International Journal of Pharmaceutics 479 (2015) 408–415
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First in vivo evaluation of particulate nasal dry powder vaccine formulations containing ovalbumin in mice Regina Scherließ a, * , Mathias Mönckedieck a , Katherine Young b , Sabrina Trows a , Simon Buske a , Sarah Hook b a b
Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany School of Pharmacy, University of Otago, 18, Frederick Street, Dunedin, New Zealand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 November 2014 Received in revised form 8 January 2015 Accepted 10 January 2015 Available online 13 January 2015
In this study three different dry powder vaccine formulations containing the model antigen ovalbumin were evaluated for their immune effects after nasal administration to C57Bl/6 mice in an adoptive cell transfer model. The formulations were chitosan nanoparticles in a mannitol matrix, chitosan microparticles and agarose nanoparticles in a mannitol matrix. Dry powder administration to mice was well tolerated and did not result in any adverse reactions. No translocation of the dry powder formulations to the lung could be detected. The local cellular immune response in the cervical lymph nodes was modest and only for the chitosan microparticles and the agarose nanoparticles was there a significant difference compared to s.c. injection of ovalbumin in alum. No humoral response could be measured after nasal administration. The results provide some evidence that nasal administration of dry powder formulations can stimulate an immune response, but the response was modest. This is probably due to a low antigen dose and low immunogenicity of the formulations. Further studies will aim at enhancing the antigen load and improving adjuvant activity. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Chitosan Nano-in-microparticle formulation Microparticles Powder insufflation Local cellular response
1. Introduction Mucosal vaccination is an attractive alternative to parenteral vaccination and offers some advantages such as avoidance of needle-stick injuries, removing the requirement for medical personnel for vaccine administration and an improved local immune response at the site of delivery (Lycke, 2012). The respiratory tract, particularly the nose, is well suited for mucosal vaccination as it is the normal route of entry for many pathogens; hence, it is well equipped with immunocompetent cells (Davis, 2001). Further, it is easily accessible for (self-) administration. Vaccines for mucosal vaccination via the respiratory tract have been successfully developed, for example the influenza vaccines FluMist/Fluenz (Medimmune/Astra Zeneca), which make use of a cold-adapted influenza strain and are administered via a nasal spray. Further nasal vaccines under development are formulated in polymeric particles made of poly-lactid-co-glycolid (Csaba et al., 2009; Slütter et al., 2010) or biopolymers such as chitosan (Amidi et al., 2007; Gordon et al., 2008; van der Lubben et al., 2003) and
* Corresponding author. Tel.: +49 431 8801340. E-mail address: [email protected] (R. Scherließ). http://dx.doi.org/10.1016/j.ijpharm.2015.01.015 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
alginate (Borges et al., 2008), virus-like particles (Vodak et al., 2012) or lipid particles (Alpar et al., 2001, 2005; Saraf et al., 2006). So far all successful respiratory vaccines are live attenuated vaccines (Lycke, 2012) with subunit vaccines providing more challenges for mucosal administration. This is due to the low immunogenicity of subunit antigens which is associated with increased safety compared to live-attenuated vaccines (WilsonWelder et al., 2009). In parenteral administration this drawback is overcome by the addition of adjuvants such as alum, which increase immunogenicity and may modulate the resulting immune response to a certain degree (Chen and Cerutti, 2010). The same is needed for mucosal subunit vaccines, but understanding the appropriate adjuvant strategy and finding an effective and safe mucosal adjuvant is still the subject of much research (Chadwick et al., 2010; Holmgren et al., 2003). One strategy is to administer the antigen in particulate form, as soluble antigens tend to result in tolerance rather than immunity at mucosal surfaces (Mestecky et al., 2007; Neutra and Kozlowski, 2006). To interact with the immune system the particulate antigen has to be taken up first. This can be mediated by cells such as dendritic cells (DCs) or M-cells (Neutra and Kozlowski, 2006). This creates size restrictions for possible formulations as DCs are generally thought to take up particles in the nano- to low micrometre size range (Bachmann and Jennings, 2010; De Temmerman et al., 2011; Li et al., 2011), whereas M-cells
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may take up particles up to 10 mm (Tafaghodi et al., 2004). The antigen will then be processed and activated antigen presenting cells (APC) will access local lymph nodes (LN), where local immune reactions and systemic response are generated. In this study two types of formulations were developed as nasal subunit vaccines. The first utilised polymeric nanoparticles (NP) of either chitosan (Scherließ and Buske, 2012) or agarose (Trows and Scherließ, 2012) as antigen carriers; the other type were chitosan microparticles (MP) (Westmeier, 2010). All particles were formulated as dry powder by spray drying. Particles of two different sizes (nanometre versus micrometre) were investigated as antigen carriers. The nanometre particles are believed to be taken up to a larger extent by dendritic cells (De Temmerman et al., 2011), but the amount of antigen which can be carried per particle is higher for the microparticles. Further, nanoparticles were formulated from chitosan, which has been reported to have adjuvant activity (Boonyo et al., 2007; van der Lubben et al., 2001) and from agarose which has no adjuvant activity. As the nanoparticles could not be dried without the use of a bulking agent, they were incorporated in a water soluble matrix of mannitol producing a nano-in-microparticle (NiM) formulation, which stabilises the nanoparticles and allows redispersion of individual nanoparticles upon matrix dissolution. A great advantage of dry powder formulations in comparison to liquid preparations is their increased storage stability (Amorij et al., 2008). Not only is the particulate form stabilised and aggregation prevented, but also the antigen itself is stabilised once transferred successfully to the dried state. This allows storage at room temperature and prolonged shelf life (Ohtake et al., 2010). 1.1. Aim of the study The aim of the study was to investigate whether the developed nano- and microparticulate dry powder vaccine formulations were tolerated in vivo and whether an immune effect to the model subunit antigen ovalbumin could be generated following nasal administration. 2. Materials and methods 2.1. Formulations Chitosan (Chitosan, Sigma–Aldrich, Germany) nanoparticles were produced by ionic gelation with bile salts (Scherließ and Buske, 2012) using chitosan with high adjuvant activity as determined earlier (Scherließ et al., 2013a). For antigen-loaded nanoparticles, the antigen was added to the chitosan solution prior to addition of the bile salt solution. The nanoparticle dispersion was subsequently spray dried (Büchi B-290, Büchi, Flawil, Switzerland) with the addition of mannitol (Pearlitol, Roquette, Lestrem, France) as matrix resulting in nano-inmicroparticles (NiMs). An antigen-free placebo powder formulation of chitosan nanoparticles in a mannitol matrix was similarly prepared. Chitosan microparticles were produced by direct spray drying of a chitosan solution (Scherließ and Trows, 2011). Agarose (AppliChem GmbH, Germany) nanoparticles were produced by solvent-change precipitation utilising 2-propanol (Trows and Scherließ, 2012). Similarly to the chitosan nanoparticles, mannitol was added to the dispersion and the formulation was spray dried to form NiMs. All formulations were stored dry under refrigeration until used. Controls used included a liquid preparation of 10 mg ovalbumin in 50 mL sterile PBS and a subcutaneous formulation of 10 mg ovalbumin in 200 mL alum (Alu-Gel-S, Serva, Germany). Both control formulations were prepared directly before administration.
