Two dual-specific (anti-IgG and anti-dsDNA) monoclonal autoantibodies derived from the NZB/NZW F1 recognize an epitope in the hinge region

The anti-IgG properties of two dual-specific (anti-dsDNA and anti-IgG) monoclonal NZB/NZW F1-derived autoantibodies, BV 17–45 and BV 16–13, were studied to resolve the location and possible commonality of the IgG epitope. To determine if BV 17–45 and BV 16–13 recognized the same IgG epitope, the relative temperature sensitivity of the conformational IgG epitopes were evaluated using the conformational sensitive immunoassay. Comparison of the temperature sensitivity of the conformational immunoglobulin epitopes over a temperature range of 25–100°C suggested that the epitope recognized by BV 17–45 was the same as the IgG epitope recognized by BV 16–13. Further studies with papain- and pepsin-generated F(ab′)₂, Fab, and Fc fragments of BV 17–45 and BV 16–13 revealed that the dual-specific autoantibodies BV 17–45 and BV 16–13 both bound an epitope in the hinge region of the IgG molecule. The potential correlation between these studies and the pathogenic nature of dual-specific autoantibodies is discussed.     

Dual Specificity and the Formation of Stable Autoimmune Complexes

Since dual specificity at the antibody active-site level involves new principles relative to monospecific antigen-antibody interactions and may be a general property of autoantibodies, it was important to further characterize such antibodies. Four lupus derived autoantibodies were studied to understand parameters and mechanisms involved in the participation of dual-specific antibody molecules in the formation of highly stable immune complexes. Because the dual-specific binding properties of selected lupus-related murine autoantibodies had been previously described using a solid-phase polystyrene-based ELISA, a conformational sensitive membrane based assay (CSI) was used on a comparative basis to further characterize NZB/NZW F₁ murine monoclonal anti-DNA autoantibodies BV 04–01 (anti-ssDNA), BV 16–19 (anti-ssDNA), BV 17–45 (anti-dsDNA), and BV 16–13 (anti-dsDNA). All four monoclonal autoantibodies exhibited anti-IgG binding in the solid-phase ELISA. However in the CSI assay, only anti-dsDNA monoclonal autoantibodies BV 17-45 and BV 16-13 demonstrated anti-IgG binding, while anti-ssDNA autoantibodies BV 04–01 and BV 16–19 did not. Upon subjection to time-dependent thermal denaturation, with and without thiol reduction at 100°C in the CSI, the self-binding activities of BV 17–45 and BV 16–13 were abrogated demonstrating that the recognized IgG autoepitope(s) possessed conformational or discontinuous three-dimensional properties. The immunological implications of dual specificity are discussed on a structure–function basis and its correlation with formation of pathogenic immune complexes. © 1997 John Wiley & Sons, Ltd.     

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.     

Antibodies to pathogenic epitopes on type XVII collagen cause skin fragility in a complement-dependent and -independent manner

In bullous pemphigoid (BP), the most prevalent autoimmune blistering disease, type XVII collagen (COL17) is targeted by circulating autoantibodies. BP is thought to be an autoantibody-mediated complement-fixing blistering disease, and a juxtamembranous noncollagenous 16A (NC16A) domain spanning Glu(490) to Arg(566) was proved to be the main pathogenic region on COL17, although precise pathogenic epitopes within NC16A have not been elucidated. In this study, we showed that injection of rabbit IgG Abs targeting Asp(522) to Gln(545) induced skin fragility associated with in vivo deposition of IgG and complement in neonatal COL17-humanized mice. Notably, immunoadsorption of rabbit anti-NC16A IgG Ab with this epitope (Asp(522) to Gln(545)) or the anti-NC16A IgG administered together with the peptides of this epitope as a decoy ameliorated skin fragility in the injected neonatal COL17-humanized mice compared with the anti-NC16A IgG alone even though all of the mice showed both IgG and complement deposition. These results led us to investigate an additional, complement-independent mechanism of skin fragility in the mice injected with anti-COL17 Abs. The rabbit anti-NC16A IgG depleted the expression of COL17 in cultured normal human keratinocytes, whereas immunoadsorption of the same IgG with this epitope significantly suppressed the depletion effect. Moreover, passive transfer of F(ab')(2) fragments of the human BP or rabbit IgG Abs against COL17 demonstrated skin fragility in neonatal COL17-humanized mice. In summary, this study reveals the importance of Abs directed against distinct epitopes on COL17, which induce skin fragility in complement-dependent as well as complement-independent ways.     

