Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Class-B CpG-ODN Formulated With a Nanostructure Induces Type I Interferons-Dependent and CD4+ T Cell-Independent CD8+ T-Cell Response Against Unconjugated Protein Antigen.

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.

A liquid crystal of ascorbyl palmitate, used as vaccine platform, provides sustained release of antigen and has intrinsic pro-inflammatory and adjuvant activities which are dependent on MyD88 adaptor protein.

Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1ß, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design.

Adjuvant activity of CpG-ODN formulated as a liquid crystal.

The adjuvants approved in human vaccine with recombinant/purified antigens induce weak cellular immune response and so the development of new adjuvant strategies is critical. CpG-ODN has successfully been used as an adjuvant (phase I-III clinical trials) but its bioavailability needs to be improved. We investigated the adjuvant ability of CpG-ODN formulated with a liquid crystal nanostructure of 6-O-ascorbyl palmitate (Coa-ASC16). Mice immunized with OVA/CpG-ODN/Coa-ASC16 elicited a potent specific IgG1, IgG2a, Th1 and Th17 cellular response without systemic adverse effects. These responses were superior to those induced by OVA/CpG-ODN (solution of OVA with CpG-ODN) and to those induced by the formulation OVA/CpG-ODN/Al(OH)3. Immunization with OVA/CpG-ODN/Coa-ASC16 resulted in a long-lasting cell-mediated immune response (at least 6.5 months). Furthermore, Coa-ASC16 alone allows a controlled release of CpG-ODN in vitro and induces local inflammatory response, independent of TLR4 signaling, characterized by an influx of neutrophils and Ly6C(high) monocytes and pro-inflammatory cytokines. Remarkably, the adjuvant capacity of CpG-ODN co-injected with Coa-ASC16 (OVA/CpG-ODN plus Coa-ASC16) was similar to the adjuvant activity of OVA/CpG-ODN, supporting the requirement for whole formulation to help CpG-ODN adjuvanticity. These results show the potential of this formulation, opening a new avenue for the development of better vaccines.