Development Of Peptide Vaccines Research Articles

COBEpro: a novel system for predicting continuous B-cell epitopes

Accurate prediction of B-cell epitopes has remained a challenging task in computational immunology despite several decades of research. Only 10% of the known B-cell epitopes are estimated to be continuous, yet they are often the targets of predictors because a solved tertiary structure is not required and they are integral to the development of peptide vaccines and engineering therapeutic proteins. In this article, we present COBEpro, a novel two-step system for predicting continuous B-cell epitopes. COBEpro is capable of assigning epitopic propensity scores to both standalone peptide fragments and residues within an antigen sequence. COBEpro first uses a support vector machine to make predictions on short peptide fragments within the query antigen sequence and then calculates an epitopic propensity score for each residue based on the fragment predictions. Secondary structure and solvent accessibility information (either predicted or exact) can be incorporated to improve performance. COBEpro achieved a cross-validated area under the curve (AUC) of the receiver operating characteristic up to 0.829 on the fragment epitopic propensity scoring task and an AUC up to 0.628 on the residue epitopic propensity scoring task. COBEpro is incorporated into the SCRATCH prediction suite at http://scratch.proteomics.ics.uci.edu.

A phase I study to evaluate safety, immunogenicity and antitumor activity of a HER2 multi-peptide virosome vaccine in patients with metastatic breast cancer

3055 Background: Anti-HER2 directed immunotherapy has proven to be beneficial in patients with HER2 overexpressing breast cancer. This makes HER2 an attractive target for a therapeutic vaccine. We have shown that vaccination of rodents with three HER2-peptides representing B-cell epitopes of the extracellular domain of HER2 can induce elevated HER2-specific IgG levels and result in a delayed tumor onset and reduced tumor growth progression in c-neu transgenic mice. We have now initiated a phase I clinical trial with an anti-HER2 vaccine- construct of immunopotentiating reconstituted influenza virosomes with the three peptides (PEV6A) in patients with metastatic breast cancer (MBC). Methods: 9 patients with MBC were enrolled in a single arm, single-centre study. The endpoints were safety, production of peptide- and HER2-specific antibodies, and T-cell responses. Patients were required to have low HER2 expressing tumors (+ or ++) and positive hormone receptors. All patients had stable disease at the time of study entry and continued with their anti-hormonal treatment until progression. Patients received 3 intramuscular vaccinations in monthly intervals each consisting of PEV6A containing 10ug of each peptide. Results: All possibly drug-related adverse events were mild to moderate. No patient experienced any possibly drug-related serious adverse events. Injection site reactions such as erythema and edema were the most frequent possibly drug-related AEs. One patient died one month after the last vaccination due to disease progression. All the remaining 8 patients are alive, 6 are stable for a period of up to 12 months and 2 had disease progression after 5 and 7 months respectively. In seven patients immunogenicity was proven by solid peptide specific antibody responses. Conclusions: PEV6A is safe, very well tolerated and immunogenic. The production of HER2-peptide specific antibody could imply clinical benefit comparable to passive anti-HER2 antibody therapy. These data clearly support the further development of virosomal peptide vaccines to induce an antitumor antibody immune response. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration BioLife-Science Inc., Vienna, Pevion Biotech Ltd. BioLife-Science Inc., Vienna, Pevion Biotech Ltd. BioLife-Science Inc., Vienna BioLife-Science Inc., Vienna, Pevion Biotech Ltd., Pevion Biotech Ltd. Berne

Cancer antigen WT1-targeting treatment for the malignancies -Development of WT1 peptide vaccine-

Wilm's tumor gene WT1, which has an oncogenic function, is expressed in various kinds of hematological malignancies and solid cancers. WT1 antibodies at higher titers and WT1-specific cytotoxic T lymphocytes (CTLs) at higher frequencies were detected in cancer patients than in healthy donors, indicating that WT1 protein was immunogenic. Furthermore, WT1-specific immune responses are considered to be involved with Graft versus Leukemia effect in the context of hematopoietic stem cell transplantation. These findings provided us with the rationale for cancer immunotherapy targeting WT1. Clinical trials of WT1 peptide vaccination for cancer patients were started, and WT1 vaccination-driven immunological responses and clinical responses, including reduction of leukemic cells, reduction of M-protein amount in myeloma, and shrinkage of solid cancer, were observed. Further enhancement of efficacy of WT1 peptide vaccine can be expected by co-administration of WT1-specific helper peptide or anti-cancer chemotherapy agent. WT1 peptide vaccination in the setting of MRD (minimal residual disease) may prolong "progression-free survival time", or decrease "relapse rate".

Design and development of synthetic peptide vaccines: past, present and future

Synthetic peptide vaccines aiming at the induction of a protective CD8+ T-cell response against infectious or malignant diseases are widely used in the clinic but, despite their success in animal models, they do not yet live up to their promise in humans. This review assesses the development of synthetic peptide vaccines, weighs it against the immunological concepts that have emerged, and identifies the key issues that play a role in the failure or success of a synthetic peptide vaccine. The current state-of-the-art peptide vaccine is a complete synthetic inflammatory product that is ingested by professional antigen-presenting cells and stimulates both CD4+ and CD8+ T cells.

Development of antibody-targeted peptide vaccines as novel reagents for combination immunotherapy in cancer patients

Development of antibody-targeted peptide vaccines as novel reagents for combination immunotherapy in cancer patients

Prediction of continuous B‐cell epitopes in an antigen using recurrent neural network

B-cell epitopes play a vital role in the development of peptide vaccines, in diagnosis of diseases, and also for allergy research. Experimental methods used for characterizing epitopes are time consuming and demand large resources. The availability of epitope prediction method(s) can rapidly aid experimenters in simplifying this problem. The standard feed-forward (FNN) and recurrent neural network (RNN) have been used in this study for predicting B-cell epitopes in an antigenic sequence. The networks have been trained and tested on a clean data set, which consists of 700 non-redundant B-cell epitopes obtained from Bcipep database and equal number of non-epitopes obtained randomly from Swiss-Prot database. The networks have been trained and tested at different input window length and hidden units. Maximum accuracy has been obtained using recurrent neural network (Jordan network) with a single hidden layer of 35 hidden units for window length of 16. The final network yields an overall prediction accuracy of 65.93% when tested by fivefold cross-validation. The corresponding sensitivity, specificity, and positive prediction values are 67.14, 64.71, and 65.61%, respectively. It has been observed that RNN (JE) was more successful than FNN in the prediction of B-cell epitopes. The length of the peptide is also important in the prediction of B-cell epitopes from antigenic sequences. The webserver ABCpred is freely available at www.imtech.res.in/raghava/abcpred/.

Peptide vaccines against arthritis

In this article, four distinct approaches to the development of peptide vaccines against arthritis will be reviewed. One is based on evidence that antagonizing proinflammatory cytokines can benefit patients with arthritis. The second stems from the observation that heat-shock proteins are essential arthritogenic agents in the development of arthritis. The third derives from research on the mechanisms by which specific HIV-1 peptides produce T-cell suppression, while the fourth has developed from clinical reports that children with juvenile rheumatoid arthritis (Still’s disease) experience long-term remission following infection with the measles virus. The four sets of anti-arthritis peptides bear no sequence similarities but have all been tested successfully on adjuvant arthritis models, thereby providing multiple paths to the development of peptide vaccines against arthritis. Specific comparisons between methods of delivery, adjuvant requirements and dosages are reported and future directions for researc...

