In silico design of a novel multi-epitope vaccine against HCV infection through immunoinformatics approaches.

Infection with the hepatitis C virus (HCV) is one of the causes of liver cancer, which is the world's sixth most prevalent and third most lethal cancer. The current treatments do not prevent reinfection; because they are expensive, their usage is limited to developed nations. Therefore, a prophylactic vaccine is essential to control this virus. Hence, in this study, an immunoinformatics method was applied to design a multi-epitope vaccine against HCV. The best B- and T-cell epitopes from conserved regions of the E2 protein of seven HCV genotypes were joined with the appropriate linkers to design a multi-epitope vaccine. In addition, cholera enterotoxin subunit B (CtxB) was included as an adjuvant in the vaccine construct. This study is the first to present this epitopes-adjuvant combination. The vaccine had acceptable physicochemical characteristics. The vaccine's 3D structure was predicted and validated. The vaccine's binding stability with Toll-like receptor 2 (TLR2) and TLR4 was confirmed using molecular docking and molecular dynamics (MD) simulation. The immune simulation revealed the vaccine's efficacy by increasing the population of B and T cells in response to vaccination. In silico expression in Escherichia coli (E. coli) was also successful.

Structural and functional implications of leucine-rich repeats in toll-like receptor1 subfamily.

Leucine-rich repeats (LRRs) - the protein-protein and protein-ligand interaction motif of proteins participating in a plethora of functions in plants, vertebrates, invertebrates, and prokaryotes - are a fascinating piece of conserved yet versatile structural motif. In toll-like receptors (TLRs), this domain forms the extracellular part that is preceded by an intracellular toll/interleukin-1 receptor (TIR) domain. The extracellular part is crucial for recognizing a structurally diverse set of viral, bacterial, fungal, and parasite-derived components, while the TIR domain is recruited for activation of downstream signaling following recognition. The distinct ability of the paralogs TLR1 and TLR6 to dimerize with TLR2 and recognize different ligands intrigued and motivated us to exchange the dimerizing and ligand-binding residues between TLR1/6 and note the effect on dimer formation and ligand binding. The appreciable sequence modification brought about no significant alteration in the native scaffold of the motif, as revealed from the comparison of simulations with wild-type dimers. Moreover, docking of the exchanged ligands to the variant proteins supported favorable binding. Thus, the structural stability and the functional plasticity offered by the motif might be the reason for its extensive use across cellular functions and life forms, a feature crucial for coevolution and the knowledge essential for therapeutics.

In Silico Designing of a Multitope Vaccine against Rhizopus microsporus with Potential Activity against Other Mucormycosis Causing Fungi.

During the current era of the COVID-19 pandemic, the dissemination of Mucorales has been reported globally, with elevated rates of infection in India, and because of the high rate of mortality and morbidity, designing an effective vaccine against mucormycosis is a major health priority, especially for immunocompromised patients. In the current study, we studied shared Mucorales proteins, which have been reported as virulence factors, and after analysis of several virulent proteins for their antigenicity and subcellular localization, we selected spore coat (CotH) and serine protease (SP) proteins as the targets of epitope mapping. The current study proposes a vaccine constructed based on top-ranking cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B cell lymphocyte (BCL) epitopes from filtered proteins. In addition to the selected epitopes, ß-defensins adjuvant and PADRE peptide were included in the constructed vaccine to improve the stimulated immune response. Computational tools were used to estimate the physicochemical and immunological features of the proposed vaccine and validate its binding with TLR-2, where the output data of these assessments potentiate the probability of the constructed vaccine to stimulate a specific immune response against mucormycosis. Here, we demonstrate the approach of potential vaccine construction and assessment through computational tools, and to the best of our knowledge, this is the first study of a proposed vaccine against mucormycosis based on the immunoinformatics approach.

In-silico design of envelope based multi-epitope vaccine candidate against Kyasanur forest disease virus.

