Human TLR4 Research Articles

Intraoperative Guidance of Pancreatic Cancer Resection Using a Toll-like Receptor 2-Targeted Fluorescence Molecular Imaging Agent.

Human TLR2 is broadly expressed among pancreatic adenocarcinomas, and the highly specific TLR2L-800 fluorescence molecular imaging agent has potential for use in fluorescence-guided surgery to increase R0 margins and improve patient survival.

Fentanyl enhances immune cell response through TLR4/MD-2 complex.

Opioids have been shown to induce neuroinflammation and immune cell activation, that might contribute to some of the opioid side effects, such as opioid-induced tolerance and paradoxical hyperalgesia. In this context, TLR4/MD-2 complex has been proposed as an off-target site for opioid action. This study was aimed at investigating the effect of fentanyl on lipopolysaccharide (LPS)-induced TLR4/MD-2 activation in rat primary microglia and human monocyte-derived macrophages (MDM). The effect of fentanyl was first explored by measuring the expression and release of different proinflammatory mediators in primary rat microglia and human MDM by real-time PCR and ELISA. Then, the involvement of TLR4/MD-2 signaling was investigated studying NF-κB activation in HEK293 cells stably transfected with human TLR4, MD-2, and CD14 genes (HEK-Blue hTLR4 cells) and in human MDM. Fentanyl increased mRNA levels, as well as the LPS-induced secretion of proinflammatory mediators in primary microglia and MDM. Two inhibitors of TLR4/MD-2 signaling, namely the oxazoline derivative of N-palmitoylethanolamine (PEA-OXA) and CLI-095, blocked the production and release of proinflammatory cytokines by microglia stimulated with LPS and fentanyl, suggesting that TLR4/MD-2 could be the target of the proinflammatory activity of fentanyl. Finally, we showed that fentanyl in combination with LPS activated NF-κB signaling in human MDM and in HEK-Blue hTLR4 cells and this effect was blocked by inhibitors of TLR4/MD-2 complex. These results provide new insight into the mechanism of the proinflammatory activity of fentanyl, which involves the activation of TLR4/MD-2 signaling. Our findings might facilitate the development of novel inhibitors of TLR4/MD-2 signaling to combine with opioid-based analgesics for effective and safe pain management.

Programming Bordetella pertussis lipid A to promote adjuvanticity

BackgroundBordetella pertussis is the causative agent of whooping cough or pertussis. Although both acellular (aP) and whole-cell pertussis (wP) vaccines protect against disease, the wP vaccine, which is highly reactogenic, is better at preventing colonization and transmission. Reactogenicity is mainly attributed to the lipid A moiety of B. pertussis lipooligosaccharide (LOS). Within LOS, lipid A acts as a hydrophobic anchor, engaging with TLR4-MD2 on host immune cells to initiate both MyD88-dependent and TRIF-dependent pathways, thereby influencing adaptive immune responses. Lipid A variants, such as monophosphoryl lipid A (MPLA) can also act as adjuvants. Adjuvants may overcome the shortcomings of aP vaccines.ResultsThis work used lipid A modifying enzymes from other bacteria to produce an MPLA-like adjuvant strain in B. pertussis. We created B. pertussis strains with distinct lipid A modifications, which were validated using MALDI-TOF. We engineered a hexa-acylated monophosphorylated lipid A that markedly decreased human TLR4 activation and activated the TRIF pathway. The modified lipooligosaccharide (LOS) promoted IRF3 phosphorylation and type I interferon production, similar to MPLA responses. We generated three other variants with increased adjuvanticity properties and reduced endotoxicity. Pyrogenicity studies using the Monocyte Activation Test (MAT) revealed that these four lipid A variants significantly decreased the IL-6, a marker for fever, response in peripheral blood mononuclear cells (PBMCs).ConclusionThese findings pave the way for developing wP vaccines that are possibly less reactogenic and designing adaptable adjuvants for current vaccine formulations, advancing more effective immunization strategies against pertussis.

