TLR Agonists Research Articles

Neoadjuvant vidutolimod and nivolumab in high-risk resectable melanoma: A prospective phase II trial

Intratumoral TLR9 agonists and anti-PD-1 produce clinical responses and broad immune activation. We conducted a single-arm study of neoadjuvant TLR9 agonist vidutolimod combined with anti-PD-1 nivolumab in high-risk resectable melanoma. In 31 evaluable patients, 55% major pathologic response(MPR) was observed, meeting primary endpoint. MPR was associated with necrosis, and melanophagocytosis with increased CD8+ tumor-infiltrating lymphocytes and plasmacytoid dendritic cells (pDCs) in the tumor microenvironment, and increased frequencies of Ki67+CD8+ Tcells peripherally. MPRs had an enriched pre-treatment gene signature of myeloid cells, and response to therapy was associated with gene signatures of immune cells, pDCs, phagocytosis, and macrophage activation. MPRs gut microbiota were enriched for Gram-negative bacteria belonging to the Bacteroidaceae and Enterobacteriaceae families and the small subgroup of Gram-negative Firmicutes. Our findings support that combined vidutolimod and nivolumab stimulates a broad anti-tumor immune response and is associated with distinct baseline myeloid gene signature and gut microbiota. ClinicalTrials.gov identifier: NCT03618641.

Intratumoral vidutolimod in combination with PD-1 blockade in locoregionally advanced melanoma

Combinatorial immunotherapy may improve the efficacy of neoadjuvant checkpoint inhibitors in locoregionally advanced melanoma. In this issue of Cancer Cell, Davar and colleagues report a promising phase 2 neoadjuvant trial of the TLR9 agonist vidutolimod in combination with nivolumab. Analyses suggest a unique myeloid expression signature is associated with response.

Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages

Abstract Objectives M2-polarized macrophages and interleukin (IL)-6 significantly alter the tumor microenvironment and promote the malignant behaviors of tumor cells. This study aimed to establish M2-type macrophages from THP-1 cells, which are human leukemia monocytes, and investigate the significance of toll-like receptor 2 (TLR2) signaling in IL-6 production. Methods THP-1 cells were treated with phorbol 12-myristate 13-acetate, IL-4, and IL-13 to stimulate their differentiation into M2 macrophages. Cell differentiation was confirmed by cytokine production, marker expression, and morphological alterations. Treatment with TLR agonists induced TLR stimulation in M2 macrophages. Subsequently, secretion and expression levels of IL-6 in M2 macrophages were measured using enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, and western blotting. Results Myeloid differentiation factor 88, tumor necrosis factor-associated factor 6, and IL-1 receptor-associated kinase-1/4 signaling pathways contributed to IL-6 production upon TLR2 activation in M2 macrophages. While both TLR2 and TLR4 activated NF-κB in M2 macrophages, IL-6 production was mainly dependent on TLR2, not TLR4, suggesting the involvement of major mechanisms other than NF-κB in IL-6 production. Notably, TLR2-stimulated extracellular signal-regulated kinase (ERK) was necessary for abundant IL-6 production, indicating that TLR2-mediated ERK signaling plays an essential role in M2 macrophages. Conclusions These results highlight the significance of TLR2 signaling in IL-6 production by M2 macrophages and provide insights into the underlying regulatory mechanisms.

Toll-Like Receptor 2 Attenuates the Formation and Progression of Angiotensin II-Induced Abdominal Aortic Aneurysm in ApoE−/− Mice

Introduction: We demonstrated Toll-like receptor (TLR) 4 in the pathogenesis of angiotensin II (AngII)-mediated abdominal aortic aneurysm (AAA) formation. Here, we study TLR2 in the AAA formation. Methods: Male ApoE−/− and ApoE−/−TLR2−/− mice were treated with AngII. Mice were injected with the TLR2 agonist Pam3CSK4. The incidence and severity of AAA were determined. MCP-1, MCP-5, RANTES, CXCL10, CCR5, and CXCR3 were analyzed. M1 and M2 macrophages in the aorta were detected by flow cytometry. Results: These studies demonstrated an increase in AAA formation in TLR2−/− mice and a decrease by Pam3CSK4. Pam3CSK4 decreased the ratio of M1/M2 and the levels of RANTES, CXCL10, CCR5, and CXCR3. Furthermore, Pam3CSK4 treatment 1 week following AngII retarded the progression of AAA. Conclusion: These data demonstrated a protective effect of TLR2 signaling on AAA in association with a decrease in the ratio of M1 to M2 macrophages and the expression of chemokines and their receptors. Furthermore, the treatment of Pam3CSK4 after AngII demonstrated a marked retardation of lesion progression. Given the fact that most AAA patients are detected late in the disease process, these findings suggest that TLR2 stimulation may play a therapeutic role in retarding disease progression.

A TLR7 Agonist Conjugated to a Nanofibrous Peptide Hydrogel as a Potent Vaccine Adjuvant.

Toll-like receptors (TLRs) recognize pathogen- and damage-associated molecular patterns and, in turn, trigger the release of cytokines and other immunostimulatory molecules. As a result, TLR agonists are increasingly being investigated as vaccine adjuvants. Many of these agonists are small molecules that quickly diffuse away from the vaccination site, limiting their co-localization with antigens and, thus, their effect. Here, the small-molecule TLR7 agonist 1V209 is conjugated to a positively-charged multidomain peptide (MDP) hydrogel, K2, which was previously shown to act as an adjuvant promoting humoral immunity. Mixing the 1V209-conjugated K2 50:50 with the unfunctionalized K2 produces hydrogels that retain the shear-thinning and self-healing physical properties of the original MDP while improving the solubility of 1V209 more than 200-fold compared to the unconjugated molecule. When co-delivered with ovalbumin as a model antigen, 1V209-functionalized K2 produces a robust Th2 immune response and an antigen-specific Th1 immune response superior to alum, a widely used vaccine adjuvant. Together, these results suggest that K2 MDP hydrogels functionalized with 1V209 are a promising adjuvant for vaccines against infectious diseases, especially those benefiting from a combined Th1 and Th2 immune response.

Long-term Immunity of a Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Irradiated by Gamma Rays in Mice.

COVID-19 vaccines effectively prevent symptomatic infection and severe disease, including hospitalization and death. However, unequal vaccine distribution during the pandemic, especially in low- and middle-income countries, has led to the emergence of vaccine-resistant strains. This underscores the need for alternative, safe, and thermostable vaccine platforms, such as dissolved microneedle array patches (MAP) delivering a subunit vaccine, which eliminate the need for cold chain and trained healthcare personnel. This study demonstrates that the SARS-CoV-2 S1 monomer with RS09, a TLR-4 agonist peptide, serves as an optimal protein subunit immunogen. This combination stimulates a stronger S1-specific immune response, resulting in binding to the membrane-bound spike on the cell surface and ACE2-binding inhibition, compared to the monomer S1 alone or trimer S1, regardless of RS09. MAP delivery of the rS1RS09 subunit vaccine elicited higher and longer-lasting immunity compared to conventional intramuscular injection. S1-specific IgG levels remained significantly elevated for up to 70 weeks post-administration. Additionally, different doses of 5, 15, and 45 μg of MAP vaccines induced robust and sustained Th2-prevalent immune responses, suggesting a dose-sparing effect and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants at 15 and 45 μ g dose. Moreover, gamma irradiation as a terminal sterilization method did not significantly affect immunogenicity, with irradiated vaccines maintaining comparable efficacy to non-irradiated ones. The stability of MAP vaccines was evaluated after long-term storage at room temperature and refrigeration for 19 months, showing minimal protein degradation. Further, after an additional one-month of storage at elevated temperature (42°C), rS1RS09 in both non-irradiated and irradiated MAP degraded less than 3%, while the liquid subunit vaccine degraded over 23%. Overall, these results indicate that gamma irradiation sterilized MAP-rS1RS09 vaccines maintain stability during extended storage without refrigeration, supporting their potential for mass production and widespread use in global vaccination efforts.

WIF-1 contributes to lupus-induced neuropsychological deficits via the CRYAB/STAT4-SHH axis

BackgroundNeuropsychiatric systemic lupus erythematosus (NPSLE) often manifests as cognitive deterioration, with activated microglia and blood-brain barrier (BBB) disruption implicated in these neurological complications. Wnt-inhibitory factor-1 (WIF-1), a secreted protein, has been detected in the cerebrospinal fluid (CSF) of NPSLE patients. However, the contribution of WIF-1 in contributing to lupus cognitive impairment remains poorly understood.MethodsUsing MRL/MpJ-Faslpr (MRL/lpr) lupus-prone mice and TLR7 agonist imiquimod (IMQ)-induced lupus mice, recombinant WIF-1 protein (rWIF-1) and adeno-associated virus (AAV) encoding sh-WIF-1 were administered via intracerebroventricular injection. Behavioral tests, histopathological examinations, flow cytometry, and molecular biology techniques were employed to investigate the underlying mechanisms.ResultsMicroinjection of rWIF-1 exacerbated cognitive deficits and mood abnormalities, increased BBB leakage and neuronal degeneration, and caused aberrant activation of microglia and synaptic pruning in the hippocampus. Conversely, lupus mice injected with AAV-shWIF-1 exhibited significant remission. In vitro, rWIF-1 induced overactivation of microglia with an increased CD86+ pro-inflammatory subpopulation, upregulated phagocytic activity, and excessive synaptic engulfment, contributing to increased BBB permeability. Furthermore, WIF-1 exerted its biological effects through the CRYAB/STAT4 pathway, transcriptionally decreasing SHH production. We also identified that symmetric dimethylarginine (SDMA) could alleviate rWIF-1-induced microglial activation and BBB damage, thereby restoring SHH levels.ConclusionsIn conclusion, WIF-1 exacerbates lupus-induced cognitive dysfunction in mice by triggering aberrant microglial activation and BBB disruption through the CRYAB/STAT4-SHH axis, highlighting the potential therapeutic effects of SDMA for the treatment of NPSLE.

MINCLE and TLR9 agonists synergize to induce Th1/Th17 vaccine memory and mucosal recall in mice and non-human primates

Development of new vaccines tailored for difficult-to-target diseases is hampered by a lack of diverse adjuvants for human use, and none of the currently available adjuvants induce Th17 cells. Here, we develop a liposomal adjuvant, CAF®10b, that incorporates Mincle and Toll-like receptor 9 agonists. In parallel mouse and non-human primate studies comparing to CAF® adjuvants already in clinical trials, we report species-specific effects of adjuvant composition on the quality and magnitude of the responses. When combined with antigen, CAF®10b induces Th1 and Th17 responses and protection against a pulmonary infection with Mycobacterium tuberculosis in mice. In non-human primates, CAF®10b induces higher Th1 responses and robust Th17 responses detectable after six months, and systemic and pulmonary Th1 and Th17 recall responses, in a sterile model of local recall. Overall, CAF®10b drives robust memory antibody, Th1 and Th17 vaccine-responses via a non-mucosal immunization route across both rodent and primate species.

Differences in Susceptibility to SARS-CoV-2 Infection Among Transgenic hACE2-Hamster Founder Lines.

Animal models that are susceptible to SARS-CoV-2 infection and develop clinical signs like human COVID-19 are desired to understand viral pathogenesis and develop effective medical countermeasures. The golden Syrian hamster is important for the study of SARS-CoV-2 since hamsters are naturally susceptible to SARS-CoV-2. However, infected hamsters show only limited clinical disease and resolve infection quickly. In this study, we describe development of human angiotensin-converting enzyme 2 (hACE2) transgenic hamsters as a model for COVID-19. During development of the model for SARS-CoV-2, we observed that different hACE2 transgenic hamster founder lines varied in their susceptibility to SARS-CoV-2 lethal infection. The highly susceptible hACE2 founder lines F0F35 and F0M41 rapidly progress to severe infection and death within 6 days post-infection (p.i.). Clinical signs included lethargy, weight loss, dyspnea, and mortality. Lethality was observed in a viral dose-dependent manner with a lethal dose as low as 1 × 100.15 CCID50. In addition, virus shedding from highly susceptible lines was detected in oropharyngeal swabs on days 2-5 p.i., and virus titers were observed at 105.5-6.5 CCID50 in lung and brain tissue by day 4 p.i.. Histopathology revealed that infected hACE2-hamsters developed rhinitis, tracheitis, bronchointerstitial pneumonia, and encephalitis. Mortality in highly susceptible hACE2-hamsters can be attributed to neurologic disease with contributions from the accompanying respiratory disease. In contrast, virus challenge of animals from less susceptible founder lines, F0M44 and F0M51, resulted in only 0-20% mortality. To demonstrate utility of this SARS-CoV-2 infection model, we determined the protective effect of the TLR3 agonist polyinosinic-polycytidylic acid (Poly (I:C)). Prophylactic treatment with Poly (I:C) significantly improved survival in highly susceptible hACE2-hamsters. In summary, our studies demonstrate that hACE2 transgenic hamsters differ in their susceptibility to SARS-CoV-2 infection, based on the transgenic hamster founder line, and that prophylactic treatment with Poly (I:C) was protective in this COVID-19 model of highly susceptible hACE2-hamsters.

RIPK2 is crucial for the microglial inflammatory response to bacterial muramyl dipeptide but not to lipopolysaccharide.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is a kinase that plays an essential role in the modulation of innate and adaptive immune responses. As a downstream signaling molecule for nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs), it is implicated in the signaling triggered by recognition of microbe-associated molecular patterns by NOD1/2 and TLRs. Upon activation of these innate immune receptors, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). However, whether RIPK2 is essential for downstream inflammatory signaling following the activation of NOD1/2, TLRs, or both remains controversial. In this study, we examined the role of RIPK2 in NOD2-and TLR4-dependent signaling cascades following stimulation of microglial cells with bacterial muramyl dipeptide (MDP), a NOD2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist. We utilized a highly specific proteolysis targeting chimera (PROTAC) molecule, GSK3728857A, and found dramatic degradation of RIPK2 in a concentration- and time-dependent manner. Importantly, the PROTAC completely abolished MDP-induced increases in iNOS and COX-2 protein levels and pro-inflammatory gene transcription of Nos2, Ptgs2, Il-1β, Tnfα, Il6, Ccl2, and Mmp9. However, increases in iNOS and COX-2 proteins and pro-inflammatory gene transcription induced by the TLR4 agonist, LPS, were only slightly attenuated with the GSK3728857A pretreatment. Further findings revealed that the RIPK2 PROTAC completely blocked the phosphorylation and activation of p65 NF-κB and p38 MAPK induced by MDP, but it had no effects on the phosphorylation of these two mediators triggered by LPS. Collectively, our findings strongly suggest that RIPK2 plays an essential role in the inflammatory responses of microglia to bacterial MDP but not to LPS.

A Polysaccharide-Based Oral-Vaccine Delivery System and Adjuvant for the Influenza Virus Vaccine.

Influenza virus enters the host body through the mucosal surface of the respiratory tract. An efficient immune response at the mucosal site can interfere with virus entry and prevent infection. However, formulating oral vaccines and eliciting an effective mucosal immune response including at respiratory mucosa presents numerous challenges including the potential degradation of antigens by acidic gastric fluids and the risk of antigen dilution and dispersion over a large surface area of the gut, resulting in minimal antigen uptake by the immune cells. Additionally, oral mucosal vaccines have to overcome immune tolerance in the gut. To address the above challenges, in the current study, we evaluated inulin acetate (InAc) nanoparticles (NPs) as a vaccine adjuvant and antigen delivery system for oral influenza vaccines. InAc was developed as the first polysaccharide polymer-based TLR4 agonist; when tailored as a nanoparticulate vaccine delivery system, it enhanced antigen delivery to dendritic cells and induced a strong cellular and humoral immune response. This study compared the efficacy of InAc-NPs as a delivery system for oral vaccines with Poly (lactic-co-glycolic acid) (PLGA) NPs, utilizing influenza A nucleoprotein (Inf-A) as an antigen. InAc-NPs effectively protected the encapsulated antigen in both simulated gastric (pH 1.1) and intestinal fluids (pH 6.8). Moreover, InAc-NPs facilitated enhanced antigen delivery to macrophages, compared to PLGA-NPs. Oral vaccination studies in Balb/c mice revealed that InAc-Inf-A NPs significantly boosted the levels of Influenza virus-specific IgG and IgA in serum, as well as total and virus-specific IgA in the intestines and lungs. Furthermore, mice vaccinated with InAc-Inf-A-NPs exhibited notably higher hemagglutination inhibition (HI) titers at mucosal sites compared to those receiving the antigen alone. Overall, our study underscores the efficacy of InAc-NPs in safeguarding vaccine antigens post-oral administration, enhancing antigen delivery to antigen-presenting cells, and eliciting higher virus-neutralizing antibodies at mucosal sites following vaccination.

