Key antigens of Leishmania species identified in the context of host responses in Leishmania-exposed individuals from disease-endemic areas were prioritized for the development of a subunit vaccine against visceral leishmaniasis (VL), the most deadly form of leishmaniasis. Two Leishmania proteins—nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted— were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population. When formulated with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE), a Toll-like receptor 4 TH1 (T helper 1) promoting nanoemulsion adjuvant, the LEISH-F3 polyprotein induced potent protection against both L. donovani and L. infantum in mice, measured as significant reductions in liver parasite burdens. A robust immune response to each component of the vaccine with polyfunctional CD4 TH1 cell responses characterized by production of antigen-specific interferon-γ, tumor necrosis factor and interleukin-2 (IL-2), and low levels of IL-5 and IL-10 was induced in immunized mice. We also demonstrate that CD4 T cells, but not CD8 T cells, are sufficient for protection against L. donovani infection in immunized mice. Based on the sum of preclinical data, we prepared GMP materials and performed a phase 1 clinical study with LEISH-F3+GLA-SE in healthy, uninfected adults in the United States. The vaccine candidate was shown to be safe and induced a strong antigen-specific immune response, as evidenced by cytokine and immunoglobulin subclass data. These data provide a strong rationale for additional trials in Leishmania-endemic countries in populations vulnerable to VL.

Safe, effective adjuvants that enhance vaccine potency, including induction of neutralizing Abs against a broad range of variant strains, is an important strategy for the development of seasonal influenza vaccines which can provide optimal protection, even during seasons when available vaccines are not well matched to circulating viruses. We investigated the safety and ability of Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE), a synthetic Toll-like receptor (TLR)4 agonist formulation, to adjuvant Fluzone® in mice and non-human primates. The GLA-SE adjuvanted Fluzone vaccine caused no adverse reactions, increased the induction of T helper type 1 (TH1)-biased cytokines such as IFNγ, TNF and IL-2, and broadened serological responses against drifted A/H1N1 and A/H3N2 influenza variants. These results suggest that synthetic TLR4 adjuvants can enhance the magnitude and quality of protective immunity induced by influenza vaccines.

+T-cell induction via GLA-SE. Thus, we demonstrate that IL-18 and caspase-1/11 are components of the response to immunization with the TLR4 agonist/squalene oil-inwater based adjuvant, GLA-SE, providing implications for other adjuvants that combine oils with TLR agonists. Additional supporting information may be found in the online version of this article at the publisher's web-site Keywords: Adjuvants IntroductionVaccines rely on triggering the innate immune system to generate protective immunity. Many empirically established vaccines serendipitously contain molecules known as pathogen-associated molecular patterns (PAMPs) that engage host-germline encoded receptors, e.g. TLRs, which contribute to their immunogenicity Correspondence: Dr. Anthony L. Desbien e-mail: Anthony.Desbien@IDRI.org [1]. In the case where antigen preparations alone fail to be immunogenic, extrinsic adjuvants, such as alum or oil emulsions, are added to make vaccines effective. Such extrinsic adjuvants were empirically developed and despite their use in billions of vaccine doses, their mechanisms of action are not completely understood [2,3]. Knowledge of the salient attributes of empirical adjuvants would facilitate the design of the next generation of vaccines.In order to function as adjuvants, deliberately incorporated PAMPs require proper formulation [4,5]. For unknown reasons, squalene oil-in-water emulsions (SEs), such as AS02 and MF59, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 408Anthony L. Desbien et al. Eur. J. Immunol. 2015. 45: 407-417 are excellent formulations for TLR agonists, greatly enhancing the magnitude and quality of the immune response [6][7][8][9][10][11]. While it is clear that squalene emulsions by themselves are potent adjuvants, exactly how they engage the immune system or cooperate with TLR agonists is unclear. The commercial product MF59 is the prototypical squalene adjuvant. MF59 has been shown to activate the innate immune system, alter antigen presentation, and enhance humoral responses [3,[12][13][14]. While these attributes are ostensibly useful for generation of immunity, the specific components of the immune system triggered by squalene emulsions remain to be elucidated. The inflammasome is a pathogen recognition system required for protection against many diseases [15]. Engagement of the inflammasome occurs by diverse stimuli including pathogenand host-derived molecules. Known triggers include cytoplasmic dsDNA as well as aberrantly localized host-derived molecules, referred to as danger-associated molecular patterns [16]. In particular, extracellular ATP acts as a danger-associated molecular pattern, and has been recently demonstrated to contribute to activity of MF59, suggesting that MF59 acts via the inflammasome [17]. Further, the antibody response to immunization with MF59 has been linked to the adapter protein ASC, a component of the inflammasome, but not caspase-1 or NLRP3 (where NLR is nod-like receptor), indicating that adjuvant activity of MF59 is mediated by stil...

