Toll-like Receptor Agonists Research Articles

Fluorescent nucleobase analogue for cellular visualisation and regulation of immunostimulatory CpG oligodeoxynucleotides.

In this study, we explored the chemical modification of toll-like receptor 9 (TLR9) agonist DNA using a highly fluorescent thymine analogue, ThexT, focusing on its structural and photophysical characteristics. ThexT-labelled CpG oligonucleotides effectively demonstrated intracellular localisation within macrophage cell lines. Notably, immunostimulatory activity varied depending on the site of ThexT incorporation within the TLR9 agonist sequence. The introduction of fluorescent nucleobases offers a useful approach for visualising immunostimulatory oligonucleotides and for modulating immune responses.

Pilot clinical trial of neoadjuvant toll-like receptor 7 agonist (Imiquimod) immunotherapy in early-stage oral squamous cell carcinoma

BackgroundThere is no neoadjuvant immunotherapy for early-stage oral cancer patients. We report a single-arm, open-label, pilot clinical trial assessing the efficacy and safety of topical toll-like receptor-7 (TLR-7) agonist, imiquimod, utilized in a neoadjuvant setting in early-stage oral squamous cell carcinoma (OSCC).MethodsThe primary endpoint is reduction in tumor cell counts assessed by quantitative multiplex immunofluorescence and the immune-related pathologic response. The secondary endpoint is safety.Results60% of patients experienced a 50% reduction or greater in tumor cell count post-treatment (95% CI = 32% to 84%). Similarly, 60% of patients had immune-related major pathologic response (irMPR) with two complete pathologic responses, and 40% had partial response (PR) with the percent residual viable tumor ranging from 25% to 65%. An increase in functional helper and cytotoxic T-cells significantly contributed to a reduction in tumor (R=0.54 and 0.55, respectively). The treatment was well tolerated with the application site mucositis being the most common adverse event (grades 1-3), and no grade 4 life-threatening event. The median follow-up time was 17 months (95% CI = 16 months - not reached), and one-year recurrence-free survival was 93% of evaluable patients.ConclusionNeoadjuvant imiquimod immunotherapy could be safe and promising regimen for early-stage oral cancer.Trial registrationClinicalTrials.gov, Identifier NCT04883645.

A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration

BackgroundOsteosarcoma is the most common malignant bone tumour with limited treatment options and poor outcomes in advanced metastatic cases. Current immunotherapies show limited efficacy, highlighting the need for novel therapeutic approaches. Systemic immune activation by Toll-like receptor 4 (TLR4) immunostimulants has shown great promise; however, current TLR4 agonists’ toxicity hinders this systemic approach in patients with osteosarcoma.MethodsWe compared the antitumour effect of lipopolysaccharides (LPS) with that of an innovative chemically detoxified TLR4 agonist (Lipo-MP-LPS) in a syngeneic metastatic osteosarcoma mouse model. Lipo-MP-LPS exhibited an optimal safety and solubility profile for systemic administration at an effective dose. We evaluated tumour growth, lung metastases, and immune cell infiltration in wild-type and TLR4-mutant mice and performed selective immunodepletion.ResultsLipo-MP-LPS exhibited antitumour effects against localised osteosarcoma tumours and lung metastases, like those of natural LPS. Lipo-MP-LPS promoted CD8+ T cells and M1 macrophages infiltration in primary tumours and CD8+ T cells in metastases, with an M1-phenotype macrophage shift. The Lipo-MP-LPS antitumour effects were found to depend on TLR4 and CD8+ T cells, but not on macrophages.ConclusionLipo-MP-LPS inhibited tumour growth and lung metastasis of osteosarcoma by promoting CD8 + T cell infiltration, indicating its therapeutic potential for advanced osteosarcoma.

