A potential mechanism by which aspiration of duodenogastric fluid augments the risk for bronchiolitis obliterans syndrome after lung transplantation.

OBJECTIVE: Aspiration of duodenogastric refluxate may damage the respiratory epithelium of lung allografts in transplant recipients. We sought to define a mechanism by which aspiration of duodenogastric fluid augments the risk of bronchiolitis obliterans syndrome after lung transplant in a murine model. METHODS: We analyzed the immunological effects of acute aspiration of duodenogastric fluid (0.5 mL/kg) on transplant naive (strain DBA/2J) and transplanted mice (strain B6D2F1/J to strain DBA/2J). Serum antibodies to the lung self-antigens (SAgs) K-alpha1 tubulin and collagen-V were determined by enzyme-linked immunosorbent assay. Exosomes were isolated from serum, and immunoblot membranes were probed for antibodies to lung SAgs. Lung sections were assessed for fibrotic burden and obliterative bronchiolitis lesions by histologic and immunohistochemical analyses, including trichrome staining. RESULTS: Transplanted mice that received duodenogastric fluid developed higher levels of antibodies to the lung SAgs K-alpha1 tubulin and collagen-V and exosomes with lung SAgs on posttransplant days 14 and 28 than transplanted mice with sham aspiration or transplant naive mice (with and without aspiration). All lung allografts demonstrated severe grade A4 rejection on posttransplant day 14, with the highest mean fibrotic burden and mean number of obliterative bronchiolitis-like lesions per microscopic field on day 28 in recipients with aspiration. CONCLUSIONS: This study links aspiration of duodenogastric fluid after lung transplant to higher autoimmune responses to lung SAgs and the release of circulating exosomes with lung SAgs, which together promote sustained immune responses leading to extensive lung parenchymal damage and, ultimately, severe obliterative bronchiolitis-the histologic hallmark of bronchiolitis obliterans syndrome.

Dynamic Network Biomarker of Pre-Exhausted CD8+ T Cells Contributed to T Cell Exhaustion in Colorectal Cancer.

Immunotherapy has achieved positive clinical responses in various cancers. However, in advanced colorectal cancer (CRC), immunotherapy is challenging because of the deterioration of T-cell exhaustion, the mechanism of which is still unclear. In this study, we depicted CD8+ T-cell developmental trajectories and characterized the pre-exhausted T cells isolated from CRC patients in the scRNA-seq data set using a dynamic network biomarker (DNB). Moreover, CCT6A identified by DNB was a biomarker for pre-exhausted T-cell subpopulation in CRC. Besides, TUBA1B expression was triggered by CCT6A as DNB core genes contributing to CD8+ T cell exhaustion, indicating that core genes serve as biomarkers in pre-exhausted T cells. Remarkably, both TUBA1B and CCT6A expressions were significantly associated with the overall survival of COAD patients in the TCGA database (p = 0.0082 and p = 0.026, respectively). We also observed that cellular communication between terminally differentiated exhausted T cells and pre-exhausted T cells contributes to exhaustion. These findings provide new insights into the mechanism of T-cell exhaustion and provide clue for targeted immunotherapy in CRC.

Super-Resolution Spatial Proximity Detection with Proximity-PAINT.

Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single-molecule sensitivity. Here, we introduce Proximity-PAINT (pPAINT), a variation of the super-resolution microscopy technique DNA-PAINT. pPAINT uses a split-docking-site configuration to detect spatial proximity with high sensitivity, low false-positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha- and beta-tubulin in microtubules using super-resolution detection.

COVID-19 in a lung transplant recipient: Exploring the diagnostic role of circulating exosomes and the clinical impact of advanced immunosuppression.

Exosomes isolated from plasma of lung transplant recipients with allograft injury contain donor-derived lung self-antigens (collagen V and Kα1 tubulin) and human leukocyte antigen (HLA) molecules. We present a case of a 76-year-old, female lung transplant recipient treated for acute cellular rejection with methylprednisolone and anti-thymocyte globulin, who subsequently contracted SARS-CoV-2 and developed a sharp increase in the mean fluorescent intensity of anti-HLA antibodies. Analysis of circulating exosomes during rejection, but before SARS-CoV-2 infection, revealed the presence of lung self-antigens and HLA class II molecules. After the patient contracted SARS-CoV-2, exosomes with the SARS-CoV-2 spike protein were also found. After resolution of infectious symptoms, exosomes with SARS-CoV-2 spike protein were no longer detected; however, exosomes with lung self-antigens and HLA class II molecules persisted, which coincided with a progressive decline in spirometric flows, suggesting chronic lung allograft dysfunction. We propose that the analysis of circulating exosomes may be used to detect allograft injury mediated by both rejection and infection. Furthermore, the detection of exosomes containing viral proteins may be helpful in identifying allograft injury driven by viral pathogens.

Decline in Club Cell Secretory Proteins, Exosomes Induction and Immune Responses to Lung Self-antigens, Kα1 Tubulin and Collagen V, Leading to Chronic Rejection After Human Lung Transplantation.

BACKGROUND: Chronic lung allograft dysfunction (CLAD), is a major hurdle for long-term lung allograft survival after lung transplant and roughly 50% of lung transplant recipients (LTxRs) develop CLAD within 5 years. The mechanisms of CLAD development remain unknown. Donor-specific immune responses to HLA and lung self-antigens (SAgs) are vital to the pathogenesis of CLAD. Reduction in Club cell secretory protein (CCSP) has been reported in bronchoalveolar lavage (BAL) fluid samples from LTxRs with bronchiolitis obliterans syndrome (BOS). CCSP levels in BAL fluid and development of antibodies to lung SAgs in plasma were determined by ELISA. Cytokines in BAL fluid were analyzed by 30-plex Luminex panel. Exosomes from BAL fluid or plasma were analyzed for SAgs, natural killer (NK) cells markers, and cytotoxic molecules. RESULTS: We demonstrate that LTxRs with BOS have lower CCSP levels up to 9 months before BOS diagnosis. LTxRs with antibodies to SAgs 1-year posttransplant also developed DSA (43%) and had lower CCSP. BOS with lower CCSP also induced Interleukin-8 and reduced vascular endothelial growth factor. Exosomes from BOS contained increased SAgs, NK cells markers, and cytotoxic molecules. CONCLUSIONS: We conclude lower CCSP leads to inflammation, pro-inflammatory cytokine production, immune responses to HLA and SAgs, and induction of exosomes. For the first time, we demonstrate that CCSP loss results in exosome release from NK cells capable of stimulating innate and adaptive immunity posttransplant. This increases the risk of BOS, suggesting a role of NK cell exosomes in CLAD development.

Impact and production of Non-HLA-specific antibodies in solid organ transplantation.

Organ transplantation is an effective way to treat end-stage organ disease. Extending the graft survival is one of the major goals in the modern era of organ transplantation. However, long-term graft survival has not significantly improved in recent years despite the improvement of patient management and advancement of immunosuppression regimen. Antibody-mediated rejection is a major obstacle for long-term graft survival. Donor human leucocyte antigen (HLA)-specific antibodies were initially identified as a major cause for antibody-mediated rejection. Recently, with the development of solid-phase-based assay reagents, the contribution of non-HLA antibodies in organ transplantation starts to be appreciated. Here, we review the role of most studied non-HLA antibodies, including angiotensin II type 1 receptor (AT1 R), K-α-tubulin and vimentin antibodies, in the solid organ transplant, and discuss the possible mechanism by which these antibodies are stimulated.

Antibodies in cerebral cavernous malformations react with cytoskeleton autoantigens in the lesional milieu.

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.

Chronic Lung Allograft Dysfunction: Immune Responses Induced by Circulating Exosomes with Lung-Associated Self-Antigens.

Exosomes, nanovesicles shown to regulate physiological processes in vivo, have been implicated in pathological conditions including cancer, autoimmune disease, infectious disease, neurodegenerative disease, and allograft rejection. Studies of lung transplant recipients with primary graft dysfunction, respiratory viral infection, and (acute) rejection have demonstrated circulating exosomes containing donor-mismatched human leukocyte antigen and lung-associated self-antigens, K-alpha 1 tubulin and collagen V, indicating that exosomes are originating from the transplanted organ. These circulating exosomes likely play a role in activating immune responses that lead to increased risk of chronic lung allograft dysfunction, as exosomes efficiently present their antigens to the immune system by all known pathways of antigen recognition (i.e., direct, indirect, and semidirect pathways). Here, we discuss exosome biogenesis, describe their contents, and address the mechanism of exosome-mediated activation of immune responses that lead to allograft rejection, especially after lung transplantation.

Clinical relevance of lung-restricted antibodies in lung transplantation.

Lung transplant is a definitive treatment for several end-stage lung diseases. However, the high incidence of allograft rejection limits the overall survival following lung transplantation. Traditionally, alloimmunity directed against human leukocyte antigens (HLA) has been implicated in transplant rejection. Recently, the clinical impact of non-HLA lung-restricted antibodies (LRA) has been recognized and extensive research has demonstrated that they may play a dominant role in the development of lung allograft rejection. The immunogenic lung-restricted antigens that have been identified include amongst others, collagen type I, collagen type V, and k-alpha 1 tubulin. Pre-existing antibodies against these lung-restricted antigens are prevalent in patients undergoing lung transplantation and have emerged as one of the predominant risk factors for primary graft dysfunction which limits short-term survival following lung transplantation. Additionally, LRA have been shown to predispose to chronic lung allograft rejection, the predominant cause of poor long-term survival. This review will discuss ongoing research into the mechanisms of development of LRA as well as the pathogenesis of associated lung allograft injury.

Leishmania infantum ß-Tubulin Identified by Reverse Engineering Technology through Phage Display Applied as Theranostic Marker for Human Visceral Leishmaniasis.

Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the ß-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.

The role of microtubules in the immune system and as potential targets for gut-based immunotherapy.

Microtubules (MTs) are tubular polymers of tubulin that are highly dynamic and found throughout the cytoplasm. MTs are involved in maintaining cell structure and, together with microfilaments and intermediate filaments, form the cytoskeleton. Recent findings on MT structure and function contributed to the understanding of their potential role as players in the innate and adaptive immune systems. Additionally, studies suggest an essential role for these cellular structures in the gut. Here, we review recent data on interactions between MT and various arms of the immune system and propose a model that represents gut MTs as potential targets for immunotherapy, and specifically for oral immunotherapy.

Prevalence of antibodies to lung self-antigens (Kα1 tubulin and collagen V) and donor specific antibodies to HLA in lung transplant recipients and implications for lung transplant outcomes: Single center experience.

PURPOSE: For patients with end stage lung disease, lung transplantation (LT) remains the only definitive treatment option. Long term survival post LT is limited by acute and chronic allograft dysfunction. Antibodies to lung self-antigens Kα1Tubulin and collagen V (autoantibodies) have been implicated in adverse outcomes post LT. The aim of our study was to determine the prevalence of autoantibodies in pre- and post-transplant sera, evaluate the impact on post-transplant outcomes. METHODS: In a prospective observational cohort analysis, 44 patients were enrolled who received LT between 09/01/2014 and 10/31/2015. Pre- and post-transplant sera were analyzed using enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies to collagen I, collagen V, and K-alpha 1 tubulin. The outcome variables are presence of primary graft dysfunction (PGD), cumulative acute cellular rejection (ACR), treatment with pulse steroids for clinical rejection, association with DSA, and onset of Bronchiolitis Obliterans Syndrome (BOS). RESULTS: In our cohort, 33 patients (75%) tested positive for the presence of autoantibodies. Pre-transplant autoantibodies were present in 23 patients (70%). Only a small percentage (26%) cleared these antibodies with standard immunosuppression. Some developed de novo post-transplant (n = 10). PGD was observed in 34% of our cohort, however the presence of autoantibodies did not correlate with increase in the incidence or severity of PGD. The prevalence of donor specific antibodies (DSA) in the entire cohort was 73%, with an increased prevalence of DSA noted in the autoantibody positive group (78.7% vs. 54.5%) than in the autoantibody negative group. BOS was observed in 20% of the cohort, with a median time to onset of 291 days' post-transplant. Patients with pre-transplant autoantibodies had a statistically significant decrease in BOS-free survival (p = 0.029 by log-rank test). CONCLUSIONS: In our cohort, we observed a high prevalence of autoantibodies and DSA in lung transplant recipients. Pre-transplant autoantibodies were associated with de novo development of DSA along with a decrease in BOS-free survival. Limitations to our study include the small sample size and single center enrollment, along with limited time for follow-up.

