Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis and pneumonia in infants and young children worldwide and is often the cause of infections in bone marrow, solid organ transplant, cystic fibrosis and congenital heart disease patients, as well as respiratory tract disease in elderly adults. Treatment options are limited to ribavirin, which is only marginally effective, and passive immunoprophylaxis, which is very expensive. The immunosuppressive agent leflunomide has been shown to exert potent antiviral activity against several herpesviruses and polyomavirus BK. In the current study we have tested the hypothesis that leflunomide exerts antiviral activity against RSV.
Methods
Human Hep-2 or small airway epithelial cells were inoculated with RSV and treated with A77 1726, the active metabolite of leflunomide. Syncytia formation was assessed by immunohistochemical staining, and virus yield was measured by plaque assay. Cotton rats were intranasally inoculated with RSV, treated with leflunomide by gavage, and pulmonary viral loads were measured by plaque assay of lung homogenates.
Results
Phase contrast microscopy and immunohistochemical staining demonstrated profound attenuation of RSV-induced syncytia formation in infected cultures treated with A77 1726, the active metabolite of leflunomide. Plaque assays of virus yield in RSV-inoculated cell cultures demonstrated potent, dose-dependent A77-mediated antiviral activity. Likewise, pulmonary viral loads in RSV-inoculated cotton rats were reduced by >3 log by leflunomide compared with vehicle-treated controls, even when leflunomide treatment was delayed until day 3 post-inoculation.
Conclusions
These findings suggest promise for leflunomide as a convenient, orally administered addition to the growing arsenal of antiviral therapeutics. While specific antiviral mechanisms remain to be elucidated, leflunomide shows unique bifunctional potential to both reduce viral load and, by virtue of its well-documented anti-iniflammatory activity, attenuate the destructive inflammation associated with RSV disease.
References
1.
MeissnerH.C.Selected populations at increased risk from respiratory syncytial virus infection. Pediatr Infect Dis J2003; 22Suppl 2: S40–S45.
2.
MedranoC., Garcia-GueretaL., GruesoJ.Respiratory infection in congenital cardiac disease. Hospitalizations in young children in Spain during 2004 and 2005: the CIVIC Epidemiologic Study. Cardiol Young2007; 17: 360–371.
3.
FalseyA.R., HennesseyP.A., FormicaM.A., CoxC., WalshE.E.Respiratory syncytial virus infection in elderly and high-risk adults. N Engl J Med2005; 352: 1749–1759.
4.
NairH.D., NokesD.J., GessnerB.D.Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet2010; 375: 1545–1555.
KimH.W., KimH.W., CancholaJ.G.Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol1969; 89: 422–434.
7.
KapikianA.Z., MitchellR.H., ChanockR.M., ShvedoffR.A., StewartC.E.An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. Am J Epidemiol1969; 89: 405–421.
8.
The IMpact-RSV Study Group.Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization. Pediatrics1998; 102: 531–537.
MaggonK., BarikS.New drugs and treatment for respiratory syncytial virus. Rev Med Virol2004; 14: 149–168.
11.
McChesneyL.P., XiaoF., SankaryH.N.An evaluation of leflunomide in the canine renal transplantation model. Transplantation1994; 57: 1717–1722.
12.
WilliamsJ.W., XiaoF., FosterP.Leflunomide in experimental transplantation. Control of rejection and alloantibody production, reversal of acute rejection, and interaction with cyclosporine. Transplantation1994; 57: 1223–1231.
WilliamsJ.W., MitalD., ChongA.Experiences with leflunomide in solid organ transplantation. Transplantation2002; 73: 358–366.
15.
SilvermanE., MouyR., SpiegelL.Leflunomide or methotrexate for juvenile rheumatoid arthritis. N Engl J Med2005; 352: 1655–1666.
16.
ElderR.T., XuX., WilliamsJ.W., GongH., FinneganA., ChongA.S.The immunosuppressive metabolite of leflunomide, A77 1726, affects murine T cells through two biochemical mechanisms. J Immunol1997; 159: 22–27.
17.
WaldmanW.J., KnightD.A., LurainN.S.Novel mechanism of inhibition of CMV by the experimental immunosuppressive agent leflunomide. Transplantation1999; 68: 814–825.