Abstract B030: Novel strategy to improve response of high risk HPV+ HNSCC to radiation therapy

Abstract Background: Improving understanding of head and neck squamous cell carcinoma (HNSCC) has led to the discovery of novel molecular markers to stratify risk and guide treatment decisions. Recently, we have identified two subtypes of HPV+ OPSCC – one with good prognosis and one with poor prognosis. The subtypes are distinct in many ways – somatic genes mutated, HPV viral oncogenes expression, the physical state of the viral genome (integrated vs episomal), as well as global mutation and methylation profiles. Intriguingly, all differences converge on intrinsic tumor NF-κB activity with constitutively active NF-κB (usually arising from genetic defects in NF-κB regulators, including TRAF3 and CYLD) driving significantly elevated radiation sensitivity and improved patient survival. NF-κB-based groups define an important clinically actionable prognostic biomarker in HPV+ HNSCC: one important goal is to de-escalate therapy for patients with molecularly low-risk tumors to improve quality of life amongst survivors, while another goal is to improve the cure for of patients with a high risk of treatment failure. Material and methods: We utilized HPV+ head and neck cancer cells, HPV+ HNSCC xenografted mouse model, patient-derived xenografts (PDX), as well as our novel fully immunocompetent mouse model of high risk HPV+ HNSCC; we applied targeted fractionated radiation. To activate NF-κB, we used two clinically applicable drugs with different mechanisms of action: Toll-like receptor 5 (TLR5) agonist and mitochondria-derived activator of caspases (SMAC) mimetic. Custom NF-κB NanoString panel contains our recently developed NF-κB gene signature along with HPV genes and housekeeping genes that are used to normalize the gene expression data. Results: Both drugs activated NF-κB in HPV+ HNSCC and significantly improved response of HPV+ head and neck cancer cells and tumors to radiation. Interestingly, TLR5 agonist alone had a strong tumor suppressing effect in low NF-κB HPV+ HNSCC; this effect was significantly better than 32 Gy focused radiation given in fractions of 4 Gy. Custom NF-κB NanoString panel is robustly prognostic with NF-κB active tumors having improved outcomes after chemoradiation. Conclusions: Because of the average relatively favorable prognosis of HPV+ HNSCC, urgent need for improved therapy for patients with aggressive high risk tumors is greatly undervalued. Here, we present a clinical assay to risk stratify patients with HPV+ HNSCC and begin to explore pharmacological activation of NF-κB as a strategy to sensitize high risk HPV+ HNSCC to radiation therapy. TLR5 agonist is well known for the remarkable normal tissue protective effects against radiation and other damaging conditions; the dual effect of entolimod – sensitization of HPV+ HNSCC to radiation, while simultaneously protecting normal tissues from severe side effects - may be useful for patients with high-risk HPV+ head and neck cancer in combination with radiotherapy. Citation Format: Sri Vemulamanda, Natalia Issaeva1, Aditi Kothari, Travis Schrank, Wendell G. Yarbrough. Novel strategy to improve response of high risk HPV+ HNSCC to radiation therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B030.

EXOTLR1/2-STING: A Dual-Mechanism Stimulator of Interferon Genes Activator for Cancer Immunotherapy.

As natural agonists of the stimulator of interferon genes (STING) protein, cyclic dinucleotides (CDNs) can activate the STING pathway, leading to the expression of type I interferons and various cytokines. Efficient activation of the STING pathway in antigen-presenting cells (APCs) and tumor cells is crucial for antitumor immune response. Tumor-derived exosomes can be effectively internalized by APCs and tumor cells and have excellent potential to deliver CDNs to the cytoplasm of APCs and tumor cells. Here, we leverage tumor exosomes as a delivery platform, designing an EXOTLR1/2-STING loaded with CDNs. To achieve efficient loading of CDNs onto exosomes, we chemically conjugated CDNs with Pam3CSK4, a compound featuring multiple fatty acid chains, resulting in Pam3CSK4-CDGSF. Utilizing the high lipophilicity of Pam3CSK4, Pam3CSK4-CDGSF could be efficiently loaded onto the exosomes through simple incubation. Moreover, as an agonist for Toll-like receptor 1/2, Pam3CSK4 also exhibits robust immunological synergistic effects in conjunction with CDNs. EXOTLR1/2-STING effectively induced the activation of APCs and triggered tumor cell death, producing a favorable antitumor therapeutic effect. It also demonstrated significant synergistic effects with immune checkpoint therapies.

CD209d/e promotes inflammation and lung injury during influenza virus infection.

Influenza virus infects millions each year, contributing greatly to human morbidity and mortality. Upon viral infection, pathogen-associated molecular patterns activate pattern recognition receptors on host cells, triggering an immune response. The CD209 protein family, homologs of DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin), is thought to modulate immune responses to viruses. The effects of the mouse functional DC-SIGN homolog CD209d/e on the lung immune responses during influenza viral infection are not known. Therefore, we generated mice that lack both CD209d and e isoforms to determine the role in influenza viral infection. We infected wild-type and CD209d/e gene-deficient (CD209d/e-/-) mice with influenza virus and measured the cellular response in bronchoalveolar lavage, the expression of proinflammatory cytokines, antiviral genes, toll-like receptors (TLRs) in the lung, and lung pathology. We found CD209d/e-/- mice had decreased viral burden, TLR3 and TLR9 expression, interferon response, macrophages in bronchoalveolar lavage, and parenchymal lung inflammation compared with control mice. We also found less influenza viral uptake in alveolar macrophages and bone marrow-derived macrophages isolated from CD209d/e-/- mice when compared with control mice. We further investigated the role CD209d/e by treating bone marrow-derived macrophages from control and CD209d/e-/- mice with TLR agonists. We found that lacking CD209d/e decreased the expression of TLR3, TLR9, RIG1, STAT1, and STAT2 compared with controls. Collectively these results show that CD209d/e plays an important role in viral sensing/uptake and inflammatory immune responses during influenza viral infection.

P0105 Evaluation of Stimuli Driving Oncostatin M Expression in Bone Marrow-Derived Macrophages

Abstract Background Oncostatin M (OSM) is a cytokine linked to inflammatory diseases, including inflammatory bowel disease (IBD), where it contributes to inflammation, tissue remodeling, and fibrosis. Despite its established role, the specific signals driving OSM expression in colitis remain unclear. Methods Bone marrow-derived macrophages (BMDMs) were differentiated using M-CSF and stimulated for 36 hours with a range of Toll-like receptor (TLR) agonists, damage-associated molecular patterns (DAMPs), cytokines, and bacterial lysates. OSM secretion was quantified using enzyme-linked immunosorbent assays (ELISA). To dissect key signaling pathways, we applied chemical inhibitors and utilized macrophages derived from gene-deficient mice. Results Myd88-dependent stimuli, including TLR1/2 (Pam3CSK4), TLR4 (LPS), TLR2/6 (FSL-1), TLR7/8 (R848), IL-1α, and IL-33, significantly increased OSM expression in BMDMs. Gram-negative (E. coli, C. rodentium) and Gram-positive (S. aureus) bacterial lysates also enhanced OSM secretion. Pro-inflammatory cytokine IFN-γ further boosted OSM levels, consistent with in vivo findings of OSM upregulation in inflammatory monocytes. Additionally, IL-4, which induces a tissue repair phenotype in macrophages, markedly increased OSM secretion. Conclusion OSM expression in BMDMs is driven by multiple signaling pathways, indicating a synergistic effect of diverse stimuli. Identifying key stimulatory and inhibitory molecules, alongside macrophage-specific knockout analyses, will help clarify the upstream regulators of OSM. Understanding these drivers may uncover new therapeutic targets in IBD and offer insights into the onset of fibrotic processes.

Chitosan/γ-PGA nanoparticles and IFN-γ immunotherapy: A dual approach for triple-negative breast cancer treatment.

Interferon-γ (IFN-γ) is a key mediator in antitumor immunity and immunotherapy responses, yet its clinical applications remain restricted to chronic granulomatous disease and malignant osteopetrosis. IFN-γ effectiveness as a standalone treatment has shown limited success in clinical trials and its potential for synergistic effects when combined with immunotherapies is under clinical exploration. A particularly compelling combination is that of IFN-γ with Toll-like receptor (TLR) agonists that holds significant promise for cancer treatment. Previously, we demonstrated chitosan (Ch)/poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs), known to activate TLRs, as adjuvants to radiotherapy by remodeling breast tumor microenvironment and systemic immunosuppression. These immunomodulatory abilities make Ch/γ-PGA NPs promising adjuvants to IFN-γ-based therapies. Here, we addressed the synergistic therapeutic potential of combining Ch/γ-PGA NPs with IFN-γ therapy in the 4T1 orthotopic breast tumor mouse model. While control animals (placebo-treated) had progressive tumor growth and lung metastases, those treated with either NPs or IFN-γ alone had a significant slower tumor growth. Remarkably, primary tumor growth was halted throughout the duration of the treatment when both treatments were combined. Although the animals did not achieve durable complete responses upon treatment withdrawal, it was notable that the NPs plus IFN-γ group presented a lower lung metastatic burden compared to other groups. Systemically, the combination therapy slightly attenuated immunosuppression and the percentage of splenic myeloid cells, while increased the percentage of T helper 1 cells and of cytotoxic T cells. Overall, this proof-of-concept study suggests that Ch/γ-PGA NPs potentiate IFN-γ effects to reduce tumor progression, presenting a novel approach for anticancer strategies.