Kyasanur forest disease virus (KFDV) causing tick-borne hemorrhagic fever which was earlier endemic to western Ghats, southern India, it is now encroaching into new geographic regions, but there is no approved medicine or effective vaccine against this deadly disease. In this study, we did in-silico design of multi-epitope subunit vaccine for KFDV. B-cell and T-cell epitopes were predicted from conserved regions of KFDV envelope protein and two vaccine candidates (VC1 and VC2) were constructed, those were found to be non-allergic and possess good antigenic properties, also gives cross-protection against Alkhurma hemorrhagic fever virus. The 3D structures of vaccine candidates were built and validated. Docking analysis of vaccine candidates with toll-like receptor-2 (TLR-2) by Cluspro and PatchDock revealed strong affinity between VC1 and TLR2. Ligplot tool was identified the intermolecular hydrogen bonds between vaccine candidates and TLR-2, iMOD server confirmed the stability of the docking complexes. JCAT sever ensured cloning efficiency of both vaccine constructs and in-silico cloning into pET30a (+) vector by SnapGene showed successful translation of epitope region. IMMSIM server was identified increased immunological responses. Finally, multi-epitope vaccine candidates were designed and validated their efficiency, it may pave the way for up-coming vaccine and diagnostic kit development.

SARS-CoV-2 infection as a trigger of humoral response against apolipoprotein A-1.

BACKGROUND: Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes. DESIGN: Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti-SARS-CoV-2 and anti-apoA-1 IgG as well as cytokines were assessed by immunoassays on a case-control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post-pandemic period. RESULTS: Using bioinformatics modelling, linear sequence homologies between apoA-1, TLR2 and Spike epitopes were identified but without experimental evidence of cross-reactivity. Overall, anti-apoA-1 IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (P < .0001). Significant and similar associations were noted between anti-apoA-1, anti-SARS-CoV-2 IgG, cytokines and lipid profile. In ICU patients, anti-SARS-CoV-2 and anti-apoA-1 seroconversion rates displayed similar 7-day kinetics, reaching 82% for anti-apoA-1 seropositivity. In the general population, SARS-CoV-2-exposed individuals displayed higher anti-apoA-1 IgG seropositivity rates than nonexposed ones (34% vs 16.8%; P = .004). CONCLUSION: COVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.

Structure based design and synthesis of novel Toll-like Receptor 2 (TLR 2) lipid antagonists.

Toll-like receptors (TLRs) play key role in innate immune response to Damage Associated Molecular Patterns (DAMPs) and Pathogen Associated Molecular Patterns (PAMPs). DAMP/PAMP-mediated activation of TLRs triggers NFκB signaling resulting in pro-inflammatory cytokine release. Using TLR2-Pam2CSK4 agonist co-crystal structure information, we designed and synthesized a novel series of Toll-like Receptor 2 (TLR2) lipid antagonists and identified compounds 14, 15 and 17 with sub-micromolar potency. TLR2 antagonists that we identified are stable for > 1.0 h in both gastric juice and PBS buffer and could be used as research tools.

Co-stimulatory effect of TLR2 and TLR4 stimulation on megakaryocytic development is mediated through PI3K/NF-ĸB and XBP-1 loop.

Toll-like receptors (TLRs) are a class of proteins (patterns recognition receptors-PRRs) capable of recognizing molecules frequently found in pathogens (that are so-called pathogen-associated molecular patterns-PAMPs), they play a key role in the initiation of innate immune response by detecting PAMPs. Our findings show that the functional effects of TLRs co-stimulation on megakaryocytopoiesis. A single cell may receive multiple signal inputs and we consider that multiple TLRs are likely triggered during infection by multiple PAMPs that, in turn, might be involved in infection driven megakaryocytopoiesis, and the present study provide the evidence for the megakaryocytic effects of TLRs co-stimulation.

Computational formulation and immune dynamics of a multi-peptide vaccine candidate against Crimean-Congo hemorrhagic fever virus.

