on day 5 postinfection (60% survival) compared with those treated via the i.p. route (20% survival). An increase in therapeutic efficacy against other influenza virus subtypes (H5N1) was also observed when a local route of administration was used. Our findings demonstrate that local administration significantly decreases the amount of broadly neutralizing monoclonal antibody required for protection against influenza, which highlights the potential use of MAbs as a therapeutic agent for influenza-associated disease.