Lack of TLR9 exacerbates ocular impairment and visual loss during systemic Cryptococcus gattii infection.

Cryptococcus gattii is a saprophytic basidiomycete that grows in the environment and can cause systemic cryptococcosis. Ocular cryptococcosis causes blindness and is commonly associated with central nervous system (CNS) infection. Toll-like receptor 9 (TLR9) can control cryptococcosis and another mycosis. Here, using C57BL/6 TLR9 knockout mice (TLR9-/-), we evaluated the role of TLR9 signaling in ocular involvement during systemic C. gattii-infection. We observed ocular impairment and found a high fungal burden in the retina, vitreous humor (VH), and optic nerve (ON) of TLR9-/- mice three weeks after infection. Capsular polysaccharide glucuronoxylomannan (GXM) deposition, astrogliosis and morphological alterations in retina lead to progressive blindness of TLR9-/- mice. The phenomenon observed in our work has not yet been explored in a murine model. These results contribute to the understanding of the role of TLR9 during ocular cryptococcosis. Therapies using TLR9 agonists may be important for the treatment of ocular cryptococcosis.

Galloyl Dialkyl Lipids Drive Encapsulation of Peptides into Lipid Nanoparticles by Hydrogen Bonding.

The intracellular delivery of peptides and proteins is crucial for various biomedical applications. Lipid nanoparticles (LNPs) have emerged as a promising strategy for delivering peptides to phagocytic cells. However, the diverse physicochemical properties of peptides necessitate tailored formulations. This study introduces a generic approach using galloyl (GA)-functionalized lipids for the encapsulation of peptides in LNPs via hydrogen bonding between the ubiquitously present amides in peptides and the multivalently displayed galloyl phenol groups in GA-LNPs. In vitro studies showed that GA-LNPs significantly improved the cellular uptake of peptides and activated immune responses when combined with Toll-like receptor (TLR) agonists MPLA and IMDQ. In vivo, GA-LNPs accumulated in the spleen and enhanced peptide delivery to antigen-presenting cells. GA-LNPs coencapsulating peptide antigens and TLR agonists elicited robust antigen-specific CD8+ T-cell responses in mice.

Prx5 overexpression protect against doxorubicin-induced cardiotoxicity by inhibiting oxidative stress and inflammation via the TLR4/NF-κB pathway.

The clinical application of Doxorubicin (DOX) is constrained due to its cardiotoxic side effects. Oxidative stress and inflammation are crucial mechanisms driving doxorubicin-induced cardiotoxicity (DIC). Peroxiredoxin 5 (Prx5) is central to these inflammatory responses. However, the specific role of Prx5 in DIC remains unclear. This study aims to investigate the impacts of Prx5 on DIC and the underlying mechanisms. A cardiac-specific Prx5-overexpressing mice was used to establish a doxorubicin (DOX)-induced cardiotoxicity (DIC) model. Neonatal mouse cardiomyocytes (NMCMs) were cultured and stimulated with DOX. Prx5 overexpression or knockdown in cardiomyocytes was achieved using a Prx5-overexpressing adenovirus or small interfering RNA (siRNA), respectively. Echocardiography, histopathological assessments, and molecular techniques were employed to examine the effects and mechanisms of Prx5 on DIC. Prx5 expression is upregulated in cardiac tissues following DOX administration. In DOX-exposed mice, overexpression of Prx5 significantly improved cardiac function and reduced myocardial injury. It inhibited myocardial hypertrophy and fibrosis, and diminished oxidative stress and inflammatory responses. Conversely, Prx5 knockdown in vitro aggravated DOX-induced cardiomyocyte inflammation and oxidative stress. Mechanistically, overexpression of Prx5 also resulted in the downregulation of Toll-like receptor 4 (TLR4) and phosphorylated P65 expression. Moreover, the protective effects of Prx5 were significantly abrogated by a TLR4 agonist. Prx5 overexpression could protect against DOX-induced cardiac oxidative stress and inflammation. Mechanistically, Prx5 overexpression potentially inhibits the TLR4/NF-κB signaling pathway to improve DOX-induced myocardial injury. These findings strongly suggest that Prx5 could be a potential candidate target for the treatment of DOX-induced myocardial injury.

