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.

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.

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.

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.

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.

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).

The novel small-molecule antagonist MMG-11 preferentially inhibits TLR2/1 signaling.

Toll-like receptor 2 (TLR2) forms heterodimers with either TLR1 or TLR6 to induce protective early inflammatory responses to pathogen- and damage-associated molecular patterns. However, excessive activation is associated with inflammatory and metabolic diseases. Several TLR2 antagonists have been described but pharmacological characterization is still at an early stage. Previously, we identified the potent and selective TLR2 antagonist MMG-11 by computational modelling and experimental validation. Here, we characterized the TLR2 antagonists MMG-11 and CU-CPT22 as well as the TIR-domain binding TLR2 antagonist C29 in TLR-overexpressing promoter cells as well as human and mouse macrophages. In line with our recent studies, MMG-11 abrogated pro-inflammatory cytokine secretion and NF-κB activation induced by different bacterial TLR2 agonists. MMG-11 preferentially inhibited TLR2/1 signaling in promoter cells stably expressing TLR2 heterodimers and mouse macrophages. Furthermore, the TLR2 antagonist blocked ligand-induced interaction of TLR2 with MyD88 and reduced MAP kinase and NF-κB activation. MMG-11 and CU-CPT22 but not C29 displaced Pam3CSK4 in an indirect binding assay confirming the competitive mode of action of MMG-11 and CU-CPT22. Isobologram analysis revealed additive and synergistic effects when the non-competitive antagonist C29 was combined with the competitive antagonist MMG-11 or CU-CPT22, respectively. In conclusion, we provide evidence that MMG-11 acts as a competitive antagonist with a predominance for the TLR2/1 heterodimer in human and mouse cells. Our results also indicate that MMG-11 is a model compound for studying TLR2 signaling.

Designing a multi-epitopic vaccine against the enterotoxigenic Bacteroides fragilis based on immunoinformatics approach.

Enterotoxigenic Bacteroides fragilis is an enteric pathogen which is described as a causative agent of various intestinal infections and inflammatory diseases. Moreover, various research studies have reported it to be a leading factor in the development of colorectal cancer. As a part of the normal human microbiome, its treatment has become quite a challenge due to the alarming resistance against the available antibiotics. Although, this particular strain of B. fragilis shows susceptibility to few antibiotics, it is pertinent to devise an effective vaccine strategy for its elimination. There is no vaccine available against this pathogen up to date; therefore, we systematically ventured the outer membrane toxin producing proteins found exclusively in the toxigenic B. fragilis through the in-silico approaches to predict a multi-epitopic chimeric vaccine construct. The designed protein constitutes of epitopes which are predicted for linear B cells, Helper and T cells of outer membrane proteins expected to be putative vaccine candidates. The finalized proteins are only expressed in the enterotoxigenic B. fragilis, thus proving them to be exclusive. The 3D structure of the protein was first predicted followed by its refinement and validation via utilizing the bioinformatic approaches. Docking of the designed protein with the TLR2 receptor forecasted apt binding. Upon immune simulation, notable levels were observed in the expression of the immune cells.

Development of N-Acetylated Dipalmitoyl-S-Glyceryl Cysteine Analogs as Efficient TLR2/TLR6 Agonists.

Cancer vaccine is a promising immunotherapeutic approach to train the immune system with vaccines to recognize and eliminate tumors. Adjuvants are compounds that are necessary in cancer vaccines to mimic an infection process and amplify immune responses. The Toll-like receptor 2 and 6 (TLR2/TLR6) agonist dipalmitoyl-S-glyceryl cysteine (Pam2Cys) was demonstrated as an ideal candidate for synthetic vaccine adjuvants. However, the synthesis of Pam2Cys requires expensive N-protected cysteine as a key reactant, which greatly limits its application as a synthetic vaccine adjuvant in large-scaled studies. Here, we report the development of N-acetylated Pam2Cys analogs as TLR2/TLR6 agonists. Instead of N-protected cysteine, the synthesis utilizes N-acetylcysteine to bring down the synthetic costs. The N-acetylated Pam2Cys analogs were demonstrated to activate TLR2/TLR6 in vitro. Moreover, molecular docking studies were performed to provide insights into the molecular mechanism of how N-acetylated Pam2Cys analogs bind to TLR2/TLR6. Together, these results suggest N-acetylated Pam2Cys analogs as inexpensive and promising synthetic vaccine adjuvants to accelerate the development of cancer vaccines in the future.

