B-Cell and T-Cell Epitopes in Anti-factor VIII Immune Responses

Adequate hemostasis is achieved for many hemophilia A patients by infusion of plasma-derived or recombinant factor VIII (FVIII), but unfortunately, a significant subset of patients develop an immune response in which anti-FVIII antibodies, referred to clinically as “inhibitors,” interfere with its procoagulant activity. Inhibitors are the subset of anti-FVIII antibodies that bind to surfaces on FVIII (B-cell epitopes) that are important for its proper functioning in coagulation. Less antigenic FVIII molecules may be designed by identifying and then modifying the amino acid sequences of inhibitor B-cell epitopes. Conversely, characterization of these epitopes can yield important information regarding functionally important surfaces on FVIII. The production of inhibitor antibodies is driven by T cells. T cells recognize FVIII as foreign when FVIII-derived peptides bind to major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells. The class II–peptide complexes must then be recognized by T-cell receptors (TCRs). T-cell stimulation requires sustained association of antigen-presenting cells and T cells through formation of a class II–peptide–TCR complex, and peptide sequences that mediate this association are termed “T-cell epitopes.” MHC class II tetramers that bind FVIII-derived peptides and recognize antigen-specific TCRs are proving useful in the characterization of human leukocyte antigen-restricted T-cell responses to FVIII.     

B Cell Hybridoma Presents Both B-Cell and T-Cell Epitopes for Stimulating Antibody Production Via CD23 Pathway

CD23+ B cell hybridoma 17A11, pulsed with IgE:TNP-KLH triggered IgA, IgG, and IgE antibody production via CD23-mediated presentation. Prior anti-CD23 treatment abrogated 95% of the humoral antibody responses. Both B and T cell epitopes were presented by 17A11. B cell epitopes as recognized by IgG but not T cell epitopes were sensitive to treatment with 0.2 M acetic acid. Efficacy of antigen presentation via CD23 on 17A11 was comparable to that mediated via surface immunoglobulins (sIg) on a CD23 negative 4.5 parental fusion partner B cell line. This is the first demonstration that IgE:TNP-KLH pulsed B cell hybridomas present both B-and T-cell epitopes in stimulating IgA, IgG, and IgE antibody production, and raise a pertinent issue whether IgE antibodies produced under pathophysiological conditions may serve as positive feedback signal for sustaining production of different classes of antibodies.     

B Cell Hybridoma Presents Both B-Cell and T-Cell Epitopes for Stimulating Antibody Production Via CD23 Pathway

CD23+ B cell hybridoma 17A11, pulsed with IgE:TNP-KLH triggered IgA, IgG, and IgE antibody production via CD23-mediated presentation. Prior anti-CD23 treatment abrogated 95% of the humoral antibody responses. Both B and T cell epitopes were presented by 17A11. B cell epitopes as recognized by IgG but not T cell epitopes were sensitive to treatment with 0.2 M acetic acid. Efficacy of antigen presentation via CD23 on 17A11 was comparable to that mediated via surface immunoglobulins (sIg) on a CD23 negative 4.5 parental fusion partner B cell line. This is the first demonstration that IgE:TNP-KLH pulsed B cell hybridomas present both B-and T-cell epitopes in stimulating IgA, IgG, and IgE antibody production, and raise a pertinent issue whether IgE antibodies produced under pathophysiological conditions may serve as positive feedback signal for sustaining production of different classes of antibodies.     

MPB 64 Possesses Tuberculosis-Complex''-Specific B- and T-Cell Epitopes

We have developed monoclonal antibodies (MoAb) reactive with a protein from Mycobacterium tuberculosis of apparent molecular mass 24 kDa. This protein was shown to be identical with MPB 64 (Harboe et al.,) MoAb bound to four different epitopes of which two were restricted to the 'tuberculosis complex' and two were also found in mycobacteria not belonging to the 'tuberculosis complex'. The cross-reactive MoAb demonstrate that MPB 64 is present in more mycobacterial species than previously assumed. MPB 64 was shown to induce strong delayed type hypersensitivity (Dth) reactions in outbred guinea pigs immunized with M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). No reaction was observed in animals immunized with mycobacteria not belonging to the 'tuberculosis complex'. The Dth-inducing capacity of MPB64 was compared with that of another 24 kDa protein purified from M. tuberculosis and of the previously described 38 kDa protein. The Dth responses to these three antigens were further analysed in four inbred guinea pig strains. A genetic restriction of the ability of the animals to respond to MPB 64 as well as to the 38 kDa protein was observed.     