Randomized, observer-blind, controlled Phase 1 study of the safety and immunogenicity of the Na-GST-1/Alhydrogel hookworm vaccine with or without a CpG ODN adjuvant in hookworm-naïve adults.

Recombinant Necator americanus Glutathione-S-Transferase-1 (Na-GST-1) formulated on Alhydrogel (Na-GST-1/Alhydrogel) is being developed to prevent anemia and other complications of N. americanus infection. Antibodies induced by vaccination with recombinant Na-GST-1 are hypothesized to interfere with the blood digestion pathway of adult hookworms in the host. Phase 1 trials have demonstrated the safety of Na-GST-1 formulated on Alhydrogel, but further optimization of the vaccine adjuvant formulation may improve humoral immune responses, thereby increasing the likelihood of vaccine efficacy. A randomized, observer-blind, dose escalation Phase 1 trial was conducted in 24 healthy, hookworm-naïve adults. In each cohort of 12 participants, 4 were randomized to receive 100 µg of Na-GST-1/Alhydrogel and 8 to receive 30 µg or 100 µg of Na-GST-1/Alhydrogel plus the Cytosine-phospho-Guanine (CpG) oligodeoxynucleotide Toll-like receptor-9 agonist, CpG 10104, in the first and second cohorts, respectively. Progression to the second cohort was dependent upon evaluation of 7-day safety data after all participants in the first cohort had received the first dose of vaccine. Three intramuscular injections of study product were administered on days 0, 56, and 112, after which participants were followed for 6 months. IgG and IgG subclass antibody responses to Na-GST-1 were measured by qualified indirect ELISAs at pre- and post-vaccination time points. Na-GST-1/Alhydrogel administered with or without CpG 10104 was well-tolerated. The most common solicited adverse events were mild injection site tenderness and pain, and mild headache. There were no vaccine-related serious adverse events or adverse events of special interest. Both dose concentrations of Na-GST-1/Alhydrogel plus CpG 10104 had significantly higher post-vaccination levels of antigen-specific IgG antibody compared to Na-GST-1/Alhydrogel without CpG, starting after the second injection. Peak anti-Na-GST-1 IgG levels were observed between 2 and 4 weeks following the third dose, regardless of Na-GST-1 formulation. IgG levels decreased but remained significantly above baseline in all groups by day 290, at which point all participants (20 of 20 evaluable participants) still had detectable IgG. Longitudinal antigen-specific IgG1 and IgG3 subclass responses mirrored those of total IgG, whereas IgG4 responses were lower in the groups that received the vaccine with the CpG adjuvant compared to the non-CpG group. Vaccination of hookworm-naïve adults with Na-GST-1/Alhydrogel plus CpG 10104 was safe and minimally reactogenic. Addition of CpG 10104 to Na-GST-1/Alhydrogel resulted in significant improvement in IgG responses against the vaccine antigen. These promising results have led to inclusion of the CpG 10104 formulation of Na-GST-1/Alhydrogel in a Phase 2 proof-of-concept controlled human infection trial.

Plant Cell Culture-Derived Saponin Adjuvant Enhances Immune Response Against a Stabilized Human Metapneumovirus Pre-Fusion Vaccine Candidate.

