Development of a nasal spray formulated with antiviral drug against influenza

Picture1By: Albert F. Kabore, Laurie Schofield, and Jonathan Wong

Defence R&D Canada (DRDC) aims to advance the development of nasal spray formulated with liposome-encapsulated Poly ICLC (LE Poly ICLC), a potent and broad-spectrum antiviral drug, which has been shown in animal studies to protect against pandemic and seasonal influenza viruses.  In a published study, LE Poly ICLC has been shown to provide complete protection to mice when administered 21 days prior to challenge with multiple lethal doses of influenza A (H1N1) virus (Wong et al.,  Vaccine 19:2001). To meet regulatory requirements for clinical development of LE Poly ICLC, efficacy testing of nasal spray delivered LE Poly ICLC against influenza A virus was conducted using a ferret infection model, which is widely accepted as “gold standard” animal model for influenza infection.  Ferrets pre-treated with nasal sprayed LE Poly ICLC showed significantly lower virus titers in nasal washings compared to control untreated ferrets.  Furthermore, this study also suggests that nasal spray does not disrupt the integrity of the liposomes nor the Poly ICLC.  Together, these results affirmed the effectiveness of nasal spray delivered LE Poly ICLC in protection of ferrets against influenza A virus infection.

Introduction

Influenza pandemics including the1917 – 18 Spanish flu pandemic have historically killed millions of people worldwide. Despite advances in vaccinology and antiviral drug development, the world is ill equipped to defend against future pandemics. This is largely due to the fact that influenza viruses causing these pandemics are highly unpredictable, constantly mutating, and rapidly developing drug resistance. Currently, commercial antiviral drugs against influenza target virus structures and proteins, which make them ineffective when the virus undergoes mutations in these sites. To circumvent the problem of drug resistance, novel approaches, which specifically stimulate the host’s innate immunity to provide broad antiviral responses, are becoming hot areas in antiviral drug development.

Toward this end, Defence R&D Canada (DRDC) has pioneered the preclinical development of LE Poly ICLC, a potent antiviral agent that activates the toll-like receptor-3 (TLR-3) signaling pathway, which in turn results in the induction of protective antiviral immunity in the host.

DRDC aims to champion the advance development of a nasal spray formulated with LE Poly ICLC, potentially able to protect Canadian Forces, first responders and civilians against a variety of deadly viruses. This nasal spray represents a safe, effective and needle free means for delivering a potent and broad-spectrum LE Poly ICLC. When fully developed, it will enhance our ability to defend against deadly viruses, regardless of whether they are natural in origin, bioengineered, or resistant to conventional antiviral drugs.

Overview of Mechanism of Drug Action and Efficacy Testing

Poly ICLC is a synthetic double-stranded RNA (dsRNA) polyriboinosinic-polyribocytidilic acid stabilized with poly-L-lysine and carboxymethylcellulose. Poly ICLC and LE Poly ICLC induced a broad antiviral immune response in the host by activating the toll-like receptor-3 signaling pathway, enabling the host to respond to viral threats.   The distinct advantage of this approach over conventional antiviral drugs is that LE Poly ICLC elicits a broad-spectrum antiviral effect against a wide range of viral pathogens regardless of their genetic makeup, zoonotic origin, or drug resistance status (Wong, Nagata et al. 2005; Christopher and Wong 2008; Wong, Christopher et al. 2009). Furthermore, Poly ICLC can also work effectively either as a stand-alone immune-modulating agent or as a potent adjuvant to influenza vaccine candidates.   Li et al have recently demonstrated that intranasal administration of LE Poly ICLC, either before or shortly after influenza infection, was effective as a prophylactic and therapeutic agent , and also as a vaccine adjuvant against highly pathogenic avian influenza A/H5N1 virus (Li, Hu et al. 2011). According the authors, nasal vaccination using Poly ICLC or LE Poly ICLC as an adjuvant is perceived as advantageous because it is less invasive and elicits both local and systemic immune responses, and the mucosal immune response is known to be faster compared with that from systemic routes of vaccination.

Existing drugs against influenza viruses have limitations in terms of toxicity, drug resistance, and virus mutations, leaving first responders, defense personnel, and civilians vulnerable to influenza outbreaks. The prototype nasal spray formulated with LE Poly ICLC will provide a needle free and safe delivery with ease and convenience of self-administration.  When fully developed, this nasal spray has the potential to confer rapid protection to human against a wide range of deadly viruses including Ebola, western equine encephalitis, and influenza infections.  One of the most significant milestone achievements for this project  is the successful completion of the “proof of concept” efficacy study demonstrating  the effectiveness of nasally sprayed LE Poly ICLC for the protection of experimental animals against respiratory influenza A virus (H1N1) infection. In collaboration with University of Saskatchewan, a study was conducted in ferrets that mimic more of natural influenza virus infection in humans. Seventy five percent of animals pre-treated with nasally delivered LE Poly ICLC against influenza A/PR/8/34 virus showed below detectable levels of virus in nasal washes in comparison of control ferrets that showed high levels of virus (Figure 1 and 2).

 

Screen Shot 2016-04-25 at 7.17.10 PMFigure 1. Efficacy of nasal sprayed LE Poly ICLC against influenza A/PR/8/34 virus in ferrets. Groups of ferret were treated with two nasal spray of LE Poly ICLC or PBS, administered at day -3 and -1 at day 0; ferrets were intranasally challenged with 1X107 plaque forming units of influenza A/PR/8/34. At day 4 post infection, nasal washes were collected from each animal, and were assayed for influenza virus using a standard plaque assay

Picture1Figure 2. Plaque assay of nasal washing from ferrets treated with nasally sprayed LE Poly ICLC (top panel) compared to ferrets treated with nasally sprayed PBS (control, bottom panel) group.  Plaques are areas of clearing in the cell monolayer whose numbers are indicative of the number of infectious virus particles.

This study represents an important breakthrough as it clearly indicated that LE Poly ICLC is not disrupted by nasal spray delivery, and that it provides efficacy data from second animal species in addition to current mouse data as required for regulatory approval. These results are promising in demonstrating potency of LE Poly ICLC against influenza A virus.

Conclusion

Influenza pandemic preparedness is necessary to control bioterrorism-related or natural outbreaks of the virus. LE Poly ICLC is a broad-spectrum antiviral agent shown to be effective in animals for prophylactic therapy of deadly viral diseases involving H5N1, Ebola, and alphavirus infections. LE poly ICLC-formulated nasal spray will protect first responders, medical and security personnel, and the public against these viruses. Therefore, developing this novel prophylactic approach will significantly improve existing chemical, biological, radiological, nuclear and explosive (CBRNE) preparedness and prevention capabilities.

Acknowledgements

The authors wish to acknowledge the financial assistance from Chemical Biological and Nuclear Research and Technologies Initiatives for project (CRTI 06-0301TD) on the development of liposome encapsulated Poly ICLC. Thanks are also due to Major Van Loon, and Captain Godbout, formerly program manager and deputy program manager respectively.

References:

Christopher, M. E. and J. P. Wong (2008). “Broad-spectrum drugs against viral agents.” Int J Mol Sci 9(9): 1561-1594.

Ichinohe, T., A. Ainai, et al. (2010). “Induction of cross-protective immunity against influenza A virus H5N1 by an intranasal vaccine with extracts of mushroom mycelia.” J Med Virol 82(1): 128-137.

Li, Y., Y. Hu, et al. (2011). “Prophylactic, therapeutic and immune enhancement effect of liposome-encapsulated PolyICLC on highly pathogenic H5N1 influenza infection.” J Gene Med 13(1): 60-72.

Saravolac, E. G., D. Sabuda, et al. (2001). “Immunoprophylactic strategies against respiratory influenza virus infection.” Vaccine 19(17-19): 2227-2232.

Wong, J. P., M. E. Christopher, et al. (2007). “Nucleic acid-based antiviral drugs against seasonal and avian influenza viruses.” Vaccine 25(16): 3175-3178.

Wong, J. P., M. E. Christopher, et al. (2009). “Antiviral role of toll-like receptor-3 agonists against seasonal and avian influenza viruses.” Curr Pharm Des 15(11): 1269-1274.

Wong, J. P., M. E. Christopher, et al. (2009). “Activation of toll-like receptor signaling pathway for protection against influenza virus infection.” Vaccine 27(25-26): 3481-3483.

Wong, J. P., L. P. Nagata, et al. (2005). “Prophylaxis of acute respiratory virus infections using nucleic acid-based drugs.” Vaccine 23(17-18): 2266-2268.

Wong, J. P., E. G. Saravolac, et al. (1995). “Prophylactic and therapeutic efficacies of poly(IC.LC) against respiratory influenza A virus infection in mice.” Antimicrob Agents Chemother 39(11): 2574-2576.

Wong, J. P., H. Yang, et al. (1999). “Liposome-mediated immunotherapy against respiratory influenza virus infection using double-stranded RNA poly ICLC.” Vaccine 17(13-14): 1788-1795.

Captain Kabore is a Bioscience Officer in the Canadian Forces, at 1 Field Ambulance Detachment Suffield. He is currently assigned to DRDC Military Support Unit Suffield, Biotechnology Section. Currently, Captain Kabore takes on responsibilies as the program manager for the Antiviral Program (AVP).

Captain Albert F. Kabore, Ph.D., Bioscience Officer, Defense Research and Development Canada (DRDC) Suffield

 Email: albert.kabore@drdc-rddc.gc.ca