The sole objective of this research is to devise an epitope-based vaccine candidate as prophylaxis for the Crimean-Congo hemorrhagic fever virus (CCHFV) using the knowledge of immunoinformatics and structural biology. Importantly, CCHFV outbreaks have increased in several countries resulting in increased mortality up to 40% due to the lack of prospective medication and an efficient vaccine. In this study, we have used several immunoinformatic tools and servers to anticipate potent B-cell and T-cell epitopes from the CCHFV glycoprotein with the highest antigenicity. After a comprehensive evaluation, a vaccine candidate was designed using 6 CD8+, 3 CD4+, and 7 B-cell epitopes with appropriate linkers. To enhance the vaccine's efficiency, we added Mycobacterium tuberculosis lipoprotein LprG (Rv1411c) to the vaccine as an adjuvant. The final construct was composed of a total of 468 amino acid residues. The epitope included in the construct showed 98% worldwide population coverage. Importantly, the construct appeared as antigenic, immunogenic, soluble, and non-allergenic in nature. To explore further, we modelled the three-dimensional (3D) structure of the constructed vaccine. Our chimeric vaccine showed stable and strong interactions for toll-like receptor 2 (TLR2) found on the cell surface. Moreover, the dynamics simulation of immune response showed elevated levels of cellular immune activity and faster clearance of antigen from the body upon repetitive exposure. Finally, the optimized codon (CAI≈1) ensured the marked translation efficiency of the vaccine protein in E. coli strain K12 bacterium followed by the insertion of construct DNA into the cloning vector pET28a (+). We believe that the designed vaccine chimera could be useful in vaccine development to fight CCHFV outbreaks.

Simplified Monopalmitoyl Toll-like Receptor 2 Ligand Mini-UPam for Self-Adjuvanting Neoantigen-Based Synthetic Cancer Vaccines.

Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3 CysSK4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.

A mono-acyl phospholipid (20:1 lyso-PS) activates Toll-Like Receptor 2/6 hetero-dimer.

Toll-like receptor 2 (TLR2) is an important pattern recognition receptor on the surface of host immune cells that binds a variety of ligands that are released by microorganisms as well as by damaged or dying host cells. According to the current concept, TLR2/1 and TLR2/6 heterodimers are activated by tri- or di-acylated ligands, respectively. However, also mono-acyl phospholipid containing lipid fractions derived from parasites, were reported to be able to activate TLR2. In order to provide conclusive evidence for the TLR2 activating capacity of mono-acyl phospholipids derived from pathogens, we developed a biosynthetic method to enzymatically convert commercially available phospholipids into several mono-acyl-phospholipid variants that were examined for their TLR2 activating capacity. These investigations demonstrated that 1-(11Z-eicosenoyl)-glycero-3-phosphoserine 20:1 (20:1 lyso-PS) is a true agonist of the TLR2/6 heterodimer and that its polar headgroup as well as the length of the acyl chain are crucial for TLR2 activation. In silico modelling further confirmed 20:1 mono-acyl PS as a ligand for TLR2/6 heterodimer, as this predicted that multiple hydrogen bonds are formed between the polar headgroup of 20:1 mono-acyl PS and amino acid residues of both TLR2 and TLR6. Future studies can now be performed to further assess the functions of 20:1 lyso-PS as an immunological mediator, because this enzymatic method enables its preparation in larger quantities than is possible by isolation from the parasite that naturally produces this compound, Schistosoma mansoni, the source of the original discovery (Van der Kleij et al., 2002).

An alternative model for type I interferon induction downstream of human TLR2.

Surface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines, including type I interferons (IFN-I). Downstream of TLR4, IFNß secretion is only vigorously triggered in macrophages when the receptor undergoes endocytosis and switches signaling adaptor; surface TLR4 engagement predominantly induces proinflammatory cytokines via the signaling adaptor MyD88. It is unclear whether this dichotomy is generally applicable to other TLRs, cell types, or differentiation states. Here, we report that diverse TLR2 ligands induce an IFN-I response in human monocyte-like cells, but not in differentiated macrophages. This TLR2-dependent IFN-I signaling originates from the cell surface and depends on MyD88; it involves combined activation of the transcription factors IRF3 and NF-κB, driven by the kinases TBK1 and TAK1-IKKß, respectively. TLR2-stimulated monocytes produced modest IFNß levels that caused productive downstream signaling, reflected by STAT1 phosphorylation and expression of numerous interferon-stimulated genes. Our findings reveal that the outcome of TLR2 signaling includes an IFN-I response in human monocytes, which is lost upon macrophage differentiation, and differs mechanistically from IFN-I-induction through TLR4. These findings point to molecular mechanisms tailored to the differentiation state of a cell and the nature of receptors activated to control and limit TLR-triggered IFN-I responses.

Designing Multi-Epitope Vaccines to Combat Emerging Coronavirus Disease 2019 (COVID-19) by Employing Immuno-Informatics Approach.

A recent pandemic caused by a single-stranded RNA virus, COVID-19, initially discovered in China, is now spreading globally. This poses a serious threat that needs to be addressed immediately. Genome analysis of SARS-CoV-2 has revealed its close relation to SARS-coronavirus along with few changes in its spike protein. The spike protein aids in receptor binding and viral entry within the host and therefore represents a potential target for vaccine and therapeutic development. In the current study, the spike protein of SARS-CoV-2 was explored for potential immunogenic epitopes to design multi-epitope vaccine constructs. The S1 and S2 domains of spike proteins were analyzed, and two vaccine constructs were prioritized with T-cell and B-cell epitopes. We adapted a comprehensive predictive framework to provide novel insights into immunogenic epitopes of spike proteins, which can further be evaluated as potential vaccine candidates against COVID-19. Prioritized epitopes were then modeled using linkers and adjuvants, and respective 3D models were constructed to evaluate their physiochemical properties and their possible interactions with ACE2, HLA Superfamily alleles, TLR2, and TLR4.

Designing a vaccine for fascioliasis using immunogenic 24 kDa mu-class glutathione s-transferase.

Fascioliasis, caused by the liver fluke Fasciola gigantica, is a significant zoonotic disease of the livestock and human, causing substantial economic loss worldwide. Triclabendazole (TCBZ) is the only drug available for the management of the disease against which there is an alarming increase in drug resistance. No vaccine is available commercially for the protection against this disease. Increasing resistance to TCBZ and the lack of a successful vaccine against fascioliasis demands the development of vaccines. In the present study, a structural immunoinformatics approach was used to design a multi-epitope subunit vaccine using the glutathione S-transferase (GST) protein of Fasciola gigantica. The GST antigen is a safe, non-allergic, highly antigenic, and effective vaccine candidate against various parasitic flukes and worms. The cytotoxic T lymphocytes, helper T lymphocytes, and B-cell epitopes were selected for constructing the vaccine based on their immunogenic behavior and binding affinity. The physicochemical properties, allergenicity, and antigenicity of the designed vaccine were analyzed. To elucidate the tertiary structure of the vaccine, homology modeling was performed, followed by structure refinement and docking against the TLR2 immune receptor. Molecular dynamics simulations showed a stable interaction between the vaccine and the receptor complex. Finally, in silico cloning was performed to evaluate the expression and translation of the vaccine construct in the E. coli expression system. Further studies require experimental validation for the safety and immunogenic behavior of the designed vaccine.

Endogenous TLR2 ligand embedded in the catalytic region of human cysteinyl-tRNA synthetase 1.

BACKGROUND: The generation of antigen-specific cytotoxic T lymphocyte (CTL) responses is required for successful cancer vaccine therapy. In this regard, ligands of Toll-like receptors (TLRs) have been suggested to activate adaptive immune responses by modulating the function of antigen-presenting cells (APCs). Despite their therapeutic potential, the development of TLR ligands for immunotherapy is often hampered due to rapid systemic toxicity. Regarding the safety concerns of currently available TLR ligands, finding a new TLR agonist with potent efficacy and safety is needed. METHODS: A unique structural domain (UNE-C1) was identified as a novel TLR2/6 in the catalytic region of human cysteinyl-tRNA synthetase 1 (CARS1) using comprehensive approaches, including RNA sequencing, the human embryonic kidney (HEK)-TLR Blue system, pull-down, and ELISA. The potency of its immunoadjuvant properties was analyzed by assessing antigen-specific antibody and CTL responses. In addition, the efficacy of tumor growth inhibition and the presence of the tumor-infiltrating leukocytes were evaluated using E.G7-OVA and TC-1 mouse models. The combined effect of UNE-C1 with an immune checkpoint inhibitor, anti-CTLA-4 antibody, was also evaluated in vivo. The safety of UNE-C1 immunization was determined by monitoring splenomegaly and cytokine production in the blood. RESULTS: Here, we report that CARS1 can be secreted from cancer cells to activate immune responses via specific interactions with TLR2/6 of APCs. A unique domain (UNE-C1) inserted into the catalytic region of CARS1 was determined to activate dendritic cells, leading to the stimulation of robust humoral and cellular immune responses in vivo. UNE-C1 also showed synergistic efficacy with cancer antigens and checkpoint inhibitors against different cancer models in vivo. Further, the safety assessment of UNE-C1 showed lower systemic cytokine levels than other known TLR agonists. CONCLUSIONS: We identified the endogenous TLR2/6 activating domain from human cysteinyl-tRNA synthetase CARS1. This novel TLR2/6 ligand showed potent immune-stimulating activity with little toxicity. Thus, the UNE-C1 domain can be developed as an effective immunoadjuvant with checkpoint inhibitors or cancer antigens to boost antitumor immunity.

