The identification of potentially pathogenic and therapeutic epitopes from common human allergens

ObjectivesTo outline the processes involved in large-scale T-cell epitope identification from common allergens and illustrate their relevance to development of allergy specific immunotherapy.Data SourcesA set of studies recently published by our laboratory illustrating high-throughput identification of allergen specific T-cell epitopes.Study SelectionT-cell responses contribute both directly and indirectly to allergy-related disease. However, the molecular targets (epitopes) recognized by allergen-specific T cells are largely undefined. We review several different studies in the last 2 years that identified novel T-cell epitopes from a panel of 32 different allergen sources.ResultsAllergen-specific T-cell responses are highly heterogeneous. Epitopes prevalently recognized in allergic patients are often capable of binding to multiple HLA class II molecules. This feature can be used to predict these promiscuous epitopes by bioinformatic predictions. This approach was validated in the Timothy grass system and then applied to a panel of 31 other allergen sources.ConclusionT-cell epitopes for common allergens have been identified, and a general method to identify epitopes from additional allergens has been validated. Characterization of epitopes for common allergens might enable new diagnostics and immunotherapy regimens. These data will also allow the study of T-cell responses in different patient populations and throughout disease progression.     

In silico identification of novel bacterial ADP-ribosyltransferases

With the advent of the genomic era, identification of bacterial factors involved in virulence is a different challenge. Given the vast amount of information available on toxins, in terms of sequence and 3D structure, and thanks to the growing number of sequenced bacterial genomes, it is possible to proceed by homology criteria to predict novel toxins in different microorganisms. ADP-ribosyltransferases constitute a class of functionally conserved enzymes, which display toxic activity in a variety of bacterial pathogens. Since these proteins play a key role in pathogenesis, they have been extensively characterized and successfully used as vaccine components and mucosal adjuvants. Therefore, the application of in silico analyses to identify novel members of this class of enzymes represents an important challenge in the genomic era. To address this subject, different groups have recently pursued homology-based procedures to screen bacterial genomes for novel, yet undiscovered ADP-ribosyltransferases (ADPRTs) and have identified more than twenty novel ADPRTs in Gram-positive and Gram-negative bacteria. We have developed a novel pattern-based computational approach, which, flanked by secondary structure prediction tools, has allowed the identification of previously unrecognised putative ADPRTs. One of them, identified in Neisseria meningitidis has been extensively characterized and shown to possess the predicted enzymatic activity, suggesting a possible role of this protein in the virulence of Meningococcus.     

In silico identification of Gram-negative bacterial secreted proteins from primary sequence

In this study, we focus on different types of Gram-negative bacterial secreted proteins, and try to analyze the relationships and differences among them. Through an extensive literature search, 1612 secreted proteins have been collected as a standard data set from three data sources, including Swiss-Prot, TrEMBL and RefSeq. To explore the relationships among different types of secreted proteins, we model this data set as a sequence similarity network. Finally, a multi-classifier named SecretP is proposed to distinguish different types of secreted proteins, and yields a high total sensitivity of 90.12% for the test set. When performed on another public independent dataset for further evaluation, a promising prediction result is obtained. Predictions can be implemented freely online at http://cic.scu.edu.cn/bioinformatics/secretPv2_1/index.htm.     

In silico identification of potential new latex allergens

BACKGROUND: Allergy to latex of Hevea brasiliensis is a frequent problem. In spite of the significant progress of research, the identity and cross-reactivity of some latex allergens are unknown. OBJECTIVE: To identify, among the fully characterized latex proteins, those with a higher probability to be allergenic. METHODS: We used in silico techniques (amino acid sequence comparison and molecular modelling) to identify potential new allergens among the known proteins of H. brasiliensis. RESULTS: Cu/Zn superoxide dismutase, heat shock protein and calmodulin of H. brasiliensis show highly significant (E < 10(-9)) amino acid sequence homologies with known allergens. CONCLUSION: On the basis of our data, Cu/Zn superoxide dismutase, heat shock protein and calmodulin are the most probable allergens among fully characterized proteins of H. brasiliensis, and could potentially explain, at least in part, the multiple cross-reactivities of latex with vegetable foods and other plant-derived products. Consequently, we think that the above proteins should be particularly considered in the future laboratory and clinical research.     