Intrahepatic Exhausted Antiviral Immunity in an Immunocompetent Mouse Model of Chronic Hepatitis B

Targeting exhausted immune systems would be a promising therapeutic strategy to achieve a functional cure for HBV infection in patients with chronic hepatitis B (CHB). However, animal models recapitulating the immunokinetics of CHB are very limited. We aimed to develop an immunocompetent mouse model of CHB for intrahepatic immune profiling. CHB mice were created by intrahepatic delivery of the Sleeping Beauty transposon vector tandemly expressing the hepatitis B virus (HBV) genome and fumarylacetoacetate hydrolase (FAH) cDNA into C57BL/6J congenic FAH knockout mice via hydrodynamic tail vein injection. We profiled the viral and intrahepatic immune kinetics in CHB mice with or without treatment with recombinant IFNα or the hepatotropic Toll-like receptor 7 agonist SA-5 using single-cell RNA-seq. CHB mice exhibited sustained HBV viremia and persistent hepatitis. They showed intrahepatic expansion of exhausted CD8+ T (Tex) cells, the frequency of which was positively associated with viral load. Recruited macrophages increased in number but impaired inflammatory responses in the liver. The cytotoxicity of mature natural killer (NK) cells also increased in CHB mice. IFNα and SA-5 treatment both resulted in viral suppression with mild hepatic flares in CHB mice. Although both treatments activated NK cells, SA-5 had the capacity to revitalize the impaired function of Tex cells and liver-recruited macrophages. Our novel CHB mouse model recapitulated the intrahepatic exhausted antiviral immunity in patients with CHB, which might be able to be reinvigorated by a hepatotropic TLR7 agonist.

Lipopolysaccharide Induces Trained Innate Immune Tolerance in the Heart Through Interferon Signaling in a Model of Stress-Induced Cardiomyopathy.

Although the ability of the heart to adapt to environmental stress has been studied extensively, the molecular and cellular mechanisms responsible for cardioprotection are not yet fully understood. We administered Toll-like receptor (TLR) agonists or a diluent to wild-type mice and assessed their potential to induce cardiac protection against injury from a high intraperitoneal dose of isoproterenol (ISO) administered 7 days later. Cardioprotective effects were analyzed through serum cardiac troponin I levels, immune cell profiling via flow cytometry, echocardiography, and multiomic single-nuclei RNA and ATAC sequencing. Pretreatment with the TLR4 agonist lipopolysaccharide (LPS), but not TLR1/2 or TLR3 agonists, conferred cardioprotection against ISO, as demonstrated by reduced cardiac troponin I leakage, decreased inflammation, preservation of cardiac structure and function, and improved survival. Remarkably, LPS-induced tolerance was reversed by β-glucan treatment. Multiomic analysis showed that LPS-tolerized hearts had greater chromatin accessibility and upregulated gene expression compared to hearts treated with LPS and β-glucan (reverse-tolerized). The LPS tolerance was associated with upregulation of interferon response pathways across various cell types, including cardiac myocytes and stromal cells. Blocking both type 1 and type 2 interferon signaling eliminated LPS-induced tolerance against ISO, while pretreatment with recombinant type 1 and 2 interferons conferred cardiac protection. Multiomic sequencing further revealed enhanced cytoprotective signaling in interferon-treated hearts. Analysis of cell-cell communication networks indicated increased autocrine signaling by cardiac myocytes, as well as greater paracrine signaling between stromal cells and myeloid cells, in LPS-tolerized versus reverse-tolerized hearts. LPS pretreatment confers cardiac protection against ISO-induced injury through TLR4 mediated type 1 and 2 interferon signaling, consistent with trained innate immune tolerance. The observation that LPS-induced protection in cardiac myocytes involves both cell-autonomous and non-cell-autonomous mechanisms underscores the complexity of innate immune tolerance in the heart, warranting further investigation into this cardioprotective phenotype. What is new?: The Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) confers cardiac protection against isoproterenol-mediated injury in a manner consistent with trained innate immune tolerance, which is reversed by β-glucan treatment.Activation of type 1 and 2 interferon signaling, which is downstream of Toll-like receptor 4, is necessary and sufficient for LPS-induced cardiac protection.LPS-tolerized hearts show heightened autocrine signaling by cardiac myocytes and, to a greater degree, increased cell-cell communication between cardiac myocytes and stromal and myeloid cells compared to reverse-tolerized hearts.What are the clinical implications?: TLR4 and interferon signaling play key roles in the establishment of cardiac protection and LPS-induced trained innate immune tolerance.The protective effects of LPS are mediated by cell-autonomous and non-cell-autonomous mechanisms, suggesting that a deeper understanding of the molecular and cellular signatures of innate immune tolerance is required for the development of targeted approaches to modulate trained innate immunity, and consequently cytoprotection, in the heart.

Remimazolam ameliorates postoperative cognitive dysfunction after deep hypothermic circulatory arrest through HMGB1-TLR4-NF-κB pathway

BackgroundPostoperative cognitive dysfunction (POCD) is a complication of deep hypothermic circulatory arrest (DHCA). Various amounts of neurologic dysfunctions have been shown after DHCA, which has often been attributed to systemic inflammatory response syndrome and cerebral ischemia/reperfusion injury. Remimazolam is one of the commonly used anesthetic drugs with protective actions against inflammatory diseases, such as sepsis and cerebral ischemia/reperfusion injury. Here, we determined the protective effect and potential mechanism of action of remimazolam against neuronal damage after DHCA. MethodsA rat model of DHCA was established, and a gradient dosage of remimazolam was administered during cardiopulmonary bypass (CPB). The cognitive function of rats was evaluated by Morris water maze. Hematoxylin and eosin and TUNEL staining were performed to assess hippocampus tissue injury and neuronal apoptosis. Inflammatory cytokines concentration were analyzed by enzyme-linked immunosorbent assay. The protein expression was analyzed using automated electrophoresis western analysis and immunohistochemical analysis. ResultsThe appropriate dosage of remimazolam reduced histologic injury, neuronal apoptosis, microglia activation, and secondary inflammatory cascades, as well as the downregulation of the expression of the HMGB1-TLR4-NF-κB pathway after DHCA, improved the memory and learning abilities in DHCA rats. Further, administration of a TLR4 antagonist TAK-242 had a similar effect to remimazolam, while the TLR4 agonist LPS attenuated the effect of remimazolam. ConclusionsRemimazolam could ameliorate POCD after DHCA through the HMGB1-TLR4-NF-κB signaling pathway.

Regional lymph node changes on breast MRI in patients with early-stage breast cancer receiving neoadjuvant chemo-immunotherapy

Abstract Purpose Establishing breast MRI imaging patterns associated with neoadjuvant immunotherapy is needed to monitor response. We analyzed serial breast MRIs in patients receiving neoadjuvant chemo-immunotherapy on the I-SPY2 clinical trial. Methods Patients with stage 2–3 HER2-negative breast cancer were randomized to weekly paclitaxel (control), weekly paclitaxel and pembrolizumab, or weekly paclitaxel, pembrolizumab and intra-tumoral injection of SD-101, a TLR9 agonist. All patients received AC. Regional lymph nodes were retrospectively evaluated on breast MRI at baseline, 3, 12 and 20 weeks by a single blinded radiologist. MRIs were assessed for development of new regional lymphadenopathy, or increase in the longest diameter or cortical thickness of the largest abnormal regional lymph node. Results Between 12/2015 and 4/2021, a total of 43 patients enrolled in the control (n = 16) and paclitaxel + pembrolizumab ± SD-101 (n = 27) arms. 12 of 27 patients (44.4%) receiving chemo-immunotherapy experienced increased lymphadenopathy within the first 12 weeks compared to 1 of 16 patients (6.3%) in the control group (p = 0.014). Most patients with increased lymphadenopathy were in the SD101/pembro arm (n = 10, p = 0.002). Increased lymphadenopathy was observed despite concomitant decrease in breast tumor size at all time points. 11 of 12 patients with increased lymphadenopathy had pathologically negative nodes at surgery. There was no association between lymphadenopathy and lower residual cancer burden or immune-related toxicity. Conclusions The combination of neoadjuvant paclitaxel and pembrolizumab ± SD101 intratumoral injection was associated with early increases in regional lymphadenopathy on MRI despite decreased breast tumor size. Increased lymphadenopathy was not associated with node positive disease at surgery.

