Molecular area involved in the in-vitro dimerization of the murine p55 IL-2 receptor.

In order to study structure-function relationships of the M(r) 55,000 subunit of the murine IL-2R (p55 IL-2R) by epitope mapping, we have expressed the p55 IL-2R molecule in a cell-free translation system. Under these in vitro conditions, we detected the expected p55 IL-2R polypeptide initiated at Met 1 and, in addition, two internally initiated molecules at Met 64 and Met 105. In this report we describe that from such a mixture, containing three molecular species of p55 IL-2R, mAb 135D5 immunoprecipitated the polypeptide initiated at Met 105 although this N-terminally truncated form of p55 IL-2R does not contain its epitope located between amino acids 72-88. This observation can be taken as a further evidence for the formation of p55 IL-2R dimers. Finally, we identified the region implicated in the formation of p55 IL-2R dimers close to the transmembrane region of the molecule.     

IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.     

The mapping of an antibody-binding region on the Mycobacterium tuberculosis 19 kilodalton antigen.

To localize the epitopes of four independently derived murine mAb IT-10, IT-12, IT-16, and IT-19 on the 19-kDa Ag protein of Mycobacterium tuberculosis, expression plasmids were constructed containing deletions of the gene encoding the 19-kDa protein. Reaction of the 4 mAb with Western blots of the truncated recombinant proteins revealed two epitope specificities in the recognition of the 19-kDa protein. IT-10 was found to be dependent only on the presence of amino acids surrounding the first cysteine residue, whereas IT-12, IT-16, and IT-19 all required the presence of both the first and third cysteine residues. These two cysteine residues are separated by 135 amino acids, and are considered to be brought together by tertiary folding of the protein to form an assembled epitope for IT-12, IT-16, and IT-19. These three mAb demonstrated differing sensitivities to the modification of reduced 19-kDa protein using iodoacetamide: a treatment that should have prevented the reformation of disulfide bonds within the protein. This result suggests that, although IT-12, IT-16, and IT-19 appear to be specific for the same epitope, there are probably fine-specificity differences in this recognition. IT-10 was not sensitive to the absence of disulfide bonds within the 19-kDa protein, suggesting that the epitope is not conformationally sensitive, and is likely to be linear in nature.     

Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

The receptor for interleukin-5 (IL-5) is composed of two different subunits. The IL-5 receptor alpha (IL-5R alpha) is required for ligand-specific binding while association with the beta-chain results in increased binding affinity. Murine IL-5 (mIL-5) has similar activity on human and murine cells, whereas human IL-5 (hIL-5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL-5 by their respective murine counterpart yields a molecule which is as potent as mIL-5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL-5R alpha subunit, we have used chimeric IL-5R alpha molecules to define regions of hIL-5R alpha involved in species-specific hIL-5 ligand binding. We found that this property is largely determined by the NH2-terminal module of hIL-5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2-terminal module and the membrane proximal (WSXWS-containing) module of hIL-5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta-strands on the proposed tertiary structure of the NH2-terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discrimination between rat thiostatin (T-kininogen) and one of its cystatin-like inhibitory fragments by a monoclonal antibody, and localization of the epitope.

A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats.     

Functional analysis of DR17(DR3)-restricted mycobacterial T cell epitopes reveals DR17-binding motif and enables the design of allele-specific competitor peptides.

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.     

Epitope characterization of ovalbumin in BALB/c mice using different entry routes

Ovalbumin (OVA) is known as a major allergen in egg white. A number of studies have reported the partial T and B cell epitope mapping of OVA using murine models and allergic patients' sera. Recently, we have reported the IgE-binding regions of the entire OVA molecule using egg allergic patients' sera. However, the entire epitope mapping of OVA in a murine model has not been completed yet. In the present study, BALB/c mice were administered a solution of OVA using three different entry routes (oral, intraperitoneal and subcutaneous) with their respective adjuvant (cholera toxin, aluminum hydroxide and Freund's adjuvant). Two nitrocellulose membranes containing 188 overlapping synthetic peptides (with a length of 12 amino acids and an offset of two amino acids) covering the primary sequence of OVA, were probed with the three different BALB/c mice antisera. Antisera obtained from orally challenged mice identified eight IgE epitope regions, i.e. I53D60; V77R84; S103E108; G127T136; E275V280; G301F306; I323A332 and A375S384, while sera raised by intraperitoneal and subcutaneous injections exhibited two (K55D60 and K277L282) and five (K55R58; G127T136; K279L282; T303S308 and I323A332) IgE binding sequences, respectively. The residues critical for the epitope-paratope interactions were finely characterized using the oral immunization serum. Analysis of IgE binding epitopes in mice provides us with potential strategies for design of specific immunotherapy.     

Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.

Methods and Results

To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope ¹⁷³LPAPTS¹⁷⁸ is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.

Conclusions and Significance

We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.     

A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype.

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC-binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site-specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC.     

Molecular identification of the human melanocortin-2 receptor responsible for ligand binding and signaling

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why alpha-MSH was not able to bind hMC2R.     

DiSSiMiL: Diverse Small Size Mini-Libraries applied to simple and rapid epitope mapping of a monoclonal antibody.

Methods for screening protein-protein interactions are useful in protein science and for the generation of drug leads. We set out to develop a simplified assay to rapidly test protein-protein interactions, with a library of 400 pentapeptides comprising the 20 natural amino acids at two variable positions followed by three glycines (NH2-X1X2GGG). The library was used to identify the epitope of monoclonal antibody (mAb) 10D11 directed against the HOXD4 protein. Three pentapeptide 'hits' were selected (VYGGG, PWGGG and WKGGG) from direct binding assays screening for pentapeptide-mAb interactions; and from assays using pentapeptides in solution to competitively block HOXD4-mAb interactions. Alignment of the three 'hit' pentapeptides to the HOXD4 sequence predicts the mAb 10D11 epitope as NH2-VYPWMK. Synthesis of NH2-VYPWMK hexapeptide confirmed this prediction; and an alanine scan of HOXD4 ablated binding by mAb 10D11 when amino acids in the putative epitope were mutated. We propose that these simplified but diverse libraries can be used for rapid epitope mapping of some mAbs, and for generating lead small peptide analogs that interfere with receptor-ligand or other protein-protein interactions, or with enzymatic activity.     

Structure-function analysis of human IL-6. Epitope mapping of neutralizing monoclonal antibodies with amino- and carboxyl-terminal deletion mutants.

To study the active site(s) of IL-6 we combined mutagenesis of IL-6 with epitope mapping of IL-6 specific mAb. In addition to amino-terminal deletion mutants we described previously, carboxyl-terminal deletion mutants were prepared. Functional analysis showed that deletion of only five carboxyl-terminal amino acids already reduced the bioactivity 1000-fold. A panel of mAb to IL-6 was subsequently analyzed by antibody competition experiments and binding to the amino- and carboxyl-terminal deletion mutants. On the basis of the competition experiments the six neutralizing mAb were divided in two groups (I and II). The binding pattern with the deletion mutants suggested that the region recognized by the four mAb in group I is composed of residues of amino- and carboxyl-terminus: binding of two mAb was abolished after deletion of amino acid Ala I-Ile26, of the third mAb after deletion of the four carboxyl-terminal amino acids whereas the fourth mAb did not bind to either mutant. Group II mAb retained binding to these mutants. Taken together these data suggest that in the native IL-6 molecule amino acid residues of amino and carboxyl terminus are in close proximity and that together they constitute an active site. Furthermore our data suggest that the part of the molecule recognized by group II antibodies is a second site involved in biologic activity.     

Presence of a p70 IL-2-binding peptide on leukemic cells from various hemopoietic lineages

Recent studies have shown that IL-2R are composed of at least two polypeptide chains of 55 kDa (Tac or alpha-chain) and 70 to 75 kDa (p70 or beta-chain). The association of both chains forms high affinity IL-2R, whereas each chain alone binds IL-2 with a low (alpha-chain) or intermediate (beta-chain) affinity. So far, the p70 peptide has been found, in the absence of the Tac peptide, on the surface of lymphoid cells of T, B, or NK lineage. In this study, we investigated whether leukemic cells of various hemopoietic lineages expressed the p70 IL-2-binding protein. We found that both fresh leukemic cells obtained from patients, and cells from established leukemic lines of T cells, B cell, and myeloid origin constitutively expressed a p70 IL-2-binding protein on their surface, as detected by affinity cross-linking of radioiodinated IL-2. IL-2 binding and cross-linking to these cells was completely inhibited in the presence of an excess unlabeled rIL-2, but not with an anti-Tac mAb. Binding experiments on pre-B and myeloid cell lines revealed intermediate affinity IL-2R, whereas both high and intermediate affinity IL-2R were detected in T leukemic cells. The intermediate affinity binding of 125I-rIL-2 to the leukemic cell lines MOLT4 and Reh6 was inhibited by the TU27 mAb, which recognized the p75 chain of IL-2R. Moreover, the TU27 mAb could stain the K562, KM3, and MOLT4 (weakly) cell lines by indirect immunofluorescence. A high dose of rIL-2 (400 U/ml) enhanced the proliferation of cells from one out of three patients with acute myeloblastic leukemia, but it did not induce differentiation of the cells in any of three cases. Thus the finding of p70 IL-2-binding molecules on immature lymphoid and nonlymphoid hemopoietic cells should disclose new biologic functions for IL-2.     

Monoclonal antibodies to the calcium-binding region peptide of human C-reactive protein alter its conformation.

Five mouse mAb were generated against a synthetic peptide corresponding to the proposed Ca(2+)-binding region of human C-reactive protein (CRP). The peptide consists of amino acids 134 to 148 and possesses a calmodulin Ca(2+)-binding sequence. The mAb reacted with a surface epitope(s) on native, intact CRP as well as the closely related pentraxin protein, serum amyloid P-component. Three of the 5 mAb inhibited the Ca(2+)-dependent phosphorylcholine-(PC) binding activity of CRP, but did not bind to the PC-binding region itself. Four of the five mAb also inhibited the recognition of an epitope in the PC-binding site of CRP. Four of the mAb partially, or completely, protected CRP from selective cleavage by pronase between residues 146 and 147. The findings suggest that the Ca(2+)-binding region is on the surface of CRP, has substantial flexibility, and is probably responsible for the allosteric effects of Ca2+ ions on CRP.     

Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr(32)Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe(94)Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe(94)Ala up to the level of the e-pep affinity to the wild-type scFv.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope.     

A consensus tetrapeptide selected by phage display adopts the conformation of a dominant discontinuous epitope of a monoclonal anti-VWF antibody that inhibits the von Willebrand factor-collagen interaction

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides

Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.     

Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

Background

The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported.

Methods and Results

This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched ²¹³SVQYHPL²¹⁹ of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that ²¹³SVQYHPL²¹⁹ was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group.

Conclusions and Significance

We identified ²¹³SVQYHPL²¹⁹ as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.