Characterization of human variable domain antibody fragments against the U1 RNA-associated A protein,selected from a synthetic and a patient-derived combinatorial V gene library

This is the first study describing recombinant human antibody fragments directed to the U1 RNA-associated A protein (U1A). Three anti-U1A antibody fragments (Fab) were isolated from a semi-synthetic human Fab library and one anti-U1A single-chain variable fragment (scFv) was isolated from a library which was derived from the IgG-positive splenic lymphocytes of an autoimmune patient. Competition studies with autoantibodies against the U1 small nuclear ribonucleoprotein (snRNP) particle from patients with systemic lupus erythematosus (SLE) and SLE-overlap syndromes revealed that U1A binding of these antibody fragments can be inhibited by about 40% of the patient sera. All antibody fragments recognized the native U1 snRNP in immunoprecipitation assays. Two of three Fab clones as well as the scFv clone derived from the repertoire of an autoimmune patient use the same heavy chain germ-line gene DP-65. Epitope mapping revealed that these three clones appear to recognize an identical epitope domain present on the C-terminal RNP motif of the U1A protein. The DP-65 heavy chain gene is used in less than 1% of the B cells in healthy individuals, while three out of four anti-U1A antibody fragments use this gene. This points to a restricted V_H gene usage in the case of U1A, suggesting that the DP-65 heavy chain has a natural shape complementarity to the U1A protein.     

B cell epitope on the U1 snRNP-C autoantigen contains a sequence similar to that of the herpes simplex virus protein

The mechanism of autoantibody production in autoimmune diseases is not well understood. In the present study we performed the B cell epitope mapping of the U1 small nuclear ribonucleoprotein (snRNP)-C, one of the target molecules of anti-nRNP autoantibody to investigate how B cells respond to the autoantigen. After cloning and expression of a full length complementary DNA (cDNA) encoding the U1-C protein, several truncated mutants of the cDNA were constructed and expressed in E. coli. Although a few epitopes were distributed on the whole molecule, all anti-C protein antibody-positive patients' sera tested recognized the region between amino acid residues 102 and 125 of the coding sequence. This universal epitope region contains an amino acid sequence similar to that of the herpes simplex virus type 1 ICP4 protein. The peptides representing each molecule were cross-reactive to each other. In addition this region cross-reacted to the B/B′ protein. These observations suggest that molecular mimicry might be involved in the initiation of autoantibody production, followed by cross-reactive events between the autoantigens and by antigen-driven mechanisms to generate more complicated autoantibody patterns against the U1 snRNP complexes.     

Characterization of recombinant human autoantibody fragments directed toward the autoantigenic U1-70K protein

The U1-70K protein is specifically bound to stemloop I of the U1 small nuclear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snRNP), which is involved in the splicing of pre-mRNA. All components of the U1 snRNP complex, including the U1-70K protein, are important autoantigens in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Here we describe for the first time the selection and characterization of recombinant human anti-U1-70K single chain autoantibody fragments (anti-hU1-70K scFv) from autoimmune patient-derived phage display antibody libraries. All scFv specifically recognize parts of the hU1-70K protein and its apoptotic 40-kDa cleavage product. In Western blotting assays a number of scFv preferentially recognize the 40-kDa apoptotic cleavage fragment of the U1-70K protein, suggesting a possible involvement of this apoptotic cleavage product in the autoimmune response of patients. The germline gene usage of these recombinant autoantibodies was also determined. Using several U1-70K deletion and point mutants of both human (h) and Drosophila melanogaster (Dm) origin, it was established that the U1-70K epitope that is recognized by the anti-hU1-70K scFv is located within the RNA binding domain.     