Interaction of rabbit IgG with anti-IgG: localization of epitopes and absence of immunoreactivity of the pFc'-fragment]

To localize essential epitopes of rabbit IgG, a series of proteolytic IgG fragments obtained by papain (Fab, Fc) or pepsin (pFc', F(ab')2) proteolysis have been prepared and their interaction with sheep antibodies against rabbit IgG has been studied. The data obtained suggest that essential immunoreactive epitopes of rabbit IgG are located in the CH2 domain and hinge region. This finding is in line with the results obtained by computing the antigenic sites of immunoglobulins. However, the deviation from the computed antigenic structure was deduced from the complete lack of immunoreactivity of the pFc fragment, it being a dimer of the terminal CH3 domain of the Fc fragment. The hinge region comparable in size with the dimensions of the epitope reveals high affinity binding to anti-IgG, thus testifying to the localization of the expressed epitope or its essential part in the hinge region. Proteolytic cleavage of this region leads to a significant decrease in the binding of the IgG fragment to anti-IgG. In addition to the CH2 domain and hinge region, a relatively low interaction of the antigen-binding antibody fragments with anti-IgG was found.     

Islet cell antigens and autoantigen(s): Monoclonal antibodies and further characterization

A novel series of murine monoclonal antibodies to islet cells (1–45, 1–51, 1–52 and 1–39) have been generated using human insulinoma homogenate as the immunogen in order to characterize pathogenetically relevant islet cell autoantigen(s). Differentiation antigens recognized by these islet cell monoclonal antibodies displayed varied cytological distribution (pan-islet or peripheral mantle only). Monoclonal antibody 1–45 reacted with all endocrine subsets of the pancreatic islet, similar to the reactivity of islet cell autoantibody positive sera from type I diabetes subjects. Preexposure to pH2 abolished the immunoreactivity of the autoantigen; 1–45 antigen was also sensitive to low pH. Preexposure to 100° C for 1 h did not significantly alter the immunoreactivity of islet antigens recognized by ICAb positive patient sera and monoclonal antibody 1–39, thus demonstrating the extraordinary heat stability of the corresponding epitopes; those recognized by 1–45 were less heat stable. Islet cells were found to share 1–45 differentiation antigen(s)/epitope(s) with other neuroendocrine cells,viz. amerior pituitary, adrenal medulla and gut endocrine cells.     

The PGK epitope of human thyroglobulin: A molecular marker of alternatively spliced thyroglobulin molecules?

Summary Human thyroglobulin (hTg) is the 2748 aa precursor of the thyroid hormones T3 and T4. In autoimmune thyroid diseases, autoantibodies to hTg appeared which showed a restricted epitope specificity for the central region of the molecule (residues 1149–1251). Our hypothesis to explain why this particular region becomes autoantigenic is presented, which involves the existence of truncated, alternatively spliced forms of hTg in the bloodstream. To try to prove this hypothesis, we have undertaken the identification of the peptide epitopes recognized by monoclonal antibodies on the thyroglobulin molecule by multiple peptide synthesis methods; we report here on the identification of the three-residue epitope, Pro-Gly-Lys in position 1282–1284 of the hTg sequence which is recognized by monoclonal antibodies Tg2 and Tg8. Due to their sequence specificity, these antibodies could provide a means to tag the region of the hTg sequence which is suggested to be the site of an alternative processing phenomenon. Our results are discussed in terms of both the specificity of anti-hTg monoclonal antibodies and of the mechanism of appearance of autoantibodies recognizing the central region of hTg.     

Identification of two novel B-cell epitopes on the nucleocapsid protein of porcine deltacoronavirus

Highlights
1. Two monoclonal antibodies against newly emerged porcine deltacoronavirus nucleocapsid protein were prepared.
2. The epitopes that these two monoclonal antibodies recognized on nucleocapsid protein were identified.
3. The monoclonal antibody 6B7 recognized a linear epitope of N protein, while the 7F8 recognized a conformational epitope.
4. Conservation of the identified epitopes between different coronaviruses was analyzed.     

Monoclonal antibodies to the bovine immunoglobulin G2a allotypes A1 and A2.

Production of murine monoclonal antibodies (Mabs) to bovine IgG2a-A1 and A2 allotypes resulted in three Mabs being selected as anti-IgG2a-A1 and A2 reagents. Two Mabs recognize the A1 allotype and 1 recognizes the A2 allotype. Initial epitope mapping with the Mabs indicates that one of the A1 epitopes resides in the hinge region and the other epitope resides more toward the C-terminus of the immunoglobulin. The A2 epitope recognized by the A2 Mab does not appear to reside in the hinge region of the immunoglobulin but apparently resides more C-terminal.     

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。     

Epitopes recognized by serum anti-α-galactoside antibody are present on brain glycoproteins in man

Naturally occurring serum IgG against terminal α-galactoside epitopes (anti-Gal), present exclusively in man, apes and old world monkeys, was used as probe for these epitopes in human brain. Human brain grey matter soluble glycoproteins enriched inα galactosyl groups by affinity chromatography on jacalin-sepharose, specifically binds to human anti-Gal in immuno dot blots. Anti-Gal recognized exclusively the terminal α galactoside epitope in human brain glycoproteins since binding was abolished by the presence of 1-0-methyl α-D-galactopyranoside as well as by pretreatment of glycoproteins with coffee bean α-galactosidase. Anti-Gal-peroxidase staining of jacalin-binding human brain glycoproteins in western immuno blots revealed mainly five anti-Gal-binding polypeptides withM _r (in kDa) of 94, 108, 180, 210 and 230 respectively. Since the presence of anti-Gal in higher animals accompanies suppression of the corresponding epitope in most tissues, apparently to maintain immunological balance, possible implications of the above observation for autoimmunity, tumor metastasis and infection are discussed.     

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.     

Antigenic determinants of the Ku (p70/p80) autoantigen are poorly conserved between species.

The Ku (p70/p80) autoantigen is a DNA-protein complex recognized by sera from certain patients with SLE and related diseases. Although human autoantibodies react with at least eight different epitopes of the human Ku complex, they had little reactivity with rodent Ku Ag on immunoblots. Small amounts of 70- and 80-kDa proteins were immunoprecipitated from murine cell extracts, however, suggesting that the Ku particle is not unique to human cells. This was confirmed by isolating cDNA clones encoding murine Ku Ag by plaque hybridization with a human p70 cDNA probe. The murine p70 cDNA clones had a deduced amino acid sequence 82.9% identical to that of human p70, and comparable amounts of murine and human p70 mRNA were detected in 3T3 and K562 cells, respectively. The poor reactivity of human autoantibodies with murine p70 was attributable to specific amino acid substitutions in an immunodominant conformational epitope located on amino acids 560-609 of human p70. Several amino acids critical for antigenicity of this region were defined by mutagenesis studies. Other conformational epitopes of Ku were also antigenically poorly conserved among species. Species-specific epitopes recognized by lupus autoantibodies are unusual but not unique to Ku. In general, poorly conserved autoepitopes have been conformational, rather than sequential, suggesting that the antigenicity of conformational epitopes may be particularly sensitive to evolutionary change.     

Induction of determinant spreading and of Th1 responses by in vitro stimulation with HER-2 peptides

Immunization with tumor antigens induces cellular and humoral immune responses. These responses by T cells are specific for defined epitopes (determinants) in the molecule of the immunizing tumor antigen. Extension of such responses to self-antigens requires induction of autoimmunity to the tumor. As with systems of autoimmune disease, expression of T cell autoimmunity is charaterized by diversification of responses from the inducer determinant to other responder (cryptic) determinants. Since similar strategies may be useful for therapy of human cancers, we investigated whether the induction of response to a HER-2 peptide F7 (776–789) induces enhanced reactivity of other HER-2 peptides. We found that stimulation with F7 can expand a response to another epitope F13 (884–899) in both an ovarian cancer patient with progressive disease and a healthy donor who shared HLA-DR11. This response was characterized mainly by increased interferon γ secretion, and proliferation, but was not observed with another donor who shared HLA-DR14 and HLA-DQ5 with the patient. Since repeated vaccination with the same epitope may lead to a decline of primary cell reactivity caused by apoptosis spreading the response to other epitopes, the tumor antigen may provide an approach for maintaining an inflammatory Th1 response during cancer vaccination. Received: 10 April 2000 / Accepted: 12 July 2000     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles

Introduction

Fibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA.

