Mapping of dominant B-cell epitopes of a human zona pellucida protein (ZP1)

Zona pellucida (ZP) glycoproteins contain numerous antigenic determinants including carbohydrate, protein, and conformational epitopes; and the immunogenicity of these complex glycoproteins varies in different mammalian hosts. Studies have now shown that antibodies from primates immunized with a cDNA-expressed recombinant rabbit ZP protein (the homologue of the human ZP1 [hZP1]) inhibit sperm binding to the ZP without altering ovarian function, unlike immunization with ZP3 and ZP2 protein families. The ZP1 protein or peptides derived from it (recombinant or synthetic) are therefore primary candidates for use in designing safe and reversible human and animal contraceptive vaccines. In order to define peptide epitope(s) that may be critical for eliciting an immune response sufficient to effect immunological contraception without causing any adverse effects on ovarian physiology, studies have been carried out to identify immunodominant B-cell epitopes of the ZP1 protein. The amino acid sequence of the hZP1 was used to design a set of 94 (15-mer) biotinylated peptides having an overlap of 9 amino acids. Using these peptides in a modified enzyme-linked immunoassay, antibodies in sera from rabbits or baboons immunized with native porcine ZP protein were screened for ZP1 peptide recognition. These studies demonstrate that there are a limited number of peptides recognized by primate antibodies but that the overlapping peptides sharing the sequence GPLTLELQI are recognized by both rabbit and baboon antibodies regardless of the adjuvant system used to induce the immune response. This peptide is 100% conserved in amino acid sequence between the human and pig, although the rabbit protein has two conserved amino acid substitutions (100% similar, 77% identical). Because this peptide is immunogenic as well as antigenic in primates, it could play a major role in the development of human contraceptive vaccines.     

Use of the multipin peptide synthesis technique for the generation of antipeptide sera.

The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes. What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants. A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies. This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pin-synthesized peptides and beta-amide peptides and the protein antigen coated to microtiter plates.     

Use of the multipin peptide synthesis technique for the generation of antipeptide sera

The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes. What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants. A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies. This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pinsynthesized peptides and β-amide peptides and the protein antigen coated to microtiter plates.     

UreB蛋白B细胞抗原表位快速筛选与鉴定

以幽门螺旋杆菌(Helicobacterpylori,Hp)主要抗原蛋白尿素酶B(ureaseB,UreB)为靶蛋白,建立一种新的B细胞抗原表位筛选与鉴定方法.运用Fmoc固相肽合成法合成11条HpUreB蛋白的单表位抗原肽片段,在其氨基端标记FITC荧光素,应用荧光偏振方法(fluorescencepolarization,FP)快速鉴定这些肽片段的抗原性,并通过FP法在大规模样品中快速筛选相应抗体滴度高、分布人群广的优势抗原表位肽.结果表明,合成的11条UreB蛋白线性抗原肽中,10条具有较强的抗原性,其中No.2、No.5和No.11抗原肽相应的特异性抗体在感染Hp的人群中分布较广,抗体滴度较高,为UreB的优势抗原表位肽.对抗原表位进行多参数综合分析与设计,通过FP技术快速鉴定抗原肽,并筛选优势抗原表位肽,对于疾病的抗原表位谱研究具有重要的意义,同时在疾病的诊断、分型及治疗中具有重要的应用前景.     

The concept and operational definition of protein epitopes

The antigenic determinants or epitopes of a protein correspond to those parts of the molecule that are specifically recognized by the binding sites or paratopes of certain immunoglobulin molecules. Epitopes are thus relational entities that require complementary paratopes for their operational recognition. Some authors consider that the concept of epitope necessarily involves the two properties of antigenic reactivity (ability to bind to a paratope) and immunogenicity (ability to induce an immune response). Such a view creates difficulties because it makes the existence of epitopes in a protein depend on immunogenetic and regulatory mechanisms of the immunized host. The delineation of epitopes can be achieved by antigenic cross-reactivity studies or by X-ray crystallography. Both approaches require specific criteria for deciding which residues of the antigen are in contact with the paratope and are functionally part of the epitope. The relative contribution of static accessibility, segmental mobility and induced fit to immune recognition remains controversial. Each of the methods used for analysing antigenic specificity is subject to various operational constraints originating from the type of experimental probe and from the format sensitivity and specificity of the immunoassay used. If a protein is assumed to contain as many epitopes as the number of different monoclonal antibodies that can be raised against it, the delineation of epitopes corresponds to the summation in various hosts of the immune repertoire specific for the antigen. Neutralization epitopes are a special subclass of the epitopes of infectious agents and toxins that are specifically recognized by antibody molecules able to neutralize the biological activity of the antigen. The identification of neutralization epitopes is important for the development of synthetic vaccines because it is this type of epitope that should be mimicked by synthesis and used as a vaccine for eliciting protective immunity. The first demonstration that synthetic peptides could elicit antibodies that neutralized viral infectivity was made by Anderer and his colleagues in the 1960s in their work with tobacco mosaic virus. Nearly 20 years passed before it was shown that antibodies to synthetic peptides were also able to neutralize the infectivity of other viruses such as foot-and-mouth disease, polio and hepatitis B viruses.     

