Immunogenic and antigenic epitopes of immunoglobulins. VIII. A human monoclonal rheumatoid factor having specificity for a discontinuous epitope determined by histidine/arginine interchange as residue 435 of immunoglobulin G

A monoclonal human rheumatoid factor (RF-AN) reactive with human Fc gamma is shown to recognise a discontinuous epitope dependent on the presence of histidine (His) at residue 435. RF-AN is not reactive with IgG1 paraproteins having C gamma 2 or C gamma 3 domain deletions or IgG3m (5) or Ig3m (21) proteins having arginine (Arg) at residue 435. RF-AN is reactive with IgG1, 2, 4 and IgG3m (15,16) proteins having histidine at 435.     

A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein

Truncated NS3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved B-cell epitopes on this non-structural bovine viral diarrhoea virus (BVDV) protein. A goat anti-pestivirus antiserum, and a panel of anti-NS3 monoclonal antibodies, including the BVDV-1 specific antibody P1D8, were used to verify the presence or absence of the epitopes. Interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain of NS3. Expression of the B-cell epitopes was dependent on, but not within, a 57 amino acid sequence at the carboxy-terminal end of this protein, supporting observations that these conserved epitopes are conformational in nature. A comparison of deduced amino acid sequences of the helicase domain from BVDV-1, BVDV-2, BDV and CSFV isolates highlighted a single amino acid that appeared to be unique to P1D8-reactive BVDV-1 isolates. Site-directed mutagenesis studies confirmed that this amino acid is critical for the expression of the BVDV-1 specific NS3 epitope recognised by the P1D8 monoclonal antibody. Surprisingly, the amino acid was also important for an epitope recognised by two group-specific monoclonal antibodies, P1H11 and P4A11. Protein modelling studies, based on the structure of the hepatitis C NS3 helicase domain, indicated that this amino acid occupies a prominent position on the surface of the protein.     

Mapping the functional topography of Fc gamma with monoclonal antibodies: localization of epitopes interacting with the binding sites of Fc receptor on human K cells

A panel of monoclonal antibodies (mAb) specific for the C gamma 2, C gamma 3 or inter C gamma 2/C gamma 3 domain epitopes was tested for inhibition of antibody-dependent cellular cytotoxicity (ADCC) specific for anti-D IgG-coated erythrocytes. Significant inhibition of ADCC was demonstrable for some antibodies having specificity for C gamma 2 or C gamma 3 domain epitopes, while others gave no inhibition. Fab fragments of a representative C gamma 2-specific antibody (A55) and C gamma 3-specific antibody (x3a8) retained their inhibitory capacity in lymphocyte-mediated ADCC, but only A55 Fab inhibited monocyte-mediated lysis. Furthermore, the Fab portion of A55 completely abolished the complement-dependent enhancement of ADCC mediated by concanavalin A-stimulated cells, while x3a8 Fab had no effect in this system. On the other hand, x3a8 Fab inhibited the binding of anti-D IgG-sensitized erythrocytes to lymphocytes while A55 Fab did not influence this latter interaction. The results suggest that C gamma 2 domain-FcR interaction is essential for the triggering of lytic process both in lymphocyte and in monocyte-mediated ADCC, while C gamma 3 domain has no role in the latter but is responsible for the appropriate contact between effector lymphocytes and target cells. A site in the region of Lys274 appears to be critical for triggering of both lymphocyte and monocyte-mediated ADCC.     

Immunogenic and antigenic epitopes of immunoglobulins. XIV. Antigenic variants of IgG4 proteins revealed with monoclonal antibodies

Two IgG4 paraproteins having heavy chains of normal molecular weight are shown to be antigenically distinct in their reactivity profiles with 18 monoclonal antibodies having specificity for the IgG2 or IgG4 subclasses. One protein expresses IgG2 and IgG4 epitopes within the C gamma 2 domain. A second protein is deficient in the expression of IgG4 Fc-specific epitopes. These proteins will be of value in defining the structural basis of Fc effector functions and individual epitope expression.     

Immunogenic and antigenic epitopes of immunoglobulins--V. Reactivity of a panel of monoclonal antibodies with sub-fragments of human Fc gamma and abnormal paraproteins having deletions

The specificity of a panel of 27 monoclonal antibodies reactive with human Fc gamma has been further defined through reactivity profiles with sub-fragments of Fc gamma and IgG paraproteins having deletions within C gamma 2 or C gamma 3. Antibodies are identified that are directed to discontinuous epitopes requiring native Fc gamma for expression. Other antibodies are reactive with isolated C gamma 2 or C gamma 3 domains. Sub-class specific and sub-class restricted antibodies allow correlation of epitope expression with primary structure.     

抗人P185~(erbB_2)单克隆抗体的制备及特性分析

目的制备可特异识别 P185 erb B2 胞外区的单抗 ,选出亲和力较高并具有抑瘤作用的克隆。方法以高表达 erb B2 的人乳腺癌细胞系 SKBR3免疫 BABL / c小鼠 ,用纯化的重组 DNA表达的 P185胞外区蛋白作为抗原 ,以间接 EL ISA方法筛选阳性克隆。结果获得 10株稳定分泌抗 P185胞外区单抗的杂交瘤细胞株。 4株为 Ig G1、1株为 Ig G3、5株为 Ig M,分别可识别 P185胞外区 3个不同表位 ,且只与天然形式的 P185胞外区特异结合。其中 4株 Ig G1类抗体可明显抑制 SKBR3及 SKOV3细胞增殖。免疫组化实验可使 SKBR3及 SKOV3细胞膜特异性染色 ,且可检出乳腺癌、卵巢癌等石蜡切片标本中 erb B2 高表达的阳性细胞。结论所获单抗可特异识别 P185胞外区抗原 ,具有肿瘤诊断、判断预后及人源化改造后用于治疗的应用潜力。     

Modulation of the murine immune response to human IgG by complexing with monoclonal antibodies. I. Antibody responses to determinants on the constant region of light chains and gamma chains.

Complexing a human IgG lambda paraprotein with a monoclonal antibody (McAb) to a lambda-chain determinant markedly depressed the anti-lambda response of immunized mice. No anti-lambda was found in the serum after two or four injections of the complex, whereas high titres of anti-lambda chain antibody were found in the sera of mice immunized with the IgG paraprotein complexed with any single anti-gamma chain McAb or with a pool of anti-gamma chain McAbs. Responses to the highly immunogenic Fc gamma portion of the molecule were not suppressed by complexing with a single anti-gamma chain McAb and were only slightly suppressed after complexing with a pool of anti-gamma chain McAbs. Some anti-Fc gamma antibody was produced by all animals receiving a single injection of the immunogen complexed to any McAb, but free immunogen did not generate a primary response. Complexing the IgG with an anti-Fc gamma McAb enhanced the response to to the poorly immunogenic C gamma 1 domain but no anti-C-gamma 1 was produced by mice receiving the IgG complexed with an anti-C-gamma 1 McAb. In immunizations with 'free' (Bence-Jones) lambda chain, all the antibody produced was directed against 'free'-specific determinants. Complexing the Bence-Jones protein with McAbs to 'free' or 'general' determinants on the lambda chain did not enhance or suppress the response. It is concluded that the response to weakly or moderately immunogenic, but not to strongly immunogenic, regions of the molecule may be suppressed by complexing the immunogen with a McAb of appropriate specificity. Possible reasons for this result are discussed.     