No vaccine for the prevention of HMPV is currently licensed, although several subunit vaccines are in development. Saponin-based adjuvant systems (AS), including QS-21, have transformed the field of subunit vaccines by dramatically increasing their potency and efficacy, leading to the development of several licensed vaccines. However, naturally sourced tree bark-extracted QS-21 faces supply and manufacturing challenges, hindering vaccine development. This study reports on an alternative plant cell culture system for the consistent production of highly pure QS-21. We evaluated the efficacy of cultured plant cell (cpc)-produced QS-21 in a novel HMPV vaccine, formulating a recombinant pre-fusion stabilized HMPV F protein (preF) with cpcQS-21 and a synthetic toll-like receptor 4 (TLR4) agonist adjuvant formulation. In mice, TLR4 agonist containing adjuvant formulations with plant cell-produced QS-21 performed equally to licensed adjuvant AS01 containing tree-bark-extracted QS-21 and demonstrated a significant increase in immunogenicity against HMPV preF compared to the unadjuvanted control. Our findings pave the way for a reliable, scalable, and sustainable source of pure QS-21, enabling the development of highly effective HMPV and other vaccines with significant public health impact.

Astrocytes contribute to toll-like receptor 2-mediated neurodegeneration and alpha-synuclein pathology in a human midbrain Parkinson’s model

BackgroundParkinson’s disease (PD) is characterised by degeneration of ventral midbrain dopaminergic (DA) neurons and abnormal deposition of α-synuclein (α-syn) in neurons. Activation of the innate immune pathogen recognition receptor toll-like receptor 2 (TLR2) is associated with exacerbation of α-syn pathology. TLR2 is increased on neurons in the PD brain, and its activation results in the accumulation and propagation of α-syn through autophagy inhibition in neurons. In addition to the aggregation and propagation of pathological α-syn, dysfunction of astrocytes may contribute to DA neuronal death and subsequent clinical progression of PD. However, the role of astrocytes in TLR2-mediated PD pathology is less explored but important to address, given that TLR2 is a potential therapeutic target for PD.MethodsInduced pluripotent stem cells from three controls and three PD patients were differentiated into a midbrain model comprised of neurons (including DA neurons) and astrocytes. Cells were treated with or without the TLR2 agonist Pam3CSK4, and α-syn pathology was seeded using pre-formed fibrils. Confocal imaging was used to assess lysosomal function and α-syn pathology in the different cell types, as well as DA neuron health and astrocyte activation.ResultsTLR2 activation acutely impaired the autophagy lysosomal pathway, and potentiated α-syn pathology seeded by pre-formed fibrils in PD neurons and astrocytes, leading to degeneration and loss of DA neurons. The astrocytes displayed impaired chaperone-mediated autophagy reducing their ability to clear accumulated α-syn, and increases of A1 neurotoxic phenotypic proteins SerpinG1, complement C3, PSMB8 and GBP2. Moreover, the phenotypic changes in astrocytes correlated with a specific loss of DA neurons.ConclusionsTaken together, these results support a role for astrocyte dysfunction in α-syn accumulation and DA neuronal loss following TLR2 activation in PD.

Bovine Lactoferrin Enhances Toll-like Receptor 7 Response in Plasmacytoid Dendritic Cells and Modulates Cellular Immunity.

Plasmacytoid dendritic cells (pDCs) express Toll-like receptor 7 (TLR7) in the endosomes, recognize viral single-stranded RNA (ssRNA), and produce significant amounts of interferon (IFN)-α. Bovine lactoferrin (LF) enhances the response of IFN regulatory factors followed by the activation of IFN-sensitive response elements located in the promoter regions of the IFN-α gene and IFN-stimulated genes in the TLR7 reporter THP-1 cells in the presence of R-848, a TLR7 agonist. In ex vivo experiments using human peripheral blood mononuclear cells, LF enhances IFN-α levels in the supernatant in the presence of R-848. Additionally, it increases the expression of IFN-α, human leukocyte antigen (HLA)-DR, and CD86 in pDCs; HLA-DR and CD86 in myeloid dendritic cells; CD69 in CD56 dim natural killer and T killer cells; and IFN-γ in T helper type 1 and B cells in the presence of R-848. The inhibition of phagocytosis or neutralization of nucleolin, a receptor of LF, suppresses LF incorporation into pDCs. These results suggest that pDCs incorporate LF through phagocytosis or nucleolin-mediated endocytosis, and LF enhances TLR7 response in the endosome and subsequent IFN signaling pathway and activates innate and adaptive immune cells. We anticipate that LF modulates antiviral immunity against environmental ssRNA viruses and contributes to homeostasis.

