TLR Agonists Research Articles

Regulation of IL-10 production in dendritic cells is controlled by the co-activation of TLR2 and Mincle by Lactiplantibacillus plantarum OLL2712.

We showed that Lactiplantibacillus plantarum OLL2712 (OLL2712) strongly induces interleukin (IL)-10 production in immune cells. Although beneficial effects of this strain have been observed in both mice and humans, the mechanisms underlying IL-10 induction remain unclear. In this study, we found that OLL2712 co-activates two pattern recognition receptors, leading to IL-10 production in the mouse-derived thermosensitive dendritic cell line, tsDC. We first revealed the involvement of the Toll-like receptor (TLR)2-Myeloid differentiation primary response gene (MYD) 88 pathway in OLL2712-induced IL-10 production in tsDCs. However, stimulation with the TLR2 agonist alone was insufficient to induce IL-10 production. Consequently, we explored additional signaling pathways and found that the phosphorylation of spleen tyrosine kinase (Syk) was important in response to OLL2712, which was not triggered by a TLR2 agonist alone. Notably, the activation of Syk was found to depend on macrophage-inducible C-type lectin receptor (Mincle), one of the C-type lectin receptors. However, the surface-expressed Mincle is not responsible for the IL-10 production by OLL2712. Instead, it depends on the incorporation of OLL2712 into tsDCs, suggesting that Mincle recognizes incorporated OLL2712 intracellularly. In summary, OLL2712 is initially recognized by TLR2, which subsequently induces the expression of Mincle to recognize incorporated OLL2712, ultimately inducing IL-10 production.IMPORTANCEThe objective of this study is to elucidate the mechanism by which Lactiplantibacillus plantarum OLL2712 (OLL2712), previously identified by our research group as a potent stimulator of interleukin-10 production in immune cells, exerts its immunomodulatory effects. Our findings indicate that OLL2712 acts in synergy with two pattern-recognition receptors: Toll-like receptor 2 and Macrophage inducible C-type lectin receptor (Mincle). Additionally, we observed that OLL2712 needs to be internalized intracellularly to be recognized by Mincle. These findings represent the first insights into the detailed mechanism underlying the anti-inflammatory effects of OLL2712.

PfCSP-ferritin nanoparticle malaria vaccine antigen formulated with aluminum-salt and CpG 1018® adjuvants: Preformulation characterization, antigen-adjuvant interactions, and mouse immunogenicity studies

ABSTRACT Circumsporozite protein (CSP), the most abundant surface protein in parasitic Plasmodium falciparum (Pf) sporozoite and an attractive target for malaria vaccine design, has been shown to induce protective humoral response in humans. In this work, we characterized and formulated a promising recombinant PfCSP immunogen (155) candidate consisting of two PfCSP epitopes (i.e. junction, NANP repeat) fused to H. pylori apoferritin forming a 24-mer nanoparticle. In addition, two N-linked glycans were engineered to mitigate possible anti-apoferritin immune responses, and a universal T-cell epitope was included to further enhance immunogenicity. Physicochemical characterization of the 155 antigen was performed including primary structure, post-translational modifications, conformational stability, and particle size. A competitive ELISA was developed to assess antigen binding to a PfCSP-specific mAb. The in vitro antigenicity of the 155 antigen was measured upon formulation with adjuvants, including aluminum-salts (i.e. AlhydrogelTM, Adju-PhosTM) and the TLR-9 agonist CpG 1018®, when freshly combined and after storage at different temperatures over 3 months. The in vivo immunological impact of various adjuvanted formulations of the 155 antigen was investigated in mice. The results support the formulation of 155 with AlhydrogelTM + CpG 1018® adjuvants as a promising recombinant malaria vaccine candidate from both a pharmaceutical and immunological perspective.

A VZV-gE subunit vaccine decorated with mPLA elicits protective cellular immmune responses against varicella-zoster virus.

