From hit to vial: Precision discovery and development of an imidazopyrimidine TLR7/8 agonist adjuvant formulation.

Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)-activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037's in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.

TAK1 is essential for endothelial barrier maintenance and repair after lung vascular injury.

TGF-ß-activated kinase 1 (TAK1) plays crucial roles in innate and adaptive immune responses and is required for embryonic vascular development. However, TAK1's role in regulating vascular barrier integrity is not well defined. Here we show that endothelial TAK1 kinase function is required to maintain and repair the injured lung endothelial barrier. We observed that inhibition of TAK1 with 5Z-7-oxozeaenol markedly reduced expression of ß-catenin (ß-cat) and VE-cadherin at endothelial adherens junctions and augmented protease-activated receptor-1 (PAR-1)- or toll-like receptor-4 (TLR-4)-induced increases in lung vascular permeability. In inducible endothelial cell (EC)-restricted TAK1 knockout (TAK1i∆EC) mice, we observed that the lung endothelial barrier was compromised and in addition, TAK1i∆EC mice exhibited heightened sensitivity to septic shock. Consistent with these findings, we observed dramatically reduced ß-cat expression in lung ECs of TAK1i∆EC mice. Further, either inhibition or knockdown of TAK1 blocked PAR-1- or TLR-4-induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which in turn increased phosphorylation, ubiquitylation, and degradation of ß-cat in ECs to destabilize the endothelial barrier. Importantly, we showed that TAK1 inactivates GSK3ß through AKT activation in ECs. Thus our findings in this study point to the potential of targeting the TAK1-AKT-GSK3ß axis as a therapeutic approach to treat uncontrolled lung vascular leak during sepsis.

Towards Precision Vaccines: Lessons From the Second International Precision Vaccines Conference.

Other than clean drinking water, vaccines have been the most effective public health intervention in human history, yet their full potential is still untapped. To date, vaccine development has been largely limited to empirical approaches focused on infectious diseases and has targeted entire populations, potentially disregarding distinct immunity in vulnerable populations such as infants, elders, and the immunocompromised. Over the past few decades innovations in genetic engineering, adjuvant discovery, formulation science, and systems biology have fueled rapid advances in vaccine research poised to consider demographic factors (e.g., age, sex, genetics, and epigenetics) in vaccine discovery and development. Current efforts are focused on leveraging novel approaches to vaccine discovery and development to optimize vaccinal antigen and, as needed, adjuvant systems to enhance vaccine immunogenicity while maintaining safety. These approaches are ushering in an era of precision vaccinology aimed at tailoring immunization for vulnerable populations with distinct immunity. To foster collaboration among leading vaccinologists, government, policy makers, industry partners, and funders from around the world, the Precision Vaccines Program at Boston Children's Hospital hosted the 2nd International Precision Vaccines Conference (IPVC) at Harvard Medical School on the 17th-18th October 2019. The conference convened experts in vaccinology, including vaccine formulation and adjuvantation, immunology, cell signaling, systems biology, biostatistics, bioinformatics, as well as vaccines for non-infectious indications such as cancer and opioid use disorder. Herein we review highlights from the 2nd IPVC and discuss key concepts in the field of precision vaccines.

Neutrophil Activation of Endothelial Cell-Expressed TRPM2 Mediates Transendothelial Neutrophil Migration and Vascular Injury.

RATIONALE: TRPM2 (transient receptor potential melastatin-2) expressed in endothelial cells (ECs) is a cation channel mediating Ca2+ entry in response to intracellular generation of adenosine diphosphoribose-the TRPM2 ligand. OBJECTIVE: Because polymorphonuclear neutrophils (PMN) interaction with ECs generates reactive oxygen species, we addressed the possible role of TRPM2 expressed in ECs in the mechanism of transendothelial migration of PMNs. METHODS AND RESULTS: We observed defective PMN transmigration in response to lipopolysaccharide challenge in adult mice in which the EC expressed TRPM2 is conditionally deleted (Trpm2iΔEC ). PMN interaction with ECs induced the entry of Ca2+ in ECs via the EC-expressed TRPM2. Prevention of generation of adenosine diphosphoribose in ECs significantly reduced Ca2+ entry in response to PMN activation of TRPM2 in ECs. PMNs isolated from gp91phox-/- mice significantly reduced Ca2+ entry in ECs via TRPM2 as compared with wild-type PMNs and failed to induce PMN transmigration. Overexpression of the adenosine diphosphoribose insensitive TRPM2 mutant channel (C1008→A) in ECs suppressed the Ca2+ entry response. Further, the forced expression of TRPM2 mutant channel (C1008→A) or silencing of poly ADP-ribose polymerase in ECs of mice prevented PMN transmigration. CONCLUSIONS: Thus, endotoxin-induced transmigration of PMNs was secondary to TRPM2-activated Ca2+ signaling and VE-cadherin phosphorylation resulting in the disassembly of adherens junctions and opening of the paracellular pathways. These results suggest blocking TRPM2 activation in ECs is a potentially important means of therapeutically modifying PMN-mediated vascular inflammation.

Pyk2 phosphorylation of VE-PTP downstream of STIM1-induced Ca2+ entry regulates disassembly of adherens junctions.

Vascular endothelial protein tyrosine phosphatase (VE-PTP) stabilizes endothelial adherens junctions (AJs) through constitutive dephosphorylation of VE-cadherin. Here we investigated the role of stromal interaction molecule 1 (STIM1) activation of store-operated Ca2+ entry (SOCE) in regulating AJ assembly. We observed that SOCE induced by STIM1 activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. Pyk2-induced tyrosine phosphorylation of VE-PTP promoted Src binding to VE-PTP, Src activation, and subsequent VE-cadherin phosphorylation and thereby increased the endothelial permeability response. The increase in permeability was secondary to disassembly of AJs. Pyk2-mediated responses were blocked in EC-restricted Stim1 knockout mice, indicating the requirement for STIM1 in initiating the signaling cascade. A peptide derived from the Pyk2 phosphorylation site on VE-PTP abolished the STIM1/SOCE-activated permeability response. Thus Pyk2 activation secondary to STIM1-induced SOCE causes tyrosine phosphorylation of VE-PTP, and VE-PTP, in turn, binds to and activates Src, thereby phosphorylating VE-cadherin to increase endothelial permeability through disassembly of AJs. Our results thus identify a novel signaling mechanism by which STIM1-induced Ca2+ signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin and hence increase endothelial permeability. Therefore, targeting the Pyk2 activation pathway may be a potentially important anti-inflammatory strategy.

TNFα-stimulated gene-6 (TSG6) activates macrophage phenotype transition to prevent inflammatory lung injury.

TNFα-stimulated gene-6 (TSG6), a 30-kDa protein generated by activated macrophages, modulates inflammation; however, its mechanism of action and role in the activation of macrophages are not fully understood. Here we observed markedly augmented LPS-induced inflammatory lung injury and mortality in TSG6-/- mice compared with WT (TSG6+/+) mice. Treatment of mice with intratracheal instillation of TSG6 prevented LPS-induced lung injury and neutrophil sequestration, and increased survival in mice. We found that TSG6 inhibited the association of TLR4 with MyD88, thereby suppressing NF-κB activation. TSG6 also prevented the expression of proinflammatory proteins (iNOS, IL-6, TNFα, IL-1ß, and CXCL1) while increasing the expression of anti-inflammatory proteins (CD206, Chi3l3, IL-4, and IL-10) in macrophages. This shift was associated with suppressed activation of proinflammatory transcription factors STAT1 and STAT3. In addition, we observed that LPS itself up-regulated the expression of TSG6 in TSG6+/+ mice, suggesting an autocrine role for TSG6 in transitioning macrophages. Thus, TSG6 functions by converting macrophages from a proinflammatory to an anti-inflammatory phenotype secondary to suppression of TLR4/NF-κB signaling and STAT1 and STAT3 activation.

Cooperative signaling via transcription factors NF-κB and AP1/c-Fos mediates endothelial cell STIM1 expression and hyperpermeability in response to endotoxin.

Stromal interacting molecule 1 (STIM1) regulates store-operated Ca(2+) entry (SOCE). Here we show that STIM1 expression in endothelial cells (ECs) is increased during sepsis and, therefore, contributes to hyperpermeability. LPS induced STIM1 mRNA and protein expression in human and mouse lung ECs. The induced STIM1 expression was associated with augmented SOCE as well as a permeability increase in both in vitro and in vivo models. Because activation of both the NF-κB and p38 MAPK signaling pathways downstream of TLR4 amplifies vascular inflammation, we studied the influence of these two pathways on LPS-induced STIM1 expression. Inhibition of either NF-κB or p38 MAPK activation by pharmacological agents prevented LPS-induced STIM1 expression. Silencing of the NF-κB proteins (p65/RelA or p50/NF-κB1) or the p38 MAPK isoform p38α prevented LPS-induced STIM1 expression and increased SOCE in ECs. In support of these findings, we found NF-κB and AP1 binding sites in the 5'-regulatory region of human and mouse STIM1 genes. Further, we demonstrated that LPS induced time-dependent binding of the transcription factors NF-κB (p65/RelA) and AP1 (c-Fos/c-Jun) to the STIM1 promoter. Interestingly, silencing of c-Fos, but not c-Jun, markedly reduced LPS-induced STIM1 expression in ECs. We also observed that silencing of p38α prevented c-Fos expression in response to LPS in ECs, suggesting that p38α signaling mediates the expression of c-Fos. These results support the proposal that cooperative signaling of both NF-κB and AP1 (via p38α) amplifies STIM1 expression in ECs and, thereby, contributes to the lung vascular hyperpermeability response during sepsis.

The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation.

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.