Immunoreactivity to Putative B-Cell Epitopes of Hepatitis G Virus Polyprotein in Viremic and Nonviremic Subjects

The hepatitis G virus (HGV) polyprotein was scanned by computer-aided prediction of antigenicity to search for B-cell epitopes. Four polypeptide sequences, V37D (amino acids [aa] 1685 to 1721), V36S (aa 2102 to 2137), P37R (aa 2156 to 2192), and C40P (aa 2280 to 2319), were identified and synthesized for use in immunoassays. Antibodies to these peptides were searched for in a panel of 239 serum samples, which were also tested for anti-E2 antibodies and HGV RNA. Furthermore, the course of HGV markers was studied prospectively in four patients who had been transfused with HGV RNA-positive blood. There was a negative association between immunoreactivity to V37D and P37R and presence of HGV RNA (2 of 53 and 1 of 53, respectively; P < 0.05); none of the subjects with dual antibody positivity was HGV RNA positive. Anti-V37D and anti-P37R antibodies compared favorably with anti-E2 antibodies as markers of recovery from HGV infection. These results might be useful for the development of new, more sensitive diagnostic assays.     

Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing

BK virus (BKV) infection causing end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep-sequencing methodology and bioinformatics pipeline that identify BKV variants across the genome and at BKV-specific HLA-A2-, HLA-B0702-, and HLA-B08-restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets, and fragmentation libraries were sequenced on the Ion Torrent Personal Genome Machine (PGM). An error model and variant-calling algorithm were developed to accurately identify rare variants. A total of 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range, 2 to 37; interquartile range, 10), with the majority of variants (77%) detected at a frequency of <5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for the BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies.     

Conformational Dependence of Anaplasma marginale Major Surface Protein 5 Surface-Exposed B-Cell Epitopes

The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940–2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denatured A. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.     

The T-Cell Repertoire in Myasthenia Gravis Involves Multiple Cholinergic Receptor Epitopes

Antibodies against the alpha-subunit of the acetylcholine receptor (AChR) are found in most patients with myasthenia gravis and are considered to contribute to the receptor damage which leads to the characteristic signs and symptoms of the disease. This B-cell response is T-cell driven. Elevated T-cell reactivities to AChR and its alpha-subunit have been described in myasthenia gravis, and AChR alpha-subunit peptide reactive T-cell lines and clones preferentially recognizing certain defined sequence segments have been reported, thereby disclosing the possibility of specific immunotherapy. We have defined the T-cell repertoire to AChR, its alpha-subunit and the synthetic peptide sequences 100-117, 113-130, 143-163, 161-179, 207-225, 221-240, and 235-255 of the alpha-subunit in an immunospot assay which is based on secretion of interferon-gamma (IFN-gamma) by individual memory T cells upon stimulation with specific antigen in short-term cultures. Most patients with myasthenia gravis displayed T-cell reactivities to 1 to 6 different peptides. The mean numbers of T cells recognizing individual peptides varied in the myasthenia gravis patients between 1 per 77,000 and 1 per 167,000 peripheral blood mononuclear cells. None of the seven peptides evaluated could be identified as an immunodominant T-cell epitope, and any of them was found to dominate in individual patients. The numbers of T cells reacting with AChR and recombinant human AChR alpha-subunit were slightly higher (mean numbers 1 per 26,000 and 1 per 50,000 mononuclear cells, respectively). Such cells, as well as AChR alpha-subunit peptide reactive T cells, were also found in patients with other neurological diseases and in healthy subjects, but at lower frequencies and numbers. In myasthenia gravis, the elevated numbers of memory T cells recognizing multiple AChR alpha-subunit peptides may be crucial for the development of the disease, and the IFN-gamma released by such T cells might be important for its perpetuation.     

Human T-Cell Epitopes on the Mycobacterium tuberculosis Secreted Protein MPT64

Mycobacterium tuberculosis secretes a number of proteins into the extracellular envirornment, some of which are restricted to the M. tuberculosis complex. These proteins are targets for T- and B-cell immune responses in tuberculosis (TB) patients and their contacts. The authors have mapped the immunogenic regions of the MPT64 protein of M. tuberculosis using peripheral blood mononuclear cells (PBMC) from TB patients and a set of overlapping peptides encompassing the complete sequence of the protein. T-cell epitopes which induced proliferation or interferon-gamma (IFN-γ) release were distributed over the full length of the protein. A C-terminal region of the protein, however, contains sequences recognized in the context of multiple HLA-DR phenotypes by more than 80% of the subjects tested. The nature of the T-cell response was further investigated by generating MPT64-specific T-cell lines. These lines also identified the T-cell epitopes in the C-terminal region of the protein. On stimulation with antigen the lines secreted IFN-γ, but not interleukin 4 (IL-4). A minority of TB patients (6/32) mounted an antibody response to MPT64. Sera from half (3/6) of these identified two linear antibody binding sites. These results confirm the significance of this protein in the immune response to tuberculosis infection.     

B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

Peptide Mapping of Bovine T-Cell Epitopes for the 38 kDa Tuberculosis Antigen

Mycobacterium bovis infection in cattle continues to be a problem in several regions, partly due to inadequate diagnostic tests. The aim of this study was to use an experimental model of the natural disease to identify T-cell epitopes from the mycobacterial 38 kDa antigen as potentially specific diagnostic reagents. A panel of overlapping synthetic peptides (16-mers with a five-residue overlap) were produced from the published amino acid sequence. It was found that peripheral blood lymphocytes from at least three of four experimentally infected animals, which were considered to be in either T^h1- or T^hl/T^h2-dominated stages of anti-mycobacterial immunity, proliferated in response to five epitopes (residues 1–27, 88–107, 122–138, 243–260 and 307–328). However, in vitro production of IFN-γ was detected only in response to epitope 122–138, indicating a role in protective immunity. The peptides were not recognized by control, uninfected animals, but all epitopes showed various degrees of recognition by animals which were field reactors to intradermal tuberculin testing. Furthermore, epitopes 1–27, 88–107 and 122–138 were recognized by four breeds of cattle and by animals from separate herds, suggesting genetic permissiveness in recognition which would be essential in the development of a diagnostic test.     

Heterogeneity of Degradation of B-Cell Endocytosed Monoclonal Antibodies Reacting with Different sIgM Epitopes

The degradation of a panel of monoclonal antibodies (MoAb) bound to surface IgM (sIgM) was studied in three human Burkitt's lymphoma cell lines. The panel included MoAb that recognize several distinct epitopes associated with the F(c mu)5 domain, the c mu 2 domain and kappa or lambda light chains. The amount of degraded MoAb and the rate of their degradation varied considerably between the various antibodies. Properties of MoAb such as avidity or ability to cross-link sIgM did not significantly influence their degradation. The most consistent correlation between rate of degradation and MoAb used was the location of the epitope recognized by the individual MoAb. Thus, 7 out of 8 anti-light chain MoAb were degraded at a higher rate than 5 out of 5 anti-F(c mu)5 MoAb. One anti-c mu 2 MoAb was degraded at a rate similar to the majority of anti-light chain MoAb. The intracellular transport of an anti-kappa light chain MoAb and an anti-F(c mu)5 MoAb was studied in detail by subcellular fractionation in sucrose gradients. We found that the anti-kappa light chain MoAb was transported more rapidly to lysosomes than the anti-F(c mu)5 MoAb, showing that they were sorted differently intracellularly.     

微小隐孢子虫SA35与SA40蛋白的B细胞抗原表位预测

目的预测微小隐孢子虫SA35和SA40蛋白的B细胞表位。方法以单参数（亲水性、可及性、柔韧性及抗原性）预测为基础,通过二级结构预测初步筛选,并以ABCpred方案作为最终验证,预测微小隐孢子虫SA35和SA40蛋白的B细胞表位。结果SA35蛋白N端103～115和129～146和以及SA40蛋白的N端77～89、127～136、156～174和200～209区段为预测的B细胞表位。结论所得表位为这两种蛋白以后应用于合成肽检测、制备相应的抗体、发展高特异性和敏感性的诊断系统以及研制疫苗等提供了理论基础。     

A Model System for the Analysis of B-Cell Activation and Effector T-Cell Functions

We have attempted to develop an in vitro system where polyclonal B lymphocyte responses could be induced in 'antigen-like' conditions, that is, where surface immunoglobulin-dependent binding mediates interaction with a mitogen. Monoclonal anti-mu and anti-delta antibodies were covalently bound to lipopolysaccharide (LPS) and these complexes were shown to display mitogenic activity. Polyclonal plaque-forming cell (PFC) responses, however, were diminished in cultures stimulated by anti-mu-LPS (but not by anti-delta-LPS) indicating that 'anti-mu inhibition' of terminal B-cell differentiation also applies to 'specific' antibody responses. Moreover, the analysis of the functional activity of monoclonal antibodies to major histocompatibility complex (MHC) class II molecules revealed a surprising synergy between low, non-stimulatory concentrations of anti-mu-LPS (but not anti-delta-LPS) with anti-I-A antibodies. These responses are T-cell dependent and synergy with anti-mu-LPS conjugates can also be obtained with 'naturally' activated CD4+ cells isolated from normal donors. A model of molecular and cellular interactions was derived, which accounts for the present findings and is applicable in antigen-dependent lymphocyte collaboration.     

Multiple ICAM-1 (CD54) Epitopes are Involved in Homotypic B-Cell Adhesion

Two monoclonal antibodies (MoAbs), F10.2 and F10.3, were selected for their ability to interfere in homotypic adhesion of human B cells. Precipitation studies and binding to intercellular adhesion molecule 1 (ICAM-1, CD54) cDNA transfected COS cells revealed that both MoAbs are directed against ICAM-1. The binding of MoAb F10.2 was inhibited by LB-2, a MoAb recognizing the NH2-terminal immunoglobulin-like domain of ICAM-1. This suggests that the epitope recognized by F10.2 is located on the first domain of the ICAM-1 molecule. Binding of the other MoAb, F10.3, was not inhibited by F10.2 nor by two other MoAbs mapping to the first domain of the ICAM-1 molecule. The ability of F10.3 to bind to ICAM-1 is influenced by glycosylation, suggesting that this epitope is located on one of the domains carrying possible glycosylation sites, i.e. domain 2, 3 or 4. The ICAM-1 epitopes recognized by F10.3 and LB-2 or F10.2 co-operated in homotypic adhesion of cells from the EBV cell line ML1. These results suggest that in addition to an epitope located on domain 1 of the ICAM-1 molecule, another epitope whose exposure can be regulated by glycosylation is involved in homotypic B-cell adhesion of cell line ML1.     

Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.     

Control of Trypanosoma cruzi Infection in Mice Deprived of T-Cell Help

The role of CD4+ T lymphocytes in the resistance of BALB/c mice to Trypanosoma cruzi was examined by in vivo depletion using monoclonal anti-CD4 antibodies (MoAbs). When the administration of MoAbs was initiated 2 days before, or 5 to 12 days after the infection (dpi) with 50 bloodstream-form trypomastigotes of the Tulahuén strain, mice showed an enhanced susceptibility to the parasite. Specific IgG, but not IgM responses, were inhibited in anti-CD4-treated and infected mice. However, when anti-CD4 treatment of mice was delayed until the 8th week of infection, neither a reactivation of the infection as determined by mortality or parasitaemia, nor a modulation of the titre of anti-T. cruzi IgG antibodies was detected. Furthermore, mice chronically infected with T. cruzi and deprived of CD4+ T cells resisted the challenge with 50,000 trypomastigotes (approximately 1000 LD50). Secondary antibody responses against parasite antigens were inhibited after in vitro depletion of CD4+ cells in chronically infected mice before boosting with T. cruzi antigens. However, recipients of CD4 or T-cell-depleted spleen cells from mice chronically infected with T. cruzi were protected when challenged with the parasite. The possibility that the parasite control is maintained by long-lived B cells capable of rapid differentiation into IgG-secreting plasma cells in the absence of T helper cells is discussed considering the present data.     

Evidence for Multiple B- and T-Cell Epitopes in Plasmodium falciparum Liver-Stage Antigen 3

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.Preerythrocytic malaria antigens are critical targets of protective immune responses induced by irradiated sporozoites in humans (9, 22). The demonstration of T-cell-mediated protection in mice immunized by this means (10, 16), the acquisition of a significant level of protection against homologous Plasmodium falciparum challenge in human volunteers (22), and the induction by liver-stage antigens of a high level of protection against P. falciparum infection in chimpanzees (19, 31) all point to a major role for preerythrocytic stage antigens as vaccine candidates.Liver-stage antigen 3 (LSA-3) is a novel antigen expressed at the preerythrocytic stages (4). LSA-3 was selected by the differential immune response found between protected and nonprotected volunteers, both similarly immunized with irradiated sporozoites (4, 9). The gene encoding LSA-3 is unusually well-conserved (4), in contrast with many other malaria vaccine candidates (11, 19, 23). More than 10 dominant T-helper (Th), cytotoxic T-lymphocyte, and B-cell epitopes have already been characterized in LSA-3 (20), some of them displaying cross-reactivity with an homologous antigen in Plasmodium yoelii (2). The protective potential of LSA-3 was demonstrated by a series of experiments in chimpanzees and Aotus monkeys challenged with P. falciparum (4, 21) and in mice challenged by P. yoelii following immunization either by recombinant proteins with adjuvant or by formulations without adjuvant, such as recombinant proteins adsorbed on microparticles or lipopeptides in phosphate-buffered saline (PBS) (4), or DNA-based immunization (29). These convergent results stress the potential of LSA-3 as a prime vaccine candidate.We therefore decided to further analyze the antigenicity of LSA-3 and to investigate immune responses to discrete regions of the protein in exposed individuals living in areas where malaria is endemic.T- and B-cell responses were evaluated in subjects living in two villages in Senegal, West Africa, where malaria is endemic, using three small synthetic peptides and a series of 17 overlapping long synthetic peptides (LSP) encompassing most of the LSA-3 protein. In keeping with preliminary results (20), we found a high prevalence of responses to most regions of this preerythrocytic stage antigen in individuals of different age groups. These results bring additional arguments in favor of the potential of the LSA-3 protein for vaccine development.     

Antibodies to Both Terminal and Internal B-Cell Epitopes of Francisella tularensis O-Polysaccharide Produced by Patients with Tularemia

Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals.