Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics.

Here, we investigated the influence of the variable fragment (Fv) of IgG antibodies on the binding to the neonatal Fc receptor (FcRn) as well as on FcRn-dependent pharmacokinetics (PK). FcRn plays a key role in IgG homeostasis, and specific manipulation in the crystallizable fragment (Fc) is known to affect FcRn-dependent PK. Although the influence of the antigen-binding fragment (Fab) on FcRn interactions has been reported, the underlying mechanism is hitherto only poorly understood. Therefore, we analyzed the two IgG1 antibodies, briakinumab and ustekinumab, that have similar Fc parts but different terminal half-lives in human and systematically engineered variants of them with cross-over exchanges and varied charge distribution. Using FcRn affinity chromatography, molecular dynamics simulation, and in vivo PK studies in human FcRn transgenic mice, we provide evidence that the charge distribution on the Fv domain is involved in excessive FcRn binding. This excessive binding prevents efficient FcRn-IgG dissociation at physiological pH, thereby reducing FcRn-dependent terminal half-lives. Furthermore, we observed a linear correlation between FcRn column retention times of the antibody variants and the terminal half-lives in vivo. Taken together, our study contributes to a better understanding of the FcRn-IgG interaction, and it could also provide profound potential in FcRn-dependent antibody engineering of the variable Fab region.

Investigation of the immunogenicity of different types of aggregates of a murine monoclonal antibody in mice.

PURPOSE: The potential contribution of protein aggregates to the unwanted immunogenicity of protein pharmaceuticals is a major concern. In the present study a murine monoclonal antibody was utilized to study the immunogenicity of different types of aggregates in mice. Samples containing defined types of aggregates were prepared by processes such as stirring, agitation, exposure to ultraviolet (UV) light and exposure to elevated temperatures. METHODS: Aggregates were analyzed by size-exclusion chromatography, light obscuration, turbidimetry, infrared (IR) spectroscopy and UV spectroscopy. Samples were separated into fractions based on aggregate size by asymmetrical flow field-flow fractionation or by centrifugation. Samples containing different types and sizes of aggregates were subsequently administered to C57BL/6 J and BALB/c mice, and serum was analyzed for the presence of anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies. In addition, the pharmacokinetic profile of the murine antibody was investigated. RESULTS: In this study, samples containing high numbers of different types of aggregates were administered in order to challenge the in vivo system. The magnitude of immune response depends on the nature of the aggregates. The most immunogenic aggregates were of relatively large and insoluble nature, with perturbed, non-native structures. CONCLUSION: This study shows that not all protein drug aggregates are equally immunogenic.

Toward intradermal vaccination: preparation of powder formulations by collapse freeze-drying.

Intradermal powder immunization is an emerging technique in vaccine delivery. The purpose of this study was to generate powder particles for intradermal injection by freeze-drying and subsequent cryo-milling. Two different freeze-drying protocols were compared, a moderate freeze-drying cycle and an aggressive freeze-drying cycle, which induced a controlled collapse of the sugar matrix. Ovalbumin served as model antigen. The influence of collapse drying and cryo-milling on particle morphology and protein stability was investigated. Cryo-milling generated irregularly shaped particles of size 20-70 µm. The recovery of soluble monomer of ovalbumin was not changed during freeze-drying and after cryo-milling, or after 12 months of storage at 2-8 °C. A slight increase in higher molecular weight aggregates was found in formulations containing the polymer dextran after 12 months of storage at 50 °C. Light obscuration measurements showed an increase in cumulative particle counts after cryo-milling that did not further increase during storage at 2-8 °C for 12 months. The applicability of the cryo-milling process to other therapeutic proteins was shown using recombinant human granulocyte-colony stimulating factor. Collapse freeze-drying and subsequent cryo-milling allows the generation of particles suitable for intradermal powder injection.

Shape Characterization of Subvisible Particles Using Dynamic Imaging Analysis.

Protein aggregates and subvisible particles (SbvP), inherently present in all marketed protein drug products, have received increasing attention by health authorities. Dynamic imaging analysis was introduced to visualize SbvP and facilitate understanding of their origin. The educational United States Pharmacopeia chapter <1787> emphasizes that dynamic imaging analysis could be used for morphology measurements in the size range of 4-100 µm. However, adequate morphology characterization, as suggested in the United States Pharmacopeia <1787> proposed size range, remains challenging as nonspherical size standards are not commercially available. In this study, a homogenous and well-defined nonspherical particle standard was fabricated and used to investigate the capabilities of 2 dynamic imaging analysis systems (microflow imaging (MFI) and FlowCAM) to characterize SbvP shape in the size range of 2-10 µm. The actual aspect ratio of the SbvP was measured by scanning electron microscopy and compared to the results obtained by dynamic imaging analysis. The test procedure was used to assess the accuracy in determining the shape characteristics of the nonspherical particles. In general, dynamic imaging analysis showed decreasing accuracy in morphology characterization for 5 µm and 2 µm particles. The test procedure was also capable to compare and evaluate differences between the 2 dynamic imaging methods. The present study should help to define ranges of operation for dynamic imaging analysis systems.

Test models for the evaluation of immunogenicity of protein aggregates.

Protein aggregates have been discussed for a long time as a potential risk factor for immunogenicity in patients. Meanwhile, many research groups have investigated the immunogenicity of differently produced aggregates using in vitro or in vivo models. Despite all knowledge gained in these studies still little is known about the mechanisms of immunogenicity and the kind of protein aggregates bearing the greatest risk for immunogenicity. The choice of a suitable test model regarding the predictability of immunogenicity of protein aggregates in humans plays a major role and influences results and conclusions substantially. In this review we will provide an overview of the test models recently used for the evaluation of immunogenicity of protein aggregates; we will discuss advantages and drawbacks regarding their usability and predictive power for immunogenicity in humans.

Nonspherical Nanoparticle Shape Stability Is Affected by Complex Manufacturing Aspects: Its Implications for Drug Delivery and Targeting.

The shape of nanoparticles is known recently as an important design parameter influencing considerably the fate of nanoparticles with and in biological systems. Several manufacturing techniques to generate nonspherical nanoparticles as well as studies on in vitro and in vivo effects thereof have been described. However, nonspherical nanoparticle shape stability in physiological-related conditions and the impact of formulation parameters on nonspherical nanoparticle resistance still need to be investigated. To address these issues, different nanoparticle fabrication methods using biodegradable polymers are explored to produce nonspherical nanoparticles via the prevailing film-stretching method. In addition, systematic comparisons to other nanoparticle systems prepared by different manufacturing techniques and less biodegradable materials (but still commonly utilized for drug delivery and targeting) are conducted. The study evinces that the strong interplay from multiple nanoparticle properties (i.e., internal structure, Young's modulus, surface roughness, liquefaction temperature [glass transition (Tg ) or melting (Tm )], porosity, and surface hydrophobicity) is present. It is not possible to predict the nonsphericity longevity by merely one or two factor(s). The most influential features in preserving the nonsphericity of nanoparticles are existence of internal structure and low surface hydrophobicity (i.e., surface-free energy (SFE) > ≈55 mN m-1 , material-water interfacial tension <6 mN m-1 ), especially if the nanoparticles are soft (<1 GPa), rough (Rrms > 10 nm), porous (>1 m2 g-1 ), and in possession of low bulk liquefaction temperature (<100 °C). Interestingly, low surface hydrophobicity of nanoparticles can be obtained indirectly by the significant presence of residual stabilizers. Therefore, it is strongly suggested that nonsphericity of particle systems is highly dependent on surface chemistry but cannot be appraised separately from other factors. These results and reviews allot valuable guidelines for the design and manufacturing of nonspherical nanoparticles having adequate shape stability, thereby appropriate with their usage purposes. Furthermore, they can assist in understanding and explaining the possible mechanisms of nonspherical nanoparticles effectivity loss and distinctive material behavior at the nanoscale.

Evaluation of a 3D Human Artificial Lymph Node as Test Model for the Assessment of Immunogenicity of Protein Aggregates.

The immunogenicity of protein aggregates has been investigated in numerous studies. Nevertheless, it is still unknown which kind of protein aggregates enhance immunogenicity the most. The ability of the currently used in vitro and in vivo systems regarding their predictability of immunogenicity in humans is often questionable, and results are partially contradictive. In this study, we used a 2D in vitro assay and a complex 3D human artificial lymph node model to predict the immunogenicity of protein aggregates of bevacizumab and adalimumab. The monoclonal antibodies were exposed to different stress conditions such as light, heat, and mechanical stress to trigger the formation of protein aggregates and particles, and samples were analyzed thoroughly. Cells and culture supernatants were harvested and analyzed for dendritic cell marker and cytokines. Our study in the artificial lymph node model revealed that bevacizumab after exposure to heat triggered a TH1- and proinflammatory immune response, whereas no trend of immune responses was seen for adalimumab after exposure to different stress conditions. The human artificial lymph node model represents a new test model for testing the immunogenicity of protein aggregates combining the relevance of a 3D human system with the rather easy handling of an in vitro setup.

New insights into process understanding of solid lipid extrusion (SLE) of extruded lipid implants for sustained protein delivery.

The aim of this work is a better understanding of solid lipid extrusion (SLE) for protein depot production using a lab-scale twin-screw (tsc)-extruder. In this context, little is known about the relationship of process parameters such as extrusion temperature, screw speed, or formulation on implant characteristics. It is difficult to attribute release characteristics to only one parameter, since the release will always be influenced by a combination of parameters. In this study, we describe the use of an online pressure measurement tool which allows to characterize pressure profiles during an extrusion run. We systematically investigated the impact of various process parameters on implant properties as well as release patterns using a monoclonal antibody (mAb). Solid lipid implants (SLIs) were produced by tsc-extrusion using the low melting triglyceride H12 and the high melting triglyceride Dynasan® D118. A mAb available in a freeze-dried matrix containing hydroxypropyl-ß-cyclodextrine (HP-ß-CD) was used as incorporated active pharmaceutical ingredient. Extrusion temperature (33-37 °C), screw speed (40-80 rpm) and the lipid composition (30-70% of each triglyceride) were modified. Additionally, freshly extruded SLIs were ground and extruded again as a preparation technique to optimize properties of SLIs. Using the pressure monitoring tool, four characteristic phases were defined for an extrusion run. We found that both, sufficient pressure and adequately molten material, is needed to form a suitable implant. Using the double extrusion technique, release rates could substantially be slowed down without changing formulation.

Application of water-soluble polyvinyl alcohol-based film patches on laser microporated skin facilitates intradermal macromolecule and nanoparticle delivery.

The intradermal delivery of biologics has long been recognized as attractive approach for cutaneous immunotherapy, particularly vaccination. Although intradermal (i.d.) or subcutaneous (s.c.) injection provide reproducible dosing and good cost- and delivery efficiency, the major objective to avoid sharps and the need for enhanced storage stability have renewed the interest in alternative needle-free delivery strategies. This study presents a new concept for the delivery of macromolecules and nanoparticles to viable skin layers with a high density of professional antigen-presenting cells (APCs). Stable polyvinyl alcohol (PVA) polymer films as well as PVA blends with carboxymethyl cellulose (CMC) or cross-linked carbomer were prepared using an easily-scalable film casting technique. Fluorescein isothiocyanate (FITC) and rhodamine B-labeled dextrane 70 kDa (RD70), used as small and macromolecular model substances, or polystyrene (PS)-nano- and microparticles with diameters of 0.5 µm and 5 µm were directly incorporated into the polymer formulations at varying concentrations. The assembly of the polymer films with an occlusive backing tape created a film patch that provided a fast drug release upon dissolution of the water-soluble film and facilitated an intradermal drug delivery on laser microporated skin. The minimally-invasive P.L.E.A.S.E.® laser poration system (Pantec Biosolutions, Ruggell, Liechtenstein) provided access to viable skin layers by thermally ablating the superficial tissue with a pulsed Er:YAG laser (λ = 2.94 µm). In our in vitro study using excised pig skin, laser microporation induced a 4- to 5-fold increase of water transport (TEWL) through excised skin in a Franz diffusion cell compared to intact skin. The TEWL values detected were comparable to in vivo human skin. The increased water transport facilitated the dissolution of all topically applied dry PVA-based film formulations within 6 h. No dissolution of the films was seen on intact skin. The incubation of the film patches on laser microporated skin for 24 h led to a considerable intradermal delivery of RD70 or PS-nanoparticles, which was superior for pure PVA films compared to PVA-CMC or PVA-carbomer blend formulations. No intradermal delivery was observed on intact skin or when larger PS-microparticles with a diameter of 5 µm were investigated. The presented concept provides a unique opportunity to exploit the improved storage stability of sensitive drug molecules in dry film formulations while providing protection and functionality.

A pilot study using a novel pyrotechnically driven prototype applicator for epidermal powder immunization in piglets.

Epidermal powder immunization (EPI) is an alternative technique to the classical immunization route using needle and syringe. In this work, we present the results of an in vivo pilot study in piglets using a dried influenza model vaccine which was applied by EPI using a novel pyrotechnically driven applicator. A liquid influenza vaccine (Pandemrix®) was first concentrated by tangential flow filtration and hemagglutinin content was determined by RP-HPLC. The liquid formulation was then transformed into a dry powder by collapse freeze-drying and subsequent cryo-milling. The vaccine powder was attached to a membrane of a novel pyrotechnical applicator using oily adjuvant components. Upon actuation of the applicator, particles were accelerated to high speed as determined by a high-speed camera setup. Piglets were immunized twice using either the novel pyrotechnical applicator or classical intramuscular injection. Blood samples of the animals were collected at various time points and analyzed by enzyme-linked immunosorbent assay. Our pilot study shows that acceleration of a dried vaccine powder to supersonic speed using the pyrotechnical applicator is possible and that the speed and impact of the particles is sufficient to breach the stratum corneum of piglet skin. Importantly, the administration of the dry vaccine powder resulted in measurable anti-H1N1 antibody titres in vivo.

Engineered hybrid spider silk particles as delivery system for peptide vaccines.

The generation of strong T-cell immunity is one of the main challenges for the development of successful vaccines against cancer and major infectious diseases. Here we have engineered spider silk particles as delivery system for a peptide-based vaccination that leads to effective priming of cytotoxic T-cells. The recombinant spider silk protein eADF4(C16) was fused to the antigenic peptide from ovalbumin, either without linker or with a cathepsin cleavable peptide linker. Particles prepared from the hybrid proteins were taken up by dendritic cells, which are essential for T-cell priming, and successfully activated cytotoxic T-cells, without signs of immunotoxicity or unspecific immunostimulatory activity. Upon subcutaneous injection in mice, the particles were taken up by dendritic cells and accumulated in the lymph nodes, where immune responses are generated. Particles from hybrid proteins containing a cathepsin-cleavable linker induced a strong antigen-specific proliferation of cytotoxic T-cells in vivo, even in the absence of a vaccine adjuvant. We thus demonstrate the efficacy of a new vaccine strategy using a protein-based all-in-one vaccination system, where spider silk particles serve as carriers with an incorporated peptide antigen. Our study further suggests that engineered spider silk-based vaccines are extremely stable, easy to manufacture, and readily customizable.

Scale-up of water-based spider silk film casting using a film applicator.

Spider silk proteins for applications in drug delivery have attracted an increased interest during the past years. Some possible future medical applications for this biocompatible and biodegradable material are scaffolds for tissue engineering, implantable drug delivery systems and coatings for implants. Recently, we reported on the preparation of water-based spider silk films for drug delivery applications. In the current study, we describe the development of a manufacturing technique for casting larger spider silk films from aqueous solution employing a film applicator. Films were characterized in terms of morphology, water solubility, protein secondary structure, thermal stability, and mechanical properties. Different post-treatments were evaluated (phosphate ions, ethanol, steam sterilization and water vapor) to increase the content of ß-sheets thereby achieving water insolubility of the films. Finally, the mechanical properties of the spider silk films were improved by incorporating 2-pyrrolidone as plasticizer.

Long-term release and stability of pharmaceutical proteins delivered from solid lipid implants.