TgVax452, an epitope-based candidate vaccine targeting Toxoplasma gondii tachyzoite-specific SAG1-related sequence (SRS) proteins: immunoinformatics, structural simulations and experimental evidence-based approaches

BackgroundThe highly expressed surface antigen 1 (SAG1)-related sequence (SRS) proteins of T. gondii tachyzoites, as a widespread zoonotic parasite, are critical for host cell invasion and represent promising vaccine targets. In this study, we employed a computer-aided multi-method approach for in silico design and evaluation of TgVax452, an epitope-based candidate vaccine against T. gondii tachyzoite-specific SRS proteins.MethodsUsing immunoinformatics web-based tools, structural modeling, and static/dynamic molecular simulations, we identified and screened B- and T-cell immunodominant epitopes and predicted TgVax452’s antigenicity, stability, safety, adjuvanticity, and physico-chemical properties.ResultsThe designed protein possessed 452 residues, a MW of 44.07 kDa, an alkaline pI (6.7), good stability (33.20), solubility (0.498), and antigenicity (0.9639) with no allergenicity. Comprehensive molecular dynamic (MD) simulation analyses confirmed the stable interaction (average potential energy: 3.3799 × 106 KJ/mol) between the TLR4 agonist residues (RS09 peptide) of the TgVax452 in interaction with human TLR4, potentially activating innate immune responses. Also, a dramatic increase was observed in specific antibodies (IgM and IgG), cytokines (IFN-γ), and lymphocyte responses, based on C-ImmSim outputs. Finally, we optimized TgVax452’s codon adaptation and mRNA secondary structure for efficient expression in E. coli BL21 expression machinery.ConclusionOur findings suggest that TgVax452 is a promising candidate vaccine against T. gondii tachyzoite-specific SRS proteins and requires further experimental studies for its potential use in preclinical trials.

Extending the IMQ Model: Deep Characterization of the Human TLR7 Response for Early Drug Development.

Imiquimod (IMQ; brand name Aldara®) is a registered topical agent that has been proven to induce local inflammation via the Toll-like receptor (TLR)7 pathway. The purpose of this study was to characterize TLR7-mediated inflammation following 7days (168h) of topical IMQ exposure in healthy volunteers, and to compare the effects of short exposure (48h-72h) with prolonged exposure (120h-168h). IMQ (100mg) was applied under occlusion to 5 different tape-stripped treatment sites on the back of 10 healthy participants for a maximum of 7 consecutive days. Erythema and skin perfusion were measured daily up to 168h. Biopsies for immunohistochemical staining and RNA sequencing were collected at 0h, 48h, 72h, 120h and 168h post IMQ application. IMQ triggered an inflammatory response starting at 48h after application, including erythema and perfusion of the skin. At the transcriptomic level, IMQ induced TLR7 signalling, IRF involvement and activation of TNF signalling via NF-κB. Furthermore, an enhanced inflammatory response at the cellular level was observed after prolonged IMQ exposure, with cellular infiltration of dendritic cells, macrophages and T cells which was also corroborated by transcriptomic profiles. No difference was found in the erythema and perfusion response after 168h of IMQ exposure compared to 72h. Prolonged IMQ exposure revealed enhanced cellular responses and additional pathways with modulated activity compared to short exposure and can therefore be of interest as a model for investigational compounds targeting innate and adaptive immune responses.

Genome-level therapeutic targets identification and chimeric Vaccine designing against the Blastomyces dermatitidis