Salmonella Detection in Food Using a HEK-hTLR5 Reporter Cell-Based Sensor.

The development of a rapid, sensitive, specific method for detecting foodborne pathogens is paramount for supplying safe food to enhance public health safety. Despite the significant improvement in pathogen detection methods, key issues are still associated with rapid methods, such as distinguishing living cells from dead, the pathogenic potential or health risk of the analyte at the time of consumption, the detection limit, and the sample-to-result. Mammalian cell-based assays analyze pathogens' interaction with host cells and are responsive only to live pathogens or active toxins. In this study, a human embryonic kidney (HEK293) cell line expressing Toll-Like Receptor 5 (TLR-5) and chromogenic reporter system (HEK dual hTLR5) was used for the detection of viable Salmonella in a 96-well tissue culture plate. This cell line responds to low concentrations of TLR5 agonist flagellin. Stimulation of TLR5 ligand activates nuclear factor-kB (NF-κB)-linked alkaline phosphatase (AP-1) signaling cascade inducing the production of secreted embryonic alkaline phosphatase (SEAP). With the addition of a ρ-nitrophenyl phosphate as a substrate, a colored end product representing a positive signal is quantified. The assay's specificity was validated with the top 20 Salmonella enterica serovars and 19 non-Salmonella spp. The performance of the assay was also validated with spiked food samples. The total detection time (sample-to-result), including shortened pre-enrichment (4 h) and selective enrichment (4 h) steps with artificially inoculated outbreak-implicated food samples (chicken, peanut kernel, peanut butter, black pepper, mayonnaise, and peach), was 15 h when inoculated at 1-100 CFU/25 g sample. These results show the potential of HEK-DualTM hTLR5 cell-based functional biosensors for the rapid screening of Salmonella.

Safety and efficacy of toll-like receptor agonists as therapeutic agents and vaccine adjuvants for infectious diseases in animals: a systematic review.

Strengthening global health security relies on adequate protection against infectious diseases through vaccination and treatment. Toll-like receptor (TLR) agonists exhibit properties that can enhance immune responses, making them potential therapeutic agents or vaccine adjuvants. We conducted an extensive systematic review to assess the efficacy of TLR agonists as therapeutic agents or vaccine adjuvants for infectious diseases and their safety profile in animals, excluding rodents and cold-blooded animals. We collected qualitative and available quantitative data on the efficacy and safety outcomes of TLR agonists and employed descriptive analysis to summarize the outcomes. Among 653 screened studies, 51 met the inclusion criteria. In this review, 82% (42/51) of the studies used TLR agonists as adjuvants, while 18% (9/51) applied TLR agonist as therapeutic agents. The predominant TLR agonists utilized in animals against infectious diseases was CpG ODN, acting as a TLR9 agonist in mammals, and TLR21 agonists in chickens. In 90% (46/51) of the studies, TLR agonists were found effective in stimulating specific and robust humoral and cellular immune responses, thereby enhancing the efficacy of vaccines or therapeutics against infectious diseases in animals. Safety outcomes were assessed in 8% (4/51) of the studies, with one reporting adverse effects. Although TLR agonists are efficacious in enhancing immune responses and the protective efficacy of vaccines or therapeutic agents against infectious diseases in animals, a thorough evaluation of their safety is imperative to in-form future clinical applications in animal studies. https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=323122.