Determining Whether Agonist Density or Agonist Number Is More Important for Immune Activation via Micoparticle Based Assay.

It is unknown if surface bound toll-like-receptor (TLR) agonists activate cells via density or total molecular number. To answer this question, we developed a TLR agonist surface conjugated polystyrene microparticle (MP) system. Using a library of MPs with varying TLR agonist density and number, we simultaneously observed innate immune cell MP uptake and TNFα expression using ImageStream flow cytometry on a cell by cell basis. The data shows that total TLR number and not density drives cellular activation with a threshold of approximately 105-106 TLR agonists. We believe that this information will be crucial for the design of particulate vaccine formulations.

Identification and validation of a novel dual small-molecule TLR2/8 antagonist.

Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC50 values in the low micromolar range for both TLRs, selectivity towards other TLRs and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam3CSK4 or Pam2CSK4 indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLRs. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation.

Volatile Anesthetic Sevoflurane Attenuates Toll-Like Receptor 1/2 Activation.

BACKGROUND: Although immunomodulatory effects of anesthetics have been increasingly recognized, their underlying molecular mechanisms are not completely understood. Toll-like receptors (TLRs) are one of the major receptors to recognize invading pathogens and danger signals from damaged host tissues to initiate immune responses. Among the TLR family, TLR2 and TLR4 recognize a wide range of ligands and are considered to be important players in perioperative pathophysiology. Based on our recent finding that volatile anesthetics modulate TLR4 function, we tested our hypothesis that they would also modulate TLR2 function. METHODS: The effect of anesthetics isoflurane, sevoflurane, propofol, and dexmedetomidine on TLR2 activation was examined by reporter assays. An anesthetic that affected the activation was subjected to in silico rigid docking simulation on TLR2. To test our prediction that sevoflurane and a TLR1/TLR2 ligand Pam3CSK4 would compete for the same pocket of TLR2, we performed Pam3CSK4 competitive binding assay to TLR2 using HEK cells stably transfected with TLR2 (HEK-TLR2) with or without sevoflurane. We examined the effect of different anesthetics on the functions of human neutrophils stimulated with TLR2 ligands. Kruskal-Wallis test and Mann-Whitney U test were used for statistical analysis. RESULTS: We observed that the attenuation of TLR1/TLR2 activation was seen on sevoflurane exposure but not on isoflurane, propofol, or dexmedetomidine exposure. The attenuation of TLR2/TLR6 activation was not seen in any of the anesthetics tested. The rigid docking simulation predicted that sevoflurane and Pam3CSK4 bound to the same pocket of TLR1/TLR2 complex. The binding of Pam3CSK4 to HEK-TLR2 cells was impaired in the presence of sevoflurane, indicating that sevoflurane and Pam3CSK4 competed for the pocket, as predicted in silico. The stimulation of neutrophils with Pam3CSK4 induced L-selection shedding but did not affect phagocytosis and reactive oxygen species production. L-selectin shedding from neutrophils was attenuated only by sevoflurane, consistent with the result of our reporter assays. CONCLUSIONS: We found that TLR1/TLR2 activation was attenuated by sevoflurane, but we found no evidence for attenuation by isoflurane, propofol, or dexmedetomidine at clinically relevant concentrations. Our structural analysis and competition assay supported that sevoflurane directly bound to TLR2 at the interphase of the TLR1/TLR2 complex. Sevoflurane attenuated neutrophil L-selectin shedding, an important step for neutrophil migration.