人CD226 (PTA1)分子胞膜外区截短体真核表达载体的构建、表达及其识别结构域的分析

目的：确定一套CD226单克隆抗体（McAb）识别CD226分子胞膜外区结构域的部位，方法：应用计算机软件分析CD226子蛋白质序列疏水性，确定其亲水性区域。采用PCR方法扩增CD226分子基因序列，分别缺失相应亲水性区域，构建CD226截短体重组真核组细胞表达载体pcDNA3-PTA1T1和pcDNA3-PTA1T2。测序正确后，转染COS7细胞，抗CD226分子克隆抗体间接免疫荧光染色，流式细胞术检测CD226分子截短体的表达，表达成功后，采用间接免疫荧光染色和流式细胞术分析，用一套CD226McAb检测与截短突变体的免疫反应性，从而确定CD226McAb识别CD226分子结构域的部位。结果：PCR扩增出CD226分子相应目的片段序列，定向克隆入pcDNA3真核表达。CD226McAb Leo A1,New E1T -7均可识别CD226全长分子；Leo A1,New E1,FMU1,FMU2,FMU4和MFU5与截短突变体PTA1T1和PTA1T2均无反应性，FMU3可结合PTA1T1分子，不结合PTA1T2分子；FMU6和FMU7均可结合PTA1T2均无反应性；FMU3可结合PTA1T1分子，不结合PTA1T2分子；FMU6和FMU7均可结合PTA1T1及PTA1T2分子，结论：CD226McAb Leo A1,New E1,FMU1,FMU2,FMU3,FMU4和FMU5识别CD226分子膜膜外区D1结构域，其中FMU3识别的位点位于V1和V2环之间；FMU6和FMU7识别D2结构域。     

Immunogenic and Antigenic Epitopes of Immunoglobulins

The expression of idiotypic and variable region-associated isotypic determinants on a panel of human monoclonal rheumatoid factors (RF) was studied by means of murine hybridoma antibodies produced to two IgM-RF paraproteins. Fourteen RF paraproteins from patients with cryoglobulinaemia and one (RF-AN) from an Epstein-Barr virus (EBV)-established B-cell line from a patient with rheumatoid arthritis (RA) were studied. Nine RF paraproteins expressed a VkIIIb light chain sub-subgroup-associated cross-reactive idiotype and seven of these nine also expressed a heavy chain-associated cross-reactive idiotype. The reactivity of the monoclonal RF with human IgG subclass paraproteins revealed four patterns of molecular specificities: (1) RF reactive with an epitope common to all IgG subclasses; (2) RF reactive with an epitope expressed on IgG1, 2, 4 and G3m(s,t) which has histidine at 435, but not G3m(b) or G3m(g) which have arginine at 435; (3) RF reactive with an epitope expressed on IgG1, 2, and 4, but not IgG3 irrespective of allotypic markers; (4) RF reactive with epitopes expressed on some, but not all paraproteins within the subclasses. Four of five RF paraproteins that expressed both the heavy and light chain-associated idiotopes showed a similar pattern of reactivity with IgG subclass proteins.     

Efficient purification of unique antibodies using peptide affinity-matrix columns

Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85-99. Several peptide epitopes were identified and all of them recognised specifically MK16. One peptide, ER6.1, was selected and linked to beaded agarose and demonstrated excellent performance as a peptide affinity chromatography matrix. This epitope matrix was efficient in the purification of MK16 Fab fragments and had no affinity for other antibodies. Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells.Investigations of the fine specificity of the ER6.1 peptide demonstrated that it recognised a unique epitope within the heavy chain CDR3 region of the MK16 antibody. Thus, variants of MK16 antibody, which had retained the specificity and affinity of the original antibody but had slightly different amino acid composition in the CDR3 region, were not recognised by the ER6.1 peptide.     

The monocyte binding domain(s) on human immunoglobulin G

Monocyte binding has previously been assigned to the C gamma 3 domain of human immunoglobulin G (IgG) largely on the ability of the pFc' fragment to inhibit the monocyte-IgG interaction. This ability is markedly reduced compared to the intact parent IgG. We find this result with a conventional pFc' preparation but this preparation is found to contain trace contamination of parent IgG as demonstrated by reactivity with monoclonal antibodies directed against C gamma 2 domain and light-chain epitopes of human IgG. Extensive immunoaffinity purification of the pFc' preparation removes its inhibitory ability indicating that this originates in the trace contamination of parent IgG (or Fc). Neither of the human IgG1 paraproteins TIM, lacking the C gamma 2 domain, or SIZ, lacking the C gamma 3 domain, are found to inhibit the monocyte-IgG interaction. The hinge-deleted IgG1 Dob protein shows little or no inhibitory ability. Indirect evidence for the involvement of the C gamma 2 domain in monocyte binding is considered. We suggest finally that the site of interaction is found either on the C gamma 2 domain alone or between the C gamma 2 and C gamma 3 domains.     

