Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1–252) and a portion of domain Ib (amino acids 365–380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274–283, 470–492, 531–540, 555–564, and the C-terminal amino acids 596–609. Two minor epitopes were identified as well: amino acids 501–510 and 582–589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.     

Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins.

The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen binding affinity; internalization rate, intracellular processing and intrinsic toxin domain potency. This study examines the influence of antigen binding affinity on potency in various immunotoxin fusion proteins where target antigen binding is mediated by single chain antibody variable region fragments (scFv). Firstly, the relationship between affinity and potency was examined in a panel of four scFv immunotoxins generated from different anti-CD3 monoclonal antibodies fused to the 38 kDa fragment of Pseudomonas aeruginosa exotoxin A (PE38). Of these four scFv-PE38 immunotoxins, the one derived from the anti-CD3 monoclonal antibody UCHT1 has highest cell killing potency. Analysis of these four scFv-PE38 immunotoxins indicated a correlation between antigen binding affinity and immunotoxin potency in the cell killing assay with the exception of the scFvPE38 immunotoxin derived from the antibody BC3. However this scFv appeared to suffer a greater drop in affinity ( approximately 100x), relative to the parent Mab than did the other three scFvs used in this study (2-10x). Secondly, the scFv(UCHT1)-PE38 immunotoxin was then compared with a further panel of scFv(UCHT1)-derived immunotoxins including a divalent PE38 version and both monovalent and divalent Corynebacterium diphtheriae toxin (DT389) fusion proteins. When the scFv-UCHT1 domain was amino-terminally positioned relative to the toxin, as in the scFv(UCHT1)-PE38, an approximately 10-fold higher antigen-binding affinity was observed than with the C-terminal fusion, used in the DT389-scFv(UCHT1) molecule. Despite this lower antigen-binding activity, the DT389-scFv immunotoxin had a 60-fold higher potency in the T-cell-killing assay. Thirdly, a divalent form of the DT389-scFv construct, containing tandem scFv domains, had a 10-fold higher binding activity, which was exactly reflected in a 10-fold increase in potency. Therefore, when comparing immunotoxins in which scFvs from different antibodies are fused to the same toxin domain (DT or PE) a broad correlation appears to exist between binding affinity and immunotoxin potency. However, no correlation between affinity and potency appears to exist when different toxin domains are combined with the same scFv antibody domain.     

Antigenic Domains of the Open Reading Frame 2-Encoded Protein of Hepatitis E Virus

The antigenic composition of the hepatitis E virus (HEV) protein encoded by open reading frame 2 (ORF2) was determined by using synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer peptides, with each set spanning the entire ORF2 protein of the HEV Burma strain, were synthesized. All synthetic peptides were tested by enzyme immunoassay against a panel of 32 anti-HEV-positive serum specimens obtained from acutely HEV-infected persons. Six antigenic domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that can be efficiently modeled with peptides of different sizes. In contrast, antigenic epitopes identified within the two central domains (3 and 4) were modeled more efficiently with 30-mer peptides than with either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides derived from domain 5 identified at aa 490 to 579 demonstrated strong IgM antigenic reactivity. Several 30-mer synthetic peptides derived from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than 70% of anti-HEV-positive serum specimens. Thus, the results of this study demonstrate the existence of six diagnostically relevant antigenic domains within the HEV ORF2 protein.     

Identification of a conserved B-cell epitope in the equine arteritis virus (EAV) N protein using the pepscan technique

The nucleocapsid (N) gene of equine arteritis virus (EAV) is highly conserved between isolates, and the N protein is an important antigen that induces immunity when horses are infected with EAV. This study describes the identification of a linear B-cell epitope on the N protein using the pepscan technique with a monoclonal antibody (mAb) 2B1 directed against the N protein. The N protein was divided into 11 overlapping peptides, each containing 16 amino acids associated with six overlapping amino acids. The fragments were expressed as MBP fusion proteins that were then used to probe the 2B1 mAb. The minimal epitope sequence was confirmed step-by-step using single amino acid residue deletion. One completely conserved linear epitope (³⁸KPPAQP⁴³) was identified that matched with EAV-positive serum in Western blots, thereby revealing the importance of these six amino acids of the epitope for antibody-epitope binding activity. This finding not only contributes to our understanding of the antigenic structure of the N protein of EAV but also has potential for the development of diagnostic techniques.     

