Equine Polyclonal Antibodies Prevent Acute Chikungunya Virus Infection in Mice.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen that causes chikungunya disease (CHIK); the disease is characterized by fever, muscle ache, rash, and arthralgia. This arthralgia can be debilitating and long-lasting, seriously impacting quality of life for years. Currently, there is no specific therapy available for CHIKV infection. We have developed a despeciated equine polyclonal antibody (CHIKV-EIG) treatment against CHIKV and evaluated its protective efficacy in mouse models of CHIKV infection. In immunocompromised (IFNAR-/-) mice infected with CHIKV, daily treatment for five consecutive days with CHIKV-EIG administered at 100 mg/kg starting on the day of infection prevented mortality, reduced viremia, and improved clinical condition as measured by body weight loss. These beneficial effects were seen even when treatment was delayed to 1 day after infection. In immunocompetent mice, CHIKV-EIG treatment reduced virus induced arthritis (including footpad swelling), arthralgia-associated cytokines, viremia, and tissue virus loads in a dose-dependent fashion. Collectively, these results suggest that CHIKV-EIG is effective at preventing CHIK and could be a viable candidate for further development as a treatment for human disease.

Potent inhibition of arenavirus infection by a novel fusion inhibitor.

Several arenaviruses, including Lassa and Lujo viruses in Africa and five New World arenavirus (NWA) species in the Americas, cause life-threatening viral hemorrhagic fevers. In the absence of licensed antiviral therapies, these viruses pose a significant public health risk. The envelope glycoprotein complex (GPC) mediates arenavirus entry through a pH-dependent fusion of the viral and host endosomal membranes. It thus is recognized as a viable target for small-molecule fusion inhibitors. Here, we report on the antiviral activity and pre-clinical development of the novel broad-spectrum arenavirus fusion inhibitors, ARN-75039 and ARN-75041. In Tacaribe virus (TCRV) pseudotyped and native virus assays, the ARN compounds were active in the low to sub-nanomolar range with selectivity indices exceeding 1000. Pharmacokinetic analysis of the orally administered compounds revealed an extended half-life in mice supporting once-daily dosing, and the compounds were well tolerated at the highest tested dose of 100 mg/kg. In a proof-of-concept prophylactic efficacy study, doses of 10 and 35 mg/kg of either compound dramatically improved survival outcome and potently inhibited TCRV replication in serum and various tissues. Additionally, in contrast to surviving mice that received ribavirin or placebo, animals treated with ARN-75039 or ARN-75041 were cured of TCRV infection. In a follow-up study with ARN-75039, impressive therapeutic efficacy was demonstrated under conditions where treatment was withheld until after the onset of disease. Taken together, the data strongly support the continued development of ARN-75039 as a candidate therapeutic for the treatment of severe arenaviral diseases.

Strain-dependent disease and response to favipiravir treatment in mice infected with Chikungunya virus.

Antiviral countermeasures are needed to reduce the morbidity associated with Chikungunya virus (CHIKV) infection. This arbovirus reemerged in 2004 and causes periodic outbreaks in various areas throughout the world. While infection is rarely lethal, the majority of people infected with the virus develop a hallmark arthralgia as well as other disease manifestations. The virus is classified within three phylogenetic groups, namely, West African, East/Central/South African (ECSA), and Asian. Six strains of CHIKV covering the three phylogenetic groups were studied for their replication in cell culture, their ability to cause disease in susceptible mouse strains and susceptibility to antiviral treatment. Differential replication kinetics were observed for various CHIKV isolates in cell culture, which coincided with a decreased sensitivity to antiviral treatment as compared with ECSA and Asian clade viruses. This was confirmed in mouse infection studies with severe disease observed in mice infected with West African clade viruses, mild disease phenotype after infection with Asian clade viruses and an intermediate disease severity associated with ECSA virus infection. We also tested a broadly active antiviral, Favipiravir (T-705), which activity was inversely proportional to disease severity. These data suggest that some clades of CHIKV may cause more severe disease and may be more difficult to treat.

Vascular Leak and Hypercytokinemia Associated with Severe Fever with Thrombocytopenia Syndrome Virus Infection in Mice.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral hemorrhagic fever (VHF) endemic to China, South Korea, Japan, and Vietnam. Here we characterize the pathogenesis and natural history of disease in IFNAR-/- mice challenged with the HB29 strain of SFTS virus (SFTSV) and demonstrate hallmark features of VHF such as vascular leak and high concentrations of proinflammatory cytokines in blood and tissues. Treatment with FX06, a natural plasmin digest product of fibrin in clinical development as a treatment for vascular leak, reduced vascular permeability associated with SFTSV infection but did not significantly improve survival outcome. Further studies are needed to assess the role of vascular compromise in the SFTS disease process modeled in IFNAR-/- mice.

Combinations of L-NG-monomethyl-arginine and oseltamivir against pandemic influenza A virus infections in mice.

L-NG-monomethyl-arginine (L-NMMA) is an experimental compound that suppresses nitric oxide production in animals. The compound was combined with oseltamivir to treat lethal influenza A/California/04/2009 (H1N1) pandemic virus infections in mice. Treatments were given twice a day for five days starting 4 h (oseltamivir, by oral gavage) or three days (L-NMMA, by intraperitoneal route; corresponding to the time previously reported for nitric oxide induction in the animals) after infection. Low doses of oseltamivir were used in order to demonstrate synergy or antagonism. Oseltamivir monotherapy protected 70% of mice from death at 1 mg/kg/day. L-NMMA (40 and 80 mg/kg/day) was ineffective alone in preventing mortality. Compared to oseltamivir treatment alone, L-NMMA combined with oseltamivir was synergistically effective (as evaluated by three-dimensional MacSynergy analysis), resulting in survival increases from 20 to 70% when 40 or 80 mg/kg/day of L-NMMA was combined with 0.3 mg/kg/day of oseltamivir, and from 70 to 100% survival increases when these doses were combined with 1 mg/kg/day of oseltamivir. These data demonstrate that a nitric oxide inhibitor such as L-NMMA has the potential to be beneficial when combined with oseltamivir in treating influenza virus infections.

A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response.

Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza.

Low-dose ribavirin potentiates the antiviral activity of favipiravir against hemorrhagic fever viruses.

Favipiravir is approved in Japan to treat novel or re-emerging influenza viruses, and is active against a broad spectrum of RNA viruses, including Ebola. Ribavirin is the only other licensed drug with activity against multiple RNA viruses. Recent studies show that ribavirin and favipiravir act synergistically to inhibit bunyavirus infections in cultured cells and laboratory mice, likely due to their different mechanisms of action. Convalescent immune globulin is the only approved treatment for Argentine hemorrhagic fever caused by the rodent-borne Junin arenavirus. We previously reported that favipiravir is highly effective in a number of small animal models of Argentine hemorrhagic fever. We now report that addition of low dose of ribavirin synergistically potentiates the activity of favipiravir against Junin virus infection of guinea pigs and another arenavirus, Pichinde virus infection of hamsters. This suggests that the efficacy of favipiravir against hemorrhagic fever viruses can be further enhanced through the addition of low-dose ribavirin.

Enhanced efficacy of cidofovir combined with vaccinia immune globulin in treating progressive cutaneous vaccinia virus infections in immunosuppressed hairless mice.

The treatment of progressive vaccinia in individuals has involved antiviral drugs, such as cidofovir (CDV), brincidofovir, and/or tecovirimat, combined with vaccinia immune globulin (VIG). VIG is costly, and its supply is limited, so sparing the use of VIG during treatment is an important objective. VIG sparing was modeled in immunosuppressed mice by maximizing the treatment benefits of CDV combined with VIG to determine the effective treatments that delayed the time to death, reduced cutaneous lesion severity, and/or decreased tissue viral titers. SKH-1 hairless mice immunosuppressed with cyclophosphamide and hairless SCID mice (SHO strain) were infected cutaneously with vaccinia virus. Monotherapy, dual combinations (CDV plus VIG), or triple therapy (topical CDV, parenteral CDV, and VIG) were initiated 2 days postinfection and were given every 3 to 4 days through day 11. The efficacy assessment included survival rate, cutaneous lesion severity, and viral titers. Delays in the time to death and the reduction in lesion severity occurred in the following order of efficacy: triple therapy had greater efficacy than double combinations (CDV plus VIG or topical plus parenteral CDV), which had greater efficacy than VIG alone. Parenteral administration of CDV or VIG was necessary to suppress virus titers in internal organs (liver, lung, and spleen). The skin viral titers were significantly reduced by triple therapy only. The greatest efficacy was achieved by triple therapy. In humans, this regimen should translate to a faster cure rate, thus sparing the amount of VIG used for treatment.

