Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are non-protective

Avian hepatitis E virus (avian HEV) was recently discovered in chickens with hepatitis-splenomegaly syndrome in the United States. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, and immunodominant antigenic epitopes on avian HEV ORF2 protein were identified in the predicted antigenic domains by synthetic peptides. However, whether these epitopes are protective against avian HEV infection has not been investigated. In this study, groups of chickens were immunized with keyhole limpet hemocyanin (KLH)-conjugated peptides and recombinant avian HEV ORF2 antigen followed by challenge with avian HEV virus to assess the protective capacity of these peptides containing the epitopes. While avian HEV ORF2 protein showed complete protection against infection, viremia and fecal virus shedding were found in all peptide-immunized chickens. Using purified IgY from normal, anti-peptide, and anti-avian HEV ORF2 chicken sera, an in-vitro neutralization and in-vivo monitoring assay was performed to further evaluate the neutralizing ability of anti-peptide IgY. Results showed that none of the anti-peptide IgY can neutralize avian HEV in vitro, as viremia, fecal virus shedding, and seroconversion appeared similarly in chickens inoculated with avian HEV mixed with anti-peptide IgY and chickens inoculated with avian HEV mixed with normal IgY. As expected, chickens inoculated with the avian HEV and anti-avian HEV ORF2 IgY mixture did not show detectable avian HEV infection. Taken together, the results of this study demonstrated that immunodominant epitopes on avian HEV ORF2 protein identified by synthetic peptides are non-protective, suggesting protective neutralizing epitope on avian HEV ORF2 may not be linear as is human HEV.     

IgM and IgG antibodies to hepatitis E virus (HEV) detected by an enzyme immunoassay based on an HEV-specific artificial recombinant mosaic protein

To develop an enzyme immunoassay (EIA) for IgM antibody to hepatitis E virus (HEV) (IgM anti-HEV) and IgG antibody to HEV (IgG anti-HEV), a synthetic gene encoding several liner immuno-dominant antigenic epitopes from HEV structural proteins was assembled as a chimeric recombinant mosaic protein (Mpr) with glutathione S-transferase and used as an immunodiagnostic target. In addition, a neutralization confirmation test was developed using individual synthetic peptides. Among 614 patients with acute hepatitis from 10 geographically distinct outbreaks, IgG anti-HEV was found in 546 (88.9%), with a range of 77–100% depending on the outbreak. Of 130 patients tested for IgM anti-HEV, 126 (96.9%) were positive. Among patients tested within 4 months of onset of jaundice, 37/37 (100%) were IgG anti-HEV positive. For patients from whom sera were collected 1–16 days after onset of jaundice, the geometric mean IgG titer (GMT) was 1:47,000; the GMT increased to 1:70,710 30–40 days after onset of jaundice and decreased to 1:1,778 3–4 months after the onset of jaundice. For patients tested 6–8 months after onset of jaundice, 11/12 (92%) were IgG anti-HEV positive, and the GMT was 1:2,908. IgM anti-HEV was detected in 43/43 (100%) sera collected 1–40 days after onset of jaundice, and the GMT for IgM anti-HEV was 1:10,000 at that time. For sera collected 3–4 and 6–12 months after onset of jaundice, 7/14 (50%) and 5/12 (40%) respectively, were IgM anti-HEV positive. In conclusion, an artificial mosaic protein composed of linear antigenic epitopes from open reading frame 2 (ORF2) and ORF3 of HEV has been successfully applied to the development of a sensitive and specific EIA for the detection of IgG and IgM anti-HEV activity. These assays were used for the verification of HEV infection in outbreak settings and for the diagnosis of HEV infection in sporadic cases. © 1996 Wiley-Liss, Inc.     

Antigenic Epitopes of the Hepatitis A Virus Polyprotein

Forty-two antigenic domains were identified across the hepatitis A virus (HAV) polyprotein by using a set of 237 overlapping 20-mer synthetic peptides spanning the entire HAV polyprotein and a panel of serum samples from acutely HAV-infected patients. The term "antigenic domain" is used in this study to define a protein region spanned with consecutive overlapping immunoreactive peptides. Nineteen antigenic domains were found within the structural proteins, and 22 were found within the nonstructural proteins, with 1 domain spanning the junction of VP1 and P2A proteins. Five of these domains were considered immunodominant, as judged by both the breadth and the strength of their immunoreactivity. One domain is located within the VP2 protein at position 57-90 aa. A second domain, located at position 767-842 aa, contains the C-terminal part of the VP1 protein and the entire P2A protein. A third domain, located at position 1403-1456 aa, comprises the C-terminal part of the P2C protein and the N-terminal half of the P3A protein. The fourth domain, located at position 1500-1519 aa, includes almost the entire P3B, and the last domain, located at position 1719-1764 aa, contains the C-terminal region of the P3C protein and the N-terminal region of the P3D protein. It is interesting to note that four of the five most immunoreactive domains are derived from small HAV proteins and/or encompass protein cleavage sites separating different HAV proteins. The HAV-specific immunoreactivity of each antigenically reactive peptide was confirmed by using seven HAV seroconversion panels. Collectively, these data demonstrate that HAV structural and nonstructural proteins contain antigenic epitopes that can be efficiently modeled with short synthetic peptides.     

Identification and characterization of the neutralization epitope(s) of the hepatitis E virus

The neutralization epitope(s) of the hepatitis E virus (HEV) was studied by an in vitro neutralization assay using antibodies obtained by immunization of mice with 51 overlapping 30-mer synthetic peptides spanning the region 221-660 amino acids (aa) of the HEV open reading frame 2 encoded protein (pORF2) and 31 overlapping recombinant proteins of different sizes derived from the entire pORF2 of the HEV Burma strain. Antibodies against synthetic peptides and short recombinant proteins of approximately 100 aa did not neutralize HEV, suggesting the HEV neutralization epitope(s) is conformation-dependent. However, one recombinant protein of approximately 400 aa in length comprising the pORF2 sequence at position 274-660 aa as well as all truncated derivatives of this protein containing region 452-617 aa elicited antibodies, demonstrating HEV neutralizing activity. These findings establish for the first time that the minimal size fragment, designated pB166, that can efficiently model the neutralization epitope(s) is 166 aa in length and is located at position 452-617 aa of the HEV pORF2. Additionally, antibodies against pB166 were found to cross-neutralize three different HEV genotypes, suggesting that a common neutralization epitope(s) may exist within the different HEV genotypes. Thus, recombinant proteins constructed in this study may be considered as potential candidates for the development of an HEV subunit vaccine as well as for the development of highly sensitive and specific diagnostic tests.     

Hepatitis E virus in Cuba.

The hepatitis E virus (HEV) has a global distribution and is known to have caused large waterborne epidemics of icteric hepatitis. The transmission is primarily fecal-oral. Some reports have suggested parenteral transmission of HEV from its association to hepatitis B or hepatitis C infection, or due to the development of hepatitis E after blood transfusion. Though most of the developing countries in Asia and Africa have been shown to be endemic for HEV infection, studies in the Latin American countries have been limited to Mexico, Brazil and Venezuela. We have developed an enzyme immunoassay (EIA) for IgM and IgG antibodies to a recombinant protein containing antigenic epitopes of the ORF3 region of the HEV. This system, as well as a commercial kit that includes ORF2 and ORF3 antigenic epitopes, were used to study the prevalence of anti-HEV antibodies in a sample of Cuban blood donors, acute hepatitis cases and individuals subjected to plasmapheresis. The incidence of anti-HEV IgM was compared with other viral hepatitis markers. Our findings suggest that infections due to HEV are an important viral cause of sporadic hepatitis in Cuba, and that HEV is endemic to this region of the world.     

