TAP Transport Research Articles

An immunological glimpse of human virus peptides: Distance from self, MHC class I binding, proteasome cleveage, TAP transport and sequence composition entropy

Adaptive immune response is triggered when specific pathogen peptides called epitopes are recognised as exogenous according to the paradigm of self/non-self. To be recognized by immune cells, epitopes have to be exposed (presented) on the surface of the cell. Predicting if a peptide is exposed is important to shed light on the rules that govern immune response and, thus, identify potential targets and design vaccine and drugs. We focused on peptides exposed on cell surface and made accessible to immune system through the MHC Class I complex. Before this can happen, three successive selection steps have to take place: a) Proteasome cleveage, b) TAP Transport, and c) binding to MHC-class I. Starting from a set of 211 host human reference viruses, we computed the set of unique peptides occurring in the correspondent proteomes. Then, we obtained the probability values of Proteasome Cleveage, TAP Transport and Binding to MHC Class I associated to those peptides through established prediction software tools. Such values were analysed in conjunction with two other features that could play a major role: the distance from self, strictly linked to the concept of nullomers, and the sequence entropy, measuring the complexity of the peptide amino acid composition. The analysis confirmed and extended previous results on a larger, more significant and consistent data set; we showed that the higher the distances from self, the higher the score of TAP Transport and binding to MHC class I; no significant association was instead found between distance from self and Proteasome Cleveage. Additionally, amino acid peptide composition entropy was significantly associated with the other features. In particular, higher entropies were linked with higher scores of Proteasome Cleveage, TAP Transport, Binding to MHC Class I, and higher distance from self. The relationship among the three selection steps provided evidence of a tight inter-correlation, clearly suggesting it could be the product of a co-evolutive process. We believe that these results give new insights on the complex processes that regulate peptide presentation through MHC class I, and unveil the mechanisms the allow the immune system to distinguish self and viral non-self peptides.

Combination of human papillomaviruses L1 and L2 multiepitope constructs protects mice against tumor cells

Different types of cancer including cervical (>90%), anal (~88%), vaginal (~40%), and penile (~40%) cancers are associated with human papillomaviruse (HPV) infections. Three prophylactic vaccines (Cervarix, Gardasil, and Gardasil-9) were approved to provide immuno-protection against certain types of HPVs. Currently, next-generation HPV vaccines such as L1/L2-based vaccines are being developed to provide broad-type HPV protection. In this study, we introduced a comprehensive framework for design of L1/L2 polyepitope-based HPV vaccine candidate. This framework started with protein sequence retrieval and followed by conservancy analysis between high-risk HPVs, MHC-I and MHC-II epitope mapping, and B-cell and T-cell epitope mapping. Subsequently, we performed Tap transport and proteasomal cleavage, population coverage, antigenicity, allergenicity and cross-reactivity. After that, peptide-MHCI/II flexible docking and comprehensive conservancy analysis against all HPV types were carried out. The next steps were prediction of interferon-gamma and interleukin-10 inducing epitopes, epitope selection and construct design, tertiary structure prediction, refinement and validation, discontinuous B-cell epitope prediction, vaccine-TLR4 molecular docking, and codon optimization. Our data showed that two designed vaccine constructs harboring 8 L1 peptides or 7 L2 peptides, individually were highly conserved between all well-known HPV types. In addition, the combination of in silico/in vivo approaches indicated the potential ability of L1 and L2 polyepitope constructs for development of next generation prophylactic/therapeutic HPV vaccine.

Designing of Potential Polyvalent Vaccine Model for Respiratory Syncytial Virus by System Level Immunoinformatics Approaches.

