Toll-like receptor 4 signaling in hematopoietic-lineage cells contributes to the enhanced activity of the human vaccine adjuvant AS01.

The 3-O-desacyl-4'-monophosphoryl lipid A (MPL) activates immunity through Toll-like receptor 4 (TLR4) signaling. The Adjuvant System AS01 contains MPL and is used in the candidate malaria vaccine and the licensed zoster vaccine. Recent studies reported that AS01 adjuvant activity depends on a transient inflammation at the site of vaccination, but the role of stromal or structural cells in the adjuvant effect is unknown. We investigated this question in mouse models by assessing the role of TLR4 on hematopoietic versus resident structural cells during immunization with AS01-adjuvanted vaccines. We first established that TLR4-deficient animals had a reduced immune response to an AS01-adjuvanted vaccine. Using bone marrow chimera, we consistently found that Tlr4 expression in radio-sensitive cells, i.e., hematopoietic cells, was required for an optimal adjuvant effect on antibody and T-cell responses. At day 1 after injection, the pro-inflammatory reaction at the site of injection was strongly dependent on TLR4 signaling in hematopoietic cells. Similarly, activation of dendritic cells in muscle-draining lymph nodes was strictly associated with the radio-sensitive cells expressing Tlr4. Altogether, these data suggest that MPL-mediated TLR4-signaling in hematopoietic cells is critical in the mode of action of AS01.

Innate Immune Responses to Chimpanzee Adenovirus Vector 155 Vaccination in Mice and Monkeys.

Replication-deficient chimpanzee adenovirus (ChAd) vectors represent an attractive vaccine platform and are thus employed as vaccine candidates against several infectious diseases. Since inducing effective immunity depends on the interplay between innate and adaptive immunity, a deeper understanding of innate immune responses elicited by intramuscularly injected ChAd vectors in tissues can advance the platform's development. Using different candidate vaccines based on the Group C ChAd type 155 (ChAd155) vector, we characterized early immune responses in injected muscles and draining lymph nodes (dLNs) from mice, and complemented these analyses by evaluating cytokine responses and gene expression patterns in peripheral blood from ChAd155-injected macaques. In mice, vector DNA levels gradually decreased post-immunization, but local transgene mRNA expression exhibited two transient peaks [at 6 h and Day (D)5], which were most obvious in dLNs. This dynamic pattern was mirrored by the innate responses in tissues, which developed as early as 1-3 h (cytokines/chemokines) or D1 (immune cells) post-vaccination. They were characterized by a CCL2- and CXCL9/10-dominated chemokine profile, peaking at 6 h (with CXCL10/CCL2 signals also detectable in serum) and D7, and clear immune-cell infiltration peaks at D1/D2 and D6/D7. Experiments with a green fluorescent protein-expressing ChAd155 vector revealed infiltrating hematopoietic cell subsets at the injection site. Cell infiltrates comprised mostly monocytes in muscles, and NK cells, T cells, dendritic cells, monocytes, and B cells in dLNs. Similar bimodal dynamics were observed in whole-blood gene signatures in macaques: most of the 17 enriched immune/innate signaling pathways were significantly upregulated at D1 and D7 and downregulated at D3, and clustering analysis revealed stronger similarities between D1 and D7 signatures versus the D3 signature. Serum cytokine responses (CXCL10, IL1Ra, and low-level IFN-α) in macaques were predominantly observed at D1. Altogether, the early immune responses exhibited bimodal kinetics with transient peaks at D1/D2 and D6/D7, mostly with an IFN-associated signature, and these features were remarkably consistent across most analyzed parameters in murine tissues and macaque blood. These compelling observations reveal a novel aspect of the dynamics of innate immunity induced by ChAd155-vectored vaccines, and contribute to ongoing research to better understand how adenovectors can promote vaccine-induced immunity.

CCR2- and Flt3-Dependent Inflammatory Conventional Type 2 Dendritic Cells Are Necessary for the Induction of Adaptive Immunity by the Human Vaccine Adjuvant System AS01.

The Adjuvant System AS01 contains monophosphoryl lipid A (MPL) and the saponin QS-21 in a liposomal formulation. AS01 is included in recently developed vaccines against malaria and varicella zoster virus. Like for many other adjuvants, induction of adaptive immunity by AS01 is highly dependent on the ability to recruit and activate dendritic cells (DCs) that migrate to the draining lymph node for T and B cell stimulation. The objective of this study was to more precisely address the contribution of the different conventional (cDC) and monocyte-derived DC (MC) subsets in the orchestration of the adaptive immune response after immunization with AS01 adjuvanted vaccine. The combination of MPL and QS-21 in AS01 induced strong recruitment of CD26+XCR1+ cDC1s, CD26+CD172+ cDC2s and a recently defined CCR2-dependent CD64-expressing inflammatory cDC2 (inf-cDC2) subset to the draining lymph node compared to antigen alone, while CD26-CD64+CD88+ MCs were barely detectable. At 24 h post-vaccination, cDC2s and inf-cDC2s were superior amongst the different subsets in priming antigen-specific CD4+ T cells, while simultaneously presenting antigen to CD8+ T cells. Diphtheria toxin (DT) mediated depletion of all DCs prior to vaccination completely abolished adaptive immune responses, while depletion 24 h after vaccination mainly affected CD8+ T cell responses. Vaccinated mice lacking Flt3 or the chemokine receptor CCR2 showed a marked deficit in inf-cDC2 recruitment and failed to raise proper antibody and T cell responses. Thus, the adjuvant activity of AS01 is associated with the potent activation of subsets of cDC2s, including the newly described inf-cDC2s.

