Grass pollen allergoids conjugated with mannan for subcutaneous and sublingual immunotherapy: a dose-finding study.

Background: Polymerized allergoids conjugated with mannan represent a novel approach of allergen immunotherapy targeting dendritic cells. In this study, we aimed to determine the optimal dose of mannan-allergoid conjugates derived from grass pollen (Phleum pratense and Dactylis glomerata) administered via either the subcutaneous or sublingual route. Methods: A randomized, double-blind, placebo-controlled trial with a double-dummy design was conducted, involving 162 participants across 12 centers in Spain. Subjects were randomly allocated to one of nine different treatment groups, each receiving either placebo or active treatment at doses of 500, 1,000, 3,000, or 5,000 mTU/mL over four months. Each participant received five subcutaneous (SC) doses of 0.5 mL each, every 30 days, and a daily sublingual (SL) dose of 0.2 mL. Participants who received active treatment through SC, received placebo through SL. Participants who received active treatment through SL, received placebo SC. One Group, as control, received bot SC and SL placebo. The primary efficacy outcome was the improvement in titrated nasal provocation tests (NPT) at the end of the study compared to baseline. Secondary outcomes included specific antibody (IgG4, IgE) and cellular (IL-10 producing and regulatory T cell) responses. All adverse events and side reactions were recorded and assessed. Results: Post-treatment, the active groups showed improvements in NPT ranging from 33% to 53%, with the highest doses showing the greatest improvements regardless of the administration route. In comparison, the placebo group showed a 12% improvement. Significant differences over placebo were observed at doses of 3,000 mTU/mL (p=0.049 for SL, p=0.015 for SC) and 5,000 mTU/mL (p=0.011 for SL, p=0.015 for SC). A dose-dependent increase in IgG4 was observed following SC administration, and an increase in IL-10 producing cells for both routes of administration. No serious systemic or local adverse reactions were recorded, and no adrenaline was required. Conclusion: Grass pollen immunotherapy with mannan-allergoid conjugates was found to be safe and efficacious in achieving the primary outcome, whether administered via the subcutaneous or sublingual routes, at doses of 3,000 and 5,000 mTU/mL. Clinical trial registration: https://www.clinicaltrialsregister.eu/ctr-search (EudraCT), identifier 2014-005471-88; https://www.clinicaltrials.gov, identifier NCT02654223.

Activated gut-homing CD8+ T cells for coeliac disease diagnosis on a gluten-free diet.

BACKGROUND: The diagnosis of coeliac disease (CD) in individuals that have started a gluten-free diet (GFD) without an adequate previous diagnostic work-out is a challenge. Several immunological assays such as IFN-γ ELISPOT have been developed to avoid the need of prolonged gluten challenge to induce the intestinal damage. We aimed to evaluate the diagnostic accuracy of activated gut-homing CD8+ and TCRγδ+ T cells in blood after a 3-day gluten challenge and to compare it with the performance of IFN-γ ELISPOT in a HLA-DQ2.5 subsample. METHODS: A total of 22 CD patients and 48 non-CD subjects, all of them following a GFD, underwent a 3-day 10-g gluten challenge. The percentage of two T cell subsets (CD8+ CD103+ ß7hi CD38+/total CD8+ and TCRγδ+ CD103+ ß7hi CD38+/total TCRγδ+) in fresh peripheral blood drawn baseline and 6 days after the challenge was determined by flow cytometry. IFN-γ ELISPOT assays were also performed in HLA-DQ2.5 participants. ROC curve analysis was used to assess the diagnostic performance of the CD8+ T cell response and IFN-γ ELISPOT. RESULTS: Significant differences between the percentage of the two studied subsets of CD8+ and TCRγδ+ cells at days 0 and 6 were found only when considering CD patients (p < 10-3 vs. non-CD subjects). Measuring activated CD8+ T cells provided accurate CD diagnosis with 95% specificity and 97% sensitivity, offering similar results than IFN-γ ELISPOT. CONCLUSIONS: The results provide a highly accurate blood test for CD diagnosis in patients on a GFD of easy implementation in daily clinical practice.

Characterization of Conserved and Promiscuous Human Rhinovirus CD4 T Cell Epitopes.