Designing modern vaccine adjuvants depends on understanding the cellular and molecular events that connect innate and adaptive immune responses. The synthetic TLR4 agonist GLA formulated in a stable emulsion (GLA-SE) augments both cellular and humoral immune responses to vaccine antigens. This adjuvant is currently included in several vaccines undergoing clinical evaluation including those for tuberculosis, leishmaniasis, and influenza. Delineation of the mechanisms of adjuvant activity will enable more informative evaluation of clinical trials. Early after injection, GLA-SE induces substantially more antigen-specific B cells, higher serum antibody titers and greater numbers of T follicular helper (TFH) and TH1 cells than alum, the squalene-in-water emulsion (SE) alone, or GLA without SE. GLA-SE augments antigen-specific B cell differentiation into germinal center and memory precursor B cells as well as pre-plasmablasts that rapidly secrete antibodies. CD169+ SIGNR1+ subcapsular medullary macrophages are the primary cells to take up GLA-SE after immunization and are critical for the innate immune responses, including rapid IL-18 production, induced by GLA-SE. Depletion of subcapsular macrophages (SCMϕ) or abrogation of IL-18 signaling dramatically impairs the antigen-specific B cell and antibody responses augmented by GLA-SE. Depletion of SCMϕ also drastically reduces the TH1 but not TFH response. Thus the GLA-SE adjuvant operates through interaction with IL-18-producing SCMϕ for the rapid induction of B cell expansion and differentiation, antibody secretion and TH1 responses, whereas augmentation of TFH numbers by GLA-SE is independent of SCMϕ.

Vaccine development for vector-borne pathogens may be accelerated through the use of relevant challenge models, as has been the case for malaria. Because of the demonstrated biological importance of vector-derived molecules in establishing natural infections, incorporating natural challenge models into vaccine development strategies may increase the accuracy of predicting efficacy under field conditions. Until recently, however, there was no natural challenge model available for the evaluation of vaccine candidates against visceral leishmaniasis. We previously demonstrated that a candidate vaccine against visceral leishmaniasis containing the antigen LEISH-F3 could provide protection in preclinical models and induce potent T-cell responses in human volunteers. In the present study, we describe a next generation candidate, LEISH-F3+, generated by adding a third antigen to the LEISH-F3 di-fusion protein. The rationale for adding a third component, derived from cysteine protease (CPB), was based on previously demonstrated protection achieved with this antigen, as well as on recognition by human T cells from individuals with latent infection. Prophylactic immunization with LEISH-F3+formulated with glucopyranosyl lipid A adjuvant in stable emulsion significantly reduced both Leishmania infantum and L. donovani burdens in needle challenge mouse models of infection. Importantly, the data obtained in these infection models were validated by the ability of LEISH-F3+/glucopyranosyl lipid A adjuvant in stable emulsion to induce significant protection in hamsters, a model of both infection and disease, following challenge by L. donovani–infected Lutzomyia longipalpis sand flies, a natural vector. This is an important demonstration of vaccine protection against visceral leishmaniasis using a natural challenge model.

CD4 + T cells have been observed to acquire APC-derived membrane and membrane-associated molecules through trogocytosis in diverse immune settings. Despite this, the consequences of trogocytosis on the recipient T cell remain largely unknown. We previously reported that trogocytosed molecules on CD4 + T cells engage their respective surface receptors, leading to sustained TCR signaling and survival after APC removal. Using peptide-pulsed bone marrow-derived dendritic cells and transfected murine fibroblasts expressing antigenic MHC:peptide complexes as APC, we show that trogocytosis-positive CD4 + T cells display effector cytokines and transcription factor expression consistent with a T H 2 phenotype. In vitro-polarized T H 2 cells were found to be more efficient at performing trogocytosis than T H 1 or nonpolarized CD4 + cells, whereas subsequent trogocytosis-mediated signaling induced T H 2 differentiation in polarized T H 1 and nonpolarized cells. Trogocytosis-positive CD4 + T cells generated in vivo also display a T H 2 phenotype in both TCR-transgenic and wild-type models. These findings suggest that trogocytosismediated signaling impacts CD4 + T cell differentiation and effector cytokine production and may play a role in augmenting or shaping a T H 2-dominant immune response.

Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli –expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs.

Trogocytosis is the intercellular transfer of membrane and membrane-associated molecules. This underappreciated process has been described in a variety of biological settings including neuronal remodeling, fertilization, viral and bacterial spread, and cancer, but has been most widely studied in cells of the immune system. Trogocytosis is performed by multiple immune cell types, including basophils, macrophages, dendritic cells, neutrophils, natural killer cells, B cells, γδ T cells, and CD4+ and CD8+ αβ T cells. Although not expressed endogenously, the presence of trogocytosed molecules on cells has the potential to significantly impact an immune response and the biology of the individual trogocytosis-positive cell. Many studies have focused on the ability of the trogocytosis-positive cells to interact with other immune cells and modulate the function of responders. Less understood and arguably equally important is the impact of these molecules on the individual trogocytosis-positive cell. Molecules that have been reported to be trogocytosed by cells include cognate ligands for receptors on the individual cell, such as activating NK cell ligands and MHC:peptide. These trogocytosed molecules have been shown to interact with receptors on the trogocytosis-positive cell and mediate intracellular signaling. In this review, we discuss the impact of this trogocytosis-mediated signaling on the biology of the individual trogocytosis-positive cell by focusing on natural killer cells and CD4+ T lymphocytes.