Kie-Hoon Jung

In Vitro and In Vivo Activities of T-705 against Arenavirus and Bunyavirus Infections

ABSTRACT There is a need for the development of effective antivirals for the treatment of severe viral diseases caused by members of the virus families Bunyaviridae and Arenaviridae. The pyrazine derivative T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has demonstrated remarkable antiviral activity against influenza virus and, to a lesser degree, against some other RNA viruses (Y. Furuta, K. Takahashi, Y. Fukuda, M. Kuno, T. Kamiyama, K. Kozaki, N. Nomura, H. Egawa, S. Minami, Y. Watanabe, H. Narita, and K. Shiraki, Antimicrob. Agents Chemother., 46:977-981, 2002). Here, we report that T-705 is highly active against a panel of bunyaviruses (La Crosse, Punta Toro, Rift Valley fever, and sandfly fever viruses) and arenaviruses (Junin, Pichinde, and Tacaribe viruses) by cytopathic effect and virus yield reduction cell-based assays. The 50% effective concentrations for T-705 ranged from 5 to 30 μg/ml and 0.7 to 1.2 μg/ml against the bunyaviruses and arenaviruses examined, respectively. We also demonstrate that orally administered T-705 is efficacious in treating Punta Toro virus in the mouse and hamster infection models, as well as Pichinde virus infection in hamsters. When administered twice daily for 5 to 6 days, beginning 4 h pre- or 24 h post-Punta Toro virus challenge, a 30-mg/kg of body weight/day dose provided complete protection from death and limited viral burden and liver disease. A dose of 50 mg/kg/day was found to be optimal for treating Pichinde infection and limiting viral replication and disease severity. In general, T-705 was found to be more active than ribavirin in cell-based assays and in vivo, as reflected by substantially greater therapeutic indexes. Our results suggest that T-705 may be a viable alternative for the treatment of life-threatening bunyaviral and arenaviral infections.

TLR3 deletion limits mortality and disease severity due to phlebovirus infection

TLR3 was the first member of the TLR family of pattern recognition receptors found to detect a conserved viral molecular pattern, dsRNA, yet supporting evidence for a major role in host defense against viral pathogens is limited. Punta Toro virus (PTV) has been shown to produce severe infection in mice, modeling disease caused by the related highly pathogenic Rift Valley fever phlebovirus in humans and domesticated ungulates. Using TLR3-deficient mice, we investigated the involvement of TLR3 in host defense against PTV infection. Compared with wild-type, TLR3−/− mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic PTV infection. Infectious challenge produced comparable peak liver and serum viral loads; however, TLR3−/− mice were able to clear systemic virus at a slightly faster rate. Cytokine profiling suggests that TLR3 plays an important role in PTV pathogenesis through the overproduction of inflammatory mediators, which may be central to the observed differences in survival and disease severity. Compared with TLR3-deficient mice, IL-6, MCP-1, IFN-γ, and RANTES were all present at higher levels in wild-type animals. Most dramatic was the exaggerated levels of IL-6 found systemically and in liver tissue of infected wild-type mice; however, IL-6-deficient animals were found to be more susceptible to lethal PTV infection. Taken together, we conclude that the TLR3-mediated response to PTV infection is detrimental to disease outcome and propose that IL-6, although critical to establishing antiviral defense, contributes to pathogenesis when released in excess, necessitating its controlled production as is seen with TLR3−/− mice.

TLR3 Is Essential for the Induction of Protective Immunity against Punta Toro Virus Infection by the Double-Stranded RNA (dsRNA), Poly(I:C12U), but not Poly(I:C): Differential Recognition of Synthetic dsRNA Molecules

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C12U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C12U) therapy and failed to produce IFN-α, IFN-β, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3−/− mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C12U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.

Treatment of Late Stage Disease in a Model of Arenaviral Hemorrhagic Fever: T-705 Efficacy and Reduced Toxicity Suggests an Alternative to Ribavirin

A growing number of arenaviruses are known to cause viral hemorrhagic fever (HF), a severe and life-threatening syndrome characterized by fever, malaise, and increased vascular permeability. Ribavirin, the only licensed antiviral indicated for the treatment of certain arenaviral HFs, has had mixed success and significant toxicity. Since severe arenaviral infections initially do not present with distinguishing symptoms and are difficult to clinically diagnose at early stages, it is of utmost importance to identify antiviral therapies effective at later stages of infection. We have previously reported that T-705, a substituted pyrazine derivative currently under development as an anti-influenza drug, is highly active in hamsters infected with Pichinde virus when the drug is administered orally early during the course of infection. Here we demonstrate that T-705 offers significant protection against this lethal arenaviral infection in hamsters when treatment is begun after the animals are ill and the day before the animals begin to succumb to disease. Importantly, this coincides with the time when peak viral loads are present in most organs and considerable tissue damage is evident. We also show that T-705 is as effective as, and less toxic than, ribavirin, as infected T-705-treated hamsters on average maintain their weight better and recover more rapidly than animals treated with ribavirin. Further, there was no added benefit to combination therapy with T-705 and ribavirin. Finally, pharmacokinetic data indicate that plasma T-705 levels following oral administration are markedly reduced during the latter stages of disease, and may contribute to the reduced efficacy seen when treatment is withheld until day 7 of infection. Our findings support further pre-clinical development of T-705 for the treatment of severe arenaviral infections.

Efficacy of favipiravir (T-705) and T-1106 pyrazine derivatives in phlebovirus disease models

Several studies have reported favipiravir (T-705) to be effective in treating a number of viral diseases modeled in rodent systems. Notably, the related pyrazine derivative, T-1106, was found to be more effective than T-705 in treating yellow fever virus infection in hamsters. Based on these findings, we hypothesized that T-1106 may be more effective in treating hepatotropic Punta Toro virus (PTV, Phlebovirus) infection in rodents. In cell culture, the inhibitory concentrations of the compounds against various phleboviruses ranged from 3 to 55microM for T-705 and from 76 to 743microM for T-1106. In PTV-challenged hamsters, a model that generally presents with high liver viral loads, T-1106 was more effective at reducing mortality. However, in mice infected with PTV, a model wherein systemic infection is more prominent, the greater efficacy exhibited by T-1106 in the hamster system was not apparent. In contrast, T-705 was superior in preventing mortality in hamsters challenged with Pichinde virus (PICV, Arenavirus), an infection characterized as diffuse and pantropic. Remarkably, T-1106 has proven more active in vivo than would have been expected from our cell culture results, and our in vivo findings suggest that it is more effective in infections characterized predominantly by high levels of hepatic viral burden.

Interferon Alfacon-1 Protects Hamsters from Lethal Pichinde Virus Infection

ABSTRACT Hemorrhagic fever of arenaviral origin is a frequently fatal infectious disease of considerable priority to the biodefense mission. Historically, the treatment of arenaviral infections with alpha interferons has not yielded favorable results. Here we present evidence that interferon alfacon-1, a nonnaturally occurring bioengineered alpha interferon approved for the treatment of chronic hepatitis C, is active against Pichinde and Tacaribe arenaviruses in cell culture. In the hamster model of Pichinde virus (PCV) infection, interferon alfacon-1 treatment significantly protected animals from death, prolonged the survival of those that eventually died, reduced virus titers, and limited liver damage characteristic of PCV-induced disease. Moreover, interferon alfacon-1 also demonstrated therapeutic activity, to a lesser degree, when the initiation of treatment was delayed up to 2 days post-virus challenge. Despite the observed advantages of interferon alfacon-1 therapy, efforts to stimulate the immune system with the known interferon inducer poly(I:C12U) (Ampligen) offered only limited protection against lethal PCV challenge. Taken together, these data suggest that the increased potency of the bio-optimized interferon alfacon-1 molecule may be critical to the observed antiviral effects. These data are the first report demonstrating efficacious treatment of acute arenaviral disease with alpha interferon therapy, and further study is warranted.

Development of a new tacaribe arenavirus infection model and its use to explore antiviral activity of a novel aristeromycin analog

Background A growing number of arenaviruses can cause a devastating viral hemorrhagic fever (VHF) syndrome. They pose a public health threat as emerging viruses and because of their potential use as bioterror agents. All of the highly pathogenic New World arenaviruses (NWA) phylogenetically segregate into clade B and require maximum biosafety containment facilities for their study. Tacaribe virus (TCRV) is a nonpathogenic member of clade B that is closely related to the VHF arenaviruses at the amino acid level. Despite this relatedness, TCRV lacks the ability to antagonize the host interferon (IFN) response, which likely contributes to its inability to cause disease in animals other than newborn mice. Methodology/Principal Findings Here we describe a new mouse model based on TCRV challenge of AG129 IFN-α/β and -γ receptor-deficient mice. Titration of the virus by intraperitoneal (i.p.) challenge of AG129 mice resulted in an LD50 of ∼100 fifty percent cell culture infectious doses. Virus replication was evident in the serum, liver, lung, spleen, and brain 4–8 days after inoculation. MY-24, an aristeromycin derivative active against TCRV in cell culture at 0.9 µM, administered i.p. once daily for 7 days, offered highly significant (P<0.001) protection against mortality in the AG129 mouse TCRV infection model, without appreciably reducing viral burden. In contrast, in a hamster model of arenaviral hemorrhagic fever based on challenge with clade A Pichinde arenavirus, MY-24 did not offer significant protection against mortality. Conclusions/Significance MY-24 is believed to act as an inhibitor of S-adenosyl-L-homocysteine hydrolase, but our findings suggest that it may ameliorate disease by blunting the effects of the host response that play a role in disease pathogenesis. The new AG129 mouse TCRV infection model provides a safe and cost-effective means to conduct early-stage pre-clinical evaluations of candidate antiviral therapies that target clade B arenaviruses.