Prospects for Vaccines for Allergic and Other Immunologic Skin Disorders

The human skin hosts a variety of immune response-associated components that together form the skin immune system. Any abnormality in the functioning of the skin immune system leads to a variety of dermatologic complications, including dermatitis, psoriasis, and eczema. Exposure to antigens/allergens can lead to allergic skin disorders such as atopic dermatitis, urticaria, and allergic contact dermatitis. Recent investigations have provided new insights into the immunologic processes leading to the development of skin diseases. T cells play a central role in the activation and regulation of immune responses by recognizing antigen and inducing cytokine production. Despite advances in the understanding of the immunologic events leading to the development of skin diseases, no effective prevention measure exists. Current therapeutic treatments are based on either alleviating the symptoms or suppressing the immune system with immunosuppressive drugs. Allergen-specific immunotherapy is expected to induce specific T cells that abolish allergen-induced proliferation of T helper cells, as well as their cytokine production. Recent approaches using recombinant protein, polycytosine guanine oligonucleotides, and plasmid DNA for vaccination suggest the possibility of protection against these skin disorders. The involvement of T cells in psoriasis indicates that the development of a T-cell receptor peptide vaccine may be beneficial. Dendritic cell-based vaccines using tolerogenic dendritic cells that can induce T-cell tolerance have been shown to be useful in dealing with autoimmune disorders and allergic conditions. In the light of these developments, this article presents the current status and prospects of developing vaccines for allergic and other immunologic skin disorders.

An ingenious design for peptide vaccines

For humoral immunization, it may be possible to make effective and safe peptide vaccines for various diseases by selection of proper B-cell epitopes. However, a lack of T-cell epitopes on short peptides, such as those associated with major histocompatibility complex (MHC)-restriction, is a major problem for peptide vaccine development. We propose a solution for the design of peptide vaccines that involves induction of broadly reactive T-cell epitopes via agretopes. The strategy involves positioning multi-agretope type peptides on the N-terminal side of a di-lysine linker and B-cell epitopes on the C-terminal side. The addition of the arginine–glysine–aspartate (RGD)-motif to the N terminus of the peptide enhances its immunogenicity, and enables nasal immunization without adjuvants.

Allergy and Dermatophytes

Tinea pedis (athlete's foot) and onychomycosis (infection of the toenails) caused by the dermatophyte fungus Trichophyton are highly prevalent in adults. Several Trichophyton allergens have been identified based on elicitation of immunoglobulin E antibody-mediated immediate-hypersensitivity (IH) responses. Evidence of an etiologic role for Trichophyton in asthma in some subjects with IH and chronic dermatophytosis is provided by bronchial reactivity to Trichophyton. Improvement of asthma after systemic antifungal treatment corroborates this link. A unique feature of Trichophyton allergens is the ability of the same antigen to elicit delayed-type hypersensitivity (DTH) in individuals who lack IH reactivity. Delayed responses appear to confer protection, while IH responses do not, based on the association with acute versus chronic skin infection. The amino acid sequence identity of Trichophyton allergens with diverse enzyme families supports a dual role for these proteins in fungal pathogenesis and allergic disease. Characterizing the immunologic properties of Trichophyton allergens and defining immune mechanisms which drive dichotomous responses are pivotal to understanding the dermatophyte-allergy relationship. Recent studies have identified DTH-associated major T-cell epitopes which could facilitate the development of peptide vaccines. Characterization of additional molecular targets by using new techniques may aid not only in the eradication of infection but also in the resolution of allergic symptoms.

Anchor profiles of HLA‐specific peptides: Analysis by a novel affinity scoring method and experimental validation

The study of intermolecular interactions is a fundamental research subject in biology. Here we report on the development of a quantitative structure-based affinity scoring method for peptide-protein complexes, named PepScope. The method operates on the basis of a highly specific force field function (CHARMM) that is applied to all-atom structural representations of peptide-receptor complexes. Peptide side-chain contributions to total affinity are scored after detailed rotameric sampling followed by controlled energy refinement. A de novo approach to estimate dehydration energies was developed, based on the simulation of individual amino acids in a solvent box filled with explicit water molecules. Transferability of the method was demonstrated by its application to the hydrophobic HLA-A2 and -A24 receptors, the polar HLA-A1, and the sterically ruled HLA-B7 receptor. A combined theoretical and experimental study on 39 anchor substitutions in FxSKQYMTx/HLA-A2 and -A24 complexes indicated a prediction accuracy of about two thirds of a log-unit in Kd. Analysis of free energy contributions identified a great role of desolvation and conformational strain effects in establishing a given specificity profile. Interestingly, the method rightly predicted that most anchor profiles are less specific than so far assumed. This suggests that many potential T-cell epitopes could be missed with current prediction methods. The results presented in this work may therefore significantly affect T-cell epitope discovery programs applied in the field of peptide vaccine development.

Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy.

A method has been developed for measurement of antibody affinity and cross-reactivity by surface plasmon resonance spectroscopy using the EK-coil heterodimeric coiled-coil peptide capture system. This system allows for reversible capture of synthetic peptide ligands on a biosensor chip surface, with the advantage that multiple antibody-antigen interactions can be analyzed using a single biosensor chip. This method has proven useful in the development of a synthetic peptide anti-Pseudomonas aeruginosa (PA) vaccine. Synthetic peptide ligands corresponding to the receptor binding domains of pilin from four strains of PA were conjugated to the E-coil strand of the heterodimeric coiled-coil domain and individually captured on the biosensor chip through dimerization with the immobilized K-coil strand. Polyclonal rabbit IgG raised against pilin epitopes was injected over the sensor chip surface for kinetic analysis of the antigen-antibody interaction. The kinetic rate constants, k(on) and k(off), and equilibrium association and dissociation constants, KA and KD, were calculated. Antibody affinities ranged from 1.14 x 10(-9) to 1.60 x 10(-5) M. The results suggest that the carrier protein and adjuvant used during immunization make a dramatic difference in antibody affinity and cross-reactivity. Antibodies raised against the PA strain K pilin epitope conjugated to keyhole limpet haemocyanin using Freund's adjuvant system were more broadly cross-reactive than antibodies raised against the same epitope conjugated to tetanus toxoid using Adjuvax adjuvant. The method described here is useful for detailed characterization of the interaction of polyclonal antibodies with a panel of synthetic peptide ligands with the objective of obtaining high affinity and cross-reactive antibodies in vaccine development.

Animal models of insulin-dependent diabetes.

Animal models have contributed enormously to study in the field of type 1 diabetes. Perhaps the most intensively studied model is the nonobese diabetic (NOD) mouse, which develops an autoimmune-mediated spontaneous diabetes associated with the development of insulin autoantibodies and insulitis. Accurate measurement of antiislet autoantibodies by radioassay and detection of antigen-specific T cells using major histocompatibility complex tetramers are possible. Various strategies have been developed in preventing diabetes in animal models; a peptide-induced model of type 1 diabetes has been described. Finally, the development of peptide vaccines is hampered by the risk of anaphylaxis in both mouse and humans. In this chapter, methods and strategies to measure antiinsulin autoantibodies, to detect antigen-specific T cells by tetramer analysis, and to prevent diabetes using peptide vaccines are discussed. Along with these topics, a protocol of peptide-induced diabetes and peptide vaccine-induced anaphylaxis are described, serving as a reminder of the potential dangers that could exist in human trials. In summary, animal models have become necessary in the study of type 1 diabetes and provide researchers important tools to conduct studies that could not otherwise be performed in humans.

Mimotopes of the Vi Antigen ofSalmonella entericaSerovar Typhi Identified from Phage Display Peptide Library

The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.

Combinatorial peptide library methods for immunobiology research.

The field of combinatorial peptide chemistry has emerged as a powerful tool in the study of many biological systems. This review focuses on combinatorial peptide library methodology, which includes biological library methods, spatially addressable parallel library methods, library methods requiring deconvolution, the "one-bead one-compound" library method, and affinity chromatography selection method. These peptide libraries have successfully been employed to study a vast array of cell surface receptors, as well as have been useful in identifying protein kinase substrates and inhibitors. In recent immunobiological applications, peptide libraries have proven monumental in the definition of MHC anchor residues, in lymphocyte epitope mapping, and in the development of peptide vaccines. Peptides identified from such libraries, when presented in a chemical microarray format, may prove useful in immunodiagnostics. Combinatorial peptide libraries offer a high-throughput approach to study limitless biological targets. Peptides discovered from such studies may be therapeutically and diagnostically useful agents.

Synthetic peptide vaccine and antibody therapeutic development: prevention and treatment of Pseudomonas aeruginosa.