Herpes zoster is an acute infectious skin disease caused by the reactivation of latent varicella-zoster virus, vaccination, such as subunit vaccine with good safety, can effectively prevent shingles through increasing immunity of the body. However, protein antigens are prone to degradation and inactivation, which alone is generally not sufficient to induce potent immune effect. In this study, the liposomal vaccine platform modified with mPLA (TLR4 agonist) was developed to improve the immunogenicity of glycoprotein E (VZV-gE) derived from herpes zoster virus. The thin-film dispersion and freeze-drying methods were employed to encapsulate VZV-gE against degradation, enhance liposomal stability, and achieve better redissolution effects with an optimized cryoprotectant. The in vitro results presented that mPLA could effectively enhance the uptake of VZV-gE with DC2.4. In vivo immune effect evaluation showed that the prepared subunit vaccines could induce stronger IgG, IgG1, and IgG2a antibody levels in the mouse serum, improving humoral immune effects. And the secretion levels of Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10) in the splenocytes were significantly increased, inducing protective cellular immune responses. Overall, this work presented that combining immunomodulatory adjuvants decorated nanocarriers to develop subunit vaccine platforms was a promising strategy to prevent the occurrence of herpes zoster effectively.

Huanglian Ganjiang Decoction alleviates DSS-induced colitis through suppressing inflammation and protecting intestinal barrier: From the perspective of disassembling prescriptions.

Huanglian Ganjiang decoction (HGD), which is composed of Chinese medicines with cold, warm, and astringent properties, has demonstrated significant therapeutic efficacy in ulcerative colitis (UC). However, the underlying mechanisms remain unclear, highlighting the need for a multi-faceted investigation. Disassembling prescriptions is a crucial approach for investigating compatibility mechanisms. Analyzing the interactions among Chinese herbs through this method can refine treatment focus and provide novel insights into TCM prescription compatibility research. Therefore, HGD was disassembled into three groups: Cold Medicine Removed (C-R), which warm medicine and astringent medicine are combined; Warm Medicine Removed (W-R), which cold medicine and astringent medicine are combined; and Astringent Medicine Removed (A-R), which cold medicine and warm medicine are combined. To elucidate the therapeutic effects of HGD and its three disassembled prescriptions against UC and to uncover their compatibility mechanisms. The chemical composition of HGD and its disassembled prescriptions (HGDADPs) was qualitatively analyzed using UPLC-MS/MS. 3% dextran sulfate sodium (DSS) was used to induce a UC mouse model. The efficacy in treating UC was evaluated by body weight loss, disease activity index (DAI), colon length, spleen index, thymus index, and histopathological score. A compound-UC target network was established utilizing the network pharmacology. The underlying mechanisms were then investigated by assessing intestinal barrier function and inflammatory responses, as well as the APOC1/P38 MAPK and TLR4/NF-κB signaling pathways through qRT-PCR, Western blotting, and immunofluorescence. Subsequently, anisomycin, a P38 MAPK agonist, was used to confirm whether C-R protects the gut barrier via the APOC1/P38 MAPK pathway. Monophosphoryl lipid A (MPLA), a TLR4 agonist, was employed to investigate whether W-R mediates its anti-inflammatory effects via the TLR4/NF-κB signaling pathway. HGDADPs improved colitis symptoms, increasing ZO-1, Occludin, Claudin-1, and E-cadherin levels while reducing blood cell counts and IL-6 and IL-1β levels. HGD was the most effective in reducing inflammation and repairing the intestinal barrier, with A-R showing similar effects. C-R excelled in repairing the barrier, while W-R was better at reducing inflammation. Network pharmacology indicated C-R inhibits the APOC1/P38 MAPK pathway, and W-R suppresses the TLR4/NF-κB pathway, aligning with Western blotting results. ANI and MPLA reversed the effects of C-R and W-R, respectively. The mechanism by which HGDADPs treat colitis is demonstrated for the first time in our study. In HGD, cold medicine serves as the "Jun", primarily exerting anti-inflammatory effects; Warm medicine acts as the "Chen", mainly protecting the intestinal mucosal barrier; And astringent medicine functions as the "Zuo", playing a synergistic role in the treatment of colitis. In addition, the combination of cold and warm medicines (A-R) was the most crucial for HGD' s compatibility. The pairing of warm and astringent medicines (C-R) significantly contributed to the restoration of the gut barrier and the inhibition of the APOC1/P38 MAPK signaling pathway. Meanwhile, the combination of cold and astringent medicines (W-R) primarily contributed to alleviating inflammation and inhibiting the TLR4/NF-κB pathway.

