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1.
Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.  相似文献   

2.
Mutations in capillary morphogenesis gene 2 (CMG2), one of the two closely related proteins that act as anthrax toxin receptors, cause two rare human autosomal recessive conditions, juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH). Here we demonstrate that CMG2 proteins with certain JHF- and ISH-associated single amino acid substitutions in their von Willebrand factor A domain or transmembrane region do not function as anthrax toxin receptors. However, an ISH-associated CMG2 variant having a truncated cytosolic domain does still function as an anthrax receptor, and in fact makes cells hyper-sensitive to toxin, distinguishing the roles of CMG2 in physiology and anthrax pathology. Site-specific mutagenesis was used to characterize the role that domain 2 of the anthrax toxin protective antigen (PA) plays in interaction with CMG2, focusing on the interaction between the PA 2beta(3)-2beta(4) loop and a pocket (Glu-122 pocket) adjacent to the metal ion-dependent adhesion site in CMG2. Substitutions that disrupted the salt bridge between PA Arg-344 and CMG2 Glu-122 decreased the affinity of PA to CMG2 three- to fourfold. Furthermore, mutation of CMG2 Tyr-119 (within the Glu-122 pocket) to His lowered the pH threshold for PA prepore-to-pore conversion in the endocytic pathway.  相似文献   

3.
Gao M  Schulten K 《Biophysical journal》2006,90(9):3267-3279
Protective antigen (PA) is the anthrax toxin protein recognized by capillary morphogenesis gene 2 (CMG2), a transmembrane cellular receptor. Upon activation, seven ligand-receptor units self-assemble into a heptameric ring-like complex that becomes endocytozed by the host cell. A critical step in the subsequent intoxication process is the formation and insertion of a pore into the endosome membrane by PA. The pore conversion requires a change in binding between PA and its receptor in the acidified endosome environment. Molecular dynamics simulations totaling approximately 136 ns on systems of over 92,000 atoms were performed. The simulations revealed how the PA-CMG2 complex, stable at neutral conditions, becomes transformed at low pH upon protonation of His-121 and Glu-122, two conserved amino acids of the receptor. The protonation disrupts a salt bridge important for the binding stability and leads to the detachment of PA domain II, which weakens the stability of the PA-CMG2 complex significantly, and subsequently releases a PA segment needed for pore formation. The simulations also explain the great strength of the PA-CMG2 complex achieves through extraordinary coordination of a divalent cation.  相似文献   

4.
Cai C  Che J  Xu L  Guo Q  Kong Y  Fu L  Xu J  Cheng Y  Chen W 《PloS one》2011,6(6):e20646
Anthrax toxin is the major virulence factor produced by Bacillus anthracis. The toxin consists of three protein subunits: protective antigen (PA), lethal factor, and edema factor. Inhibition of PA binding to its receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) can effectively block anthrax intoxication, which is particularly valuable when the toxin has already been overproduced at the late stage of anthrax infection, thus rendering antibiotics ineffectual. Receptor-like agonists, such as the mammalian cell-expressed von Willebrand factor type A (vWA) domain of CMG2 (sCMG2), have demonstrated potency against the anthrax toxin. However, the soluble vWA domain of TEM8 (sTEM8) was ruled out as an anthrax toxin inhibitor candidate due to its inferior affinity to PA. In the present study, we report that L56A, a PA-binding-affinity-elevated mutant of sTEM8, could inhibit anthrax intoxication as effectively as sCMG2 in Fisher 344 rats. Additionally, pharmacokinetics showed that L56A and sTEM8 exhibit advantages over sCMG2 with better lung-targeting and longer plasma retention time, which may contribute to their enhanced protective ability in vivo. Our results suggest that receptor decoys based on TEM8 are promising anthrax toxin inhibitors and, together with the pharmacokinetic studies in this report, may contribute to the development of novel anthrax drugs.  相似文献   