Recombinant Eimeria Protozoan Protein Elicits Resistance to Acute Phlebovirus Infection in Mice but Not Hamsters

ABSTRACT A protein antigen from an Eimeria protozoan has recently been reported to induce antitumor activity in mice. This activity most likely results from the strong induction of interkeukin-12 (IL-12) and gamma interferon (IFN-γ), which are also essential factors in the establishment of protective immunity against viral infection. We evaluated recombinant Eimeria antigen (rEA) as a potential immunotherapeutic agent in mouse and hamster models of acute phleboviral disease. Punta Toro virus (PTV) was highly sensitive to a single dose of nanogram quantities of rEA in the mouse infection model. Intraperitoneal treatment with rEA also reduced virus load and liver damage associated with PTV infection. IL-12 was elicited following exposure of uninfected mice to quantities of rEA of 10 ng or greater, and the levels peaked at between 3 and 8 h postexposure. IFN-γ release was induced more slowly and required less rEA (1 ng) to produce a significant rise in systemic levels. The induction of IL-12 and IFN-γ involved in the coordination of innate and adaptive immune responses to microbial pathogens required myeloid differentiation factor 88, a signaling adaptor shared by most members of the Toll-like receptor (TLR) family. Despite encouraging results in the murine system, rEA failed to protect hamsters challenged with PTV. Our findings suggest that hamsters may lack functional TLR11, which has recently been shown to recognize a profilin-like protein homologous to rEA from the protozoan Toxoplasma gondii. Further investigation into the immunostimulatory capacity of rEA in other mammalian systems is necessary.

Maporal virus as a surrogate for pathogenic New World hantaviruses and its inhibition by favipiravir.

Background: Pathogenic hantaviruses geographically distributed in the Old World cause haemorrhagic fever with renal syndrome (HFRS), whereas New World hantaviruses are the aetiological agents of hantavirus cardiopulmonary syndrome (HCPS). Ribavirin, a drug associated with toxicities, is presently indicated for treatment of HFRS, whereas treatment of the more frequently lethal HCPS is limited to supportive care. Because of the need for safe and effective antivirals to treat severe hantaviral infections, we evaluated favipiravir (T-705) against Dobrava and Maporal viruses as representative Old World and New World hantaviruses, respectively. Dobrava virus causes HFRS in Europe. Maporal virus (MPRLV), recently isolated from western Venezuela, is phylogenetically similar to Andes virus, the principal cause of HCPS in Argentina. Methods: Hantavirus replication in the presence of various inhibitors was measured by focus-forming unit assays and quantitative reverse transcriptase PCR. Phylogenetic relationships were assessed by the neighbour-joining and bootstrap consensus methods. Results: Here, we show that infection of Vero E6 cells with MPRLV is dependent on β3 integrins, similar to that reported for pathogenic hantaviruses. Furthermore, by analysis of molecular determinants associated with the G1 glycoprotein cytoplasmic tail, we show the close genetic proximity of MPRLV to other HCPS-causing hantaviruses in these regions predictive of pathogenicity. We also demonstrate anti-hantavirus activity by favipiravir with inhibitory concentrations ranging from 65 to 93 μM and selectivity indices >50. Conclusions: Our data suggest that MPRLV may serve as a safer alternative to modelling infection caused by the highly lethal Andes virus and that hantaviruses are sensitive to the effects of favipiravir in cell culture.

Combinatorial ribavirin and interferon alfacon-1 therapy of acute arenaviral disease in hamsters

Several arenaviruses endemic to South America (Junin, Machupo, and Guanarito) and Africa (Lassa) are known to cause frequently fatal haemorrhagic fever. With the exception of ribavirin, which has demonstrated efficacy in cases of Lassa fever, there is no other effective therapeutic for the treatment of arenaviral haemorrhagic fever. We have recently reported that consensus interferon-α (IFN alfacon-1) can protect hamsters from lethal Pichinde virus (PCV) infection, which serves as a model for acute arenaviral disease in humans. Here we demonstrate highly effective therapy through the combined use of ribavirin with IFN alfacon-1 for the treatment of PCV infection in hamsters. Ribavirin was given orally, twice per day for 7 days, and IFN alfacon-1 was administered intraperitoneally once per day for 10 days. Treatments were initiated 1–5 days post-virus challenge using various dose combinations, many of which were less than optimal when the drugs were given independently. Combining suboptimal doses of ribavirin (5–10mg/kg/day) with IFN alfacon-1 (5–10 µg/kg/day), we were able to demonstrate increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. Our data indicate that combination therapy results in synergistic activity that may slow down the progression of the disease and decrease fatality rates associated with severe arenaviral infections in humans. Further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity.