Marijke Dierendonck

β-Glucan microparticles are good candidates for mucosal antigen delivery in oral vaccination

Continuously improving the developmental process and the efficacy of oral vaccines is essential in the fight against intestinal pathogens. A promising strategy for vaccination applying safe, biodegradable and non-replicating antigen delivery systems has gained increased interest for eliciting cellular and humoral immune responses. The current study evaluates the potential of β-glucan particles (GP) as an oral antigen delivery system and their adjuvant characteristics. GP are efficiently internalized by human intestinal epithelial cell lines (Caco-2 and HT-29 cells), without exerting negative effects on cell viability. GP triggered the expression of pro-inflammatory cytokines IL-23p19, IL-8 and the β-glucan receptors dectin-1 and TLR2 by activated Caco-2 cells, and CCL20 in HT-29 cells. In contrast, the expression level of TGF-β, an important mediator of oral tolerance, was significantly downregulated in HT-29 cells. Additionally, adoptive transfer experiments showed proliferating ovalbumin (OVA)-specific CD4(+) T cells mainly in the spleens of GP-OVA-fed mice. Furthermore, we detected a significantly increased IL-17 and a trend towards increased IFN-γ production in the spleen of GP-OVA-fed mice upon antigen restimulation. Oral administration of GP-OVA induced increased OVA-specific IgA, secretory-IgA (S-IgA) and secretory component (SC) production in intestinal fluids. Our data show that GP vehicles are able to deliver OVA via an oral route allowing efficient antigen presentation alongside adaptive immune activation, resulting in a Th17-biased response and the production of OVA-specific IgA, secretory-IgA and secretory component antibodies.

Facile Two-Step Synthesis of Porous Antigen-Loaded Degradable Polyelectrolyte Microspheres**

Without doubt, the development of vaccines constitutes one of themajor breakthroughs of humanmedicine, allowing us to prevent numerous infectious diseases. Nevertheless, for major killers such as HIV, malaria, and tuberculosis, no effective vaccine is currently available. The failure of current vaccination strategies to elicit cellular immune responses, especially CD8 cytotoxic T cells (CTLs) that can recognize and eliminate infected cells, is considered to be one of the major reasons for this failure. Consequently, there is an urgent need to develop new vaccine formulations that can induce such CTL responses. One of the most promising approaches to achieve this goal is the encapsulation of antigens in particulate carriers with dimensions between 0.1 and 10 mm. A plethora of studies has now demonstrated that such carriers can strongly enhance antigen presentation by dendritic cells (DCs), the most potent antigen presenting cells capable of priming effector T cell responses, not only quantitatively but also qualitatively. Antigen being presented by a DC as an MHC/peptide complex (MHC=major histocompatibility complex) to the T cell receptor indeed constitutes the first step in the initiation of T cell responses. Two different pathways occur for antigen presentation by MHC I and MHC II to CD8 and CD4 T-cells, respectively. MHC I presentation is responsible for the processing and presentation of cytosolic proteins, which are cleaved by the proteasome, transported to the endoplasmatic reticulum, and subsequently loaded onto MHC I molecules. By contrast, MHC II presentation occurs for endocytosed proteins, which are degraded in endolysosomal compartments, loaded onto MHC II molecules and subsequently presented at the cell surface. The way in which antigen is internalized by a DC, however, strongly affects how the antigen is processed and presented by a DC, and consequently also the type and strength of immune response induced. Although soluble antigens are almost exclusively presented by MHC II to CD4 T cells, particulate antigens not only are far more efficiently taken up by DCs, but are also presented by MHC I to CD8 T cells, thus enabling the induction of CTL responses. Although particulate carriers have the capacity to evoke potent cellular immunity, their clinical application has been impeded largely by practical problems involving their generation, including a low antigen encapsulation efficiency, the use of chemical solvents and physical stresses that negatively affect antigen stability, and the involvement of complex and labor-intensive multistep processes to generate them. Polymeric multilayer capsules have emerged as promising microscopic carriers for the delivery of antigens to DCs, overcoming some of the problems described above. These capsules are based on alternate deposition of polymers (so-called layer-by-layer technology), either through electrostatic interaction or hydrogen bonding, onto a sacrificial template, followed by decomposition of the template, resulting in hollow capsules and allowing efficient antigen encapsulation under non-denaturing conditions. Several papers have now demonstrated the potential of these capsules to target antigens to APCs both in vitro and in vivo, resulting in strongly enhanced antigen presentation to CD4 and CD8 T cells and the induction of broad and strong immune responses. The major advantage for their success is presumably threefold: 1) They protect the antigen from degradation before reaching DCs; 2) Because of their size (1–10 mm), they are preferably targeted to DCs; and 3) Because of their soft thin shell, which is prone to the reductive conditions or endosomal proteases depending on the nature of the capsule shell, they allow the antigen to be readily processed upon internalization by DCs. Moreover, these capsules have been shown to be biocompatible and degradable, both in vitro and in vivo. Notwithstanding their excellent performance, polymeric multilayer capsules are fabricated in multiple steps, which is a rather cost-inefficient fashion involving the use of a large excess of polymer and several centrifugation steps during deposition of each single layer. Therefore, a simple and versatile strategy involving a minimum of process steps that mimics polymeric multilayer capsules would be of uttermost importance to allow this type of antigen carriers to reach the clinical stage. Herein, we report on the synthesis of porous antigenloaded degradable polyelectrolyte microspheres using spray drying as a simple, yet efficient, and scalable production [*] M. Dierendonck, Dr. S. De Koker, Prof. Dr. C. Vervaet, Prof. Dr. J.-P. Remon, Dr. B. G. De Geest Laboratory of Pharmaceutical Technology Department of Pharmaceutics, Ghent University Harelbekestraat 72, 9000 Ghent (Belgium) E-mail: br.degeest@ugent.be