Solid lipid implants (SLIs) prepared by twin-screw (tsc) extrusion represent a promising technology platform for the sustained release of pharmaceutical proteins. In this work, we report on two aspects, long-term release and stability of released protein. First, SLIs were produced by tsc-extrusion containing the low melting triglyceride H12 and the high melting triglyceride Dynasan D118. Two different proteins available in a freeze-dried matrix containing hydroxypropyl-ß-cyclodextrine (HP-ß-CD) were incorporated into the lipid matrix: a monoclonal antibody (mAb) from the IgG1 class and the fab-fragment Ranibizumab (Lucentis®). SLIs, composed of 10% protein lyophilizate and both triglycerides, were extruded at 35°C and 40rpm. Sustained release of both proteins was observed in a sustained manner for approximately 120days. Protein load per implant was increased by three different approaches resulting in a protein load of 3.00mg per implant without affecting the release profiles. The incubation medium containing the released protein was collected, concentrated and analyzed including liquid chromatography (SE-HPLC, IEX, HIC), electrophoresis (SDS-PAGE, on-chip gel electrophoresis) and FT-IR spectroscopy. The mAb showed a monomer loss of up to 7% (SE-HPLC) and IEX analysis revealed the formation of 16% acidic subspecies after 18weeks. FT-IR spectra of mAb indicated the formation of random coil structures towards the end of the release study. Ranibizumab was mainly released in its monomeric form (>95%), and approximately 5% hydrophobic subspecies were formed after 18weeks of release. FT-IR analysis revealed no changes in secondary structure. The release and stability profiles of both proteins underline the potential of SLIs as a delivery system. SLIs provide a promising platform for applications where really long-term release is needed, for example for intraocular delivery of anti-vascular endothelial growth factor (VEGF) drugs for age related macular degeneration (AMD).

Twin-screw extruded lipid implants containing TRP2 peptide for tumour therapy.

Much effort has been put in the development of specific anti-tumour immunotherapies over the last few years, and several studies report on the use of liposomal carriers for tumour-associated antigens. In this work, the use of lipid implants, prepared using two different extruders, was investigated for sustained delivery in tumour therapy. The implants consisted of cholesterol, soybean lecithin, Dynasan 114, trehalose, ovalbumin (OVA) or a TRP2 peptide, and Quil-A. Implants were first produced on a Haake Minilab extruder, and then a scale-down to minimal quantities of material on a small scale ZE mini extruder was performed. All formulations were characterised in terms of extrudability, implant properties and in vitro release behaviour of the model antigen ovalbumin. The type of extruder used to produce the implants had a major influence on implant properties and the release behaviour, demonstrating that extrusion parameters and lipid formulations have to be individually adapted to each extrusion device. Subsequently, lipid implants containing TRP-2 peptide were extruded on the ZE mini extruder and investigated in vitro and in vivo. The in vivo study showed that mice having received TRP2 loaded implants had delayed tumour growth for 3days compared to groups having received no TRP2.

Water-based preparation of spider silk films as drug delivery matrices.

The main focus of this work was to obtain a drug delivery matrix characterized by biocompatibility, water insolubility and good mechanical properties. Moreover the preparation process has to be compatible with protein encapsulation and the obtained matrix should be able to sustain release a model protein. Spider silk proteins represent exceptional natural polymers due to their mechanical properties in combination with biocompatibility. As both hydrophobic and slowly biodegrading biopolymers, recombinant spider silk proteins fulfill the required properties for a drug delivery system. In this work, we present the preparation of eADF4(C16) films as drug delivery matrices without the use of any organic solvent. Water-based spider silk films were characterized in terms of protein secondary structure, thermal stability, zeta-potential, solubility, mechanical properties, and water absorption and desorption. Additionally, this study includes an evaluation of their application as a drug delivery system for both small molecular weight drugs and high molecular weight molecules such as proteins. Our investigation focused on possible improvements in the film's mechanical properties including plasticizers in the film matrix. Furthermore, different film designs were prepared, such as: monolayer, coated monolayer, multilayer (sandwich), and coated multilayer. The release of the model protein BSA from these new systems was studied. Results indicated that spider silk films are a promising protein drug delivery matrix, capable of releasing the model protein over 90 days with a release profile close to zero order kinetic. Such films could be used for several pharmaceutical and medical purposes, especially when mechanical strength of a drug eluting matrix is of high importance.

The effect of steam sterilization on recombinant spider silk particles.

In this work, the recombinant spider silk protein eADF4(C16) was used to fabricate particles in the submicron range using a micromixing method. Furthermore, particles in the micrometer range were produced using an ultrasonic atomizer system. Both particle species were manufactured by an all-aqueous process. The submicroparticles were 332 nm in average diameter, whereas 6.70 µm was the median size of the microparticles. Both particle groups showed a spherical shape and exhibited high ß-sheet content in secondary structure. Submicro- and microparticles were subsequently steam sterilized and investigated with respect to particle size, secondary structure and thermal stability. Sterilization temperature and time were increased to assess the thermal stability of eADF4(C16) particles. Actually, particles remained stable and their properties did not change even after autoclaving at 134°C. Both, the untreated and the autoclaved submicroparticles showed no overt cytotoxicity on human dermal fibroblasts after incubation for 72 h. The eADF4(C16) particles were already loaded with proteins and small molecules in previous studies. With that, we can provide a highly promising parenteral drug delivery system based on a defined polypeptide carrier, manufactured with an all-aqueous process and being fully sterilizable.

