Intranasal H5N1 vaccines, adjuvanted with chitosan derivatives, protect ferrets against highly pathogenic influenza intranasal and intratracheal challenge.

Alex J Mann,Giulia Lapini,Andrew Catchpole,Alan Smith,Albert D M E Osterhaus,Leon De Waal,Koert J Stittelaar,Emanuele Montomoli,Michael Hinchcliffe,Alastair Knight,John S Oxford,Edwin J B Veldhuis Kroeze,Nicolas Noulin,Rob Lambkin-Williams,Simona Piccirella,Rebecca Cox

doi:10.1371/journal.pone.0093761

Abstract

We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model.Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings.In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals.This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant mortality and morbidity arising from infection with HPAI H5N1 virus.

Highlights

Avian Influenza (H5N1) continues to present a significant risk to human health [1],[2],[3],[4], and recent genetic studies of H5 Hemagglutinin (HA) in an H1N1 virus backbone identified only four mutations in the HA protein were required to facilitate transmission in the ferret model emphasizing this threat [5]
Sera from vaccinated ferrets were tested with hemagglutination inhibition (HAI), Single Radial Haemolysis (SRH), and viral neutralization assays (VN) assays against the homologous challenge virus as well as representatives from clade 2.1 and clade 2.2 (Tables S1,S2,& S3 in File S1 respectively)
After a single dose of chitosan glutamate (CSN) adjuvanted vaccine 2 out of 12 ferrets seroconverted by HAI to homologous virus, and after two vaccinations 7 out of 12 seroconverted by the HAI assay, 7 attained seroprotective levels in the SRH assay, with 8 out of 12 seroconverting as measured by the VN assay

Summary

Introduction

Avian Influenza (H5N1) continues to present a significant risk to human health [1],[2],[3],[4], and recent genetic studies of H5 Hemagglutinin (HA) in an H1N1 virus backbone identified only four mutations in the HA protein were required to facilitate transmission in the ferret model emphasizing this threat [5]. Most H5N1 vaccines that have demonstrated high immunogenicity required co-administration of an adjuvant and administration by the intramuscular route [8], [9], [10]. Various adjuvanted vaccines have been shown to be able to reduce mortality in ferret challenge models but have not a) induced 100% seroconversion, or b) completely prevented virus replication in the respiratory tract. While protection from death is the most critical attribute for a pandemic vaccine, Group 1

Methods

Results

Conclusion