Epitope Profiles Research Articles

Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method

To characterize single-cell-derived mouse clonal mesenchymal stem cells (mcMSCs) established with bone marrow samples from three different mouse strains. We established mcMSC lines using subfractionation culturing method from bone marrow samples obtained from long bones. These lines were characterized by measuring cell growth, cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability. Nonclonal MSCs isolated by the conventional gradient centrifugation method were used as controls. All mcMSC lines showed typical nonclonal MSC-like spindle shape morphology. Lines differed in optimal growth density requirement. Cell surface epitope profiles of these mcMSC lines were similar to those of nonclonal MSCs. However, some lines exhibited different expression levels in a few epitopes, such as CD44 and CD105. Differentiation assays showed that 90% of the mcMSC lines were capable of differentiating into adipogenic and/or chondrogenic lineages, but only 20% showed osteogenic lineage differentiation. T-cell suppression analysis showed that 75% of the lines exhibited T-cell suppression capability. mcMSC lines have similar cell morphology and cell growth rate but exhibit variations in their cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability.

Prevalence and epitope specificity of non-neutralising antibodies in a large cohort of haemophilia A patients without inhibitors

Antibodies (inhibitors and non-neutralising antibodies [NNA]) directed against factor VIII (FVIII) remain the main iatrogenic complication in haemophilia A (HA) patients. Inhibitors reduce FVIII pro-coagulant properties, whereas NNA are directed against non-functional epitopes. NNA are poorly studied and their prevalence, epitope specificity and physiopathology inadequately defined. The aim of this study was first to evaluate NNA prevalence in a French retrospective multicentric series of 210 patients without inhibitors, then to determine their epitope specificity (against the heavy chain [HC] or the light chain [LC] of FVIII) and particularly to assess the prevalence of anti-B domain NNA using specifically designed x-MAP assays. NNA occurred in 18.1% of patients (38/210) and their prevalence was not influenced by the severity of the disease. Among the 38 patients with NNA, 73.7% had anti-FVIII Abs against the HC, 13.2% against the LC and 13.2% had anti-FVIII Abs against both chains. There is thus a clear immuno-dominance of the HC of FVIII in the epitope profile of NNA, whatever the severity of HA. The proportion of NNA that recognised the B domain was 18.4% (n=7/38). A multivariate analysis did not highlight differences in NNA occurrence between patients treated with recombinant FVIII or with plasma- derived FVIII (19.6% vs. 14.9%, p=0.53).

Antibody responses elicited through homologous or heterologous prime-boost DNA and protein vaccinations differ in functional activity and avidity

Using a gp120 envelope glycoprotein from the JR-FL strain of human immunodeficiency virus-1 (HIV-1) as a model antigen, the goal of the current study was to evaluate the level and quality of antibody responses elicited by different prime-boost vaccination regimens (protein only, DNA only, DNA plus protein) in rabbits. Our data demonstrated that incorporating DNA immunization as a prime in a heterologous prime-boost regimen was able to elicit a more diverse and conformational epitope profile, higher antibody avidity, and improved neutralizing activity than immunization with only protein. Additionally, this improved neutralizing activity was observed in spite of similar antibody specificities and avidities seen when only DNA vaccination was used, providing additional evidence that the use of a combination immunization regimen increases the protective antibody response. Insights gained from the current study confirmed that the heterologous DNA prime-protein boost approach is effective in eliciting not only high level but also improved quality of antigen-specific antibody responses, and thus may offer a new technology platform to develop better and safer subunit vaccines.

Immune response towards the amino-terminus of desmoglein 1 prevails across different activity stages in nonendemic pemphigus foliaceus

Pemphigus foliaceus (PF) is a blistering skin disease mediated by antibodies to desmoglein (Dsg) 1. The two major subtypes are nonendemic and endemic PF. A previous study in endemic PF demonstrated that changes in antibody epitope could modulate disease relapse and remission. To characterize the frequency of immunoreactivity to various Dsg1 extracellular (EC) domains in nonendemic PF and to study if there is any change in epitope profile across various activity stages. Sera from 34 patients with nonendemic PF were selected. To map the conformational epitopes by immunoprecipitation-immunoblotting, we constructed five Dsg1/Dsg2 domain-swapped molecules, with each molecule representing one EC domain of Dsg1 on a backbone of Dsg2. Dsg1 EC1, EC2, EC3, EC4 and EC5 domains were recognized by 88%, 50%, 13%, 22% and 0% of active PF sera, respectively. Immunoreactivity to EC3 or EC4 often cosegregated with that to either EC1 or EC2. Longitudinal follow-up of 21 patients with PF for a median of 16 months revealed that, in most cases, immunoreactivity to the amino-terminus of Dsg1 persisted across various activity stages; only two patients lost their EC1 reactivity upon remission and changed their major epitope(s) to EC2 ± EC3. Most of the anti-Dsg1 antibodies in nonendemic PF bind to the amino-terminus of Dsg1, a region critical for intercellular adhesion of cadherins, and this skewed amino-terminal immunoreactivity prevails across various activity stages in most patients, even upon remission. These findings are valuable for understanding the biology of Dsg-mediated cellular adhesion as well as for the development of epitope-based monitoring and therapeutic strategies.

Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120

CD4(+) helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4(+) T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this work, three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the local three-dimensional structure and enhance the presentation of nearby weakly immunogenic epitopes. However, upon immunization of groups of BALB/c mice, the CD4(+) T-cell response was broadly reduced for all three variants, and distinct epitope profiles emerged. For one variant, antibody titers were sharply increased, and the antibody exhibited significant CD4-blocking activity.

The genetic backbone modulates the phenotype of hepatitis B surface antigen mutants

Hepatitis B virus (HBV) vaccine and diagnostic escape mutants are a growing concern. The principle target of detection, hepatitis B surface antigen (HBsAg), encoded by S, is completely overlapped by the reverse transcriptase encoding P. With the increased incidence of nucleos(t)ide analogue resistance altering P, the concurrent impact on S must be assessed. HBV DNA from 59 HBsAg-positive plasma samples was sequenced across the polymerase/surface region and the amino acid sequence of HBsAg was inferred. ELISAs were formatted containing individually bound monoclonal antibodies directed against three discrete epitopes on HBsAg. Similar point mutations occurring in different genotypes were shown to influence epitope conformation differently, indicating that the genetic backbone is a major factor in predicting phenotype. C-terminal changes associated with antiviral resistance were found to modulate epitope profiles of HBsAg. Treatment options which may promote drug resistance should be avoided to both protect antiviral treatment and prevent facilitation of vaccine and diagnostic escape mutants.

Phage display selection, analysis, and prediction of B cell epitopes.

Combinatorial phage display libraries of random peptides can be used to discover the epitopes of antibodies through a procedure termed "biopanning." The affinity isolation of phage-displayed epitope peptidomimetics allows molecular definition of the epitopes of monoclonal antibodies (MAbs). Panels of MAb-specific peptides allow computational prediction of B cell epitopes. Epitope profiles recognized by polyclonal serum samples can also be generated. Detailed step by step protocols and discussion of applications are provided.

Mesenchymal progenitor cells derived from traumatized human muscle

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate cell type for cell-based tissue engineering and regenerative medicine. Currently, clinical applications for MSCs require additional surgical procedures to harvest the autologous MSCs (i.e. from bone marrow) or commercial allogeneic alternatives. We have recently identified a population of mesenchymal progenitor cells (MPCs) in traumatized muscle tissue that has been surgically debrided from traumatic orthopaedic extremity wounds. The purpose of this study was to evaluate whether MPCs derived from traumatized muscle may provide a clinical alternative to bone-marrow MSCs, by comparing their morphology, proliferation capacity, cell surface epitope profile and differentiation capacity. After digesting the muscle tissue with collagenase, the MPCs were enriched by a direct plating technique. The morphology and proliferation rate of the muscle-derived MPCs was similar to bone-marrow derived MSCs. Both populations expressed cell surface markers characteristic for MSCs (CD 73, CD 90 and CD105), and did not express markers typically absent on MSCs (CD14, CD34 and CD45). After 21 days in specific differentiation media, the histological staining and gene expression of the MPCs and MSCs was characteristic for differentiation into osteoblasts, chondrocytes and adipocytes, but not into myoblasts. Our findings demonstrate that traumatized muscle-derived MPCs exhibit a similar phenotype and resemble MSCs derived from the bone marrow. MPCs harvested from traumatized muscle tissue may be considered for applications in tissue engineering and regenerative medicine following orthopaedic trauma requiring circumferential debridement.