Stability of collapse lyophilized influenza vaccine formulations.

A clear limitation of many liquid vaccines is the obligatory cold-chain distribution system. Therefore, distribution of a dried vaccine formulation may be beneficial in terms of vaccine stability, handling and transport. Collapse freeze-drying is a process which utilizes fairly aggressive but at the same time economic lyophilization cycles where the formulation is dried above its glass transition temperature. In this study, we used collapse freeze-drying for a thermosensitive model influenza vaccine (Pandemrix(®)). The dried lyophilizates were further cryo-milled to engineer powder particles in the size range of approximately 20-80 µm which is applicable for epidermal powder immunization. Vaccine potency and stability were neither affected by high temperature input during collapse lyophilization nor over a storage period of six months. Furthermore, cryo-milled vaccine lyophilizates showed good storage stability of up to three months at high storage temperature (40 °C). This technique can provide a powerful tool for the worldwide distribution of vaccine and for new application technologies such as engineered powder immunization.

Non-spherical micro- and nanoparticles: fabrication, characterization and drug delivery applications.

INTRODUCTION: Micro- and nanoparticles in drug and vaccine delivery have opened up new possibilities in pharmaceutics. In the past, researchers focused mainly on particle size, surface chemistry and the use of various materials to control particle characteristics and functions. Lately, shape has been acknowledged as an important design parameter having an impact on the interaction with biological systems. AREAS COVERED: In this review, we report on the latest developments in fabrication methods to tailor particle geometry, summarize analytical techniques for non-spherical particles and highlight the most important findings regarding their interaction with biological systems and their potential applications in drug delivery. EXPERT OPINION: The impact of shape on particle internalization into different cell types and particle biodistribution has been extensively studied in the past. Current research focuses on shape-dependent uptake mechanisms and applications for tumour therapy and vaccination. Different fabrication methods can be used to produce a variety of different particle types and shapes. Key challenges will be the transfer of new non-spherical particle fabrication methods from lab-scale to industrial large-scale production. Not all techniques may be scalable for the production of high quantities of particles. It will also be challenging to transfer the promising in vitro findings to suitable in vivo models.

Recent insights into cutaneous immunization: How to vaccinate via the skin.

Technologies and strategies for cutaneous vaccination have been evolving significantly during the past decades. Today, there is evidence for increased efficacy of cutaneously delivered vaccines allowing for dose reduction and providing a minimally invasive alternative to traditional vaccination. Considerable progress has been made within the field of well-established cutaneous vaccination strategies: Jet and powder injection technologies, microneedles, microporation technologies, electroporation, sonoporation, and also transdermal and transfollicular vaccine delivery. Due to recent advances, the use of cutaneous vaccination can be expanded from prophylactic vaccination for infectious diseases into therapeutic vaccination for both infectious and non-infectious chronic conditions. This review will provide an insight into immunological processes occurring in the skin and introduce the key innovations of cutaneous vaccination technologies.

Influence of particle size, an elongated particle geometry, and adjuvants on dendritic cell activation.

Modern subunit vaccines have many benefits compared to live vaccines such as convenient and competitive large scale production, better reproducibility and safety. However, the poor immunogenicity of subunit vaccines usually requires the addition of potent adjuvants or drug delivery vehicles. Accordingly, researchers are investigating different adjuvants and particulate vaccine delivery vehicles to boost the immunogenicity of subunit vaccines. Despite the rapidly growing knowledge in this field, a comparison of different adjuvants is sparsely found. Until today, little is known about efficient combinations of the different adjuvants and particulate vaccine delivery vehicles. In this study we compared three adjuvants with respect to their immune stimulatory potential and combined them with different particulate vaccine delivery vehicles. For this reason, we investigated two types of polyI:C and a CL264 base analogue and combined these adjuvants with differently sized and shaped particulate vaccine delivery vehicles. A high molecular weight polyI:C combined with a spherical nano-sized particulate vaccine delivery vehicle promoted the strongest dendritic cells activation.