Background Respiratory syncytial virus (RSV) infection is a public health epidemic, leading to around 3 million hospitalization and about 66,000 deaths each year. It is a life-threatening condition exclusive to children with no effective treatment. Methods In this study, we used system-level and vaccinomics approaches to design a polyvalent vaccine for RSV, which could stimulate the immune components of the host to manage this infection. Our framework involves data accession, antigenicity and subcellular localization analysis, T cell epitope prediction, proteasomal and conservancy evaluation, host-pathogen-protein interactions, pathway studies, and in silico binding affinity analysis. Results We found glycoprotein (G), fusion protein (F), and small hydrophobic protein (SH) of RSV as potential vaccine candidates. Of these proteins (G, F, and SH), we found 9 epitopes for multiple alleles of MHC classes I and II bear significant binding affinity. These potential epitopes were linked to form a polyvalent construct using AAY, GPGPG linkers, and cholera toxin B adjuvant at N-terminal with a 23.9 kDa molecular weight of 224 amino acid residues. The final construct was a stable, immunogenic, and nonallergenic protein containing cleavage sites, TAP transport efficiency, posttranslation shifts, and CTL epitopes. The molecular docking indicated the optimum binding affinity of RSV polyvalent construct with MHC molecules (-12.49 and -10.48 kcal/mol for MHC classes I and II, respectively). This interaction showed that a polyvalent construct could manage and control this disease. Conclusion Our vaccinomics and system-level investigation could be appropriate to trigger the host immune system to prevent RSV infection.

Exploring the cancer-testis antigen BORIS to design a novel multi-epitope vaccine against breast cancer based on immunoinformatics approaches

Recently, cancer immunotherapy has gained lots of attention to replace the current chemoradiation approaches and multi-epitope cancer vaccines are manifesting as the next generation of cancer immunotherapy. Therefore, in this study, we used multiple immunoinformatics approaches along with other computational approaches to design a novel multi-epitope vaccine against breast cancer. The most immunogenic regions of the BORIS cancer-testis antigen were selected according to the binding affinity to MHC-I and II molecules as well as containing multiple cytotoxic T lymphocyte (CTL) epitopes by multiple immunoinformatics servers. The selected regions were linked together by GPGPG linker. Also, a T helper epitope (PADRE) and the TLR-4/MD-2 agonist (L7/L12 ribosomal protein from mycobacterium) were incorporated by A(EAAAK)3A linker to form the final vaccine construct. Then, its physicochemical properties, cleavage sites, TAP transport efficiency, B cell epitopes, IFN-γ inducing epitopes and population coverage were predicted. The final vaccine construct was reverse translated, codon-optimized and inserted into pcDNA3.1 to form the DNA vaccine. The final vaccine construct was a stable, immunogenic and non-allergenic protein that contained numerous CTL epitopes, IFN-γ inducing epitopes and several linear and conformational B cell epitopes. Also, the final vaccine construct formed stable and significant interactions with TLR-4/MD-2 complex according to molecular docking and dynamics simulations. Moreover, its world population coverage for HLA-I and HLA-II were about 93% and 96%, respectively. Taking together, these preliminary results can be used as an appropriate platform for further experimental investigations. Communicated by Ramaswamy H. Sarma

Structural determinants of peptide-dependent TAP1-TAP2 transit passage targeted by viral proteins and altered by cancer-associated mutations

The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8–10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport.

Design of novel multiepitope constructs-based peptide vaccine against the structural S, N and M proteins of human COVID-19 using immunoinformatics analysis.

