Pulmonary immunization of chickens using non-adjuvanted spray-freeze dried whole inactivated virus vaccine completely protects against highly pathogenic H5N1 avian influenza virus

Vaccine 32 (2014) 6445–6450

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Pulmonary immunization of chickens using non-adjuvanted spray-freeze dried whole inactivated virus vaccine completely protects against highly pathogenic H5N1 avian influenza virus Ben Peeters a,∗ , Wouter F. Tonnis d , Senthil Murugappan d , Peter Rottier b , Guus Koch a , Henderik W. Frijlink d , Anke Huckriede c , Wouter L.J. Hinrichs d a

Central Veterinary Institute of Wageningen University and Research Centre, P.O. Box 65, 8200 AB Lelystad, The Netherlands Virology Division, Department of Infectious Diseases and Immunology, Utrecht University, Faculty of Veterinary Medicine, Yalelaan 1, 3584 CL Utrecht, The Netherlands c University Medical Center Groningen, Department of Medical Microbiology, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands d University of Groningen, Department of Pharmaceutical Technology and Biopharmacy, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands b

a r t i c l e

i n f o

Article history: Received 18 April 2014 Received in revised form 29 August 2014 Accepted 22 September 2014 Available online 5 October 2014 Keywords: Avian flu Powder vaccine Inhalation Poultry vaccine Mass vaccination

a b s t r a c t Highly pathogenic avian influenza (HPAI) H5N1 virus is a major threat to public health as well as to the global poultry industry. Most fatal human infections are caused by contact with infected poultry. Therefore, preventing the virus from entering the poultry population is a priority. This is, however, problematic in emergency situations, e.g. during outbreaks in poultry, as there are currently no mass application methods to effectively vaccinate large numbers of birds within a short period of time. To evaluate the suitability of needle-free pulmonary immunization for mass vaccination of poultry against HPAI H5N1, we performed a proof-of-concept study in which we investigated whether non-adjuvanted spray-freeze-dried (SFD) whole inactivated virus (WIV) can be used as a dry powder aerosol vaccine to immunize chickens. Our results show that chickens that received SFD-WIV vaccine as aerosolized powder directly at the syrinx (the site of the tracheal bifurcation), mounted a protective antibody response after two vaccinations and survived a lethal challenge with HPAI H5N1. Furthermore, both the number of animals that shed challenge virus, as well as the level of virus shedding, were significantly reduced. Based on antibody levels and reduction of virus shedding, pulmonary vaccination with non-adjuvanted vaccine was at least as efficient as intratracheal vaccination using live virus. Animals that received aerosolized SFD-WIV vaccine by temporary passive inhalation showed partial protection (22% survival) and a delay in time-to-death, thereby demonstrating the feasibility of the method, but indicating that the efficiency of vaccination by passive inhalation needs further improvement. Altogether our results provide a proof-ofconcept that pulmonary vaccination using an SFD-WIV powder vaccine is able to protect chickens from lethal HPAI challenge. If the efficacy of pulmonary vaccination by passive inhalation can be improved, this method might be suitable for mass application. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Avian influenza is a serious disease of poultry that is caused by highly pathogenic strains of avian influenza virus (HPAI). Since 1997, H5N1 subtype viruses have caused large outbreaks in poultry and wild birds in South-East Asia and more recently in Europe, the Middle-East and Africa [1–3]. These outbreaks are the primary source of human infections of which approximately 60% have

∗ Corresponding author. Tel.: +31 320 238693; fax: +31 320 238668. E-mail addresses: [email protected], [email protected] (B. Peeters). http://dx.doi.org/10.1016/j.vaccine.2014.09.048 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

resulted in a fatal outcome. Each wave of human infections by HPAI H5N1 has been preceded by outbreaks in poultry and, although there is some indication for human to human transmission [4,5], it is generally assumed that the majority of human cases is the result of contact with H5N1 infected poultry. To combat avian influenza outbreaks, culling of all poultry on infected holdings and pre-emptive culling on neighbouring noninfected holdings (‘stamping out’) has been used successfully [6]. However, this approach is not applicable in developing countries for several reasons and counters resistance from the society in developed countries. To stop transmission and achieve efficient control of the disease, vaccination may be used as an alternative

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to pre-emptive culling, provided that sufficient quantities of vaccine are available and can be applied shortly after the start of the outbreak [7]. Unfortunately, currently used inactivated influenza vaccines for poultry are not suitable for mass application methods such as aerosol vaccination due to a lack of efficacy [8,9]. Furthermore, the use of live influenza vaccines for mass application has not yet been approved due to safety concerns [7]. We have shown previously that spray-freeze drying of subunit vaccines or of whole inactivated virus (WIV) vaccines yields highly stable vaccine preparations [10,11]. Furthermore, we have demonstrated the induction of systemic and mucosal immune responses in mice upon pulmonary vaccination with spray freeze-dried (SFD) subunit as well as SFD-WIV vaccine [10,12,13]. To examine whether pulmonary vaccination is also suitable for the protection of chickens against avian influenza, we performed vaccination-challenge experiments in which we administered SFD-WIV influenza H5N1 vaccine via the pulmonary route to chickens either by direct administration at the syrinx or by passive inhalation. Here we show that pulmonary vaccination using an SFD-WIV powder vaccine is able to protect chickens against HPAI H5N1.

Fig. 1. Schematic overview of the vaccination-challenge experiments. The day of first vaccination is designated day 0 (d0). The animals in groups 1–3 were vaccinated once at d21, whereas the animals in groups 4 and 5 were vaccinated 3 times (d0, d14 and d28). The animals in group 6 were not vaccinated. All animals were challenged with HPAI A/Turkey/Turkey/1/05 (H5N1) at d42 and the experiment was terminated at d56. IM: intramuscular; IT: intratracheal; WIV: whole inactivated virus; adjv: adjuvant; SFD-WIV: spray-freeze dried WIV; TCID50: median tissue culture infectious dose; insufflator: delivery device. See text for further details.

2. Materials and methods 2.1. Virus Influenza A strain NIBRG-23, is a reassortant virus prepared by reverse genetics from A/turkey/Turkey/1/2005 (H5N1) virus (in which the polybasic haemagglutinin (HA) cleavage site has been excised) and A/PR/8/34 (H1N1) virus (NIBSC code: 08/156; http://www.nibsc.org/documents/ifu/08-156.pdf). A seed strain of NIBRG-23 virus was obtained from the National Institute of Biological Standards and Controls, Potters Bar, Hertfordshire, UK. The virus was grown in embryonated chicken eggs and purified and inactivated as previously described [13]. The wild-type HPAI influenza strain A/turkey/Turkey/1/2005 (H5N1) (Clade 2.2.1) was obtained from the Animal Health and Veterinary Laboratories Agency, Weybridge, UK. For use as antigen in haemagglutination assays, the virus was inactivated with 0.02% paraformaldehyde (Merck) for 16 h at 37 ◦ C. 2.2. Vaccine preparation NIBRG-23 whole inactivated virus (WIV) preparations were mixed with adjuvant (Stimune® ; Prionics A.G., Zurich, Switzerland) in a ratio of 4: 5 (v/v). Spray-freeze drying of NIBRG-23 WIV in the presence of inulin was performed as described previously [13]. In brief, a solution of 45 mg/mL inulin and WIV (inulin: HA = 200: 1) was sprayed into a vessel of liquid nitrogen and subsequently freeze dried as follows: first, 24 h of drying at −35 ◦ C and 0.220 mbar; next, the pressure was immediately lowered to 0.050 mbar while the temperature was gradually increased to 20 ◦ C during 8 h. At these settings freeze drying was continued for 16 more hours. Reconstituted WIV vaccines were prepared by dissolving appropriate amounts of SFD-WIV in PBS followed by the addition of adjuvant as specified above. In order to compare the performance of the different inactivated vaccines with that of a live vaccine, we also used untreated NIBRG-23 virus, which is non-pathogenic for chickens. 2.3. Vaccination-challenge experiments in chickens Thirty-five specific-pathogen-free chickens were housed in the Animal Biosafety Level 3+ high containment facility of the Central Veterinary Institute of Wageningen UR. The animals were randomly divided over 5 groups of 5 animals and one group of 10 animals. In the latter group, 1 animal died for unknown reasons before the start of the experiment.

