Structural and functional variations in skeletal-muscle and scallop muscle actins.

Structural and functional properties in two striated-muscle actins, one from a vertebrate, the other from an invertebrate (scallop), were compared in relation to a smooth-muscle actin isoform (aortic actin). In spite of differences in the variable N-terminal region, the two striated-muscle isoactins showed, in contrast with aortic actin, a large structural homology revealed by proteinase-susceptibility and interaction with the myosin head. Thus the myosin head may bind to the two striated-muscle actins in constant parts of the 18-113 sequence. In contrast, antigenic reactivity of conformational epitopes of these actins strongly differentiated scallop actin from the two others. The behaviour of the scallop actin appears to be related to several amino acid substitutions located near or at functional domains such as monomer-monomer binding site, DNAase-I-dependent actin-actin binding site and actin-severing domain, which modified the polypeptide chain exposure.     

Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model.

A model of the tick-borne encephalitis virus envelope protein E is presented that contains information on the structural organization of this flavivirus protein and correlates epitopes and antigenic domains to defined sequence elements. It thus reveals details of the structural and functional characteristics of the corresponding protein domains. The localization of three antigenic domains (composed of 16 distinct epitopes) within the primary structure was performed by (i) amino-terminal sequencing of three immunoreactive fragments of protein E and (ii) sequencing the protein E-coding regions of seven antigenic variants of tick-borne encephalitis virus that had been selected in the presence of neutralizing monoclonal antibodies directed against the E protein. Further information about variable and conserved regions was obtained by a comparative computer analysis of flavivirus E protein amino acid sequences. The search for potential T-cell determinants revealed at least one sequence compatible with an amphipathic alpha-helix which is conserved in all flaviviruses sequenced so far. By combining these data with those on the location of disulfide bridges (T. Nowak and G. Wengler, Virology 156:127-137, 1987) and the structural characteristics of epitopes, such as dependency on conformation or on intact disulfide bridges or both, a model was established that goes beyond the location of epitopes in the primary sequence and reveals features of the folding of the polypeptide chain, including the generation of discontinuous protein domains.     

Anti-sm autoantibodies in systemic lupus target highly basic surface structures of complexed spliceosomal autoantigens

Autoantibodies directed against spliceosomal proteins are a common and specific feature of systemic lupus erythematosus. These autoantibodies target a collection of proteins, including Sm B, B', D1, D2, and D3. We define the common antigenic targets of Sm D2 and D3 and examine their role in spliceosomal autoimmunity. Our results define nine major common epitopes, five on Sm D2 and four on Sm D3. These epitopes have significantly higher (more basic) isoelectric points than do nonantigenic regions. In fact, this association is of sufficient power to make isoelectric point an excellent predictor of spliceosomal antigenicity. The crystallographic structure of Sm D2 and D3 is now partially described. The anti-Sm D2 and D3 antigenic targets are located on the surface of the respective three-dimensional complexed proteins, thereby suggesting that these epitopes are accessible in the native configuration. All but one of these nine epitopes conspicuously avoid the specific regions involved in intermolecular interactions within the spliceosomal complex. One of the D3 epitopes (RGRGRGMGR) has significant sequence homology with a major antigenic region of Sm D1 (containing a carboxyl-terminal glycine-arginine repeat), and anti-D3 Abs cross-react with this epitope of Sm D1. These results demonstrate that spliceosomal targets of autoimmunity are accessible on native structure surfaces and that cross-reactive epitopes, as well as structural associations of various spliceosomal Ags, may be involved in the induction of autoimmunity in systemic lupus.     

Peptide epitope mapping in vaccine development: Introduction

Protection from infectious disease by the host immune response requires specific molecular recognition of unique antigenic determinants of a given pathogen. An epitope is an antigenic determinant which: 1) specifically stimulates the immune response (either B or T cell mediated); and 2) is acted upon by the products of these protective mechanisms. In B cell immunity, antibodies produced from stimulation by specific epitopes recognize and bind to these same antigenic structures. Identification of protective epitopes is extremely valuable to successful vaccine development. In order to be protective these antibodies must, in addition to recognition and binding, interfere with some vital step in pathogenesis such as adherence or toxin action. Protein B cell epitopes are frequently composed of the side chains (R-groups) of the amino acids found at solvent-exposed surfaces. These epitopes are classified as continuous (also linear or sequential) if composed of a single antibody-recognizing element located at a single locus of the primary structure. They are discontinuous (or assembled) if more than one physically separated entity is involved. T cell epitopes are peptides on the surface of antigen-presenting cells (macrophages, dendritic cells, and B cells) that are bound to major histocompatibility proteins; the T cell recognizes this peptide-MHC complex. Received 12 August 1996/ Accepted in revised form 03 November 1996     

