Cross reactivity between IA-2 and phogrin/IA-2β in binding of autoantibodies in IDDM

Summary Patients with insulin-dependent diabetes mellitus (IDDM) possess antibodies to the cytoplasmic domains of two closely related tyrosine phosphatase-like proteins, IA-2 and phogrin, previously detected as 40 kDa and 37 kDa tryptic fragments, respectively. A higher proportion of IDDM patients possess antibodies to IA-2 than to phogrin, and autoimmunity to phogrin might arise through cross-reactivity with the highly homologous IA-2. In this study, we have investigated the major regions of IA-2 recognized by antibodies in IDDM patients and examined the ability of phogrin to block antibody binding to these regions as a measure of cross-reactivity. Analysis of antibody binding to in vitro transcribed and translated polypeptides representing different regions of the cytoplasmic domain of IA-2 identified five different patterns of reactivity with antibodies in IDDM. Protein footprinting analysis, whereby polypeptide fragments generated on protease treatment of immune complexes are studied, indicated considerable heterogeneity in antibody recognition of IA-2, even between sera with similar reactivity to deletion mutants. Blocking studies with recombinant phogrin indicated that IA-2 antibodies recognize epitopes that are both unique to IA-2 and shared with phogrin. The amino-terminal 150 amino acids of the cytoplasmic domain of IA-2 encompass epitopes that are not represented on phogrin, whereas shared epitopes are localized within the carboxy-terminal 220 amino acids. The results demonstrate considerable heterogeneity between IDDM patients in autoantibody recognition of IA-2 in IDDM, whereas antibody recognition of phogrin is restricted in most patients to epitopes also present on IA-2. [Diabetologia (1997) 40: 1327–1333] Received: 4 April 1997 and in revised form: 2 July 1997     

登革2型病毒E蛋白免疫优势表位的筛选鉴定

目的 用噬菌体展示肽库筛选登革2型病毒(DEN2)E蛋白的抗原表位，并确定该抗原表位性质。方法 以DEN2型特异的E单克隆抗体作为筛选分子，生物淘洗噬菌体随机12肽库，将筛选的噬菌体阳性克隆进行ELISA检测、DNA序列测定及展示肽的氨基酸序列推导，通过噬菌体展示肽序列与DEN2E蛋白的氨基酸一级结构的对比，初步确定E蛋白的抗原表位；用模拟该表位线性序列的合成十肽进行抗体结合试验、噬菌体竞争抑制试验及与DEN感染患者的血清学试验，确定其为免疫优势线性表位。结果 肽库淘洗获得的11个ELISA阳性的噬菌体克隆有相似的结构基序WFKKGSS，其展示肽与DEN2E蛋白390～398 AA序列有3～5个氨基酸相同。对应于DEN2E蛋白390～399AA的合成十肽能与淘洗单抗特异反应，并可抑制噬菌体阳性克隆与该单抗结合。该合成肽与DEN2感染患者血清有较高的免疫反应性。结论 本实验通过噬菌体随机肽库的生物淘洗确定的DEN2E蛋白(E390～398AA)线性序列为免疫优势表位，其对应的合成肽E10可望用于DEN2感染的快速诊断。     

Pichia pastoris displaying ZIKV protein epitopes from the Envelope and NS1 induce in vitro immune activation

The severe consequences of ZIKV infection and its emergence and re-emergence in several countries have boosted vaccines' development. Yeasts such as Pichia pastoris has been widely employed as antigen carriers for immunization against infectious agents. Components of the yeast cell wall have immunostimulatory properties, and recombinant antigens can be anchored to the cell surface to enhance the presentation to the immune system. Here we aimed at producing and anchoring ZIKV proteins in the P. pastoris surface as a vaccine approach. Expression cassettes were designed with epitopes of the Envelope and NS1 proteins. Immunofluorescence microscopy confirmed the anchoring of recombinant proteins. Yeasts' ability to stimulate immune cells was evaluated in vitro by incubation with lymphocytes and monocytes isolated from mouse spleen. P. pastoris expressing EnvNS1 epitopes promoted increased levels of IL-6, IL-10, and TNF-α cytokines and an increase in the number of CD4+, CD8+, and CD16+ lymphocytes, similarly to ZIKV. This profile is indicative of the activation of immunological cells and suggests an immunogenic potential of the proposed yeast vaccines against ZIKV, reinforcing the possibility of P. pastoris as adjuvant and carrier of antigens.     

Epitopes of human brain acetylcholinesterase

The main purpose of the present work was to identify B-cell epitopes on human brain acetylcholinesterase (AChE) by the synthetic peptide approach. Five hundred and seventy-four decapeptides comprising amino acids No. n to n+9 (where n denotes the residue number of the 583 amino acids in the primary structure of human brain AChE and is an integer in the range 1-574) were synthesized, using the multipin combinatorial chemical synthesis technique, and biotinylated. Epitopes of human brain AChE were detected by enzyme-linked immunosorbent assay (ELISA) and compared with the predicted epitopes of human AChE by 'Goldkey' software. Among 574 synthetic decapeptides, 47 decapeptides at 11 antigenic regions showed immunoreactivity with mouse anti-human brain AChE polyclonal antibodies. The minimum sequence of epitope was defined at every antigenic region explored. The locations and sequences of the former ten continuous epitopes at the 11 antigenic regions of the human brain AChE had been identified as follows: TPVLVWIY (112-119), RTVLVSMNY (143-151), LLDQRLALQW (173-182), RRATQLAH (246-253), VFRFSFVPV (294 approximately 302), KDEGSYFLVY (332-341), RVYA (424-427), LMRY (476-479), KAPQWPPY (496-503), GLRAQACAFW (523-532). The rate of hits of the predicted epitopes from the software came out at 33%. In our work, the epitopes of human AChE have been mapped by purified polyclonal antibody at eleven distinct sites in the primary structure.     

Searching for the Cartilage-associated Mimicry Epitope in Adjuvant Arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis ( Mt ) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein ( Mt. hsp65 178-186 ). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65 178-186 specific T cells.     

Methodological advances in the design of peptide-based vaccines

The tremendous advances in genomics, recombinant DNA technology, bioengineering and nanotechnology, in conjunction with the development of high-end computations, have been instrumental in the process of rational design of peptide-based vaccines. The use of peptide vaccines was limited owing to their inherent instability when systemically administered; however, advanced formulation techniques have been developed for their systemic delivery, thereby overcoming their degradation, clearance, cellular uptake and off-target effects. With the rise of sophisticated immunological predictors and experimental techniques, several methodological advances have occurred in this field. This review examines contemporary methods to identify and optimize epitopes, engineer their immunogenic properties and develop their safe and efficient delivery into the host.     

Altered gut microbiota and mucosal immunity in patients with schizophrenia

Evidence shows that gut microbiota may play important roles in schizophrenia pathogenesis via the “gut-brain” axis, but the mechanisms remain unclear. Here, eighty-four patients with schizophrenia and 84 sex- and age-matched healthy controls were enrolled. Shotgun metagenomic sequencing and 16S rRNA sequencing were performed, and the gut microbiota-associated epitopes (MEs) were predicted, which, together with IgA content, were used to determine the gut microbiota composition associated with gut immune status. Patients with schizophrenia had significantly reduced gut microbiota richnesses compared with those of the healthy controls, and the gut microbiota compositions clearly distinguished the patients with schizophrenia from the healthy controls. Based on two-stage metagenomic-wide association studies, nineteen gut microbiota taxonomies were associated with schizophrenia, and the microbial dysbiosis (MD) index was calculated based on the abundance of differential taxonomies. We found that MD index was positively correlated with MEs diversity and gut IgA levels, and negatively correlated with gut microbiota richness. Glutamate synthase (GOGAT) was more active in the guts of patients with schizophrenia than in those of healthy controls, and high GOGAT activity was associated with altered gut microbiota taxonomies associated with gut IgA levels. Our results may imply a role of the microbiome in the etiology of schizophrenia and contribute to the development of microbiome targeted interventions for schizophrenia.     

Characterization of HCV Genotype 5a Envelope Proteins: Implications for Vaccine Development and Therapeutic Entry Target

Background:

Hepatitis C virus (HCV) is one of the major causes of cirrhosis and hepatocellular carcinoma with an estimation of 185 million people with infection. The E2 is the main target for neutralizing antibody responses and the variation of this region is related to maintenance of persistent infection by emerging escape variants and subsequent development of chronic infection. While both E1 and E2 are hypervariable in nature, it is difficult to design vaccines or therapeutic drugs against them.