Blastomyces dermatitidis is a thermally dimorphic fungus that can cause serious and sometimes fatal infections, including blastomycosis. After spore inhalation, a pulmonary infection develops, which can be asymptomatic and have lethal effects, such as acute respiratory distress syndrome. Its most common extra-pulmonary sites are the central nervous system, bones, skin, and genito-urinary systems. Currently, no vaccine has been approved by the FDA to prevent this infection. In the study, a peptide-based vaccine was developed against blastomycosis by using subtractive proteomics and reverse vaccinology approaches. It focuses on mining the whole genome of B. dermatitidis, identifying potential therapeutic targets, and pinpointing potential epitopes for both B- and T-cells that are immunogenic, non-allergenic, non-toxic, and highly antigenic. Multi-epitope constructs were generated by incorporating appropriate linker sequences. A linker (EAAAK) was also added to incorporate an adjuvant sequence to increase immunological potential. The addition of adjuvants and linkers ultimately resulted in the formation of a vaccine construct in which the number of amino acids was 243 and the molecular weight was 26.18 kDa. The designed antigenic and non-allergenic vaccine constructs showed suitable physicochemical properties. The vaccine's structures were predicted, and further analysis verified their interactions with the human TLR-4 receptor through protein-protein docking. Additionally, MD simulation showed a potent interaction between prioritized vaccine-receptor complexes. Immune simulation predicted that the final vaccine injections resulted in significant immune responses for the T- and B-cell immune responses. Moreover, in silico cloning ensured a high expression possibility of the lead vaccine in the E. coli (K12) vector. This study offers an initiative for the development of effective vaccines against B. dermatitidis; however, it is necessary to validate the designed vaccine's immunogenicity experimentally.

Immune targeting of filarial glutaredoxin through a multi-epitope peptide-based vaccine: A reverse vaccinology approach

Despite the efforts of global programme to eliminate lymphatic filariasis (GPELF), the threat of lymphatic filariasis (LF) still looms over humanity in terms of long-term disabilities, and morbidities across the globe. In light of this situation, investigators have chosen to focus on the development of immunotherapeutics targeting the physiologically important filarial-specific proteins. Glutaredoxin (16.43 kDa) plays a pivotal role in filarial redox biology, serving as a vital contributor. In the context of the intra-host survival of filarial parasites, this antioxidant helps in mitigating the oxidative stress imposed by the host immune system. Given its significant contribution, the development of a vaccine targeting glutaredoxin holds promise as a new avenue for achieving a filaria-free world. Herein, multi-epitope-based vaccine was designed using advanced immunoinformatics approach. Initially, 4B-cell epitopes and 6 T-cell epitopes (4 MHC I and 2 MHC II) were identified from the 146 amino acid long sequence of glutaredoxin of the human filarid, Wuchereria bancrofti. Subsequent clustering of these epitopes with linker peptides finalized the vaccine structure. To boost TLR-mediated innate immunity, TLR-specific adjuvants were incorporated into the designed vaccine. After that, experimental analyses confirm the designed vaccine, Vac4 as anefficient ligand of human TLR5 to elicit protective innate immunity against filarial glutaredoxin. Immune simulation further demonstrated abundant levels of IgG and IgM as crucial contributors in triggering vaccine-induced adaptive responses in the recipients. Hence, to facilitate the validation of immunogenicity of the designed vaccine, Vac4 was cloned in silico in pET28a(+) expression vector for recombinant production. Taken together, our findings suggest that vaccine-mediated targeting of filarial glutaredoxin could be a future option for intervening LF on a global scale.

Comparative transcriptomic analysis reveals the molecular changes of acute pancreatitis in experimental models

