Paving the way for pulmonary influenza vaccines:Exploring formulations, models and site of deposition

Vaccination against influenza is considered to be the cornerstone in the prevention and control of influenza outbreaks. However, except for Flumist®, influenza vaccines are currently administered by injection, which induces sub-optimal immune responses. In this respect, an inhalable influenza vaccine could be advantageous as the respiratory tract has abundant immune cells and is the portal of influenza virus entry [3,4]. Despite that, several issues related to administration of influenza vaccines via the respiratory tract are still unaddressed and need further investigation. In this thesis, we explored two different approaches to enhance immunogenicity of influenza vaccine candidates upon administration via the respiratory tract. In the first approach, the site of deposition in the respiratory tract that elicits optimal immune responses was investigated. The second approach deals with the adjuvantation of influenza vaccine candidate; whether or not adjuvantation has the potential to augment immune responses which ultimately leads to enhanced protective efficacy

The effects of oxygen concentration on cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in precision-cut lung slices

Although animal models are often used in drug research, alternative experimental models are becoming more popular as they reduce animal use and suffering. Of particular interest are precision-cut lung slices, which refer to explants - with a reproducible thickness and diameter - that can be cultured ex vivo. Because lung slices (partially) reflect functional and structural features of whole tissue, they are often applied in the field of immunology, pharmacology, toxicology, and virology. Nevertheless, previous research failed to adequately address concerns with respect to the viability of lung slices. For instance, the effect of oxygen concentration on lung slice viability has never been thoroughly investigated. Therefore, the main goal of this study was to investigate the effect of oxygen concentration (20 vs. 80% O-2) on the degree of cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in lung slices. According to the results, slices incubated at 20% O-2 displayed less cell death, antioxidant transcription, and acute inflammation, as well as more cell proliferation, demonstrating that these slices were considerably more viable than slices cultured at 80% O-2. These findings expand our knowledge on lung slices and their use as an alternative experimental model in drug research

Chemical modifications of silicon surfaces for the generation of a tunable surface isoelectric point

The aim of this work was to generate a tunable surface isoelectric point (sIEP), where the surface is modified with two molecules: a weak base (pyridine), carrying a pH dependent positive charge, and a derivative of a strong acid (sulfate), carrying a permanent negative charge in a physiologically relevant pH range. To this end, silicon surfaces were modified with 3-aminopropyltriethoxysilane. These amine-modified surfaces were subsequently derivatized into pyridine- or sulfate-modified surfaces. Then, the surface pKa of pyridine-modified surfaces was determined by a fluorescent nanoparticle adhesion assay (FNAA). Next, these values were used to calculate in which ratio the chemicals must be present in the reaction mixture to generate a mixed pyridine/sulfate-modified surface with a target sIEP. After preparing surfaces with a target sIEP, an FNAA with positively and negatively charged nanoparticles was used to verify the sIEP of the generated surfaces. The FNAA revealed that pyridine-modified surfaces had a pKa of 6.69 ± 0.18. When an sIEP was generated, negative nanoparticles bound to surfaces at pH values below the sIEP and positive nanoparticles bound at pH values above the sIEP. Furthermore, we found sIEP values of 5.97 ± 0.88 when we aimed for an sIEP of 6.2, and 7.12 ± 0.21 when we aimed for an sIEP of 7.1. Finally, the pH dependent binding and release of a negatively and positively charged (bio)polymer was investigated for a target sIEP of 7. A negatively charged polymer (poly(I:C)) was bound at a pH < sIEP and released at a pH > sIEP with a release efficiency of 85 ± 9% and a positively charged polymer (trimethyl chitosan) bound at a pH > sIEP and released at a pH < sIEP with a release efficiency of 72 ± 9%. In conclusion, we established a method for preparing modified silicon surfaces with a tunable sIEP, which can be used for pH-dependent binding and release of biomacromolecules

Dry influenza vaccines:towards a stable, effective and convenient alternative to conventional parenteral influenza vaccination

Cold-chain requirements, limited stockpiling potential and the lack of potent immune responses are major challenges of parenterally formulated influenza vaccines. Decreased cold chain dependence and stockpiling can be achieved if vaccines are formulated in a dry state using suitable excipients and drying technologies. Furthermore, having the vaccine in a dry state enables the development of non-parenteral patient friendly dosage forms: microneedles for transdermal administration, tablets for oral administration, and powders for epidermal, nasal or pulmonary administration. Moreover, these administration routes have the potential to elicit an improved immune response. This review highlights the rationale for the development of dried influenza vaccines, as well as processes used for the drying and stabilization of influenza vaccines; it also compares the immunogenicity of dried influenza vaccines administered via non-invasive routes with that of parenterally administered influenza vaccines. Finally, it discusses unmet needs, challenges and future developments in the field of dried influenza vaccines

Tailored protein release from biodegradable poly(ε-caprolactone-PEG)- b-poly(ε-caprolactone) multiblock-copolymer implants

In this study, the in vitro release of proteins from novel, biodegradable phase-separated poly(ε-caprolactone-PEG)-block-poly(ε-caprolactone), [PCL-PEG]-b-[PCL]) multiblock copolymers with different block ratios and with a low melting temperature (49-55 °C) was studied. The effect of block ratio and PEG content of the polymers (i.e. 22.5, 37.5 and 52.5 wt%) as well as the effect of protein molecular weight (1.2, 5.8, 14, 29 and 66 kDa being goserelin, insulin, lysozyme, carbonic anhydrase and albumin, respectively) on protein release was investigated. Proteins were spray-dried with inulin as stabilizer to obtain a powder of uniform particle size. Spray-dried inulin-stabilized proteins were incorporated into polymeric implants by hot melt extrusion. All incorporated proteins fully preserved their structural integrity as determined after extraction of these proteins from the polymeric implants. In general, it was found that the release rate of the protein increased with decreasing molecular weight of the protein and with increasing the PEG content of the polymer. Swelling and degradation rate of the copolymer increased with increasing PEG content. Hence, release of proteins of various molecular weights from [PCL-PEG]-b-[PCL] multi-block copolymers can be tailored by varying the PEG content of the polymer. © 2014 Elsevier B.V. All rights reserved

Pulmonary immunization: deposition site is of minor relevance for influenza vaccination but deep lung deposition is crucial for hepatitis B vaccination

Vaccination via the pulmonary route could be an attractive alternative to parenteral administration. Research towards the best site of antigen deposition within the lungs to induce optimal immune responses has conflicting results which might be dependent on the type of vaccine and/or its physical state. Therefore, in this study, we explored whether deep lung deposition is crucial for two different vaccines, i.e., influenza and hepatitis B vaccine. In view of this, influenza subunit vaccine and hepatitis B surface antigen were labeled with a fluorescent dye and then spray-dried. Imaging data showed that after pulmonary administration to mice the powders were deposited in the trachea/central airways when a commercially available insufflator was used while deep lung deposition was achieved when an in-house built aerosol generator was used. Immunogenicity studies revealed that comparable immune responses were induced upon trachea/central airways or deep lung targeting of dry influenza vaccine formulations. However, for hepatitis B vaccine, no immune responses were induced by trachea/central airways deposition whereas they were considerable after deep lung deposition. Thus, we conclude that deep lung targeting is not a critical parameter for the efficacy of pulmonary administered influenza vaccine whereas for hepatitis B vaccine it is