The causative agent of severe acute respiratory syndrome (SARS) reported by the Chinese Center for Disease Control (China CDC) has been identified as a novel Betacoronavirus (SARS-CoV-2). A computational approach was adopted to identify multiepitope vaccine candidates against SARS-CoV-2 based on S, N and M proteins being able to elicit both humoral and cellular immune responses. In this study, the sequence of the virus was obtained from NCBI database and analyzed with in silico tools such as NetMHCpan, IEDB, BepiPred, NetCTL, Tap transport/proteasomal cleavage, Pa3P, GalexyPepDock, I-TASSER, Ellipro and ClusPro. To identify the most immunodominant regions, after analysis of population coverage and epitope conservancy, we proposed three different constructs based on linear B-cell, CTL and HTL epitopes. The 3D structure of constructs was assessed to find discontinuous B-cell epitopes. Among CTL predicted epitopes, S257-265, S603-611 and S360-368, and among HTL predicted epitopes, N167-181, S313-330 and S1110-1126 had better MHC binding rank. We found one putative CTL epitope, S360-368 related to receptor-binding domain (RBD) region for S protein. The predicted epitopes were non-allergen and showed a high quality of proteasomal cleavage and Tap transport efficiency and 100% conservancy within four different clades of SARS-CoV-2. For CTL and HTL epitopes, the highest population coverage of the world’s population was calculated for S27-37 with 86.27% and for S196-231, S303-323, S313-330, S1009-1030 and N328-349 with 90.33%, respectively. We identified overall 10 discontinuous B-cell epitopes for three multiepitope constructs. All three constructs showed strong interactions with TLRs 2, 3 and 4 supporting the hypothesis of SARS-CoV-2 susceptibility to TLRs 2, 3 and 4 like other Coronaviridae families. These data demonstrated that the novel designed multiepitope constructs can contribute to develop SARS-CoV-2 peptide vaccine candidates. The in vivo studies are underway using several vaccination strategies.

In silico/In vivo analysis of high-risk papillomavirus L1 and L2 conserved sequences for development of cross-subtype prophylactic vaccine

Human papillomavirus (HPV) is the most common sexually transmitted infection in the world and the main cause of cervical cancer. Nowadays, the virus-like particles (VLPs) based on L1 proteins have been considered as the best candidate for vaccine development against HPV infections. Two commercial HPV (Gardasil and Cervarix) are available. These HPV VLP vaccines induce genotype-limited protection. The major impediments such as economic barriers especially gaps in financing obstructed the optimal delivery of vaccines in developing countries. Thus, many efforts are underway to develop the next generation of vaccines against other types of high-risk HPV. In this study, we developed DNA constructs (based on L1 and L2 genes) that were potentially immunogenic and highly conserved among the high-risk HPV types. The framework of analysis include (1) B-cell epitope mapping, (2) T-cell epitope mapping (i.e., CD4+ and CD8+ T cells), (3) allergenicity assessment, (4) tap transport and proteasomal cleavage, (5) population coverage, (6) global and template-based docking, and (7) data collection, analysis, and design of the L1 and L2 DNA constructs. Our data indicated the 8-epitope candidates for helper T-cell and CTL in L1 and L2 sequences. For the L1 and L2 constructs, combination of these peptides in a single universal vaccine could involve all world population by the rate of 95.55% and 96.33%, respectively. In vitro studies showed high expression rates of multiepitope L1 (~57.86%) and L2 (~68.42%) DNA constructs in HEK-293T cells. Moreover, in vivo studies indicated that the combination of L1 and L2 DNA constructs without any adjuvant or delivery system induced effective immune responses, and protected mice against C3 tumor cells (the percentage of tumor-free mice: ~66.67%). Thus, the designed L1 and L2 DNA constructs would represent promising applications for HPV vaccine development.

In silico analysis of transmembrane protein 31 (TMEM31) antigen to design novel multiepitope peptide and DNA cancer vaccines against melanoma

