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

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.

Figure 1. Antibody responses of immunised ferrets to A/Vietnam/1194/2004 (clade 1), A/Indonesia/05/2005 (clade 2.1), & A/Turkey/Turkey/1/2005 (clade 2.2) H5N1 viruses by HAI (turkey and horse erythrocytes), VN, and SRH assays.

All animals were intranasally immunised on days 0 and 21. Data presented for each of the vaccine groups before and after 1 vaccination (Day 0 and Day 21), 2 vaccinations (Day 42), and just prior to challenge (Day 48). Treatment groups were Placebo (PBS) n = 12; unadjuvanted/antigen alone (0.075 mg/mL HA) n = 6; CSN adjuvanted vaccine (0.075 mg/mL HA+5 mg/mL CSN) n = 12; & TM-CSN adjuvanted vaccine (0.075 mg/mL HA+5 mg/mL CSN) n = 6. Bars represent geometric mean group values (horizontal bars) and ±SD (vertical bars). Vaccine responses are as follows: graphs A, D, G, & J are responses to A/Vietnam/1194/2004, as measured by HAI (turkey erythrocytes), HAI (horse erythrocytes), VN & SRH respectively; B, E, H, & K are responses to A/Indonesia/05/2005; C, F, I, & L are responses to A/Turkey/Turkey/1/2005. Seroconverting ferrets (threshold represented by blue horizontal dotted line) were defined as those animals with an equal or greater than 4 fold increase in titre from baseline for the HAI and VN assays. Those that attain an area of 25 mm² or more for the SRH assay were defined as seroprotected. Seroprotection levels for the HAI assay were defined as equal or greater than 40HAI (threshold represented by red horizontal dotted line).

Figure 2. Morbidity and mortality in in control and vaccinated ferrets post challenge with homologous H5N1 HPAI virus.

2A. Kaplan Meyer survival plot for vaccinated ferrets post challenge. The following groups were challenged with HPAI virus by the intratracheal route and the data is shown in 2A: red line = placebo group challenged, green line = unadjuvanted/antigen alone vaccine group, all other groups are represented by the black line. 2B.daily percentage weight change post challenge. Treatment group mean weight changes (%) on each day post challenge are represented with ± SEM. 2C. Temperature change (°C) post challenge. Ferret temperature was monitored every 10 minutes with implantable thermometers allowing analysis of temperature change from baseline (average of Days 45–48 temperatures for each animal). Treatment group mean temperature changes every 12 hrs (00∶00 and 12∶00) are represented with ± SD. The following groups were challenged with HPAI virus by the intratracheal route and the data is shown in 2B and 2C: red circle = placebo group challenged, green squares = unadjuvanted/antigen alone vaccine group, blue pyramids = CSN adjuvanted vaccine, orange diamonds = TM-CSN adjuvanted vaccine. The following groups were challenge by the intranasal route: black diamonds = placebo, and blue circles = CSN adjuvanted vaccine.

Figure 3. Virus Titres in Throat- and Nasal Swabs in control and vaccinated ferrets post challenge.

Both throat swabs (3A) and nasal swabs (3B) were analysed for viral loads by cell culture (log₁₀TCID₅₀/mL) and plotted daily after challenge. Plots contain mean daily mean titres with ± SD for each treatment. The following treatment groups were challenged with HPAI virus by the intratracheal route: red circle = placebo group challenged, green squares = unadjuvanted/antigen alone vaccine group, blue triangles = CSN adjuvanted vaccine, orange diamonds = TM-CSN adjuvanted vaccine. The following groups were challenge by the intranasal route: black diamonds = placebo, and blue circles = CSN adjuvanted vaccine.

Figure 4. Virus Titres in Nasal Turbinates, Lungs, Brain, and Olfactory bulb in control and vaccinated ferrets post challenge.

On the day that each ferret was euthanised samples taken from the turbinates (A), lung (B), brain (C), and olfactory bulb (D) were analysed for viral loads by cell culture (log₁₀TCID50/gram) and plotted as a scatter plot with geometric mean titres for each group. The following groups were challenged with HPAI virus by the intratracheal route: red circle = placebo group challenged, green squares = unadjuvanted/antigen alone vaccine group, blue triangles = CSN adjuvanted vaccine, orange diamonds = TM-CSN adjuvanted vaccine. The following groups were challenge by the intranasal route: black diamonds = placebo, and blue circles = CSN adjuvanted vaccine.

Figure 5. Histopathology in control and vaccinated ferrets post challenge.

Histopathology was performed on ferrets that were euthanised according to schedule as well as animals euthanized prematurely on welfare groups and any decedents. In those ferrets that were not euthanised according to the schedule all had acute severe pneumonia or diffuse alveolar damage, which was attributed to the likely cause of death. None of those animals that were affected by encephalitis had to be euthanised prematurely. Each panel represents: (A) extent of alveolitis, (B) severity of alveolitis, (C) relative weight of lung, (D) percentage lung affected, and (E) severity of rhinitis.