Detection of free kappa chains in human serum and urine using pairs of monoclonal antibodies reacting with C kappa epitopes not available on whole immunoglobulins.

The properties of five monoclonal antibodies (McAbs) which react with free kappa chains but not with IgG kappa, IgM kappa or IgA kappa have been studied. IgG preparations stored for long periods sometimes contained low and high molecular weight material reacting as free kappa. This could be removed by passing the preparation through an affinity column to which two of the McAbs had been coupled, but reactive material reappeared when the purified IgG was heated at 65 degrees C. Two of the McAbs from one fusion reacted with F(ab')2 fragments of IgG whereas two from another fusion did not. Pairs of McAbs (one from each fusion) were much more sensitive in the detection of free kappa, particularly monomer, than a single McAb. This provided the basis for a very sensitive method of detection of free kappa chains in human body fluids and culture supernates.     

Immunogenic and antigenic epitopes of immunoglobulins I. Cross-reactivity of murine monoclonal antibodies to human IgG with the immunoglobulins of certain animal species

Antibody-producing hybridoma clones have been isolated following immunization of mice with human IgG. Twenty-five monoclonal antibodies (nine anti-C gamma 3, fourteen anti-C gamma 2, one anit-kappa and one anti-lambda) were selected for study of their cross-reactivity with the IgG of fifteen mammalian species and chicken immunoglobulin. Each antibody exhibited a unique reaction profile suggesting that human IgG expresses a very large repertoire of immunogenic epitopes. Whilst some antibodies showed a very restricted cross-reactivity profile for others a very wide reactivity profile was observed-including two clones producing autoantibodies. Antibodies demonstrating cross-reactivity between human Fc gamma and 7S chicken immunoglobulin allow its definitive assignment as a homologue of human IgG. Four clones demonstrated specificity for bovine IgG subclass gamma 1 and gamma 2 and the degree of reactivity allows their application to qualitative and quantitative assay systems. These studies suggest new perspectives for the characterization of immunoglobulins and the standardization of anti-immunoglobulin reagents.     

噬菌体随机肽库分析HIV-1 p24抗原表位

目的：利用噬菌体随机肽库分析抗HIV－1核心区抗原p24单抗体在抗原上的识别位点。方法：用抗HIV－1 p24单抗2C7和3H10作为筛选分子，对噬菌体肽库进行生物洗（biopanning)，并通过DN测序、ELISA效价测定等对所获得的噬菌休克隆进行鉴定，最后对合成的7肽位点通过间接ELISA及免疫抑制试验进行血清学分析。结果：序列分析结果表明，单抗2C7和3H10在HIV－1 p24上的抗原识别表位的保守序列分别为DHPXPXX和XXXXKAF。分别合成这2个7肽氨基酸序列P－C1（DHPSPWG）和P－H3（SPWLKAFGGS），并分析其免疫学结合特性，结果表明与P－H3相比，单抗2C7的抗原识别表位P－C1的固相结合特性较好，固相P－C1检测血样，13份抗HIV阳性本中，12份为阳性（检出率为92．3％），19份抗HIV阴性样本中，仅1份为假阳性结果（特异性为94．7％），与P－C1相比，单抗3H10的抗原表位P－H3的固相结合能力极差，但液相结合活性较好，血样与P－H3的抑制试验表明，13份抗HIV阳性样本中12份样本对P－H3的抑制率大于60％（12／13），而9份抗HIV阴性样本中仅1份对P－H3的抑制率大于50％，结论：用抗HIV－1 p24单抗筛选噬菌体随机肽库，获得单抗在p24抗原上的识别表位的氨基酸序列，血清学结果表明这2个抗原表位存在于p24自然抗原上，在抗HIV－1的感染检测中具有潜在的应用价值。     

Fate of the mutated IgG2 heavy chain: lack of expression of mutated membrane-bound IgG2 on the B cell surface in selective IgG2 deficiency