Receptor Heterodimerization and Co-Receptor Engagement in TLR2 Activation Induced by MIC1 and MIC4 from Toxoplasma gondii.

The microneme organelles of Toxoplasma gondii tachyzoites release protein complexes (MICs), including one composed of the transmembrane protein MIC6 plus MIC1 and MIC4. In this complex, carbohydrate recognition domains of MIC1 and MIC4 are exposed and interact with terminal sialic acid and galactose residues, respectively, of host cell glycans. Recently, we demonstrated that MIC1 and MIC4 binding to the N-glycans of Toll-like receptor (TLR) 2 and TLR4 on phagocytes triggers cell activation and pro-inflammatory cytokine production. Herein, we investigated the requirement for TLR2 heterodimerization and co-receptors in MIC-induced responses, as well as the signaling molecules involved. We used MICs to stimulate macrophages and HEK293T cells transfected with TLR2 and TLR1 or TLR6, both with or without the co-receptors CD14 and CD36. Then, the cell responses were analyzed, including nuclear factor-kappa B (NF-κB) activation and cytokine production, which showed that (1) only TLR2, among the studied factors, is crucial for MIC-induced cell activation; (2) TLR2 heterodimerization augments, but is not critical for, activation; (3) CD14 and CD36 enhance the response to MIC stimulus; and (4) MICs activate cells through a transforming growth factor beta-activated kinase 1 (TAK1)-, mammalian p38 mitogen-activated protein kinase (p38)-, and NF-κB-dependent pathway. Remarkably, among the studied factors, the interaction of MIC1 and MIC4 with TLR2 N-glycans is sufficient to induce cell activation, which promotes host protection against T. gondii infection.

HHV-5 epitope: A potential vaccine candidate with high antigenicity and large coverage.

Outbreak of Human Herpes virus-5 (HHV-5) infection in emerging countries has raised worldwide health concern owing to prevalence of congenital impairments and life threatening consequences in immunocompromised individuals. Thus, there lies an impending need to develop vaccine against HHV-5. HHV-5 enters into host cells with the help of necessary components glycoprotein B (gB) and H/L. In this study, the conformational linear B-cell and T-cell epitopes for gB of HHV-5 have been predicted using conformational approaches, for their possible collective use as vaccine candidates. We examined epitope's interactions with major histocompatibility complexes using molecular docking and also investigated their stable binding with specific toll like receptor-2 (TLR2), present on host cells during HHV-5 infection. Predicted MHC-I epitope 'LVAIAVVII' with high antigenicity and large coverage of HLA alleles was found to superimpose on MHC-II epitope (Rank 1) and was also identified to be the core sequence of putative B cell epitope 'ILVAIAVVIITYLI'. Resulting epitope was found to have consistent interaction with TLR2 during long term (100 ns) MD run. We also validated this nonamer epitope for its dissimilarity with human genome and high population coverage, suggesting it to be a potential vaccine candidate with higher coverage for both the MHC alleles of Indian population. Communicated by Ramaswamy H. Sarma.

Design of novel multi-epitope vaccines against severe acute respiratory syndrome validated through multistage molecular interaction and dynamics.