A major part of the polypeptide chain of tobacco mosaic virus protein is antigenic

Eighteen synthetic peptides representing virtually the entire length of the polypeptide chain of tobacco mosaic virus coat protein (TMVP) have been analyzed for their ability to bind in an enzyme immunoassay to 30 monoclonal antibodies raised against the dissociated viral subunits. Only five of the monoclonal antibodies were able to bind a number of peptides while the other 25 antibodies recognized only the complete molecule and seemed to be specific for conformational features that are absent in the peptide fragments. The 18 peptides were also tested for their ability to bind to several antisera to TMVP. Virtually the entire sequence of TMVP possessed antigenic activity. Four new epitopes were identified in the vicinity of residues 19–32, 90–95, 115–134 and 134–146. These results bring to 11 the number of continuous epitopes that have been identified in the TMVP molecule and show that the entire surface of the molecule is antigenic. When peptides of TMVP of a length of 6–8 residues were tested for antigenic activity previously a correlation was found between the location of short continuous epitopes and mobile segments of the protein. In the present study, in which longer peptides as well as monoclonal antibodies were used to probe the antigenicity of TMVP, additional conformation-dependent epitopes were shown to be present. Our results illustrate the operational nature of any definition of antigenicity and caution against the use of any single criterion for distinguishing between antigenic and non-antigenic regions of a protein.     

Diagnostic epitope variability within Taenia solium 8 kDa antigen family: implications for cysticercosis immunodetection

To study diagnostic epitopes within the Taenia solium 8 kDa antigen family, six overlapping synthetic peptides from an 8 kDa family member (Ts8B2) were synthesized and evaluated by ELISA and MABA with sera from patients with neurocysticercosis (NCC), from infected pigs and from rabbits immunized with recombinant Ts8B2 protein. The pre-immune rabbit sera and the Ts8B2 recombinant protein served as negative and positive controls, respectively. A similar analysis was done with the already described antigenic peptides from another member of the 8 kDa family, highly similar to Ts8B2, the CyDA antigen. Surprisingly, neither the Ts8B2 peptides nor the CyDA peptides were recognized by infected human and porcine sera. However, the entire Ts8B2 recombinant, as well as amino and carboxy-terminal halves were recognized by the positive serum samples. The observed lack of recognition of linear Ts8B2 peptides suggests that the principal serological response to the Ts8B2 family is focused on conformational epitopes in contrast to the previously observed antigenicity of the CyDA peptides. This differential antigenicity of 8 kDa family peptides could be related with parasite antigenic variability. The fact that rabbits experimentally immunized with Ts8B2 did make anti-peptide antibodies to peptides Ts8B2-6 and CyDA-6, located in the carboxy-terminal region demonstrated that the Ts8B2 peptides are not intrinsically non-immunogenic.     

Anti-La antibody production by MRL-1pr/1pr mice. Analysis of fine specificity

In evaluating the origin of autoantibodies, patterns of self-Ag recognition have been interpreted to reflect the relative role of Ag in stimulating a response. Few studies, however, have assessed whether human autoantibodies display patterns of autoantigen recognition similar to those of SLE-prone mice. In previous studies, anti-La antibodies from humans have been shown to bind multiple epitopes on recombinant human La Ag, including immunoreactivity with a large fragment, termed La C, representing the middle portion of the La sequence. We report herein for the first time that MRL-1pr mice also spontaneously produce antibodies to recombinant human La protein and resemble human autoantibodies in their reactivity with La C. To further investigate the fine specificity of this response, we tested for antibody binding to six synthetic La peptides representing sequences within La C. Whereas two of the synthetic La peptides reacted with MRL-1pr sera containing anti-La binding, low reactivity was observed with a large panel of human anti-La sera. Our results therefore show that patterns of La antigen recognition displayed by MRL-1pr antibodies differ from those of human autoantibodies, possibly reflecting differences between mouse and man in the induction of these responses.     

Identification of T-cell epitopes on E2 protein of rubella virus, as recognized by human T-cell lines and clones.

T-cell epitopes on the E2 protein of rubella virus were studied by using 15 overlapping synthetic peptides covering the E2 protein sequence. The most frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 tested T-cell lines responding to it. Two CD4+ cytotoxic T-cell cloned isolated from one T-cell line responded strongly in proliferation assays with peptide E2-4 and were cytotoxic to target cells presenting the E2-4 determinant. Truncated peptides contained within the E2-4 peptide sequence were used to define the T-cell determinants. Results indicated that amino acid residues 54 to 65 were directly involved. Human cell lines with different HLA phenotypes were tested for the capacity to present the antigenic determinants. The results suggested that recognition of peptide E2-4 by T-cell clones was associated with HLA DR7.     

Induction of potent antitumor CTL responses by recombinant vaccinia encoding a melan-A peptide analogue

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.     

Assessment of synthetic chimeric multiple antigenic peptides for diagnosis of GB virus C infection

The use of synthetic peptides of both structural and nonstructural proteins of GB virus C (GBV-C) has been studied for the development of new systems to diagnose infection caused by this virus. In an attempt to increase the antigenicity of linear peptide sequences, chimeric multiple antigenic peptides (MAPs) containing epitopes from E2, NS4, and NS5 GBV-C proteins have been synthesized. The synthetic constructs were evaluated by ELISA to establish whether the epitopes in chimeric branched peptides are more efficiently recognized by the specific antibodies compared to the monomeric linear sequences. Moreover, we have investigated the application of a commercial biosensor instrument for the detection of antibodies against the GBV-C in human serum samples. The results of the immunoassays reported in this work highlight the usefulness of synthetic tetrameric branched peptides containing sequences from envelope and nonstructural GBV-C proteins for the diagnosis of GBV-C infection. The potential clinical value of the MAP₄(E2-NS5a) for the serodiagnosis of GBV-C infection was demonstrated, thus providing the basis for performing prevalence studies of the infection among the hemodialyzed and hepatitis C virus (HCV)-infected population.     

Function in the Immune System of Antigenic Peptides from S-Antigen (Arrestin)

A significant amount of information concerning immunologic domains of an antigenic molecule can be obtained by studying its peptides. We describe a method for identifying and characterizing immunologically relevant T-cell and B-cell epitopes in S-antigen, a well-characterized, highly pathogenic retinal autoantigen for the induction of experimental autoimmune uveitis. The method involves the generation of peptide fragments by enzymatic treatment of native S-antigen and by the simultaneous synthesis of large numbers of peptides in small quantities for screening and testing. Peptides demonstrating T- or B-cell activity are then synthesized in large quantity for additional studies. Although useful information was obtained by the use of enzymatically generated peptides, synthetic peptides provided the greatest flexibility and specificity, allowing the precise localization of amino acid sequences of S-antigen required for a particular immunological function such as antibody binding, T-cell proliferative responses, pathogenicity, and the induction of tolerance. These studies have wide applicability to the study of other antigenic molecules and have led to a better understanding of the immune mechanisms involved in the pathogenesis of experimental autoimmune uveitis. This, in turn, provides a basis for the processes that may be occurring in certain forms of human uveitis.     

Epitope mapping and evaluation of specificity of T-helper sites in four major antigenic peptides of chicken riboflavin carrier protein in outbred rats

This paper reviews our studies on synthetic peptides spanning the major antigenic determinants of the chicken riboflavin carrier protein (RCP; 219 AA). These determinants are composed of residues 4-24 (YGC), 64-83 (CED), 130-147 (GEN), and 200-219 (HAC) and function as minivaccines in terms of eliciting anti-peptide antibodies which recognize the native protein and are particularly promising contraceptive vaccine candidates. We have used 15-residue synthetic peptides to define short sequences involved in interaction with antibody and with T-cells. We have mapped the boundaries of T-cell epitopes of these peptides in outbred rats by immunizing the animals with each peptide and assaying the popliteal lymph node cell proliferation against a series of overlapping synthetic 15-mers covering the entire length of the individual peptides. The peptides YGC, GEN, and HAC harboured a single T-cell epitope each whereas the peptide CED exhibited bimodal response possessing two epitopes, one at N-terminus and the other at the C-terminus. These studies provide insight into the way in which an immunogen is viewed by the immune system. In addition, preferential T-cell helper function for B cells recognizing unique determinants on the same molecule was demonstrated. This information helps in exploiting synthetic peptides in the construction of designer immunogens which have potential as candidate vaccines.     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) immuno-dominant and immuno-recessive area

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno-dominant N-terminus and an immuno-recessive C-terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley-Liss, Inc.     

Identification of a rabies virus T cell epitope on the basis of its similarity with a hepatitis B surface antigen peptide presented to T cells by the same MHC molecule (HLA-DPw4).