Multiepitope cancer vaccines are announcing themselves as the future of melanoma treatment. Herein, high immunogenic regions of transmembrane protein 31 (TMEM31) antigen were selected according to cytotoxic T lymphocytes’ (CTL) epitopes and major histocompatibility complex (MHC) binding affinity through in silico analyses. The 32–62, 77–105, and 125–165 residues of the TMEM31 were selected as the immunodominant fragments. They were linked together by RVRR and HEYGAEALERAG motifs to improve epitopes separation and presentation. In addition, to activate helper T lymphocytes (HTL), Pan HLA DR-binding epitope (PADRE) peptide sequence and tetanus toxin fragment C (TTFrC) were incorporated into the final construct. Also, the Beta-defensin conserved domain was utilized in the final construct as a novel adjuvant for Toll-like receptor 4/myeloid differentiation factor (TLR4-MD) activation. The CTL epitopes, cleavage sites, post-translational modifications, TAP transport efficiency, and B cells epitopes were predicted for the peptide vaccine. The final construct contained multiple CTL and B cell epitopes. In addition, it showed 93.55% and 99.13% population coverage in the world for HLA I and HLA II, respectively. According to these preliminary results, the multiepitope cancer vaccine can be an appropriate choice for further experimental investigations.

Abstract B073: A pan-HLA predictor of neoantigen processing and presentation to the tumor cell surface

Abstract Currently, neoantigens are often predicted using algorithms predominantly based on knowledge of the key peptide binding affinity difference between HLA alleles. Although HLA binding algorithms predict binding affinity of a peptide to HLA reasonably well, they do not predict processing and presentation of to the cell surface (i.e., the immunopeptidome). In fact, only 15%–20% of “predicted” peptide binders are processed or presented, and therefore contribute to the immunopeptidome. Erroneous predictions may be addressed with time-consuming and laborious experiments, such as mass-spectrometry (MS). However, in silico predictions may also prove to be very useful in prioritizing therapeutically relevant immunogenic peptides. Previous in silico studies that predict naturally processed and presented peptides to the cell surface have focused on only one of the many steps in the antigen processing and presentation pathway (such as TAP transport or proteasome cleavage, etc.). Additionally, previous antigen processing prediction tools have been trained and are therefore applicable to specific HLA alleles, making it challenging to make predictions for not so well-characterized alleles. Here, we outline a machine learning approach trained on MS elution data that predicts, in a pan-HLA manner, natural processing and presentation of neoantigens to the cell surface. The predictor is integrated with multiple immune parameters in a deep learning layer to predict neoantigens, and may be used for more accurate neoantigen predictions for any HLA allele, in both the class I and class II systems. Further, by analyzing previously published clinical data we illustrate that its application leads to a significantly improved identification of neoantigen targets for personalized cancer immunotherapy. Citation Format: Trevor Clancy, Richard Stratford. A pan-HLA predictor of neoantigen processing and presentation to the tumor cell surface [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B073.

Prediction of cross-clade HIV-1 T-cell epitopes using immunoinformatics analysis.

Epitope mapping has emerged as a powerful tool to develop peptide vaccines against hypervariable viruses such as HIV. This method has led to stimulate a specific immune response and achieve advanced vaccine formulations. In this study, we identified peptides that were potentially immunostimulatory and highly conserved in HIV-1 main group. The analyses included were CTL assay, Tap transport, and the potential allergenicity. The highest population coverage rate was also found for all potential T-cell epitopes in 16 specified geographic regions of the world. The current study is the first attempt to explore peptide-protein flexible docking across all the major epitopes of HIV-1. Our data indicated that REV54-63 and VPU58-66 with the highest epitope identification scores, GP16037-46 and VPR38-47 with the highest conservation (98.89%), and NEF134-144 and GP16037-46 epitopes with a higher quality of peptide-protein interaction models in docking procedure were chosen as putative epitopes among all HLA class I epitopes. TAT40-67 , VPR65-82 , and VPU30-44 with the highest score of binding affinity, VPR65-82 with the highest conservation (97.55%), and GP160481-498 epitope with a higher quality of peptide-protein interaction models in docking procedure were determined as putative epitopes among all HLA-DR epitopes. Furthermore, two epitopes of GP160481-498 and VIF144-159 were predicted to bind 22 and 21 HLA-II alleles, respectively. Accumulative population coverage of potential helper T-cell epitopes and CTL epitopes varied between 90.82% and 100%. Generally, these predicted highly immunogenic T-cell epitopes can contribute to design HIV-1 peptide vaccine candidates. Combination of bioinformatics tools with in vivo methods will be necessary for HIV-1 vaccine development.