Dysfunction and regulatory interplay of T and B cells in chronic hepatitis B: immunotherapy and emerging antiviral strategies.

In the context of chronic hepatitis B virus (HBV) infection, the continuous replication of HBV within host hepatocytes is a characteristic feature. Rather than directly causing hepatocyte destruction, this replication leads to immune dysfunction and establishes a state of T-B immune tolerance. Successful clearance of the HBV virus is dependent on the close collaboration between humoral and cellular immunity. Humoral immunity, mediated by B-cell subpopulations, and cellular immunity, dominated by T-cell subpopulations show varying degrees of dysfunction during chronic hepatitis B (CHB). Notably, not all T- and B-cells produce positive immune responses. This review examine the most recent developments in the mutual regulation of T-B cells during chronic HBV infection. Our focus is on the prevailing immunotherapeutic strategies, such as T cell engineering, HBV-related vaccines, PD-1 inhibitors, and Toll-like receptor agonists. While nucleos(t)ide analogues (NUCs) and interferons have notable limitations, including inadequate viral suppression, drug resistance, and adverse reactions, several HBV entry inhibitors have shown promising clinical efficacy. To overcome the challenges posed by NUCs or monotherapy, the combination of immunotherapy and novel antiviral agents presents a promising avenue for future CHB treatment and potential cure.

Polyphenol-mediated assembly of toll-like receptor 7/8 agonist nanoparticles for effective tumor immunotherapy.

Toll-like receptor (TLR) 7/8 agonists have shown significant potential in tumor immunotherapy. However, the limited pharmacokinetic properties and systemic toxicity resulting from off-target effects limits their biomedical applications. We here report the polyphenol-mediated assembly of resiquimod (R848, a TLR7/8 agonist) nanoparticles (RTP NPs) to achieve tumor-selective immunotherapy while avoiding systemic adverse effects. Upon intravenous administration, the prepared RTP NPs are effectively accumulated at tumor sites, which increase their bioavailability and reduce systemic inflammation. RTP NPs can trigger a potent antitumor immune response in a mouse tumor model to inhibit tumor growth. Additionally, after subcutaneous injection at the tail base, RTP NPs efficiently migrate to the lymph nodes, where they elicit immune memory to prevent tumorigenesis. This study underscores the potential application of polyphenol-mediated assembly in developing nanomedicines with reduced toxicity for tumor-specific immunotherapy. STATEMENT OF SIGNIFICANCE: Toll-like receptor agonist (R848) nanoparticles for tumor-selective immunotherapy were synthesized through polyphenol-mediated assembly, a method that simplifies preparation process and minimizes potential side effects. Intravenously administered these nanoparticles effectively extended circulation time, enhanced tumor enrichment, and reduced systemic inflammation, thus augmenting the bioavailability and minimizing the side effects of R848. The nanoparticles significantly inhibited tumor growth by triggering a potent antitumor immune response, including dendritic cell maturation, macrophage polarization, T-cell infiltration, and cytokine secretion. Moreover, after subcutaneous injection at the tail base, they can elicit immune memory to prevent tumorigenesis.

Cooperative tumor inhibition by CpG-oligodeoxynucleotide and cyclic dinucleotide in head and neck cancer involves T helper cytokine and macrophage phenotype reprogramming