BACKGROUND Acute pancreatitis (AP) encompasses a spectrum of pancreatic inflammatory conditions, ranging from mild inflammation to severe pancreatic necrosis and multisystem organ failure. Given the challenges associated with obtaining human pancreatic samples, research on AP predominantly relies on animal models. In this study, we aimed to elucidate the fundamental molecular mechanisms underlying AP using various AP models. AIM To investigate the shared molecular changes underlying the development of AP across varying severity levels. METHODS AP was induced in animal models through treatment with caerulein alone or in combination with lipopolysaccharide (LPS). Additionally, using Ptf1α to drive the specific expression of the hM3 promoter in pancreatic acinar cells transgenic C57BL/6J- hM3/Ptf1α(cre) mice were administered Clozapine N-oxide to induce AP. Subsequently, we conducted RNA sequencing of pancreatic tissues and validated the expression of significantly different genes using the Gene Expression Omnibus (GEO) database. RESULTS Caerulein-induced AP showed severe inflammation and edema, which were exacerbated when combined with LPS and accompanied by partial pancreatic tissue necrosis. Compared with the control group, RNA sequencing analysis revealed 880 significantly differentially expressed genes in the caerulein model and 885 in the caerulein combined with the LPS model. Kyoto Encyclopedia of Genes and Genomes enrichment analysis and Gene Set Enrichment Analysis indicated substantial enrichment of the TLR and NOD -like receptor signaling pathway, TLR signaling pathway, and NF-κB signaling pathway, alongside elevated levels of apoptosis-related pathways, such as apoptosis, P53 pathway, and phagosome pathway. The significantly elevated genes in the TLR and NOD -like receptor signaling pathways, as well as in the apoptosis pathway, were validated through quantitative real-time PCR experiments in animal models. Validation from the GEO database revealed that only MYD88 concurred in both mouse pancreatic tissue and human AP peripheral blood, while TLR1 , TLR7 , RIPK3 , and OAS2 genes exhibited marked elevation in human AP. The genes TUBA1A and GADD45A played significant roles in apoptosis within human AP. The transgenic mouse model hM3/Ptf1α(cre) successfully validated significant differential genes in the TLR and NOD -like receptor signaling pathways as well as the apoptosis pathway, indicating that these pathways represent shared pathological processes in AP across different models. CONCLUSION The TLR and NOD receptor signaling pathways play crucial roles in the inflammatory progression of AP, notably the MYD88 gene. Apoptosis holds a central position in the necrotic processes of AP, with TUBA1A and GADD45A genes exhibiting prominence in human AP.

MiR-574-5p activates human TLR8 to promote autoimmune signaling and lupus

Endosomal single-stranded RNA-sensing Toll-like receptor-7/8 (TLR7/8) plays a pivotal role in inflammation and immune responses and autoimmune diseases. However, the mechanisms underlying the initiation of the TLR7/8-mediated autoimmune signaling remain to be fully elucidated. Here, we demonstrate that miR-574-5p is aberrantly upregulated in tissues of lupus prone mice and in the plasma of lupus patients, with its expression levels correlating with the disease activity. miR-574-5p binds to and activates human hTLR8 or its murine ortholog mTlr7 to elicit a series of MyD88-dependent immune and inflammatory responses. These responses include the overproduction of cytokines and interferons, the activation of STAT1 signaling and B lymphocytes, and the production of autoantigens. In a transgenic mouse model, the induction of miR-574-5p overexpression is associated with increased secretion of antinuclear and anti-dsDNA antibodies, increased IgG and C3 deposit in the kidney, elevated expression of inflammatory genes in the spleen. In lupus-prone mice, lentivirus-mediated silencing of miR-574-5p significantly ameliorates major symptoms associated with lupus and lupus nephritis. Collectively, these results suggest that the miR-574-5p-hTLR8/mTlr7 signaling is an important axis of immune and inflammatory responses, contributing significantly to the development of lupus and lupus nephritis.

Trypanosoma cruzi killing and immune response boosting by novel phenoxyhydrazine-thiazole against Chagas disease

Trypanosoma cruzi (T. cruzi) causes Chagas, which is a neglected tropical disease (NTD). WHO estimates that 6 to 7 million people are infected worldwide. Current treatment is done with benznidazole (BZN), which is very toxic and effective only in the acute phase of the disease. In this work, we designed, synthesized, and characterized thirteen new phenoxyhydrazine-thiazole compounds and applied molecular docking and in vitro methods to investigate cell cytotoxicity, trypanocide activity, nitric oxide (NO) production, cell death, and immunomodulation. We observed a higher predicted affinity of the compounds for the squalene synthase and 14-alpha demethylase enzymes of T. cruzi. Moreover, the compounds displayed a higher predicted affinity for human TLR2 and TLR4, were mildly toxic in vitro for most mammalian cell types tested, and LIZ531 (IC50 2.8 μM) was highly toxic for epimastigotes, LIZ311 (IC50 8.6 μM) for trypomastigotes, and LIZ331 (IC50 1.9 μM) for amastigotes. We observed that LIZ311 (IC50 2.5 μM), LIZ431 (IC50 4.1 μM) and LIZ531 (IC50 5 μM) induced 200 μg/mL of NO and JM14 induced NO production in three different concentrations tested. The compound LIZ331 induced the production of TNF and IL-6. LIZ311 induced the secretion of TNF, IFNγ, IL-2, IL-4, IL-10, and IL-17, cell death by apoptosis, decreased acidic compartment formation, and induced changes in the mitochondrial membrane potential. Taken together, LIZ311 is a promising anti-T. cruzi compound is not toxic to mammalian cells and has increased antiparasitic activity and immunomodulatory properties.