The epitope characterisation and the osteogenic differentiation potential of human fat pad-derived stem cells is maintained with ageing in later life

Some clinical settings are deficient in osteogenic progenitors, e.g. atrophic nonunited fractures, large bone defects, and regions of scarring and osteonecrosis. These benefit from the additional use of bone marrow-derived mesenchymal stem cells, but these cells exhibit an age-related decline in lifespan, proliferation and osteogenic potential. Therapeutic approaches for the repair of bone could be optimised by the identification of a stem cell source that does not show age-related changes. Fat pad-derived stem cells are capable of osteogenesis, but a detailed study of the effect of ageing on their epitope profile and osteogenic potential has so far not been performed. Fat pad-derived cells were isolated from 2 groups of 5 patients with a mean age of 57 years (S.D. 3 years) and 86 years (S.D. 3 years). The proliferation, epitope profile and osteogenic differentiation potential of cells from the 2 groups were compared. Cells isolated from the fat pad of both groups showed similar proliferation rates and exhibited a cell surface epitope profile similar but not identical to that of bone marrow-derived stem cells. The cells from both groups cultured in osteogenic medium exhibited osteogenesis as shown by a significant upregulation of alkaline phosphatase and osteocalcin genes, and significantly greater alkaline phosphatase enzyme activity compared to cells cultured in the control medium. The cells cultured in the osteogenic medium also showed greater calcium phosphate deposition on alizarin red staining. There was no significant difference between the osteogenic potential of the two age groups for any of the parameters studied. The fat pad is a consistent and homogenous source of stem cells that exhibits osteogenic differentiation potential with no evidence of any decline with ageing in later life. This has many potential therapeutic tissue engineering applications for the repair of bone defects in an increasingly ageing population.

The use of factor VIII/von Willebrand factor concentrate for immune tolerance induction in haemophilia A patients with high‐titre inhibitors: association of clinical outcome with inhibitor epitope profile

A retrospective chart review of 11 subjects with severe haemophilia A and high-titre inhibitors who received a von Willebrand factor-containing FVIII concentrate (VWF/FVIII) for immune tolerance induction (ITI) was accompanied by B cell inhibitor epitope mapping during 10/11 treatment courses. ITI was successful or partially successful in all seven subjects who received VWF/FVIII for initial ITI, and failed in all four subjects whose ITI with this product was initiated following treatment failure using recombinant factor VIII. Variables including age at inhibitor development and age at ITI initiation, interval between inhibitor detection and ITI initiation, titre at start of ITI, and peak historical titres prior to and during ITI were not statistically significant outcome predictors in this cohort. However, the B cell epitope specificity in all four successful and in one of two partially successful ITI subjects for whom information was available included the C2 and excluded the A2 domains. Conversely, FVIII B cell epitopes in one partially successful ITI and in all three failed ITI subjects for whom data were available mapped to both the C2 and the A2 domains. The FVIII B cell epitope profile was associated with ITI outcome in this VWF/FVIII-treated cohort. Its role in predicting ITI outcome and guiding choice of FVIII product for ITI requires further study.

Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques

RhD discrepancies between current and historical results are problematic to resolve. The investigation of 10 discrepancies is reported here. Samples identified were those that reacted by automated gel technology and were negative with an FDA-approved reagent. Reactivity with a commercially available panel of monoclonal anti-D was performed. Genomic DNA was evaluated for RHD alleles with multiplex RHD exon polymerase chain reaction (PCR), weak D PCR-restriction fragment length polymorphism, and RHD exon 5 and 7 sequence analyses. The monoclonal anti-D panel identified two samples as DVa, yet possessed the DAR allele. Two weak D Type 1 samples had a similar monoclonal anti-D profile, but only one reacted directly with one of two FDA-approved anti-D. Only two of four weak D Type 2 samples reacted directly with one FDA-approved anti-D, and their D epitope profile differed. The monoclonal anti-D reagents did not distinguish between partial and weak D Types 1 and 2. Weak D Types 1 and 2 do not show consistent reactivity with FDA-approved reagents and technology. To limit anti-D alloimmunization, it is recommended that samples yielding an immediate-spin tube test cutoff score of not more than 5 (i.e., < or =1+ agglutination) or a score of not more than 8 (i.e., < or =2+ hemagglutination) by gel technology be considered D- for transfusion and Rh immune globulin prophylaxis. That tube test anti-D reagents react poorly with some Weak D Types 1 and 2 red cells is problematic, inasmuch as they should be considered D+ for transfusion and prenatal care. Molecular tests that distinguish common partial and Weak D types provide the solution to resolving D antigen discrepancies.

