Vaccine 29 (2011) 4345–4352 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Development of a dried influenza whole inactivated virus vaccine for pulmonary immunization Sandrine A.L. Audouy a , Gieta van der Schaaf b , Wouter L.J. Hinrichs b , Henderik W. Frijlink b , Jan Wilschut a , Anke Huckriede a,∗ a Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands b Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands article info Article history: Received 28 January 2011 Received in revised form 27 March 2011 Accepted 4 April 2011 Available online 21 April 2011 Keywords: Influenza Dry powder vaccine Pulmonary immunization abstract Stabilization and ease of administration are two ways to substantially improve the use of current vaccines. In the present study an influenza whole inactivated virus (WIV) vaccine was freeze-dried or spray-freeze dried in the presence of inulin as a cryoprotectant. Only spray-freeze drying rendered powders compat- ible with administration to the lungs by insufflation. Pulmonary administration of the powder vaccine obtained by this method to BALB/c mice led to a transient influx of neutrophils and a concomitant decrease in the number of macrophages as did administration of liquid vaccine. Inflammatory reactions to both vaccines were mild and short-lived. Immunization studies showed that the immunogenic properties of WIV vaccine were not affected by drying. Pulmonary administration of the powder WIV vaccine induced a systemic immune response of the same magnitude as liquid vaccine while mucosal IgA responses were higher for powder WIV. In a challenge study where immunized mice were exposed to a lethal dose of live virus, two pulmonary doses of either liquid or powder WIV vaccine were equally effective as a single intramuscular injection of subunit vaccine in terms of reduction of the viral load in the lungs. To con- clude, in the models employed for these studies the use of a dry powder WIV vaccine for pulmonary immunization was shown to be safe and efficient. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Intramuscular (i.m.) injection is the most widely applied route for delivery of influenza vaccines. In most cases this vaccination method proves efficient to induce protective immunity. However, the use of new administration routes may be of great interest with regard to efficacy, safety, ease of administration and costs [1–3]. Nasal or dermal administrations are examples of alternative routes to i.m. injection. Another approach is the delivery of vaccines via the pulmonary route. In addition to ease of access, the lungs present a large surface and, due to the abundance of antigen-presenting cells, a large interface area with the immune system [2]. Recent and on- going clinical studies on inhalable measles vaccines in Mexico and India demonstrate the safety and feasibility of pulmonary immu- nization in a clinical context and its suitability for mass vaccination campaigns [4–6]. The pulmonary route has also been explored for vaccination against influenza. In several animal models, both systemic and mucosal immune responses have been successfully induced by ∗ Corresponding author. Tel.: +31 50 3632714; fax: +31 50 3638171. E-mail address: a.l.w.huckriede@med.umcg.nl (A. Huckriede). pulmonary immunization and protective efficacy has been demon- strated [7–9]. Pulmonary immunization against influenza has also been explored in several small scale clinical studies with promising results (reviewed in Amorij et al. [1]). So far, most pulmonary vaccines have been administered in soluble form. Yet, liquid vaccines are prone to degradation either during storage and distribution or during generation of aerosols. Formulation of dry products has proven a good approach for stabi- lization of a broad range of compounds, including vaccines [10,11]. Drying of vaccines is usually performed by spray-drying (SD) or lyophilization, i.e. freeze-drying (FD) or spray-freeze drying (SFD), in the presence of a proper (cryo-)protectant. Inulin, due to its high glass transition temperature and low crystallization rate, possesses excellent stabilizing properties [12]. We have shown earlier that the immunogenicity of influenza subunit and WIV vaccine is conserved after SD, FD or SFD with inulin [13–15] and reported on the induc- tion of systemic and mucosal immune responses upon pulmonary vaccination with SFD subunit vaccine in mice [16]. In the present paper, a whole inactivated virus vaccine was used to optimize powder preparation for delivery to the lung and to investigate different aspects of vaccination with a powder for- mulation in vivo. The choice for a WIV vaccine was based on the observation that, upon intramuscular and intranasal admin- 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.04.029