5.
炭疽保护性抗原(PA)是炭疽毒素的重要组分,同时也是现有炭疽疫苗的主要有效成分,在炭疽杆菌的致病与免疫中发挥关键作用。以重组PA为免疫原,采用B淋巴细胞杂交瘤技术,结合炭疽毒素敏感细胞的毒性中和试验,大量筛选抗PA单克隆抗体,获得了9株炭疽毒素中和性单抗。进一步分析表明这些单抗以IgG1亚类为主,分别识别PA 3个结构域的4个不同中和表位区。针对结构域2的4株单抗识别同一表位区,其中3株单抗的中和活性强于抗PA多抗;针对结构域4的4株单抗识别两个不同表位区;另有1株单抗识别位于结构域3的表位。实验结果提示PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。实验中获得的针对不同表位的中和性单抗为深入研究PA的免疫保护机理提供了工具,也为研制针对炭疽毒素的被动免疫制剂和治疗药物打下基础。  相似文献   

6.
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.  相似文献   

7.
The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates entry of the two enzymatic moieties of the toxin into the cytosol. Two PA receptors, anthrax toxin receptor (ATR)/tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been identified. We expressed and purified the von Willebrand A (VWA) domain of CMG2 and examined its interactions with monomeric and heptameric forms of PA. Monomeric PA bound a stoichiometric equivalent of CMG2, whereas the heptameric prepore form bound 7 eq. The Kd of the VWA domain-PA interaction is 170 pm when liganded by Mg2+, reflecting a 1000-fold tighter interaction than most VWA domains with their endogenous ligands. The dissociation rate constant is extremely slow, indicating a 30-h lifetime for the CMG2.PA monomer complex. CMG2 metal ion-dependent adhesion site (MIDAS) was studied kinetically and thermodynamically. The association rate constant (approximately 10(5) m(-1) s(-1)) is virtually identical in the presence or absence of Mg2+ or Ca2+ , but the dissociation rate of metal ion liganded complex is up to 4 orders of magnitude slower than metal ion free complex. Residual affinity (Kd approximately 960 nm) in the absence of divalent metal ions allowed the free energy for the contribution of the metal ion to be calculated as 5 kcal mol(-1), demonstrating that the metal ion-dependent adhesion site is directly coordinated by CMG2 and PA in the binding interface. The high affinity of the VWA domain for PA supports its potency in neutralizing anthrax toxin, demonstrating its potential utility as a novel therapeutic for anthrax.  相似文献   

8.
目的:构建炭疽受体CMG2和人IgGl Fc片段融合基因载体,转染CHO细胞并通过毒素中和试验检测CMG2-Fc拮抗炭疽毒素(PA+LF)的能力。方法-将含有CMG2胞外区1-217AA片度基因和人IgGl的Fc片段基因共同连接入pcDNA3.1载体转染CHO细胞并筛选高表达CMG2-Fc的CHO细胞系,通过小鼠RAW264.7巨噬细胞保护试验检测CMG2-Fc拮抗炭疽毒素的能力。结果:获得了表达CMG2-Fc的细胞株,毒素中和实验显示该蛋白可以有效抑制炭疽毒素引起的细胞损伤。结论:CMG2-Fc能够保护小鼠巨噬细胞免受炭疽毒素攻击,提示其可以作为抗毒素治疗炭疽感染。  相似文献   

9.
The anthrax protective antigen (PA) is a key component of the tripartite anthrax toxin. Monoclonal antibody (mAb) 14B7 and its engineered, affinity-matured variants have been shown to be effective in blocking PA binding to cellular receptors and mitigating anthrax toxicity. Here, we perform computational structural modeling of the mAb 14B7-PA interaction. Our objectives are to determine the structure of the 14B7-PA complex, to deduce a structural explanation for the affinity maturation from the docking models, and to study the effect of inaccuracies in the antibody homology model on docking. We used the RosettaDock program to dock PA with the mAb 14B7 crystal structure or homology model. Our simulations generate two distinct binding orientations consistent with experimental residue mutations that diminish 14B7-PA binding. Furthermore, the models suggest new site-directed mutations to positively identify one of these two solutions as the correct 14B7-PA docking orientation. The models indicate that PA regions 648-660 and 712-720 may be important for 14B7 binding in addition to the known PA epitope, and the binding interfaces are similar to that seen in the PA complex with cellular receptor CMG2. Antibody residues involved in affinity maturation do not contact the antigen in the docking models, suggesting that affinity maturation in the 14B7 family does not result from direct enhancements of antibody-antigen contacts. Docking the homology model produces low-resolution representations of the crystal structure docking orientations, but homology model docking is frustrated by antibody H3 loop conformation errors. This work demonstrates the usefulness and limitations of computational structure prediction for the development of antibody therapeutics, and reemphasizes the need for flexible backbone docking algorithms to achieve high-resolution docking using homology models.  相似文献   

10.
The major immunogenic component of the current anthrax vaccine, anthrax vaccine adsorbed (AVA) is protective antigen (PA). We have shown recently that the thermodynamic stability of PA can be significantly improved by binding to the Von‐Willebrand factor A (VWA) domain of capillary morphogenesis protein 2 (CMG2), and improvements in thermodynamic stability may improve storage and long‐term stability of PA for use as a vaccine. In order to understand the origin of this increase in stability, we have isolated the receptor binding domain of PA, domain 4 (D4), and have studied the effect of the addition of CMG2 on thermodynamic stability. We are able to determine a binding affinity between D4 and CMG2 (~300 nM), which is significantly weaker than that between full‐length PA and CMG2 (170–300 pM). Unlike full‐length PA, we observe very little change in stability of D4 on binding to CMG2, using either fluorescence or 19F‐NMR experiments. Because in previous experiments we could observe a stabilization of both domain 4 and domain 2, the mechanism of stabilization of PA by CMG2 is likely to involve a mutual stabilization of these two domains.  相似文献   

11.
目的:构建炭疽受体CMG2和人IgG1 Fc片段融合基因载体,转染CHO细胞并通过毒素中和试验检测CMG2-Fc拮抗炭疽毒素(PA+LF)的能力。方法:将含有CMG2胞外区1-217AA片度基因和人IgG1的Fc片段基因共同连接入pcDNA3.1载体转染CHO细胞并筛选高表达CMG2-Fc的CHO细胞系,通过小鼠RAW264.7巨噬细胞保护试验检测CMG2-Fc拮抗炭疽毒素的能力。结果:获得了表达CMG2-Fc的细胞株,毒素中和实验显示该蛋白可以有效抑制炭疽毒素引起的细胞损伤。结论:CMG2-Fc能够保护小鼠巨噬细胞免受炭疽毒素攻击,提示其可以作为抗毒素治疗炭疽感染。  相似文献   

12.
The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity, single-chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here, we report the high-resolution X-ray structures of three high-affinity, single-chain antibodies in the 14B7 family; 14B7 and two high-affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 Å resolution. These structures provide insights into the mechanism of neutralization, and the effect of various mutations on antibody affinity, and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.  相似文献   

13.
炭疽是由炭疽芽孢杆菌引起的严重威胁人类健康的传染病。炭疽毒素包括3种蛋白质成分:保护性抗原(PA)、致死因子(LF)和水肿因子(EF)。PA与LF形成致死毒素(LT),与EF形成水肿毒素(ET)。由于致死毒素(LT)在感染者损伤及死亡中发挥主要作用,因此在炭疽感染晚期单纯使用抗生素治疗难以发挥疗效,治疗性中和抗体成为目前最有效的炭疽治疗药物。目前国外获得的炭疽毒素抗体多为炭疽PA抗体,美国FDA已批准瑞西巴库(人源PA单抗)用于吸入性炭疽的治疗。一旦炭疽芽孢杆菌被人为改构或PA中和表位发生突变,针对PA单一表位的抗体将可能失效,因此针对LF的抗体将成为炭疽治疗的有效补充。目前国外已有的LF抗体多为鼠源抗体和嵌合抗体,而全人源抗体可以避免鼠源抗体免疫原性高等缺点。本研究首先用LF抗原免疫人抗体转基因小鼠,利用流式细胞仪从小鼠脾淋巴细胞中分选抗原特异的记忆B细胞,通过单细胞PCR方法快速获得两株具有结合活性的抗LF单抗1D7和2B9。瞬时转染Expi 293F细胞制备抗体,通过毒素中和实验(TNA)发现1D7和2B9在细胞模型中均显示较好的中和活性,并且与PA单抗联合使用时,表现出较好的协同作用。总之,本文利用转基因小鼠、流式分选技术和单细胞PCR技术的优势,快速筛选到全人源LF抗体,为快速筛选全人源单克隆抗体开辟了新的思路与方法。  相似文献   

14.
A panel of Fabs that neutralize anthrax toxin in vitro was selected from libraries generated from human donors vaccinated against anthrax. At least two of these antibodies protect rats from anthrax intoxication in vivo. Fabs 83K7C and 63L1D bind with subnanomolar affinity to protective antigen (PA) 63, and Fab 63L1D neutralizes toxin substoichiometrically, inhibits lethal factor (LF) interaction with PA63 and binds to a conformational epitope formed by PA63.  相似文献   

15.
We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.  相似文献   

16.
Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (312)SFFD(315), in the 2beta(2)-2beta(3) loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2beta(2)-2beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.  相似文献   

17.
使用分泌型表达载体,实现了重组炭疽毒素受体胞外区 (rATR(CMG2)-EXCELL) 在毕赤酵母 KM71H 培养物上清中的分泌表达 . 表达量约占培养物上清总蛋白质的 20%. 经过螯合柱初步纯化,每升诱导培养物可获得约 1 mg 电泳纯的 rATR(CMG2)-EXCELL. 体外与配基 PA 结合试验和细胞保护试验显示, rATR(CMG2)-EXCELL 具有很好的生物活性 . rATR(CMG2)-EXCELL 的成功表达为今后研究炭疽毒素受体的作用机理、发展新型炭疽治疗药物打下基础 .  相似文献   

18.
The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional 13C‐edited 1H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.  相似文献   

19.
Anthrax toxin, a three-component protein toxin secreted by Bacillus anthracis, assembles into toxic complexes at the surface of receptor-bearing eukaryotic cells. The protective antigen (PA) protein binds to receptors, either tumor endothelial cell marker 8 (TEM8) or CMG2 (capillary morphogenesis protein 2), and orchestrates the delivery of the lethal and edema factors into the cytosol. TEM8 is reported to be overexpressed during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues. To extend prior work on targeting of tumor with modified anthrax toxins, we used phage display to select PA variants that preferentially bind to TEM8 as compared with CMG2. Substitutions were randomly introduced into residues 605-729 of PA, within the C-terminal domain 4 of PA, which is the principal region that contacts receptor. Candidates were characterized in cellular cytotoxicity assays with Chinese hamster ovary (CHO) cells expressing either TEM8 or CMG2. A PA mutant having the substitutions R659S and M662R had enhanced specificity toward TEM8-overexpressing CHO cells. This PA variant also displayed broad and potent tumoricidal activity to various human tumor cells, especially to HeLa and A549/ATCC cells. By contrast, the substitution N657Q significantly reduced toxicity to TEM8 but not CMG2-overexpressing CHO cells. Our results indicate that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.  相似文献   

20.
Protective antigen (PA), the binding subunit of anthrax toxin, is the major component in the current anthrax vaccine, but the fine antigenic structure of PA is not well defined. To identify linear neutralizing epitopes of PA, 145 overlapping peptides covering the entire sequence of the protein were synthesized. Six monoclonal antibodies (mAbs) and antisera from mice specific for PA were tested for their reactivity to the peptides by enzyme-linked immunosorbent assays. Three major linear immunodominant B-cell epitopes were mapped to residues Leu156 to Ser170, Val196 to Ile210, and Ser312 to Asn326 of the PA protein. Two mAbs with toxin-neutralizing activity recognized two different epitopes in close proximity to the furin cleavage site in domain 1. The three-dimensional complex structure of PA and its neutralizing mAbs 7.5G and 19D9 were modeled using the molecular docking method providing models for the interacting epitope and paratope residues. For both mAbs, LeTx neutralization was associated with interference with furin cleavage, but they differed in effectiveness depending on whether they bound on the N- or C-terminal aspect of the cleaved products. The two peptides containing these epitopes that include amino acids Leu156–Ser170 and Val196–Ile210 were immunogenic and elicited neutralizing antibody responses to PA. These results identify the first linear neutralizing epitopes of PA and show that peptides containing epitope sequences can elicit neutralizing antibody responses, a finding that could be exploited for vaccine design.Bacillus anthracis is a Gram-positive, facultatively anaerobic, rod-shaped bacterium that secretes a variety of toxins, including anthrax toxin. This toxin is made up of three proteins as follows: protective antigen (PA),3 edema factor (EF), and lethal factor (LF). Like other binary toxins, anthrax toxin follows the same pattern where distinct subunits are involved in the different steps at which it can act. The B subunit (PA) is involved in receptor binding and cellular internalization into the cytoplasm, whereas the A subunit (EF and/or LF) bears the enzymatic activity (1). Anthrax can occur naturally in animals by spore transmission via ingestion, inhalation, or an open skin wound, but it can also be a result of bioterrorism and biological warfare (2).PA is the component of the currently licensed anthrax vaccine that elicits protective antibodies. Recent studies have demonstrated that a strong humoral response to truncated recombinant PA contributes to a protective immune response to anthrax (3, 4). Accordingly, there is considerable interest in ascertaining the location and immunogenicity of B-cell epitopes on the PA molecule. The development of numerous monoclonal antibodies (mAbs) to different epitopes on the PA molecule that influence PA functions, in conjunction with epitope mapping, has provided an opportunity to study the fine antigenic structure of PA. Most of these epitopes have been defined in mice (58), in macaques (9), in rabbits (10), as well as in vaccinated humans (11). Consequently, the epitopes described thus far are localized to three discrete regions within the PA. These regions correspond to the 2β2–2β3 loop of domain 2, the domain 3-domain 4 boundary, and the small loop of domain 4. The 2β2–2β3 loop of domain 2 is responsible for mediating membrane insertion, and many neutralizing murine mAbs target this region (5, 8). The boundary between domains 3 and 4, which does not have a known functional activity, has been suggested as a region recognized by polyclonal antibodies from vaccinated humans and rabbits (6, 12). The cellular receptor binding region is localized to the small loop of domain 4, and this region has been described to be recognized by two neutralizing mAbs (7, 9). With the exception of a neutralizing mAb that bound to PA20 (13), no B-cell epitopes have been reported in domain 1. Therefore, identification of further dominant antigenic epitopes is pivotal for understanding the minimal immunogenic region of PA that will allow for precise direction of potent immune responses.Two mAbs to PA have been reported previously by our laboratory, one known as 7.5G binds to domain 1 and can neutralize the cytotoxic activity of lethal toxin (LeTx) (13). The other, mAb 10F4, binds to domain 4 and has weak neutralizing activity. In addition, we now describe four new anti-PA mAbs, of which only one neutralizes LeTx. The characterization of the B-cell epitopes in PA recognized by protective and nonprotective mAbs is important to better understand the antigenic structure of this toxin, and such information is potentially useful for the design of vaccines and passive immune therapies against B. anthracis. Because PA has been identified as an effective subunit vaccine, we wanted to identify the specific epitopes that provide the protection and use them as immunogens. Using mAbs and 145 overlapping peptides covering the entire sequence of PA, we identify the first linear neutralizing epitopes in domain 1 of PA, and we demonstrate that two peptides containing epitopes in domain 1 were capable of inducing strong LeTx-neutralizing antibody responses.  相似文献   

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