The use of sialidase treatment in influenza virus infection
Session VII – John Nicholls
Title of Contribution: The use of sialidase treatment in influenza virus infection
Author(s): Nicholls JM1, Chan MCW2, Chan Renne WY2, Peiris JSM2 , Michael Malakhov3, Henry Li3, Yan Huang3, Shahparak Zaltash3, Stephen Hawley3, F Fang 3
Affiliation(s): 1 Department of Pathology, Hong Kong University, Hong Kong China, 2 Department of Microbiology, Hong Kong University, Hong Kong, China, 3 NexBio, San Diego, California, USA
Abstract:
Evidence accumulated over fifty years has indisputably demonstrated the key role of sialic acid (Sia) as the receptor for influenza viral (IFV) infection. Though much attention has been given to the determinants of IFV- Sia interaction there have been few investigations into the potential therapeutic role of sialidase therapy for the inhibition of IFV infection. DAS181 (FludaseTM) is a sialidase fusion protein currently being developed as a potential broad-spectrum prophylactic and therapeutic agent against influenza virus (IFV) infection. DAS181 combines the sialidase from Actinomyces viscosus with a heparin binding sequence derived from the human protein amphiregulin to act as an epithelial anchoring binding domain. Two papers reported potent in vitro and in vivo efficacy of DAS181 against IFV, one investigating the human IFV A & B strains in a human airway epithelial cell culture and MDCK cells and the other focusing on the highly pathogenic H5N1 IFV A strain A/Vietnam/1203/2004 (VN/1203) in mice. Previously we have demonstrated the utility of an ex vivo lung model for H1N1 and H5N1 infection and therefore wished to investigate the potential therapeutic effect of DAS181 in this ex-vivo model.
Human lung tissues were obtained from patients undergoing biopsy and open thoracotomy for the investigation of pulmonary disease. Tissues were cultured according to previously published methods and cultured with H5N1, H3N2 and H1N1 viruses for up to 48 hours at 37oC. Two methods of DAS181 treatment were used: the first was incubation of the lung tissues with 100 - 500 U/ml of DAS181 followed by washing and then viral infection. In this setting no additional DAS181 was added to the medium. In the second method DAS181 at 100 - 500 U/ml was added to the medium before and after IFV infection and remained in the medium for up to 48 hours. Tissues were removed at 24 and 48 hours and fixed in formalin for examination for the detection of IFV infection or preserved for RNA extraction . The supernatant was also stored for PCR analysis of IFV infection. Mass spectrometric analysis was also performed on normal lung tissues after 2 hours incubation with DAS181.
We found that using lectin binding studies DAS181 was effective at desialyation when SNA binding was used for human tissues and when MAA was used for duck intestines as a control. After 48 hours of ex vivo organ culture there was significant reduction in IFV replication in tissues continuously cultured with 2 hours of DAS181 and those exposed to DAS181 for 2 hours as detected by immunohistochemistry and M-gene analysis. Mass spectrometric analysis showed a partial reduction in Sia profiles.
The results indicate that desialyation of tissues by DAS181 does result in a reduction in IFV replication in human tissues. A phase I study is now in progress to determine the safety of an inhalational form of DAS181.