Further characterization and subcellular localization of Sm and U1 ribonucleoprotein antigens

Sera from patients with systemic autoimmune diseases often contain antibodies against small nuclear ribonucleoprotein (snRNP) particles. Anti-Sm antibodies react with the entire set of U1, U2, U4, U5 and U6 (U1-U6) RNP particles whereas anti-(U1)RNP sera specifically recognize particles containing U1 RNA. Here we performed semi-quantitative immunoblotting using 16 human anti-Sm, 15 human anti-(U1)RNP sera and two mouse monoclonal antibodies to establish which snRNA-associated proteins carry antigenic determinants. Almost every (15/16) human anti-Sm sera recognized epitopes present on a 28-kDa (B/B') protein doublet and on a 16-kDa (D) polypeptide. Nine anti-(U1)RNP sera also recognized the B/B' doublet, but in all cases a much stronger reaction was observed with one or more of the specifically U1 RNA-associated 70 kDa, A or C antigens. With affinity-purified antibody fractions eluted from individual antigen bands on nitrocellulose blots it is shown that the anti-Sm-reactive polypeptides B/B' and D contain common epitopes. We also report the finding of one human anti-Sm serum with exclusive specificity for the B/B' doublet and a mouse monoclonal anti-Sm antibody recognizing only the D protein, indicating that these antigens also carry unique epitopes. In immunoprecipitation assays, purified anti-B/B' and -D antibodies react with (U1-U6) RNP while purified anti-70 kDa, anti-A and anti-C antibodies precipitate exclusively U1 RNP particles. Finally, we established the subcellular localization of Sm and U1 RNP antigens using a biochemical cell fractionation procedure. Part of the 70 kDa and B/B' antigens were found in a nuclease and high salt-resistant nuclear substructure, usually referred to as nuclear matrix, while the A and D antigens could be extracted completely from HeLa nuclei by ribonuclease treatment and subsequent high salt extraction.     

Identification and characterization of human genes encoding Hprp3p and Hprp4p, interacting components of the spliceosome

Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome. U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome. U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins. We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively. Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p. Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe. Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over- produced in Escherichia coli . This splicing factor can co- immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP. Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p. Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G- proteins. These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat. The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p. Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading. Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin. Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction. These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.      

Apoptotic modifications affect the autoreactivity of the U1 snRNP autoantigen

A hallmark of systemic autoimmune diseases is the presence of high titers of serum autoantibodies targeting a diversity of autoantigens. Most components of the U1 snRNP complex are autoantigenic in systemic lupus erythematosus (SLE) and SLE overlap syndrome, which is also called mixed connective tissue disease (MCTD). It is hypothesized that posttranslational modifications, in particular cell death-associated modifications, play an important role in breaking tolerance to self-antigens. Recently, it became clear that the U1 snRNP particle, more specifically its U1-70K protein component, displays a new epitope during apoptosis. This review intends to give an overview of the modifications that occur on the U1 snRNP autoantigens, especially those arising during cell death, to summarize recent data describing autoantibody reactivities with apoptosis-specific epitopes on the U1 snRNP complex, and to provide some insight into the mechanisms that might underlie the immune response to self-antigens.     

T cell lines recognizing the 70-kD protein of U1 small nuclear ribonucleoprotein (U1snRNP).

In sera of patients with mixed connective tissue disease (MCTD) high titres of IgG autoantibodies to U1snRNP-specific proteins (70 kD, A, C) are found, suggesting an antigen-driven and T-cell-dependent process. In order to establish U1snRNP-specific T cell lines we cultured under various culture conditions mononuclear cells from MCTD patients and healthy donors with a highly purified UsnRNP preparation from HeLa cells. Nine T cell lines were established by limiting dilution cloning from two MCTD patients and five T cell lines from a healthy individual. All T cell lines expressed the TCR alpha beta/CD3 complex. Surprisingly, most of the T cells lines exhibited the CD8 phenotype. Irrespective of this phenotype, all T cell lines showed a proliferative response to an N-terminal part (aa 51-195) of recombinant U1-specific 70-kD protein. One CD8+ T cell clone exhibited cytotoxic activity against an autologous B cell line pulsed with snRNP or recombinant fragments (aa 51-95 and aa 51-88). Interestingly, two T cell lines proliferated in response to four recombinant polypeptides representing different parts of the U1snRNP 70-kD protein. Since regions of sequence homology are distributed over the 70-kD molecule, it is suggested that conserved motifs may be recognized by the T cell lines.     