C-terminal invariable domain of VlsE is immunodominant but its antigenicity is scarcely conserved among strains of Lyme disease spirochetes

VlsE, the variable surface antigen of Borrelia burgdorferi, contains two invariable domains located at the amino and carboxyl terminal ends, respectively, and a central variable domain. In this study, both immunogenicity and antigenic conservation of the C-terminal invariable domain were assessed. Mouse antiserum to a 51-mer synthetic peptide (Ct) which reproduced the entire sequence of the C-terminal invariable domain of VlsE from B. burgdorferi strain B31 was reacted on immunoblots with whole-cell lysates extracted from spirochetes of 12 strains from the B. burgdorferi sensu lato species complex. The antiserum recognized only VlsE from strain B31, indicating that epitopes of this domain differed among these strains. When Ct was used as enzyme-linked immunosorbent assay (ELISA) antigen, all of the seven monkeys and six mice that were infected with B31 spirochetes produced a strong antibody response to this peptide, indicating that the C-terminal invariable domain is immunodominant. None of 12 monkeys and only 11 of 26 mice that were infected with strains other than B31 produced a detectable anti-Ct response, indicating a limited antigenic conservation of this domain among these strains. Twenty-six of 33 dogs that were experimentally infected by tick inoculation were positive by the Ct ELISA, while only 5 of 18 serum samples from dogs clinically diagnosed with Lyme disease contained detectable anti-Ct antibody. Fifty-seven of 64 serum specimens that were collected from American patients with Lyme disease were positive by the Ct ELISA, while only 12 of 21 European samples contained detectable anti-Ct antibody. In contrast, antibody to the more conserved invariable region IR(6) of VlsE was present in all of these dog and human serum samples.     

The Immunological Reactivity of the Three Homologous Repetitive Tetrapeptides in the Region 41–64 of Allergen M from Cod

The CD-calcium binding loop of Allergen M encompasses the two homologous tetrapep-tides Asp-Glu-Asp-Lys and Asp-Glu-Leu-Lys. These tetrapeptides were recently suggested to be mutually critical for the immunological specificity of this region. To obtain evidence of the validity of this hypothesis, four peptide analogues encompassing these tetrapeptides at the termini were produced by manual solid-phase peptide synthesis. The structures of these peptides were ascertained by amino acid analyses and end-group sequence determination. The crude preparations were prepared by high-performance liquid chromatography and high-voltage electrophoresis. The immunological reactivity was investigated by using the preliminary purified peptides in the IgE and IgG test systems used. The results of radioallergosorbent test inhibition showed that all the synthetic peptides encompassing a minimum of two of the tetrapeptides have the capacity to bind specific IgE of the serum pool. The composition and sequence of the six spacer amino acids between these tetrapeptides were of no importance for the acitivity. The highest inhibition percentage was obtained by the peptides derived from the primary structure of Allergen M. No activity was found for the control peptides synthesized under identical conditions. Similar results were shown by PIC tests, using two cod-allergic sera and two recipients. In the IgG test system, all the four peptides containing the two terminal tetrapeptides showed an antigenic reactivity. They deflected allergen M line in rocket line immunoelectrophoresis. No deflection was given by the control peptides or proteins used. We conclude that the immunological reactivity of region 41–64 of allergen M is determined by 3 homologous tetrapeptides, repeated in three sites interspaced by six amino acids in a segment of 24 residues.     

Analysis of Pseudomonas exotoxin activation and conformational changes by using monoclonal antibodies as probes.

Pseudomonas exotoxin (PE) is a protein toxin composed of three structural domains. In its native form, the toxin is a 66,000-Mr proenzyme that must be activated to express full ADP-ribosylating activity. To study the process of activation and accompanying conformational changes, we have isolated 10 monoclonal antibodies to a 40,000-Mr fragment of the toxin (PE40) that exhibits full enzyme activity but lacks the toxin's cell-binding domain and contains amino acids 253 to 613 (comprising domains II, Ib, and III). By using mutant PE molecules in which all of domain I and portions of domains II, Ib, and III were deleted, the locations of the epitopes for each of the antibodies were determined. Eight of these monoclonal antibodies were further characterized. Of these eight, all reacted with soluble PE40 and an interleukin-2-PE40 conjugate, but only two reacted strongly with native soluble PE. However, all eight reacted with PE after it had been immobilized on nitrocellulose or after it had been activated to express full ADP-ribosylating activity. Antibodies were also assessed for their ability to neutralize the cytotoxic activity of either PE or interleukin-2-PE40. These antibodies should be useful as probes for monitoring the activation and processing of PE that occur during endocytosis and in determining the location of epitopes that are important for toxin activity.     

Mapping of antigenic determinant regions of the Bor56 protein of Orientia tsutsugamushi.

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.     

丙型肝炎病毒NS5a高免疫原区人工合成多肽抗原性的研究

目的 检测不同基因型丙型肝炎病毒(HCV)非结构5a区(NS5a)高免疫原区短链多肽的抗原性，确定该区的主要线性抗原表位并探讨基因异质性与免疫原性间的关系。方法 设计并合成特异性多肽；DNA Star软件对多肽氨基酸同源性进行比较；间接ELISA法检测多肽的抗原性。结果多肽氨基酸的同源性在不同区及不同基因型间变化较大。氨基酸残基2212～2241、2272-2301和2302-2331的多肽抗原性最强。18个来自保守区的多肽可以与不同基因型抗-HCV阳性血清起反应(阳性率高达96％)；而来自不同基因型间氨基酸序列保守性较低区的多肽抗原性与同基因血清的反应性较高。结论 HCV NS5a高免疫原区主要的线性抗原位于氨基酸残基2212-2241、2272-2301和2302-2331的位置；人工合成的多肽可以有效地用于检测抗-HCV抗体；一些多肽的抗原性具有基因型特异性。     

Comparison and fine mapping of both high and low neutralizing monoclonal antibodies against the principal neutralization domain of HIV-1

Summary Monoclonal antibodies raised against viral lysate of HIV-1 (strain LAV-1) and against recombinant gp 160 of HIV-1 (strain HTLV IIIB) which neutralized HIV-1 in a type specific manner were mapped with the aid of peptides (Pepscan analysis). Each of these monoclonal antibodies bound to peptides located on the principal neutralizing domain (PND) of HIV-1. We found that the antigenic sites of the MAbs described in this paper are represented by linear peptides of at least 10 amino acids long. The affinity of the MAbs is high for these peptides and in the same order of magnitude as for native gp 160. The fine mapping of the epitopes may reflect structural features of the PND, for instance which amino acid side chains are exposed and which are buried in the protein. Furthermore the fine mapping of the epitopes explained the HIV type-specific neutralizing activity of the MAbs. Antibodies that bound to the tip of the loop (amino acids QRGPGRAF) have a higher neutralizing activity than antibodies that bound to amino acids towards the N-terminal side of the loop (amino acids KSIRI). Furthermore, MAbs that bound to virtually the same amino acids on the tip of the loop (amino acids IQRGPGRAF and RGPGRAFV) had different neutralizing activities due to different affinities for native gp 160. These data reveal that neutralizing activity not only is determined by the affinity of an antibody to the neutralizing site but also by its fine binding specificities to the V 3 loop of gp 120.     

The allergenic structure of allergen M from cod. III. Studies on the antigenicity of long-sequence peptides.

Fragments TM 1 (75 amino acid residues) and TM 2 (38 amino acid residues), and 3 other polypeptides (range 16-25 amino acid residues) of Allergen M from cod were shown to be active in rabbit IgG-mediated reactions. The same peptides were previously found to possess reactivity in IgE-mediated reactions, thus suggesting a structural relationship between their antigenic and allergenic determinants.     

Cytotoxic necrotizing factor type 1-neutralizing monoclonal antibody NG8 recognizes three amino acids in a C-terminal region of the toxin and reduces toxin binding to HEp-2 cells

Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are toxins of pathogenic Escherichia coli that share 85% identity over 1,014 amino acids. Although both of these toxins modify GTPases, CNF1 is a more potent inducer of multinucleation in HEp-2 cells, binds more efficiently to HEp-2 cells, and, despite the conservation of amino acids (C866 and H881) required for enzymatic activity of the toxins, deamidates RhoA and Cdc42 better than CNF2. Here we exploited the differences between CNF1 and CNF2 to define the epitope on CNF1 to which the CNF1-specific neutralizing monoclonal antibody (MAb) (MAb NG8) binds and to determine the mechanism by which MAb NG8 neutralizes CNF1 activity on HEp-2 cells. For these purposes, we generated a panel of 21 site-directed mutants in which amino acids in CNF1 were exchanged for the amino acids in CNF2 between amino acids 546 and 869 and vice versa. This region of CNF1 not only is recognized by MAb NG8 but also is involved in binding of this toxin to HEp-2 cells. All the mutants retained the capacity to induce multinucleation of HEp-2 cells. However, the CNF1 double mutant with D591E and F593L mutations (CNF1(D591E F593L)) and the CNF1(H661Q) single mutant displayed drastically reduced reactivity with MAb NG8. A reverse chimeric triple mutant, CNF1(E591D L593F Q661H), imparted MAb NG8 reactivity to CNF2. MAb NG8 neutralized CNF2(E591D L593F Q661H) activity in a dose-dependent manner and reduced the binding of this chimeric toxin to HEp-2 cells. Taken together, these results pinpoint three amino acids in CNF1 that are key amino acids for recognition by neutralizing MAb NG8 and further help define a region in CNF1 that is critical for full toxin binding to HEp-2 cells.     

Sera from patients with rheumatic diseases recognize different epitope regions on the 52-kD Ro/SS-A protein.

Patients suffering from systemic lupus erythematosus (SLE) or Sjögren's syndrome (SS) often contain autoantibodies directed to the Ro(SS-A) complex. In this study the antigenic determinants on two of the components of the Ro complex, i.e. the Ro60 and the Ro52 polypeptides, were investigated. Anti-Ro+ sera were selected by counter-immunoelectrophoresis. Depending on the detection method, 59-68% of the SLE patients produced anti-Ro but not anti-La antibody, while 72-81% of the SS patients produced both anti-Ro and anti-La antibody. Immunoprecipitation of recombinant Ro-proteins showed that 61 sera (87%) were reactive with both Ro proteins, seven sera with Ro60 only, one serum with Ro52 only, and one serum did not precipitate the proteins at all. The anti-Ro60 reactivity of human sera is strongly associated with the native form of Ro60, suggesting that conformational autoepitopes are an important feature of Ro60. In the case of Ro52, frequently the residues located between amino acids 216 and 292 were essential for reactivity with the antibodies. With 70% of the lupus sera tested this appeared to be the only region important for reactivity. The antibodies of SS patients generally recognized multiple B cell epitopes located between amino acids 55 and 292. The results of this study indicate that the antigenic determinants on Ro52 are different for autoantibodies produced by lupus patients compared with those of SS patients.     

Human cytomegalovirus structural proteins: immune reaction against pp150 synthetic peptides.

In the present study, several peptides of the major structural antigen (pp150) of human cytomegalovirus (CMV) have been chemically synthesized and tested by a modified slot blotting procedure for their ability to bind CMV-specific immunoglobulin G (IgG) and IgM present in human sera. The sequences of the peptides were deduced on the basis of either (i) their presence in a fusion protein already known to be frequently recognized by human antibody or (ii) their high content of hydrophilic amino acids as deduced from the published nucleotide sequence. An important IgM-binding epitope was found to be located in the last 38 amino acids at the carboxy terminus of the molecule. This region reacts with anti-CMV IgM present in the great majority (83.3%) of IgM-positive human sera, and adsorption experiments have shown that IgM titers to the entire pp150 decrease 25 to 50% in most sera previously absorbed with this region. The overall results obtained endorse the continued synthesis of other sequences in order to define a group of peptides sensitive and specific enough to replace the virus and infected cells as an antigenic substrate in the serological evaluation of anti-CMV antibody.     

Real-time biospecific interaction analysis of antibody reactivity to peptides from the envelope glycoprotein, gp160, of HIV-1.

A new assay designed to quantitate antibody reactivity to specific peptides using biospecific interaction analysis (BIAcore) has been developed. Peptides of various lengths (15-40 amino acids) and isoelectric points (pI = 4.5-13) were covalently linked (immobilized) to a biosensor and interacted with polyclonal human sera. The immobilization procedure was highly reproducible, with bound peptides retaining high antibody reactivity. The assay was rapid, requiring only 25-30 min to immobilize the peptide and 2-8 min for each subsequent peptide/serum binding interaction. The same peptide surface has been used for up to 90 cycles of serum binding and regeneration with only a 0.3% decay in reactivity over cycle number. The quantitative BIAcore signal, measuring peptide/antibody binding interactions, was directly related to the antigen/antibody concentrations within the biosensor. The assay allowed interactants to be studied in their native form and without the need of additional secondary detection antibodies. Correlation between conventional peptide ELISA and BIAcore was obtained. The BIAcore linear range persisted over a series of eight two-fold dilutions. This extended linear dynamic response range is an improvement over conventional ELISA measurements. The sensitivity for monoclonal antibody detection is similar to conventional ELISAs and 4.9 ng/ml was readily detected.     