酵母表达的重组戊型肝炎病毒结构区ORF2蛋白的抗原性鉴定

目的 对应用巴氏毕赤酵母表达系统制备的重组戊型肝炎病毒(HEV)结构区ORF2蛋白的抗原性进行鉴定。方法 以原核细胞表达的重组HEV ORF2抗原作为对照，应用间接ELISA方法鉴定重组HEV ORF2蛋白在HEV IgM和IgG抗体检测中的特异性和敏感性；并测定不同保存时间对抗原稳定性的影响。同时，比较了两种重组抗原与国外5株HEV ORF2单克隆抗体的反应特性。结果 应用酵母抗原可检测到HEV抗体的最低包被量为12．5ng／ml，可检测到HEV IgM、IgG抗体的血清最大稀释度皆为l：51200重组蛋白与其他类型肝炎患者血清无交叉反应，37℃加速试验证实重组蛋白4℃保存12个月可保持良好的抗原性。各株单克隆抗体与酵母表达的HEV ORF2蛋白有更好的反应性，其中4B2、2E2与酵母表达的HEV ORF2蛋白的反应性比与原核表达抗原的反应性分别高出125倍和25倍。结论 应用巴氏毕赤酵母表达系统制备的重组HEV ORF2蛋白可能包含了更为广泛的构象依赖性抗原表位，具有良好抗原性、特异性和稳定性。提示其可作为开发戊型肝炎诊断试剂的一个独具优势的候选抗原。     

Rheumatoid-Factor-Reactive Sites on CH2 Established Analysis of Overlapping Peptides of Primary Sequence

Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis (RA) isolated on IgG affinity columns were studied for their reactivity with overlapping 7-mer peptides representative of solvent accessible regions of the CH₂ domains of IgG using a pin-ELISA assay. A panel of nine monoclonal RF from RA patients' B cells were studied in parallel. Four peptides SVFLFPP (239–245), KFNWYVD (274–280), NSTYRVVSV (297–305) and VLTVLHQNWL (305–314) reacted with most RF. Glycine substitution showed that tryptophanes at 277 and 313, tyrosine at 278, valines at 279 and 305, and leucine 314 represented important residues for RF reactivity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a restricted spectrum of reactivity for CH₂ epitopes, which often were identical to those binding to polyclonal IgM RF. Combinations of synthetic 7-mer peptides representing RF-reactive CH₂ or CH₃ epitopes produced as much as 60–90% inhibition of RF binding to IgG when peptides were preincubated with RF in free solution before completion of the reaction of RF with IgG on the ELISA plate.     

Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies

Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs.     

Varying abilities of recombinant polypeptides from different regions of hepatitis E virus ORF2 and ORF3 to detect anti‐HEV immunoglobulin M

Following infection with hepatitis E virus (HEV), anti‐HEV immunoglobulin (Ig) M is thought to develop before anti‐HEV IgG and to be a better marker for differentiating between the acute and convalescent phases of infection. In order to select polypeptides for improved detection of anti‐HEV IgM, six and three overlapping polypeptides from open reading frames (ORFs) 2 and 3, respectively, of HEV genotypes 1 and 4 were expressed as fusion proteins in Escherichia coli. The reactivities of the polypeptides with anti‐HEV IgM were evaluated using immunoblotting and enzyme immunoassays (EIAs). The data indicated that polypeptides from the N‐terminus of ORF3 and middle region of ORF2 were weakly or not reactive with anti‐HEV IgM, while those from the remaining regions of ORF2 and ORF3 contained reactive epitopes. Anti‐HEV IgM against the N‐ or C‐terminus of ORF2 appeared earlier and disappeared faster than that against polypeptides from the C‐terminus of ORF3, based on serum samples from rhesus monkeys infected experimentally, and from patients infected naturally, with HEV. The N‐ and C‐terminal polypeptides from ORF2 complemented one another in detecting anti‐HEV IgM and EIA sensitivity was improved significantly with a combination of these polypeptides. The reactivities of ORF2 polypeptides from genotypes 1 and 4 were similar but that of ORF3 differed with sera from monkeys infected by the two genotypes. Thus, a combination of N‐ and C‐terminal polypeptides of ORF2 from one genotype may be effective in EIAs to detect anti‐HEV IgM. J. Med. Virol. 81:1052–1061, 2009. © 2009 Wiley‐Liss, Inc.     

Fine specificity of antibodies against AQP4: epitope mapping reveals intracellular epitopes

The autoantibody to aquaporin-4 (AQP4) is a marker and a pathogenetic factor in Neuromyelitis Optica (NMO) (Devic’s syndrome). Our aim was to identify B-cell antigenic linear epitopes of the AQP4 protein and investigate similarities with other molecules. To this end, we screened sera from 21 patients positive for anti-AQP4 antibodies (study group), from 23 SLE and 23 pSS patients without neurologic involvement (disease controls) and from 28 healthy individuals (normal controls). Eleven peptides, spanning the entire intracellular and extracellular domains of the AQP4 molecule, were synthesized, and all sera were screened for anti-peptide antibodies by ELISA. Specificity was evaluated by homologous inhibition assays. NMO positive sera exhibited reactivity against 3 different peptides spanning the sequences aa1–22 (AQPpep1) (42.9% of patients), aa88–113 (AQPpep4) (33%) and aa252–275 (AQPpep8) (23.8%). All epitopes were localized in the intracellular domains of AQP4. Homologous inhibition rates were ranging from 71.1% to 84.3%. A 73% sequence homology was observed between AQPpep8′ aa257–271, a 15-mer peptide part of the AQPpep8 aa252–275, and the aa219–233 domain of the Tax1-HTLV-1 binding protein (TAX1BP1), a host protein associated with replication of the Human T-Lymphotropic Virus 1 (HTLV-1). Antibodies against the AQP4 and the TAX1BP1 15-mer peptides were detected in 26.3% (N = 5) and 31.6% (N = 6) of NMO positive sera (r_s = 0.81, P < 0.0001). Healthy controls did not react with these peptides, while homologous and cross-inhibition assays confirmed binding specificity. This first epitope mapping for AQP4 reveals that a significant proportion of anti-AQP4 antibodies target linear epitopes localized in the intracellular domains of the channel. One of the epitopes displays high similarity with a portion of TAX1BP1 protein.     

Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides.

Four recombinant antigens representing two distinct antigenic domains from two different strains of hepatitis E virus (HEV), were used individually to develop four ELISAs designed to detect antibodies to HEV. Both IgG and IgM class antibodies to HEV were detected in 7 of 8 pedigreed serum/plasma from known outbreaks of HEV in Mexico, Burma, Somalia and Pakistan. In addition, specific HEV-antibodies were detected in cynomolgus macaques following inoculation with various HEV strains. Anti-HEV was also detected in 8 of 386 (2.1%) randomly selected American blood donors. Supplemental tests utilizing both synthetic peptides and specific blocking assays provided additional serologic data confirming the presence of anti-HEV. Similar prevalence studies on a limited number of available sera from other geographical regions (Alaska, Japan, Germany, New Zealand, Thailand and Mexico) confirmed the presence of anti-HEV in at least 1.1 to 7.6% of the specimens.     

Expression of a hepatitis E virus (HEV)-trpE fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates

Summary A 1700 base cDNA fragment coding for the putative structural gene(s) of hepatitis E virus (HEV) was inserted into the pATH 10 expression vector. The fusion protein (C2) expressed by this plasmid was found to contain epitopes recognized by anti-HEV antibodies. C 2 protein was used in a Western blot format to examine its usefulness in detecting anti-HEV antibodies in well documented human cases of HEV and non-human primates infected with HEV. Both IgM and IgG anti-HEV could be detected in our Western blot assay. This Western blot assay was found not to detect antibodies from acute-phase sera from patients with either HAV or HBV. The C 2 protein contains broadly cross-reactive epitopes, and the Western blot assay was able to detect anti-HEV antibodies in patient sera from Asia, Africa, and North America. The optimum serum dilution for the detection of both IgM and IgG was 1:25.     

Mapping of B-cell epitopes of hemagglutinin protein of rinderpest virus

Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus HN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination.     

Study of antibody and T cell responses in rabbits immunized with synthetic human B cell epitope analogues of La (SSB) autoantigen

The aim of this study was to investigate the immunogenicity of four synthetic peptides, representing linear B cell epitopes of the human La/SSB autoantigen: 145-164 aa (p1), 289-308 aa (p2), 301-318 aa (p3) and 349-364 aa (p4), in rabbits. New Zealand White rabbits were immunized with each of the above peptides attached in four copies on tetrameric sequential oligopeptide carriers (SOC) in duplicate. Control immunizations were also performed (one rabbit each, immunized with Freud's complete adjuvant alone or with the SOC carrier alone). Animals were bled at regular intervals and sera were analysed for anti-La/SSB activity by ELISA assays using as antigen the various synthetic peptides, as well as the whole La/SSB protein. Four months after the last immunization, the animals were killed and peripheral blood mononuclear and spleen cells were co-cultured with either the peptides, the SOC carrier, or 27 peptides, covering the entire length of the human La/SSB molecule (23 amino acids long, overlapping by eight residues to each other). A specific, IgG, anti-peptide antibody response was detected, initially directed against the priming peptide, and subsequently expanded to the other La/SSB synthetic peptides. The antibody titres remained high, even 4 months after the last immunization. Sera from rabbits immunized with either p2 or p3 reacted also with the whole La/SSB protein, as was demonstrated by ELISA and immunoblot assays. No reactivities against either Ro60 or Ro52 autoantigen were found. Rabbit spleen cell reacted not only with the epitope used for the immunization but also with other La/SSB peptides. Immunization of rabbits with the major human La/SSB B cell antigenic determinants, linked to SOC carrier, induces strong and sustained antibody and T cell responses against multiple epitopes of the human La/SSB protein. Thus, La/SSB B cell linear epitopes are probably capable also of functioning as T cell epitopes, in this experimental animal.     

Mapping of linear epitopes on fibre knob of human adenovirus serotype 5

Linear antigenic epitopes on the Ad5 fibre knob (FK5) were characterised with fibre- and virion-specific antisera, using 15-mer overlapping peptides covering the knob of the fibre. They were compared with epitopes on the Ad2 fibre knob (FK2) domain. The stronger reactive FK5 epitopes were represented by peptides P3 (amino acids (aa A419-L433)), P6 (aa S449-E463), P7 (aa I459-L473), P12 (aa G509-N523), P14 (aa P529-G543) and P16 (aa A549-Y563). P3 spans the B beta-strand and the left portion of the C beta-strand, P6 and P7 the D beta-strand and the adjacent parts of the CD and DE loops, P12, P14 and P16 the G, H and I beta strands and the adjacent parts of the loops, respectively. The stronger reactive epitopes on FK2 were located in P2 (aa P409-L423), P6 (aa T449-Q463), P8 (aa E469-G483), P13 (aa Q519-T533) and P16 (aa S549-K563). The positions of FK5 and FK2 derived peptides, representing epitopes, are either identical or overlapping or adjacent, as determined by amino acid sequence alignment. Antisera obtained against several longer peptides showed virus neutralising capacity, indicating neutralising epitopes in these peptides.     

戊型肝炎病毒新潜在中和性表位的发现

目的 探讨戊型肝炎病毒(HEV)衣壳蛋白是否存在除主要免疫优势中和表位aa459～606以外的其他中和表位.方法 通过对几株单克隆抗体及其表位的性质进行分析,对比位于主要免疫优势表位区aM59～606和位于该表位N端序列aa394～458区域的数个表位的中和活性.结果 发现位于aa423～437的表位对应的单抗具有潜在中和活性,不同于已知的HEV中和性表位(aa459～606),该表位是一个线性非免疫优势表位.结论 HEV ORF2 aa423～437为新的潜在的线性非免疫优势中和表位,该发现丰富了对HEV衣壳结构的认识,为HEV预防与治疗提供了新的针对靶点.     

Occurrence of hepatitis E virus IgM, low avidity IgG serum antibodies, and viremia in sporadic cases of non-A, -B, and -C acute hepatitis.

Serum samples were taken from 57 patients with sporadic non-A, -B, and -C (Non A, B, C) acute hepatitis at different times after onset of the disease and tested for the presence of the hepatitis E virus (HEV) RNA, IgM, and low avidity IgG antibodies. The viral antibodies were detected using two ELISA. One assay (GL) was produced using a mixture of recombinant peptides specified by ORF2 and ORF3 of the viral genome. The other was produced with an ORF2 specified peptide, pE2. The latter occurs naturally as homodimer, it is recognized strongly in its dimeric form by human sera and, in the primate model, it confers protection against experimental HEV infection. Nineteen samples were positive for one or more of these acute markers of HEV infection, 14 of which were acute sera with elevated ALT levels and 5 were convalescent sera with normal ALT level. The results showed that icteric phase of sporadic hepatitis lasts for about 17 days and it coincides with a period when viremia is subsiding as HEV antibodies are developing. Viremia was intermittent and all but one of the 5 instances were confined to the icteric phase with elevated ALT levels. On two of these occasions, viremia preceded detection of HEV antibody, on another 2 occasions it was concurrent with the detection of pE2 specific IgM and/or low avidity IgG and only in one case of protracted viremia was the viral genome detected concurrently with avid pE2 IgG antibody. Ten (71%) of the 14 acute sera were reactive for pE2 IgM, eight (57%) were reactive for low avidity pE2 IgG, and six (43%) for the GL IgM. The sensitivity for the diagnosis of acute hepatitis E may be increased to 87% by combining pE2 IgM and viremia. GL IgM was detected later, but persisted for a longer period of time than the pE2 antibodies, and it was the only acute antibody detected in the convalescent sera.     

Salivary sIgA response in HIV-1 infection.

Local and systemic production of total and HIV-1 specific IgA was determined in whole saliva and serum from 45 HIV-1-infected individuals and 15 healthy controls. The antigenic domains important for sIgA and IgG binding, respectively, was investigated with epitope mapping using synthetic peptides of HIV-1 proteins. Decreased levels of total sIgA in saliva were found among patients with low CD4+ cell counts in advanced stages of acquired immunodeficiency. HIV-1 specific IgA response, predominantly directed to the envelope proteins, was found in saliva and serum also at later stages of disease. Analyses using peptide enzyme-linked immunosorbent assays (ELISA) showed that the sIgA antibody response in saliva was mainly directed to the V4 region (aa 385-409) and a more C-terminal part of the V3-region (aa 325-344) compared with the IgG response, which predominantly was directed to a more central part of the V3 loop (aa 308-325). A similar picture was seen for immunoglobulins of the two isotypes derived from serum. We have in this study shown IgA epitope-specific immune response within HIV-1 gp160, indicating that antibodies of IgA isotype may recognize somewhat different antigenic domains compared to IgG antibodies.     

The humoral immune response to the kinetoplastid membrane protein-11 in patients with American leishmaniasis and Chagas disease: prevalence of IgG subclasses and mapping of epitopes

The kinetoplastid membrane protein-11 (KMP-11) is a major target of the humoral immune response during Leishmania-infections. The majority of sera from visceral leishmaniasis, mucocutaneous leishmaniasis and even some cutaneous leishmaniasis patients contain detectable IgG antibodies against KMP-11. We also provide evidence that this protein may act as a potent antigen in T. cruzi infections, since most Chagas sera show immunological cross-reactivity. Therefore, KMP-11 cannot be used as a specific diagnostical tool for the serodiagnosis of leishmaniasis in those regions where both, Leishmania and T. cruzi infections overlap geographically. When analyzing the subclass specificity of the antibody response to KMP-11 we observed the following order of reactivity: IgG1 > > IgG3 > IgG2 > IgG4, which is similiar to that seen in crude parasite extract. The mapping of antigenic determinants by using synthetic 20-mer peptides revealed the existence of predominantly conformational epitopes in leishmaniasis, while 50% of sera from Chagas patients reacted with a particular KMP-11 peptide. These results therefore suggest the presence of disease-specific B-cell epitopes.