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PubMed2003

Comparative analysis of linear antibody epitopes on human and mycobacterial 60-kDa heat shock proteins using samples of healthy blood donors.

Growing evidence suggests that anti-Hsp60 antibodies might be involved in the pathogenesis of different autoimmune diseases. Attempts were reported also on the characterization of epitope specificity of anti-Hsp60 antibodies in different infectious and autoimmune diseases. However, there is a lack of data on the occurrence and epitope specificity of anti-Hsp60 antibodies in the healthy human antibody repertoire. Therefore, the aim of our study was to demonstrate the presence of anti-Hsp60 antibodies and to investigate the epitope specificity of these antibodies using a large set of synthetic 10 mer peptides covering regions of Hsp60 with high probability of antigenicity. Here we report the identification of several linear epitopes using serum Ig (IVIG) and sera from healthy subjects by ELISA assay. We have identified two epitopes 'specific' for human Hsp60 and two different epitopes 'specific' for Hsp65. In addition, six epitopes were cross-reactive in nature, detected on both proteins. The presence of these 'specific' epitopes may explain the differences in epitope structure between Hsp60 and Hsp65 observed earlier in patients with cardiovascular disease. The binding of the IVIG preparation to Hsp60 epitopes might indicate that anti-Hsp60 autoantibodies are present in the healthy human natural autoantibody repertoire.

Amino Acid Sequence↗
PubMed2004

Impact of intrapeptide epitope location on CD8 T cell recognition: implications for design of overlapping peptide panels.

BACKGROUND: Antigen-specific CD8 T cells following infection or immunization are typically assessed by measuring interferon-gamma production after stimulation with overlapping peptides spanning the region of interest. The effect of epitope location within such peptides is not known but may influence recognition. OBJECTIVE: To examine if peptides containing the appropriate C-terminal anchor amino acid residue would provide more sensitive detection of T cell responses. The impact was examined of epitope location within overlapping peptides on recognition of epitope-specific CD8 T cell responses. METHODS: C-terminal amino acid residues were analyzed in well-defined optimal epitopes for HIV, Epstein-Barr virus, cytomegalovirus and influenza and in peptide-binding motifs. Recognition of known epitopes within longer synthesized peptides by peripheral blood mononuclear cells or CD8 T cell lines was tested using interferon-gamma Elispot at various peptide concentrations. RESULTS: Only 9 of 20 amino acids served as the C-terminal anchor position in 96% of described optimal epitopes and in 95% of peptide-binding motifs. A CD8 T cell response to an epitope within a longer peptide is best detected when the epitope is situated at the C-terminal end of the longer peptide, both when using peptides designed to include the optimal epitope at every possible position and when comparing responses towards optimal epitopes and corresponding overlapping peptides in a larger group of subjects. CONCLUSION: When using overlapping peptides to screen for CD8 T cell responses, more sensitive detection will be achieved using known C-terminal anchor amino acid residues at the C-terminus.

Amino Acid Motifs↗
PubMedDate not supplied

CD4+ T-cell epitope determination using unexposed human donor peripheral blood mononuclear cells.

The engineering of protein therapeutics to improve their stability, their efficacy, or to create "humanized" versions introduces changes to the amino acid sequence that are potential T-cell epitopes. Until now, there has been no available assay to detect primary T-cell responses to novel epitopes in humans. Currently available in vitro protocols for epitope determination rely on peripheral blood lymphocytes from environmentally exposed or disease-bearing donors. This severely limits the opportunity to confirm T-cell epitopes in novel proteins, because exposed donors are not available to novel or engineered proteins. Other methods for determining T-cell epitopes are either computer-modeled predictions based on potential binding to HLA molecules or the identification of peptides presented by HLA molecules removed from the surface of tumor cells or protein-pulsed antigen-presenting cells. Because HLA binding is necessary, but not sufficient, for T-cell responses, these methods must be validated by in vitro presentation assays. The authors describe a dendritic cell-based assay that identifies CD4+ T-cell epitopes in novel proteins using unexposed donors. Predicted T-cell epitopes in the protein of interest were confirmed using cells from two verified exposed donors. The major CD4+ T-cell epitope of the novel protein examined in this study associated with the expression of HLA DRb1*15. This assay reflects de novo priming in vitro, and it accurately identifies primary T-cell epitopes. This assay is a powerful tool for determining relevant immunostimulatory T-cell epitopes for all types of immunoregulatory applications.

Antigen Presentation↗
PubMed2003

A new epitope-based HLA-DPB matching approach for cadaver kidney retransplants.

BACKGROUND: Several years ago a significant impact of matching for HLA-DPB1 alleles on the survival of cadaver kidney retransplants was shown. Here we report the results of a new approach, based on matching for HLA-DPB1 epitopes. METHODS: The analysis is based on 1,478 patients who received a cadaver kidney retransplant between 1988 and 1998. DNA methodology (polymerase chain reaction, sequence-specific oligonucleotides) was used to perform HLA-DPB1 typing. Epitope matching was facilitated with the aid of sequence databases and computer calculations. RESULTS: Matching for the HLA-DP epitopes A, B, E, and F, corresponding to the homonymous hypervariable regions of the second exon of the DPB1 gene, seems to have a greater influence on graft survival than matching for the epitopes C and D. Within a group of 529 retransplants with exactly one allelic HLA-DPB1 mismatch, a significantly better graft outcome was observed when less than two epitope mismatches were found, compared with the group with more than three epitope mismatches (at 2 years: 77.8% vs. 65.8%, P=0.0112). Importantly, patients with two DPB1 allele mismatches who had less than or equal to two epitope mismatches exhibited a significantly better graft outcome than recipients who had one HLA-DPB1 allelic mismatch but more than three epitope mismatches (at 2 years: 77.1% vs. 65.8%, P=0.0488). CONCLUSIONS: The findings indicate that the impact of HLA-DPB1 matching on the outcome of kidney retransplants is a result of the predominant immunogenicity of certain epitopes of the HLA-DP molecule. Matching for immunogenic HLA-DPB1 epitopes seems to be functionally more relevant than classical matching at the allelic level.

Cadaver↗
PubMed2006

Public epitope specificity of HLA class I antibodies induced by a failed kidney transplant: alloantibody characterization by flow cytometric techniques.

BACKGROUND: Patients whose kidney grafts fail develop alloantibodies that react with many HLA molecules. We analyzed the epitope specificity of HLA class I alloantibodies in the sera of 55 patients who had been sensitized by kidney grafts, and investigated the immunogenicity of various polymorphic epitopes. METHODS: HLA class I alloantibodies were detected and characterized by flow cytometry (FlowPRA beads). Potential "immunizing epitopes" were identified by comparing the amino acid sequences of HLA class I antigens/alleles of the donor, recipient and the antibody-reactivity pattern. RESULTS: In the 55 anti-HLA class I-positive patients, 82 different antibody reactivity patterns were identified; all but 5 (94%) were determined by a "public epitope" of donor HLA-A and/or -B molecules. Forty-five of 50 patients who showed HLA-A Res-MMs with their donors produced HLA-A antibodies, but only 31 of 51 subjects with HLA-B Res-MMs produced HLA-B antibodies (P=0.001; O.R.=5.81). The antibody patterns were specific for a "single" epitope of the mismatched donor molecules in 91% of patients. Forty-three of the 120 (36%) mismatched HLA-A and/or -B epitopes were positively correlated with antibody production. The polymorphic determinants of higher immunogenic capacity were b80N (Bw6-associated) and ab82-83LR (Bw4-associated) public epitopes. CONCLUSIONS: The humoral immune response against a kidney graft mainly produces HLA class I antibodies specific for "public epitopes" of mismatched donor molecules. A "single" donor-epitope may determine the production of a spread antibody pattern. In renal transplantation, epitope matching is better than HLA antigen matching for avoiding or minimizing development of HLA antibodies.

Antibody Specificity↗
PubMed1990

Structural and functional analysis of orthopoxvirus epitopes with neutralizing monoclonal antibodies.

Neutralizing monoclonal antibodies (MAbs) were produced in BALB/c mice immunized with live modified vaccinia virus Ankara or infected with sublethal doses of the neurovirulent vaccinia virus strain Munich 1. The immunization scheme proved to be important for obtaining MAbs of different specificity. The MAbs could be classified into three epitope groups (1 A, 1 B and 2). Immunogold electron microscopy demonstrated that the epitopes were localized on the virus surface. In immunoblotting, MAbs were reactive with polypeptides of 14K, 16K and 30K. Purified MAbs binding to the epitopes 1 A and 2 showed a 50% reduction of 100 p.f.u./0.05 ml vaccinia virus M1 with respectively 3.9 and 5.9 ng of immunoglobulin/0.05 ml. MAbs binding to the epitope 1 B neutralized the virus at a concentration of 250 ng/0.05 ml. In intraperitoneal challenge experiments, MAbs binding to the epitopes 1 A and 2 protected mice against 4 LD50 of vaccinia virus M1, but not against local lesions by subcutaneous application. MAbs against epitope 1 B had no protective effect in vivo. The three epitopes were present in 14 of 16 orthopoxviruses tested but with quantitative differences. Maximal binding (Vmax) and the antibody concentration at half-maximal binding (Km) which were calculated as for Michaelis-Menten kinetics from regression analysis of the ELISA data and the MAb concentration giving 50% plaque reduction were the basis for the evaluation. In monkey-pox virus Kopenhagen the epitopes 1 A and 1 B were absent. MAbs binding to epitope 2 reacted just as well as with vaccinia viruses. Ectromelia virus lacked all the epitopes.

Animals↗
PubMed1991

Epitope changes on the haemagglutinin molecule of recently isolated H1N1 influenza viruses.

We have studied changes of epitopes on the haemagglutinin molecule (HA) of H1N1 influenza viruses isolated between 1977 and 1986. For this purpose monoclonal antibodies (MAbs) were raised against the HA of the influenza A/England/333/80 and A/Yamagata/120/86 strain viruses. In order to define the amino acid residues responsible for the change of epitopes, we prepared several HA cDNAs modified by site-directed mutagenesis and cloned them into a simian virus 40 expression vector (SVHA). The substitution of glycine with serine at position 125c (suffix indicates presence in H1 but not in H3 subtype HAs) on the HA of the influenza A/USSR/90/77 strain virus resulted in the loss of epitope 110 (epitopes were named after MAbs) and created new epitopes 139 and 15, which were observed on the HA of A/England/333/80 and a few isolates from 1983. These new epitopes disappeared from the HA in some of the isolates in 1983 and most of the isolates in 1984 and 1986. The disappearance of epitopes 139 and 15 seems to be associated with the loss of epitope W18, which was identified on the HA of A/USSR/90/77. We suggested previously that amino acid residue 189 was involved in epitope W18. We therefore expressed an HA protein with two amino acid substitutions at positions 189 and 125c and found that the conversion of glutamine to lysine at position 189 in SVHA-67 prevented the expression of epitopes 139 and 15.

Animals↗
PubMed2005

Identification of IgE-binding epitopes on gliadins for patients with food allergy to wheat.

BACKGROUND: Food allergy to wheat induces different symptoms as atopic eczema/dermatitis syndrome (AEDS), urticaria and more severe reactions as wheat-dependent exercise-induced anaphylaxis (WDEIA). Different gliadin classes are involved in this allergy but IgE-binding epitopes are known only on omega5-gliadins and for WDEIA cases. OBJECTIVES: The aim of the study was to identify IgE-binding epitopes on several gliadin classes and for several patients with different symptoms and ages. METHODS: Eleven sera were analysed by pepscan with overlapping synthetic peptides. RESULTS: Sera from five patients with anaphylaxis, urticaria or WDEIA, displayed strong IgE-binding to sequential epitopes of the repetitive domains of alphabeta, gamma, omega2 or omega5-gliadins with two immunodominant epitopes on omega5-gliadin and a consensus motif of the type QQX1PX2QQ (X1 being L, F, S or I and X2 Q, E or G). One patient allergic to deamidated wheat proteins also had IgE to a repetitive peptide of gamma and omega2-gliadins of the type QPQQPFP. Sera from four patients with AEDS detected no linear epitopes on gliadins, despite the fact that they contained specific IgE to alpha, beta, gamma or omega-gliadins. One child with AEDS recognized cysteine-containing sequences in the nonrepetitive domains of alphabeta and gamma-gliadins. CONCLUSION: B epitopes in wheat allergy were different from B epitopes of coeliac disease. Differences exist in IgE-binding epitopes between patients with food allergy to wheat. IgE from those suffering from WDEIA, anaphylaxis and urticaria detected sequential epitopes in the repetitive domain of gliadins whereas IgE from AEDS patients probably recognized conformational epitopes.

Adult↗
PubMed1992

The use of multiple-pin peptide synthesis in an analysis of the continuous epitopes recognised by various anti-(recombinant bovine growth hormone) sera. Comparison with predicted regions of immunogenicity and location within the three-dimensional structure of the molecule.

A recently developed technology called epitope scanning permits the rapid and accurate delineation of continuous stretches of amino acids in a protein which constitute the sequential epitopes recognised by an antiserum raised to that protein. In the present report, we describe the use of this technique to identify the epitopes in the recombinant bovine growth-hormone (rbGH) molecule recognised by three polyclonal guinea-pig antisera and two polyclonal rabbit antisera. The results obtained show that, for guinea-pig antisera, 3 or 4, very-well-defined major continuous epitopes are present. As would be expected given the intrinsic genetic factors (major histocompatibility restriction, antigen processing and presentation) controlling the immune response in individual animals, subtle differences are evident in the precise location and relative reactivities of these epitopes in different guinea-pig antisera. Nevertheless, there is a large degree of overlap in these epitopes, such that immunodominant regions of the antigen can be clearly delineated. In a structural sense, these epitopes share a common motif in that they are sited in areas of the protein antigen with little secondary structure (loop/coil), although there is some contribution by neighbouring alpha-helices. For the two rabbit antisera, the response tends to be rather more heterogeneous, with recognition of more peptides and less clearly defined epitopes than was the case with the guinea-pig antiserum. Comparison of the four guinea-pig epitopes, identified by our experimental methods with computer predictions for this molecule (Jameson-Wolf antigenic index), indicate that two are strongly predicted, one is weakly predicted and one is not predicted. These observations, together with the displayed intraspecies and interspecies variation clearly indicate the limitations of these predictive methods. In conclusion, we have demonstrated that, despite the expected variation in the exact location of continuous epitopes defined by different anti-rbGH sera, there are large regions of overlap defining immunogenic core regions within the molecule. We believe that studies of this nature, together with further understanding of antigen processing and peptide presentation to immune cells, may have a role to play in the development of candidate peptide vaccines.

Amino Acid Sequence↗
PubMed1990

The specificity of 130-kDa leech sensory afferent proteins is encoded by their carbohydrate epitopes.

From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130-kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3-2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2-369, Laz7-79, and Laz6-212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6-297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9-84) is located on connective tissue surrounding the CNS, as well as in the peripheral tissues. The hydrophilic-hydrophobic nature of the 130-kDa sensory afferent and glial proteins was determined by phase separation with Triton X-114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130-kDa glycoproteins with N-Glycanase or preincubation of their respective mAbs with alpha-methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose-containing Lan3-2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.

Animals↗
PubMed1997

Distribution of immunogenic epitopes on the two major immunodominant proteins (rOmpA and rOmpB) of Rickettsia conorii among the other rickettsiae of the spotted fever group.

Forty-four monoclonal antibodies were raised against strain Seven, the type strain of Rickettsia conorii. Of these 44 monoclonal antibodies, 13, 27, and 4 were demonstrated to be directed against the 116-kDa protein (rOmpA), the 124-kDa protein (rOmpB), and lipopolysaccharide-like antigen, respectively. The antiprotein monoclonal antibodies were found to be directed against 29 distinct epitopes, which were located on the two major immunodominant proteins discussed above. Further analysis showed that strain-specific epitopes were located on the rOmpA protein and species- and subgroup-specific epitopes were located on the rOmpB protein. R. conorii Manuel, Indian tick typhus rickettsia, and Kenya tick typhus rickettsia also possessed all 29 epitopes, whereas the other rickettsiae of the spotted fever group (SFG) expressed between 3 and 25 epitopes, with the exception of Rickettsia helvetica, R. akari, and R. australis which did not possess any epitopes. Additional analyses by Western immunoblotting confirmed that the epitopes shared among the SFG rickettsiae were located on the same two high-molecular-mass proteins as on R. conorii. However, although epitopes on the R. conorii rOmpB protein were expressed on the rOmpB proteins of most other SFG rickettsiae, some were found on the rOmpA proteins of R. aeschlimannii, R. rickettsii, and R. rhipicephali. Both proteins possessing the common epitopes were found to have different sizes in the SFG rickettsial species. The different distributions of common epitopes in the SFG rickettsiae were also used to build a taxonomic dendrogram, which demonstrated that all the R. conorii strains formed a relatively independent cluster within the SFG rickettsiae and was generally consistent with previously proposed taxonomies.

Antibodies, Monoclonal↗
PubMed1989

Human immune response to meningococcal outer membrane protein epitopes after natural infection or vaccination.

Antibody levels in 41 sets of human acute- and convalescent-phase meningococcal sera were compared with those in 23 sets of human prevaccination and 2-week postvaccination sera. We used a modification of a solid-phase radioimmunoassay (SPRIA) technique to test each of the human serum samples as inhibitors of monoclonal antibodies (MAbs) that bind (HIMSPRIA) to the outer membrane complex from a 2a:P1.2:P5.1 strain. We used three murine MAbs specific for the 2a, P1.2, and P5.1 epitopes on meningococcal class 1, 2, and 5 proteins, respectively, to detect antibodies with similar specificities in human sera. Each of 40 available matching strains from patients were also screened with the three MAbs in a nitrocellulose spot blot assay. A total of 37 (92%) were positive for the 2a epitope, 36 (90%) were positive for the P1.2 epitope, and 16 (40%) were positive for the P5.1 epitope. Of 38 available convalescent-phase sera, 27 (71%) matched with these strains and had detectable inhibiting antibody for each of the MAb-defined protein epitopes of the infecting strain. Three convalescent-phase sera had no HIMSPRIA activity for MAb-defined epitopes that were present on the infecting strain; others had activity for one or two of the epitopes. The results were similar for pre- and postvaccination sera. The average level of HIMSPRIA activity for the P1.2 epitope was greater than fivefold higher in postvaccination sera compared with that in convalescent-phase sera. Sera with distinct patterns of HIMSPRIA activity also were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis and showed a correlation between the HIMSPRIA activity for particular epitopes and the level of antibody binding to the immunoblotted proteins possessing those epitopes. A comparison of the HIMSPRIA and the bactericidal activity of selected postvaccination sera indicated a possible correlation between HIMSPRIA and bactericidal activity, but it also suggested the presence of bactericidal antibodies with specificities other than those defined by the MAbs.

Antibodies, Bacterial↗
PubMed1994

Meningococcal group A lipooligosaccharides (LOS): preliminary structural studies and characterization of serotype-associated and conserved LOS epitopes.

Structural studies indicate that the neisserial lipooligosaccharides (LOS) are composed of an oligosaccharide (OS) portion with a phosphorylated diheptose (Hep) core attached to the toxic lipid A moiety. A conserved meningococcal LOS epitope, defined by monoclonal antibody (MAb) D6A, is expressed on group A and many group B and C meningococci of different LOS serotypes (J. J. Kim, R. E. Mandrell, H. Zhen, M. A. Apicella, J. T. Poolman, and J. M. Griffiss, Infect. Immun. 56:2631-2638, 1988). This MAb-defined D6A epitope is immunogenic in humans (M. M. Estabrook, R. E. Mandrell, M. A. Apicella, and J. M. Griffiss, Infect. Immun. 58:2204-2213, 1990; M. M. Estabrook, C. J. Baker, and J. M. Griffiss, J. Infect. Dis. 197:966-970, 1993). In this study, we characterize this important MAb-defined LOS epitope. Serotype L10 and L11 group A meningococal LOS were chemically modified and used to investigate what portion of the LOS molecule is important for expression of the conserved (D6A) epitope and serotype-associated LOS epitopes by use of immunoblotting techniques and selected MAbs as probes. Preliminary structural characterization of the LOS was also accomplished by electrospray ionization-mass spectrometry. Our results indicate the following. (i) Antibodies that recognize the serotype-associated or conserved LOS epitopes recognize the OS portion of the LOS. (ii) The phosphorylated diheptose core region of the OS is essential for expression of the conserved D6A epitope. (iii) The lipid portion of the molecule is important for optimum expression of the LOS epitopes. (iv) The proposed compositions of the O-deacylated LOS are consistent with the presence of a phosphorylated diheptose core and are as follows: for O-deacylated L10 LOS, 3Hex (hexose), 1HexNAc (N-acetylhexosamine), 2KDO (2-keto-3-deoxy-D-manno-octulosonic acid), 2Hep (heptose), 1PEA or 2PEA (phosphoethanolamine), and O-deacylated lipid A; and for O-deacylated L11 LOS, 2Hex, 1HexNAc, 2KDO, 2Hep, 2PEA, and O-deacylated lipid A. Because the phosphorylated diheptose core region of the LOS is essential for the formation of a conserved LOS epitope (D6A) that is immunogenic in humans, care should be taken to maintain stereochemical requirements for the expression of this conserved epitope in the design of effective, nontoxic LOS vaccines.

Animals↗
PubMed1995

T-cell, adhesion, and B-cell epitopes of the cell surface Streptococcus mutans protein antigen I/II.

The T-cell and antibody responses to a cell surface streptococcal antigen (SA I/II) were investigated in naturally sensitized humans. Serum antibody responses were directed predominantly to the N-terminal (residues 39 to 481) and central (residues 816 to 1213) regions of SA I/II which may be involved in bacterial adhesion to salivary receptors. T-cell responses were also directed predominantly towards the central region. The linear peptide relationship of the immunodominant and minor T- and B-cell as well as adhesion epitopes was mapped within residues 816 to 1213. Immunodominant T-cell and B-cell epitopes were identified within residues 803 to 853, which were separated in linear sequence from the adhesion epitopes (residues 1005 to 1044). Adhesion epitopes overlapped with minor B- and T-cell epitopes (residues 1005 to 1054 and 1085 to 1134). An immunodominant promiscuous T-cell epitope (residues 985 to 1004) was adjacent to an adhesion epitope (residues 1005 to 1024). The limited B-cell response to adhesion epitopes is consistent with the success of Streptococcus mutans in colonizing the oral cavity. The strategy of T-cell, adhesion, and B-cell epitope mapping has revealed a general approach for identifying components of subunit vaccines which may focus responses to critical functional determinants. Such epitopes of SA I/II may constitute the components of a subunit vaccine against dental caries.

Amino Acid Sequence↗
PubMed1996

Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.

Adhesins, Bacterial↗
PubMed1998

Conformational dependence of Anaplasma marginale major surface protein 5 surface-exposed B-cell epitopes.

The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940-2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denatured A. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.

Anaplasma↗
PubMed2004

Identification of the Entamoeba histolytica galactose-inhibitable lectin epitopes recognized by human immunoglobulin A antibodies following cure of amebic liver abscess.

Immunity to Entamoeba species intestinal infection is associated with the presence of intestinal IgA antibodies against the parasite's galactose-inhibitable adherence lectin. We determined the epitope specificity of serum and intestinal antilectin IgA antibodies by enzyme-linked immunosorbent assay using overlapping fragments of a recombinant portion of the lectin heavy subunit, designated LC3. These findings were correlated with the effects of epitope-specific murine antilectin immunoglobulin A (IgA) monoclonal antibodies (MAbs) on amebic in vitro galactose-specific adherence. LC3 is a highly antigenic and immunogenic cysteine-rich protein (amino acids [aa] 758 to 1150) that includes the lectin's carbohydrate binding domain. The study subjects, from Durban, South Africa, were recently cured of amebic liver abscess (ALA) with or without concurrent Entamoeba histolytica intestinal infection or were infection free 1 year after cure. We also studied seropositive subjects that were infected with E. histolytica, disease free, and asymptomatic. Serum anti-LC3 IgA antibodies from all study groups exclusively recognized the third (aa 868 to 944) and the seventh (aa 1114 to 1134) LC3 epitopes regardless of clinical status; epitope 6 (aa 1070 to 1114) was also recognized by serum anti-LC3 IgG antibodies. However, IgG antibody recognition of epitope 6 but not 3 or 7 was lost 1 year following cure of ALA. We produced 14 murine anti-LC3 IgA MAbs which collectively recognized five of the seven LC3 epitopes. The majority of the murine MAbs recognized the first epitope (aa 758 to 826), which was not recognized by human IgA antibodies. Interestingly, adherence of E. histolytica trophozoites to CHO cells was inhibited by MAbs against epitopes 1, 3, 4 (aa 944 to 987), and 6 (P < 0.01). The LC3 epitopes recognized by human IgA antibodies (3 and 7) were further characterized by use of overlapping synthetic peptides. We identified four peptides (aa 891 to 903, 918 to 936, 1114 to 1134, and 1128 to 1150) that in linear or cyclized form were recognized by pooled intestinal IgA antibodies and serum IgG antibodies from subjects with ALA and asymptomatic, seropositive infected subjects. This study identifies the lectin epitopes to be studied in an amebiasis subunit vaccine designed to elicit mucosal immunity mimicking that of humans cured of ALA.

Animals↗
PubMed1985

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.

Antibodies, Monoclonal↗