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2.2. Characterisation of formulations Nanoparticle size was determined by dynamic laser scattering utilising a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) and is given as mean z-average of a triple measurement. Microparticle size was measured by laser diffraction upon dry dispersion at 3 bar (Helos, Sympatec, Clausthal-Zellerfeld, Germany) and is given as median (50) particle size (n = 3). To assess particle morphology, scanning electron microscopy (Zeiss DSM 940, Carl Zeiss AG (Oberkochen, Germany) pictures have been taken. Antigen content was determined with a protein assay (Micro BCA assay, Thermo Scientific, Rockford, IL, USA) utilising an OVA calibration curve. Protein integrity has been confirmed by SDS-PAGE, which was performed using a Mini Protean Tetra cell system (BIO-RAD Laboratories Inc. USA) with hand casted 10% polyacrylamide gels (90 70 1 mm). Samples were dissolved in 0.01 N HCl pH 2.6 and incubated with loading buffer containing 5% mercaptoethanol at 90 C for 5 min. 10 mL sample volume was transferred to the gels resulting in 4 mg of BSA per lane. A molecular weight marker (PageRuler Prestained Protein ladder, Thermo Scientific, Rockford, IL, USA) and pure protein were used as reference. The electrophoresis was run for 50 min at 200 V. Gels were stained with Coomassie Brilliant Blue (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and destained with a mixture of acetic acid and isopropanol until protein bands were visible clearly. 2.3. Animals 6–8-week-old C57BL/6J mice, OT-I mice and OT-II mice, were obtained from the HTRU, University of Otago, and were bred and maintained under specific-pathogen free conditions. In OT-I transgenic mice a significant percentage of the CD8 T cells express a T cell receptor specific for the MHC-I restricted CD8 epitope of the OVA protein cell (OVA 257–264), whereas OT-II transgenic mice exhibit a significant population of CD4 T cells expressing the TCR for the MHC-II restricted CD4 epitope of OVA (OVA323–339) (McBurney et al., 2008). All mice had ad libitum access to food and water. All experiments were performed with the approval of the University of Otago Animal Ethics Committee (AEC#D94/11). At day 1, OT-I mice and OT-II mice were killed by cervical dislocation and lymph nodes and spleens were removed. Single cell preparations were washed, counted and prepared for injection. All animals in the study received an intravenous injection of 2 107 OTI and OTII cells dispersed in 200 mL sterile PBS buffer in their tail vein. The animals were separated into 6 subgroups and on day 0, 14 and 28 of the study, the animals were anaesthetised and received ovalbumin-loaded chitosan NiM, agarose NiM, chitosan microparticles or blank chitosan NiM as dry powder insufflation into the left nostril using a PennCentury DP4-MTM (powder amount equal to 10 mg antigen). The nasal control group received 10 mg ovalbumin in 50 mL PBS and the subcutaneous control group got an injection of 10 mg ovalbumin with alum in the dorsal skin fold (without previous anaesthesia). The weight and condition of the mice was monitored regularly during the study and all animals were examined for possible adverse reactions. On day 31 all mice of the study were euthanized and blood, lymph nodes (cervical, mediastinal) and spleens were removed. Nose and lung washes were collected by repeatedly flushing the respective organs with PBS (0.2 mL per nose, 1 mL per lung). 2.4. FACS analysis For FACS-staining 1 mL of lymph node or spleen preparations (2 106 cell/mL) were washed twice with FACS buffer (PBS pH 7.4 + 10 g/L BSA) and incubated with a 24G2 antibody to inhibit
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non-specific antibody binding. Cells were washed and then stained with anti-CD4, anti-CD8, anti-Va2 and anti-Vb5 antibodies (all antibodies from BD Biosciences). Sample analysis was done on a BD FACS Canto II using propidium iodide (BD Biosciences) to gate on live cells. Data analysis was performed using FlowJo software (Version 9.2; TreeStar, Inc. Ashland, Oregon, USA). 2.5. Anti-OVA IgG antibody determination The blood samples were allowed to clot and the serum was separated and stored at 20 C until further analysis. OVA specific antibody titres were determined by ELISA as described previously (McBurney et al., 2008). 2.6. Total IgA antibody determination Nasal and lung washes were centrifuged in order to separate cells from the supernatant containing IgA. The supernatant was stored at 20 C until further evaluation. Antibody quantification was performed via an ELISA utilising a standard (purified mouse IgA, k isotype control from BD Biosciences PharmingenTM, Heidelberg, Germany) for calibration and quantification. For this, 96 well ELISA plates were coated overnight with 1 mg IgA capture antibody/well. The plates were washed and blocked to avoid nonspecific binding. Subsequently, the wash fluid samples were pipetted on the plate in a serial dilution and were incubated at 37 C for 2 h. The plates were washed again and the secondary antibody (Biotin Rat anti-mouse IgA) was added and the plates were incubated at 37 C for 30 min. After another washing step, streptavidin-HRP was added and the plates were incubated at 37 C for 20 min. Finally, the HRP substrate (1 mM ABTS (2,20 -azino-di-3ethylbenzthiazolinesulfonic acid)) was added and the plates were incubated in the dark for 5 min. The reaction was stopped by addition of 50 mL of a 2 mM sodium azide to each well and the plates were read at 414 nm using a POLARStar Omega plate reader (BMG labtech). IgA quantification was calculated using the standard curve. 2.7. Restimulation and cytokine assay For each sample, 3 wells of a 96 well plate were coated with anti-CD3 and incubated overnight. The plate was washed and IL-2 (2 ng/mL) containing media was added to 9 wells per sample. Three wells also contained OVA (200 mg/mL) and 3 wells contained only IL-2 as a negative control. 100 mL of the samples were added to each well and the plates were incubated at 37 C for 3 days. The