Intralesional injection of CpG ODNs complexed with glatiramer acetate mitigates systemic cytokine toxicities and synergistically advances checkpoint blockade efficacy.

PD-L1/PD-1 checkpoint inhibitors (CPIs) are mainstream agents for cancer immunotherapy, but the prognosis is unsatisfactory in solid tumor patients lacking preexisting T-cell reactivity. Adjunct therapy strategies including the intratumoral administration of immunostimulants aim to address this limitation. CpG oligodeoxynucleotides (ODNs), TLR9 agonists that can potentiate adaptive immunity, have been widely investigated to tackle PD-L1/PD-1 resistance, but clinical success has been hindered by inconsistent efficacy and immune-related toxicities caused by systemic exposure. Here, we utilized glatiramer acetate (GA), the FDA-approved, lysine-rich polypeptidesto complex CpG into polycationic nanoparticles (R4B) and investigated the safety and antitumor efficacy of CpG ODNs in the murine CT26 colorectal carcinoma model. In a maximum tolerated dose study, repetitive R4B treatment displayed comparable antitumor efficacy to CpG alone treatment within a dose range from 15µg to 150µg while significantly attenuating systemic proinflammatory cytokine IL-6 release. A pharmacokinetic and biodistribution analysis confirmed that R4B localized and gradually released CpG around the lesions within 96h while 'naked' CpG quickly diffused from the injection site. Genome-wide transcriptome analysis validated that R4B treatment activated prominent TLR9-driven immune system responses in both lesions and spleens. In a CT26 multiple tumor model, intratumoral administration of R4B generated systemic immune efficacy, evidenced by an abscopal effect on untreated tumors. Notably, R4B treatment accomplished these effects with mitigated systemic proinflammatory cytokines when compared with CpG alone. We further discovered that combining R4B with anti-PD-1 treatment led to the most pronounced effects on tumor growth and longest benefits to survival time. Our investigation into possible mechanisms underlying this phenomenon included increased recruitment of cytotoxic CD8+ T cells and natural killer (NK) cells to the tumor microenvironments and the reversal of PD-L1/PD-1 axis inhibition. In summary, these results warrant further investigation for safely improving clinical responses in CPI-resistant solid tumor patients with localized CpG ODN therapy.

Epstein-Barr Virus-Induced 3 Attributes to TLR7-Mediated Splenomegaly.

Epstein-Barr virus-induced 3 (EBI3) functions as a component of the heterodimer cytokine IL-27, which regulates innate and acquired immune responses. The expression of EBI3 gene is induced by Toll-like receptors (TLRs). Repeated treatment with imiquimod (IMQ), a TLR7 agonist, induces splenomegaly and cytopaenia due to increased splenic function. Although immune cell activation is speculated to play a role in chronic infection-mediated splenomegaly, the detailed mechanisms remain unknown. This study shows that IMQ treatment induces marked splenomegaly and severe bicytopaenia (anaemia and thrombocytopaenia) in wild-type mice. In IMQ-treated mice, myeloid cell populations in the spleen increased, and extramedullary haematopoiesis was observed. RNA-seq analysis revealed the upregulation of type I interferon (IFN)-related genes in the spleens of IMQ-treated mice. IMQ-induced pathological changes were partially mitigated by EBI3 deficiency. To investigate the mechanism of the improved phenotypes in the Ebi3 KO mice, we examined the involvement of IL-27, a heterodimer of EBI3 and IL-27p28. The expression of Il27a, which encodes IL-27p28, was increased in the spleen and peripheral blood by IMQ treatment. Furthermore, IL-27 stimulation upregulated type I IFN-related genes in bone marrow-derived macrophage cultures without type I IFN. These findings suggest that EBI3 deficiency mitigated IMQ-mediated pathological changes, presumably via a lack of IL-27 formation. Our study thus provides insights into the molecular mechanisms underlying chronic infection-mediated splenomegaly.