The antigenic determinant recognized by a HLA-DPw4-restricted human T cell clone specific for rabies virus was identified by using a vaccinia-rabies nonstructural phosphoprotein recombinant virus and synthetic peptides of the sequence of rabies nonstructural Ag. These peptides were selected on the basis of three models that predict T cell epitopes. The antigenic determinant recognized by the rabies virus-specific T cell clone contained a five-amino acid segment highly homologous to a sequence found in a hepatitis B surface Ag epitope that stimulates human T cells in the context of the HLA-DPw4. A preliminary model of DPw4-restricted T cell determinants is elaborated based on a hypothesis of how the 2 alpha-helical peptides may bind to this MHC molecule. Results are further discussed in the context of the usefulness in identifying DPw4-restricted T cell epitopes for the production of synthetic vaccines because this MHC class II molecule is found with high frequency in the population.     

The structure of an antigenic determinant in a protein

The immunogenic and antigenic determinants of a synthetic peptide and the corresponding antigenic determinants in the parent protein have been elucidated. Four determinants have been defined by reactivity of a large panel of antipeptide monoclonal antibodies with short, overlapping peptides (7-28 amino acids), the immunizing peptide (36 amino acids), and the intact parent protein (the influenza virus hemagglutinin, HA). The majority of the antipeptide antibodies that also react strongly with the intact protein recognize one specific nine amino acid sequence. This immunodominant peptide determinant is located in the subunit interface in the HA trimeric structure. The relative inaccessibility of this site implies that antibody binding to the protein is to a more unfolded HA conformation. This antigenic determinant differs from those previously described for the hemagglutinin and clearly demonstrates the ability of synthetic peptides to generate antibodies that interact with regions of the protein not immunogenic or generally accessible when the protein is the immunogen.     

Determination of protein-derived epitopes by mass spectrometry

Mass spectrometry has evolved as a technique suitable for the characterization of peptides and proteins beyond their linear sequence. The advantages of mass spectrometric sample analysis are high sensitivity, high mass accuracy, rapid analysis time and low sample consumption. In epitope mapping, the molecular structure of an antigen (the epitope or antigenic determinant) that interacts with the paratope (recognition surface) of the antibody is identified. To obtain information on linear, conformational and/or discontinuous epitopes, various approaches have been developed in conjunction with mass spectrometry. These methods include limited proteolysis and epitope footprinting, epitope excision and epitope extraction for linear epitopes and probing the surface accessibility of residues by differential chemical modifications of specific amino acid side chains or by differential hydrogen/deuterium exchange of the protein backbone amides for conformational and discontinuous epitopes. Epitope mapping by mass spectrometry is applicable in basic biochemical research and, with increasing robustness, should soon find its implementation in routine clinical diagnosis.     

Selection of HIV-specific immunogenic epitopes by screening random peptide libraries with HIV-1-positive sera

Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing Ab responses. In fact, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. To overcome these obstacles, we screened random peptide libraries using sera from HIV-infected subjects to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counterscreening with HIV-negative sera, we isolated peptides specifically recognized by Abs from HIV-1-infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 epitopes generated in vivo in infected subjects. Consistent with these findings, sera of simian HIV-infected monkeys also recognized the HIV-specific epitopes. The selected peptides were immunogenic in mice, where they elicited HIV-specific Abs that effectively neutralized HIV-1 isolates. These results demonstrate that pools of HIV-1 mimotopes can be selected from combinatorial peptide libraries by taking advantage of the HIV-specific Ab repertoire induced by the natural infection.     

Determination of protein-derived epitopes by mass spectrometry

Mass spectrometry has evolved as a technique suitable for the characterization of peptides and proteins beyond their linear sequence. The advantages of mass spectrometric sample analysis are high sensitivity, high mass accuracy, rapid analysis time and low sample consumption. In epitope mapping, the molecular structure of an antigen (the epitope or antigenic determinant) that interacts with the paratope (recognition surface) of the antibody is identified. To obtain information on linear, conformational and/or discontinuous epitopes, various approaches have been developed in conjunction with mass spectrometry. These methods include limited proteolysis and epitope footprinting, epitope excision and epitope extraction for linear epitopes and probing the surface accessibility of residues by differential chemical modifications of specific amino acid side chains or by differential hydrogen/deuterium exchange of the protein backbone amides for conformational and discontinuous epitopes. Epitope mapping by mass spectrometry is applicable in basic biochemical research and, with increasing robustness, should soon find its implementation in routine clinical diagnosis.     

Artificial mosaic protein containing antigenic epitopes of hepatitis E virus.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.