Preparation of peptides which mimic glycosphingolipids by using phage peptide library and their modulation on beta-galactosidase activity

We describe the use of a phage-displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD-1 and AD-2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9-mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD-2 except for one amino acid substitution. Pentadecamer, 9-mer and point mutated 9-mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9-mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean beta-galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.     

Characteristics and determinants of sumatriptan-associated chest pain

Analysis of epitopes recognized by therapeutic monoclonal antibodies (mAb) is critical in clinical applications and in structure/function studies of target antigen. mAb MGr6 recognizes the extracellular domain of the p185HER2 oncoprotein and is a promising candidate for cancer immunodiagnosis and immunotherapy. Thus, epitope location and structure on p185HER2 need to be investigated. The use of MGr6-selected phage-displayed peptides for epitope analysis served to dissect the MGr6 epitope into at least two subregions, mimicked by CHSDC- and (L)P-(L)K(L) phage displayed peptides, respectively. Comparison of amino acid sequences of CHSDC peptides with the p185HER2 protein sequence and analysis of MGr6 reactivity with p185HER2 deletion mutants identified the linear subregion CCHEQCAAG of the MGr6 epitope, corresponding to amino acids 235-243 of the p185HER2 protein. This continuous subregion is part of a larger conformational epitope, and other amino acids, including a proline, a lysine and leucine residues contained in (L)P-(L)K(L) phage-displayed peptides appear to contribute to the formation of the MGr6 epitope surface.     

Laparoscopic evaluation of liver disease in chronic renal failure prior to renal transplantation

It is not rare that controversial indications about the presence or the expression level of multidrug-resistant (MDR) proteins come out from different laboratories upon examination of identical tumor specimens. Distinct aspects, including the use of weakly discriminating monoclonal antibodies (MAbs) and/or unsuitable techniques and procedures, contribute in generating differences in the MDR phenotype evaluation of cancer cells. In this regard we describe here an innovative immunohistochemical approach for the determination of P-glycoprotein expression in cells and tissues. The method is based on the ability of phage-displayed peptides to mimic antibody epitopes. For this purpose we utilized the phage clone #55, which was affinity-purified from a phage-displayed random-peptide library using the MAb MM4.17 (specific for MDR1-P-glycoprotein) as previously described. This clone has been chosen since it clearly and undoubtedly reacts with its cognate MAb, as was determined by ELISA and dot blot tests. Inhibition of the MAb MM4.17 binding to MDR1-P-glycoprotein-expressing cells could be performed by adding a calibrated concentration of phage clone #55 particles, which mimic MDR1-P-glycoprotein antigen. This methodology can eliminate misleading interpretations concerning the presence and expression level of MDR1-P-glycoprotein and might well contribute in routine clinical determinations of MDR in tumor specimens, thus contributing to our understanding of the basis of the mechanisms of tumor cell resistance to drugs.     

The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily

The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and > 95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.     

Identification of HCV core mimotopes: improved methods for the selection and use of disease-related phage-displayed peptides

Disease-specific epitope discovery from random peptide libraries displayed on phage using sera from patients involves a number of screening steps with many immune and non-immune sera. To rapidly identify mimotopes of the human hepatitis C virus (HCV) core protein, we have used an anti-core human monoclonal antibody (mAb; B12.F8) as a probe in screening phage that were affinity-selected using a serum from an HCV infected patient. Three different positive phage were isolated displaying low or no homology with the natural antigen, but which still efficiently bound to the antigen binding site of the B12.F8 antibody. Testing the reactivity of these phage with forty-five sera from HCV infected patients showed that antibodies recognizing them are present in more than 80% of this population. These antibodies showed distinct fine specificity, as they bound the selected phage in a mutually exclusive fashion. Co-expression of two mimotopes in the same cells led to chimeric particles which were recognized by antibodies of different specificity. These data provide novel information on the potential use of the phage display technology for the characterization of antibody specificity as well as disease diagnosis and prevention.     