Objectives:

The objective of this study was to characterize genotype 5a E1 and E2 sequences to determine possible glycosylation sites, conserved B-cell epitopes and peptides in HCV that could be useful targets in design of vaccine and entry inhibitors.

Patients and Methods:

This study was conducted through PCR amplification of E1 and E2 regions, sequencing, prediction of B-cell epitopes, analysis of N-linked glycosylation and peptide design in 18 samples of HCV genotype 5a from South African.

Results:

Differences in the probability of glycosylation in E1 and E2 regions were observed in this study. Three conserved antigenic B-cell epitopes were predicted in the E2 regions and also 11 short peptides were designed from the highly conserved residues.

Conclusions:

This study provided conserved B-cell epitopes and peptides that can be useful for designing entry inhibitors and vaccines able to cover a global population, especially where genotype 5a is common.     

重组CYP2D26主要B细胞表位在II型自身免疫性肝炎小鼠模型构建中的效应研究

目的 研究在大肠杆菌中以重组病毒样颗粒形式表达的人Ⅱ型自身免疫性肝炎标志蛋白 CYP2D6主要抗原表位aa262-270免疫小鼠后对构建小鼠自身免疫性肝炎模型的效应.方法 设计人Ⅱ型自身免疫性肝炎标志蛋白主要抗原表位aa262-270 (WDPAQPPRD)的下负链寡核苷酸片段,经退火后插入表达乙肝核心蛋白 HBcAg的质粒pNP中(pNP质粒由pThioHisA质粒插入HBcAg基因片段构建而成).构建成质粒pNP-AIH2;转染大肠杆菌DH5α感受态细胞;以IPTG诱导重组蛋白表达,经硫酸铵沉淀、洗涤,密度梯度离心纯化和脱盐后,肌肉注射免疫小鼠3次,以ELISA和western blot检测免疫后小鼠特异性抗体的产生和水平,通过转氨酶检测和肝脏石蜡切片HE染色观察炎症反应.结果 SDS-PAGE结果显示重组菌诱导表达的目的蛋白分子量为19kD,与预期相符;电镜证实其以可溶性的病毒样颗粒形式存在;ELISA显示,免疫小鼠血.清与包被的肝脏匀浆蛋白特异反应,检测到持续存在的特异抗体,滴度至少为1∶2000; western blot检测表明,抗血清在分子量约为55 kD的地方产生了一个特异的条带,与小鼠CYP2D6蛋白大小一致.与正常对照相比,实验组小鼠转氨酶水平未明显升高,肝切片HE染色没有观测到明显的病理特征.结论 携带人Ⅱ型自身免疫性肝炎标志蛋白 CYP2D26主要表位的病毒样颗粒免疫小鼠后,可刺激产生较强的特异抗体水平,但在观测时间内,未见诱导小鼠产生明显的肝脏炎症反应.     

The Th1 immune response against HIV-1 Gag p24-derived peptides in mice expressing HLA-A02.01 and HLA-DR1

Using HLA-DR1-transgenic H-2 class II knockout mice, we identified two new HLA-DR1-restricted HIV-1 Gag p24-derived epitopes (Gag(321-340 )and Gag(331-350)) and confirmed the immunogenicity of seven that have been previously described. The human relevance was confirmed for the two new ones (Gag(321-340 )and Gag(331-350)) assaying peripheral blood mononuclear cells from HLA-DR1(+) HIV-1-infected long-term asymptomatic subjects and showing that Gag(331-350) could prime CD4(+) T cells from two HLA-DR1(+) HIV-1 seronegative donors in vitro. Seven of these epitopes, structurally conserved among HIV-1 clade B isolates, were selected for a comparative evaluation of their Th1 helper potential by immunizing HLA-A02.01/HLA-DR1-transgenic, H-2 class I/class II knockout mice with recombinant mouse invariant chain constructs in which each helper epitope was inserted in association with two reporter HIV-1-derived HLA-A02.01-restricted CD8(+) T cell epitopes. A T helper effect was demonstrated in all cases, and was particularly strong with epitopes Gag(301-320),Gag(321-340 )and Gag(271-290), which should, therefore, be considered in the design of new vaccines.