Head and neck cancer ranks as the sixth most common cancer worldwide, highlighting the critical need for the development of new therapies to enhance treatment efficacy. The activation of innate immune receptors given their potent immune stimulatory properties aid in the eradication of cancer cells. In this study, we investigated the immune mechanism and anti-tumor function of a Toll-like receptor 9 (TLR9) agonist, CpG-oligodeoxynucleotide-2722 (CpG-2722), in combination with cyclic dinucleotides, which are agonists of stimulator of interferon genes (STING). Our results revealed that CpG-2722 stimulation increased the expression of Th1 pro-inflammatory cytokines. Stimulation by STING agonists exhibited lower expression of Th1 cytokines but higher expression of Th2 cytokines compared to CpG-2722. However, the combination of these two agonists significantly enhanced Th1 cytokines while reducing Th2 cytokines. Moreover, in vivo experiment showed that both CpG-2722 and 2'3'-c-di-AMP suppressed head and neck tumor growth, with their combination proving more effective than the use of these agonists alone. The combined treatment cooperatively promoted the production of Th1 cytokines and type I interferons, while suppressing Th2 cytokines in the tumors as observed in vitro. Additionally, it led to the accumulation of M1 macrophages, dendritic cells, and T cells, shaping a favorable tumor microenvironment for T cell-mediated tumor killing. The anti-tumor activity of the CpG-2722 and 2'3'-c-di-AMP combination depends on the macrophage presence but does not directly activate M1 macrophage polarization, instead working through a reprogrammed cytokine profile. Furthermore, this combination shows a cooperative anti-tumor activity with anti-PD-1 in treating head and neck tumors. Overall, these findings highlight a Th response and macrophage phenotype reprograming involved functional mechanism underlying the cooperative activity of the combination of TLR9 and STING agonists in the immunotherapy of head and neck cancer.

Discovery and development of INNA-051, a TLR2/6 agonist for the prevention of complications resulting from viral respiratory infections.

Viral respiratory infection is associated with significant morbidity and mortality. The diversity of viruses implicated, coupled with their propensity for mutation, ignited an interest in host-directed antiviral therapies effective across a wide range of viral variants. Toll-like receptors (TLRs) are potential targets for the development of broad-spectrum antivirals given their central role in host immune defenses. Synthetic agonists of TLRs have been shown to boost protective innate immune responses against respiratory viruses. However, clinical success was hindered by short duration of benefit and/or induction of systemic adverse effects. INNA-051, a TLR2/6 agonist, is in development as an intranasal innate immune enhancer for prophylactic treatment in individuals at risk of complications resulting from respiratory viral infections. In vivo animal demonstrated the efficacy as prophylaxis against multiple viruses including SARS-CoV-2, influenza, and rhinovirus. Early clinical trials demonstrated an acceptable safety and tolerability profile. Intranasal delivery to the primary site of infection in humans induced a local innate host defense response characterized by innate immune cell infiltration into the nasal epithelium and activation and antiviral response genes . Taken together, the preclinical and clinical data on INNA-051 support further investigation of its use in community infection setting.

Recombinant African swine fever virus p30-flagellin fusion protein promotes p30-specific humoral and cellular immune responses in mice.

African swine fever (ASF) is a transmissible and deadly viral disease caused by the African swine fever virus (ASFV) that has considerably affected the global pig industry. Vaccination is considered a potentially effective method to control ASF. However, live attenuated vaccines can't protect against all circulating virus isolates. Subunit vaccines can induce both cellular and humoral immune responses, but often require the addition of adjuvants. Flagellin, a stimulator of Toll-like receptor 5 (TLR5), functions as a potent adjuvant by enhancing cellular and humoral immune responses. However, its high antigenicity may cause severe systemic inflammation. In this study, an Escherichia coli expression system was used to express ASFV p30 protein (p30) fused with Salmonella Typhimurium FliCΔD2D3 (without the D2 and D3 domains of FliC). The immunological effect of p30-FlicΔD2D3 protein in mice was evaluated. Results revealed that the ASFV p30 protein and the p30-FlicΔD2D3 fusion protein were effectively expressed by the E. coli expression system. In vitro activity analysis showed that the p30-FlicΔD2D3 fusion protein could be recognized by ASFV-positive serum, had good immunoreactivity, and remarkably promoted IL-8 secretion related to TLR5 activity in HEK293-mTLR5 cells. However, p30-FlicΔD2D3 induced significantly lower levels of inflammatory factor IL-8 than that induced by wild-type flagellin. Immunization with the p30-FlicΔD2D3 fusion protein considerably promoted cellular and humoral immune responses in mice. Therefore, the p30-FlicΔD2D3 protein retained good immune reactivity and TLR5 agonist efficacy. It also enhanced humoral and cellular immune responses in mice. This work offered valuable information that will be helpful to develop ASF subunit vaccines.