At the age of 3 weeks (day 0), the animals were vaccinated according to the scheme shown in Fig. 1. The animals in group 1 (n = 5) were vaccinated once by intramuscular injection of 0.5 ml of adjuvanted WIV vaccine (30 ␮g/ml HA) in the upper leg muscle. The animals in group 2 (n = 5) were vaccinated once by intramuscular injection of 0.5 ml of reconstituted SFD-WIV (dissolved in PBS and mixed with adjuvant to a final concentration of 30 ␮g/ml HA). The animals in group 3 (n = 5) were inoculated once intratracheally with 0.1 ml of live attenuated NIBRG-23 virus at a dose of 5.6 log10 TCID50 (15 ␮g HA) per animal. The animals in group 4 (n = 9), were placed in a custommade box with a volume of 0.5 m3 and exposed to aerosolized non-adjuvanted SFD-WIV at a concentration of 1 mg/m3 HA for 13–15 min. Based on a respiration rate of 44 l/h/kg body weight [14,15] and 3 applications (see below), this would theoretically result in a total dose of maximally 15 ␮g HA per animal. Aerosolization was performed through a hole in the closed box by inserting an Eppendorf tube filled with SFD-WIV that was pierced with a 30 Gauge hypodermic needle and by applying a short pulse of pressurized air through an opening in the lid of the tube. This procedure was repeated 2 and 4 weeks later (day 14 and 28, respectively). The animals in group 5 (n = 5) were inoculated with nonadjuvanted SFD-WIV (5 ␮g HA) using a DP-4-C Dry Powder Insufflator (Penn-Century Inc., Philadelphia, USA) with a custom length delivery tube designed to deliver the powder at the syrinx. The powder was aerosolized by using an air puff of 1 ml. This procedure was repeated 2 and 4 weeks later (day 14 and 28, respectively). The animals in group 6 (n = 5) did not receive any vaccination. For groups 1–3, blood samples were taken 3 weeks after vaccination (day 21), at the day of challenge (day 42), and at the end of the experiment (2 weeks after challenge; day 56). For groups 4 and 5, blood samples were taken at the day of the second (day 14) and third vaccination (day 28), at the day of challenge (day 42), and at the end of the experiment (day 56). At the day of challenge (day 42), all animals were inoculated with 5.6 log10 TCID50 of HPAI strain A/turkey/Turkey/1/2005 H5N1 by the combined intranasal/intratracheal route (liquid suspension; 0.1 ml each). The animals were observed for clinical signs twice daily, and at days 1, 3, 5, 7 and 10 post challenge (dpc) swabs were taken from the choana and the cloaca. Surviving animals were sacrificed by bleeding at 14 dpc (day 56). The study was approved by the institutional Animal Experiment Committee (project number 2013069) in accordance with the guidelines provided by the Dutch Animal Protection Act.

B. Peeters et al. / Vaccine 32 (2014) 6445–6450

Percentage survival

100

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Groups 1, 2, 3, 5

80 60 40 Group 4

20 Group 6

0

0

2

4 6 8 10 Days post challenge

12

14

Fig. 2. Kaplan–Meier survival curves. For details of the different groups see Fig. 1 and text.

2.4. Evaluation of systemic immune response and virus shedding Sera prepared from blood samples taken at the indicated time points (see above, and Fig. 1) were analyzed for influenza virus specific antibody levels by using a haemagglutination inhibition (HI) assay [16] using 8 haemagglutination units of formalin-inactivated virus A/turkey/Turkey/1/05 (H5N1). The assay was performed in duplicate. Virus in swabs from choana and cloaca was quantified using a real-time RT-PCR as described by van der Goot et al. [17]. A standard curve consisting of serial 10-fold dilutions of the challenge strain was used to convert the PCR data into equivalent virus titres (equivalent median tissue infective dose; eqTCID50/ml). 3. Results 3.1. Clinical observations Fig. 2 shows the percentage survival of vaccinated chickens after challenge. As expected, all animals in the non-vaccinated control group (group 6) succumbed to the challenge virus infection within 2 to 3 dpc. Animals that received non-adjuvanted SFD-WIV vaccine by direct application at the syrinx (group 5) all survived and did not show any signs of disease, demonstrating that pulmonary vaccination using a non-adjuvanted SFD-WIV powder vaccine is able to completely protect chickens from a lethal HPAI challenge. In the group of animals that received non-adjuvanted SFD-WIV vaccine by passive inhalation (group 4) 2 out of 9 animals survived. However, there was a clear delay in time-to-death and a difference in clinical symptoms compared to the non-vaccinated group. A Cox–Mantel log rank test [18] revealed that the difference in survival between this group and the control (group 6) was significant (2 (1) = 6.57, p = 0.010).The animals that died at 2–4 dpc showed signs of depression, but no other clinical signs. The animals that survived until 5 and 6 dpc showed severe neurological signs and were euthanized. The two surviving animals did not show any signs of disease. As expected, a single intramuscular vaccination using either an adjuvanted WIV vaccine (group 1) or a reconstituted adjuvanted SFD-WIV vaccine (group 2) resulted in complete protection against disease. Also, a single intratracheal vaccination with live attenuated NIBRG-23 virus (group 3) resulted in complete protection.

Fig. 3. Haemagglutination inhibition (HI) titres (log2) of sera collected at the indicated time points. The values shown are the mean log2 values of two independent measurements for the responders. For details see Table S1.

Animals that received non-adjuvanted SFD-WIV vaccine by direct application at the syrinx (group 5) did not show a measurable antibody response at 2 weeks after the first vaccination. However, a clear antibody response (mean HI titre of 5.5 log2) was seen after the second vaccination. The antibody level increased only slightly after the third vaccination (mean HI titre of 6.6 log2). All animals in this group were protected from morbidity and mortality after HPAI challenge (Fig. 2). Animals that received SFD-WIV vaccine by passive inhalation (group 4) did not show a measurable antibody response at 2 weeks after the first vaccination. After the second vaccination, two animals (nr. 23 and 24) developed an HI titre of 4 log2, whereas the other animals developed a very low response (nr. 18, 21) or no detectable response at all (nr. 16, 17, 19, 20, 22). After the third vaccination, low but readily detectable (i.e. ≥2 log2) HI titres were observed in 3 additional animals (nr. 20–22). The HI titres of animals 23 and 24 did not further increase after the third vaccination. Only the latter two animals (with an HI titre of >3 log2) survived the HPAI challenge, whereas all animals with either an HI titre of <3 log2 or no detectable HI titre succumbed to the infection after HPAI challenge. Animals that were vaccinated once intratracheally with live attenuated vaccine (group 3) developed an HI titre at 3 weeks after vaccination (mean HI titre of 5.5 log2). At the time of challenge, i.e. 6 weeks after vaccination, the HI titre had increased only slightly (mean HI titre of 5.9 log2). All animals survived after HPAI challenge. Animals that were vaccinated once with adjuvanted WIV vaccine (group 1) or reconstituted adjuvanted SFD-WIV vaccine (group 2) developed high HI titres at 3 weeks after vaccination (mean HI titres of 10.8 log2 and 10.3 log2, respectively). At the time of challenge, i.e. 6 weeks after vaccination, the HI titres had not (or only slightly) increased (mean HI titres of 10.8 log2 and 10.7 log2, respectively). All animals survived after HPAI challenge. 3.3. Shedding of challenge virus

3.2. Systemic immune response The development of a systemic influenza-specific immune response was examined by means of an HI assay. The results are presented in Fig. 3 and Table S1 (Supplemental information).

Only one of the animals (nr. 30) that received non-adjuvanted SFD-WIV vaccine by direct application at the syrinx (group 5) showed virus shedding in choanal swabs (Table 1). No virus could be detected in cloacal swabs from this animal or from any

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Table 1 Virus shedding after challenge. Virus shedding (eq. TCID50/ml) Group

Vaccine

1

WIV IM

2

Animal nr

Choana swabs

Cloaca swabs

1

3

5

7

10

1

3

5

7

10

1 2 3 4 5

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

WIV-R IM

6 7 8 9 10

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

3

NIBRG-23 IT

11 12 13 14 15

0.0 0.0 0.0 0.9 0.0

0.0 0.0 2.2 2.8 0.0

0.0 0.0 2.8 2.4 0.0

0.0 0.0 2.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

4

SFD-WIV box

16 17 18 19 20 21 22 23 24

2.8 3.7 3.8 3.1 0.0 1.8 2.9 1.7 1.5

5.3

3.8

4.1 3.0 5.2 2.3 1.1

4.2 3.4 0.0 0.0

0.0 0.0

0.0 0.3 2.6 0.0 0.0

2.4 2.1

2.4 0.0

0.0 2.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.2 0.0

0.0 0.0

0.0 0.0

0.0 0.0 0.0 0.0 0.6

0.0 0.0 0.0 0.0 1.5

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

5

SFD-WIV IT insufflator

26 27 28 29 30

0.0 0.0 0.0 0.0 1.1

6

Control

36 37 38 39 40

2.7 3.3 2.7 2.8 0.3

4.9

2.5

1.0 0.0 2.8 1.6 0.0

The values are based on quantitative real-time RT-PCR data. A standard curve consisting of serial 10-fold dilutions of the challenge strain was used to convert the PCR data (Ct -values) into equivalent virus titres (equivalent median tissue infective dose; eqTCID50/ml). IM: intramuscular; IT: intratracheal; WIV: whole inactivated virus; adjv: adjuvant; SFD-WIV: spray-freeze dried WIV; WIV-R: reconstituted SFD-WIV. See Fig. 1 and text for further details.

of the other animals in this group. The level of virus shedding was relatively low compared to the levels observed in nonvaccinated animals (group 6) or non-surviving animals in group 4. All animals that received non-adjuvanted SFD-WIV vaccine by passive inhalation (group 4) showed shedding of challenge virus in choanal swabs. In animals that succumbed to the infection, virus shedding was observed up till the day of death. In the two surviving animals (nr. 23 and 24), virus shedding was observed until 5 dpc and 3 dpc, respectively. The level of virus shedding seemed to be inversely related to the HI titre at the day of challenge. Most birds also showed virus shedding in cloacal swabs, although the levels were lower and shedding was delayed relative to the choanal swabs. Two animals (nr. 13 and 14) that were vaccinated intratracheally with live attenuated vaccine (group 3) showed shedding of challenge virus in choanal swabs. Virus shedding was observed until 5 (nr. 13) and 7 dpc, (nr. 14). In both animals, the level of virus shedding was somewhat higher than that observed in animal nr. 30 of group 5. No virus was detected in cloacal swabs. No virus could be detected in either choanal or cloacal swabs from animals vaccinated with adjuvanted WIV vaccine (group 1) or adjuvanted reconstituted SFD-WIV vaccine (group 2).

4. Discussion In this paper we demonstrated that pulmonary vaccination with influenza WIV powder vaccine prepared by SFD can induce an immune response in chickens, even when administered without an adjuvant. Direct delivery of powder vaccine at the syrinx resulted in an immune response that protected 100% of the animals from lethal infection with HPAI H5N1. Based on the HI assays, already after two vaccinations the maximum attainable antibody level seems to be reached in most animals. The apparent increase in mean HI titres when comparing sera taken 2 weeks after the second vaccination and 2 weeks after the third vaccination is mainly accounted for by one animal reacting poorly after the first two vaccinations (Fig. 3, Table S1). Vaccination by administration of the powder vaccine at the syrinx not only protected from morbidity and mortality but it also significantly reduced replication of the challenge virus as shown by the fact that only 1 out of 5 animals shed low amounts of virus after HPAI challenge (Table 1). Judging from the HI titres and virus shedding data, pulmonary vaccination using SFD-WIV powder vaccine is at least as efficient as intratracheal vaccination using a low-pathogenic live vaccine. Our data suggest that pulmonary vaccination with non-adjuvanted WIV powder vaccine results in even better protection from challenge

