Further studies on the soluble form (gs) of rabies virus glycoprotein (g): molecular structure of gs protein and possible mechanism of the shedding

In this study, we investigated the antigenic structures and maturation of some C-terminal-deficient derivatives of rabies virus glycoprotein (G). The Gs protein, a soluble form of G protein shed from infected cells, displayed antigenicity to most of our conformational epitope-specific anti-G mAbs, but took the 1-30-44 epitope-deficient conformation (termed G(C) form). (The 1-30-44 epitope was acid-sensitive and dependent on two separate regions, the Lys-202-containing and Asn-336-containing regions; Kankanamge et al., Microbiol. Immunol., 47: 507-519). Intact G proteins took the 1-30-44 epitope-positive form (referred to as G(B) form) on the cell surface, but not inside the cell. A deletion mutant G(1-429) (termed GDeltaTC), lacking the transmembrane (TM) and cytoplasmic domains, was shown to be accumulated in the rough endoplasmic reticulum (rER) with BiP and did not seem to be shed. Another C-terminal-deficient mutant G(1-462) (termed CT1) was deprived of the whole cytoplasmic domain except for a basic amino acid left at the C-terminus, but was transported to the cell surface, where it showed pH-dependent cell fusion activity and almost full antigenicity to most of the anti-G mAbs with the exception of very weak antigenicity to mAb #1-30-44. No Gs protein could be detected in the CT1-producing cultures. Based on these results, we think that the cytoplasmic domain was not necessary for the G protein to be transported to the cell surface, but was necessary to keep its 1-30-44 epitope-positive G(B) conformation. Gs proteins might have lost the C-terminal regions during the maturation process after being exported from the rER.     

Studies on the escape mutants of rabies virus which are resistant to neutralization by a highly conserved conformational epitope-specific monoclonal antibody #1-46-12

We investigated a virus-neutralizing conformational epitope of the rabies virus glycoprotein (G) that is recognized by an anti-G monoclonal antibody (mAb; #1-46-12) and shared by most of the laboratory strains of the virus. To investigate the epitope structure, we isolated escape mutants from the HEP-Flury virus (wild-type; wt) after repeated passages in culture in the presence of the mAb. Immunofluorescence studies indicated that the mutants could be classified into two groups; the Group I lacked the epitope, while Group II preserved the epitope. The latter was dominant under the passage conditions, since Group I disappeared during the continuous passages. G proteins showed different electrophoretic mobilities; G protein of Group I migrated at the same rate as wt G protein, while that of Group II migrated at a slower rate, which was shown to be due to acquisition of an additional oligosaccharide side chain. Nucleotide sequencing of the G gene strongly suggested that amino acid substitutions at Thr-36 by Pro and Ser-39 by Thr of the G protein are responsible for the escape mutations of Groups I and II, respectively. The latter is a unique mutation of the rabies virus that allows the G protein to be glycosylated additionally at Asn-37, a potential glycosylation site that is not glycosylated in the parent virus, in preserving the epitope-positive conformation. These results suggest that to keep the 1-46-12 epitope structure is of greater survival advantage for the virus to escape the neutralization than to destroy it, which could be achieved by acquiring an additional oligosaccharide chain at Asn-37.     

Two different conformations of rabies virus glycoprotein taken under neutral pH conditions

We previously reported that the rabies virus glycoprotein (G) takes either of two different conformations (referred to as B and C forms) under neutral pH conditions, that could be differentiated by their reactivity to a monoclonal antibody (mAb), #1-30-44, that recognizes the acid-sensitive conformational epitope, and the formation taken is dependent on two separate regions containing Lys-202 and Asn-336 of the protein (Kankanamge et al., Microbiol. Immunol., 47, 507-519, 2003). Semi-quantitative antibody-binding assays demonstrated that only one-third to one-fourth of mature G proteins on the cell surface were taking the 1-30-44 epitope-positive B form even at pH 7.4. The ratio of B to C varied, depending on the environmental pH, but did not decrease to zero even at pH 5.8-6.2, preserving a certain content (about 15-20%) of B form. Immunoprecipitation studies demonstrated that a portion of G proteins were intimately associated with a dimer form of matrix (M) protein in terms of resistance to treatment with a mixture of 1% deoxycholate and 1% Nonidet P-40, and seemed to preserve the B form even at lower pHs. Similar results were also obtained with the virion-associated G proteins, including the intimate association of a portion of the G proteins with the M protein dimer. From these results, we assume that a certain portion of the rabies virion-associated G proteins are associated with a dimer form of M protein, keeping the 1-30-44 epitope-positive B conformation under various pH conditions, which might possibly assure the virion's recognition of host cell receptor molecules in the body.     

Further characterization of the rabies virus glycoproteins produced by virus-infected and G cDNA-transfected cells using a monoclonal antibody, #1-30-44, which recognizes an acid-sensitive epitope

Expression of rabies virus glycoprotein (G) by G cDNA-transfected mammalian cells resulted in the production of only a fusion-negative form. Low pH-dependent fusion activity, however, was seen when the expression was done under control of the T7 promoter with the help of recombinant vaccinia virus (RVV-T7) that provided T7 RNA polymerase. Fusion-inactive G proteins were transported to the cell surface as being detected by a conformational epitope-specific monoclonal antibody (mAb; #1-46-12). The fusion-inactive G proteins were recognized by most of our 13 conformation-specific mAbs, except for one mAb, #1-30-44, that recognized the low pH-sensitive conformational epitope. When the G gene expression was done with the help of RVV-T7, although most G proteins remained in the epitope-negative form, a small fraction of G gene products were 1-30-44 epitope-positive, and cell fusion activity could be seen when cells were exposed to low pH conditions. From these results, we conclude that acquisition of low pH-dependent fusion activity is closely related to structural maturation of the G protein to form the low pH-sensitive 1-30-44 epitope. Such maturation seems to be dependent on certain rabies virus-induced cellular conditions or functions, which might also be provided in part by the vaccinia virus infection. We further assume that expression of G cDNA alone mostly results in the production of mis-folded and/or differently folded forms of G protein, and only a small fraction is correctly folded even under RVV-T7-mediated expression conditions.     

Structural difference recognized by a monoclonal antibody #404-11 between the rabies virus nucleocapsid (NC) produced in virus infected cells and the NC-like structures produced in the nucleoprotein (N) cDNA-transfected cells

We investigated structural changes in the rabies virus (HEP-Flury strain) nucleocapsid (NC) during the virus replication, for which we used two anti-nucleoprotein (N) monoclonal antibodies (mAbs), #404-11 (specific for a conformation-dependently exposed linear epitope) and #1-7-11 (specific for a conformational epitope which is exposed after the nucleocapsid formation). Both mAbs recognized the N protein of the viral NC, but not of the RNA-free N-P complex. The 1-7-11 and 404-11 epitopes could be mapped to the N-terminal and the C-terminal regions of N protein, respectively. Immunoprecipitation studies demonstrated that treatment of the NC either with the alkaline phosphatase or sodium deoxycholate (DOC) resulted in dissociation of most P proteins from the NC and in the reduced reactivity to mAb #404-11, but not to mAb #1-7-11. NC-like structures produced in the N cDNA-transfected cells displayed strong reactivity to mAb #1-7-11; however, reactivity to mAb #404-11 was very weak. And, coexpression with viral phosphoprotein (P) resulted in little increase in reactivity to mAb #404-11 of the NC-like structures, while the reactivity was significantly increased by cotransfection with P and the viral minigenome whose 3'- and 5'-end structures were derived from the viral genome. From these results, we assume that, although the 404-11 epitope is a linear one, the epitope-containing region is exposed only when N proteins encapsidate properly the viral RNA in collaboration with the P protein. Further, exposure of the 404-11 epitope region might be function-related, and be regulated by association and dissociation of the P protein.     

Studies on the conditions required for structural and functional maturation of rabies virus glycoprotein (G) in G cDNA-transfected cells

When the rabies virus G cDNA was expressed with the help of T7 RNA polymerase provided by a recombinant vaccinia virus (RVV-T7), functional G proteins were produced in terms of their ability to induce low pH-dependent syncytium formation and the formation of conformational epitopes, including the acid-sensitive epitope recognized by mAb #1-30-44. Such an ability and the 1-30-44 epitope formation, however, were not associated with the G gene products when G cDNA was expressed without the help of RVV-T7 using a tetracycline-regulated expression vector (pTet-G), although they were normally transported to the surface of established G protein-producing BHK-21 (G-BHK) cells. But, when the G-BHK cells were treated with 2.5 m M sodium butyrate (NaB) after the removal of tetracycline, we could observe not only a much increased frequency of G protein-producing cells, but also the greatly enhanced maturation of the protein. Another short acylate, sodium propionate (NaP), similarly induced increased G protein synthesis at a concentration of 2.5 m M as NaB; however, such proteins were mostly not endowed with the fusion activity nor the 1-30-44 epitope, while NaP at a higher concentration as 5.0 m M did induce similarly the increased production and enhanced maturation of G protein, including the 1-30-44 epitope formation. From these results, we conclude that functional maturation of G protein to acquire fusogenic activity is correlated with 1-30-44 epitope formation, and 2.5 m M NaB not only stimulates G protein production, but also provides such cellular conditions as are required for the structural and functional maturation of the protein.     

Identification of a Phosphatase-Sensitive Epitope of Rabies Virus Nucleoprotein Which Is Recognized by a Monoclonal Antibody 5-2-26

We have investigated a phosphatase-sensitive sequential epitope of the nucleoprotein (N), one of the phosphoproteins of rabies virus, which is recognized by the monoclonal antibody (MAb) #5-2-26. The epitope was shared in common by all of the rabies virus strains we tested, including the HEP, ERA, CVS and Japanese strains (Nishigahara and Komatsukawa). Thin layer chromatography of the acid hydrolyzates of ³²P-labeled N protein showed that the protein contained phosphoserine and phospho-threonine at a molar ratio of about 4 to 1, while no phosphotyrosine was detected. Immunoprecipitation studies with several deletion mutants of the N protein showed that the epitope is located in a region spanning from amino acid 344 to 415. If the phosphatase-sensitive epitope is located at or near the phosphoamino acid, the location of the latter could be narrowed further to a region from amino acid 354 to 389 by comparing the amino-acid sequences among the viral strains. To examine this assumption, point mutation was introduced by amino-acid substitution with alanine at either of five potential phosphorylation sites (i.e., positions 354, 375, 377, 386 and 389) in the 354–389 region. Among those, only one substitution, at position 389, greatly affected the antigenicity. Substitution of serine-389 by threonine also reduced the antigenicity. These results strongly suggest that serine-389 is a phosphorylation site and essential for constructing or stabilizing the antigenic structure for MAb 5-2-26.     

Monoclonal antibody #3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3-9-16, that recognized a linear epitope located at the N-terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Malpha) whose 3-9-16 epitope is hidden, and an N-terminal-exposed epitope-positive form (Mbeta). The Mbeta protein accounted for about 25-30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mbeta antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mbeta antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb-dependent complement-mediated cytolysis. Similarly, the Mbeta antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mbeta) of which exposes the 3-9-16 epitope located in the N-terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Malpha), probably through its intimate association with the Golgi area and/or the cell membrane.     

Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody,RG719

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

Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine.

We have mapped a linear epitope recognized by the virus-neutralizing monoclonal antibody 6-15C4 within the primary sequence of the G protein from the Evelyn-Rokitnicki-Abelseth strain of rabies virus. This was accomplished by using fragments of the rabies virus G protein and deduced amino acid sequences of neutralization-resistant variant rabies viruses. The monoclonal antibody 6-15C4 specifically recognized a synthetic peptide (peptide G5-24) which resembles the 6-15C4 epitope in structure. In addition, a tandem peptide constructed from the G5-24 peptide and a dominant TH cell epitope of the rabies virus N protein induced protective immunity against lethal rabies virus challenge infection in mice.     

汉滩病毒S基因的分段表达及其线性和构象型抗原表位分析

构建汉滩病毒76—118N蛋白及其分别从N-端和C-端缺失的共6个突变体,在大肠杆菌BL-21中进行表达,并对其中一些蛋白进行了纯化。通过Western blot、酶联免疫吸附试验(ELISA)进行汉滩病毒N蛋白的抗原表位分析,N蛋白及6个缺失突变体都与组特异性抗体L13F3呈阳性反应,而缺失突变体与型特异性抗体AH30呈阴性反应。构建汉滩病毒76—118N蛋白及其6个缺失突变体的真核表达载体,并在COS-7细胞中进行表达。通过间接免疫荧光试验(IFA)进行汉滩病毒N蛋白的抗原表位分析,病人血清与真核表达的N蛋白及6个缺失突变体呈阳性反应。而仅有N蛋白及缺失N端1～30位氨基酸序列的NPN30与型特异性抗体AH30呈阳性反应。证实组特异性抗体L13F3结合的抗原表位位于N端1～30位氨基酸;而C端抗原表位对于型特异性抗体AH30与N蛋白的识别和结合具有重要意义,缺失N端100位氨基酸序列可能破坏羧基端构象型表位,也可以影响N蛋白与AH30的结合。     

Further studies on the mechanism of rabies virus neutralization by a viral glycoprotein-specific monoclonal antibody, #1-46-12

We previously reported that a conformational epitope-specific monoclonal antibody (mAb; #1-46-12) neutralized the rabies virus by binding only a small number (less than 20) of the antibody molecules per virion, while a linear epitope-specific mAb (#7-1-9) required more than 250 IgG molecules for the neutralization. We also isolated both the epitope-negative (R-31) and-positive (R-61) escape mutants that resisted mAb #1-46-12. Co-infection studies with wild type (wt) and R-61 mutant have shown that although the infectivity of R-61 mutant was not affected by the binding of about 300 IgG molecules per virion, incorporation of a small number of wt G protein into the R-61 virion resulted in dramatic loss of the resistance. In this study, we further investigated properties of the mutant G proteins. The R-61 G protein lost reactivity to the mAb when solubilized, even keeping a trimer form, suggesting that membrane-anchorage is essential for the maintenance of its epitope-positive conformation. On the other hand, incorporation of wt G proteins into the R-31 virions did not affect their resistance to the mAb very much. Although we have not so far found the presumed conformational changes induced by the mAb-binding, we think that these results are not inconsistent with our previously proposed novel model (referred to as a domino effect model) for the virus neutralization by mAb #1-46-12 other than a classical spike-blocking model, which implicates successive spreading of the postulated antibody-induced conformational changes of G protein to the neighboring spikes until abolishing the host cell-binding ability of the virion.     

A Unique Mutation of Glycoprotein Gene of the Attenuated RC-HL Strain of Rabies Virus,a Seed Virus Used for Production of Animal Vaccine in Japan

Although the RC-HL strain of rabies virus is avirulent in adult mice, the amino acid at position 333 of its G protein is arginine, which is thought to be necessary for virulence in adult mice upon intracerebral inoculation of the virus. This result suggests that besides arginine at position 333, some other positions of G protein might also be involved in determining the virulence of rabies virus.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.     

Epitope diversity of hepatitis B virus capsids: quasi-equivalent variations in spike epitopes and binding of different antibodies to the same epitope

To investigate the range of antigenic variation of HBV capsids, we have characterized the epitopes for two anti-capsid antibodies by cryo-electron microscopy and image reconstruction of Fab-labeled capsids to approximately 10A resolution followed by molecular modeling. Both antibodies engage residues on the protruding spikes but their epitopes and binding orientations differ. Steric interference effects limit maximum binding to approximately 50% average occupancy in each case. However, the occupancies of the two copies of a given epitope that are present on a single spike differ, reflecting subtle distinctions in structure and hence, binding affinity, arising from quasi-equivalence. The epitope for mAb88 is conformational but continuous, consisting of a loop-helix motif (residues 77-87) on one of the two polypeptide chains in the spike. In contrast, the epitope for mAb842, like most conformational epitopes, is discontinuous, consisting of a loop on one polypeptide chain (residues 74-78) combined with a loop-helix element (residues 78-83) on the other. The epitope of mAb842 is essentially identical with that previously mapped for mAb F11A4, although the binding orientations of the two monoclonal antibodies (mAbs) differ, as do their affinities measured by surface plasmon resonance. From the number of monoclonals (six) whose binding had to be characterized to give the first duplicate epitope, we estimate the total number of core antigen (cAg) epitopes to be of the order of 20. Given that different antibodies may share the same epitope, the potential number of distinct anti-cAg clones should be considerably higher. The observation that the large majority of cAg epitopes are conformational reflects the relative dimensions of a Fab (large) and the small size and close packing of the motifs that are exposed and accessible on the capsid surface.     

应用噬菌体展示随机肽库淘筛mAb 5H5识别的抗原表位

本文利用噬菌体随机9肽库探索汉滩病毒（HTNV）核衣壳蛋白（NP）B细胞抗原表位。以抗HTNV NP单克隆抗体(mAb）5H5作为筛选分子,生物淘洗噬菌体递呈的随机9肽库。阳性克隆经夹心ELISA、竞争ELISA鉴定后,随机挑取10个克隆,DNA测序,与HTNV76－118株S基因进行同源性分析。结果显示筛选到的噬菌体能特异地与5H5结合,这种结合可被天然抗原所抑制。10个克隆的氨基酸序列相同,均为VRDAEEQYE,与76－118株NP氨基端的aa25-33一致。证实了该线性表位是mAb 5H5识别的表位,噬菌体肽库有助于病毒抗原表位的确定。     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.     

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.     

The mapping and reconstitution of a conformational discontinuous B-cell epitope of HIV-1

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.