IgG2 deficiency is clinically characterized by sinopulmonary infections caused by pneumococcus and Hemophilus. We reported homozygous one-base insertion (1793insG) in the C(gamma)2 gene in two Japanese siblings in whom serum IgG2 levels were under detection limits. The 1793insG was present in exon 4, just upstream from the alternative splice site for M exons; the result being a complete amino acid change in transmembrane and cytosolic parts of membrane-bound gamma2 heavy chain (m gamma 2HC). To determine why this mutation caused selective and complete IgG2 deficiency, we constructed expression vectors of normal and mutant membrane-bound chimeric IgG heavy chain cDNAs. Stable transformants, Ag8N-L and Ag8M-L, expressing either normal and mutant chimeric IgG heavy chain with light chain respectively were obtained using P3X63Ag8653 as recipient cells. Of the Ag8N-L, 22.1% were surface IgG+; however, none of the Ag8M-L were surface IgG+. Addition of an anti-human IgG antibody induced cell death of Ag8N-L and we considered that the expressed chimeric IgG protein on Ag8N-L might function as the Ig receptor for signal transduction. However, Ag8M-L did not express mutant IgG on its surface nor did it secrete this mutant into culture medium. The mutant chimeric IgG protein was rapidly degraded within Ag8M-L. Thus, the mutated IgG2 heavy chain in our patient could not be expressed on the cell surface because of loss of the transmembrane domain and the evolutionally conserved cytoplasmic domain. In humans, B cells expressing surface IgG are indispensable for secretion of IgG.     

Iscom佐剂增强含前S表位重组乙肝表面抗原的免疫原性

目的 探讨Iscom佐剂对含前S表位重组乙肝表面抗原(SS1S2)免疫原性的影响.方法 用纯化的SSIS2抗原与Al(OH)3和Iscom佐剂分别配制疫苗,在0 d和14 d时采用肌肉和皮下注射方式免疫BALB/c小鼠,免后14 d各取一半小鼠采血,测定乙肝病毒S、前S1和前S2抗体滴度以及总的IgG1和IgG2a抗体滴度,并计算IgG2a和IgG1抗体比例.同时分离脾淋巴细胞,进行IFN-γ酶联斑点(ELISPOT)测定.结果 单针免疫时,两组疫苗血清样品乙肝病毒S、前S1和前S2抗体阳转率、抗体滴度及IFN-γ分泌细胞数相当,但Iscom佐剂疫苗组IgG2a抗体比例较高;加强免疫后,两组疫苗抗体滴度均呈上升趋势,Iscom疫苗组抗体滴度上升幅度大于Al(OH)3疫苗组,两者S、前S1和前S2抗体滴度差异均有统计学意义.加强免疫后Iscom疫苗组IgG2a抗体比例保持平衡,而Al(OH)3疫苗组IgG2a抗体比例进一步降低.在细胞免疫方面,Iscom疫苗组在加强免疫后产生的特异性IFNγ分泌细胞数显著超过Al(OH)3疫苗组.结论 本研究初步显示,Iscom佐剂对含前S表位重组乙肝表面抗原具有比Al(OH)3佐剂更强地免疫增强作用.

Abstract：

Objective To investigate the effect of Iscom matrix on the immunogenicity of recombinant hepatitis B surface antigen containing PreS epitopes(SS1S2). Methods SS1S2+ Al(OH)3 and SS1S2+Iscom vaccines were made by combining purified SS1S2 antigen with Al( OH)3 adjuvant or Iscom matrix.Groups of BALB/c mice were injected i. m or s. c by either of the two vaccines at day 0 and day 14. Half of the mice were sacrificed and sera were taken and spleen cells separated from the mice 14 days after each injection. Anti-S, anti-PreS1, and anti-PreS2 antibody titers were measured, and total IgG1 and IgG2a titers were further detected for each serum sample. IFN-γ ELISPOT assay was performed to detect IFN-γsecreting cells from the pooled spleen cells for each vaccine group. Results The seroconversion rates and geometric mean titers(GMTs) and the numbers of IFN-γ secreting cells were approximately at the same level for the differently formulated vaccines after the first injection except that the ratio of IgG2a to IgG1 in the Iscom group was higher than the Al(OH)3 group. After boost injection, the GMTs of total IgG rise slightly in the Al(OH)3 group but significantly in the Iscom group. The IgG2a to IgG1 ratio in the Iscom group kept balanced while dropped further in the Al(OH)3 group. The number of specific IFN-γsecreting cells triggered by the Iscom vaccine exceeded significantly the number of Al( OH)3 vaccine, showinga stronger cellular response. Conclusion The results in this study shows that Iscom matrix is more potent in enhancing the immunogenicity of recombinant hepatitis B virus surface antigen containing PreS epitopes than Al( OH)3 adjuvant.     