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virus replication than live vaccine. This is based on the observation that fewer animals shed virus and that the level of shedding was lower after intratracheal vaccination with powder vaccine than with live vaccine. Furthermore, only one of the animals vaccinated with powder vaccine (the one that showed virus shedding; i.e. nr. 30) showed a substantial boost in HI titre (here defined as an increase of >2 log2) after challenge, whereas such a boost was observed in 3 of the animals (nr. 12–14) vaccinated with live vaccine. A boost in antibody response after challenge is assumed to be indicative of challenge virus replication. Taken together, these observations indicate that pulmonary SFD-WIV vaccination may result in better local immunity than live virus vaccination. It has been shown previously that vaccination of chickens with dispersed dry powder Newcastle disease virus vaccines can be used as an alternative for liquid spray or aerosol vaccination [19–21]. However, in those cases the vaccine consisted of a live attenuated vaccine as opposed to inactivated vaccine that was used here. de Geus et al. [9] used whole inactivated influenza virus in the form of a liquid aerosol, but no induction of influenza-specific antibodies was observed after aerosol vaccination of chickens, neither in the presence nor absence of adjuvants. The latter may be due to inefficient delivery of the vaccine to the lungs, and/or to the fact that the animals received only a single vaccination. Delivery of SFD-WIV vaccine by passive inhalation resulted in partial protection. This vaccination route resulted in survival of 2 out of 9 animals and a delay in time-to-death for the non-surviving animals. This lower level of protection when compared to group 5 is most probably the result of inefficient delivery of the powder vaccine to the lungs, resulting in a sub-optimal vaccine dose. Our ‘inhalation-box’ pilot experiment leaves ample room for improvement and we expect that the efficiency of vaccine delivery can be significantly enhanced. This could for instance be achieved by increasing the concentration of the aerosolized powder vaccine, by increasing the retention time of the animals in the inhalation box, or by optimizing the particle size of the powder to ensure more efficient delivery to the lungs. Another option would be to include an adjuvant that is specifically suited to stimulate the mucosal immune response of powder vaccines in the lungs. Successful inclusion of adjuvants in SFD influenza vaccines has recently been demonstrated for various Toll-like receptor ligand adjuvants and a saponin-derived adjuvant [22,23]. Certainly, the aim of such improvements would be to eventually achieve full protection by a single, low-dose immunization. Our results show that ‘classical’ parenteral adjuvanted inactivated vaccines (groups 1 and 2) yield a much higher antibody titre than either a live vaccine (group 3) or a non-adjuvanted inactivated powder vaccine administered via the pulmonary route (group 5). However, our results also show that antibody levels induced by the latter two vaccines are sufficient to completely protect animals from morbidity and mortality after HPAI challenge. Furthermore, pulmonary vaccination may result in better local (mucosal) immunity, and can be applied to chickens of all ages, including one-dayold birds at hatcheries. Therefore, it may become a suitable alternative for ‘classical’ vaccination once the efficiency of immunization has been optimized. This would open the way to needle-free vaccination, a method that is amenable to large scale mass application. Currently, due to production costs and the need for special equipment, the practical use of pulmonary vaccination might be limited to its usage in emergency situations as an alternative for preemptive culling. Whether pulmonary vaccination may also become suitable for large-scale prophylactic vaccination remains to be seen. Acknowledgements We thank Henk Lommers from the Dutch Ministry of Economic Affairs for his initiative to set up this collaboration, and for his