In Silico Analysis of Synaptonemal Complex Protein 1 (SYCP1) and Acrosin Binding Protein (ACRBP) Antigens to Design Novel Multiepitope Peptide Cancer Vaccine Against Breast Cancer

Multiepitope cancer vaccines are manifesting as the future of breast cancer immunotherapy. In the present study, high immunogenic fragments of synaptonemal complex protein 1 (SYCP1) and acrosin binding protein (ACRBP) antigens were selected according to MHCs binding affinity and CD8+ cytotoxic T lymphocytes’ (CTL) epitopes by various immunoinformatic servers. (RCQHKIAEMVALMEKHKHQYDKI) residue from p702–724 SYCP1 and the (YIQYPNYCSFKSQQCL) residue from p481–496 ACRBP were selected as the immunodominant fragments. Then, the selected fragments were fused together with a furin-sensitive linker to form the final peptide vaccine construct. The cleavage sites, TAP transport efficiency, CTL epitopes, post-translational modifications, and B cells epitopes were predicted for the final construct. The final construct contained appropriate CTLs epitopes and also, several linear and conformational B cell epitopes. Also, it exhibited 90.37% HLA population coverage in the world. Therefore, these preliminary results require validation by in vitro and in vivo experiments.

Identification and Validation of HLA-A24 XBP1us, XBP1sp, CD138, and CS1 Peptides to Generate Antigens Specific-Cytotoxic T Lymphocytes: Preclinical Basis for Vaccine Therapy in HLA-A24 Patients with Multiple Myeloma and Other Cancers

XBP1 (X-box binding protein 1), CD138 (Syndecan-1), and CS1 (CD2 subset 1, CRACC, SLAMF7, CD319) are highly expressed antigens associated with tumor pathogenesis in multiple myeloma cells and various solid tumor cells. We have previously reported on the use of HLA-A2 peptides specific to these antigens to generate antigen-specific and HLA-2-restricted cytotoxic T cells (CTL) in preclinical studies and phase I/II clinical trials in patients with smoldering MM. These studies extend this concept to HLA-A24 patients. Immunogenic HLA-A24 peptides specific to both the spliced and unspliced isoforms of XBP1, CD138, and CS1 were identified, synthesized, and evaluated in vitro for their ability to generate the antigen-specific cytotoxic T lymphocytes (CTL) targeting tumor cells. The specific epitopes were selected based on their binding affinity to HLA-A24, extended half-time disassociation rates, proteasomal C terminal cleavage, and TAP transport. We report here on novel highly immunogenic HLA-A24-specific peptides XBP1 US185 -193 (I S P W I L A V L), XBP1 SP223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F), and CS1240-248 (L F V L G L F L W), which induce antigen-specific CD3+CD8+ CTL against MM and various solid tumors. Each of the single peptide-specific CTL displayed immune functional activities including IFN-g/IL-2 cytokine production and cytotoxic activity against HLA-A24+ MM cells. Furthermore, our studies demonstrated that a cocktail of the four HLA-A24-specific peptides induced multipeptide-specific CD3+CD8+ CTL with functional anti-tumor properties against myeloma cells. Phenotypically, the multipeptide-specific CTL were central memory (CM; CCR7+CD45RO+/CD3+CD8+), effector memory (EM; CCR7-CD45RO+/CD3+CD8+) and terminal effector (TE; CCR7-CD45RO-/CD3+CD8+) cells expressing high levels of cell activation markers CD69, CD38, and CD40L. The multipeptide-specific effector and memory CTL subsets demonstrated HLA-A24-restricted and peptide-specific anti-tumor activities, and the highest activities were detected within the CM CTL subset. Treatment of the multipeptide-specific CTL or tumor target cells with different types of checkpoint inhibitors (a-PD1, a-TIM3, a-PDL-1, a-Galectin-9) or the immune modulator drug, lenalidomide, enhanced their anti-myeloma activities and antigen-specific CD3+CD8+ T cell proliferation to HLA-A24+ tumor cells. Therefore, a cocktail of the four immunogenic HLA-A24 epitopes provides the framework for development of a cancer vaccine based targeted immunotherapy. In conclusion, we report here on novel immunogenic HLA-A24-specific XBP1 unspliced, XBP1 spliced, CD138, and CS1 peptides that elicit CTL with anti-myeloma activities. These peptides will allow us to extend our immunotherapeutic vaccine approach beyond the current HLA-A2-specific peptides to HLA-A24 patients with smoldering myeloma, multiple myeloma, other plasma cell disorders and potentially with solid tumors. These preclinical studies also demonstrate that incorporation of immunomodulatory agents and/or checkpoint inhibitors can amplify and maintain antigen-specific memory CTL responses against tumor. DisclosuresBae:OncoPep Inc.: Consultancy, Equity Ownership. Raje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Chauhan:Stemline Therapeutics: Consultancy. Munshi:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Anderson:Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

In vitro bioacessibility and transport across Caco-2 monolayers of haloacetic acids in drinking water

Water disinfection plays a crucial role in water safety but it is also a matter of concern as the use of disinfectants promotes the formation of disinfection by-products (DBPs). Haloacetic acids (HAAs) are one of the major classes of DBPs since they are frequently found in treated water, are ubiquitous, pervasive and have high water solubility, so a great concern emerged about their formation, occurrence and toxicity. Exposure to HAAs is influenced by consumption patterns and diet of individuals thus their bioavailability is an important parameter to the overall toxicity.In the current study the bioacessibility of the most representative HAAs (chloroacetic acid – MCAA, bromoacetic acid – MBAA, dichloroacetic acid – DCAA, dibromoacetic acid – DBAA, and trichloroacetic acid – TCAA) after simulated in vitro digestion (SIVD) in tap water and transport across Caco-2 monolayers was evaluated. Compounds were monitored in 8 points throughout the digestion phases by an optimized LC-MS/MS methodology.MCAA and MBAA were not bioaccessible after SIVD whereas DCAA, DBAA and TCAA are highly bioaccessible (85 ± 4%, 97 ± 4% and 106 ± 7% respectively). Concerning transport assays, DCAA and DBAA were highly permeable throughout the Caco-2 monolayer (apparent permeability and calculated fraction absorbed of 13.62 × 10−6 cm/s and 90% for DCAA; and 8.82 × 10−6 cm/s and 84% for DBAA), whereas TCAA showed no relevant permeability.The present results may contribute to efficient risk analysis studies concerning HAAs oral exposure from tap water taking into account the different biological behaviour of these chemically similar substances.

Prediction and Identification of HLA-A*0201 Restricted CTL Epitopes from Eps8

To find and identify HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes from epidermal growth factor pathway substrate number 8 (Eps8) for specific immunotherapy based on Eps8-derived epitopes in clinic. Online biological softwares involved C-proteasomal cleavage, MHC class I binding affinity and TAP transport efficiency were used for prediction of HLA-A*0201 restricted epitopes from Eps8. Then, T2-binding assays and peptide/MHC complex stability tests were used to further verify the predicted epitopes. Specific secretion of IFN-γ from human CTL was assayed using the IFN-γ ELISPOT kit, and cytolytic activity was measured by a 4-h lactate dehydrogenase (LDH) release assay. Finally, the functional effects in vivo were measured in HLA-A*0201/Kb transgenic (Tg) mice. Four natural epitopes were designed through online biological softwares. Of the four epitopes selected, p360-368 was found to have the high binding affinity to HLA-A*0201, while p101-109 and p276-284 showed moderate affinities. DC50 of peptide/MHC complexes of the natural epitopes mentioned were all longer than 8 h. In functional assays with human PBMNC in vitro and in HLA-A*0201/Kb transgenic mice in vivo, CTLs primed by each epitope (p101-109, p276-284 and p360-368) secreted IFN-γ and were toxic to cancer cells from a variety of tissue types in an HLA-A*0201-restricted and Eps8-specific manner. Natural epitopes (p101-109, p276-284 and p360-368) may be the HLA-A*0201 restricted epitope derived from Eps8.

Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma.

IntroductionInterferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.ResultsWe could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.ConclusionWe suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.

Computational Approach to Identify the Major Histocompatibility Complex Binding Antigenic Peptides from 'Ascariasis'

The Ascaris lumbricoides cause ‘ascariasis’ in human and its elimination is the major public concern. In this current investigation, we predicted the MHC (Major Histocompatibility Complex) class I & II binding peptides with computational approach like PSSM (Position Specific Scoring Matrices) and SVM (Support Vector Machine) algorithms. We predict the peptide binders of Cytochrome b (mitochondrion) protein from Ascaris lumbricoide sequence to MHC-I molecules are as 11mer_H2_Db, 10mer_H2_Db, 9mer_H2_Db, 8mer_H2_Db. Also study integrates prediction of peptide MHC class I binding; proteasomal C terminal cleavage and TAP transport efficiency by using sequence and properties of the amino acids. We also found the binding of peptides to different alleles by using Position Specific Scoring Matrix. Cytochrome B from Ascaris lumbricoides (365 residues long) with 357 nonamers having antigenic MHC binding peptides. PSSM based server will predict the peptide binders from sequence to MHCII molecules are as I_Ab.p, I_Ad.p, I_Ag7, which are found antigenic epitopes region in Cytochrome B from Ascaris lumbricoides. This investigation can be useful in rational vaccine design and simultaneously increase the understanding the role of the immune system against antigenic.

Prediction of MHC Binding Peptides and Antigenic Peptides from Ascaris lumbricoides

The parasitic disease Acariasis is the major concern because of its morbidity and mortality issue. In this investigation, we predicted the binding peptides of the MHC class I and MHC class II by Position Specific Scoring Matrices (PSSM) and Support Vector Machine (SVM) algorithms. We predicted the binding affinity of Cytochrome c oxidase subunit 2 (mitochondrion) having a 232 amino acids long residue sequence, which shows 224 nonamers. We predicted the peptide binding affinity to MHC I moles are as 8mer_H2_Db(The binding thresholds: 33.04,optimal score:52.494), 9mer_H2_Db(Optimal Score: 50.232, Binding Threshold: 17.96),10mer_H2_Db (The Optimal Score: 58.858, Binding Threshold: 41.32), 11mer_H2_Db(Optimal Score: 79.495, Binding Threshold: 56.96) and MHC- II molecules are as I_Ab.p, I_Ad.p, I_Ag7. We also study integrates prediction of peptide MHC class I binding; proteasomal C terminal cleavage and TAP transport efficiency by using sequence and properties of the amino acids. We also found the binding of peptides to different alleles by using Position Specific Scoring Matrix. PSSM based server will predict the peptide binders of Cytochrome c oxidase subunit 2 from Ascaris lumbricoides sequence to, which are found antigenic epitopes region in protein.