Architecture of the nervous system in metacercariae of Diplostomum pseudospathaceum Niewiadomska, 1984 (Digenea).

The development of metacercariae of Diplostomum pseudospathaceum Niewiadomska, 1984 is accompanied by profound morphological transformations often characterized as metamorphosis, which makes these metacercariae an interesting case for studying the morphogenesis of the digenean nervous system. Although the nervous system of D. pseudospathaceum is one of the most extensively studied among digeneans, there are still gaps in our knowledge regarding the distribution patterns of some neuroactive substances, most notably neuropeptides. The present study addresses these gaps by studying pre-infective metacercariae of D. pseudospathaceum using immunochemical staining and confocal microscopy to characterize the distribution patterns of serotonin (5-HT) and two major groups of flatworm neuropeptides, FMRFamide-related (FaRPs) and substance P-related (SP) peptides. The general morphology of the nervous system was examined with antibodies to alpha-tubulin. The nervous system of the metacercariae was shown to conform to the most common morphology of the nervous system in the hermaphroditic generation, with three pairs of posterior nerve cords and four pairs of anterior nerves. The patterns of FaRP- and 5-HT immunoreactivity (IR) were similar to those revealed in earlier studies by cholinesterase activity, which is in accordance with the known role of these neurotransmitters in controlling muscle activity in flatworms. The SP-IR nervous system was significantly different and consisted of mostly bipolar cells presumably acting as mechanoreceptors. The architecture of the nervous system in D. pseudospathaceum metacercariae is discussed in comparison to that in cercariae of D. pseudospathaceum and metacercariae of related digenean species.

Amplified Expansion Stimulated Emission Depletion Microscopy.

Expansion microscopy (ExM) enhances spatial resolution by using a swellable polymer that expands the sample volume by a factor of ≈4 in one dimension and a factor of ≈64 in volume. Combining ExM with stimulated emission depletion (STED) microscopy, referred to as ExSTED, increases the resolution to up to 10 nm. However, photobleaching is a critical issue in ExSTED because the sample expansion lowers the fluorophore density whereas high-resolution STED requires high depletion intensity. To overcome these issues, we developed extremely bright expansion nanoscopy by using biotin-avidin signal amplification to increase the labeling density. Our method provides up to sevenfold increases in fluorescence signal intensity in expanded samples, thus enabling the use of STED imaging with maximum depletion intensities of a commercial microscope in the order of GW cm-2 . We demonstrated the method by using biotinylated antibodies and genetic incorporation approaches that allow localization of biotin in a specific molecule or organelle.

Plant and human aquaporins: pathogenesis from gut to brain.

Corn, soybean, spinach leaf, and tomato aquaporins have been shown to share homology with human aquaporin-4, which is abundantly expressed by brain astrocytic endfeet. Thus, antibodies formed against the dietary aquaporins may potentially cross-react with brain aquaporin, leading to blood-brain barrier permeability and setting the stage for neuroautoimmunity and neurodegeneration. Here, we review the role of aquaporins in plants and humans in maintaining a healthy organism and mechanisms by which dietary aquaporins may contribute to neurological disorders. We include clinical data on the correlation between four real-world, dietary aquaporin and five neurological tissue antibodies. Our findings showed the percent of neurological tissue antibody production increased with the number of positive food aquaporins. Of the four food aquaporins, spinach was the most common reactive. Of the neurological tissues assessed, tubulin was the most common positive. Patients with antibody reactivity to dietary aquaporins may consider abstaining from the aquaporin-containing food in order to prevent neurological tissue damage.

Diagnostic application of recombinant Leishmania proteins and evaluation of their in vitro immunogenicity after stimulation of immune cells collected from tegumentary leishmaniasis patients and healthy individuals.