沙眼衣原体多形态膜蛋白D基因序列分析及其B细胞抗原表位预测

分析沙眼衣原体多形态膜蛋白D(PmpD)的基因序列并预测PmpD蛋白的B细胞抗原表位。在GenBank中检索沙眼衣原体不同血清型的PmpD基因序列,进行序列比对分析。以L2血清型PmpD基因序列为材料,采用Karplus-Schulz、Chou-Fasman和Gamier-Robson方案预测蛋白质的二级结构和柔性区;按Jamesonv-Wolf方案预测抗原指数,运用Kyte-Doolittle方案预测PmpD氨基酸的亲水性,利用Emini方案预测蛋白质的表面可及性。检索到20个沙眼衣原体不同菌株的PmpD基因序列,分析发现其核苷酸序列非常保守,一致性高达99.14%~100%;对预测结果综合分析,推测最有可能的B细胞表位位于PmpD N端的67~74、132~140、335~340、851~861、972~988及1091~1097。多参数方案综合预测PmpD蛋白的B细胞抗原表位,为进一步实验鉴定PmpD抗原表位及其多表位疫苗设计和研究奠定基础。     

扁豆过敏原LenC3蛋白B细胞抗原表位预测及同源建模

目的：通过应用生物信息学软件模拟、分析预测扁豆过敏原Len c 3蛋白的结构、性质及B 细胞抗原表位, 为探索基于扁豆过敏原Len c 3 抗原表位的改造提供依据。方法：从Uniprot 蛋白质数据库中得到扁豆过敏原Len c 3的氨基酸序列,通过生物信息学软件Swiss-Model及Swiss-PdbViewer 4.0 模拟和分析扁豆过敏原Len c 3蛋白的结构,使用DNAStar软件对其B细胞抗原表位进行模拟、分析预测。结果：扁豆过敏原Len c 3蛋白为疏水性蛋白,在氨基酸残基第7-26 位有一跨膜区,Ramachandran 图评估扁豆过敏原Len c 3蛋白的三维结构显示其空间构象稳定,Len c 3蛋白潜在的B细胞抗原表位为35-36,48-50,66-71,87-90。结论：本研究通过对扁豆过敏原Len c 3蛋白进行生物信息学分析获得了该过敏原的结构、性质及潜在的B 细胞抗原表位,为进一步了解和掌握扁豆过敏原Len c 3蛋白的结构功能以及抗原性改造、单克隆抗体制备、表位疫苗设计等提供重要的线索。     

Genetic analysis of type-specific antigenic determinants of herpes simplex virus glycoprotein C.

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) elicits a largely serotype-specific immune response directed against previously described determinants designated antigenic sites I and II. To more precisely define these two immunodominant antigenic regions of gC-1 and to determine whether the homologous HSV-2 glycoprotein (gC-2) has similarly situated antigenic determinants, viral recombinants containing gC chimeric genes which join site I and site II of the two serotypes were constructed. The antigenic structure of the hybrid proteins encoded by these chimeric genes was studied by using gC-1- and gC-2-specific monoclonal antibodies (MAbs) in radioimmunoprecipitation, neutralization, and flow cytometry assays. The results of these analyses showed that the reactivity patterns of the MAbs were consistent among the three assays, and on this basis, they could be categorized as recognizing type-specific epitopes within the C-terminal or N-terminal half of gC-1 or gC-2. All MAbs were able to bind to only one or the other of the two hybrid proteins, demonstrating that gC-2, like gC-1, contains at least two antigenic sites located in the two halves of the molecule and that the structures of the antigenic sites in both molecules are independent and rely on limited type-specific regions of the molecule to maintain epitope structure. To fine map amino acid residues which are recognized by site I type-specific MAbs, point mutations were introduced into site I of the gC-1 or gC-2 gene, which resulted in recombinant mutant glycoproteins containing one or several residues from the heterotypic serotype in an otherwise homotypic site I background. The recognition patterns of the MAbs for these mutant molecules demonstrated that (i) single amino acids are responsible for the type-specific nature of individual epitopes and (ii) epitopes are localized to regions of the molecule which contain both shared and unshared amino acids. Taken together, the data described herein established the existence of at least two distinct and structurally independent antigenic sites in gC-1 and gC-2 and identified subtle amino acid sequence differences which contribute to type specificity in antigenic site I of gC.     

猪瘟病毒强弱毒株和野毒株E2全基因序列测定及比较分析

为了比较猪瘟病毒 (HCV)野毒株、疫苗株及标准株之间E2基因抗原区域的差异 ,采用RT PCR扩增了HCV石门株、兔化弱毒疫苗株、野毒 0 3及 0 7株的囊膜糖蛋白E2 (gp55)全基因的cDNA片段 ,分别克隆于pGEM T载体中并对其进行了核苷酸序列测定及氨基酸序列的推导 ,同时进行了同源性比较及E2结构与功能的分析。所测 4株HCVE2基因的长度均为1 2 73bp,所编码的氨基酸序列均包括部分信号肽序列和完整的跨膜区序列 ,共由 381个氨基酸组成 ;4个毒株E2蛋白N末端的 683位至 690位信号肽序列 (WLLLVTGA)和C末端 1 0 30～1 0 63位跨膜区均为保守序列 ,而且具疏水性 ;N末端抗原功能区中 ,4个E2蛋白与其它所比较序列在位于第 753位至 759位氨基酸处 ,均有一段保守序列RYLASLH ,无一氨基酸发生变异 ,为亲水性 ,在整个E2蛋白抗原谱中抗原性峰值为最高 ,推测对抗原性产生起重要作用 ;4个E2蛋白的氨基酸序列中均含有 1 5个半胱氨酸 (Cys)残基 ,其数量及位置与国外五株HCV(Brescia ,C ,Alfort.ALD和GPE)完全一致。表明…     

Characterization of antigenic properties of horseradish peroxidase with monoclonal antibodies

Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO ₁ and 36F ₉ were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.     

Structural and functional studies with antibodies to the integrin beta 2 subunit. A model for the I-like domain

To establish a structure and function map of the beta2 integrin subunit, we mapped the epitopes of a panel of beta2 monoclonal antibodies including function-blocking, nonblocking, and activating antibodies using human/mouse beta2 subunit chimeras. Activating antibodies recognize the C-terminal half of the cysteine-rich region, residues 522-612. Antibodies that do not affect ligand binding map to residues 1-98 and residues 344-521. Monoclonal antibodies to epitopes within a predicted I-like domain (residues 104-341) strongly inhibit LFA-1-dependent adhesion. These function-blocking monoclonal antibodies were mapped to specific residues with human --> mouse knock-out or mouse --> human knock-in mutations. Combinatorial epitopes involving residues distant in the sequence provide support for a specific alignment between the beta-subunit and I domains that was used to construct a three-dimensional model. Antigenic residues 133, 332, and 339 are on the first and last predicted alpha-helices of the I-like domain, which are adjacent on its "front." Other antigenic residues in beta2 and in other integrin beta subunits are present on the front. No antigenic residues are present on the "back" of the domain, which is predicted to be in an interface with other domains, such as the alpha subunit beta-propeller domain. Most mutations in the beta2 subunit in leukocyte adhesion deficiency are predicted to be buried in the beta2 subunit I-like domain. Two long insertions are present relative to alpha-subunit I-domains. One is tied down to the back of the I-like domain by a disulfide bond. The other corresponds to the "specificity-determining loop" defined in beta1 and beta3 integrins and contains the antigenic residue Glu(175) in a disulfide-bonded loop located near the "top" of the domain.     

Selection of HIV-specific immunogenic epitopes by screening random peptide libraries with HIV-1-positive sera

Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing Ab responses. In fact, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. To overcome these obstacles, we screened random peptide libraries using sera from HIV-infected subjects to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counterscreening with HIV-negative sera, we isolated peptides specifically recognized by Abs from HIV-1-infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 epitopes generated in vivo in infected subjects. Consistent with these findings, sera of simian HIV-infected monkeys also recognized the HIV-specific epitopes. The selected peptides were immunogenic in mice, where they elicited HIV-specific Abs that effectively neutralized HIV-1 isolates. These results demonstrate that pools of HIV-1 mimotopes can be selected from combinatorial peptide libraries by taking advantage of the HIV-specific Ab repertoire induced by the natural infection.     

Paramyxovirus membrane protein enhances antibody production to new antigenic determinants in the actin molecule: a model for virus-induced autoimmunity.

Paramyxovirus membrane (M) protein specifically binds to cellular actin but not to bovine serum albumin or myoglobin, as determined by affinity chromatography and enzyme-linked immunosorbent assay. The binding site for M protein on actin is different from the binding sites for antiactin antibodies. The interaction of M protein with actin resulted in production of antibodies to several new antigenic sites on the actin molecule. Five rabbits immunized with actin alone produced antibodies against the N-terminal sequence (residues 1 to 39). Another five rabbits immunized with a mixture of M protein and actin produced antibodies against a C-terminal fragment and a central region as well as the N-terminal fragment. By immunoblotting with proteolytic fragments of actin, the new antigenic sites were located between amino acid residues 40 to 113, 114 to 226, and 227 to 375. Antisera taken from some patients with recent measles virus infections demonstrated antiactin antibodies to sites other than the N-terminal fragment of actin (amino acids 1 to 39). The interaction of paramyxovirus M protein with actin and the subsequent production of antibodies to new antigenic sites may serve as a model for one of the mechanisms of virus-induced autoimmunity.     

Antigenic probes locate a serum-gelsolin-interaction site on the C-terminal part of actin.

The implication of part of the C-terminal of actin (within the 285-375 sequence) in the interaction of serum gelsolin was investigated by the use of specific antibodies. These antibodies were directed against two or three discrete epitopes, one of which was specific for skeletal-muscle actin. Some of these epitopes were found to be near the serum gelsolin-actin interface. Thus it can be assumed that part of the C-terminal of actin is exposed at the barbed end of the actin filament. The interaction between tropomyosin and actin was also studied.     

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF

Abstract The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membraine protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae . Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeuroginosa for phagocytosis. These epitopes were partially masked by lipopolysacharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.     

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.     

The nucleotide sequence of herpes simplex virus type 2 (333) glycoprotein gB2 and analysis of predicted antigenic sites

The gene coding for glycoprotein B2 (gB2) of herpes simplex virus type 2 (HSV-2) strain 333 was mapped and its nucleotide sequence determined. Open reading frame analysis deduced a polypeptide consisting of 902 amino acids and having close homology to gB1 of HSV type 1. Several predicted features of gB2 are consistent with a membrane-bound glycoprotein, i.e., a signal peptide sequence, a hydrophilic extracellular domain containing possible N-linked glycosylation sites, a hydrophobic membrane spanning sequence, and a cytoplasmic domain. Computer analysis on hydrophilicity, accessibility, and flexibility of the gB2 amino acid sequence, produced a composite surface value plot. At least nine major antigenic regions were predicted on the extracellular domain. The amino acids between residues 59-74, 127-139, 199-205, 460-476, and 580-594 exhibited the highest surface values. Comparison of the primary sequence with gB1 revealed localized regions showing amino acid diversity. Several of these locations correspond to major antigenic regions. Chou and Fasman analyses indicated that the amino acid substitutions, between positions 57-66, 461-472, and 473-481, induced changes in the secondary structure of gB. These sites could represent site-specific epitopes in the gB polypeptide.     

Prediction of B-cell epitopes forSalmonella typhi OmpC

The possible B-cell epitopes of the outer membrane porin OmpC ofSalmonella typhi have been identified, using the primary structure of the protein, by means of multiple sequence alignment and the known molecular structure of two other porins. From the analysis, 8 regions were identified as immunodominant and these were ranked based on antigenic index and the ratio of the number of nonconserved residues to the fragment length. Model building of the top two ranked regions show the tendency to form loop structures supporting the possibility of these being candidate epitopes.     

