Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a ...versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.
Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process ...is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.
Integrin signalling triggers cytoskeletal rearrangements, including endocytosis and exocytosis of integrins and other membrane proteins. In addition to recycling integrins, this trafficking can also ...regulate intracellular signalling pathways. Here we describe a role for αv integrins in regulating Toll-like receptor (TLR) signalling by modulating intracellular trafficking. We show that deletion of αv or β3 causes increased B-cell responses to TLR stimulation in vitro, and αv-conditional knockout mice have elevated antibody responses to TLR-ligand-associated antigens. αv regulates TLR signalling by promoting recruitment of the autophagy component LC3 (microtubule-associated proteins 1 light chain 3) to TLR-containing endosomes, which is essential for progression from NF-κB to IRF signalling, and ultimately for traffic to lysosomes where signalling is terminated. Disruption of LC3 recruitment leads to prolonged NF-κB signalling and increased B-cell proliferation and antibody production. This work identifies a previously unrecognized role for αv and the autophagy components LC3 and atg5 in regulating TLR signalling and B-cell immunity.
Innate immunity is vital for protection from microbes and is mediated by humoral effectors, such as cytokines, and cellular immune defenses, including phagocytic cells (e.g., macrophages). After ...internalization by phagocytes, microbes are delivered into a phagosome, a complex intracellular organelle with a well-established and important role in microbial killing. However, the role of this organelle in cytokine responses and microbial sensing is less well defined. In this study, we assess the role of the phagosome in innate immune sensing and demonstrate the critical interdependence of phagocytosis and pattern recognition receptor signaling during response to the Gram-positive bacteria Staphylococcus aureus. We show that phagocytosis is essential to initiate an optimal MyD88-dependent response to Staphylococcus aureus. Prior to TLR-dependent cytokine production, bacteria must be engulfed and delivered into acidic phagosomes where acid-activated host enzymes digest the internalized bacteria to liberate otherwise cryptic bacterial-derived ligands that initiate responses from the vacuole. Importantly, in macrophages in which phagosome acidification is perturbed, the impaired response to S. aureus can be rescued by the addition of lysostaphin, a bacterial endopeptidase active at neutral pH that can substitute for the acid-activated host enzymes. Together, these observations delineate the interdependence of phagocytosis with pattern recognition receptor signaling and suggest that therapeutics to augment functions and signaling from the vacuole may be useful strategies to increase host responses to S. aureus.
Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases: Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic ...proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines. The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive. Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity. Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen. In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86. However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide. Mutant BMDC had defective activation of NF-κB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact. A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naïve CD4(+) T cells. In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.
An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are ...formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3
h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17
μm to 3
μm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10
μm in diameter.
Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may ...predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.
BackgroundSYNB1891 is a live, modified strain of probiotic E. coli Nissle engineered to produce cyclic dinucleotides under hypoxia leading to stimulator of interferon genes (STING)-activation in ...phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways.MethodsThis first-in-human study (NCT04167137) enrolled patients with refractory advanced solid tumors to receive intratumoral (IT) injections of SYNB1891 monotherapy or in combination atezolizumab. Patients enrolled in the monotherapy arms received doses of 1x106 - 3x108 live cells on Days 1, 8 and 15 of the first 21-day cycle and then on Day 1 of each subsequent cycle. Patients enrolled in the 2 combination cohorts received doses of 1x107 - 3x107 live cells in combination with atezolizumab administered on a 21-day cycle. The primary objective of the study was to evaluate safety and tolerability of SYNB1891 alone and in combination with atezolizumab. Other objectives include SYNB1891 kinetics in blood and injected tumor, STING-target engagement as assessed by IT gene expression and serum cytokines, and tumor responses.ResultsThis interim analysis includes 23 patients across 6 monotherapy cohorts dosed at 1x106, 3x106, 1x107, or 3x107, 1x108 and 3x108 live cells, and 7 patients dosed in 2 combination therapy cohorts (1x107 and 3x107 live cells). The mean (range) age was 61 (25–82); 19 patients were female. There were 4 cytokine release syndrome events in monotherapy cohorts, including one grade 3 event which met the criterion for dose limiting toxicity at 3x108 live cells; there were no other SYNB1891-related serious adverse events. There were no SYNB1891-related infections. SYNB1891 was not detected in the blood at 6 or 24 hours after the first dose or intratumorally 7 days following the first dose. Treatment with SYNB1891 demonstrated activation of the STING pathway and target engagement as assessed by upregulation of interferon-stimulated genes (ISG15, IFIT1, IFIt2), chemokines/cytokines (CXCL9, CXCL10, TNFRS18, TNFSF10) and T-cell response genes (GZMA, CD4, PD-L2) in core biopsies obtained pre-dose and 7 days following the third weekly dose. In addition, there was a dose-response increase in serum cytokines. Durable, stable disease was observed in two patients treated with SYNB1891 monotherapy refractory to prior PD-1/L1 antibodies with vulvar melanoma (1x106 live cells; RECIST -28%) and small cell lung cancer (1x107 live cells; RECIST -12%).ConclusionsRepeat IT injection of SYNB1891 as monotherapy and in combination atezolizumab in this ongoing study is safe and well-tolerated up to at least 1x108 live cells, and shows evidence of STING pathway target engagement.AcknowledgementsWe thank Inessa Vulfova for her clinical support in conduct of this study.Trial Registration clinicaltrials.gov (NCT04167137)Ethics ApprovalThe study protocol, the informed consent form (ICF), and printed subject information materials were reviewed and approved by the institutional review board (IRB) at the investigational site before any study procedures were performed. Written informed consent to participate in the study was obtained from each subject before any study-specific procedures were performed.The Ohio State University Cancer Institutional Review Board; Approval ID: 2020C0194MD Anderson Cancer Center Institutional Review Board; Approval ID: 2019–0576Mary Crawley Medical Research Center Institutional Review Board; Approval ID: 19–31 SYNB1891-CP-001North Texas Institutional Review Board; Approval ID: 2019.040WIRB Approval ID: 20192779University of Pittsburgh Institutional Review Board Approval ID: STUDY20010116
Phagocytes are an important part of host defense, playing a critical role in innate immune responses against pathogens and in the initiation of adaptive immunity. One of the main characteristics of ...these cells is their ability to recognize and internalize invading microorganisms into a phagosome. The internalized microbe is rapidly delivered into a mature phagolysosome where it is killed and degraded. However, numerous pathogens have evolved complex mechanisms to manipulate these intracellular organelles to establish a survival niche. Here, we describe several methods to assess important properties of phagosomes in macrophages, such as phagocytosis, acidification of the phagosome contents during the maturation process, and the ability of phagosomes to inactivate and kill pathogens. Phagocytosis and phagosome acidification assays are FACS-based assays where labeled bacteria are used as probes to monitor internalization into a phagosome and to detect the pH of the phagosome environment. The killing assay is based on the counting of bacterial colonies after recovery of internalized bacteria from macrophages.
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