Co-Delivery of a Novel Lipidated TLR7/8 Agonist and Hemagglutinin-Based Influenza Antigen Using Silica Nanoparticles Promotes Enhanced Immune 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.

Acromioclavicular joint Stabilization: Our experience with the lockdown technique.

Background: Acromioclavicular joint (AC joint) disruption is a common injury with considerable variation with regards to surgical management. The Lockdown™ procedure (previously known as Surgilig™), Modified Weaver-Dunn procedure, Arthroscopic AC joint stabilization and Ligament Augmentation and Reconstruction system (LARS) procedure have all been described for treatment of this injury with varying outcomes. Purpose: To measure the functional and radiological outcomes following all cases of AC joint reconstruction using the Lockdown™ technique over the last 10 years at Medway Maritime Hospital. Methods: Data on a total of 53 patients who underwent AC joint reconstruction between 2012 and 2021 were collected. Electronic records were used to extract data regarding patient characteristics, surgery details, and duration of follow-up. Telephonic interviews of patients were conducted to collect data on hand dominance, surgical complications, and responses to Oxford shoulder score (OSS) and QuickDASH score questionnaires. Hospital PACS data were reviewed to record preoperative injury severity and postoperative acromioclavicular joint reduction. Results: Results for 42 patients were available and analysed as 10 could not be contacted and 1 patient sadly passed away. Of the patients reviewed, 92.9% were males with a mean age of 42.2 years (Range 16-67 years) and mean follow up of 68 months (12-119.1 months). The injury involved the dominant arm in 59.5%cases. Majority of the cases were Rockwood type V injuries (71.43%) while the rest were either type III(19.05%) or type IV(9.52%).The mean preoperative OSS was 21.3/48 which improved to 44.3/48 in the postoperative period. Similarly, the mean QuickDASH score was 50.6 preoperatively, that improved to 9.1 postoperatively.The most common patient reported complication was prominent metalwork seen in 5 cases (11.6%) followed by stiffness seen in 3 cases (7%) and superficial infection seen in 1 case (2.3%). The AC joint remained reduced radiologically in 81% of cases, while a resubluxation between 50 and 100% was seen in the rest of the cases. Patients with radiological resubluxation did not report this as a complication in 87.5% of the cases. Overall, 38 patients reported their outcome as excellent, 2 patients rated it good while 1 patient reported it as fair and 1 as poor. Conclusion: The Lockdown™ technique for stabilization of AC joint has excellent or good patient satisfaction in 95.2% of cases in long term follow up of more than 5.7 years. Prominent metalwork and stiffness are the commonest clinical complications. Radiological resubluxation can be seen in a fifth of the cases but does not directly lead to patient dissatisfaction. Level of evidence: Level IV Retrospective case series.

Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx.

Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca2+ elevation enhances EV release, we screened 80 hit compounds and identified compound 634 as a Ca2+ influx inducer. 634 enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from 634-treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca2+ elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of 634. The analogues that retained the ability to induce Ca2+ influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca2+ influx. The levels of Ca2+ induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, 634, enhances the release of EVs with immunostimulatory potency via induction of Ca2+ influx. This agent is a novel tool for EV-based immune studies and vaccine development.

A Dual Adjuvant System for Intranasal Boosting of Local and Systemic Immunity for Influenza Vaccination.

Systemically vaccinated individuals against COVID-19 and influenza may continue to support viral replication and shedding in the upper airways, contributing to the spread of infections. Thus, a vaccine regimen that enhances mucosal immunity in the respiratory mucosa is needed to prevent a pandemic. Intranasal/pulmonary (IN) vaccines can promote mucosal immunity by promoting IgA secretion at the infection site. Here, we demonstrate that an intramuscular (IM) priming-IN boosting regimen with an inactivated influenza A virus adjuvanted with the liposomal dual TLR4/7 adjuvant (Fos47) enhances systemic and local/mucosal immunity. The IN boosting with Fos47 (IN-Fos47) enhanced antigen-specific IgA secretion in the upper and lower respiratory tracts compared to the IM boosting with Fos47 (IM-Fos47). The secreted IgA induced by IN-Fos47 was also cross-reactive to multiple influenza virus strains. Antigen-specific tissue-resident memory T cells in the lung were increased after IN boosting with Fos47, indicating that IN-Fos47 established tissue-resident T cells. Furthermore, IN-Fos47 induced systemic cross-reactive IgG antibody titers comparable to those of IM-Fos47. Neither local nor systemic reactogenicity or adverse effects were observed after IN delivery of Fos47. Collectively, these results indicate that the IM/IN regimen with Fos47 is safe and provides both local and systemic anti-influenza immune responses.

A Triple High Throughput Screening for Extracellular Vesicle Inducing Agents With Immunostimulatory Activity.

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.

Glyco-Nanoadjuvants: Impact of Linker Length for Conjugating a Synthetic Small-Molecule TLR7 Ligand to Glyco-Nanoparticles on Immunostimulatory Effects.

Immunotherapy has become a powerful clinical strategy for treating infectious diseases and cancer. Synthetic small-molecule toll-like receptor 7 (TLR7) ligands are attractive candidates as immunostimulatory agents for immunotherapy. TLR7 is mainly localized in intracellular endosomal compartments so that the formulation of their small-molecule ligands with macromolecules enhances endocytic uptake of TLR7 ligands and improves the pharmaceutical properties. Previously, we demonstrated that gold nanoparticles co-immobilized with a TLR7 ligand derivative, that is, a conjugate of synthetic small-molecule TLR7 ligand (1V209) and thioctic acid (TA) via 4,7,10-trioxa-1,13-tridecanediamine, and α-mannose (1V209-αMan-GNPs: glyco-nanoadjuvants) significantly enhances immunostimulatory effects. In the present study, we designed a second-generation glyco-nanoadjuvant that possesses a poly(ethylene glycol) (PEG) chain as a spacer between 1V209 and GNPs and investigated the impact of linker length in 1V209 derivatives on the immunostimulatory activities. We used different chain lengths of PEG (n = 3, 5, 11, or 23) as spacers between 1V209 and thioctic acid to prepare four 1V209-αMan-GNPs. In the in vitro study using primary mouse bone-marrow-derived dendritic cells, 1V209-αMan-GNPs that immobilized with longer 1V209 derivatives, especially the 1V209 derivative possessing PEG23 (1V209-PEG23-TA), showed the highest potency toward induction both for interleukin-6 and type I interferon production than those derivatives with shorter PEG chains. Furthermore, 1V209-αMan-GNPs that immobilized with 1V209-PEG23-TA showed significantly higher adjuvant effects for inducing both humoral and cell-mediated immune responses against ovalbumin in the in vivo immunization study. These results indicate that the linker length for immobilizing small-molecule TLR7 ligand on the GNPs significantly affects the adjuvant activity of 1V209-αMan-GNPs and that 1V209-αMan-GNPs immobilized with 1V209-PEG-23-TA could be superior adjuvants for immunotherapies.

Small Molecule Calcium Channel Activator Potentiates Adjuvant Activity.

There remains an unmet need for reliable fully synthetic adjuvants that increase lasting protective immune responses from vaccines. We previously reported a high-throughput screening for small molecules that extended nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) activation after a Toll-like receptor 4 (TLR4) ligand, lipopolysaccharide (LPS), stimulation using a human myeloid reporter cell line. We identified compounds with a conserved aminothiazole scaffold including 2D216 [N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide], which increased murine antigen-specific antibody responses when used as a co-adjuvant with LPS. Here, we examined the mechanism of action in human cells. Although 2D216 activated the major mitogen-activated protein kinases, it did not interact with common kinases and phosphatases and did not stimulate many of the pattern recognition receptors (PRRs). Instead, the mechanism of action was linked to intracellular Ca2+ elevation via Ca2+ channel(s) at the plasma membrane and nuclear translocation of the nuclear factor of activated T-cells (NFAT) as supported by RNA-seq data, analysis by reporter cells, Ca2+ flux assays, and immunoblots. Interestingly, 2D216 had minimal, if any, activity on Jurkat T cells but induced cytokine production and surface expression of costimulatory molecules on cells with antigen-presenting functions. A small series of analogs of 2D216 were tested for the ability to enhance a TLR4 ligand-stimulated autologous mixed lymphocyte reaction (MLR). In the MLR, 2E151, N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide, was more potent than 2D216. These results indicate that a small molecule that is not a direct PRR agonist can act as a co-adjuvant to an approved adjuvant to enhance human immune responses via a complementary mechanism of action.

Small Molecule Potentiator of Adjuvant Activity Enhancing Survival to Influenza Viral Challenge.

As viruses continue to mutate the need for rapid high titer neutralizing antibody responses has been highlighted. To meet these emerging threats, agents that enhance vaccine adjuvant activity are needed that are safe with minimal local or systemic side effects. To respond to this demand, we sought small molecules that would sustain and improve the protective effect of a currently approved adjuvant, monophosphoryl lipid A (MPLA), a Toll-like receptor 4 (TLR4) agonist. A lead molecule from a high-throughput screen, (N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide, was identified as a hit compound that sustained NF-κB activation by a TLR4 ligand, lipopolysaccharide (LPS), after an extended incubation (16 h). In vitro, the resynthesized compound (2D216) enhanced TLR4 ligand-induced innate immune activation and antigen presenting function in primary murine bone marrow-derived dendritic cells without direct activation of T cells. In vivo murine vaccination studies demonstrated that compound 2D216 acted as a potent co-adjuvant when used in combination with MPLA that enhanced antigen-specific IgG equivalent to that of AS01B. The combination adjuvant MPLA/2D216 produced Th1 dominant immune responses and importantly protected mice from lethal influenza virus challenge. 2D216 alone or 2D216/MPLA demonstrated minimal local reactogenicity and no systemic inflammatory response. In summary, 2D216 augmented the beneficial protective immune responses of MPLA as a co-adjuvant and showed an excellent safety profile.

Structure-activity relationship studies in substituted sulfamoyl benzamidothiazoles that prolong NF-κB activation.

In the face of emerging infectious diseases, there remains an unmet need for vaccine development where adjuvants that enhance immune responses to pathogenic antigens are highly desired. Using high-throughput screens with a cell-based nuclear factor κB (NF-κB) reporter assay, we identified a sulfamoyl benzamidothiazole bearing compound 1 that demonstrated a sustained activation of NF-κB after a primary stimulus with a Toll-like receptor (TLR)-4 agonist, lipopolysaccharide (LPS). Here, we explore systematic structure-activity relationship (SAR) studies on compound 1 that indicated the sites on the scaffold that tolerated modification and yielded more potent compounds compared to 1. The selected analogs enhanced release of immunostimulatory cytokines in the human monocytic cell line THP-1 cells and murine primary dendritic cells. In murine vaccination studies, select compounds were used as co-adjuvants in combination with the Food and Drug Administration approved TLR-4 agonistic adjuvant, monophosphoryl lipid A (MPLA) that showed significant enhancement in antigen-specific antibody titers compared to MPLA alone. Additionally, our SAR studies led to identification of a photoaffinity probe which will aid the target identification and mechanism of action studies in the future.

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection.

Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors-such as aging, pregnancy, and genetic polymorphisms-influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure-activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.

Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release.

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.

Glyco-Nanoadjuvants: Sugar Structures on Carriers of a Small Molecule TLR7 Ligand Affect Their Immunostimulatory Activities.

Toll-like receptors (TLRs) are pattern recognition receptors that activate innate immunity, and their ligands are promising adjuvants for vaccines and immunotherapies. Small molecule TLR7 ligands are ideal vaccine adjuvants as they induce not only proinflammatory cytokines but also type I interferons. However, their application has only been approved for local administration due to severe systemic immune-related adverse events. In a previous study, we prepared the gold nanoparticles coimmobilized with synthetic small molecule TLR7 ligand, 1V209, and α-mannose (1V209-αMan-GNPs). 1V209-αMan-GNPs were selectively delivered via a cell surface sugar-binding protein, mannose receptor, which enabled selective delivery of TLR7 ligands to immune cells. Besides the mannose receptor, immune cells express various sugar-binding proteins such as macrophage galactose binding lectins and sialic acid-binding immunoglobulin-type lectins and recognize distinct sugar structures. Hence, in the present study, we investigated whether sugar structures on GNPs affect the efficiency and selectivity of intracellular delivery and subsequent immunostimulatory potencies. Five neutral sugars and two sialosides were selected and each sugar was coimmobilized with 1V209 onto GNPs (1V209-SGNPs) and their innate immunostimulatory potencies were compared to that of 1V209-αMan-GNPs. The in vitro study using mouse bone marrow derived dendritic cells (BMDCs) demonstrated that α-glucose, α-N-acetylglucosamine, or α-fucose immobilized 1V209-SGNPs increased interleukin-6 and type I interferon release similar to that of 1V209-αMan-GNPs, whereas galacto-type sugar immobilized 1V209-SGNPs predominantly enhanced type I interferon release. In contrast, sialoside immobilized 1V209-SGNPs did not enhance the potency of 1V209. In the in vivo immunization study using ovalbumin as a model antigen, neutral sugar immobilized 1V209-SGNPs induced comparable T helper-1 immune response to that of 1V209-αMan-GNPs and by 10-fold higher than that of sialoside immobilized 1V209-SGNPs. These results indicate that the sugar structures on 1V209-SGNPs affect their immunostimulatory activities, and functionalization of the carrier particles is important to shape immune responses.

A Novel Synthetic Dual Agonistic Liposomal TLR4/7 Adjuvant Promotes Broad Immune Responses in an Influenza Vaccine With Minimal Reactogenicity.

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, N-cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)thio)acetamide (C25H26N4O2S; 1Z105), and identified the 8-(furan-2-yl) substituted pyrimido[5,4-b]indole analog (C29H28N4O3S; 2B182C) 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], 2B182C used as adjuvant induced higher serum anti-HA and anti-NA IgG1 levels compared to 1Z105, and also increased the anti-NA IgG2a responses. In combination with a TLR7 ligand, 1V270, 2B182C induced equivalent levels of anti-NA and anti-HA IgG1 to 1V270+1Z105. However, the combination of 1V270+2B182C induced 10-fold higher anti-HA and anti-NA IgG2a levels compared to 1V270+1Z105. A stable liposomal formulation of 1V270+2B182C 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 1V270+2B182C 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.

Identification of Compounds With Glucocorticoid Sparing Effects on Suppression of Chemokine and Cytokine Production by Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

In recent years target based drug discovery has expanded our therapeutic armamentarium in the treatment of inflammatory and autoimmune diseases. Despite these advances and adverse effects, glucocorticoids remain reliable agents that are used in many of these diseases. The anti-inflammatory mechanisms of glucocorticoids include the suppression of transcription factor activity like nuclear factor kappa B (NF-κB). By reanalyzing data from two prior high throughput screens (HTS) that utilized a NF-κB reporter construct in THP-1 cells, we identified 1824 small molecule synthetic compounds that demonstrated NF-κB suppressive activities similar to the glucocorticoids included in the original >134,000 compound libraries. These 1824 compounds were then rescreened for attenuating NF-κB activity at 5 and 16 h after LPS stimuli in the NF-κB THP-1 reporter cells. After a "Top X" selection approach 122 hit compounds were further tested for toxicity and suppression of LPS induced CXCL8 release in THP-1 cells. Excluding cytotoxic compounds, the remaining active compounds were grouped into chemotype families using Tanimoto based clustering. Promising representatives from clustered chemotype groups were commercially purchased for further testing. Amongst these index compounds a lead chemotype: 1H-pyrazolo [3,4 d] pyrimidin-4-amine, effectively suppressed CXCL8, and TNF production by THP-1 cells when stimulated with LPS, TNF or IL-1ß. Extending these studies to primary cells, these lead compounds also reduced IL-6 and CXCL8 production by TNF stimulated fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. Importantly a lead 1H-pyrazolo [3,4 d] pyrimidin-4-amine compound demonstrated synergistic effects with dexamethasone when co-administered to TNF stimulated THP-1 cells and RA FLS in suppressing chemokine production. In summary, a cell based HTS approach identified lead compounds that reduced NF-κB activity and chemokine secretion induced by potent immunologic stimuli, and one lead compound that acted synergistically with dexamethasone as an anti-inflammatory agent showing a dose-sparing effect.

Synthesis and immunostimulatory activity of sugar-conjugated TLR7 ligands.

Toll-like receptors (TLRs) are a type of pattern recognition receptors (PRRs), which are activated by recognizing pathogen-associated molecular patterns (PAMPs). The activation of TLRs initiates innate immune responses and subsequently leads to adaptive immune responses. TLR agonists are effective immuomodulators in vaccine adjuvants for infectious diseases and cancer immunotherapy. In exploring hydrophilic small molecules of TLR7 ligands using the cell-targeted property of a vaccine adjuvant, we conjugated 1V209, a small TLR7 ligand molecule, with various low or middle molecular weight sugar molecules that work as carriers. The sugar-conjugated 1V209 derivatives showed increased water solubility and higher immunostimulatory activity in both mouse and human cells compared to unmodified 1V209. The improved immunostimulatory potency of sugar-conjugates was attenuated by an inhibitor of endocytic process, cytochalasin D, suggesting that conjugation of sugar moieties may enhance the uptake of TLR7 ligand into the endosomal compartment. Collectively our results support that sugar-conjugated TLR7 ligands are applicable to novel drugs for cancer and vaccine therapy.

Structure-Activity Relationship Studies To Identify Affinity Probes in Bis-aryl Sulfonamides That Prolong Immune Stimuli.

Agents that safely induce, enhance, or sustain multiple innate immune signaling pathways could be developed as potent vaccine adjuvants or coadjuvants. Using high-throughput screens with cell-based nuclear factor κB (NF-κB) and interferon stimulating response element (ISRE) reporter assays, we identified a bis-aryl sulfonamide bearing compound 1 that demonstrated sustained NF-κB and ISRE activation after a primary stimulus with lipopolysaccharide or interferon-α, respectively. Here, we present systematic structure-activity relationship (SAR) studies on the two phenyl rings and amide nitrogen of the sulfonamide group of compound 1 focused toward identification of affinity probes. The murine vaccination studies showed that compounds 1 and 33 when used as coadjuvants with monophosphoryl lipid A (MPLA) showed significant enhancement in antigen ovalbumin-specific immunoglobulin responses compared to MPLA alone. SAR studies pointed to the sites on the scaffold that can tolerate the introduction of aryl azide, biotin, and fluorescent rhodamine substituents to obtain several affinity and photoaffinity probes which will be utilized in concert for future target identification and mechanism of action studies.

Gold Nanoparticles Coimmobilized with Small Molecule Toll-Like Receptor 7 Ligand and α-Mannose as Adjuvants.