Methods

Citrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features.

Results

Five citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit_1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit_1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11).

Conclusions

Fibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.     

Autoantibodies interacting with purified native thyrotropin receptor.

Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells. An ELISA test was devised to study anti-TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor. The use of deglycosylated TSHR and of purified receptor ectodomain (alpha-subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain. Autoantibodies were detected in 45 of 48 subjects with untreated Graves' disease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (Kd = 0.25-1 x 10-10 M) and the autoantibodies belonged to the IgG class in all cases. Although the concentration of autoantibodies was higher in Graves' disease patients (3.50 +/- 0.36 mg.L-1) than in control subjects (1.76 +/- 0.21) (mean +/- SEM), there was an overlap between the groups. Receptor-stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells. Their characteristics (recognition of alpha-subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test. TSAb were only found in individuals with Graves' disease. The ELISA test measures total anti-TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation. Our observations thus disprove the hypothesis according to which Graves' disease is related to the appearance of anti-TSHR antibodies not present in normal subjects. Actually, anti-TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those found in patients with Graves' disease. What distinguishes the latter from normal subjects is the existence of subpopulation(s) of antibodies directed against specific epitope(s) of the receptor involved in its activation.     

Antitubulin antibodies. II. Natural autoantibodies and induced antibodies recognize different epitopes on the tubulin molecule

Natural and induced antitubulin antibodies were compared for their epitope recognition on alpha- and beta-tubulin subunits by immunoenzymatic assays and Western blot techniques on partially digested tubulin molecules. Our results indicated that natural autoantibodies recognized different epitopes from those recognized by induced antibodies, because: 1) all polyspecific natural autoantibodies tested so far recognized the same or very overlapping epitopes in the central part of both alpha- and beta-subunits (between positions 100 and 300 on the tubulin amino acid sequence) and that this epitope differed from the various epitopes recognized by induced antitubulin antibodies on the amino-terminal or carboxy-terminal parts of the tubulin subunits; 2) one human myeloma protein (monoclonal (m)IgA, kappa) with a monospecific antitubulin activity bound to an epitope around position 310 on both alpha- and beta-subunits and a second human mIg (mIgM, kappa) with a monospecific anti-beta activity bound to an epitope on the carboxy-terminal part of the subunit around amino acid position 350. Both epitopes differed from epitopes recognized by induced antitubulin antibodies. These results thus confirmed our previous findings indicating that natural and induced antitubulin antibodies do not share cross-reactive idiotopes.     

A Human Monoclonal Antibody against Hepatitis B Surface Antigen with Potent Neutralizing Activity

We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common “a” determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log₁₀ higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.     

Autoimmune epitopes in messenger RNA

Patients with systemic autoimmune disorders produce autoantibodies against sequence-specific conformational RNA epitopes on U1 snRNA, 28S rRNA, and transfer RNAs. The molecular basis for immunological reactivity with these highly abundant and stable RNAs is not understood. Here, we report the existence of discrete RNA epitopes in messenger RNAs that are generally less abundant and less stable than snRNAs and tRNAs. An iterative selection and amplification procedure using pooled autoimmune patient sera identified immunoreactive mRNA species. Following deconvolution of the pools to identify the reactive sera, several mRNAs recognized by these autoantibodies were cloned and sequenced. Detailed analysis using one particular serum indicated reactivity against the messages encoding alternative splicing factor (ASF/SF2) and calmodulin. Deletion and site-directed mutagenesis determined that an epitope recognized by this serum is located in a 17-base stem-loop structure common to both messages. This serum was then used to immunoprecipitate native mRNAs encoding ASF/SF2 and calmodulin from total HeLa cell RNA. Our results demonstrate that despite its low abundance and instability, messenger RNA is capable of reacting with autoantibodies generated during an autoimmune response. These data are consistent with direct presentation as a model to explain the generation of RNA conformation-specific autoantibodies.