Abstract LB081: Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma

Abstract Telomerase (TERT) is an enzyme critical for carcinogenesis expressed in about 90% of human cancers, including aggressive diffuse large B-cell lymphoma (DLBCL). Targeting TERT proven difficult due to on-target toxicities to non-malignant hematopoietic stem cells and potentially to activated immune effector cells. To overcome these challenges, we developed novel TLR9+ cell-selective CpG-oligodeoxynucleotides (ODNs) to deliver telomere-damaging nucleoside analogue, 6-thio-2’-deoxy-guanosine (6tdG), into target B cell lymphoma cells in vitro and in vivo. Human and mouse TLR9+/TERT+ lymphoma cells rapidly internalized CpG(6tdG)-oligonucleotides through active endocytosis. 6tdG molecules were released from the 3’ end of the oligonucleotide into cytosol and cell nucleus. TERT-mediated 6tdG incorporation into telomeres, led to DNA damage and lymphoma cell death. In vitro, CpG(6tdG)-oligonucleotides showed potency in targeting variety of human DLBCL cells in nanomolar range without detectable toxicity to non-malignant TERT— cells or human TERT+ hematopoietic stem cells and activated T cells. Importantly, the repeated systemic administration of CpG(6tdG)-oligonucleotides was well-tolerated by humanized hCD34/NOG mice, without detectable decrease in the percentage of hematopoietic stem cells, myeloid cells or T cells although the numbers of B cells were partly reduced as expected. Intravenously injected CpG(6tdG)-oligonucleotides, but not 6tdG nucleoside, had potent and dose-dependent antitumor effects against xenotransplanted models of human GC- or ABC-subtypes of DLBCL, such as OCI-Ly18 or OCI-Ly3, respectively. Furthermore, we assessed CpG(6tdG)-oligonucleotide activity against aggressive, syngeneic TERT+ Eμ-myc/15A lymphoma in immunocompetent mice. CpG(6tdG)-oligonucleotide, but not the CpG-oligonucleotide alone, 6tdG alone or co-injection of thereof, was effective in triggering regression of established 15A tumors with long-term survival of the majority of mice. The effect of CpG(6tdG)-oligonucleotide correlated with the activation of tumor antigen-specific T cell-mediated antitumor immune responses. In contrast, when tested in immunodeficient mice, CpG(6tdG)-oligonucleotide induced significant but transient regression of 15A lymphoma. These results suggest that the oligonucleotide elicits two-pronged activity triggering direct lymphoma cell cytotoxicity and also immune-mediated antitumor effects. Overall, this cell-selective strategy could provide an effective and safer strategy against aggressive TERT+ DLBCL cells with potential to engage and support T cell-based antitumor immunity. Citation Format: Chunsong Yu, Yong Liang, Piotr Swiderski, Larry Kwak, Marcin Kortylewski. Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB081.

Display of FliC131 on the Surface of Lactococcus lactis as a Strategy to Increase its Adjuvanticity for Mucosal Immunization

Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines.

Design and Synthesis of 3-(2H-Chromen-3-yl)-5-aryl-1,2,4-oxadiazole Derivatives as Novel Toll-like Receptor 2/1 Agonists That Inhibit Lung Cancer In Vitro and In Vivo.