Delineation of tyrosine-containing epitopes within the beta subunit of bovine thyrotropin

The epitopes recognised by two monoclonal antibodies (mAb 279 and mAb 299), specific for the beta subunit of bovine thyroid-stimulating hormone (bTSH), have been localised using a technique in which the tyrosine residues in the bTSH beta subunit were subjected to modification when the bTSH beta subunit was complexed with either mAb or in the free, unbound state. The epitope recognised by mAb 279 was localised to the C-terminal region of bTSH beta with the tyrosine residue Tyr104 protected from modification by the presence of this mAb. In addition, the experimental results indicate that the tyrosine residues Tyr18 and/or Tyr112 are also involved in the mAb 279 epitope. The epitope recognised by mAb 299 was localised to the region 59-74 of bTSH beta as both Tyr59 and Tyr74 were protected from modification by the presence of this mAb. Since both mAbs have been previously found to inhibit receptor binding, the sequence regions/amino acid positions recognised by these mAbs are likely to represent determinants for receptor binding. Moreover, these data indicate that the identified amino acid residues are located on the surface of the molecule, consistent with predictions of the tertiary structure of the bTSH beta subunit based on the recently elucidated X-ray crystal structure of human chorionic gonadotropin.     

Monoclonal antibody against bovine Lactoferricin and its epitopic site

Bovine lactoferricin (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of "QWR".     

Obstetricians'' knowledge and attitudes toward epidural analgesia in labour

A 30 kDa immunodominant surface antigen (p30) of Babesia equi has been used as a diagnostic antigen. The B cell epitopes on this molecule recognized by horse sera and monoclonal antibody (MAb) against p30, 36/133.97, were determined. A synthetic peptide of p30 with amino acid sequence of 123FYQEVLFKGFEAV135 exhibited strong positive reaction with the infected horse sera. In contrast, MAb 36/133.97 recognized different region of p30, as peptide synthesized with amino acid sequence of 27ASGAVVDFQLESI39 reacted strongly. In competitive inhibition ELISA, the binding of MAb 36/133.97 to recombinant p30 was inhibited by horse antibodies, although they did not recognize same or an overlapping epitope. The data on B cell epitopes in this study may be important in improving serodiagnostic methods of B. equi infection.     

Mapping and characterization of a sequential epitope on the rabies virus glycoprotein which is recognized by a neutralizing monoclonal antibody, RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

Identification of an epitope of alpha-enolase (a candidate plasminogen receptor) by phage display

Alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An alpha-enolase related molecule (alpha-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid cells and has the capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as a plasminogen receptor. We have generated a monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reacted with both alpha-ERM and purified human alpha-enolase in Western blotting and in enzyme linked immunosorbent assays (ELISA). mAB 9C12 detected a cell surface associated molecule on human peripheral blood neutrophils and on U937 human monocytoid cells as assessed by fluorescence activated cell sorting (FACS) analyses. In addition, mAB 9C12 recognized an intracellular pool of alpha-enolase/alpha-ERM in permeabilized U937 cells. A phage display approach was employed to identify the alpha-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human alpha-enolase cDNA, were cloned into the filamentous phage vector pComb3B, to generate a phage-displayed peptide library. Recombinant phages binding to mAB 9C12 were selected and their DNA inserts characterized by direct sequencing. All of the fragments which bound to mAB 9C12 encoded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human alpha-enolase. This sequence is located within an external loop of the molecule. These data indicate that this sequence contains the epitope recognized by mAB 9C12 and is, therefore, exposed on the cell surface, further suggesting that alpha-enolase and alpha-ERM share common amino acid sequences.     

Molecular recognition of a peptide mimic of the Lewis Y antigen by an anti-Lewis Y antibody

Peptides as mimics of carbohydrates display a distinct advantage in vaccine design because of ease of synthesis and their inherent T cell-dependent nature as immunogens. While peptides that mimic carbohydrates have been described, it is not clear how they do so. To further our insight into structural relationships between peptide-mimics and carbohydrate structures, we have analyzed a potential recognition scheme between the murine monoclonal antibody, B3, directed against the tumor-associated antigen Lewis Y oligosaccharide and a peptide identified from phage display screening with B3. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. The B3 antibody binds to the peptide sequence APWLYGPA in which the putative sequence APWLY is critical for binding to the antibody. Not having experimental structural information for B3, the crystal structure of another anti-Lewis Y antibody, BR96, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, provides a molecular basis for LeY antigen recognition and specificity, and how this binding relates to peptide binding. As a guide to place the APWLY motif in the B3 combining site, a fragment library was searched for analogous compounds that have the potential to bind to B3. Our modeling study shows that the B3-peptide complex shares similar recognition features for the difucosylated type 2 lactoseries' structure. This analysis provides a molecular perspective for peptide mimicry of a carbohydrate epitope.     

