Dry Powder Vaccine Formulation Research Articles

Nano-adjuvanted dry powder vaccine for the mucosal immunization against airways pathogens.

Nasal vaccination has been shown to provide optimal protection against respiratory pathogens. However, mucosal vaccination requires the implementation of specific immunization strategies to improve its effectiveness. Nanotechnology appears a key approach to improve the effectiveness of mucosal vaccines, since several nanomaterials provide mucoadhesion, enhance mucosal permeability, control antigen release and possess adjuvant properties. Mycoplasma hyopneumoniae is the main causative agent of enzootic pneumonia in pigs, a respiratory disease responsible for considerable economic losses in the pig farming worldwide. The present work developed, characterized, and tested in vivo an innovative dry powder nasal vaccine, obtained from the deposition on a solid carrier of an inactivated antigen and a chitosan-coated nanoemulsion, as an adjuvant. The nanoemulsion was obtained through a low-energy emulsification technique, a method that allowed to achieve nano droplets in the order of 200 nm. The oil phase selected was alpha-tocopherol, sunflower oil, and poly(ethylene glycol) hydroxystearate used as non-ionic tensioactive. The aqueous phase contained chitosan, which provides a positive charge to the emulsion, conferring mucoadhesive properties and favoring interactions with inactivated M. hyopneumoniae. Finally, the nanoemulsion was layered with a mild and scalable process onto a suitable solid carrier (i.e., lactose, mannitol, or calcium carbonate) to be transformed into a solid dosage form for administration as dry powder. In the experimental study, the nasal vaccine formulation with calcium carbonate was administered to piglets and compared to intramuscular administration of a commercial vaccine and of the dry powder without antigen, aimed at evaluating the ability of IN vaccination to elicit an in vivo local immune response and a systemic immune response. Intranasal vaccination was characterized by a significantly higher immune response in the nasal mucosa at 7 days post-vaccination, elicited comparable levels of Mycoplasma-specific IFN-γ secreting cells and comparable, if not higher, responsiveness of B cells expressing IgA and IgG in peripheral blood mononuclear cells, with those detected upon a conventional intramuscular immunization. In conclusion, this study illustrates a simple and effective strategy for the development of a dry powder vaccine formulation for nasal administration which could be used as alternative to current parenteral commercial vaccines.

Intranasal immunization with aluminum salt-adjuvanted dry powder vaccine

Intranasal vaccination using dry powder vaccine formulation represents an attractive, non-invasive vaccination modality with better storage stability and added protection at the mucosal surfaces. Herein we report that it is feasible to induce specific mucosal and systemic antibody responses by intranasal immunization with a dry powder vaccine adjuvanted with an insoluble aluminum salt. The dry powder vaccine was prepared by thin-film freeze-drying of a model antigen, ovalbumin, adsorbed on aluminum (oxy)hydroxide as an adjuvant. Special emphasis was placed on the characterization of the dry powder vaccine formulation that can be realistically used in humans by a nasal dry powder delivery device. The vaccine powder was found to have "passable" to "good" flow properties, and the vaccine was uniformly distributed in the dry powder. An in vitro nasal deposition study using nasal casts of adult humans showed that around 90% of the powder was deposited in the nasal cavity. Intranasal immunization of rats with the dry powder vaccine elicited a specific serum antibody response as well as specific IgA responses in the nose and lung secretions of the rats. This study demonstrates the generation of systemic and mucosal immune responses by intranasal immunization using a dry powder vaccine adjuvanted with an aluminum salt.

Spray dried cubosomes with ovalbumin and Quil-A as a nanoparticulate dry powder vaccine formulation

Subunit vaccine formulations are often produced as liquid dispersions through complicated processes. It is desirable, however, to have simple, cheap and up-scalable methods to produce nanoparticulate subunit vaccines in powder form. Here, a simple single-step spray drying process for production of powder cubosome precursors with the model antigen ovalbumin (OVA) and the adjuvant Quil-A is presented. The cubosomes were characterized in vitro and evaluated in vivo by subcutaneous and oral administration for their potential as a vaccine formulation. Hydrated cubosomes had average particle size of 257 ± 8 nm and zeta potential of −18.0 ± 0.6 mV. The powder contained 10.6 ± 0.7% w/w OVA prior to hydration, of which 65 ± 1% was released within the first 20 min in 9.5 mM PBS at pH 7.3, with the remaining OVA gradually released over the following 24 h. Immunization with cubosomes resulted in significantly stronger antigen-specific serum IgG responses (p < 0.01), CD8+ T cell expansion (p < 0.0001) and target T cell killing compared to controls when given s.c., and was ineffective orally. This study shows that spray drying is a suitable method for producing nanoparticulate vaccine formulations in dry powder form.

Preparation and preclinical evaluation of a freeze-dried formulation of a novel combined multivalent whole-cell/B-subunit oral vaccine against enterotoxigenic Escherichia coli diarrhea

A promising liquid killed multivalent whole-cell plus enterotoxin B-subunit oral vaccine against enterotoxigenic Escherichia coli (ETEC), the primary cause of diarrhea among children in low-income countries and travelers to these areas, has recently been developed and tested in preclinical and phase-I and phase-II clinical studies. The vaccine contains killed E. coli bacteria over-expressing the main ETEC colonization factors (CFs) CFA/I, CS3, C5 and C6, and a recombinant enterotoxin B subunit protein (LCTBA) given together with a recently developed enterotoxin-derived adjuvant, dmLT. A dry-powder vaccine formulation should be advantageous especially for use in low-income countries. Here we describe a method to produce a dry-powder formulation by freeze-drying of the vaccine using inulin as stabilizer. Although not completely preventing aggregation of bacteria during freeze-drying, the stabilizer provided both improved overall bacterial morphology and almost complete recovery of the CF and B subunit antigens. Most importantly, oral-intragastric immunization of mice with the freeze-dried vaccine together with dmLT adjuvant elicited strong intestinal mucosal and serum antibody responses against all vaccine antigens, which were comparable to those achieved with the liquid vaccine. Our results indicate the feasibility to use freeze-drying with inulin as stabilizer for preparing a dry-powder formulation of the novel ETEC vaccine with retained oral-mucosal immunogenicity compared to the liquid formulation.

Carrier-based dry powder formulation for nasal delivery of vaccines utilizing BSA as model drug

Dry powder formulations for nasal vaccine delivery offer versatile advantages compared to liquid formulations, such as increased storage stability and simplified administration. The objective of the present study was the development of a dry powder nasal vaccine formulation making use of antigen-loaded chitosan microparticles. Special emphasis was put on the development and characterization of a formulation which can realistically be used in humans by means of a nasal dry powder sprayer. Microparticles of chitosan with bovine serum albumin as model antigen were produced by spray drying and showed a particle size of about 3μm. In order to improve nasal deposition and dispersibility, powder blends with low separation tendency were prepared. A range of sugar alcohols (mannitol, sorbitol, maltitol) was evaluated as carriers. Among the tested carriers, blends with spray-granulated mannitol showed the most adequate deposition profile without simulated nasal inspiration and were very well dispersible by a nasal dry powder device. In conclusion, this study illustrates a simple and effective strategy for the development of a dry powder vaccine formulation for nasal administration.

Preclinical refinements of a broadly protective VLP-based HPV vaccine targeting the minor capsid protein, L2

An ideal prophylactic human papillomavirus (HPV) vaccine would provide broadly protective and long-lasting immune responses against all high-risk HPV types, would be effective after a single dose, and would be formulated in such a manner to allow for long-term storage without the necessity for refrigeration. We have developed candidate HPV vaccines consisting of bacteriophage virus-like particles (VLPs) that display a broadly neutralizing epitope derived from the HPV16 minor capsid protein, L2. Immunization with 16L2 VLPs elicited high titer and broadly cross-reactive and cross-neutralizing antibodies against diverse HPV types. In this study we introduce two refinements for our candidate vaccines, with an eye towards enhancing efficacy and clinical applicability in the developing world. First, we assessed the role of antigen dose and boosting on immunogenicity. Mice immunized with 16L2-MS2 VLPs at doses ranging from 2 to 25μg with or without alum were highly immunogenic at all doses; alum appeared to have an adjuvant effect at the lowest dose. Although boosting enhanced antibody titers, even a single immunization could elicit strong and long-lasting antibody responses. We also developed a method to enhance vaccine stability. Using a spray dry apparatus and a combination of sugars & an amino acid as protein stabilizers, we generated dry powder vaccine formulations of our L2 VLPs. Spray drying of our L2 VLPs did not affect the integrity or immunogenicity of VLPs upon reconstitution. Spray dried VLPs were stable at room temperature and at 37°C for over one month and the VLPs were highly immunogenic. Taken together, these enhancements are designed to facilitate implementation of a next-generation VLP-based HPV vaccine which addresses U.S. and global disparities in vaccine affordability and access in rural/remote populations.