Adjuvants enhance the immune response during vaccination. Among FDA-approved adjuvants, aluminum salts are most commonly used in vaccines. Although aluminum salts enhance humoral immunity, they show a limited effect for cell-mediated immune responses. Thus, further development of adjuvants that induce T-cell-mediated immune response is needed. Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns activate innate immunity, which is crucial to shape adaptive immunity. Using TLR ligands as novel adjuvants in vaccines has therefore attracted substantial attention. Among them a small molecule TLR7 ligand, imiquimod, has been approved for clinical use, but its use is restricted to local administration due to unwanted adverse side effects when used systematically. Since TLR7 is mainly located in the endosomal compartment of immune cells, efficient transport of the ligand into the cells is important for improving the potency of the TLR7 ligand. In this study we examined gold nanoparticles (GNPs) immobilized with α-mannose as carriers for a TLR7 ligand to target immune cells. The small molecule synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine (1V209), and α-mannose were coimmobilized via linker molecules consisting of thioctic acid on the GNP surface (1V209-αMan-GNPs). The in vitro cytokine production activity of 1V209-αMan-GNPs was higher than that of the unconjugated 1V209 derivative in mouse bone marrow-derived dendritic cells and in human peripheral blood mononuclear cells. In the in vivo immunization study, 1V209-αMan-GNPs induced significantly higher titers of IgG2c antibody specific to ovalbumin as an antigen than did unconjugated 1V209, and splenomegaly and weight loss were not observed. These results indicate that 1V209-αMan-GNPs could be useful as safe and effective adjuvants for development of vaccines against infectious diseases and cancer.

Discovery of a Novel Microtubule Targeting Agent as an Adjuvant for Cancer Immunotherapy.

For an activating immunotherapy such as adjuvants, a compound that can prolong immune stimulation may enhance efficacy. We leveraged data from two prior high throughput screens with NF-κB and interferon reporter cell lines to identify 4H-chromene-3-carbonitriles as a class of compounds that prolonged activation in both screens. We repurchased 23 of the most promising candidates. Out of these compounds we found #1 to be the most effective agent in stimulating the release of cytokines and chemokines from immune cells, including murine primary bone marrow derived dendritic cells. Mechanistically, #1 inhibited tubulin polymerization, and its effect on immune cell activation was abolished in cells mutated in the beta-tubulin gene (TUBB) encoding the site where colchicine binds. Treatment with #1 resulted in mitochondrial depolarization followed by mitogen-activated protein kinase activation. Because tubulin polymerization modulating agents have been used for chemotherapy to treat malignancy and #1 activated cytokine responses, we hypothesized that #1 could be effective for cancer immunotherapy. Intratumoral injection of #1 delayed tumor growth in a murine syngeneic model of head and neck cancer. When combined with PD-1 blockade, tumor growth slowed in the injected tumor nodule and there was an abscopal effect in an uninjected nodule on the contralateral flank, suggesting central antitumor immune activation. Thus, we identified a new class of tubulin depolymerizing agent that acts as both an innate and an adaptive immune activating agent and that limits solid tumor growth when used concurrently with a checkpoint inhibitor.

Identification of Compounds That Prolong Type I Interferon Signaling as Potential Vaccine Adjuvants.

Vaccines are reliant on adjuvants to enhance the immune stimulus, and type I interferons (IFNs) have been shown to be beneficial in augmenting this response. We were interested in identifying compounds that would sustain activation of an endogenous type I IFN response as a co-adjuvant. We began with generation of a human monocytic THP-1 cell line with an IFN-stimulated response element (ISRE)-ß-lactamase reporter construct for high-throughput screening. Pilot studies were performed to optimize the parameters and conditions for this cell-based Förster resonance energy transfer (FRET) reporter assay for sustaining an IFN-α-induced ISRE activation signal. These conditions were confirmed in an initial pilot screen, followed by the main screen for evaluating prolongation of an IFN-α-induced ISRE activation signal at 16 h. Hit compounds were identified using a structure enrichment strategy based on chemoinformatic clustering and a naïve "Top X" approach. A select list of confirmed hits was then evaluated for toxicity and the ability to sustain IFN activity by gene and protein expression. Finally, for proof of concept, a panel of compounds was used to immunize mice as co-adjuvant with a model antigen and an IFN-inducing Toll-like receptor 4 agonist, lipopolysaccharide, as an adjuvant. Selected compounds significantly augmented antigen-specific immunoglobulin responses.

Recent Advances and Perspectives in Small-molecule TLR Ligands and Their Modulators.

Activation of Toll-like receptors (TLRs) located on immune cells leads to induction of immune responses that can be useful in vaccines for infectious diseases, cancer immunotherapy, and autoimmune diseases. Novel TLR signaling pathway modulators can further enhance the efficacy of TLR ligands.