Toll-like receptor (TLR) 2 is a transmembrane receptor that participates in the innate immune response by forming a heterodimer with TLR1 or TLR6. TLR2 agonists play an important role in tumor therapy. Herein, we synthesized a series of 3-(2H-chromen-3-yl)-5-aryl-1,2,4-oxadiazole derivatives and identified WYJ-2 as a potent small and selective molecule agonist of TLR2/1, with an EC50 of 18.57 ± 0.98 nM in human TLR2 and TLR1 transient-cotransfected HEK 293T cells. WYJ-2 promoted the formation of TLR2/1 heterodimers and activated the nuclear factor kappa B (NF-κB) signaling pathway. Moreover, our study indicated that WYJ-2 could induce pyroptosis in cancer cells, mediated by activating the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. WYJ-2 exhibited effective anti-non-small cell lung cancer (NSCLC) activity in vitro and in vivo. The discovery that activating TLR2/1 induces pyroptosis in cancer cells may highlight the prospects of TLR2/1 agonists in cancer treatment in the future.

The bacterial lysate OM-85 engages Toll-like receptors 2 and 4 triggering an immunomodulatory gene signature in human myeloid cells

OM-85 is a bacterial lysate used in clinical practice to reduce duration and frequency of recurrent respiratory tract infections. Whereas knowledge of its regulatory effects in vivo has substantially advanced, the mechanisms of OM-85 sensing remain inadequately addressed. Here, we show that the immune response to OM-85 in the mouse is largely mediated by myeloid immune cells through Toll-like receptor (TLR) 4 in vitro and in vivo. Instead, in human immune cells, TLR2 and TLR4 orchestrate the response to OM-85, which binds to both receptors as shown by surface plasmon resonance assay. Ribonucleic acid-sequencing analyses of human monocyte-derived dendritic cells reveal that OM-85 triggers a pro-inflammatory signature and a unique gene set, which is not induced by canonical agonists of TLR2 or TLR4 and comprises tolerogenic genes. A largely overlapping TLR2/4-dependent gene signature was observed in individual subsets of primary human airway myeloid cells, highlighting the robust effects of OM-85. Collectively, our results suggest caution should be taken when relating murine studies on bacterial lysates to humans. Furthermore, our data shed light on how a standardized bacterial lysate shapes the response through TLR2 and TLR4, which are crucial for immune response, trained immunity, and tolerance.

Proteomics-based vaccine targets annotation and design of multi-epitope vaccine against antibiotic-resistant Streptococcus gallolyticus

Streptococcus gallolyticus is a non-motile, gram-positive bacterium that causes infective endocarditis. S. gallolyticus has developed resistance to existing antibiotics, and no vaccine is currently available. Therefore, it is essential to develop an effective S. gallolyticus vaccine. Core proteomics was used in this study together with subtractive proteomics and reverse vaccinology approach to find antigenic proteins that could be utilized for the design of the S. gallolyticus multi-epitope vaccine. The pipeline identified two antigenic proteins as potential vaccine targets: penicillin-binding protein and the ATP synthase subunit. T and B cell epitopes from the specific proteins were forecasted employing several immunoinformatics and bioinformatics resources. A vaccine (360 amino acids) was created using a combination of seven cytotoxic T cell lymphocyte (CTL), three helper T cell lymphocyte (HTL), and five linear B cell lymphocyte (LBL) epitopes. To increase immune responses, the vaccine was paired with a cholera enterotoxin subunit B (CTB) adjuvant. The developed vaccine was highly antigenic, non-allergenic, and stable for human use. The vaccine's binding affinity and molecular interactions with the human immunological receptor TLR4 were studied using molecular mechanics/generalized Born surface area (MMGBSA), molecular docking, and molecular dynamic (MD) simulation analyses. Escherichia coli (strain K12) plasmid vector pET-28a ( +) was used to examine the ability of the vaccine to be expressed. According to the outcomes of these computer experiments, the vaccine is quite promising in terms of developing a protective immunity against diseases. However, in vitro and animal research are required to validate our findings.

Combining antibiotic with anti-TLR2/TLR13 therapy prevents brain pathology in pneumococcal meningitis.

Despite effective antibiotic therapy, brain-destructive inflammation often cannot be avoided in pneumococcal meningitis. The causative signals are mediated predominantly through TLR-recruited myeloid differentiation primary response adaptor 88 (MyD88), as indicated by a dramatic pneumococcal meningitis phenotype of Myd88-/- mice. Because lipoproteins and single-stranded RNA are crucial for recognition of Gram-positive bacteria such as Streptococcus pneumoniae by the host immune system, we comparatively analyzed the disease courses of Myd88-/- and Tlr2-/- Tlr13-/- mice. Their phenotypic resemblance indicated TLR2 and -13 as master sensors of S. pneumoniae in the cerebrospinal fluid. A neutralizing anti-TLR2 antibody (T2.5) and chloroquine (CQ) - the latter applied here as an inhibitor of murine TLR13 and its human ortholog TLR8 - abrogated activation of murine and human primary immune cells exposed to antibiotic-treated S. pneumoniae. The inhibitory effect of the T2.5/CQ cocktail was stronger than that of dexamethasone, the current standard adjunctive drug for pneumococcal meningitis. Accordingly, TLR2/TLR13 blockade concomitant with ceftriaxone application significantly improved the clinical course of pneumococcal meningitis compared with treatment with ceftriaxone alone or in combination with dexamethasone. Our study indicates the importance of murine TLR13 and human TLR8, besides TLR2, in pneumococcal meningitis pathology, and suggests their blockade as a promising antibiotic therapy adjunct.

Revolutionizing and identifying novel drug targets in Citrobacter koseri via subtractive proteomics and development of a multi-epitope vaccine using reverse vaccinology and immuno-informatics

Citrobacter koseri is a gram-negative rod that has been linked to infections in people with significant comorbidities and immunocompromised immune systems. It is most commonly known to cause urinary tract infections. Thus, the development of an efficacious C. koseri vaccine is imperative, as the pathogen has acquired resistance to current antibiotics. Subtractive proteomics was employed during this research to identify potential antigenic proteins to design an effective vaccine against C. koseri. The pipeline identified two antigenic proteins as potential vaccine targets: DP-3-O-acyl-N-acetylglucosamine deacetylase and Arabinose 5-phosphate isomerase. B and T cell epitopes from the specific proteins were forecasted employing several immunoinformatic and bioinformatics resources. A vaccine was created using a combination of seven cytotoxic T cell lymphocytes (CTL), five helper T cell lymphocyte (HTL), and seven linear B cell lymphocyte (LBL) epitopes. An adjuvant (β-defensin) was added to the vaccine to enhance immunological responses. The created vaccine was stable for use in humans, highly antigenic, and non-allergenic. The vaccine’s molecular and interactions binding affinity with the human immunological receptor TLR3 were studied using MMGBSA, molecular dynamics (MD) simulations, and molecular docking analyses. E. coli (strain-K12) plasmid vector pET-28a (+) was used to examine the ability of the vaccine to be expressed. The vaccine shows great promise in terms of developing protective immunity against diseases, based on the results of these computer experiments. However, in vitro and animal research are required to validate our findings. Communicated by Ramaswamy H. Sarma

Structural vaccinology, molecular simulation and immune simulation approaches to design multi-epitopes vaccine against John Cunningham virus

The JCV (John Cunningham Virus) is known to cause progressive multifocal leukoencephalopathy, a condition that results in the formation of tumors. Symptoms of this condition such as sensory defects, cognitive dysfunction, muscle weakness, homonosapobia, difficulties with coordination, and aphasia. To date, there is no specific and effective treatment to completely cure or prevent John Cunningham polyomavirus infections. Since the best way to control the disease is vaccination. In this study, the immunoinformatic tools were used to predict the high immunogenic and non-allergenic B cells, helper T cells (HTL), and cytotoxic T cells (CTL) epitopes from capsid, major capsid, and T antigen proteins of JC virus to design the highly efficient subunit vaccines. The specific immunogenic linkers were used to link together the predicted epitopes and subjected to 3D modeling by using the Robetta server. MD simulation was used to confirm that the newly constructed vaccines are stable and properly fold. Additionally, the molecular docking approach revealed that the vaccines have a strong binding affinity with human TLR-7. The codon adaptation index (CAI) and GC content values verified that the constructed vaccines would be highly expressed in E. coli pET28a (+) plasmid. The immune simulation analysis indicated that the human immune system would have a strong response to the vaccines, with a high titer of IgM and IgG antibodies being produced. In conclusion, this study will provide a pre-clinical concept to construct an effective, highly antigenic, non-allergenic, and thermostable vaccine to combat the infection of the John Cunningham virus.