Identification of Three Non-Overlapping Epitopes in the C2 Domain of Factor VIII.

Factor VIII (fVIII) inhibitory antibodies (fVIII inhibitors) are a significant source of morbidity in patients with hemophilia A. Approximately 30% of patients with severe hemophilia A will develop inhibitors. Most inhibitors are directed against either the A2 or C2 domains of fVIII. The repertoire of antibodies to the C2 domain is functionally complex, including antibodies that inhibit fVIII binding to phospholipid and von Willebrand factor and display either type I or type II kinetics.The goal of this study was to investigate the diversity of the immune response to the C2 domain of human fVIII in a murine hemophilia A model and to identify a set of non-overlapping epitopes recognized by these fVIII inhibitors. A panel of 55 murine anti-C2 antibodies was obtained, which included 53 antibodies from anti-fVIII hybridomas produced in our laboratory and previously described antibodies ESH-4 and ESH-8. Nine of the hybridomas were cloned by limiting dilution and the corresponding monoclonal IgG antibodies were purified. IgG from the remaining 44 hybridomas was isolated directly from the hybridoma supernatants and biotinylated. The 9 monoclonal antibodies along with ESH-4 and ESH-8 were used as primary antibodies in a competition ELISA using human fVIII as the antigen. The panel of 55 biotinylated antibodies was used as secondary antibodies. Antibody pairs were classified as having non-overlapping or overlapping epitopes based on whether the binding of the secondary antibody was present or absent, respectively.A basis set of 3 antibodies, 1B5-1B, 3E6-1B, and (2)117-1B, was defined, which consisted of a set of non-overlapping antibodies that as a group competed for binding of human fVIII with all other antibodies. 1B5-1B and 3E6-1B exhibited type I kinetics with Bethesda titers of 950 and 30, respectively. The Bethesda titer of (2)117-1B could not be determined due to its type II behavior. ESH-4 and ESH-8 had the same epitope profiles as 3E6-1B and (2)117-1B, respectively. Of the 52 non-basis set antibodies, 85% overlapped with 1B5-1B, 29% with 3E6-1B, and 56% with (2)117-1B. The majority of the antibodies overlapped with more than one member of the basis set, leading to the identification of five classes of antibodies (see figure). Because of the large number of antibodies characterized, it is unlikely that the frequency of antibodies in any additional classes is high. The elucidation of the structural complexity of the anti-fVIII C2 repertoire should be useful in the characterization of the pathogenicity of C2 inhibitors. [Display omitted]

Reduced Immunogenicity of β-Lactoglobulin by Conjugating with Chitosan

Bovine beta-lactoglobulin (beta-LG) was conjugated with chitosan (CHS) by means of a water-soluble carbodiimide to reduce the immunogenicity of beta-LG. Each beta-LG-CHS conjugate was purified by ion-exchange chromatography and hydrophobic chromatography. The conjugation between beta-LG and CHS was confirmed by SDS-PAGE, the isoelectric point of the conjugate being higher than that of beta-LG. Two types of the beta-LG-CHS conjugate were obtained with molar ratios of beta-LG to CHS of 1:1 (F1) and 1:2 (F2). Structural analyses by fluorescence measurement, ELISA with monoclonal antibodies and retinol-binding activity indicated that the conjugates had almost maintained the native structure of beta-LG. The antigenicity of the beta-LG-CHS conjugates was similar to that of beta-LG in C3H/He mice. Reduction of the immunogenicity of beta-LG was achieved by conjugation with CHS. In particular, F2 showed very low immunogenicity. B cell epitopes of beta-LG and the conjugates recognized in C3H/He mice were determined with 15-mer multi-pin peptide; the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, while the antibody response to each epitope was dramatically reduced. Conjugation of beta-LG with chitosan was effective for reducing the immunogenicity of beta-LG.