18.
KnightD.A., HejmanowskiA.Q., DierksheideJ.E., WilliamsJ.W., ChongA.S., WaldmanW.J.Inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide. Transplantation2001; 71: 170–174.
19.
ChongA.S., ZengH., KnightD.A.Concurrent antiviral and immunosuppressive activities of leflunomide in vivo. Am J Transplant2006; 6: 69–75.
20.
JohnG.T., ManivannanJ., ChandyS., PeterS., JacobC.K.Leflunomide therapy for cytomegalovirus disease in renal allograft recipients. Transplantation2004; 77: 1460–1461.
21.
JohnG.T., ManivannanJ., ChandyS.A prospective evaluation of leflunomide therapy for cytomegalovirus disease in renal transplant recipients. Transplant Proc2005; 37: 4303–4305.
22.
AveryR.K., BolwellB.J., Yen-LiebermanB.Use of leflunomide in an allogeneic bone marrow transplant recipient with refractory cytomegalovirus infection. Bone Marrow Transplant2004; 34: 1071–1075.
23.
LeviM.E., MandavaN., ChanL.K., WeinbergA., OlsonJ.L.Treatment of multidrug-resistant cytomegalovirus retinitis with systemically administered leflunomide. Transpl Infect Dis2006; 8: 38–43.
24.
EhlertK., GrollA.H., KuehnJ., VormoorJ.Treatment of refractory CMV-infection following hematopoietic stem cell transplantation with the combination of foscarnet and leflunomide. Klin Padiatr2006; 218: 180–184.
25.
TeschnerS., GerkeP., GeyerM.Leflunomide therapy for polyomavirus-induced allograft nephropathy: efficient BK virus elimination without increased risk of rejection. Transplant Proc2009; 41: 2533–2538.
26.
WilliamsJ.W., JavaidB., KadambiP.V.Leflunomide for polyomavirus type BK nephropathy. N Engl J Med2005; 352: 1157–1158.
27.
ZhaoX., SullenderW.M.In vivo selection of respiratory syncytial viruses resistant to palivizumab. J Virol2005; 79: 3962–3968.
28.
ChongA.S., HuangW., LiuW.In vivo activity of leflunomide: pharmacokinetic analyses and mechanism of immunosuppression. Transplantation1999; 68: 100–109.
DeVincenzoJ.P., El SaleebyC.M., BushA.J.Respiratory syncytial virus load predicts disease severity in previously healthy infants. J Infect Dis2005; 191: 1861–1868.
34.
WelliverT.P., GarofaloR.P., HosakoteY.Severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus Is characterized by the absence of pulmonary cytotoxic lymphocyte responses. J Infect Dis2007; 195: 1126–1136.
LinY., VandeputteM., WaerM.Suppression of T-independent IgM xenoantibody formation by leflunomide during xenografting of hamster hearts in rats. Transplantation1998; 65: 332–339.
37.
KraanM.C., de KosterB.M., ElferinkJ.G., PostW.J., BreedveldF.C., TakP.P.Inhibition of neutrophil migration soon after initiation of treatment with leflunomide or methotrexate in patients with rheumatoid arthritis: findings in a prospective, randomized, double-blind clinical trial in fifteen patients. Arthritis Rheum2000; 43: 1488–1495.
38.
JosephsonM.A., GillenD., JavaidB.Treatment of renal allograft polyoma BK virus infection with leflunomide. Transplantation2006; 81: 704–710.
39.
SmolenJ.S., KaldenJ.R., ScottD.L.Efficacy and safety of leflunomide compared with placebo and sulphasalazine in active rheumatoid arthritis: a double-blind, randomised, multicentre trial. Lancet1999; 353: 259–266.
40.
SilvermanE., SpiegelL., HawkinsD.Long-term open-label preliminary study of the safety and efficacy of leflunomide in patients with polyarticular-course juvenile rheumatoid arthritis. Arthritis Rheum2005; 52: 554–562.
41.
FukushimaR., KanamoriS., HirashibaM.Teratogenicity study of the dihydroorotate-dehydrogenase inhibitor and protein tyrosine kinase inhibitor Leflunomide in mice. Reprod Toxicol2007; 24: 310–316.