Human rhinovirus (RV) is the most common cause of upper respiratory infections and exacerbations of asthma. In this work, we selected 14 peptides (6 from RV A and 8 from RV C) encompassing potential CD4 T cell epitopes. Peptides were selected for being highly conserved in RV A and C serotypes and predicted to bind to multiple human leukocyte antigen class II (HLA II) molecules. We found positive T cell recall responses by interferon gamma (IFNγ)-ELISPOT assays to eight peptides, validating seven of them (three from RV A and four from RV C) as CD4 T cell epitopes through intracellular cytokine staining assays. Additionally, we verified their promiscuous binding to multiple HLA II molecules by quantitative binding assays. According to their experimental HLA II binding profile, the combination of all these seven epitopes could be recognized by >95% of the world population. We actually determined IFNγ responses to a pool encompassing these CD4 T cell epitopes by intracellular cytokine staining, finding positive responses in 29 out of 30 donors. The CD4 T cell epitopes identified in this study could be key to monitor RV infections and to develop peptide-based vaccines against most RV A and C serotypes.

Prediction of unconventional protein secretion by exosomes.

MOTIVATION: In eukaryotes, proteins targeted for secretion contain a signal peptide, which allows them to proceed through the conventional ER/Golgi-dependent pathway. However, an important number of proteins lacking a signal peptide can be secreted through unconventional routes, including that mediated by exosomes. Currently, no method is available to predict protein secretion via exosomes. RESULTS: Here, we first assembled a dataset including the sequences of 2992 proteins secreted by exosomes and 2961 proteins that are not secreted by exosomes. Subsequently, we trained different random forests models on feature vectors derived from the sequences in this dataset. In tenfold cross-validation, the best model was trained on dipeptide composition, reaching an accuracy of 69.88% ± 2.08 and an area under the curve (AUC) of 0.76 ± 0.03. In an independent dataset, this model reached an accuracy of 75.73% and an AUC of 0.840. After these results, we developed ExoPred, a web-based tool that uses random forests to predict protein secretion by exosomes. CONCLUSION: ExoPred is available for free public use at http://imath.med.ucm.es/exopred/ . Datasets are available at http://imath.med.ucm.es/exopred/datasets/ .

Human rhinovirus-specific CD8 T cell responses target conserved and unusual epitopes.

Human Rhinovirus (HRV) is a major cause of common cold, bronchiolitis, and exacerbations of chronic pulmonary diseases such as asthma. CD8 T cell responses likely play an important role in the control of HRV infection but, surprisingly, HRV-specific CD8 T cell epitopes remain yet to be identified. Here, we approached the discovery and characterization of conserved HRV-specific CD8 T cell epitopes from species A (HRV A) and C (HRV C), the most frequent subtypes in the clinics of various pulmonary diseases. We found IFNγ-ELISPOT positive responses to 23 conserved HRV-specific peptides on peripheral blood mononuclear cells (PBMCs) from 14 HLA I typed subjects. Peptide-specific IFNγ production by CD8 T cells and binding to the relevant HLA I were confirmed for six HRV A-specific and three HRV C-specific CD8 T cell epitopes. In addition, we validated A*02:01-restricted epitopes by DimerX staining and found out that these peptides mediated cytotoxicity. All these A*02:01-restricted epitopes were 9-mers but, interestingly, we also identified and validated an unusually long 16-mer epitope peptide restricted by A*02:01, HRVC1791-1806 (GLEPLDLNTSAGFPYV). HRV-specific CD8 T cell epitopes describe here are expected to elicit CD8 T cell responses in up to 87% of the population and could be key for developing an HRV vaccine.

Identification of CD8+ T cell epitopes through proteasome cleavage site predictions.

BACKGROUND: We previously introduced PCPS (Proteasome Cleavage Prediction Server), a web-based tool to predict proteasome cleavage sites using n-grams. Here, we evaluated the ability of PCPS immunoproteasome cleavage model to discriminate CD8+ T cell epitopes. RESULTS: We first assembled an epitope dataset consisting of 844 unique virus-specific CD8+ T cell epitopes and their source proteins. We then analyzed cleavage predictions by PCPS immunoproteasome cleavage model on this dataset and compared them with those provided by a related method implemented by NetChop web server. PCPS was clearly superior to NetChop in term of sensitivity (0.89 vs. 0.79) but somewhat inferior with regard to specificity (0.55 vs. 0.60). Judging by the Mathew's Correlation Coefficient, PCPS predictions were overall superior to those provided by NetChop (0.46 vs. 0.39). We next analyzed the power of C-terminal cleavage predictions provided by the same PCPS model to discriminate CD8+ T cell epitopes, finding that they could be discriminated from random peptides with an accuracy of 0.74. Following these results, we tuned the PCPS web server to predict CD8+ T cell epitopes and predicted the entire SARS-CoV-2 epitope space. CONCLUSIONS: We report an improved version of PCPS named iPCPS for predicting proteasome cleavage sites and peptides with CD8+ T cell epitope features. iPCPS is available for free public use at https://imed.med.ucm.es/Tools/pcps/ .

