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Vaccines are highly effective in preventing the spread of communicable diseases and are critical to overall public health. As immune stimulants vaccine adjuvants augment the level and longevity of these protective responses. Seeking novel adjuvants using parallel high throughput screens and subsequent systematic structure-activity relationship studies we identified an analogue of a hit compound, , that in screening assays retained induction of calcium (Ca ) influx, tetraspanin CD63 EV reporter activity and cell viability. Here, we further our analyses of the biological activity of related its potential use as a vaccine adjuvant. was tested for activation of murine bone marrow-derived dendritic cells (mBMDC) by flow cytometry for Ca entry, levels of CD80 and CD86 expression, and stimulation of antigen-specific T cell proliferation. Cytokines and IgG responses from BALB/c mice injected with as a single agent or as an adjuvant with ovalbumen were measured by ELISA. triggered store-operated Ca entry in mBMDC as well as increases in CD80 and CD86 surface expression. In co-culture experiments, this compound amplified the stimulation of cognate T cell proliferation. Intramuscular injection of elicited minimal systemic cytokine and chemokine release. When used as an adjuvant with ovalbumen, generated a significant antigen-specific IgG1 response with a higher splenocyte T helper 2 (Th2) cytokine response. activated mBMDCs associated with EV release and a store-operated calcium entry response. Enhanced cognate T cell proliferation was mediated either through direct engagement with compound-stimulated mBMDCs or indirectly via immunostimulatory extracellular vesicles released by -activated mBMDCs. elicited minimal systemic cytokine and chemokine release, demonstrating a promising safety profile. When used as an adjuvant in a murine vaccination model, enhanced the IgG1 response with an associated T helper 2 cytokine profile. Hence this compound shows promise as an adjuvant if a Th2 response is beneficial or in combination with other agents to provide a balanced immune response in vaccines.

BackgroundPancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically in locally advanced PC, but most patients eventually develop distant recurrence. We have previously shown that IRE alone is capable of generating protective, neoantigen-specific immunity. Here, we aim to generate meaningful therapeutic immune effects by combining IRE with local (intratumoral) delivery of a CD40 agonistic antibody (CD40Ab).MethodsKPC46 organoids were generated from a tumor-bearing male KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) mouse. Orthotopic tumors were established in the pancreatic tail of B6/129 F1J mice via laparotomy. Mice were randomized to treatment with either sham laparotomy, IRE alone, CD40Ab alone, or IRE followed immediately by CD40Ab injection. Metastatic disease and immune infiltration in the liver were analyzed 14 days postprocedure using flow cytometry and multiplex immunofluorescence imaging with spatial analysis. Candidate neoantigens were identified by mutanome profiling of tumor tissue for ex vivo functional analyses.ResultsThe combination of IRE+CD40 Ab improved median survival to greater than 35 days, significantly longer than IRE (21 days) or CD40Ab (24 days) alone (p<0.01). CD40Ab decreased metastatic disease burden, with less disease in the combination group than in the sham group or IRE alone. Immunohistochemistry of liver metastases revealed a more than twofold higher infiltration of CD8+T cells in the IRE+CD40 Ab group than in any other group (p<0.01). Multiplex immunofluorescence imaging revealed a 4–6 fold increase in the density of CD80+CD11c+ activated dendritic cells (p<0.05), which were spatially distributed throughout the tumor unlike the sham group, where they were restricted to the periphery. In contrast, CD4+FoxP3+ T-regulatory cells (p<0.05) and Ly6G+myeloid derived cells (p<0.01) were reduced and restricted to the tumor periphery in the IRE+CD40 Ab group. T-cells from the IRE+CD40 Ab group recognized significantly more peptides representing candidate neoantigens than did T-cells from the IRE or untreated control groups.ConclusionsIRE can induce local tumor regression and neoantigen-specific immune responses. Addition of CD40Ab to IRE improved dendritic cell activation and neoantigen recognition, while generating a strong systemic antitumor T-cell response that inhibited metastatic disease progression.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z′ factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.

Recent studies show that redox-active small molecules are selectively cytotoxic to chronic lymphocytic leukemia (CLL). Although elevated levels of reactive oxygen species in CLL cells have been implicated, the molecular mechanism underlying this selectivity is unclear. In other cell types, the nuclear factor erythroid 2—related factor 2 (Nrf2) signaling pathway regulates the oxidative stress response. We found elevated Nrf2 signaling in untreated CLL cells compared with normal lymphocytes. Therefore, we tested 27 known electrophilic and antioxidant compounds with drug-like properties and determined their CLL-selective cytotoxicity and effect on Nrf2 signaling. The selected compounds were from five distinct structural classes; α-β unsaturated carbonyls, isothiocyanates, sulfhydryl reactive metals, flavones, and polyphenols. Our results show that compounds containing α-β unsaturated carbonyls, sulfhydryl reactive metals, and isothiocyanates are strong activators of Nrf2 in a reporter assay system and in primary human CLL based on increased expression of the Nrf2 target heme oxygenase—1. α-β Unsaturated carbonyl-containing compounds were selectively cytotoxic to CLL, and loss of the α-β unsaturation abrogated Nrf2 activity and CLL toxicity. The α-β unsaturated carbonyl containing compounds ethacrynic acid and parthenolide activated Nrf2 in normal peripheral blood mono-nuclear cells, but had a less potent effect in CLL cells. Furthermore, ethacrynic acid bound directly to the Nrf2-negative regulator Kelch-like ECH-associated protein 1 (Keap1) in CLL cells. These experiments document the presence of Nrf2 signaling in human CLL and suggest that altered Nrf2 responses may contribute to the observed selective cytotoxicity of electrophilic compounds in this disease.

3-Hydroxyanthranilic acid (HAA), a compound generated during tryptophan metabolism initiated by indoleamine 2,3-dioxygenase, is known to induce T cell death, but its molecular target is not known. Here we report that HAA inhibits NF-κB activation upon T cell antigen receptor engagement by specifically targeting PDK1. Inhibition of NF-κB by HAA leads to dysfunction and cell death of activated Th2 cells, which in turn suppresses experimental asthma. Inhibition of NF-κB and induction of apoptosis is specific to CD4 T cells because HAA does not inhibit NF-κB activation or induce cell death upon Toll-like receptor 4 stimulation in dendritic cells. Thus, HAA is a natural inhibitor that restrains T cell expansion and activation.

The limited efficacy of seasonal influenza vaccines is usually attributed to ongoing variation in the major antigenic targets for protective antibody responses including hemagglutinin (HA) and neuraminidase (NA). Hence, vaccine development has largely focused on broadening antigenic epitopes to generate cross-reactive protection. However, the vaccine adjuvant components which can accelerate, enhance and prolong antigenic immune responses, can also increase the breadth of these responses. We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus HA, inducing rapid, and sustained antibody responses that are protective against influenza viruses in homologous and heterologous murine challenge models. To further enhance adjuvant efficacy, we performed a structure-activity relationship study for the TLR4 ligand, -cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4- ]indol-2-yl)thio)acetamide (C H N O S; ), and identified the 8-(furan-2-yl) substituted pyrimido[5,4- ]indole analog (C H N O S; ) as a derivative with higher potency in activating both human and mouse TLR4-NF-κB reporter cells and primary cells. In a prime-boost immunization model using inactivated influenza A virus [IIAV; A/California/04/2009 (H1N1)pdm09], used as adjuvant induced higher serum anti-HA and anti-NA IgG1 levels compared to , and also increased the anti-NA IgG2a responses. In combination with a TLR7 ligand, , induced equivalent levels of anti-NA and anti-HA IgG1 to . However, the combination of induced 10-fold higher anti-HA and anti-NA IgG2a levels compared to . A stable liposomal formulation of was developed, which synergistically enhanced anti-HA and anti-NA IgG1 and IgG2a responses without demonstrable reactogenicity after intramuscular injection. Notably, vaccination with IIAV plus the liposomal formulation of protected mice against lethal homologous influenza virus (H1N1)pdm09 challenge and reduced lung viral titers and cytokine levels. The combination adjuvant induced a greater diversity in B cell clonotypes of immunoglobulin heavy chain (IGH) genes in the draining lymph nodes and antibodies against a broad spectrum of HA epitopes encompassing HA head and stalk domains and with cross-reactivity against different subtypes of HA and NA. This novel combination liposomal adjuvant contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

Epidemiological evidence points to the inverse relationship between microbial exposure and the prevalence of allergic asthma and autoimmune diseases in Westernized countries. The molecular basis for this observation has not yet been completely delineated. Here we report that the administration of certain toll-like receptor (TLR) ligands, via the activation of innate immunity, induces high levels of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism in various organs. TLR9 ligand–induced pulmonary IDO activity inhibits Th2-driven experimental asthma. IDO activity expressed by resident lung cells rather than by pulmonary DCs suppressed lung inflammation and airway hyperreactivity. Our results provide a mechanistic insight into the various formulations of the hygiene hypothesis and underscore the notion that activation of innate immunity can inhibit adaptive Th cell responses.

Epidemiological evidence points to the inverse relationship between microbial exposure and the prevalence of allergic asthma and autoimmune diseases in Westernized countries. The molecular basis for this observation has not yet been completely delineated. Here we report that the administration of certain toll-like receptor (TLR) ligands, via the activation of innate immunity, induces high levels of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism in various organs. TLR9 ligand-induced pulmonary IDO activity inhibits Th2-driven experimental asthma. IDO activity expressed by resident lung cells rather than by pulmonary DCs suppressed lung inflammation and airway hyperreactivity. Our results provide a mechanistic insight into the various formulations of the hygiene hypothesis and underscore the notion that activation of innate immunity can inhibit adaptive Th cell responses.

Co-delivery of antigens and adjuvants to the same antigen-presenting cells (APCs) can significantly improve the efficacy and safety profiles of vaccines. Here, we report amine-grafted silica nanoparticles (A-SNP) as a tunable vaccine co-delivery platform for TLR7/8 agonists along with the recombinant influenza antigen hemagglutinin H7 (H7) to APCs. A-SNP of two different sizes (50 and 200 nm) were prepared and coated with INI-4001 at different coating densities, followed by co-adsorption of H7. Both INI-4001 and H7 showed >90% adsorption to the tested A-SNP formulations. TNF-α and IFN-α cytokine release by human peripheral blood mononuclear cells as well as TNF-α, IL-6, and IL-12 release by mouse bone marrow-derived dendritic cells revealed that the potency of the INI-4001-adsorbed A-SNP (INI-4001/A-SNP) formulations was improved relative to aqueous formulation control. This improved potency was dependent on particle size and ligand coating density. In addition, slow-release profiles of INI-4001 were measured from INI-4001/A-SNP formulations in plasma with 30–50% INI-4001 released after 7 days. In vivo murine immunization studies demonstrated significantly improved H7-specific humoral and Th1/Th17-polarized T cell immune responses with no observed adverse reactions. Low-density 50 nm INI-4001/A-SNP elicited significantly higher IFN-γ and IL-17 induction over that of the H7 antigen-only group and INI-4001 aqueous formulation controls. In summary, this work introduces an effective and biocompatible SNP-based co-delivery platform that enhances the immunogenicity of TLR7/8 agonist-adjuvanted subunit influenza vaccines.

Extracellular vesicles (EVs) play an important role in intercellular communication and regulation of cells, especially in the immune system where EVs can participate in antigen presentation and may have adjuvant effects. We aimed to identify small molecule compounds that can increase EV release and thereby enhance the immunogenicity of vaccines. We utilized a THP-1 reporter cell line engineered to release EV-associated tetraspanin (CD63)-Turbo-luciferase to quantitatively measure EVs released in culture supernatants as a readout of a high throughput screen (HTS) of 27,895 compounds. In parallel, the cytotoxicity of the compounds was evaluated by PrestoBlue dye assay. For screening immunostimulatory potency, we performed two additional independent HTS on the same compound library using NF-κB and interferon-stimulated response element THP-1 reporter cell lines. Hit compounds were then identified in each of the 3 HTS’s, using a “Top X″ and a Gaussian Mixture Model approach to rule out false positive compounds and to increase the sensitivity of the hit selection. Thus, 644 compounds were selected as hits which were further evaluated for induction of IL-12 in murine bone-marrow derived dendritic cells (mBMDCs) and for effects of cell viability. The resulting 130 hits were then assessed from a medicinal chemistry perspective to remove compounds with functional group liabilities. Finally, 80 compounds were evaluated as vaccine adjuvants in vivo using ovalbumin as a model antigen. We analyzed 18 compounds with adjuvant activity for their ability to induce the expression of co-stimulatory molecules on mBMDCs. The full complement of data was then used to cluster the compounds into 4 distinct biological activity profiles. These compounds were also evaluated for quantitation of EV release and spider plot overlays were generated to compare the activity profiles of compounds within each cluster. This tiered screening process identified two compounds that belong to the 4-thieno-2-thiopyrimidine scaffold with identical screening profiles supporting data reproducibility and validating the overall screening process. Correlation patterns in the adjuvanticity data suggested a role for CD63 and NF-κB pathways in potentiating antigen-specific antibody production. Thus, our three independent cell-based HTS campaigns led to identification of immunostimulatory compounds that release EVs and have adjuvant activity.