Application of Modeling Approaches to Explore Vaccine Adjuvant Mode-of-Action.

Novel adjuvant technologies have a key role in the development of next-generation vaccines, due to their capacity to modulate the duration, strength and quality of the immune response. The AS01 adjuvant is used in the malaria vaccine RTS,S/AS01 and in the licensed herpes-zoster vaccine (Shingrix) where the vaccine has proven its ability to generate protective responses with both robust humoral and T-cell responses. For many years, animal models have provided insights into adjuvant mode-of-action (MoA), generally through investigating individual genes or proteins. Furthermore, modeling and simulation techniques can be utilized to integrate a variety of different data types; ranging from serum biomarkers to large scale "omics" datasets. In this perspective we present a framework to create a holistic integration of pre-clinical datasets and immunological literature in order to develop an evidence-based hypothesis of AS01 adjuvant MoA, creating a unified view of multiple experiments. Furthermore, we highlight how holistic systems-knowledge can serve as a basis for the construction of models and simulations supporting exploration of key questions surrounding adjuvant MoA. Using the Systems-Biology-Graphical-Notation, a tool for graphical representation of biological processes, we have captured high-level cellular behaviors and interactions, and cytokine dynamics during the early immune response, which are substantiated by a series of diagrams detailing cellular dynamics. Through explicitly describing AS01 MoA we have built a consensus of understanding across multiple experiments, and so we present a framework to integrate modeling approaches into exploring adjuvant MoA, in order to guide experimental design, interpret results and inform rational design of vaccines.

Erratum: Author Correction: Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity.

[This corrects the article DOI: 10.1038/s41541-017-0027-3.].

Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity.

Combining immunostimulants in adjuvants can improve the quality of the immune response to vaccines. Here, we report a unique mechanism of molecular and cellular synergy between a TLR4 ligand, 3-O-desacyl-4'-monophosphoryl lipid A (MPL), and a saponin, QS-21, the constituents of the Adjuvant System AS01. AS01 is part of the malaria and herpes zoster vaccine candidates that have demonstrated efficacy in phase III studies. Hours after injection of AS01-adjuvanted vaccine, resident cells, such as NK cells and CD8+ T cells, release IFNγ in the lymph node draining the injection site. This effect results from MPL and QS-21 synergy and is controlled by macrophages, IL-12 and IL-18. Depletion strategies showed that this early IFNγ production was essential for the activation of dendritic cells and the development of Th1 immunity by AS01-adjuvanted vaccine. A similar activation was observed in the lymph node of AS01-injected macaques as well as in the blood of individuals receiving the malaria RTS,S vaccine. This mechanism, previously described for infections, illustrates how adjuvants trigger naturally occurring pathways to improve the efficacy of vaccines.

Natural history of human T-lymphotropic virus 1-associated myelopathy: a 14-year follow-up study.

BACKGROUND: The progression of neurological disability in human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) remains undefined. OBJECTIVES: To determine the time course of disability scores and to identify predictors of outcome among patients with HAM/TSP. DESIGN: Clinical 14-year follow-up study. SETTING: University hospital. Patients One hundred twenty-three patients with HAM/TSP. MAIN OUTCOME MEASURES: We determined time from onset to the following 4 Kurtzke Disability Status Scale (DSS) end points: scores of 6 (unilateral aid required), 6.5 (bilateral aid required), 8 (wheelchair confinement), and 10 (death related to the disease). Times to reach selected DSS scores were estimated using the Kaplan-Meier method. Univariate and multivariate analyses identified variables related to the rate of progression to DSS 8. The HTLV-1 proviral loads were also assessed. RESULTS: The disability of the cohort progressed throughout the follow-up period. The median times from onset to DSS 6, 6.5, and 8 were 6, 13, and 21 years, respectively. The median time from DSS 6 to DSS 8 was 8 years; DSS 10 was reached by one fourth of the patients within 20 years. Age at onset of 50 years or older and high HTLV-1 proviral load were associated with a shorter time to DSS 8 (P = .01 and P = .02, respectively). A shorter time to DSS 6 significantly adversely affected the time to progression from DSS 6 to DSS 8. CONCLUSIONS: Human T-lymphotropic virus 1-associated myelopathy/tropical spastic paraparesis is a rapidly disabling disease. Monitoring for HTLV-1 proviral load is recommended in future therapeutic trials.

Pro-inflammatory cytokines modulate matrix metalloproteinase secretion and organic anion transport at the blood-cerebrospinal fluid barrier.

Neuroinflammation and neuroinfection trigger cytokine-mediated responses that include an increase in the cerebrospinal fluid (CSF) levels of pro-inflammatory matrix metalloproteinases (MMPs) and organic anions such as leukotrienes and prostaglandins. The choroid plexus (CP) epithelium forming the interface between the blood and the CSF regulates the CSF concentration of bioactive organic anions and is involved in neuro-immune regulation. We demonstrated that both fourth and lateral ventricle CPs are a source of pro- and active MMP-2 and MMP-9 in the brain. Using a cellular model of the blood-CSF barrier, we showed that a pro-inflammatory cytokine treatment leads to an increase in the choroidal MMP secretion at either the apical or the basolateral membrane, depending on the ventricular origin of the choroidal cells. This effect was not concomitant with an alteration in the structural blood-CSF barrier. Neither was the pool of antioxidant sulfhydryls in the choroidal cells challenged. In contrast, the efficiency of the choroidal epithelium to clear the CSF from organic anions was highly reduced. Thus, during inflammation, the CPs could be one source of MMPs found in the CSF facilitate leucocyte migration by secreting MMPs into the choroidal stroma, and promote the inflammatory process by failing in its ability to clear deleterious compounds from the brain.

Evidence of in situ cytotoxicity in American cutaneous leishmaniasis.

The role of cytotoxicity in the defense mechanisms or pathogenesis of human cutaneous leishmaniasis is not yet well known. In the present work we assessed the presence of NK, CD8+ and CD45RO+ T cells, as well as the expression of a molecule associated with cytotoxic properties (TIA-1) in the lesions of cutaneous leishmaniasis. CD8+ T cells, NK and activated T cells were found within the dermal cell infiltrate. We found a heterogeneous but usually strong expression of TIA-1, a marker of cytotoxic granules of T and NK cells, in human cutaneous leishmaniasis lesions. These data suggest that cytotoxic activity occurs in situ in American cutaneous leishmaniasis and that both NK cells and activated CD8+ T cells are involved in this reaction.

Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles.

Wrinkles are modifications of the skin associated with cutaneous ageing and develop preferentially on sun-exposed skin. The aim of the study was to analyse the clinicopathological features of wrinkles, among the different types of skin relief modifications. Despite its importance in dermato-cosmetology and skin ageing, few studies have been specifically devoted to wrinkles. In the present study, we analyzed the histological features of the pre-auricular wrinkle compared to retro-auricular skin, obtained from sixteen patients undergoing facial surgery; skin samples were immediately processed for routine histology and histochemical staining. Four types of skin depressions could be defined according to their depth: folds, permanent wrinkles, reducible wrinkles and skin micro-relief. Two different types of pre-auricular wrinkles were observed: (i) permanent wrinkles which were conserved after sampling and, (ii) reducible wrinkles which required in vivo staining to be visible at histology. Histological analysis of the epidermis and dermis of the skin forming the pre-auricular wrinkle revealed a normal skin morphology, identical to that of the skin immediately adjacent to the wrinkle. This was particularly striking for the reducible wrinkles which could not be individualized in the absence of in vivo staining. Both types of wrinkles comprised skin modifications observed in sun-exposed skin, however, in the upper dermis, permanent wrinkles showed a more pronounced accumulation of basophilic fibers, i.e. actinic elastosis, than reducible wrinkles did. These data suggest that the development of wrinkles could be secondary to actinic elastosis and to the disappearance of microfibrils and collagen fibers at the dermal-epidermal junction.

[Astrocytes, cells involved in neuro-immune interactions in the central nervous system]. / Les astrocytes, cellules relais des interactions neuro-immunes dans le système nerveux central.

The astrocyte, the major glial cell in the central nervous system, may influence many aspects of inflammation and immune reactivity within the brain. We have established a model of chronically activated T lymphocytes, interacting with neural cells of diverse origin to study the complex immune regulatory system suspected to lead to neuroinflammatory diseases. We show that human astrocytes became reactive following T cell contact, secreting proinflammatory cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinase (TIMP). The altered MMP/TIMP system was shown to be involved in deleterious effects displayed by activated T cells towards human multipotent neural precursers by controlling their sensitivity to T cell-induced Fas-mediated apoptosis. MMP/TIMP was suspected to stabilize Fas at the cell membrane. In a model of mixed rat glial cells in primary culture (astrocytes, oligodendrocytes), activated T lymphocytes induced the collapse of processes and the death of immature oligodendrocytes. These effects were associated with upregulation of Fas at the cell surface of oligodendrocytes and secretion of MMP and TIMP by astrocytes. By amplifying the expression of inflammatory molecules including the MMP/TIMP system, astrocytes appear to be a crucial relay in the deleterious molecular cascade triggered by activated T lymphocytes. Detection of altered MMP/TIMP in patients suffering from myelopathy associated with retroviral infection (HTLV-1) strongly suggests its involvement in the physiopathological process of the disease.