supernatant was then removed and cytokine production was assessed using a cytometric bead assay (CBA, BD Biosciences) according to the manufacturer’s instructions. 2.8. Statistical evaluation Statistical analyses were conducted utilising a one-way analysis of variance (ANOVA) followed by post hoc analysis using Tukey’s multiple comparison with a confidence interval of a = 0.05 (GraphPad Prism, 6.04). Significance is indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001. 2.9. Histological examination Single noses of animals from mice immunised by the intranasal and subcutaneous routes were prepared for histological examination. For this, the noses were separated, immobilised and decalcified. Afterwards the samples were sliced horizontally from the nostrils to the nasopharynx and slices were stained with HE stain for histological evaluation. Pictures of the nasal cross section and close ups of the epithelium have been taken. The figure shows a detail of the upper nasal cavity (slice number between 750 and 902). 3. Results The chitosan microparticulate formulation exhibited a median particle size of 2.09 mm (0.01 mm), whereas chitosan nanoparticles were approximately 180 nm (PDI 0.3) and agarose nanoparticles were approximately 170 nm (PDI 0.13). The final NiM formulations had a particle size of 2–5 mm and were spherical in shape (Fig. 1). To verify that nanoparticles were released from the NiM formulations upon dissolution of the mannitol matrix, NiM samples were dispersed in buffer and particle size was checked. Z-average of the nanoparticles was maintained, while PDI was slightly increased indicating that some particles might have aggregated. Antigen content was 1.2% (w/w) for the microparticulate formulation and 0.9% (w/w) for the NiM formulations. Integrity of the antigen in the formulations was confirmed by SDS-PAGE (Fig. 2). Similar formulations have been characterised due to their dispersibility in several studies and it was shown that they can be dispersed well by an active device resulting in a mean particle size of 3–5 mm and predominant nasal deposition of more than 80% of the dose in a human nasal cast model is achieved (Buske and Scherließ, 2013; Scherließ, 2011; Trows and Scherließ, 2012). For the mouse insufflator used here,
Fig. 1. Chitosan microparticles (left); agarose nanoparticles in mannitol microparticles (middle); chitosan nanoparticles in mannitol microparticles (right).
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Fig. 2. SDS-PAGE gel of OVA from chitosan MP (left) and OVA from chitosan NP and agarose NP (right). Untreated OVA and molecular weight marker have been examined for comparison.
Fig. 3. Histological pictures of nasal epithelium (a) untreated control (s.c. administration) (b) intranasal OVA chitosan MP (c) intranasal OVA agarose NiM (d) intranasal OVA chitosan NiM.
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dispersion characteristics were checked in advance and depending on the emitted dose fraction, loaded dose was calculated to ensure delivery of the correct dose. The powder dose emitted by the device is totally deposited in the nose, as the device is placed directly into the nostril. A previously performed pilot study showed general suitability of the formulations and the setup for nasal administration of powders to mice. Powder administration with the insufflator was possible and the powder was tolerated without any unwanted reactions such as increased nose licking, irritancy or difficulties in breathing. The same is true for the animals of the main study, which all gained weight throughout the study and behaved normally. Histological examination of the epithelium of the nasal cavities showed no difference between the animals having received OVA-containing powder and the control animal having received an s.c. injection instead of a nasal treatment (Fig. 3). This confirms local tolerability of the dry powder formulations. Local cellular immune responses at the lung-draining mediastinal lymph node and the nose-draining cervical lymph nodes were assessed (Figs. 4 and 5). Both the number and percentage of antigen-specific CD4+ and CD8+ cells were assessed as upon antigen stimulation the percentage of antigen specific cells increases, but also lymph nodes are enlarged which may result in an increase in the total number of cells. If the nasal formulations are effective, an increase in antigen-specific cells in the nose draining cervical lymph nodes should be seen, whereas an increase in the mediastinal LN would indicate the antigen drained to the lungs. From the s.c. formulation, no local cellular immune response is expected as alum-adjuvanted parenteral antigen predominantly results in a humoral response (Ribeiro and Schijns, 2010; Sharp et al., 2009). No changes in the percent or number of CD4+ Tg cells were observed (data not shown). Administration of antigen-carrying nanoparticles as dry powder to the nose showed a trend towards expansion of CD8+ Tg cells in the cervical lymph nodes with a significant increase in the percent CD8+ Tg cells being detected in mice immunised with OVA chitosan MP and OVA agarose NP compared to the intranasal OVA solution. Interestingly, the placebo chitosan NP also resulted in a mild increase in CD8+ Tg cells, which might be due to its adjuvant effect seen upon s.c. administration
(Scherließ et al., 2013a), which seems to also stimulate pre-existing CD8+ Tg cells (from OT1) without the presence of antigen. However, due to the modest and statistically not significant responses observed this result should not be over interpreted. For the mediastinal LN, only the liquid OVA preparation resulted in a significant increase in CD8+ Tg cells (number and percentage), whereas for all other formulations the percent and total number of CD8+ Tg cells remained at the same level. Systemic levels of IgG and local levels of IgA, in nasal washes, were investigated. A potential advantage of nasal immunisation is the production of a local IgA immune response; however, only low levels of IgA were detected (Fig. 6, left, no statistical significant differences). This is partly due to difficulties in complete recovery of the wash fluid from nasal washes. As expected, only mice given s.c. OVA in alum had significantly elevated levels of OVA-specific IgG in serum (Fig. 6, right). Restimulation of the lymph node and spleen cells in vitro did not result in antigen-specific proliferation or cytokine production (data not shown) indicating the transient nature of the immune response provoked. 4. Discussion The development of dry powder vaccine formulations that can be delivered by the intranasal route offers many potential advantages. Here the local tolerability of the formulations and their ability to stimulate both systemic and local immune responses was examined. Two of the tested formulations were dry powder nanoparticle-in-microparticle (NiM) formulations. The mannitol microparticles had a mean diameter of 3–5 mm and served as matrix for the antigen-loaded 180 nm nanoparticles (Scherließ et al., 2013b). As the mannitol matrix is soluble in aqueous media it will dissolve upon deposition on the wet nasal mucosa releasing the nanoparticles to be taken up by APCs. The third formulation was a 2–3 mm antigen loaded microparticle which would need to interact with the APC as a whole. It is believed that in the respiratory tract larger particles are cleared by macrophages which means it may be beneficial to produce vaccines in the nanometre size range (Blank et al., 2011). Nonetheless, previous in vitro experiments with the formulations tested here showed that both the nanoparticles and microparticles
Fig. 4. Percentage (left) and number (right) of CD8+ Tg cells from cervical lymph nodes. Data from individual mice (n = 3–5) and the mean are shown (*p < 0.05).
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Fig. 5. Percentage (left) and number (right) of CD8+ Tg cells from mediastinal LN. Data from individual mice (n = 3–5) and the mean are shown. **p < 0.05, ***p < 0.001 compared to all other formulations.
could be taken up by dendritic cells (Scherließ et al., 2013b); therefore, all formulations were examined in vivo. In this study, powders were delivered using a PennCentury DP4-MTM insufflator which is designed for use with mice. Mice tolerated powder doses of up to 1.1 mg without adverse reactions although it could be expected that the mucoadhesive biopolymers chitosan and agarose, respectively, stick to the mucosa and hence may obstruct the nasal cavity (Illum et al., 2001). This was not the case in this study and no formulation residue could be detected in the nasal cavity when animals were sacrificed 3 days after the last dose of vaccine was administered. Interestingly the liquid vaccine preparation given intranasally preferentially stimulated a response in the lymph node draining the lungs as opposed to the lymph nodes draining the nose indicating the vaccine ended up in the lungs. This did not occur with the dry powder formulation suggesting these did not drain to
the lungs. This shows an advantage of dry powder formulations if exclusive nasal administration is wanted. The immune response following nasal administration of antigen-containing particles as powder was limited to a modest local CD8 T cell response in the draining lymph nodes. The reason only a CD8 response was detected is likely due to differences in the affinity OT-II CD4 T cells and OT-I CD8 T cells have for their respective ovalbumin peptide epitopes (Gallegos and Bevan, 2004). Antibody responses would be of benefit for protective immunity, whereas a predominant cytotoxic T cell lymphocyte (CTL) response would be preferred for defending intracellular pathogens and in cancer therapy. A perceived advantage of nasal immunisation is the potential to stimulate protective mucosal IgA as well as systemic IgG besides a sound CTL response (Debin et al., 2002). However it has been suggested that a ten times higher mucosal dose might be needed to achieve similar IgG levels to parenteral administration
Fig. 6. Total nasal IgA response (left) and OVA-specific serum IgG. Data from individual mice (n = 1–5) and the mean are shown (*p < 0.05).
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(Haneberg and Holst, 2002). A further challenge for nasal immunisation is elimination of the vaccine from the nose by nasal clearance which will reduce the dose that will be available for uptake and processing. It will be important in future studies to increase and expand the immune responses generated by the dry powder vaccine by increasing the dose of antigen given and through the inclusion of adjuvants. In this study, the micro- and nanoparticles stimulated a similar immune response. The mucoadhesive properties of chitosan will impact on residence time in the nose and chitosan has been reported to have adjuvant activity (Scherließ et al., 2013a), which should increase the immune response. However, there was no difference observed in immune responses induced by the agarose and chitosan nanoparticles. It is known that the choice of adjuvant may play a critical role for mucosal vaccines. Well-known parenteral adjuvants such as alum are not effective on mucosal surfaces (Lawson et al., 2011), whereas others such as the cholera enterotoxin (CT) work very well but are associated with unacceptable side effects such as neurotoxicity when administered intranasally (Lycke, 2012). Chitosan appears to be a promising adjuvant for mucosal administration as it serves as particle forming agent, is mucoadhesive, is reported to mediate uptake due to an effect on tight junctions (Vllasaliu et al., 2010) and has been reported to activate the NALP3 inflammasome (Sharp et al., 2009). In the present study, chitosan NP seem to have some intrinsic activity as seen by the slightly elevated CD8+ Tg levels in the cervical lymph nodes after chitosan placebo NP treatment, which has not been seen with antigen-free preparations of agarose NP (data not shown). However, the adjuvant effect was too low to significantly boost the immune effect, to result in strong T-cell activation and for the induction of memory cells being capable for restimulation indicating that this adjuvant alone is not sufficient for a nasal dry powder vaccine. Future studies should hence examine the inclusion of an adjuvant that activates APCs through another pathway. It would e.g. be of interest to see if the inclusion of a toll-like receptor dependent adjuvant activating the MyDd88 pathway produces synergistic effects when combined with a chitosan particle. 5. Conclusion The results show that the immune system can be stimulated with the developed formulations, but the immune responses were modest. This is probably due to a low antigen dose and low immunogenicity of the formulations. Future experiments will aim to optimise the immunogenicity of the powder formulations by addition of other adjuvants and an increase in antigen load. The formulations will then be screened in vitro for uptake and their effectiveness to induce DC activation and maturation in order to select the best candidates for in vivo studies. Acknowledgements The authors would like to acknowledge that parts of this study were funded by the Deutsche Forschungsgemeinschaft (DFG) and the Wissenschaftlerinnenförderung of the Faculty of Natural Sciences, Kiel University. References Alpar, H.O., Eyles, J.E., Williamson, E.D., Somavarapu, S., 2001. Intranasal vaccination against plague, tetanus and diphtheria. Adv. Drug Deliv. Rev. 51, 173–201. Alpar, H.O., Somavarapu, S., Atuah, K.N., Bramwell, V.W., 2005. Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery. Adv. Drug Deliv. Rev. 57, 411–430. Amidi, M., Romeijn, S.G., Verhoef, J.C., Junginger, H.E., Bungener, L., Huckriede, A., Crommelin, D., Jiskoot, J.A., 2007. N-trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model. Vaccine 25, 144–153.
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Scherließ, R., Buske, S., Young, K., Weber, B., Rades, T., Hook, S., 2013a. In vivo evaluation of chitosan as an adjuvant in subcutaneous vaccine formulations. Vaccine 31, 4812–4819. Scherließ, R., Mönckedieck, M., Young, K., Trows, S., Buske, S., Hook, S., 2013b. Evaluation of impact of nasal dry powder vaccine formulations on uptake and activation by dendritic cells. In: Dalby, R. (Ed.), RDD Europe 2013. Davies Health Care International Publishing, Berlin, pp. 279–283. Scherließ, R., Trows, S., 2011. Novel formulation concept for particulate uptake of vaccines via the nasal associated lymphoid tissue. Procedia Vaccinol. 4, 113–119. Sharp, F.A., Ruane, D., Claass, B., Creagh, E., Harris, J., Malyala, P., Singh, M., O'Hagan, D.T., Pétrilli, V., Tschopp, J., O'Neill, L.A.J., Lavelle, E.C., 2009. Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome. Proc. Natl. Acad. Sci. U. S. A. 106, 870–875.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
First in vivo evaluation of particulate nasal dry powder vaccine formulations containing ovalbumin in mice Regina Scherließ a, * , Mathias Mönckedieck a , Katherine Young b , Sabrina Trows a , Simon Buske a , Sarah Hook b a b
Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany School of Pharmacy, University of Otago, 18, Frederick Street, Dunedin, New Zealand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 November 2014 Received in revised form 8 January 2015 Accepted 10 January 2015 Available online 13 January 2015
In this study three different dry powder vaccine formulations containing the model antigen ovalbumin were evaluated for their immune effects after nasal administration to C57Bl/6 mice in an adoptive cell transfer model. The formulations were chitosan nanoparticles in a mannitol matrix, chitosan microparticles and agarose nanoparticles in a mannitol matrix. Dry powder administration to mice was well tolerated and did not result in any adverse reactions. No translocation of the dry powder formulations to the lung could be detected. The local cellular immune response in the cervical lymph nodes was modest and only for the chitosan microparticles and the agarose nanoparticles was there a significant difference compared to s.c. injection of ovalbumin in alum. No humoral response could be measured after nasal administration. The results provide some evidence that nasal administration of dry powder formulations can stimulate an immune response, but the response was modest. This is probably due to a low antigen dose and low immunogenicity of the formulations. Further studies will aim at enhancing the antigen load and improving adjuvant activity. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Chitosan Nano-in-microparticle formulation Microparticles Powder insufflation Local cellular response
1. Introduction Mucosal vaccination is an attractive alternative to parenteral vaccination and offers some advantages such as avoidance of needle-stick injuries, removing the requirement for medical personnel for vaccine administration and an improved local immune response at the site of delivery (Lycke, 2012). The respiratory tract, particularly the nose, is well suited for mucosal vaccination as it is the normal route of entry for many pathogens; hence, it is well equipped with immunocompetent cells (Davis, 2001). Further, it is easily accessible for (self-) administration. Vaccines for mucosal vaccination via the respiratory tract have been successfully developed, for example the influenza vaccines FluMist/Fluenz (Medimmune/Astra Zeneca), which make use of a cold-adapted influenza strain and are administered via a nasal spray. Further nasal vaccines under development are formulated in polymeric particles made of poly-lactid-co-glycolid (Csaba et al., 2009; Slütter et al., 2010) or biopolymers such as chitosan (Amidi et al., 2007; Gordon et al., 2008; van der Lubben et al., 2003) and
* Corresponding author. Tel.: +49 431 8801340. E-mail address: [email protected] (R. Scherließ). http://dx.doi.org/10.1016/j.ijpharm.2015.01.015 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
alginate (Borges et al., 2008), virus-like particles (Vodak et al., 2012) or lipid particles (Alpar et al., 2001, 2005; Saraf et al., 2006). So far all successful respiratory vaccines are live attenuated vaccines (Lycke, 2012) with subunit vaccines providing more challenges for mucosal administration. This is due to the low immunogenicity of subunit antigens which is associated with increased safety compared to live-attenuated vaccines (WilsonWelder et al., 2009). In parenteral administration this drawback is overcome by the addition of adjuvants such as alum, which increase immunogenicity and may modulate the resulting immune response to a certain degree (Chen and Cerutti, 2010). The same is needed for mucosal subunit vaccines, but understanding the appropriate adjuvant strategy and finding an effective and safe mucosal adjuvant is still the subject of much research (Chadwick et al., 2010; Holmgren et al., 2003). One strategy is to administer the antigen in particulate form, as soluble antigens tend to result in tolerance rather than immunity at mucosal surfaces (Mestecky et al., 2007; Neutra and Kozlowski, 2006). To interact with the immune system the particulate antigen has to be taken up first. This can be mediated by cells such as dendritic cells (DCs) or M-cells (Neutra and Kozlowski, 2006). This creates size restrictions for possible formulations as DCs are generally thought to take up particles in the nano- to low micrometre size range (Bachmann and Jennings, 2010; De Temmerman et al., 2011; Li et al., 2011), whereas M-cells
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may take up particles up to 10 mm (Tafaghodi et al., 2004). The antigen will then be processed and activated antigen presenting cells (APC) will access local lymph nodes (LN), where local immune reactions and systemic response are generated. In this study two types of formulations were developed as nasal subunit vaccines. The first utilised polymeric nanoparticles (NP) of either chitosan (Scherließ and Buske, 2012) or agarose (Trows and Scherließ, 2012) as antigen carriers; the other type were chitosan microparticles (MP) (Westmeier, 2010). All particles were formulated as dry powder by spray drying. Particles of two different sizes (nanometre versus micrometre) were investigated as antigen carriers. The nanometre particles are believed to be taken up to a larger extent by dendritic cells (De Temmerman et al., 2011), but the amount of antigen which can be carried per particle is higher for the microparticles. Further, nanoparticles were formulated from chitosan, which has been reported to have adjuvant activity (Boonyo et al., 2007; van der Lubben et al., 2001) and from agarose which has no adjuvant activity. As the nanoparticles could not be dried without the use of a bulking agent, they were incorporated in a water soluble matrix of mannitol producing a nano-in-microparticle (NiM) formulation, which stabilises the nanoparticles and allows redispersion of individual nanoparticles upon matrix dissolution. A great advantage of dry powder formulations in comparison to liquid preparations is their increased storage stability (Amorij et al., 2008). Not only is the particulate form stabilised and aggregation prevented, but also the antigen itself is stabilised once transferred successfully to the dried state. This allows storage at room temperature and prolonged shelf life (Ohtake et al., 2010). 1.1. Aim of the study The aim of the study was to investigate whether the developed nano- and microparticulate dry powder vaccine formulations were tolerated in vivo and whether an immune effect to the model subunit antigen ovalbumin could be generated following nasal administration. 2. Materials and methods 2.1. Formulations Chitosan (Chitosan, Sigma–Aldrich, Germany) nanoparticles were produced by ionic gelation with bile salts (Scherließ and Buske, 2012) using chitosan with high adjuvant activity as determined earlier (Scherließ et al., 2013a). For antigen-loaded nanoparticles, the antigen was added to the chitosan solution prior to addition of the bile salt solution. The nanoparticle dispersion was subsequently spray dried (Büchi B-290, Büchi, Flawil, Switzerland) with the addition of mannitol (Pearlitol, Roquette, Lestrem, France) as matrix resulting in nano-inmicroparticles (NiMs). An antigen-free placebo powder formulation of chitosan nanoparticles in a mannitol matrix was similarly prepared. Chitosan microparticles were produced by direct spray drying of a chitosan solution (Scherließ and Trows, 2011). Agarose (AppliChem GmbH, Germany) nanoparticles were produced by solvent-change precipitation utilising 2-propanol (Trows and Scherließ, 2012). Similarly to the chitosan nanoparticles, mannitol was added to the dispersion and the formulation was spray dried to form NiMs. All formulations were stored dry under refrigeration until used. Controls used included a liquid preparation of 10 mg ovalbumin in 50 mL sterile PBS and a subcutaneous formulation of 10 mg ovalbumin in 200 mL alum (Alu-Gel-S, Serva, Germany). Both control formulations were prepared directly before administration.
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2.2. Characterisation of formulations Nanoparticle size was determined by dynamic laser scattering utilising a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) and is given as mean z-average of a triple measurement. Microparticle size was measured by laser diffraction upon dry dispersion at 3 bar (Helos, Sympatec, Clausthal-Zellerfeld, Germany) and is given as median (50) particle size (n = 3). To assess particle morphology, scanning electron microscopy (Zeiss DSM 940, Carl Zeiss AG (Oberkochen, Germany) pictures have been taken. Antigen content was determined with a protein assay (Micro BCA assay, Thermo Scientific, Rockford, IL, USA) utilising an OVA calibration curve. Protein integrity has been confirmed by SDS-PAGE, which was performed using a Mini Protean Tetra cell system (BIO-RAD Laboratories Inc. USA) with hand casted 10% polyacrylamide gels (90 70 1 mm). Samples were dissolved in 0.01 N HCl pH 2.6 and incubated with loading buffer containing 5% mercaptoethanol at 90 C for 5 min. 10 mL sample volume was transferred to the gels resulting in 4 mg of BSA per lane. A molecular weight marker (PageRuler Prestained Protein ladder, Thermo Scientific, Rockford, IL, USA) and pure protein were used as reference. The electrophoresis was run for 50 min at 200 V. Gels were stained with Coomassie Brilliant Blue (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and destained with a mixture of acetic acid and isopropanol until protein bands were visible clearly. 2.3. Animals 6–8-week-old C57BL/6J mice, OT-I mice and OT-II mice, were obtained from the HTRU, University of Otago, and were bred and maintained under specific-pathogen free conditions. In OT-I transgenic mice a significant percentage of the CD8 T cells express a T cell receptor specific for the MHC-I restricted CD8 epitope of the OVA protein cell (OVA 257–264), whereas OT-II transgenic mice exhibit a significant population of CD4 T cells expressing the TCR for the MHC-II restricted CD4 epitope of OVA (OVA323–339) (McBurney et al., 2008). All mice had ad libitum access to food and water. All experiments were performed with the approval of the University of Otago Animal Ethics Committee (AEC#D94/11). At day 1, OT-I mice and OT-II mice were killed by cervical dislocation and lymph nodes and spleens were removed. Single cell preparations were washed, counted and prepared for injection. All animals in the study received an intravenous injection of 2 107 OTI and OTII cells dispersed in 200 mL sterile PBS buffer in their tail vein. The animals were separated into 6 subgroups and on day 0, 14 and 28 of the study, the animals were anaesthetised and received ovalbumin-loaded chitosan NiM, agarose NiM, chitosan microparticles or blank chitosan NiM as dry powder insufflation into the left nostril using a PennCentury DP4-MTM (powder amount equal to 10 mg antigen). The nasal control group received 10 mg ovalbumin in 50 mL PBS and the subcutaneous control group got an injection of 10 mg ovalbumin with alum in the dorsal skin fold (without previous anaesthesia). The weight and condition of the mice was monitored regularly during the study and all animals were examined for possible adverse reactions. On day 31 all mice of the study were euthanized and blood, lymph nodes (cervical, mediastinal) and spleens were removed. Nose and lung washes were collected by repeatedly flushing the respective organs with PBS (0.2 mL per nose, 1 mL per lung). 2.4. FACS analysis For FACS-staining 1 mL of lymph node or spleen preparations (2 106 cell/mL) were washed twice with FACS buffer (PBS pH 7.4 + 10 g/L BSA) and incubated with a 24G2 antibody to inhibit
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non-specific antibody binding. Cells were washed and then stained with anti-CD4, anti-CD8, anti-Va2 and anti-Vb5 antibodies (all antibodies from BD Biosciences). Sample analysis was done on a BD FACS Canto II using propidium iodide (BD Biosciences) to gate on live cells. Data analysis was performed using FlowJo software (Version 9.2; TreeStar, Inc. Ashland, Oregon, USA). 2.5. Anti-OVA IgG antibody determination The blood samples were allowed to clot and the serum was separated and stored at 20 C until further analysis. OVA specific antibody titres were determined by ELISA as described previously (McBurney et al., 2008). 2.6. Total IgA antibody determination Nasal and lung washes were centrifuged in order to separate cells from the supernatant containing IgA. The supernatant was stored at 20 C until further evaluation. Antibody quantification was performed via an ELISA utilising a standard (purified mouse IgA, k isotype control from BD Biosciences PharmingenTM, Heidelberg, Germany) for calibration and quantification. For this, 96 well ELISA plates were coated overnight with 1 mg IgA capture antibody/well. The plates were washed and blocked to avoid nonspecific binding. Subsequently, the wash fluid samples were pipetted on the plate in a serial dilution and were incubated at 37 C for 2 h. The plates were washed again and the secondary antibody (Biotin Rat anti-mouse IgA) was added and the plates were incubated at 37 C for 30 min. After another washing step, streptavidin-HRP was added and the plates were incubated at 37 C for 20 min. Finally, the HRP substrate (1 mM ABTS (2,20 -azino-di-3ethylbenzthiazolinesulfonic acid)) was added and the plates were incubated in the dark for 5 min. The reaction was stopped by addition of 50 mL of a 2 mM sodium azide to each well and the plates were read at 414 nm using a POLARStar Omega plate reader (BMG labtech). IgA quantification was calculated using the standard curve. 2.7. Restimulation and cytokine assay For each sample, 3 wells of a 96 well plate were coated with anti-CD3 and incubated overnight. The plate was washed and IL-2 (2 ng/mL) containing media was added to 9 wells per sample. Three wells also contained OVA (200 mg/mL) and 3 wells contained only IL-2 as a negative control. 100 mL of the samples were added to each well and the plates were incubated at 37 C for 3 days. The
supernatant was then removed and cytokine production was assessed using a cytometric bead assay (CBA, BD Biosciences) according to the manufacturer’s instructions. 2.8. Statistical evaluation Statistical analyses were conducted utilising a one-way analysis of variance (ANOVA) followed by post hoc analysis using Tukey’s multiple comparison with a confidence interval of a = 0.05 (GraphPad Prism, 6.04). Significance is indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001. 2.9. Histological examination Single noses of animals from mice immunised by the intranasal and subcutaneous routes were prepared for histological examination. For this, the noses were separated, immobilised and decalcified. Afterwards the samples were sliced horizontally from the nostrils to the nasopharynx and slices were stained with HE stain for histological evaluation. Pictures of the nasal cross section and close ups of the epithelium have been taken. The figure shows a detail of the upper nasal cavity (slice number between 750 and 902). 3. Results The chitosan microparticulate formulation exhibited a median particle size of 2.09 mm (0.01 mm), whereas chitosan nanoparticles were approximately 180 nm (PDI 0.3) and agarose nanoparticles were approximately 170 nm (PDI 0.13). The final NiM formulations had a particle size of 2–5 mm and were spherical in shape (Fig. 1). To verify that nanoparticles were released from the NiM formulations upon dissolution of the mannitol matrix, NiM samples were dispersed in buffer and particle size was checked. Z-average of the nanoparticles was maintained, while PDI was slightly increased indicating that some particles might have aggregated. Antigen content was 1.2% (w/w) for the microparticulate formulation and 0.9% (w/w) for the NiM formulations. Integrity of the antigen in the formulations was confirmed by SDS-PAGE (Fig. 2). Similar formulations have been characterised due to their dispersibility in several studies and it was shown that they can be dispersed well by an active device resulting in a mean particle size of 3–5 mm and predominant nasal deposition of more than 80% of the dose in a human nasal cast model is achieved (Buske and Scherließ, 2013; Scherließ, 2011; Trows and Scherließ, 2012). For the mouse insufflator used here,
Fig. 1. Chitosan microparticles (left); agarose nanoparticles in mannitol microparticles (middle); chitosan nanoparticles in mannitol microparticles (right).
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Fig. 2. SDS-PAGE gel of OVA from chitosan MP (left) and OVA from chitosan NP and agarose NP (right). Untreated OVA and molecular weight marker have been examined for comparison.
Fig. 3. Histological pictures of nasal epithelium (a) untreated control (s.c. administration) (b) intranasal OVA chitosan MP (c) intranasal OVA agarose NiM (d) intranasal OVA chitosan NiM.
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dispersion characteristics were checked in advance and depending on the emitted dose fraction, loaded dose was calculated to ensure delivery of the correct dose. The powder dose emitted by the device is totally deposited in the nose, as the device is placed directly into the nostril. A previously performed pilot study showed general suitability of the formulations and the setup for nasal administration of powders to mice. Powder administration with the insufflator was possible and the powder was tolerated without any unwanted reactions such as increased nose licking, irritancy or difficulties in breathing. The same is true for the animals of the main study, which all gained weight throughout the study and behaved normally. Histological examination of the epithelium of the nasal cavities showed no difference between the animals having received OVA-containing powder and the control animal having received an s.c. injection instead of a nasal treatment (Fig. 3). This confirms local tolerability of the dry powder formulations. Local cellular immune responses at the lung-draining mediastinal lymph node and the nose-draining cervical lymph nodes were assessed (Figs. 4 and 5). Both the number and percentage of antigen-specific CD4+ and CD8+ cells were assessed as upon antigen stimulation the percentage of antigen specific cells increases, but also lymph nodes are enlarged which may result in an increase in the total number of cells. If the nasal formulations are effective, an increase in antigen-specific cells in the nose draining cervical lymph nodes should be seen, whereas an increase in the mediastinal LN would indicate the antigen drained to the lungs. From the s.c. formulation, no local cellular immune response is expected as alum-adjuvanted parenteral antigen predominantly results in a humoral response (Ribeiro and Schijns, 2010; Sharp et al., 2009). No changes in the percent or number of CD4+ Tg cells were observed (data not shown). Administration of antigen-carrying nanoparticles as dry powder to the nose showed a trend towards expansion of CD8+ Tg cells in the cervical lymph nodes with a significant increase in the percent CD8+ Tg cells being detected in mice immunised with OVA chitosan MP and OVA agarose NP compared to the intranasal OVA solution. Interestingly, the placebo chitosan NP also resulted in a mild increase in CD8+ Tg cells, which might be due to its adjuvant effect seen upon s.c. administration
(Scherließ et al., 2013a), which seems to also stimulate pre-existing CD8+ Tg cells (from OT1) without the presence of antigen. However, due to the modest and statistically not significant responses observed this result should not be over interpreted. For the mediastinal LN, only the liquid OVA preparation resulted in a significant increase in CD8+ Tg cells (number and percentage), whereas for all other formulations the percent and total number of CD8+ Tg cells remained at the same level. Systemic levels of IgG and local levels of IgA, in nasal washes, were investigated. A potential advantage of nasal immunisation is the production of a local IgA immune response; however, only low levels of IgA were detected (Fig. 6, left, no statistical significant differences). This is partly due to difficulties in complete recovery of the wash fluid from nasal washes. As expected, only mice given s.c. OVA in alum had significantly elevated levels of OVA-specific IgG in serum (Fig. 6, right). Restimulation of the lymph node and spleen cells in vitro did not result in antigen-specific proliferation or cytokine production (data not shown) indicating the transient nature of the immune response provoked. 4. Discussion The development of dry powder vaccine formulations that can be delivered by the intranasal route offers many potential advantages. Here the local tolerability of the formulations and their ability to stimulate both systemic and local immune responses was examined. Two of the tested formulations were dry powder nanoparticle-in-microparticle (NiM) formulations. The mannitol microparticles had a mean diameter of 3–5 mm and served as matrix for the antigen-loaded 180 nm nanoparticles (Scherließ et al., 2013b). As the mannitol matrix is soluble in aqueous media it will dissolve upon deposition on the wet nasal mucosa releasing the nanoparticles to be taken up by APCs. The third formulation was a 2–3 mm antigen loaded microparticle which would need to interact with the APC as a whole. It is believed that in the respiratory tract larger particles are cleared by macrophages which means it may be beneficial to produce vaccines in the nanometre size range (Blank et al., 2011). Nonetheless, previous in vitro experiments with the formulations tested here showed that both the nanoparticles and microparticles
Fig. 4. Percentage (left) and number (right) of CD8+ Tg cells from cervical lymph nodes. Data from individual mice (n = 3–5) and the mean are shown (*p < 0.05).
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Fig. 5. Percentage (left) and number (right) of CD8+ Tg cells from mediastinal LN. Data from individual mice (n = 3–5) and the mean are shown. **p < 0.05, ***p < 0.001 compared to all other formulations.
could be taken up by dendritic cells (Scherließ et al., 2013b); therefore, all formulations were examined in vivo. In this study, powders were delivered using a PennCentury DP4-MTM insufflator which is designed for use with mice. Mice tolerated powder doses of up to 1.1 mg without adverse reactions although it could be expected that the mucoadhesive biopolymers chitosan and agarose, respectively, stick to the mucosa and hence may obstruct the nasal cavity (Illum et al., 2001). This was not the case in this study and no formulation residue could be detected in the nasal cavity when animals were sacrificed 3 days after the last dose of vaccine was administered. Interestingly the liquid vaccine preparation given intranasally preferentially stimulated a response in the lymph node draining the lungs as opposed to the lymph nodes draining the nose indicating the vaccine ended up in the lungs. This did not occur with the dry powder formulation suggesting these did not drain to
the lungs. This shows an advantage of dry powder formulations if exclusive nasal administration is wanted. The immune response following nasal administration of antigen-containing particles as powder was limited to a modest local CD8 T cell response in the draining lymph nodes. The reason only a CD8 response was detected is likely due to differences in the affinity OT-II CD4 T cells and OT-I CD8 T cells have for their respective ovalbumin peptide epitopes (Gallegos and Bevan, 2004). Antibody responses would be of benefit for protective immunity, whereas a predominant cytotoxic T cell lymphocyte (CTL) response would be preferred for defending intracellular pathogens and in cancer therapy. A perceived advantage of nasal immunisation is the potential to stimulate protective mucosal IgA as well as systemic IgG besides a sound CTL response (Debin et al., 2002). However it has been suggested that a ten times higher mucosal dose might be needed to achieve similar IgG levels to parenteral administration
Fig. 6. Total nasal IgA response (left) and OVA-specific serum IgG. Data from individual mice (n = 1–5) and the mean are shown (*p < 0.05).
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(Haneberg and Holst, 2002). A further challenge for nasal immunisation is elimination of the vaccine from the nose by nasal clearance which will reduce the dose that will be available for uptake and processing. It will be important in future studies to increase and expand the immune responses generated by the dry powder vaccine by increasing the dose of antigen given and through the inclusion of adjuvants. In this study, the micro- and nanoparticles stimulated a similar immune response. The mucoadhesive properties of chitosan will impact on residence time in the nose and chitosan has been reported to have adjuvant activity (Scherließ et al., 2013a), which should increase the immune response. However, there was no difference observed in immune responses induced by the agarose and chitosan nanoparticles. It is known that the choice of adjuvant may play a critical role for mucosal vaccines. Well-known parenteral adjuvants such as alum are not effective on mucosal surfaces (Lawson et al., 2011), whereas others such as the cholera enterotoxin (CT) work very well but are associated with unacceptable side effects such as neurotoxicity when administered intranasally (Lycke, 2012). Chitosan appears to be a promising adjuvant for mucosal administration as it serves as particle forming agent, is mucoadhesive, is reported to mediate uptake due to an effect on tight junctions (Vllasaliu et al., 2010) and has been reported to activate the NALP3 inflammasome (Sharp et al., 2009). In the present study, chitosan NP seem to have some intrinsic activity as seen by the slightly elevated CD8+ Tg levels in the cervical lymph nodes after chitosan placebo NP treatment, which has not been seen with antigen-free preparations of agarose NP (data not shown). However, the adjuvant effect was too low to significantly boost the immune effect, to result in strong T-cell activation and for the induction of memory cells being capable for restimulation indicating that this adjuvant alone is not sufficient for a nasal dry powder vaccine. Future studies should hence examine the inclusion of an adjuvant that activates APCs through another pathway. It would e.g. be of interest to see if the inclusion of a toll-like receptor dependent adjuvant activating the MyDd88 pathway produces synergistic effects when combined with a chitosan particle. 5. Conclusion The results show that the immune system can be stimulated with the developed formulations, but the immune responses were modest. This is probably due to a low antigen dose and low immunogenicity of the formulations. Future experiments will aim to optimise the immunogenicity of the powder formulations by addition of other adjuvants and an increase in antigen load. The formulations will then be screened in vitro for uptake and their effectiveness to induce DC activation and maturation in order to select the best candidates for in vivo studies. Acknowledgements The authors would like to acknowledge that parts of this study were funded by the Deutsche Forschungsgemeinschaft (DFG) and the Wissenschaftlerinnenförderung of the Faculty of Natural Sciences, Kiel University. References Alpar, H.O., Eyles, J.E., Williamson, E.D., Somavarapu, S., 2001. Intranasal vaccination against plague, tetanus and diphtheria. Adv. Drug Deliv. Rev. 51, 173–201. Alpar, H.O., Somavarapu, S., Atuah, K.N., Bramwell, V.W., 2005. Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery. Adv. Drug Deliv. Rev. 57, 411–430. Amidi, M., Romeijn, S.G., Verhoef, J.C., Junginger, H.E., Bungener, L., Huckriede, A., Crommelin, D., Jiskoot, J.A., 2007. N-trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model. Vaccine 25, 144–153.
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