Protection against Chikungunya virus induced arthralgia following prophylactic treatment with adenovirus vectored interferon (mDEF201).

Recent outbreaks of Chikungunya virus (CHIKV) infection have resulted in millions of cases of disease with significant morbidity. No approved antiviral treatments exist for the prevention or treatment of this viral disease. Infection with CHIKV results in a high rate of symptomatic disease that primarily includes a debilitating arthralgia. To model this cardinal disease manifestation, adult DBA/1J mice were challenged with CHIKV by footpad injection. Viremia and hind limb virus titers increased ∼100-fold while spleen virus increased >1000-fold within 1day post-virus infection (dpi). Footpad swelling was measured over a 10-day period, with peak swelling observed between 6 and 7dpi. Histology of the hind leg at the site of virus challenge showed evidence of myositis and synovitis starting on 5dpi. Cytokine profiling of the hind limb at the site of inoculation revealed a biphasic inflammatory response represented by an increase in IL-6, MCP-1, IFN-γ, MIP-1α, RANTES, and IL-17. To investigate the prophylactic capacity of IFN, mice were treated with mDEF201, an adenovirus-vectored IFN-α. Intranasal administration of a single 10(7)pfu/ml dose of mDEF201 administered 21days to 24h prior to infection, significantly reduced footpad swelling, virus titers in the hind leg and spleen, and several inflammatory cytokines. Efficacy was not observed when treatment was initiated 24h after virus challenge. This arthralgia model of CHIKV recapitulates relevant disease features commonly observed in human disease making it applicable to preclinical testing of therapies that target both viral replication and the associated joint disease.

A mouse model of chikungunya virus with utility in antiviral studies.

Chikungunya virus (CHIKV) infection generally causes a debilitating arthritis in infected patients. Infection with CHIKV is generally not life-threatening and is associated with a mortality rate <0.1%. However, close to 100% of those infected will develop symptoms of disease, primarily involving swelling and pain of the joints, which can last for months or even years. A model that mimics these symptoms is needed for thedevelopment of therapies to ameliorate disease and control viral infection. In this chapter, we describe the establishment of a model of CHIKV infection in mice that is nonlethal and utilizes footpad swelling and virus titer of various tissues as key disease parameters. This model was developed primarily for use in evaluating the in vivo efficacy of candidate antiviral agents, although important questions regarding basic biology and pathogenesis of the disease may also be elucidated using this system.

Use of plethysmography in assessing the efficacy of antivirals in a mouse model of pandemic influenza A virus.

The recently emerged swine-origin H1N1 influenza A virus (IAV) caused a pandemic outbreak in 2009 with higher risk of severe disease among children and pregnant women in their third trimester (Van Kerkhove et al., 2011), and is continuing to be important seasonal IAV strain. Mice are commonly used in antiviral studies as models of influenza disease, which utilize morbidity and mortality to assess the efficacy of a test compound. Here, we investigated the utility of unrestrained plethysomography to quantify the lung function of IAV-infected BALB/c mice. Administration of a lethal dose (∼30X LD(50)) of pandemic H1N1 IAV resulted in a rapid decline in breath volume, as determined by a significant (P<0.001) decrease in the pressure associated with inspiration and expiration detected as early as 2 days after virus challenge. Severe disease was also accompanied by a significant (P<0.05) increase in breath time on 8 dpi. Plethysmography parameters correlated with weight loss and other parameters of disease such as gross pathology and the weight of the lung. Breath time was reduced in surviving mice challenged with a sublethal dose of virus as compared with normal controls, and is a predictive indicator of outcome in these mice. In antiviral studies, the use of plethysmography resulted in the detection of a clear and rapid treatment response, which was similar to other non-invasive parameters, such as weight change. Oseltamivir and ribavirin significantly (P<0.001) improved parameters of lung function, particularly mean breath volume, as early as 2 dpi and in a dose-dependent manner. Moreover, a combination of these two drugs further improved these parameters. Plethysmography provides a sensitive evaluation of lung function in IAV-infected mice in response to antiviral therapy.