The RHCE allele ceCF: the molecular basis of Crawford (RH43)

The Crawford antigen (RH43) was described in 1980. It occurred in African American people, as a low-prevalence Rhesus antigen, who were also VS+. Twelve blood samples were analyzed because of inquiries into discrepant reactions in routine anti-D typing. The RHCE alleles were determined by nucleotide sequencing from genomic DNA. The D epitope profile was determined with 60 monoclonal anti-D. The population frequency was estimated in four major US regional blood centers. The novel RHce(W16C, Q233E, L245V) allele, dubbed ceCF, was found to be occurring in the cde haplotype as cause of the reactivity with the immunoglobulin M anti-D GAMA401. The ceCF phenotype expressed few D epitopes resembling but not matching the reaction patterns observed with other RhCE variants, like R0 (Har), ceRT, and ceSL. The frequency of the ceCF phenotype was 0.056 percent among African American persons and 0.007 percent in the general US population. The novel RHce(W16C, Q233E, L245V) allele, which is a variant of the known ce(s) allele, RHce(W16C, L245V), occurs in a haplotype with the RHD deletion and represents the molecular basis of the Crawford antigen.

The RHCE allele ceSL: the second example for D antigen expression without D‐specific amino acids

The example of ceRT proved that the expression of some D epitopes does not require D-specific amino acids. This allele denoted as RHce(R154T) caused the "false-positive" reactions that were observed in ccddee blood donors who typed positive for the D antigen with some monoclonal anti-D. No other example exposing a similar molecular mechanism was known. Eleven donor and 1 patient ccddee samples were collected in Switzerland that typed "false-positive" with some monoclonal anti-D in bromelain technique. Their RHCE alleles were determined by nucleotide sequencing from genomic DNA and by a polymerase chain reaction with sequence-specific priming. The D epitope profile was compared to ceRT. The population frequencies were estimated in Switzerland and Germany by serology or at the molecular level, respectively. The "false-positive" reactions were caused by the RHCE allele RHce(S122L) occurring in the cde haplotype. Its ceSL phenotype expressed few D epitopes that belonged to the D epitope 6 group. The frequency of ceSL among D- donors was about 1:675 in the region of Bern, Switzerland. No ceSL donors were found elsewhere in Switzerland or in southwestern Germany. ceSL represented the second molecular mechanism for D antigen expression without any D-specific amino acids. ceSL and ceRT were useful to delineate the molecular mechanisms of D expression by RhCE proteins carrying amino acids not representative for the RhD proteins. The ceSL population frequencies differed significantly among three Swiss and German populations.

Epitope Mapping of Der p 2 by Site-directed Mutagenesis: Differential IgE Binding Epitope Profiles among Individuals Sensitized to Only Dermatophagoides spp. and those with Non-pyroglyphid Mite Responses

Epitope Mapping of Der p 2 by Site-directed Mutagenesis: Differential IgE Binding Epitope Profiles among Individuals Sensitized to Only Dermatophagoides spp. and those with Non-pyroglyphid Mite Responses

Recent Developments with B-Cell Epitope Identification for Predictive Studies

This review discusses currently available methods for predicting B-cell epitopes on proteins. The use of animals for assessing protein immunogenicity is addressed primarily to highlight the differences in B- and T-cell epitope recognition between species. These differences have to be considered when interpreting potential B-cell epitopes identified by the methods addressed here. “In vitro alternatives” focuses on the strengths and limitations of peptide-based technologies. Three types of computer-based methods for identifying potential B-cell epitopes are discussed: (i) methods applying physico-chemical and structural propensity scales for predicting linear epitopes from the primary structure of a protein, (ii) comparative methods basing prediction upon amino acid sequence and structural similarities between antigenically known and unknown proteins, and (iii) a method combining structural features with a B-cell epitope motif database for predicting linear and conformational antigenic determinants. With respect to human safety, the usefulness of antibody-based tests is limited to comparative studies between an antigenically known protein and variants thereof. Similarly, computer-based methods using data mining can address similarities in B-cell epitope profiles between related proteins, if a proper cut off can be defined for the minimal amino acid sequence similarity required for obtaining an acceptable accuracy. Among the physico-chemical and structural scales, scales identifying in a protein hairpin and non-specific turns seem useful for predicting epitopes with a continuous primary binding site. When conformational epitopes have to be identified as well, a novel computer-based tool seems to be the most promising alternative to X-ray crystallography. However, both methods remain to be extensively evaluated and validated. Thus, promising tools for B-cell epitope identification have been developed. But, no validated method for B-cell epitope identification on antigenically unknown proteins is available yet.

Mapping of FVIII inhibitor epitopes using cellulose-bound synthetic peptide arrays

Epitope mapping using antibodies against factor VIII (FVIII) has been performed using blotting techniques with truncated and/or digested FVIII molecules. Here, we focused on the precise mapping of affinity purified IgG from patients with an immune response against blood clotting FVIII using synthetic peptide arrays on cellulose membranes comprising the entire sequence of FVIII. The aim was to elucidate the epitope profile from different inhibitors and possibly detect new epitopes, which have not been described before. The epitope patterns from five patients showed reactivity with all domains in the FVIII molecule, but were different between various patients. These results included epitopes usually buried within the folded protein. However, in competition assays using FVIII as competitive agent in a mixture with inhibitor IgG, the most immunogenic regions were located in the FVIII light chain. Our results show that the C1 domain was the region with highest immunogenicity in all patients. Here, we demonstrate that the SPOT method is very well suited for the precise location of epitopes in the core of the protein, which usually cannot be detected by other methods.

Comparison of human stem cells derived from various mesenchymal tissues: Superiority of synovium as a cell source

To compare the properties of human mesenchymal stem cells (MSCs) isolated from bone marrow, synovium, periosteum, skeletal muscle, and adipose tissue. Human mesenchymal tissues were obtained from 8 donors during knee surgery for ligament injury. After collagenase digestion or gradient-density separation, nucleated cells were plated at an appropriate density for expansion at the maximum rate without colony-to-colony contact. Yield, expandability, differentiation potential, and epitope profile were compared among MSCs from the 5 different tissue sources. Colony number per 10(3) nucleated cells was lower, and cell number per colony was higher, in bone marrow than in other mesenchymal tissues. When the cells were replated at low density every 14 days, bone marrow-, synovium-, and periosteum-derived cells retained their proliferation ability even at passage 10. In chondrogenesis studies in which the cells were pelleted and cultured in vitro, pellets from bone marrow-, synovium-, and periosteum-derived cells were shown to be larger and stained more extensively for cartilage matrix. Synovium-derived cells, in particular, had the greatest ability for chondrogenesis. In adipogenesis experiments, the frequency of oil red O-positive colonies was highest in synovium- and adipose tissue-derived cells. In studies of osteogenesis, the rate of alizarin red-positive colonies was highest in bone marrow-, synovium-, and periosteum-derived cells. For epitope profiling, 15 surface antigens were measured. Most appeared to have similar epitope profiles irrespective of cell source. Our findings indicate that there are significant differences in MSC properties according to tissue source, beyond donor and experimental variation. Superiority of synovium as a potential source of MSCs for clinical applications was demonstrated.

Epitope mapping of Mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes

Phage display techniques have been widely employed to map the epitope structures which served as the basis for developing molecular vaccines. In the present study, we applied this technique to map the epitopes of Mycoplasma hyopneumoniae, the etiologic agent causing swine enzootic pneumonia, and evaluated directly the immune responses in mice of the selected phage-displayed epitopes (phagotopes). Two phage-displayed random peptide libraries were biopanned with the protein A-purified IgG of the rabbit anti- M. hyopneumoniae hyperimmune serum and the selected phage clones were sequenced and analyzed. Some of the inserts of the selected phagotopes showed a good match with the known proteins of M. hyopneumoniae. Others, which did not match with any known proteins, but shared extensive homology with each other, were clustered and classified as the conformational epitopes of M. hyopneumoniae. To evaluate the potential of using these phagotopes as effective vaccines, several phage clones were chosen to immunize mice. IgA coproantibody, IgA in bronchoalveolar lavage fluid and serum IgG responses were assayed. The serum raised by the phage clones clearly recognized several major mycoplasmal proteins indicating that the phagotope-induced immune responses were antigen-specific. The stronger IgG1 response revealed that the immune responses of the epitope-displaying phage were mainly through Th2 activation. The growth inhibition assay showed that the selected phage clones CS4 and ϕ58 are potential vaccine candidates and suggested that the mycoplasmal 97 kDa, 56 kDa, 30 kDa and 23 kDa proteins may play important roles in the immune responses. The present work demonstrates that the whole epitope profile of a microorganism can be obtained through screening the phage displayed peptide libraries with the hyperimmune serum and reveals the potential of using epitope-displaying phages as peptide vaccines.