Elucidation of the core residues of an epitope using membrane-based combinatorial peptide libraries

Combinatorial peptide libraries have proved to be a valuable tool for the study of the interaction of a functional protein with its ligand. Here, the epitope for a monoclonal antibody 201/9, raised against beta-factor XIIa, has been identified with a two-step approach using peptide libraries attached to a polymer (polyvinylidene difluoride) membrane. First, the octapeptide libraries with two amino acids defined at position 2 and 4, represented by the formula X-O2-X-O4-X-X-X-X, were synthesized on a sheet of polymer membrane in which X represents a mixture of all the natural -amino acids except cysteine, while O2 and O4 each represent a single amino acid. The libraries were probed with the antibody 201/9, and the bound antibody was detected with a sensitive chemiluminescent method. In the first cycle, the peptide mixtures X-Phe-X-Gln-X-X-X-X showed the strongest signal development. In the second cycle Phe and Gln were incorporated into new libraries consisting of sequences O1-Phe-X-Gln-X-X-X-X, X-Phe-O3-Gln-X-X-X-X, X-Phe-X-Gln-O5-X-X-X, X-Phe-X-Gln-X-O6-X-X, X-Phe-X-Gln-X-X-O7-X, and X-Phe-X-Gln-X-X-X-O8. After probing these new peptides, the residues representing the core sequence of the epitope for monoclonal antibody 201/9 were elucidated. The sequence Ser-Phe-Leu-Gln-Glu-Asn, identified as the immunodominant epitope, correlates well with the sequence Ser-Phe-Leu-Gln-Glu-Ala previously identified (Gao, B., and Esnouf, M. P. (1996) J. Immunol. 157, 183-188) in a scan of overlapping peptides based on the sequence of human beta-factor XIIa.     

Definition of essential amino acid residues in the recognition of a peptide by a mouse monoclonal antibody

A mouse monoclonal antibody reacting in ELISA with a synthetic peptide representing a linear amino acid stretch of the protein antigen was tested on all overlapping 5-mer to 9-mer fragments of the peptide, as prepared by multi-pin synthesis. Analysis of the binding data suggests that several residues in the peptide might be relatively unrelevant for recognition, while few others seem to play a critical role as key residues. On the basis of such observations, we attempted to reconstruct an alternative essential epitope by introducing multiple amino acid substitutions in the 9-mer peptide exhibiting the best binding activity, and then tested its ability to be recognized by the monoclonal antibody.     

Identification of peptides, selected by phage display technology, that inhibit von Willebrand factor binding to collagen

A repeated selection of phages from a cyclic hexapeptide phage display library resulted in an enrichment of phages that bound to the monoclonal antibody (MoAb) 82D6A3 (an anti-von Willebrand Factor [vWF] antibody that inhibits binding of vWF to collagen). Two clones were selected that bound both to MoAb 82D6A3 and to rat tail collagen type I in a specific and dose-dependent manner. The two phage clones were further used in a two-direction competition experiment with vWF. vWF was able to displace phages from collagen in a dose-dependent manner with an IC50 of 35 micrograms/mL and phages were able to inhibit vWF binding to collagen. With the use of specific primers, the sequence of the cysteine-flanked hexapeptide inserts could be deduced. The two phage clones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitution of an N for a Q at position 6 of the hexapeptide. Sequence comparison with the known vWF sequence showed the presence of a comparable sequence at position 1129-1136 (VWTLPDQC), located between the collagen-binding A3-domain and the D4-domain. The two cyclic peptides, the putative corresponding vWF peptide, and a peptide with a scrambled cyclic sequence were synthesized. The two cyclic peptides inhibited vWF binding to rat tail collagen type I in a dose-dependent manner, whereas the linear vWF peptide and the scrambled cyclic peptide were inactive. For half maximal inhibition, 100 +/- 12.7 micromol/L and 34.8 +/- 8.59 micromol/L (mean +/- SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to inhibit vWF binding to calfskin and human collagen type I, but effective concentrations were some 5 to 10 times higher.     

Activation of a murine autoreactive B cell by immunization with human recombinant autoantigen La/SS-B: characterization of the autoepitope

Immunization of Balb/c mice with a homogeneously purified recombinant human La/SS-B protein resulted in activation of an autoreactive B cell secreting a novel monoclonal anti-La antibody termed La4B6. La4B6 reacted with La protein from a variety of sources including human, bovine, rat and mouse. ATP blocked the binding of La4B6 to recombinant La protein. The human epitope was identified as consisting of the amino acid sequence SKGRRFKGKGKGN, which includes the proposed ATP-binding site of the La protein. In the human and bovine La protein, the epitope exists as a continuous amino acid sequence. In rat and mouse the epitope was found to consist of the amino acid sequence SKG interrupted by a species-specific insert of 16 amino acids, and followed by the second half of the epitope, the amino acid sequence RRFKGKGKGN. Our data suggest that in the case of the rat and mouse La proteins the two separated parts of the epitope are able to form a conformational epitope which looks similar to the continuous human epitope.     

Epitope analysis of a sperm acrosomal antigen defined by HSA-5 monoclonal antibody

Among the monoclonal antibodies recommended by the WHO Sperm Antigen Workshop for immunocontraceptive vaccine development, HSA-5 showed a high degree of sperm specificity and significantly inhibited in vitro fertilization in both humans and mice. Using a Western blot assay, HSA-5 was found to recognize a sperm antigen designated as HSAg-5 (human) or MSAg-5 (mouse) which ranged in molecular weight from 18 to 100 kDa. This monoclonal antibody was used as the probe for the immunoscreening of mouse testis cDNA libraries constructed in the lambda gt-11 expression vector. One of the positive cDNA clones was shown to have a cDNA insert of approximately 1 kb and to encode a recombinant fusion protein containing 77 amino acid residues in the C-terminal region of MSAg-5. This 1 kb cDNA insert was engineered in a pGEX vector to express a recombinant glutathione S-transferase fusion protein (GST-5). Using an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, both anti-GST-5 sera and the monoclonal antibody were shown to react with GST-5. The Northern blot of a mouse testis RNA preparation revealed that the isolated cDNA probe hybridized with a 4.0 kb mRNA. Several oligopeptides were synthesized based on the predicted C-terminal hydrophilic regions of the recombinant fusion protein. Using ELISA and a dot blot assay, peptide regions containing the immunogenic epitopes recognized by HSA-5 monoclonal antibody were identified.     

Enhancement of ELISAs for screening peptides in epitope phage display libraries

The complete analysis of epitope phage display libraries requires sensitive assays capable of detecting peptides expressed on phage that have a wide range of affinities for antibody. We have compared two ELISAs, a 'direct' assay where the phage is captured by an anti-phage antibody and the peptide detected by the antibody used for screening, and a 'reverse' assay where the antibody used for screening is first coated on the well and the binding of phage detected by the anti-phage antibody. We demonstrate, by comparing two fUSE5 derived phage bearing five peptides reacting with the anti-cryptococcal polysaccharide antibody 2H1, that the reverse ELISA is the more sensitive assay. Further, there is a limit in affinity, here around 1 microM, above which phage clones are negative by the direct ELISA. We describe an enhancement of the direct assay by mixing 2H1 with 3-fold excess of anti-heavy or anti-light chain antibody. The resulting polymerization of 2H1 induces an increase in antibody avidity that is responsible for the enhancement. The enhanced direct ELISA allowed rapid and sensitive detection of positive clones and is easily inhibited by free peptide, while the reverse ELISA is not. The enhanced ELISA has also been used successfully for immunological screening of intermediate libraries, allowing detection of rare positive clones that would otherwise be lost. The combination of the three ELISAs, reverse, direct, and enhanced direct, should provide a way to rank phage clones into three classes: very low, low, and high affinity, providing information previously obtained only by the synthesis and testing of many peptides.     

Familial brain wave patterns: study of a 12-sib family

To identify peptide-specific antibodies which define sperm surface antigens, hybridomas were derived from the splenocytes of mice immunized with swollen human spermatozoa which had been subjected to limited proteolytic cleavage under reducing conditions prior to immunization. A total of 13.7% of the hybrid clones secreted antibodies which reacted with deglycosylated human seminal plasma glycoproteins when screened by an ELISA. A monoclonal antibody, designated mAb 4A8 sp., specifying a peptide epitope of human epididymal and a sperm surface glycoprotein was selected which inhibited human sperm-egg binding in a dose-dependent manner, and totally blocked sperm penetration in vitro. This inhibition did not result from an effect of the antibody on the motility of spermatozoa, nor was it due to premature induction of the acrosome reaction. Exclusion of oligosaccharide chains by chemical hydrolysis with trifluoromethane sulphonic acid (TFMS), enzymatic degradation and binding of lectins, did not abrogate the reactivity of mAb 4A8 to the cognate epitope whereas antibody binding was precluded upon digestion with proteolytic enzymes. In Western immunoblots of human seminal plasma glycoproteins, the antigen presented as a set of immunoreactive polypeptides, a major glycoprotein of M(r) 78 kDa and less prominent bands of M(r) 56 and 44 kDa. Immunocytochemical staining of a number of human reproductive and somatic tissues revealed strong immunostaining of the luminal epithelium of the epididymis as well as of spermatozoa in the lumen. Immunolocalization to the plasma membrane of ejaculated human spermatozoa was demonstrated by immunofluorescence, although on undigested spermatozoa the antigen epitope was less accessible. Upon capacitation the antigen persisted on the sperm surface and was present on the head of capacitated acrosome-intact spermatozoa. The pronounced peripheral immunostaining of the sperm head was accentuated after DTT/trypsin treatment, implicating the dynamic accessibility of the epitope on the plasma membrane of capacitated spermatozoa. It is suggested that the protein in question appears on the sperm membrane as a consequence of its modification in the epididymis (insertion and processing), and may be involved in the processes leading to sperm attachment and interaction with the human zona pellucida.     

Variant antibody identification by peptide mapping

During development of CGP56901, a monoclonal antibody (MAb) specific for a unique epitope on human IgE, the protein A-purified IgG from one of the candidate production cell lines, showed an additional minor heavy chain (H-chain) band with a molecular weight slightly lower than that of the principal H-chain band on SDS-PAGE. The N-terminal amino acid sequence of this minor H-chain species indicated that at least the first 30 amino acids were identical to those of the antibody light-chain (L-chain) variable domain. More detailed studies using peptide mapping and amino acid sequencing analysis confirmed a crossover event between the V genes of the antibody. The position is between Arg108 of the L chain and Ala124 of the H chain. This crossover resulted in a variant H chain, which had 16 fewer amino acid residues than the normal CGP56901 H chain. These results show that peptide mapping is a useful "first-line" analytical tool in the characterization of the quality of the monoclonal antibody.     

Recognition of three epitopic regions on invasion plasmid antigen C by immune sera of rhesus monkeys infected with Shigella flexneri 2a

The invasive ability of Shigella spp. is correlated with the expression of several plasmid-encoded proteins, including invasion plasmid antigen C (IpaC). By characterizing the antigenic structure of IpaC with monoclonal antibodies and convalescent-phase sera, it may be possible to determine the physical location of specific epitopes as well as the involvement of epitopes in a protective immune response or the host's susceptibility to disease. By using overlapping octameric synthetic peptides, which together represent the entire IpaC protein, the precise linear sequence of four surface-exposed epitopes was defined for four IpaC monoclonal antibodies. Furthermore, 17 unique peptide epitopes of IpaC were mapped by using 9-day-postinfection serum samples from 13 rhesus monkeys challenged with Shigella flexneri 2a. Each individual recognized a somewhat different array of IpaC peptide epitopes after infection with shigellae. However, the epitopes were clustered within three regions of the protein: region I (between amino acid residues 1 and 61), region II (between amino acid residues 177 and 258), and region III (between amino acid residues 298 and 307). Region II was recognized by 92% of S. flexneri-infected individuals and was considered to be a highly immunogenic region. Animals asymptomatic for shigellosis after challenge with S. flexneri recognized peptide epitopes within all three epitopic regions of IpaC, whereas symptomatic animals recognized peptides in only one or two of the epitopic regions. Antibody from monkeys challenged with S. sonnei recognized IpaC peptide epitopes which fell within and outside the three S. flexneri epitopic regions. While numerous potential epitopes exist on the IpaC protein, the identification of three regions in which epitopes are clustered suggests that these regions are significant with respect to the immune response and to subsequent pathogenesis postinfection.