A (1→6)-Branched (1→4)-ß-d-Glucan from Grifola frondosa Inhibits Lipopolysaccharide-Induced Cytokine Production in RAW264.7 Macrophages by Binding to TLR2 Rather than Dectin-1 or CR3 Receptors.

Mushroom polysaccharides including ß-glucans possess various health-promoting properties and are known to be the major bioactive constituents of Grifola frondosa (GF), which is a popular edible and medicinal mushroom. Dectin-1, a pattern-recognition receptor, is responsible for recognizing ß-glucans. In this study, parental RAW264.7 macrophages and Dectin-1-expressing RAW264.7 macrophages were used to investigate the anti-inflammatory activity and receptor involvement of the water-soluble polysaccharides from GF. Results indicated that the high molecular weight fraction of GF (GF70-F1; 1260 kDa) inhibited TNF-α and IL-6 production as well as NF-κB activation in lipopolysaccharide-induced macrophages. Chemical and enzymatic linkage analyses indicated that GF70-F1 mainly contained the known (1→3),(1→6)-ß-d-glucan and a polysaccharide not previously isolated from GF, a nondigestible glucan with a ß-(1→4)-linked backbone and ß-(1→6)-linked branches. The ability of GF70-F1 to inhibit cytokine production was not affected by the expression level of Dectin-1 in cells, and a similar inhibitory activity was observed after removing the (1→3),(1→6)-ß-d-glucan from GF70-F1. Blockade of Toll-like receptor 2 (TLR2) but not Dectin-1 or complement receptor 3 (CR3) attenuated the inhibitory activity of GF70-F1. The nondigestible (1→6)-branched (1→4)-ß-d-glucan in GF70-F1 may contribute to the anti-inflammatory activity via interacting with TLR2 rather than Dectin-1 or CR3 receptors.

A Self-Adjuvanting Vaccine Platform: Optimization of Site-Specific Sortase A Mediated Conjugation of Toll-Like Receptor 2 Ligands onto the Carboxyl or Amino terminus of Recombinant Protein Antigens.

Self-adjuvanting vaccines, consisting of recombinant protein antigens and covalently attached Toll-like receptor (TLR) agonists, have the ability to simultaneously and efficiently deliver antigen and TLR adjuvant to antigen presenting cells (APCs). Here, an enzyme-mediated ligation approach was used to overcome difficulties in producing homogeneous, molecularly defined self-adjuvanting vaccine products under native conditions. This process was optimized to allow the incorporation of the lipopeptide TLR2 agonist fibroblast-stimulating lipopeptide (FSL)-1 onto the N- or C-termini of recombinant protein antigens, employing the enzyme Staphylococcus aureus sortase A (SrtAsa) penta mutant. In addition, because SrtAsa-mediated ligations are reversible, a tryptophan zipper derived sequence was introduced into both reactants, which was demonstrated to improve ligation efficiency through the formation of a ß-hairpin structure that hinders the reverse reaction. Finally, it was demonstrated that N- or C-terminal conjugation, and the incorporation of the ß-hairpin structure, did not affect the TLR2-agonist activities of protein antigen TLR agonist conjugates. Overall, this SrtAsa-mediated ligation platform enabled production of antigen TLR2 agonist conjugates with enhanced ligation efficiency, with the conjugates demonstrating potent TLR2 signaling activation (EC50 <1nM).

Molecular characterization of Bu-1 and TLR2 gene in Haringhata Black chicken.

Haringhata Black is the only registered indigenous poultry genetic resource of West Bengal till date. Molecular characterization of HB revealed that Bu-1 to be highly glycoylated transmembrane protein unlike mammalian Bu-1, whereas TLR2 of HB chicken was observed to be rich in Leucine rich repeat. HB chicken was observed to be genetically close to chicken of Japan, while distant to chicken breed of UK and Chicago. Avian species wise evolution study indicates genetic closeness of HB chicken with turkey. Differential mRNA expression profile for the immune response genes (TLR2, TLR4 and Bu1 gene) were studied for HB chicken with respect to other chicken breed and poultry birds, which reveals that HB chicken were better in terms of B cell mediated immunity and hence better response to vaccination. Hence HB chicken is one of the best poultry genetic resources to be reared under backyard system where biosecurity measures are almost lacking.