Arnaud Didierlaurent

AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant System, Induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4′-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-κB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4+ T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

Sustained desensitization to bacterial Toll-like receptor ligands after resolution of respiratory influenza infection

The World Health Organization estimates that lower respiratory tract infections (excluding tuberculosis) account for ∼35% of all deaths caused by infectious diseases. In many cases, the cause of death may be caused by multiple pathogens, e.g., the life-threatening bacterial pneumonia observed in patients infected with influenza virus. The ability to evolve more efficient immunity on each successive encounter with antigen is the hallmark of the adaptive immune response. However, in the absence of cross-reactive T and B cell epitopes, one lung infection can modify immunity and pathology to the next for extended periods of time. We now report for the first time that this phenomenon is mediated by a sustained desensitization of lung sentinel cells to Toll-like receptor (TLR) ligands; this is an effect that lasts for several months after resolution of influenza or respiratory syncytial virus infection and is associated with reduced chemokine production and NF-κB activation in alveolar macrophages. Although such desensitization may be beneficial in alleviating overall immunopathology, the reduced neutrophil recruitment correlates with heightened bacterial load during secondary respiratory infection. Our data therefore suggests that post-viral desensitization to TLR signals may be one possible contributor to the common secondary bacterial pneumonia associated with pandemic and seasonal influenza infection.

Flagellin Promotes Myeloid Differentiation Factor 88-Dependent Development of Th2-Type Response

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4+ T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-κB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide.

ABSTRACT Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-κB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-κB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-κB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1β or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1β- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1β and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1β and LPS.

Role of Toll‐like receptors in costimulating cytotoxic T cell responses

Stimulation of Toll‐like receptors (TLR) by pathogen‐derived compounds leads to activation of APC, facilitating the induction of protective immunity. This phenomenon is the basis of most adjuvant formulations currently in development. Here, we tested the ability of TLR2, 3, 4, 5, 7 and 9 signaling to enhance CTL responses upon vaccination with virus‐like particles. Stimulation of TLR2 and 4 failed to increase CTL responses, whereas ligands for TLR3, 5 and 7 exhibited moderate adjuvant function. In contrast, stimulation of TLR9 dramatically increased CTL responses, indicating that ligands for TLR9 are likely to be the most promising candidates for the development of novel adjuvant formulations for stimulating CTL responses.

An antiinflammatory role for IKKβ through the inhibition of "classical" macrophage activation

The nuclear factor κB (NF-κB) pathway plays a central role in inflammation and immunity. In response to proinflammatory cytokines and pathogen-associated molecular patterns, NF-κB activation is controlled by IκB kinase (IKK)β. Using Cre/lox-mediated gene targeting of IKKβ, we have uncovered a tissue-specific role for IKKβ during infection with group B streptococcus. Although deletion of IKKβ in airway epithelial cells had the predicted effect of inhibiting inflammation and reducing innate immunity, deletion of IKKβ in the myeloid lineage unexpectedly conferred resistance to infection that was associated with increased expression of interleukin (IL)-12, inducible nitric oxide synthase (NOS2), and major histocompatibility complex (MHC) class II by macrophages. We also describe a previously unknown role for IKKβ in the inhibition of signal transducer and activator of transcription (Stat)1 signaling in macrophages, which is critical for IL-12, NOS2, and MHC class II expression. These studies suggest that IKKβ inhibits the “classically” activated or M1 macrophage phenotype during infection through negative cross talk with the Stat1 pathway. This may represent a mechanism to prevent the over-exuberant activation of macrophages during infection and contribute to the resolution of inflammation. This establishes a new role for IKKβ in the regulation of macrophage activation with important implications in chronic inflammatory disease, infection, and cancer.

How the gut senses its content

for a human adult. These surfaces are covered by thin epi- thelial layers that are protected from potentially harmful microorganisms by innate and adaptive defence mecha- nisms. Intestinal mucosal surfaces are in continuous contact with a heterogeneous population of microorgan- isms of the endogenous flora (up to 10 11-12 per g of lumenal material in the colon) and are exposed to food and microbes. A major role for the mucosal epithelia is in barrier function, essential for preventing colonization or invasion of the host by foreign microorganisms. Epithelial tissues also provide the mucosal immune system with a continuous stream of information about the external envi- ronment. Depending on the nature and the dose of the antigens transported from the gut lumen into mucosal lym- phoid tissues, strong immune responses or unresponsive- ness can be induced. Immune responses participate in the elimination of pathogenic microorganisms, while immune tolerance prevents harmful reactions against the gut flora and food antigens. The type of immune response triggered by environmental antigens appears to depend, in part, on initial recognition by the innate immune system. Recent data underscore the importance of innate immunity in sensing the microbial environment and the role of the epithelium in releasing signals that allow recruitment of pro-inflammatory leucocytes, immune cells, or both. We shall review these various aspects of mucosal immunity, with special emphasis on the cross talk that takes place between the microflora, the epithelium and the immune cells in the gut.