PCPS: A Web Server to Predict Proteasomal Cleavage Sites.

The proteasome complex is mainly responsible for proteolytic degradation of cytosolic proteins, generating the C-terminus of MHC I-restricted peptide ligands and CD8 T cell epitopes. Therefore, prediction of proteasomal cleavage sites is relevant for anticipating CD8 T-cell epitopes. There are two different proteasomes, the constitutive proteasome, expressed in all types of cells, and the immunoproteasome, constitutively expressed in dendritic cells. Although both proteasome forms generate peptides for presentation by MHC I molecules, the immunoproteasome is the main form involved in providing peptide fragments for priming CD8 T cells. On the contrary, the proteasome provides peptides for presentation by MHC I molecules that can be targeted by already primed CD8 T cells. Proteasome cleavage prediction server (PCPS) is a server for predicting cleavage sites generated by both the constitutive proteasome and the immunoproteasome. Here, we illustrate the usage of PCPS to predict proteasome and immunoproteasome cleavage sites and compare the results with those provided by NetChop, a related tool available online. PCPS is implemented for free public use available online at http://imed.med.ucm.es/Tools/pcps/ .

Human Oral Epithelial Cells Impair Bacteria-Mediated Maturation of Dendritic Cells and Render T Cells Unresponsive to Stimulation.

The oral mucosa is a first line of defense against pathogenic organisms and yet tolerates food antigens and resident bacteria. Mucosal epithelial cells are emerging as important regulators of innate and adaptive immune responses. However, the contribution of oral epithelial cells (OECs) determining oral immunity is understudied. Here, we evaluated the ability of H413 and TR146 cells, two OEC lines derived from human oral squamous cell carcinomas, and primary OECs to modulate immune responses to a cocktail of Gram+ and Gram- bacteria known as MV130. OECs expressed CD40 constitutively and class II major histocompatibility complex (MHC II) molecules when stimulated with IFNγ, but not CD80 or CD86. Dendritic cells (DCs) treated with bacteria in co-culture with OECs did not fully mature, as judged by the expression of MHC II, CD80 and CD86, and barely released IL-12 and TNFα, compared to control DCs. Furthermore, in the presence of OECs, DCs were unable to stimulate allogenic naive CD4 T cells to produce IFNγ and TNFα. Similarly, OECs in culture with total CD4 T cells or Th1 cells stimulated with anti-CD3 and anti-CD28 antibodies abrogated CD25 and CD69 expression, T cell proliferation and the release of IFNγ and TNFα. The inhibition on T cell activation by OECs was cell-contact dependent, TGFß independent and largely irreversible. Overall, this behavior of OECs is likely key to avoid immune system over-reaction against resident bacteria.

Naturally presented HLA class I-restricted epitopes from the neurotrophic factor S100-ß are targets of the autoimmune response in type 1 diabetes.

Type 1 diabetes (T1D) results from the destruction of pancreatic ß-cells by the immune system, and CD8+ T lymphocytes are critical actors in this autoimmune response. Pancreatic islets are surrounded by a mesh of nervous cells, the peri-insular Schwann cells, which are also targeted by autoreactive T lymphocytes and express specific antigens, such as the neurotrophic factor S100-ß. Previous work has shown increased proliferative responses to whole S100-ß in both human T1D patients and the nonobese diabetic (NOD) mouse model. We describe for the first time naturally processed and presented epitopes (NPPEs) presented by class I human leukocyte antigen-A*02:01 (A2.1) molecules derived from S100-ß. These NPPEs triggered IFN-γ responses more frequently in both newly diagnosed and long-term T1D patients compared with healthy donors. Furthermore, the same NPPEs are recognized during the autoimmune response leading to diabetes in A2.1-transgenic NOD mice as early as 4 wk of age. Interestingly, when these NPPEs are used to prevent diabetes in this animal model, an acceleration of the disease is observed together with an exacerbation in insulitis and an increase in S100-ß-specific cytotoxicity in vaccinated animals. Whether these can be used in diabetes prevention needs to be carefully evaluated in animal models before use in future clinical assays.-Calviño-Sampedro, C., Gomez-Tourino, I., Cordero, O. J., Reche, P. A., Gómez-Perosanz, M., Sánchez-Trincado, J. L., Rodríguez, M. Á., Sueiro, A. M., Viñuela, J. E., Calviño, R. V. Naturally presented HLA class I-restricted epitopes from the neurotrophic factor S100-ß are targets of the autoimmune response in type 1 diabetes.

The novel oral glucan synthase inhibitor SCY-078 shows in vitro activity against sessile and planktonic Candida spp.

Objectives: We studied the antifungal activity of SCY-078 (an orally bioavailable 1,3-ß -d- glucan synthesis inhibitor), micafungin and fluconazole against the planktonic and sessile forms of 178 Candida and non- Candida isolates causing fungaemia in patients recently admitted to a large European hospital. Methods: The in vitro activity of SCY-078, micafungin and fluconazole against the planktonic form of the isolates was assessed using EUCAST EDef 7.3 and CLSI M27-A3. Antibiofilm activity was assessed using the XTT reduction assay. Results: SCY-078 and micafungin showed potent in vitro activity against Candida and non- Candida isolates. The in vitro activity of both drugs was similar, but SYC-078 displayed significantly lower MIC values than micafungin against Candida parapsilosis and non- Candida isolates, whereas micafungin displayed significantly lower MIC values for the remaining species ( P <0.001). In contrast, SCY-078 and micafungin showed essentially the same activity against the biofilms with the exception of Candida glabrata , in which the micafungin sessile MIC values were significantly lower ( P <0.001). These observations were confirmed by assessing biofilm structure by scanning electron microscopy after antifungal treatment. Conclusions: Our study showed that the high in vitro activity of SCY-078 against invasive Candida isolates in both sessile and planktonic forms is comparable to that of micafungin.

Fundamentals and Methods for T- and B-Cell Epitope Prediction.

Adaptive immunity is mediated by T- and B-cells, which are immune cells capable of developing pathogen-specific memory that confers immunological protection. Memory and effector functions of B- and T-cells are predicated on the recognition through specialized receptors of specific targets (antigens) in pathogens. More specifically, B- and T-cells recognize portions within their cognate antigens known as epitopes. There is great interest in identifying epitopes in antigens for a number of practical reasons, including understanding disease etiology, immune monitoring, developing diagnosis assays, and designing epitope-based vaccines. Epitope identification is costly and time-consuming as it requires experimental screening of large arrays of potential epitope candidates. Fortunately, researchers have developed in silico prediction methods that dramatically reduce the burden associated with epitope mapping by decreasing the list of potential epitope candidates for experimental testing. Here, we analyze aspects of antigen recognition by T- and B-cells that are relevant for epitope prediction. Subsequently, we provide a systematic and inclusive review of the most relevant B- and T-cell epitope prediction methods and tools, paying particular attention to their foundations.

Biofilm Production and Antibiofilm Activity of Echinocandins and Liposomal Amphotericin B in Echinocandin-Resistant Yeast Species.

The echinocandins and liposomal amphotericin B are active against biofilm produced by echinocandin-susceptible Candida strains. However, few data have been reported on the production of biofilm by echinocandin-resistant isolates and their antifungal susceptibility. We studied the production of biofilm by fks mutant Candida strains and intrinsically echinocandin-resistant non-Candida isolates and the susceptibility of both entities to liposomal amphotericin B and echinocandins. We analyzed the production of biofilm by isolates from patients with fungemia (fks mutant Candida, n = 5; intrinsically echinocandin-resistant non-Candida, n = 12; and Candida wild type, n = 10). Biofilm formation was measured to classify strains according to biomass (crystal violet assay) and metabolic activity (XTT reduction assay). Preformed biofilms were tested against liposomal amphotericin B, caspofungin, micafungin, and anidulafungin. The sessile MIC was defined as the antifungal concentration yielding a 50% or 80% reduction in the metabolic activity of the biofilm compared to that of the growth control (SMIC50 and SMIC80, respectively). fks mutant Candida isolates formed biofilms in a fashion similar to that of Candida wild-type strains. The echinocandins had the highest activity against biofilms formed by wild-type Candida isolates, followed by fks mutant Candida isolates and non-Candida isolates. Liposomal amphotericin B had the highest activity against fks mutant Candida biofilms. The formation of biofilm by echinocandin-resistant strains was similar to that of wild-type strains, although resistance to echinocandins remained high.