Vimentin-cross-reactive epitope of type 12 streptococcal M protein.

The NH2-terminal amino acid sequence of type 12 M protein was determined by automated Edman degradation of a 38-kilodalton polypeptide fragment purified from a limited pepsin digest of intact type 12 streptococci. The sequence of the first 13 amino acid residues of the polypeptide confirmed that predicted by the nucleotide sequence of the mature type 12 M protein. A chemically synthesized peptide copying the NH2-terminal 25 residues, SM12(1-25)C, evoked opsonic antibodies against type 12 streptococci as well as renal glomerular cross-reactive antibodies. The serum from one of six rabbits reacted in immunofluorescence tests with human glomeruli in a mesangial staining pattern. The cross-reactive antibodies were completely inhibited by the immunizing peptide and absorption with type 12 streptococci. Subpeptides of the 25-residue synthetic peptide were without inhibitory effect, suggesting that the cross-reactive antibodies are directed against a conformational epitope of SM12(1-25)C. Anti-SM12(1-25)C antisera reacted specifically with the intermediate filament protein vimentin extracted from mesangial cells. None of the cross-reactions of anti-SM12(1-25)C were inhibited by a synthetic peptide SM1(1-26)C of type 1 M protein, which was previously shown to share a cross-reactive epitope with vimentin. These results indicate that type 12 M protein contains at least one vimentin cross-reactive epitope that is clearly distinct from the tetrapeptide epitope shared with vimentin by type 1 M protein.     

MHC class II gene associations with autoantibodies to U1A and SmD1 proteins

Autoantibodies against U small nuclear ribonucleoproteins (snRNP) are frequently present in the serum of patients with systemic rheumatic diseases, and have been reported to be associated with HLA-DR and -DQ genes. To better define the role of HLA genes in the production of such antibodies, we studied immunogenetic associations with autoantibodies reacting with U1 RNP, U1A and SmD1 proteins, and synthetic peptides containing immunodominant linear epitopes of these proteins. Only two out of the 15 overlapping peptides of U1A (i.e. peptides 35-58 and 257-282) and three of 11 peptides of SmD1 (i.e. peptides 1-20, 44-67 and 97-119) were significantly recognized by patients' sera selected on the basis of their antibody positivity with RNP in immunodiffusion. The distribution of DRB1, DQB1 and DPB1 alleles among the anti-RNP antibody-positive patients (n = 28) and healthy control subjects was similar. Antibodies against U1A (tested in Western immunoblotting with HeLa cell extracts) were positively associated to DRB1*06 allele; antibodies reacting with SmD1 peptide 44-67 were negatively associated to DRB1*02 and DQB1*0602 alleles. No association was found between DPB1 alleles and antibodies reacting with U1A and SmD1 antigens. This first study reporting an association between autoantibodies reacting with U1A and SmD1 proteins (and peptides of these proteins), and immunogenetic markers suggest that the production of antibody subsets directed against different components (or regions of these proteins) bound to the same snRNP particle is associated with distinct MHC class II alleles.     

A recombinant 70K protein ELISA. Screening for antibodies against U1snRNP proteins in human sera.

Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of U1snRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic disease were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNase ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNase but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-U1snRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-U1snRNP antibody screening may not be appropriate, because antibodies against other frequently occurring U1snRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.     