Development and evaluation of AS03, an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion

AS03 is an Adjuvant System composed of α-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion. In various nonclinical and clinical studies, high levels of antigen-specific antibodies were obtained after administration of an AS03-adjuvanted vaccine, permitting antigen-sparing strategies. AS03 has been shown to enhance the vaccine antigen-specific adaptive response by activating the innate immune system locally and by increasing antigen uptake and presentation in draining lymph nodes, a process that is modulated by the presence of α-tocopherol in AS03. In nonclinical models of the AS03-adjuvanted prepandemic H5N1 influenza vaccine, increased levels of anti-influenza antibody afforded protection against disease and against virus replication of influenza strains homologous and heterologous to the vaccine strain. By incorporating AS03 in the pandemic H1N1/2009 vaccine, vaccine immunogenicity was increased compared with nonadjuvanted H1N1 vaccines. High H1N1/2009/AS03 vaccine effectiveness was demonstrated in several assessments in multiple populations. Altogether, the nonclinical and clinical data illustrate the ability of AS03 to induce superior adaptive responses against the vaccine antigen, principally in terms of antibody levels and immune memory. In general, these results support the concept of Adjuvant Systems as a plausible approach to develop new effective vaccines.

The Generation of Mature, Single-Positive Thymocytes In Vivo Is Dysregulated by CD69 Blockade or Overexpression

During development in the thymus, mature CD4+ or CD8+ cells are derived from immature CD4+CD8+ cells through a series of selection events. One of the hallmarks of this maturation process is the expression of CD69, which first appears on thymocytes as they begin positive selection. We have used blockade and overexpression of CD69 to determine the role of CD69 in thymocyte development. Blockade of CD69 led to a reduction in single-positive cells and a concomitant increase in double-positive cells in the thymus. Overexpression of a CD69 transgene in the thymus resulted in a dramatic increase in both CD8SP and CD4SP cells. Coexpression with a TCR transgene demonstrated that both positive and negative selection were enhanced by the increased levels of CD69 on thymocytes. Finally, mice overexpressing CD69 displayed a sharp reduction in the number of T cells in the spleen and lymph node. Taken as a whole, these data suggest the involvement of CD69 in the process of selection and maturation during the trafficking of thymocytes to the medulla.

Enhancement of Adaptive Immunity by the Human Vaccine Adjuvant AS01 Depends on Activated Dendritic Cells

Adjuvant System AS01 is a liposome-based vaccine adjuvant containing 3-O-desacyl-4′-monophosphoryl lipid A and the saponin QS-21. AS01 has been selected for the clinical development of several candidate vaccines including the RTS,S malaria vaccine and the subunit glycoprotein E varicella zoster vaccine (both currently in phase III). Given the known immunostimulatory properties of MPL and QS-21, the objective of this study was to describe the early immune response parameters after immunization with an AS01-adjuvanted vaccine and to identify relationships with the vaccine-specific adaptive immune response. Cytokine production and innate immune cell recruitment occurred rapidly and transiently at the muscle injection site and draining lymph node postinjection, consistent with the rapid drainage of the vaccine components to the draining lymph node. The induction of Ag-specific Ab and T cell responses was dependent on the Ag being injected at the same time or within 24 h after AS01, suggesting that the early events occurring postinjection were required for these elevated adaptive responses. In the draining lymph node, after 24 h, the numbers of activated and Ag-loaded monocytes and MHCIIhigh dendritic cells were higher after the injection of the AS01-adjuvanted vaccine than after Ag alone. However, only MHCIIhigh dendritic cells appeared efficient at and necessary for direct Ag presentation to T cells. These data suggest that the ability of AS01 to improve adaptive immune responses, as has been demonstrated in clinical trials, is linked to a transient stimulation of the innate immune system leading to the generation of high number of efficient Ag-presenting dendritic cells.