抗膀胱癌重组免疫毒素的可溶性表达及其抗肿瘤活性

目的 :构建抗膀胱癌重组免疫毒素BDI 1 PE38/KDEL的可溶性表达载体 ,并表达、纯化具有抗肿瘤活性的可溶性蛋白。方法 :将抗膀胱癌重组免疫毒素BDI 1 PE38/KDEL的基因片段 ,插入含有大肠杆菌脯氨酰顺反异构酶FkpA基因的共表达载体pTMFK中 ,构建重组共表达载体pTMFK IT。以重组质粒转化大肠杆菌BL2 1(DE3) Star,共表达目的蛋白与FkpA。表达产物以镍离子金属螯合层析柱及抗PE抗体亲和柱层析进行纯化。用ELISA检测免疫毒素的抗原结合活性 ,用噻唑蓝(MTT)法进行体外细胞杀伤实验。结果 :成功地构建了抗膀胱癌免疫毒素基因与FkpA基因的共表达载体 ,并获得纯度较高的目的表达产物。纯化后的表达产物与BIU 87膀胱癌细胞膜抗原具有良好的特异性结合活性 ,对BIU 87膀胱癌细胞有明显的体外特异性杀伤作用。结论 :获得了对膀胱癌细胞具有显著特异性杀伤活性的免疫毒素 ,为将其进一步应用于膀胱癌的靶向治疗研究奠定了基础     

Identification of major linear epitopes on the sp100 nuclear PBC autoantigen by the gene-fragment phage-display technology

Approximately 20-30% of sera from patients suffering from primary biliary cirrhosis contain autoantibodies against a nuclear protein termed sp100. By indirect cytoimmunofluorescence it was shown that the sp100 autoantigen is distributed in up to 20 dot-like structures per nucleus co-localizing with the so-called nuclear bodies. In western blots these sera react with a protein with an apparent molecular mass of 100kDa. By screening expression libraries with affinity-purified anti-sp100 antibodies we isolated a full-length sp100 cDNA whose sequence exactly matched the previously published sp100 sequence and encodes a protein of 481 amino acids with a deduced molecular mass of 53 kDa. In an attempt to determine immunoreactive regions on the sp100 antigen with the recently developed gene-fragment phage-display technology we were able to identify a stretch of sixteen amino acids (IKKEKPFSNSKVECQA) at position 296-311 as a major antigenic region (antigenic region 1) on the sp100-autoantigen. A second antigenic region (antigenic region 2) of twenty amino acids in length could be identified between amino acids 332-351 (EGSTDVDEPLEVFISAPRSE). By using immobilized synthetic peptides and various sp100-positive PBC patient sera the corresponding epitopes could be shown to be centered around epitope cores of six amino acids (SNSKVE, antigenic region 1) and nine amino acids (EPLEVFISA, antigenic region 2) respectively.     

Immunological properties of two related fragments from human and equine growth hormones

The immunological properties of a synthetic human growth hormone fragment comprising the amino acids 73 through 128 and of the homologous natural horse growth fragment formed by amino acids 73 through 123, have been comparatively studied. Antisera obtained in rabbits inoculated with the native human hormone or with the fragments, were used. By hemagglutination experiments both fragments have the same reactivity toward the anti-human growth hormone serum, but complement fixation curves detect the existence of at least two populations of antibodies presumably originated against the sequence 73-128 of human growth hormone. Of these, only one of the corresponding antigenic areas is present in the homologous region of equine growth hormone. The known cross-reactivity detected between both hormones is thus partially explained.     

PE重组免疫毒素在肿瘤导向治疗中的应用

绿脓杆菌外毒素A(PE)经过修饰后失去了与细胞结合的能力,将单克隆抗体的Fv段或细胞因子作为导向分子与修饰后的PE通过DNA重组技术融合,获得重组的免疫毒素,这种免疫毒素通过导向分子将毒素带到靶细胞,并杀伤靶细胞,因而被广泛运用于肿瘤的导向治疗。     

Monoclonal antibodies neutralize Bacillus cereus Nhe enterotoxin by inhibiting ordered binding of its three exoprotein components

The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB.