Duality of ß-glucan microparticles: antigen carrier and immunostimulants.

Designing efficient recombinant mucosal vaccines against enteric diseases is still a major challenge. Mucosal delivery of recombinant vaccines requires encapsulation in potent immunostimulatory particles to induce an efficient immune response. This paper evaluates the capacity of ß-glucan microparticles (GPs) as antigen vehicles and characterizes their immune-stimulatory effects. The relevant infectious antigen FedF was chosen to be loaded inside the microparticles. The incorporation of FedF inside the particles was highly efficient (roughly 85%) and occurred without antigen degradation. In addition, these GPs have immunostimulatory effects as well, demonstrated by the strong reactive oxygen species (ROS) production by porcine neutrophils upon their recognition. Although antigen-loaded GPs still induce ROS production, antigen loading decreases this production by neutrophils for reasons yet unknown. However, these antigen-loaded GPs are still able to bind their specific ß-glucan receptor, demonstrated by blocking complement receptor 3, which is the major ß-glucan receptor on porcine neutrophils. The dual character of these particles is confirmed by a T-cell proliferation assay. FedF-loaded particles induce a significantly higher FedF-specific T-cell proliferation than soluble FedF. Taken together, these results show that GPs are efficient antigen carriers with immune-stimulatory properties.

ß-glucan microparticles targeted to epithelial APN as oral antigen delivery system.

Enteric diseases still have a devastating impact on global health. Oral vaccination is crucial to prevent intestinal infections, since only vaccines delivered to the intestinal tract elicit potent immune responses at the site of pathogen entry. However, oral vaccines encounter multiple barriers, including poor uptake and tolerance mechanisms, preventing the immune system to react to innocuous environmental antigens. Antigen delivery systems combined with selective targeting seem a promising strategy to overcome these obstacles. The current study evaluates the capacity of aminopeptidase N (APN)-targeted ß-glucan microparticles (GPs) as antigen delivery system. Antibodies against APN, an intestinal epithelial receptor, are efficiently oriented conjugated to GPs via the biolinker protein G. The resultant microparticles were analyzed for their antigen load, adjuvanticity and interaction with enterocytes and dendritic cells (DCs). Functionalization of GPs with antibodies neither impedes antigen load nor adjuvanticity. In addition, targeting to APN increases the uptake of microparticles by enterocytes and DCs, leading to an enhanced maturation of the latter as evidenced by an upregulation of maturation markers and a strong pro-inflammatory cytokine response. Finally, oral administration of APN-targeted antigen-loaded particles to piglets elicits higher serum antigen-specific antibody responses as compared to control particles. Taken together, these data support the use of APN-targeted GPs for oral delivery of antigens.

Pseudorabies virus US3 leads to filamentous actin disassembly and contributes to viral genome delivery to the nucleus.

The conserved alphaherpesvirus US3 tegument protein induces rearrangements of the actin cytoskeleton, consisting of protrusion formation and stress fiber breakdown. Although US3 does not affect levels of total actin protein, it remains unclear whether US3 modulates the total levels of filamentous (F) actin. In this report, we show that the pseudorabies virus (PRV) US3 protein, via its kinase activity, leads to disassembly of F-actin in porcine ST cells. F-actin disassembly has been reported before to contribute to host cell entry of HIV. In line with this, in the current study, we report that US3 has a previously uncharacterized role in viral genome delivery to the nucleus, since quantitative polymerase chain reaction (qPCR) assays on nuclear fractions demonstrated a reduced nuclear delivery of US3null PRV compared to wild type PRV genomes. Treatment of cells with the actin depolymerizing drug cytochalasin D enhanced virus genome delivery to the nucleus, particularly of US3null PRV, supporting a role for F-actin disassembly during certain aspects of viral entry. In conclusion, the US3 kinase of PRV leads to F-actin depolymerization, and US3 and F-actin disassembly contribute to viral genome delivery to the nucleus.

Aflatoxins of type B and G affect porcine dendritic cell maturation in vitro.

The toxic effects of highly carcinogenic mycotoxins, especially aflatoxins (AF), on key antigen-presenting cells, such as dendritic cells (DC), are largely unknown. To elucidate the effect of AF on DC function, porcine monocyte-derived DC (MoDC) were treated with a mixture of several AF (i.e., AFB1, AFB2, AFG1, and AFG2) and the phagocytic capacity, the membrane expression level of several DC activation markers, the T-cell proliferation-inducing capacity, and the cytokine secretion pattern were assessed. As compared to untreated MoDC, AF significantly up-regulated the expression of the co-stimulatory molecules CD25 and CD80/86. However, the phagocytic activity of MoDC was not affected by AF treatment. While the cytokine secretion pattern of AF-treated MoDC was similar to control MoDC, the T-cell proliferation-inducing capacity of MoDC was increased upon aflatoxin treatment. The results indicate that a mixture of naturally occurring AF enhances the antigen-presenting capacity of DC, which could explain the observed immunotoxicity of AF by breaking down tolerance and further emphasizes the need to reduce the admissible level of AF in agricultural commodities.

Cell type-specific differences in ß-glucan recognition and signalling in porcine innate immune cells.

ß-glucans exert receptor-mediated immunomodulating activities, including oxidative burst activity and cytokine secretion. The role of the ß-glucan receptors dectin-1 and complement receptor 3 (CR3) in the response of immune cells towards ß-glucans is still unresolved. Dectin-1 is considered as the main ß-glucan receptor in mice, while recent studies in man show that CR3 is more important in ß-glucan-mediated responses. This incited us to elucidate which receptor contributes to the response of innate immune cells towards particulate ß-glucans in pigs as the latter might serve as a better model for man. Our results show an important role of CR3 in ß-glucan recognition, as blocking this receptor strongly reduced the phagocytosis of ß-glucans and the ß-glucan-induced ROS production by porcine neutrophils. Conversely, dectin-1 does not seem to play a major role in ß-glucan recognition in neutrophils. However, recognition of ß-glucans appeared cell type-specific as both dectin-1 and CR3 are involved in the ß-glucan-mediated responses in pig macrophages. Moreover, CR3 signalling through focal adhesion kinase (FAK) was indispensable for ß-glucan-mediated ROS production and cytokine production in neutrophils and macrophages, while the Syk-dependent pathway was only partly involved in these responses. We may conclude that CR3 plays a cardinal role in ß-glucan signalling in porcine neutrophils, while macrophages use a more diverse receptor array to detect and respond towards ß-glucans. Nonetheless, FAK acts as a master switch that regulates ß-glucan-mediated responses in neutrophils as well as macrophages.

Aflatoxin B1 interferes with the antigen-presenting capacity of porcine dendritic cells.

Aflatoxins (AFs) are harmful to animal and human health upon consumption of AF-contaminated feed or food. Among many forms of AFs, aflatoxin B1 (AFB1) is the most toxic and carcinogenic. In addition, AFB1 impairs cell-mediated immunity, although the exact mechanism of this immunotoxicity is currently unknown. By far the most pivotal cells in the induction of immune responses are dendritic cells (DCs). These highly specialised cells dictate T-cell polarisation depending on the nature of the encountered antigens and environmental cues. To elucidate the effect of AFB1 on the function of DCs, we used porcine monocyte-derived DCs (MoDCs) as a model system. A low dose of AFB1 transiently reduced the phagocytic capacity of MoDCs. Furthermore, as compared to untreated MoDCs, AFB1 significantly downregulated the cell surface expression of the co-stimulatory molecule CD40 at 12 h post treatment, while at 24 h the membrane expression levels of CD40 and the activation marker CD25 were significantly upregulated. Interestingly, the T-cell proliferation-inducing capacity of DCs was diminished upon AFB1 treatment. In contrast, the cytokine secretion pattern of AFB1-treated MoDCs was similar to mock-treated MoDCs. The results in this study indicate that a low level of AFB1 dysregulates the antigen-presenting capacity of DCs, which could explain the observed immunotoxicity of this mycotoxin, and further stress the need to reduce AFB1 levels in agricultural commodities.

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-1ß. 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.