First in vivo evaluation of particulate nasal dry powder vaccine formulations containing ovalbumin in mice

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.

TLR2-targeted secreted proteins from Mycobacterium tuberculosis are protective as powdered pulmonary vaccines

Despite considerable research efforts towards effective treatments, tuberculosis (TB) remains a staggering burden on global health. Suitably formulated sub-unit vaccines offer potential as safe and effective generators of protective immunity. The Mycobacterium tuberculosis antigens, cutinase-like proteins (Culp) 1 and 6 and MPT83, were conjugated directly to the novel adjuvant Lipokel (Lipotek Pty Ltd), a TLR2 ligand that delivers antigen to immune cells in a self-adjuvanting context. Protein-Lipokel complexes were formulated as dry powders for pulmonary delivery directly to the lungs of mice by intra-tracheal insufflation, leading to recruitment of neutrophils and antigen presenting cell populations to the lungs at 72h, that persisted at 7 days post immunisation. Significant increases in the frequency of activated dendritic cells were observed in the mediastinal lymph node (MLN) at 1 and 4 weeks after homologous boosting with protein-Lipokel vaccine. This was associated with the increased recruitment of effector CD4+ and CD8+ T-lymphocytes to the MLN and systemic antigen-specific, IFN-γ producing T-lymphocyte and IgG responses. Notably, pulmonary immunisation with either Culp1-6-Lipokel or MPT83-Lipokel powder vaccines generated protective responses in the lungs against aerosol M. tuberculosis challenge. The successful combination of TLR2-targeting and dry powder vaccine formulation, together with important practical benefits, offers potential for pulmonary vaccination against M. tuberculosis.

Designing CAF-adjuvanted dry powder vaccines: Spray drying preserves the adjuvant activity of CAF01

Dry powder vaccine formulations are highly attractive due to improved storage stability and the possibility for particle engineering, as compared to liquid formulations. However, a prerequisite for formulating vaccines into dry formulations is that their physicochemical and adjuvant properties remain unchanged upon rehydration. Thus, we have identified and optimized the parameters of importance for the design of a spray dried powder formulation of the cationic liposomal adjuvant formulation 01 (CAF01) composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6′-dibehenate (TDB) via spray drying. The optimal excipient to stabilize CAF01 during spray drying and for the design of nanocomposite microparticles was identified among mannitol, lactose and trehalose. Trehalose and lactose were promising stabilizers with respect to preserving liposome size, as compared to mannitol. Trehalose and lactose were in the glassy state upon co-spray drying with the liposomes, whereas mannitol appeared crystalline, suggesting that the ability of the stabilizer to form a glassy matrix around the liposomes is one of the prerequisites for stabilization. Systematic studies on the effect of process parameters suggested that a fast drying rate is essential to avoid phase separation and lipid accumulation at the surface of the microparticles during spray drying. Finally, immunization studies in mice with CAF01 in combination with the tuberculosis antigen Ag85B-ESAT6-Rv2660c (H56) demonstrated that spray drying of CAF01 with trehalose under optimal processing conditions resulted in the preservation of the adjuvant activity in vivo. These data demonstrate the importance of liposome stabilization via optimization of formulation and processing conditions in the engineering of dry powder liposome formulations.

Novel Formulation Concept for Particulate Uptake of Vaccines via the Nasal Associated Lymphoid Tissue

Aerosol vaccination via the mucosa targets an epithelium critical to host defence against inhaled pathogens, potentially avoids needle injection, and provides an exciting opportunity in the development of stable dry powder vaccine formulations. Specialised cells in the mucosa are able to take up and guide antigens directly to immune cells. In contrast to soluble antigen formulations, particles with antigen also provoke a local sIgA mediated immune response before being presented to the systemic immune system. In this study, particles containing the model antigen BSA and chitosan as stabiliser with adjuvant activity are produced by spray drying. The compatibility and uptake of these particles via the respiratory epithelium is determined in vitro on Calu-3 cells. The in vitro deposition studies are performed in a nasal cast made from CT scan data using a novel nasal dry powder device. The deposition profile is optimised by the use of interactive mixtures with a low separation capacity. The spray drying process results in spherical particles with a size in the low micrometer range (x50 3μm), which are well tolerated when administered to the cells and which are readily taken up. As the particles have to be big enough to be retained in the appropriate place in the respiratory tract (e.g. the nasal cavity) to be taken up efficiently, the primary particles are too small. Deposition studies show a high fraction of almost 56% transiting the nose and being capable of inhalation. This fraction can be reduced by utilising an interactive mixture with a carrier, where only 5% of the antigen carrying particles leave the nasal cavity. Particulate vaccine formulations are a promising formulation approach for mucosal vaccination targeting the nasal mucosa. With small antigen carrying particles immobilised on carrier particles, the antigen is delivered exclusively to the nose.

Hepatitis-B Surface Antigen (HBsAg) Powder Formulation: Process and Stability Assessment

The purpose of this study was to develop a hepatitis-B surface antigen (HBsAg) dry powder vaccine formulation suitable for epidermal powder immunization (EPI) via an efficient, scalable powder-formation process. Several HBsAg dry powder formulations were prepared using four different powder-formation methods: freeze-drying/compress/grind/sieve (FD/C/G/S), spray-drying (SD), agarose beads, and spray freeze-drying (SFD). Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Physical, chemical and biochemical stability of HBsAg was determined by dynamic light scattering, an enzyme immune assay, and immunogenicity in a mouse or hairless guinea pig model. Out of the four powder-formation methods evaluated SFD outperformed other methods in the following considerations: good process efficiency, flexible scalability, and desirable particle characteristics for skin penetration. The stress posed by SFD appeared to be mild as HBsAg in the dry form retained its potency and immunogenicity. Notably, the mechanism of fast freezing by SFD actually promoted the preservation of HBsAg nanoparticle size, in good correlation with long-term biochemical stability. Among several formulations screened, the formulation containing 10 microg HBsAg in 1-mg powder with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability performance. In conclusion, HBsAg dry powder formulations suitable for EPI were successfully prepared using SFD. Further, a systematic formulation development strategy allowed the development and optimization of an HBsAg dry powder formulation, demonstrating excellent long-term physical, biochemical, and immunological stability.

Formulation of a dry powder influenza vaccine for nasal delivery.

The purpose of this research was to prepare a dry powder vaccine formulation containing whole inactivated influenza virus (VIIV) and a mucoadhesive compound suitable for nasal delivery. Powders containing WIIV and either lactose or trehalose were produced by lyophilization. A micro-ball mill was used to reduce the lyophilized cake to sizes suitable for nasal delivery. Chitosan flakes were reduced in size using a cryo-milling technique. Milled powders were sieved between 45 and 125 μm aggregate sizes and characterized for particle size and distribution, morphology, and flow properties. Powders were blended in the micro-ball mill without the ball. Lyophilization followed by milling produced irregularly shaped, polydisperse particles with a median primary particle diameter of ≈21 μm and a yield of ≈37% of particles in the 45 to 125 μm particle size range. Flow properties of lactose and trehalose powders after lyophilization followed by milling and sieving were similar. Cryo-milling produced a small yield of particles in the desired size range (<10%). Lyophilization followed by milling and sieving produced particles suitable for nasal delivery with different physicochemical properties as a function of processing conditions and components of the formulation. Further optimization of particle size and morphology is required for these powders to be suitable for clinical evaluation.