Epitope mapping of antigenic determinants of hepatitis C virus proteins by phage display

Study of individual hepatitis C (HCV) proteins could help to find a molecular structure and conformation, localization of antigenic and immunogenic determinants, to reveal of protective epitopes. It is necessary for practical medicine - development of diagnostic test-systems, vaccines and therapeutics. Linear and conformation dependent epitopes of HCV proteins was localized in this work and immunogenic properties of phage displayed peptides screened on monoclonal antibodies to HCV proteins have been investigated. Eleven epitopes of four HCV proteins have been studied. Three epitopes was found as linear, two epitopes were dependent on secondary structure of proteins and one epitope was dependent on tertiary structure of NS3 protein. Aminoacid sequences of other determinants have been determined and the distinct localization of these determinants will be continued after discovering of tertiary structure of HCV proteins. It was shown, that phage mimotope 3f4 is immunogenic and could induce specific hu- moral immune response to NS5A HCV protein. The data obtained could be useful for improving of HCV diagnostic test-systems, studying of amino acid substitutions and its influence on antigenic properties of the HCV proteins. The results could help to study an immune response in patients infected with different genotypes of HCV. Phage displayed peptides mimicking the antigenic epitopes of HCV proteins could be applied to development of HCV vaccine.     

B细胞表位作图研究进展

抗原-抗体的特异性结合是由抗体表面的抗原决定簇与抗原表面的表位基序间的特异性互补识别决定的。B细胞表位作图既包括B细胞抗原表位基序的鉴定(即确定抗原分子上被B细胞表面受体或抗体特异性识别并结合的氨基酸基序),也包括绘制抗原蛋白的全部或接近全部的B细胞表位基序在其一级或高级结构上的分布图谱的过程。B细胞表位作图是研发表位疫苗、治疗性表位抗体药物和建立疾病免疫诊断方法的重要前提。目前,已经建立了多种B细胞表位鉴定或绘制抗原蛋白B细胞表位图谱的实验方法。基于抗原-单抗复合物晶体结构的X-射线晶体学分析的B细胞表位作图和基于抗原蛋白或抗原片段的突变体库筛选技术的B细胞表位作图可以在氨基酸水平,甚至原子水平上揭示抗原分子上与单抗特异性结合的关键基序;其它B细胞表位作图方法(如基于ELISA的肽库筛选技术)常常只能获得包含B细胞表位的抗原性肽段,因而,很少用于最小表位基序的鉴定;而改良的生物合成肽法多用于B细胞表位的最小基序鉴定和精细作图。鉴于每种B细胞作图方法都存在各自的优势与不足,B细胞表位作图往往需要多种作图方法的有机结合。本文对目前常用的B细胞表位作图的实验方法及其在动物疫病防控中的应用进行综述,以期为研究者设计最佳的表位作图方案提供参考。     

Monoclonal antibodies against different epitopes of peptide hormones. Use of photoreactive analogues in studies on vasopressin.

In order to produce monoclonal antibodies directed against different epitopes of the neurohypophyseal hormone vasopressin, the hormone was coupled to carrier proteins via photoreactive groups at different positions in the vasopressin sequence: [2-(4-azidophenylalanine), 8-arginine]vasopressin (peptide P1, photoreactive group at position 2) and desamino-[8-N6-(4-azidophenylamidino)lysine]vasopressin (peptide P2, photoreactive group at position 8) were conjugated to thyroglobulin by flash photolysis. Monoclonal antibodies against these conjugates bound ([3H]8-arginine]vasopressin with dissociation constants ranging over 40-400 nM. Epitope analysis by means of competitive ELISA showed that the monoclonal antibody obtained with peptide P1 as hapten was directed against the C-terminal acyclic tripeptide when its conformation was stabilized by interaction with the disulphide-linked cyclic hexapeptide. In contrast, the epitope analysis of three monoclonal anti-(peptide P2) antibodies demonstrated that they recognized antigenic determinants in the cyclic hexapeptide ring, mainly the hydrophobic surface formed by Tyr2 and Phe3. Our results suggest that monoclonal antibodies against different epitopes in small peptide hormones can be generated selectively by using photoreactive peptides in such a way that different antigenic sites are exposed in the hapten-carrier conjugate.