Combination adjuvant improves influenza virus immunity by downregulation of immune homeostasis genes in lymphocytes.

Adjuvants play a central role in enhancing the immunogenicity of otherwise poorly immunogenic vaccine antigens. Combining adjuvants has the potential to enhance vaccine immunogenicity compared with single adjuvants, although the cellular and molecular mechanisms of combination adjuvants are not well understood. Using the influenza virus hemagglutinin H5 antigen, we define the immunological landscape of combining CpG and MPLA (TLR-9 and TLR-4 agonists, respectively) with a squalene nanoemulsion (AddaVax) using immunologic and transcriptomic profiling. Mice immunized and boosted with recombinant H5 in AddaVax, CpG+MPLA, or AddaVax plus CpG+MPLA (IVAX-1) produced comparable levels of neutralizing antibodies and were equally well protected against the H5N1 challenge. However, after challenge with H5N1 virus, H5/IVAX-1-immunized mice had 100- to 300-fold lower virus lung titers than mice receiving H5 in AddaVax or CpG+MPLA separately. Consistent with enhanced viral clearance, unsupervised expression analysis of draining lymph node cells revealed the combination adjuvant IVAX-1 significantly downregulated immune homeostasis genes, and induced higher numbers of antibody-producing plasmablasts than either AddaVax or CpG+MPLA. IVAX-1 was also more effective after single-dose administration than either AddaVax or CpG+MPLA. These data reveal a novel molecular framework for understanding the mechanisms of combination adjuvants, such as IVAX-1, and highlight their potential for the development of more effective vaccines against respiratory viruses.

Toll-like Receptors 1, 3 and 7 Activate Distinct Genetic Features of NF-κB Signaling and γ-Protocadherin Expression in Human Cardiac Fibroblasts.

Fibroblasts play a pivotal role in key processes within the heart, particularly in cardiac remodeling that follows both ischemic and non-ischemic injury. During remodeling, fibroblasts drive fibrosis and inflammation by reorganizing the extracellular matrix and modulating the immune response, including toll-like receptor (TLR) activation, to promote tissue stabilization. Building on findings from our prior research on heart tissue from patients with advanced coronary artery disease and aortic valve disease, this study sought to explore specific effects of TLR1, TLR3, and TLR7 activation on NF-κB signaling, proinflammatory cytokine production, and γ-protocadherin expression in cardiac fibroblasts. Human cardiac fibroblasts were exposed to agonists for TLR1, TLR3, or TLR7 for 24h, followed by an analysis of NF-κB signaling, cytokine production, and γ-protocadherin expression. The activation of these TLRs triggered distinct responses in the NF-κB signaling pathway, with TLR3 showing a stronger activation profile compared to TLR1 and TLR7, particularly in downregulating γ-protocadherin expression. These findings highlight a potential role for TLR3 in amplifying inflammatory responses and reducing γ-protocadherin levels in cardiac fibroblasts, correlating with the enhanced inflammation and lower γ-protocadherin expression observed in diseased myocardium from patients with coronary artery disease and aortic valve disease. Consequently, TLR3 represents a potential therapeutic target for modulating immune responses in cardiovascular diseases.

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

COVID-19 vaccines effectively prevent severe disease, but unequal distribution, especially in low- and middle-income countries, has led to vaccine-resistant strains. This highlights the urgent need for alternative vaccine platforms that are safe, thermostable, and easy to distribute. This study evaluates the immunogenicity, stability, and scalability of a dissolved microneedle array patch (MAP) delivering the rS1RS09 subunit vaccine, comprising the SARS-CoV-2 S1 monomer and RS09, a TLR-4 agonist peptide. The rS1RS09 vaccine was administered via MAP or intramuscular injection in murine models. The immune responses of the MAP with and without gamma irradiation as terminal sterilization were assessed at doses of 5, 15, and 45 µg, alongside neutralizing antibody responses to Wuhan, Delta, and Omicron variants. The long-term storage stability was also evaluated through protein degradation analyses at varying temperatures. The rS1RS09 vaccine elicited stronger immune responses and ACE2-binding inhibition than S1 monomer alone or trimer. The MAP delivery induced sgnificantly higher and longer-lasting S1-specific IgG responses for up to 70 weeks compared to intramuscular injections. Robust Th2-prevalent immune responses were generated in all the groups vaccinated via the MAP and significant neutralizing antibodies were elicited at 15 and 45 µg, showing dose-sparing potential. The rS1RS09 in MAP has remained stable with minimal protein degradation for 19 months at room temperature or under refrigeration, regardless of gamma-irradiation. After an additional month of storage at 42 °C, cit showed less than 3% degradation, ompared to over 23% in liquid vaccines Conclusions: Gamma-irradiated MAP-rS1RS09 is a promising platform for stable, scalable vaccine production and distribution, eliminating cold chain logistics. These findings support its potential for mass vaccination efforts, particularly in resource-limited settings.

Baicalin ameliorates neuroinflammation by targeting TLR4/MD2 complex on microglia via PI3K/AKT/NF-κB signaling pathway.

This study aims to elucidate the target and mechanism of baicalin, a clinically utilized drug, in the treatment of neuroinflammatory diseases. Neuroinflammation, characterized by the activation of glial cells and the release of various pro-inflammatory cytokines, plays a critical role in the pathogenesis of various diseases, including spinal cord injury (SCI). The remission of such diseases is significantly dependent on the improvement of inflammatory microenvironment. Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD2) complex plays an important role in pathogen recognition and innate immune activation. baicalin, a natural flavonoid, is renowned for its potent anti-inflammatory property. In this study, we discovered that baicalin significantly reduced the activation of glial cells and the levels of pro-inflammatory cytokines at the lesion site of SCI mice, thereby mitigating demyelination and neuronal damage. By directly occupying the active pocket of TLR4/MD2 complex on microglia, baicalin inhibited PI3K/AKT/NF-κB pathway, thereby exerting its anti-inflammatory effect. These findings were corroborated in mice induced by lipopolysaccharide, a TLR4 agonist. Furthermore, baicalin indirectly altered phenotype of astrocytes by reducing secretion of TNF-α, IL-1α, and C1q levels from microglia. Our work demonstrated that baicalin effectively alleviated neuroinflammation by directly targeting microglia and indirectly modulating astrocytes phenotype. As a natural flavonoid, baicalin holds significant potential as a therapeutic candidate for diseases characterized by neuroinflammation.

In Silico-Guided Discovery of Polysaccharide Derivatives as Adjuvants in Nanoparticle Vaccines for Cancer Immunotherapy.

Cancer vaccines utilizing nanoparticle (NP) structures that integrate antigens and adjuvants to enhance delivery and stimulate immune responses are emerging as a promising avenue in cancer immunotherapy. However, the development of cancer vaccines has been significantly hindered by the low immunogenicity of tumor antigens. To address this challenge, substantial efforts have been made in developing innovative adjuvants to elicit effective immune responses. In this study, we develop a NP cancer vaccine assisted by a polysaccharide derivative adjuvant, designed through a computational strategy, to evoke effective antigen-specific antitumor immunity. Using TLR4 as the putative receptor, we conducted a comprehensive evaluation of a prescreening library consisting of 34 inulin derivatives through docking and molecular dynamics simulation. Consequently, a new derivative, benzoylated inulin (InBz), is selected as the most promising TLR4 agonist. The adjuvant effect of InBz is evaluated by fabricating InBz NPs encapsulating the model antigen ovalbumin (OVA). In vitro, InBz-OVA NPs effectively activate the TLR4 signaling pathways and facilitate dendritic cell maturation, thereby enhancing the antigen delivery and presentation. In vivo, InBz-OVA NPs outperform a commercial aluminum-based adjuvant, elicit robust antibody titers, induce antigen-specific cytotoxic T lymphocytes, and achieve significant tumor suppression in murine models. Besides, the adjuvant effects of other representative derivatives, namely, acetylated and chloroacetylated inulin, with moderate and low potential from the library, are also chemically synthesized and experimentally evaluated and found to be in agreement with computational predictions, confirming the credibility of the strategy. This study provides an effective platform for the pursuit of efficient polysaccharide-based vaccine adjuvants.

Novel Imidazo[4,5-c]pyridine Compounds as TLR7 Agonists for Treating Cancer

Novel Imidazo[4,5-<i>c</i>]pyridine Compounds as TLR7 Agonists for Treating Cancer

Helminth infection induces innate immune priming in plasmacytoid dendritic cells.

Heligmosomoides polygyrus co-infection is reported to have protective antiviral effects against pulmonary viral infections. To investigate a potential underlying mechanism, we infected C57BL/6 mice with H. polygyrus larvae for two weeks. Bone marrow (BM)-derived plasmacytoid dendritic cells (pDCs) were generated and stimulated with TLR agonists. We found increased expression of type 1 interferon genes (Ifnα1, Ifnα4, Ifnβ1, Mx1, Isg15), increased TNF, IL-6, IL-10 secretion, and higher expression of antigen presentation markers in BM-derived-pDCs from infected mice compared to naïve control. Our findings may partly explain the mechanism of the antiviral protection previously reported in acute helminth infections.

TLR Agonist Immunoadjuvants Provide Effective Protection Against PCV2 and PRV Infections in a Bivalent Subunit Vaccine for PCV2 and PRV.

Diseases associated with porcine circovirus type 2 (PCV2) and pseudorabies virus (PRV) significantly affect the economy of pig farms, particularly when combined infections lead to bacterial co-infections. Antigens from the pseudorabies variant strain gB and gD proteins and PCV2 (genotyped) Cap protein were mixed with the pattern recognition receptor (PRR) agonist FLICd as adjuvants and formulated with a micro-hydrogel adjuvant into PCV2 and PRV bivalent subunit vaccines. Twenty pigs, aged 30-35 days, were divided into groups A (received bivalent subunit vaccine) and B (received bivalent subunit vaccines with recombinant FLICd adjuvant), as well as C (non-vaccinated challenge control) and D (blank control). Groups A and B showed no significant difference in average daily weight gain compared to the unvaccinated controls. Fourteen days post-second vaccination, groups A and B exhibited significantly higher levels of PRV and PCV2 antibodies than groups C and D. Group B showed significantly higher average titers of PRV-specific neutralizing antibodies than group A. Fourteen days post-second vaccination, a PRV (ZJM-1 strain) challenge test was conducted. The vaccinated group achieved 100% protection. Vaccination effectively reduced virus load post-challenge and shortened the PRV shedding period. Vaccination with PCV2 and PRV bivalent subunit vaccines effectively prevents the onset of PCV2-related diseases and infections by wild pseudorabies strains.

LncRNA CARINH regulates expression and function of innate immune transcription factor IRF1 in macrophages.

The discovery of long non-coding RNAs (lncRNAs) has provided a new perspective on the centrality of RNA in gene regulation and genome organization. Here, we screened for lncRNAs with putative functions in the host response to single-stranded RNA respiratory viruses. We identify CARINH as a conserved cis-acting lncRNA up-regulated in three respiratory diseases to control the expression of its antisense gene IRF1, a key transcriptional regulator of the antiviral response. CARINH and IRF1 are coordinately increased in the circulation of patients infected with human metapneumovirus, influenza A virus, or SARS-CoV-2, and in macrophages in response to viral infection or TLR3 agonist treatment. Targeted depletion of CARINH or its mouse ortholog Carinh in macrophages reduces the expression of IRF1/Irf1 and their associated target gene networks, increasing susceptibility to viral infection. Accordingly, CRISPR-mediated deletion of Carinh in mice reduces antiviral immunity, increasing viral burden upon sublethal challenge with influenza A virus. Together, these findings identify a conserved role of lncRNA CARINH in coordinating interferon-stimulated genes and antiviral immune responses.

NF-κB-Inducing Kinase Is Essential for Effective c-Rel Transactivation and Binding to the Il12b Promoter in Macrophages.

This study investigates the role of NIK in activating specific inflammatory genes in macrophages, focusing on the effect of a mutation in NIK found in alymphoplasia (aly/aly) mice. Mouse peritoneal macrophages from aly/aly mice showed a severe defect in the production of some pro-inflammatory cytokines, such as IL-12. This effect seemed to take place at the transcriptional level, as shown by the reduced transcription of Il12b and Il12a in aly/aly macrophages after exposure to the TLR4 agonist LPS. Immunoprecipitation studies showed that the binding of NIK to c-Rel was not efficient in RAW 264.7 cells over-expressing the aly/aly mutation. In addition, the shuttling of c-Rel to the nucleus was shown to be impaired in aly/aly macrophages in response to LPS. When looking more specifically at the regulation of the Il12b promoter, we found that c-Rel bound to the NF-kB consensus sequence in macrophages from WT mice 1 hr. after LPS challenge, whereas in aly/aly macrophages, the transcription factor bound to the promoter was p65. These findings indicate that NIK is essential for efficient c-Rel activation and proper inflammatory responses. NIK dysfunction could lead to weakened immune responses, and targeting this pathway may help in developing therapies for immune-related conditions.

A phase I clinical trial adding OX40 agonism to in situ therapeutic cancer vaccination in patients with low-grade B cell lymphoma highlights challenges in translation from mouse to human studies.

Activating T cell costimulatory receptors is a promising approach for cancer immunotherapy. In preclinical work, adding an OX40 agonist to in situ vaccination (ISV) with SD101, a TLR9 agonist, was curative in a mouse model of lymphoma. We sought to test this combination in a Phase I clinical trial for patients with low-grade B cell lymphoma. We treated 14 patients with low-dose radiation, intratumoral SD101, and intratumoral and intravenous BMS986178, an agonistic anti-OX40 antibody. The primary outcome was safety. Secondary outcomes included overall response rate and progression-free survival. Adverse events were consistent with prior experience with low-dose radiation and SD101. No synergistic or dose-limiting toxicities were observed. One patient had a partial response, and 9 patients had stable disease, a result inferior to our experience with TLR9 agonism and low-dose radiation alone. Flow cytometry and single cell RNA sequencing of serial tumor biopsies revealed that T and NK cells were activated after treatment. However, high baseline OX40 expression on T follicular helper and T regulatory type 1 cells, as well as high post-treatment soluble OX40, shed from these T cells upon activation, associated with progression-free survival of less than 6 months. Clinical results of T cell costimulatory receptor agonism have now repeatedly been inferior to the motivating preclinical results. Our study highlights potential barriers to clinical translation, particularly differences in preclinical and clinical reagents and the complex biology of these coreceptors in heterogenous T cell subpopulations, some of which may antagonize immunotherapy.

Immunoregulatory and antiviral effect mediated by TLR7 and BMAP28 interaction in bovine alphaherpesvirus-infected respiratory primary cultures.

Varicellovirus bovinealpha 1 and 5 (formerly bovine alphaherpesvirus type 1 and 5, BoAHV-1 and BoAHV-5) are closely related and can be isolated from similar clinical conditions, including respiratory and nervous diseases, genital infections and abortion. Pathogens' activation of toll-like receptors (TLRs) induces the expression of proinflammatory cytokines and antimicrobial peptides such as cathelicidins. Cathelicidins are presumed to act as endogenous ligands of TLRs, stimulating, in turn, their activation. Cattle's innate immune responses mediated by TLRs and cathelicidins remain undefined. This work aimed to study the interaction mechanisms between TLR7 and a bovine cathelicidin (bovine myeloid antimicrobial peptide (BMAP)28 and their influence on the replication of BoAHV-1 and BoAHV-5. It was shown that in vitro infection by BoAHV-1 and BoAHV-5 induces the innate immune responses associated with TLR7 and BMAP28 in primary cultured bovine fetal lung cells. Pretreatment with TLR7 agonist (imiquimod) or an endogenous cathelicidin inductor (sodium butyrate) enhanced the innate immune response to BoAHV mediated by TLR7 signalling, BMAP28, TNFα, IFNβ and IFNγ. It exerted an antiviral effect, decreasing BoAHV replication. Inhibition of endosomal TLRs and downstream signalling molecules MAPK, NF-κβ, IKK-2, and JNK II counteracted these antiviral effects. Therefore, BMAP28 shows novel roles in modulating the immune response induced by TLR7 activation through signalling pathways involving kinases and NF-κβ. Enhancing TLR7 and endogenous cathelicidin expression could be a complementary tool for the prevention and/or control of BoAHV infections.

Engineering a Novel T‐Cell Epitope‐Based Vaccine Targeting PRAME in Cancer Immunotherapy

ABSTRACTPRAME, a cancer/testis antigen, is often expressed abnormally in various cancers, making it a hopeful candidate for cancer immunotherapy. This study aimed to create a vaccine targeting PRAME using immunoinformatics methods. The PRAME protein sequence was obtained from UniProtKB, and a TLR4 agonist sequence from Mycobacterium tuberculosis was included as an adjuvant. Potential T‐cell epitopes were forecasted using the IEDB server and assessed for their antigenicity, lack of allergenicity, and binding affinities to human and murine MHC molecules. Selected epitopes were then combined into a multi‐epitope structure using suitable linkers. The vaccine's physical and chemical properties, 3D structure, and interaction with TLR4 were examined through computational analyses. Five promising MHC‐I and five MHC‐II epitopes from PRAME were pinpointed and incorporated into the multi‐epitope vaccine and the TLR4 agonist adjuvant. The vaccine displayed advantageous biochemical traits, including high stability, solubility, and antigenicity. Molecular docking simulations showcased a steady interaction between the vaccine and TLR4, with a binding energy of −250.5 kcal/mol. Molecular dynamics analyses confirmed the structural stability and minimal distortion of the vaccine‐TLR4 complex. This study introduces a fresh approach to vaccine design targeting PRAME, a cancer‐related antigen. The vaccine structure exhibits promising physical, chemical, and immunological characteristics, suggesting its potential to trigger robust cellular and humoral immune responses against PRAME‐positive cancers.

Screening of Oncogenic Proteins and Development of a Multiepitope Peptide Vaccine Targeting AKT1 and PARP1 for Breast Cancer by Integrating Reverse Vaccinology and Immune-Informatics Approaches.

Breast cancer remains a significant global health challenge, requiring innovative therapeutic strategies. In silico methods, which leverage computational tools, offer a promising pathway for vaccine development. These methods facilitate antigen identification, epitope prediction, immune response modelling, and vaccine optimization, accelerating the design process. This study employed a reverse vaccinology approach combined with various bioinformatic tools to design a multi-epitope peptide vaccine. Using reverse vaccinology, AKT1 and PARP1 were identified as potential vaccine candidates, as their expression levels were significantly higher in breast cancer samples compared to healthy controls. The vaccine was designed by integrating immune cell epitopes with a TLR4 agonist as an adjuvant. It demonstrated high antigenicity, no allergenicity, and no toxicity. Validation of its 3D structure using the Ramachandran plot confirmed optimal conformation and stereochemical properties. Molecular docking and simulation studies showed the vaccine was stable and compact when interacting with TLR4. Moreover, the subunit vaccine effectively eliminated the antigen and triggered a strong IgG/IgM immune response lasting approximately one year (350 days). These findings suggest that the designed vaccine holds promise as a therapeutic option for breast cancer. However, further in vitro and in vivo studies are necessary to validate its efficacy before advancing to clinical trials.