In silico prediction of FVIII epitopes recognised by natural autoantibodies in polyvalent immunoglobulin concentrates

Inhibitory antibodies directed against blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic and autoimmune patients. Identifying B-cell FVIII epitopes and mapping them on the molecule remain important challenges. Using a combination of different algorithms, more than 30 hypothetical linear epitopes were predicted on the FVIII molecule surface. We selected several major predicted sequences, spanning all FVIII domains, for specific antibody induction in rabbits. All peptides tested successfully induced production of specific anti-FVIII rabbit antibodies, supporting the relevance of our approach. To investigate the presence of FVIII-reactive antibodies in the healthy donor population, a pooled fraction rich in all IgG subclasses was purified on peptide-Sepharose columns. Substantial amounts of Ig, specific for each FVIII peptide, were purified with yields ranging from 8 to 223 ng/mg immunoglobulins. Our results confirm the diversity of FVIII epitopes recognised by natural human anti-FVIII autoantibodies. All IgG subclasses were found in the affinity-isolated anti-peptide material, with overrepresentation of IgG2 and IgG4. Evidence was also found for new FVIII epitopes. Five human anti-peptide preparations displayed FVIII-neutralising activity, ranging from 1.3 to 5.3 BU/mg. Although the presence of naturally occurring anti-FVIII antibodies in healthy donors has been previously described, our methodology has allowed, for the first time, a fine mapping of several inhibitory and non-inhibitory epitopes. Our observations support the hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones.     

In silico analysis of tkt1 from avian pathogenic Escherichia coli and its virulence evaluation in chickens

Extraintestinal pathogenic Escherichia coli (ExPEC) contain tktA and tktB which code for transketolases involved in the pentose phosphate pathway. Recent studies demonstrated that a third gene coding for transketolase 1 (tkt1) was located in a pathogenicity island of avian and human ExPEC belonging to phylogenetic group B2. In the present study, in silico analysis of tkt1 revealed 68% and 69% identity with tktA and tktB, respectively, of ExPEC and 68% identity with tktA and tktB of E. coli MG1655. The translated tkt1 shared 69% and 68% identity with TktA and TktB proteins, respectively, of ExPEC and E. coli MG1655. Phylogenetically, it is shown that the three genes (tktA, tktB and tkt1) cluster in three different clades. Further analysis suggests that tkt1 has been acquired though horizontal gene transfer from plant-associated bacteria within the family Enterobacteriaceae. Virulence studies were performed in order to evaluate whether tkt1 played a role in avian pathogenic E. coli CH2 virulence in chickens. The evaluation revealed that mutant virulence was slightly lower based on LD50 when compared to the wild type during infection of chickens, but there were no significant differences when the two strains were compared based on the number of deaths and lesion scores.     

Towards in silico prediction of immunogenic epitopes

As torrents of new data now emerge from microbial genomics, bioinformatic prediction of immunogenic epitopes remains challenging but vital. In silico methods often produce paradoxically inconsistent results: good prediction rates on certain test sets but not others. The inherent complexity of immune presentation and recognition processes complicates epitope prediction. Two encouraging developments - data driven artificial intelligence sequence-based methods for epitope prediction and molecular modeling methods based on three-dimensional protein structures - offer hope for the future.     

Preliminary identification of mycobacteria isolated from clinical specimens

From March 1979 to June 1980, we preliminary identified mycobacteria at the time of their initial isolation by colonial morphology, pigmentation, and growth rate. The definitive identifications were predicted accurately for 92% of the mycobacteria isolated from 108 patients. For laboratories that isolate relatively few different species of mycobacteria, the advantages of this approach are its simplicity and the speed with which presumptive identifications can be made available for clinical use.     

Rapid detection and identification of pathogenic mycobacteria by combining radiometric and nucleic acid probe methods.

The combination of radiometric methodology (BACTEC 12B) and probe technology for recovery and identification of mycobacteria was studied in two large hospital laboratories. The sediment from vials with positive growth indices was tested with DNA probes specific for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. The sensitivity of the radiometric method and the specificity of the probes resulted in a marked reduction in the time to the final report. Biochemical testing could be eliminated on isolates giving a positive reaction with one of the probes. Some 176 isolates of M. tuberculosis, 110 of M. avium, and 5 of M. intracellulare were recovered. Two-thirds of these isolates were detected and identified within 2 weeks of inoculation and the remainder was detected by 4 weeks, a reduction of 5 to 7 weeks to the final report.     

H5N1型高致病性禽流感病毒血凝素蛋白及神经氨酸酶的B细胞抗原表位预测

目的预测H5N1亚型高致病性禽流感病毒HA蛋白和NA蛋白的B细胞表位,为基于B细胞表位的预防性疫苗设计提供依据。方法基于HA蛋白和NA蛋白的蛋白质序列,采用Kyte-Doolittle的亲水性方案,Emini方案,Karplus方案和Jameson-wolf抗原指数方案,并辅以MAGE蛋白的二级结构柔性区域分析,预测HA蛋白和NA蛋白的B细胞表位。结果分别预测出了6条血凝素蛋白(Hemagglutinin,HA)以及6条神经氨酸酶(Neuraminidase,NA)B细胞优势表位。结论这些B细胞表位可为禽流感疫苗的研制提供实验依据。     

Modulation of macrophage antimicrobial mechanisms by pathogenic mycobacteria

Tuberculosis remained a mysterious disease until Koch was able to demonstrate in the late 1800s that it was caused by a bacterium spread by aerosols, Mycobacterium tuberculosis. Today, tuberculosis still is a major health problem causing approximately 2 million deaths annually with about one third of the world's population being latently infected with M. tuberculosis. The secret of success for M. tuberculosis lies in its ability to persist inside host cells, the macrophages. Whereas macrophages are designed to destroy any incoming microbe, pathogenic mycobacteria have evolved strategies to survive within macrophages by preventing phagosome-lysosome fusion, thereby creating a niche that allows them to persist within an otherwise hostile environment. In this contribution, we discuss recent advances in our understanding of the interplay between the host and this pathogen that lead to survival of mycobacteria within macrophages.     

In silico identification of breast cancer genes by combined multiple high throughput analyses

Publicly available human genomic sequence data provide an unprecedented opportunity for researchers to decode the functionality of human genome. Such information is extremely valuable in cancer prevention diagnosis and treatment. Cancer Genome Anatomy Project (CGAP) and Gene Expression Omnibus (GEO) are two bioinformatic infrastructures for studying functional genomics. The goal of this study is to explore the feasibility of incorporating the Internet-available bioinformatic databases to discover human breast cancer-related genes. Several tools including the Gene Finder, Virtual Northern (vNorthern) and SAGE digital gene expression displayer (DGED) were used to analyze differential gene expression between benign and malignant breast tissues. A pilot study was performed using both EST and SAGE vNorthern to analyze the expression of a panel of known genes, including high abundance genes beta-actin and G3PDH, low abundance genes BRCA1 and p53, tissue-specific genes CEA and PSA and two breast cancer-related genes Her2/neu and MUC1. We found a high expression of beta-actin and G3PDH and a low expression of BRCA1 and p53 across different types of tissues as well as a tissue-specific expression of CEA in colon and PSA in prostate. A further analysis of 30 known breast cancer-related genes in breast cancer tissues by vNorthern demonstrated a high expression of oncogenes and low expression of tumor suppressor genes. An open-end analysis of two pools of breast cancer and benign breast tissue libraries by SAGE DGED produced 53 differentially expressed genes according to the screening criteria of a >five-fold difference and p<0.01. Further analysis by EST vNorthern and virtual microarray analysis reduced the candidate genes to six, with four down-regulated genes, ANXA1, CAV1, KRT5 and MMP7, and two up-regulated genes, ERBB2 and G1P3 in breast cancer. These findings were validated by a real-time RT-PCR analysis in eight paired human breast cancer tissue samples. We conclude that the combined multiple high throughput analyses is an effective data mining strategy in cancer gene identification. This approach may improve the usage of public available genomic data through strategic data mining of high throughput analysis.     

Rapid identification of mycobacteria by Raman spectroscopy

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.     

In silico evolution of early metabolism

We developed a simulation tool for investigating the evolution of early metabolism, allowing us to speculate on the formation of metabolic pathways from catalyzed chemical reactions and on the development of their characteristic properties. Our model consists of a protocellular entity with a simple RNA-based genetic system and an evolving metabolism of catalytically active ribozymes that manipulate a rich underlying chemistry. Ensuring an almost open-ended and fairly realistic simulation is crucial for understanding the first steps in metabolic evolution. We show here how our simulation tool can be helpful in arguing for or against hypotheses on the evolution of metabolic pathways. We demonstrate that seemingly mutually exclusive hypotheses may well be compatible when we take into account that different processes dominate different phases in the evolution of a metabolic system. Our results suggest that forward evolution shapes metabolic network in the very early steps of evolution. In later and more complex stages, enzyme recruitment supersedes forward evolution, keeping a core set of pathways from the early phase.     

Species identification of mycobacteria in paraffin-embedded tissues: frequent detection of nontuberculous mycobacteria.

Diagnosis of infections caused by mycobacteria, especially nontuberculous mycobacteria still represents a difficult task both in microbiology and pathology. The aim of this study was to determine the frequency of mycobacterial DNA detectable by PCR in formalin-fixed paraffin-embedded tissues showing suspicious granulomatous lesions. A total of 190 archival specimens were analyzed, using a nested PCR protocol, which amplifies a fragment of the mycobacterial 65-kDa heat-shock protein gene. Restriction fragment-length polymorphisms and sequencing were utilized to further analyze the obtained PCR products. Corresponding microbiological culture results were available for 41 cases. We detected mycobacterial DNA in 119 cases (63%), of which 71 (60%) were positive for Mycobacterium tuberculosis complex DNA and 41 (34%) for DNA of nontuberculous mycobacteria. Seven cases (6%) could not be subtyped for technical reasons. The largest group of nontuberculous mycobacteria comprised 29 cases (25% of the 119 positive cases), which were assigned to Mycobacterium fortuitum complex. Mycobacterium avium-intracellulare complex was detected in eight (7%) cases, Mycobacterium gordonae in three (2.5%) and Mycobacterium rhodesiae in a single case (0.8%). All cases of Mycobacterium tuberculosis were unequivocally identified by restriction fragment-length polymorphism analysis. In contrast, sequencing provided a gain of information over restriction fragment-length polymorphism analysis in 37% of the nontuberculous mycobacteria cases (15 of 41). Alignment studies on DNA of nontuberculous mycobacteria showed frequent sequence variations, supporting the existence of sequevars. Comparison of molecular data to available results of microbiological culture assays showed a good concordance of 83%. In conclusion, amplification and sequencing of the mycobacterial 65-kDa heat-shock protein gene is an excellent tool for species identification of mycobacteria, especially nontuberculous mycobacteria, in formalin-fixed paraffin-embedded tissues.     

鼠Ⅲ型肝炎病毒H-2K~K限制性CTL表位的初步鉴定

目的预测和初步鉴定鼠Ⅲ型肝炎病毒(MHV-3)的CTL表位,为基于慢性肝炎病毒抗原表位的免疫治疗奠定理论基础。方法利用超基序、量化基序及人工神经网络方案相结合预测MHV-3 S蛋白的H-2K~K限制性CTL表位;用分子模拟对候选表位肽做进一步筛选;人工合成相关待测表位肽,利用T2-K~K细胞株测定各肽与H-2K~K分子的结合力。结果结合超基序、量化基序、人工神经网络预测结果及分子模拟结果,预测出了4条候选表位肽。MHC亲和力实验表明,在候选的4条表位肽中,IEPYNGVI(141-148)、YELSGYTV(306-313)与H-2K~K呈中等亲和力结合,荧光系数分别为1.25及1.04。结论表位预测的结果与亲和力分析结果一致性较好,两者联合应用初步认为IEPYNGVI(141-148)及YELSGYTV(306-313)为MHV-3 S蛋白H-2K~K限制性CTL表位的可能性最大,为下一步体内鉴定及基于小鼠肝炎病毒抗原表位的免疫治疗奠定基础。