Dominant epitopes of the C6 diagnostic peptide of Borrelia burgdorferi are largely inaccessible to antibody on the parent VlsE molecule

Lyme borreliosis (LB) is a disease for which antibody-based detection assays are often required for diagnosis. The variable surface molecule VlsE and IR6, one of its invariable regions, are commonly targeted by the antibody response in infected individuals. A series of enzyme-linked immunosorbent assays was performed to comparatively examine the antibody responses of North American LB patients (n = 37) to VlsE and invariable segments of this molecule. Both immunoglobulin M (IgM) and IgG responses to full-length VlsE and to peptides reproducing invariable regions 2, 4, and 6, as well as the invariable domains at the amino and carboxyl termini of VlsE, were assessed. The proportions and specificities of reactivity to the invariable segments were tested by using cognate peptides as competitors for VlsE binding by patient serum antibodies. IR6 epitopes (by the C6 peptide) were found to dominate the response to invariable segments. IR6 (C6)-specific antibodies were detected in 78% of the serum specimens, whereas <40% of patients generated antibodies that bound the N- or C-terminal domain and <12% of patients responded to either IR2 or IR4. Interestingly, 15 of 37 patients generated IgG antibodies that reacted with C6 but not with VlsE. Conversely, IgM responses were frequent for VlsE but not for invariable segments. A representative number of the serum specimens (n = 8) that contained IgG antibodies reacting with both C6 and VlsE was assessed in competition experiments, using C6 as a competitor. Only half of these specimens contained IgG antibodies whose binding to VlsE could be inhibited >50% by competition with the added C6 peptide. The median percent inhibition was 45.5%. These findings indicate that IR6 epitopes are largely concealed from the VlsE molecular surface and that full-length VlsE-based diagnosis likely detects antibodies to conformational and/or variable region epitopes.     

不同基因型戊型肝炎病毒的抗原表位分析

目的 研究不同基因型戊型肝炎病毒(HEV)的抗原表位特点。方法 以HEV基因工程重组蛋白p166Bur、p166Mex为免疫原，制备单克隆抗体(McAbs)。采用间接ELISA法及免疫印迹法(Western blot)，检测所制备的McAbs与7种不同基因型和亚型p166(p166Bur、p166Pak、p166Mor、p166Mex、p166Us、p166Nz和p166Chn)的交叉反应性。结果 获得4株稳定分泌基因型相关MeAb的杂交瘤细胞株，即1B5、1D10,1G10和D4A3。经检测，1B5、1D10,1G10分泌的McAbs只与p166Bur及p166Mor特异结合，D4A3分泌的McAb只与p166Mex特异结合。结论 不同基因型HEV存在不同的抗原表位。     

寻常性天疱疮抗原EC1-2和EC3-4表位的分子克隆与蛋白表达

目的：克隆并表达寻常性天疱疮抗原（ＰＶＡ,即桥粒芯糖蛋白－３）的ＥＣ１－２和ＥＣ３－４表位,用于通过血清学方法特异性诊断天疱疮和了解ＰＶＡ的表位与抗ＰＶＡ抗体间的联系。方法：从角朊细胞抽提ＲＮＡ,逆转录合成ｃＤＮＡ,扩增目的基因ＥＣ１－２和ＥＣ３－４后与质粒载体ＰＧＥＸ－４Ｔ－１连接,电转导入大肠杆菌ＢＬ２１,经ＩＰＴＧ诱导后表达ＥＣ１－２和ＥＣ３－４融合蛋白,ＳＤＳ－ＰＳＧＥ电泳后转移至硝酸纤素     

Two monoclonal antibodies with defined epitopes of P44 major surface proteins neutralize Anaplasma phagocytophilum by distinct mechanisms

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.