Multi-epitope vaccine design against leishmaniasis using IFN-γ inducing epitopes from immunodominant gp46 and gp63 proteins

There is no currently approved human vaccine against leishmaniasis. Utilization of immunogenic antigens and their epitopes capable of enhancing immune responses against leishmaniasis is a crucial step for rational in silico vaccine design. The objective of this study was to generate and evaluate a potential vaccine candidate against leishmaniasis, designed by immunodominant proteins from gp46 and gp63 of Leishmania major, which can stimulate helper T-lymphocytes (HTL) and cytotoxic T-lymphocytes (CTL). For this aim, the IFN-γ-inducing MHC-I and MHC-II binders were predicted for each examined protein (gp46 and gp63) and connected with appropriate linkers, along with an adjuvant (Mycobacterium tuberculosis L7/L12) and a histidine tag. The vaccine’s stability, antigenicity, structure, and interaction with the TLR-4 receptor were evaluated in silico. The resulting chimeric vaccine was composed of 344 amino acids and had a molecular weight of 35.64 kDa. Physico-chemical properties indicated that it was thermotolerant, soluble, highly antigenic, and non-allergenic. Predictions of the secondary and tertiary structures were made, and further analyses confirmed that the vaccine construct could interact with the human TLR-4 receptor. Virtual immune simulation demonstrated strong stimulation of T-cell responses, particularly by an increase in IFN-γ, following vaccination. In summary, the in silico data indicated that the vaccine candidate showed high antigenicity in humans. It was also found to trigger significant levels of clearance mechanisms and other components of the cellular immune profile. Nevertheless, further wet experiments are required to properly assess the efficacy of this multi-epitope vaccine candidate against leishmaniasis.

TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth.

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) remains a deadly cancer worldwide with a need for new therapeutic approaches. A dysregulation in the equilibrium between pro- and anti-inflammatory responses with a predominant immunosuppressive inflammatory reaction in advanced stage tumors seem to contribute to tumor growth and metastasis. The current therapies do not include strategies against pro-tumorigenic inflammation in cancer patients. We have shown that the upregulated cell surface expression of Toll-like Receptor (TLR) 2 and of TLR9 inside PDAC cells maintain chronic inflammatory responses, support chemotherapeutic resistance, and mediate tumor progression in human pancreatic cancer. We further demonstrated intracellular TLR2 and TLR9 targeting using specific intrabodies, which resulted in downregulated inflammatory signaling. In this study, we tested, for the first time, an intrabody-mediated TLR blockade in human TLR2- and TLR9-expressing pancreatic cancer cells for its effects on inflammatory signaling-mediated tumor growth. Newly designed anti-TLR2- and anti-TLR9-specific intrabodies inhibited PDAC growth. Co-expression analysis of the intrabodies and corresponding human TLRs showed efficient retention and accumulation of both intrabodies within the endoplasmic reticulum (ER), while co-immunoprecipitation studies indicated both intrabodies interacting with their cognate TLR antigen within the pancreatic cancer cells. Cancer cells with attenuated proliferation expressing accumulated TLR2 and TRL9 intrabodies demonstrated reduced STAT3 phosphorylation signaling, while apoptotic markers Caspases 3 and 8 were upregulated. To conclude, our results demonstrate the TLR2 and TLR9-specific intrabody-mediated signaling pathway inhibition of autoregulatory inflammation inside cancer cells and their proliferation, resulting in the suppression of pancreatic tumor cell growth. These findings underscore the potential of specific intrabody-mediated TLR inhibition in the ER relevant for tumor growth inhibition and open up a new therapeutic intervention strategy for the treatment of pancreatic cancer.