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efforts in acquiring the necessary budget. We also thank the technicians from UMCG and RUG for production and characterization of the vaccines, the animal technicians from CVI for the execution of the animal experiments, and Diana van Zoelen for performing the laboratory tests. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2014.09.048. References [1] Sims LD, Domenech J, Benigno C, Kahn S, Kamata A, Lubroth J, et al. Origin and evolution of highly pathogenic H5N1 avian influenza in Asia. Vet Rec 2005;157(Aug (6)):159–64. [2] Joannis T, Lombin LH, De Benedictis P, Cattoli G, Capua I. Confirmation of H5N1 avian influenza in Africa. Vet Rec 2006;158(Mar (9)): 309–10. [3] de Jong MD, Hien TT. Avian influenza A (H5N1). J Clin Virol 2006;35(Jan (1)):2–13 (The Official Publication of the Pan American Society for Clinical Virology). [4] Olsen SJ, Ungchusak K, Sovann L, Uyeki TM, Dowell SF, Cox NJ, et al. Family clustering of avian influenza A (H5N1). Emerg Infect Dis 2005;11(Nov (11)):1799–801. [5] Wang H, Feng Z, Shu Y, Yu H, Zhou L, Zu R, et al. Probable limited person-toperson transmission of highly pathogenic avian influenza A (H5N1) virus in China. Lancet 2008;371(Apr (9622)):1427–34. [6] Stegeman A, Bouma A, Elbers AR, de Jong MC, Nodelijk G, de Klerk F, et al. Avian influenza A virus (H7N7) epidemic in The Netherlands in 2003: course of the epidemic and effectiveness of control measures. J Infect Dis 2004;190(Dec (12)):2088–95. [7] van den Berg T, Lambrecht B, Marche S, Steensels M, Van Borm S, Bublot M. Influenza vaccines and vaccination strategies in birds. Comp Immunol Microbiol Infect Dis 2008;31(Mar (2–3)):121–65. [8] de Geus ED, Rebel JM, Vervelde L. Induction of respiratory immune responses in the chicken; implications for development of mucosal avian influenza virus vaccines. Vet Q 2012;32(Jun (2)):75–86. [9] de Geus ED, van Haarlem DA, Poetri ON, de Wit JJ, Vervelde L. A lack of antibody formation against inactivated influenza virus after aerosol vaccination in presence or absence of adjuvantia. Vet Immunol Immunopathol 2011;143(Sep (1–2)):143–7. [10] Murugappan S, Patil HP, Kanojia G, ter Veer W, Meijerhof T, Frijlink HW, et al. Physical and immunogenic stability of spray freeze-dried influenza vaccine powder for pulmonary delivery: comparison of inulin, dextran, or a mixture of dextran and trehalose as protectants. Eur J Pharm Biopharm 2013;85(Nov (3 Pt A)):716–25 (Official Journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV). [11] Saluja V, Amorij JP, Kapteyn JC, de Boer AH, Frijlink HW, Hinrichs WL. A comparison between spray drying and spray freeze drying to produce an influenza subunit vaccine powder for inhalation. J Control Release 2010;1144(Jun (2)):127–33 (Official Journal of the Controlled Release Society). [12] Amorij JP, Saluja V, Petersen AH, Hinrichs WL, Huckriede A, Frijlink HW. Pulmonary delivery of an inulin-stabilized influenza subunit vaccine prepared by spray-freeze drying induces systemic, mucosal humoral as well as cell-mediated immune responses in BALB/c mice. Vaccine 2007;25(Dec (52)):8707–17. [13] Audouy SA, van der Schaaf G, Hinrichs WL, Frijlink HW, Wilschut J, Huckriede A. Development of a dried influenza whole inactivated virus vaccine for pulmonary immunization. Vaccine 2011;29(Jun (26)): 4345–52. [14] Calder WA. Respiratory and heart rates of birds at rest. Condor 1968;70:358–65. [15] Julian RJ. Lung volume of meat-type chickens. Avian Dis 1989;33(Jan–Mar (1)):174–6. [16] Office International des Epizooties. Avian influenza. In: Manual of diagnostictests and vaccines for terrestrial animals. Office International des Epizooties; Paris, 2008. [17] van der Goot JA, van Boven M, Stegeman A, van de Water SG, de Jong MC, Koch G. Transmission of highly pathogenic avian influenza H5N1 virus in Pekin ducks is significantly reduced by a genetically distant H5N2 vaccine. Virology 2008;382(Dec (1)):91–7. [18] Rich JT, Neely JG, Paniello RC, Voelker CC, Nussenbaum B, Wang EW. A practical guide to understanding Kaplan–Meier curves. Otolaryngol—Head Neck Surg 2010;143(Sep (3)):331–6 (Official Journal of American Academy of Otolaryngology-Head and Neck Surgery). [19] Corbanie EA, Remon JP, Van Reeth K, Landman WJ, van Eck JH, Vervaet C. Spray drying of an attenuated live Newcastle disease vaccine virus intended for respiratory mass vaccination of poultry. Vaccine 2007;25(Nov (49)): 8306–17.

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[20] Corbanie EA, Vervaet C, van Eck JH, Remon JP, Landman WJ. Vaccination of broiler chickens with dispersed dry powder vaccines as an alternative for liquid spray and aerosol vaccination. Vaccine 2008;26(Aug (35)): 4469–76. [21] Huyge K, Van Reeth K, De Beer T, Landman WJ, van Eck JH, Remon JP, et al. Suitability of differently formulated dry powder Newcastle disease vaccines for mass vaccination of poultry. Eur J Pharm Biopharm 2012;80(Apr (3)):649–56 (Official Journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV).

[22] Patil HP. Development of adjuvanted influenza vaccines for pulmonary delivery. The Netherlands: University of Groningen; 2014 (Ph.D. Thesis). [23] Patil HP, Murugappan S, Ter Veer W, Meijerhof T, de Haan A, Frijlink HW, et al. Evaluation of monophosphoryl lipid A as adjuvant for pulmonary delivered influenza vaccine. J Control Release 2013;21(Nov (174)):C:51–62 (Official Journal of the Controlled Release Society).