Pseudomonas aeruginosa and Pseudomonas maltophilia account for 80% of opportunistic infections by pseudomonads. Pseudomonas aeruginosa is an opportunistic pathogen that causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa is notable for its resistance to antibiotics, and is therefore a particularly dangerous pathogen. Only a few antibiotics are effective against Pseudomonas, including fluoroquinolones, gentamicin, and imipenem, and even these antibiotics are not effective against all strains. The difficulty treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a strain that is so resistant that it cannot be treated. Since antibiotic therapy has proved so ineffective as a treatment, we embarked on a research program to investigate the development of a synthetic peptide consensus sequence vaccine for this pathogen. In this review article we will describe our work over the last 15 years to develop a synthetic peptide consensus sequence anti-adhesin vaccine and a related therapeutic monoclonal antibody (cross-reactive to multiple strains) to be used in the prevention and treatment of P. aeruginosa infections. Further, we describe the identification and isolation of a small peptide structural element found in P. aeruginosa strain K (PAK) bacterial pili, which has been proven to function as a host epithelial cell-surface receptor binding domain. Heterologous peptides are found in the pili of all strains of P. aeruginosa that have been sequenced to date. Several of these peptide sequences have been used in the development of an consensus sequence anti-adhesin vaccine targeted at the prevention of host cell attachment and further for the generation of a monoclonal antibody capable of prevention and treatment of existing infections.

Editorial: “Current trends in peptide science”

The “Current Trends in Peptide Science” reviews in this issue feature papers on a variety of peptide approaches to combating human diseases, including peptide antimicrobials, inhibitors of enzymes, anticancer agents, and vaccines. In the first paper, Bryant, Lazarus, and their colleagues describe advances in opioid peptide research made through the introduction of the 2′6′-dimethyl-L-tyrosine (Dmt) residue into opioidmimetics, particularly in the context of Dmt-Tic compounds. The authors review the ability of Dmt—when substituted for Tyr in opioid peptides—to enhance the interaction and accompanying biological activities of the resulting compounds with respect to δ and μ-opioid receptors. Structural data on these materials are presented which have facilitated the development of pharmacophores for both δ-opioid receptor agonists and antagonists. Through studies of mixture-based peptide synthetic combinatorial libraries, Blondelle and her colleagues then describe their advances in the development of antimicrobial and antiviral peptides, and their application to the control of infectious diseases. Noting that immunization with peptides can induce humoral and cellular immune responses mediated by antibody and T lymphocytes, respectively, the authors further describe their efforts in evaluating synthetic peptides as vaccines, including applications to HIV-1 proteins. In the following paper, Cooperman reviews research into class I ribonucleotide reductases (RR's), which are established targets for cancer chemotherapeutic and antiviral agents. Oligopeptides are described which specifically inhibit the RR's from which they are derived; structural work on these peptides is presented which has led to a model compatible with binding data, from which detailed knowledge could lead to peptide inhibitors selective for tumor cells. Focusing on the key role of the growth factor receptor-bound protein 2 (Grb2) in the Ras signaling pathway, which renders Grb2 a potential target for the design of antitumor agents, Lung and Tsai review current approaches to the development of Grb2 SH2 domain peptide inhibitors intended to interrupt Grb2 recognition. The authors delineate the classes of inhibitors used in this context, which range from those containing nonhydrolyzable mimetics of phosphates to nonphosphorylated cyclic peptide ligands for the Grb2 SH2 domain. Then, Cachia and Hodges provide a comprehensive discussion of synthetic peptide vaccines which may have utility in the prevention and treatment of Pseudomonas aeruginosa—an opportunistic pathogen that causes urinary tract and respiratory system infections, and which becomes extremely difficult to treat in cystic fibrosis patients. These authors describe their progress in development of anti-adhesin peptides targeted at the prevention of host cell attachment, and toward the elucidation of rules in consensus sequence design that can facilitate synthetic peptide vaccine development. These reviews highlight and re-state the emerging importance of the diverse roles of peptides in potential treatment of human disorders ranging from cancer to HIV to cystic fibrosis. We hope that readers discover, and share, the optimism inherent in these reports.

Synthetic peptides as cancer vaccines.

Effective cancer therapy or prevention has been the dream of physicians and scientists for many years. Although we are still very far from our ultimate goal of cancer prevention, significant milestones have been realized in terms of our knowledge base and understanding of the pathogenesis of cancerous cells and the involvement of the immune system against both self- and virus-associated tumor antigens. Immunotherapeutic strategies are now accepted to being superior in terms of the exquisite specificity that they offer in targeting only tumor cells as opposed to the existent chemotherapy or radiation therapy that is more general and invasive with many associated side effects. There are several immunotherapeutic strategies that are currently under investigation. This review primarily focuses on the significant advances made in the use of synthetic peptides in the development of subunit cancer vaccines. We have attempted to highlight some of the fundamental issues regarding antigen processing and presentation, Major Histocompatibility Complex (MHC) restriction, T-cell help, structural determinants in antibody recognition, and the use of these concepts in the rational design and delivery of peptide vaccines to elicit protective humoral and cell mediated immune responses. The recent use of costimulatory molecules and cytokines to augment immune responses also has been discussed along with the contributions of our laboratory to the field of synthetic peptide vaccine development.

The Development of a Mimotope-based Synthetic Peptide Vaccine Against Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide and the development of a synthetic peptide epitope-based vaccine to induce virus-neutralising antibodies against RSV would seem to be a valid approach to the production of an effective vaccine against infection.A combinatorial solid-phase peptide library has been screened with a virus-neutralising, protective monoclonal antibody (MAb19) directed towards a conserved and conformationally-determined epitope of the Fusion (F) protein of the virus. Two of the sequences identified from the peptide library using MAb19 reacted specifically with the antibody and amino acid substitution experiments identified four sequences from one of the mimotopes which showed increased reactivity with MAb19.Immunisation of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb19 to the virus and neutralised viral infection in vitro, with titres equivalent to those in sera from RSV-infected animals. Following RSV challenge of mimotope-immunised mice, a significant reduction in the titre of virus and a greatly reduced cell infiltration into the lungs of immunised mice compared to that in controls was observed.The induction of virus-specific cytotoxic T-lymphocyte responses as well as virus-specific antibodies are likely to be necessary in an effective vaccine. The incorporation of a peptide representing a CTL epitope from the M2 protein of the virus together with peptides inducing T-helper and anti-mimotope responses in a peptide cocktail vaccine resulted in a more effective clearance of the virus from immunised, challenged mice than peptide-induced humoral or cellular immunity alone.

Identification and characterization of B-cell epitopes of a 53-kDa outer membrane protein from Porphyromonas gingivalis.

We have previously reported that Porphyromonas gingivalis FDC 381 possesses a 53-kDa protein antigen (Ag53) on its outer membrane that evokes a strong humoral immune response in many patients with periodontal disease and that the humoral immune responses to Ag53 differ greatly among patients. To understand how the individual humoral immune system against Ag53 was determined, the regions of Ag53 recognized by specific antibody (B-cell epitopes) and dominant subclasses of serum immunoglobulin G (IgG) against major B-cell epitopes were examined by enzyme-linked immunosorbent assay. This study used sera from six patients with periodontitis, which all reacted strongly with sonic extracts of P. gingivalis 381 and with purified Ag53, and sera from six periodontally healthy children, which did not react with either sonic extracts of P. gingivalis 381 or Ag53. The epitopes were identified using synthetic 5-residue overlapping decapeptides covering the entire Ag53. Thirteen of 89 synthetic decapeptides showed a strong reaction with sera from the periodontal patients, but no reaction with those from the healthy children. Four peptides of 13 exerted different immune responses among patients. Furthermore, restriction analyses of the highly antigenic regions revealed that three sequences, RAAIRAS, YYLQ and MSPARR, were identified as major B-cell epitopes. Additionally, these epitopes were recognized mainly by the IgG2 isotype. These data suggest that the difference of B-cell epitopes might influence individual differences in antibody titer against Ag53 and also that the epitopes recognized commonly by multiple antibodies are quite valuable for peptide vaccine development against P. gingivalis infection.