Peptide Handling by HLA-B27 Subtypes Influences Their Biological Behavior, Association with Ankylosing Spondylitis and Susceptibility to Endoplasmic Reticulum Aminopeptidase 1 (ERAP1)

HLA-B27 is strongly associated with ankylosing spondylitis (AS). We analyzed the relationship between structure, peptide specificity, folding, and stability of the seven major HLA-B27 subtypes to determine the role of their constitutive peptidomes in the pathogenicity of this molecule. Identification of large numbers of ligands allowed us to define the differences among subtype-bound peptidomes and to elucidate the peptide features associated with AS and molecular stability. The peptides identified only in AS-associated or high thermostability subtypes with identical A and B pockets were longer and had bulkier and more diverse C-terminal residues than those found only among non-AS-associated/lower-thermostability subtypes. Peptides sequenced from all AS-associated subtypes and not from non-AS-associated ones, thus strictly correlating with disease, were very rare. Residue 116 was critical in determining peptide binding, thermodynamic properties, and folding, thus emerging as a key feature that unified HLA-B27 biology. HLA-B27 ligands were better suited to TAP transport than their N-terminal precursors, and AS-associated subtype ligands were better than those from non-AS-associated subtypes, suggesting a particular capacity of AS-associated subtypes to bind epitopes directly produced in the cytosol. Peptides identified only from AS-associated/high-thermostability subtypes showed a higher frequency of ERAP1-resistant N-terminal residues than ligands found only in non-AS-associated/low-thermostability subtypes, reflecting a more pronounced effect of ERAP1 on the former group. Our results reveal the basis for the relationship between peptide specificity and other features of HLA-B27, provide a unified view of HLA-B27 biology and pathogenicity, and suggest a larger influence of ERAP1 polymorphism on AS-associated than non-AS-associated subtypes.

You are the Foremost Responder - What to do or not to do is the Question?

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, which is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. MHC molecules are cell surface proteins, which take active part in host immune reactions and involvement of MHC class in response to almost all antigens and it give effects on specific sites. Predicted MHC binding regions acts like red flags for antigen specific and generate immune response against the parent antigen. So a small fragment of antigen can induce immune response against whole antigen. This theme is implemented in designing subunit and synthetic peptide vaccines. In this study, we analyzed thioredoxin glutathione reductase of Schistosoma mansoni and is allows potential drug targets to identify active sites, which form antibodies against or infection. The method integrates prediction of peptide MHC class binding; proteosomal C terminal cleavage and TAP transport efficiency. Antigenic epitopes of thioredoxin glutathione reductase are important antigenic determinants against the various toxic reactions and infections.

Defense genes missing from the flight division

Birds have a smaller repertoire of immune genes than mammals. In our efforts to study antiviral responses to influenza in avian hosts, we have noted key genes that appear to be missing. As a result, we speculate that birds have impaired detection of viruses and intracellular pathogens. Birds are missing TLR8, a detector for single-stranded RNA. Chickens also lack RIG-I, the intracellular detector for single-stranded viral RNA. Riplet, an activator for RIG-I, is also missing in chickens. IRF3, the nuclear activator of interferon-beta in the RIG-I pathway is missing in birds. Downstream of interferon (IFN) signaling, some of the antiviral effectors are missing, including ISG15, and ISG54 and ISG56 (IFITs). Birds have only three antibody isotypes and IgD is missing. Ducks, but not chickens, make an unusual truncated IgY antibody that is missing the Fc fragment. Chickens have an expanded family of LILR leukocyte receptor genes, called CHIR genes, with hundreds of members, including several that encode IgY Fc receptors. Intriguingly, LILR homologues appear to be missing in ducks, including these IgY Fc receptors. The truncated IgY in ducks, and the duplicated IgY receptor genes in chickens may both have resulted from selective pressure by a pathogen on IgY FcR interactions. Birds have a minimal MHC, and the TAP transport and presentation of peptides on MHC class I is constrained, limiting function. Perhaps removing some constraint, ducks appear to lack tapasin, a chaperone involved in loading peptides on MHC class I. Finally, the absence of lymphotoxin-alpha and beta may account for the observed lack of lymph nodes in birds. As illustrated by these examples, the picture that emerges is some impairment of immune response to viruses in birds, either a cause or consequence of the host-pathogen arms race and long evolutionary relationship of birds and RNA viruses.