HSV-1 immune escapes in microglia by down-regulating GM130 to inhibit TLR3-mediated innate immune responses

BackgroundTo investigate the mechanism of Golgi matrix protein 130(GM130) regulating the antiviral immune response of TLR3 after herpes simplex virus type 1(HSV-1) infection of microglia cells. We explored the regulatory effects of berberine on the immune response mediated by GM130 and TLR3.MethodsAn in vitro model of HSV-1 infection was established by infecting BV2 cells with HSV-1.ResultsCompared to the uninfected group, the Golgi apparatus (GA) fragmentation and GM130 decreased after HSV-1 infection; TLR3 increased at 6 h and began to decrease at 12 h after HSV-1 infection; the secretion of interferon-beta(IFN-β), tumour necrosis factor alpha(TNF-α), and interleukin-6(IL-6) increased after infection. Knockdown of GM130 aggravated fragmentation of the GA and caused TLR3 to further decrease, and the virus titer also increased significantly. GM130 knockdown inhibits the increase in TLR3 and inflammatory factors induced by TLR3 agonists and increases the viral titer. Overexpression of GM130 alleviated fragmentation of the GA induced by HSV-1, partially restored the levels of TLR3, and reduced viral titers. GM130 overexpression reversed the reduction in TLR3 and inflammatory cytokine levels induced by TLR3 inhibitors. Therefore, the decrease in GM130 levels caused by HSV-1 infection leads to increased viral replication by inhibiting TLR3-mediated innate immunity. Berberine can protect the GA and reverse the downregulation of GM130, as well as the downregulation of TLR3 and its downstream factors after HSV-1 infection, reducing the virus titer.ConclusionsIn microglia, one mechanism of HSV-1 immune escape is disruption of the GM130/TLR3 pathway. Berberine protects the GA and enhances TLR3-mediated antiviral immune responses.

Peculiar transcriptional reprogramming with functional impairment of dendritic cells upon exposure to transformed HTLV-1-infected cells.

Manipulation of immune cell functions, independently of direct infection of these cells, emerges as a key process in viral pathophysiology. Chronic infection by Human T-cell Leukemia Virus type 1 (HTLV-1) is associated with immune dysfunctions, including misdirected responses of dendritic cells (DCs). Here, we interrogate the ability of transformed HTLV-1-infected T cells to manipulate human DC functions. We show that exposure to transformed HTLV-1-infected T cells induces a biased and peculiar transcriptional signature in monocyte-derived DCs, associated with an inefficient maturation and a poor responsiveness to subsequent stimulation by a TLR4 agonist. This poor responsiveness is also associated with a unique transcriptional landscape characterized by a set of genes whose expression is either conferred, impaired or abolished by HTLV-1 pre-exposure. Induction of this functional impairment requires several hours of coculture with transformed HTLV-1-infected cells, and associated mechanisms driven by viral capture, cell-cell contacts, and soluble mediators. Altogether, this cross-talk between infected T cells and DCs illustrate how HTLV-1 might co-opt communications between cells to induce a unique local tolerogenic immune microenvironment suitable for its own persistence.

Carbohydrate-Lectin Interactions Reprogram Dendritic Cells to Promote Type 1 Anti-Tumor Immunity.

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) is thought to shape immunity by directing T cell function. Controlling downstream responses, however, remains a major challenge. A critical goal in advancing vaccine development involves the identification of receptors that drive type 1 cellular immunity. The immune system monitors cells for aberrant glycosylation (a sign of a foreign entity), but potent activation occurs when a second signal, such as single-stranded RNA or lipopolysaccharide, is present to activate TLR signaling. To exploit dual signaling, we engineered a glycan-costumed virus-like particle (VLP) vaccine that displays a DC-SIGN-selective aryl mannose ligand and encapsulates TLR7 agonists. These VLPs deliver programmable peptide antigens to induce robust DC activation and type 1 cellular immunity. In contrast, VLPs lacking this critical DC-SIGN ligand promoted DC-mediated humoral immunity, offering limited tumor control. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, significantly inhibiting tumor growth in a murine melanoma model. The tailored VLPs also afforded protection against the reintroduction of tumor cells. Thus, DC lectin-driven immune reprogramming, combined with the modular programmability of VLP platforms, provides a promising framework for directing cellular immunity to advance cancer immunotherapies and vaccines.