Hydrogen bonded polymeric multilayer films assembled below and above the cloud point temperature

Polymeric multilayer films assembled via hydrogen-bonding are witnessing increased interest from the scientific community. Here we report on hydrogen bonded multilayers of tannic acid and neutral poly(2-oxazoline)s. Importantly we demonstrate, to the best of our knowledge, for the first time that a temperature responsive polymer, in this case poly(2-(n-propyl)-2-oxazline), can be assembled below and above its TCP with distinctly different growth mechanisms.

Interaction between polymeric multilayer capsules and immune cells

Polymeric multilayer capsules are emerging carrier systems in the field of drug delivery. These materials are fabricated by set-wise assembly of interaction species onto a sacrificial template followed by the decomposition of this template, yielding hollow capsules. Using bio-responsive polymers that can be triggered by pH, enzymes or reduction, several groups are exploring these systems for intracellular drug delivery. In this review we focus on the recent efforts made in investigating the in vitro and in vivo interaction between these capsules and cells of the immune system.

Spray-dried polyelectrolyte microparticles in oral antigen delivery: stability, biocompatibility, and cellular uptake.

During the past decade, extensive research has undeniably improved the formulation and delivery of oral vaccines. Nevertheless, several factors, such as the harsh gastrointestinal environment together with tolerance induction to exogenous antigens, have thus far impeded the optimal effectiveness and clinical application of oral delivery systems. The current study encompasses an initial evaluation of the stability, biocompatibility, and cellular uptake of two promising candidate systems for oral antigen delivery, that is, calcium carbonate- (CP) and mannitol-templated (MP) porous microspheres. Both spray-dried formulations were efficiently internalized by human intestinal epithelial cells (Caco-2 and HT-29) and degraded into phagolysosomal intracellular compartments. In addition, cellular particle uptake and processing significantly up-regulated the expression of (HLA) class-II and costimulatory molecules on intestinal epithelial cells. Even though the high surface-area-to-volume ratio of the microspheres was expected to favor protease access, antigen release was remarkably limited in simulated intestinal fluid and was even absent under gastric conditions. Finally, neither CP nor MP exerted cytotoxicity upon prolonged in vitro incubation with high antigen concentration. Altogether, these data support the potential of CP and MP for oral antigen delivery and motivate the further development of these promising carrier systems in in vivo studies.

Nanoporous polyelectrolyte vaccine microcarriers: a formulation platform for enhancing humoral and cellular immune responses

In this paper we report on the design, characterization and immuno-biological evaluation of nanoporous polyelectrolyte microparticles as vaccine carrier. Relative to soluble antigen, formulation of antigen as a sub-10 μm particle can strongly enhance antigen-specific cellular immune responses. The latter is crucial to confer protective immunity against intracellular pathogens and for anti-cancer vaccines. However, a major bottleneck in microparticulate vaccine formulation is the development of generic strategies that afford antigen encapsulation under benign and scalable conditions. Our strategy is based on spray drying of a dilute aqueous solution of antigen, oppositely charged polyelectrolytes and mannitol as a pore-forming component. The obtained solid microparticles can be redispersed in aqueous medium, leading to leaching out of the mannitol, thereby creating a highly porous internal structure. This porous structure enhances enzymatic processing of encapsulated proteins. After optimizing the conditions to process these microparticles we demonstrate that they strongly enhance cross-presentation in vitro by dendritic cells to CD8 T cells. In vivo experiments in mice confirm that this vaccine formulation technology is capable of enhancing cellular immune responses.

One-step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells.

Vaccination is regarded as the most efficient and cost-effective way to prevent infectious diseases. Vaccine design nowadays focuses on the implementation of safer recombinant subunit vaccines. However, these recombinant subunit antigens are often poor immunogens and several strategies are currently under investigation to enhance their immunogenicity. The encapsulation of antigens in biodegradable microparticulate delivery systems seems a promising strategy to boost their immunogenicity. Here, we evaluate the capacity of polyelectrolyte complex microparticles (PECMs), fabricated by single step spray-drying, to deliver antigens to porcine dendritic cells and how these particles affect the functional maturation of dendritic cells (DCs). As clinically relevant model antigen F4 fimbriae, a bacterial adhesin purified from a porcine-specific enterotoxigenic Escherichia coli strain was chosen. The resulting antigen-loaded PECMs are efficiently internalised by porcine monocyte-derived DCs. F4 fimbriae-loaded PECMs (F4-PECMs) enhanced CD40 and CD25 surface expression by DCs and this phenotypical maturation correlated with an increased secretion of IL-6 and IL-1β. More importantly, F4-PECMs enhance both the T cell stimulatory and antigen presentation capacity of DCs. Moreover, PECMs efficiently promoted the CD8(+) T cell stimulatory capacity of dendritic cells, indicating an enhanced ability to cross-present the encapsulated antigens. These results could accelerate the development of veterinary and human subunit vaccines based on polyelectrolyte complex microparticles to induce protective immunity against a variety of extra- and intracellular pathogens.

Beta-glucan particles as novel antigen delivery systems : towards oral vaccination

Gastro-intestinal infections are still a main cause of enteric diseases and mortality among humans and animals. Oral vaccination is crucial in generating an adequate local mucosal immune response, however the hostile environment of the intestinal tract and oral tolerance remain huge obstacles that inhibit the ability to successfully develop new mucosal vaccines. A promising strategy for vaccination with safe, biodegradable non-replicating antigen delivery systems has gained increased interest for eliciting cellular and humoral immune responses. The current study evaluates the potential of β-glucan (BGP) and calcium carbonate (CaCO3)- and mannitol-templated polyelectrolyte particles as mucosal antigen delivery systems and their adjuvant characteristics. All microparticle types are efficiently internalized by Caco-2 and HT-29 cell lines and in particular the BGP triggered the expression of pro-inflammatory cytokines IL-23p19, IL-8 and beta-glucan receptors in activated Caco-2 cells and CCL20 in HT-29 cells. In contrast, the expression level of TGF-b, an important mediator of the active component of oral tolerance, was significantly downregulated in HT-29 cells. Oral administration of BGP induced intestinal adaptive immune responses characterized by an increased sIgA and secretory component production. Interestingly, adoptive transfer experiments pointed out the proliferation of naive OVA-specific CD4+ OT-II cells and increased IL-17 production in spleens of BGP-fed mice upon antigen restimulation. These results demonstrate that BGP enhances MHC-II-presentation and promotes mucosal immune responses preferably skewed towards an Th17 response and represents a promising strategy for oral vaccination.