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.

+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...

The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant formulated in a stable emulsion (GLA-SE) promotes strong TH1 and balanced IgG1/IgG2 responses to protein vaccine antigens. This enhanced immunity is sufficient to provide protection against many diseases including tuberculosis and leishmaniasis. To better characterize the adjuvant action it is important to understand how the different cytokines and transcription factors contribute to the initiation of immunity. In the present study using T-bet-/- and IL-12-/- mice and a blocking anti-IFNαR1 monoclonal antibody, we define mechanisms of adjuvant activity of GLA-SE. In accordance with previous studies of TLR4 agonist based adjuvants, we found that TH1 induction via GLA-SE was completely dependent upon T-bet, a key transcription factor for IFNγ production and TH1 differentiation. Consistent with this, deficiency of IL-12, a cytokine canonical to TH1 induction, ablated TH1 induction via GLA-SE. Finally we demonstrate that the innate immune response to GLA-SE, including rapid IFNγ production by memory CD8+ T cells and NK cells, was contingent on type I interferon, a cytokine group whose association with TH1 induction is contextual, and that they contributed to the adjuvant activity of GLA-SE.

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.

BackgroundAdjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE).Materials and methodsIn this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations.ResultsWe identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies.ConclusionThese efforts will aid in the development of a platform formulation for use with other similar vaccines.

The involvement of innate receptors that recognize pathogen- and danger-associated molecular patterns is critical to programming an effective adaptive immune response to vaccination. The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) synergizes with the squalene oil-in-water emulsion (SE) formulation to induce strong adaptive responses. Although TLR4 signaling through MyD88 and TIR domain-containing adapter inducing IFN-β are essential for GLA-SE activity, the mechanisms underlying the synergistic activity of GLA and SE are not fully understood. In this article, we demonstrate that the inflammasome activation and the subsequent release of IL-1β are central effectors of the action of GLA-SE, as infiltration of innate cells into the draining lymph nodes and production of IFN-γ are reduced in ASC animals. Importantly, the early proliferation of Ag-specific CD4 T cells was completely ablated after immunization in ASC animals. Moreover, numbers of Ag-specific CD4 T and B cells as well as production of IFN-γ, TNF-α, and IL-2 and Ab titers were considerably reduced in ASC, NLRP3, and IL-1R mice compared with wild-type mice and were completely ablated in TLR4 animals. Also, extracellular ATP, a known trigger of the inflammasome, augments Ag-specific CD4 T cell responses, as hydrolyzing it with apyrase diminished adaptive responses induced by GLA-SE. These data thus demonstrate that GLA-SE adjuvanticity acts through TLR4 signaling and NLRP3 inflammasome activation to promote robust Th1 and B cell responses to vaccine Ags. The findings suggest that engagement of both TLR and inflammasome activators may be a general paradigm for induction of robust CD4 T cell immunity with combination adjuvants such as GLA-SE.

It is estimated that one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). This astounding statistic, in combination with costly and lengthy treatment regimens make the development of therapeutic vaccines paramount for controlling the global burden of tuberculosis. Unlike prophylactic vaccination, therapeutic immunization relies on the natural pulmonary infection with Mtb as the mucosal prime that directs boost responses back to the lung. The purpose of this work was to determine the protection and safety profile over time following therapeutic administration of our lead Mtb vaccine candidate, ID93 with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE)), in combination with rifampicin, isoniazid, and pyrazinamide (RHZ) drug treatment. We assessed the host inflammatory immune responses and lung pathology 7–22 weeks post infection, and determined the therapeutic efficacy of combined treatment by enumeration of the bacterial load and survival in the SWR/J mouse model. We show that drug treatment alone, or with immunotherapy, tempered the inflammatory responses measured in brochoalveolar lavage fluid and plasma compared to untreated cohorts. RHZ combined with therapeutic immunizations significantly enhanced TH1-type cytokine responses in the lung over time, corresponding to decreased pulmonary pathology evidenced by a significant decrease in the percentage of lung lesions and destructive lung inflammation. These data suggest that bacterial burden assessment alone may miss important correlates of lung architecture that directly contribute to therapeutic vaccine efficacy in the preclinical mouse model. We also confirmed our previous finding that in combination with antibiotics therapeutic immunizations provide an additive survival advantage. Moreover, therapeutic immunizations with ID93/GLA-SE induced differential T cell immune responses over the course of infection that correlated with periods of enhanced bacterial control over that of drug treatment alone. Here we advance the immunotherapy model and investigate reliable correlates of protection and Mtb control.

The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here we sought to determine the role of B cells in the induction of T helper 1 (TH1) CD4+ T cells upon vaccination with a TB antigen combined with a TLR4 agonist. We used B cell deficient mice (μMT-/-), tetramer-positive CD4+ T cells, markers of memory ‘precursor’ effector cells (MPEC), and T cell adoptive transfers and demonstrated that the early antigen-specific cytokine-producing TH1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory TH1 response in μMT-/- mice. We further show that antigen-presentation by B cells was necessary for their role in MPEC generation using B cell adoptive transfers from wild type or MHC class II knock-out mice into μMT-/- mice. Our study challenges the view that B cell deficiency exclusively alters the TH1 response at memory time-points. Collectively, our results provide new insightson the multifaceted roles of B cells which will have a high impact on vaccine development against several pathogens including those requiring TH1 cell-mediated immunity.