Severe acute respiratory syndrome (SARS) is endemic in South China and is continuing to spread worldwide since the 2003 outbreak, affecting human population of 37 countries till present. SARS is caused by the severe acute respiratory syndrome Coronavirus (SARS-CoV). In the present study, we have designed two multi-epitope vaccines (MEVs) composed of cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and B cell epitopes overlap, bearing the potential to elicit cellular as well as humoral immune response. We have used truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 as molecular adjuvants at N-terminal of both the MEVs. Selected overlapping epitopes of both the MEVs were further validated for stable molecular interactions with their respective human leukocyte antigen class I and II allele binders. Moreover, CTL epitopes were further studied for their molecular interaction with transporter associated with antigen processing. Furthermore, after tertiary structure modelling, both the MEVs were validated for their stable molecular interaction with Toll-like receptors 2 and 4. Codon-optimized cDNA of both the MEVs was analysed for their potential high level of expression in the mammalian cell line (Human) needed for their further in vivo testing. Overall, the present study proposes in silico validated design of two MEVs against SARS composed of specific epitopes with the potential to cause a high level of SARS-CoV specific cellular as well as humoral immune response. Communicated by Ramaswamy H. Sarma.

Phloretin as a Potent Natural TLR2/1 Inhibitor Suppresses TLR2-Induced Inflammation.

Toll-like receptor 2 (TLR2) responses are involved in various inflammatory immune disorders. Phloretin is a naturally occurring dietary flavonoid that is abundant in fruit. Here, we investigated whether the anti-inflammatory activity of phloretin is mediated through TLR2 pathways, and whether phloretin acts as an inhibitor of TLR2/1 heterodimerization using the TLR2/1 agonist Pam3CSK4. We tested the effects of phloretin on tumor necrosis factor (TNF)-α production induced by various TLRs using known TLR-specific agonists. Phloretin significantly inhibited Pam3CSK4-induced TRL2/1 signaling in Raw264.7 cells compared to TLR signaling induced by the other agonists tested. Therefore, we further tested the effects of phloretin in human embryonic kidney (HEK) 293-hTLR2 cells induced by Pam3CSK4, and confirmed that phloretin has comparable inhibition of TLR2/1 heterodimerization to that induced by the known TLR2 inhibitor CU-CPT22. Moreover, phloretin reduced the secretion of the inflammatory cytokines TNF-α and interleukin (IL)-8 in Pam3CSK4-induced HEK293-hTLR2 cells, whereas it did not significantly reduce these cytokines under Pam2CSK4-induced activation. Western blot results showed that phloretin significantly suppressed Pam3CSK4-induced TLR2 and NF-κB p65 expression. The molecular interactions between phloretin and TLR2 were investigated using bio-layer interferometry and in silico docking. Phloretin bound to TLR2 with micromolar binding affinity, and we proposed a binding model of phloretin at the TLR2⁻TLR1 interface. Overall, we confirmed that phloretin inhibits the heterodimerization of TLR2/1, highlighting TLR2 signaling as a therapeutic target for treating TLR2-mediated inflammatory immune diseases.

Antibody Responses to a Quadrivalent Hepatitis C Viral-Like Particle Vaccine Adjuvanted with Toll-Like Receptor 2 Agonists.

The development of an effective preventative hepatitis C virus (HCV) vaccine will reside, in part, in its ability to elicit neutralizing antibodies (NAbs). We previously reported a genotype 1a HCV virus like particle (VLP) vaccine that produced HCV specific NAb and T cell responses that were substantially enhanced by Toll-like receptor 2 (TLR2) agonists. We have now produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine and tested the ability of two TLR2 agonists, R4Pam2Cys and E8Pam2Cys, to stimulate the production of NAb. We now show that our vaccine with R4Pam2Cys or E8Pam2Cys produces strong antibody and NAb responses in vaccinated mice after just two doses. Total antibody titers were higher in mice inoculated with vaccine plus E8Pam2Cys compared to HCV VLPs alone. However, the TLR2 agonists did not result in stronger NAb responses compared to vaccine without adjuvant. Such a vaccine could provide a substantial addition to the overall goal to eliminate HCV.