The present study evaluated the cytokine profile in PBMC supernatants and the humoral response in mucosal leishmaniasis (ML) patients and in healthy subjects living in an endemic area. Four proteins, which had previously proven to be antigenic in the human disease, were tested: LiHyM, enolase, eukaryotic initiation factor 5a, and Beta-tubulin. Results showed that all of the proteins stimulated human cells with higher IFN-γ and lower IL-4 and IL-10 levels. The analysis of antibody isotypes correlated with cell response, since the IgG2 production was higher than IgG1 in both groups. By contrast, a Th2 response was found when an antigenic Leishmania extract was used. Serological analyses revealed high sensitivity and specificity values for the serodiagnosis of the disease, when compared to the data obtained using the antigenic preparation. In conclusion, this study presents new candidates to be evaluated as biomarkers in tegumentary leishmaniasis.

Circulating immune complexome analysis identified anti-tubulin-α-1c as an inflammation associated autoantibody with promising diagnostic value for Behcet's Disease.

BACKGROUND: Behcet's disease (BD) is a chronic, multisystem-involved vasculitis and its pathogenesis remains elusive. No specific serological markers for BD diagnosis have been established. Identification of novel diagnostic biomarkers will be helpful in timely diagnostic and treatment for Behcet's disease. OBJECTIVE: To screen novel autoantigens or autoantibodies with potential diagnostic value in circulating immune complexes (CICs) from BD patients. METHODS: A proteomic strategy for immune complexome analysis was developed, in which CICs were separated from serum sample of 10 BD patients and 10 healthy controls and then subjected to Orbitrap mass spectrometry for autoantigen profiling. Anti-tubulin-α-1c antibody levels were further determined by enzyme-linked immunosorbent assay (ELISA) in sera of patients with BD, systemic lupus erythematosus (SLE), recurrent aphthous ulcers (RAU), ANCA associated systemic vasculitis (AASV), Takayasu's arteritis (TA) and 59 healthy controls. RESULT: A total of 17 potential antigens were identified in CICs from BD patients, but not in HC. The autoantibody to one of the identified antigens, tubulin-α-1c, was significantly increased in BD patients compared with that in healthy and disease controls. The sensitivity and specificity of tubulin-α-1c antibody in the diagnosis of BD in this study were 61.36% and 88.4%, respectively. Further analysis demonstrated that anti-tubulin-α-1c was associated with complications of deep venous thrombosis and erythema nodosum in BD. The levels of anti-tubulin-α-1c were also significantly correlated with the BD inflammation and disease activity markers ESR, CRP and BVAS. CONCLUSION: Anti-tubulin-α-1c antibody is a promising biomarker in diagnosis and severity evaluation of BD and in indicating the risk of deep venous thrombosis and erythema nodosum. The immune complexome analysis by proteomic CIC autoantigen screening is a feasible way of identifying novel biomarkers in BD.

Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.

A key event required for effective resolution of inflammation is efferocytosis, which is defined as phagocytic removal of apoptotic cells mostly by macrophages acquiring an alternatively activated phenotype (M2). c-Myc has been reported to play a role in alternative activation of human macrophages and is proposed as one of the M2 macrophage markers. We found that M2-like peritoneal macrophages from zymosan A-treated mice exhibited a marked accumulation of Myc-nick, a truncated protein generated by a Calpain-mediated proteolytic cleavage of full-length c-Myc. Further, ectopic expression of Myc-nick in murine bone marrow-derived macrophages promoted the M2 polarization and, consequently, enhanced their efferocytic capability. Notably, Myc-nick-induced efferocytosis was found to be tightly associated with α-tubulin acetylation by K acetyltransferase 2a (Kat2a/Gcn5) activity. These findings suggest Myc-nick as a novel proresolving mediator that has a fundamental function in maintaining homeostasis under inflammatory conditions.-Zhong, X., Lee, H.-N., Kim, S. H., Park, S.-A., Kim, W., Cha, Y.-N., Surh, Y.-J. Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.

Antibody Repertoires Identify ß-Tubulin as a Host Protective Parasite Antigen in Mice Infected With Trypanosoma cruzi.

Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and ß-tubulin. The major protein band recognized by host IgG was T. cruzi ß-tubulin. The T. cruzi ß-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi ß-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi ß-tubulin. A single immunization of mice with recombinant T. cruzi ß-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.

Homology modeling and epitope prediction of Der f 33.

Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34-45, 63-67, 103-108, 224-230, 308-316, and 365-377) and four T cell epitopes (positions 178-186, 241-249, 335-343, and 402-410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.