Major Autoantigenic Sites of the (U1) Small Nuclear Ribonucleoprotein-Specific 68-kDa Protein

A 68-kDa protein associated with (U1)snRNP is a major target for human autoantibodies to small ribonucleoprotein particles (snRNP) prevalent in a variety of inflammatory rheumatic diseases. The epitopes recognized by these antibodies were mapped by expression of subfragments of p68 cDNA in Escherichia coli and testing of the corresponding recombinant proteins for immunoreactivity with sera of patients with autoimmune diseases. Three of four antigenic regions were analysed in detail. The immunodominant autoantigenic region was found to coincide with the RNA-binding domain of the p68 protein and was shown to contain a nested set of overlapping discontinuous epitopes. Two additional non-overlapping major antigenic domains were localized in the carboxy-terminal half of the p68 protein. Each of these two carboxy-terminal domains was shown to contain more than one conformation-dependent epitope. Taking into account previous mapping studies, the data demonstrate that p68 contains at least four antigenic regions, each of which harbours multiple epitopes which are recognized in a patient-specific manner.     

Autoantibody production against the U small nuclear ribonucleoprotein particle proteins E, F and G in patients with connective tissue diseases

The nucleoplasmic U small nuclear ribonucleoprotein particles (snRNP) have a set of seven proteins in common which are designated B', B, D, D', E, F and G. Patients suffering from rheumatoid autoimmune diseases such as systemic lupus erythematosus often develop autoantibodies against the proteins B', B, and D. Here we describe a sensitive immunoassay which allows the specific detection of autoantibodies reacting with the E, F or G snRNP proteins. We were able to identify several patient sera containing autoantibodies against one or more of these proteins. This demonstrates that all snRNP proteins described so far are potentially antigenic in systemic rheumatoid diseases. The characterization of the antibodies showed an immunological cross-reactivity between the snRNP protein G and the 70-kDa protein of U1 snRNP. Several sera contained autoantibodies which were specific for the F snRNP protein.     

IgE-binding epitopes of the American cockroach Per a 1 allergen

Cockroach is one of the major indoor allergens for IgE-mediated allergic respiratory illnesses throughout the world. The American cockroach (Periplaneta americana) Per a 1 allergen is antigenically cross-reactive with the German cockroach (Blattella germanica) Bla g 1 allergen. The aim of this study was to identify linear B cell epitopes of Per a 1 that are recognized by human IgE. Per a 1 deletion mutants were generated from the recombinant Per a 1.0104 allergen (274 amino acid residues), and antigenicities were assessed by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and binding inhibition. Human atopic sera were not able to recognize deletion mutants consisting of amino acids 1-77, 86-205, and 200-266. However, human sera did recognize the N-terminal mutant containing amino acids 1-87 and the C-terminal mutant containing amino acids 200-274, demonstrating positive IgE binding that was heterogeneously distributed among the different sera tested. Amino acid residues 78-85 and 267-274, containing internal repeats, were shown to be required for IgE binding to the Per a 1.0104 protein. Two peptides corresponding to these IgE-binding amino acid sequences were synthesized. Peptide 78-85 showed a positive IgE interaction with 80% of the sera, while peptide 267-274 was capable of IgE binding to all of the sera tested. Moreover, preincubation of atopic sera with IgE-positive recombinants and peptides resulted in marked inhibition of the IgE binding to purified Per a 1.0104 allergen. Amino acid sequences 78LIRALFGL85 and 267IRSWFGLP1274 of the American cockroach Per a 1.0104 allergen were involved in IgE binding. These findings will advance the understanding of the specific reactivity of the epitopes of cockroach allergens, thereby contributing to the development of specific immunotherapies for clinical use.     

Repetitive P68-autoantigen specific epitopes recognized by human anti-(U1) small nuclear ribonucleoprotein autoantibodies.

The major target of anti-(U1)snRNP autoantibodies, a serological marker of patients with mixed connective tissue disease and related rheumatic disorders, is a 68 kDa protein (p68) associated with (U1)RNA-containing small nuclear ribonucleoprotein particles. With recombinant p68 fusion proteins, multiple autoepitopes have been identified, and one of these has been mapped to the pentamer sequence ERKRR, which is located within antigenic domain A in the amino-terminal half of p68. The lysine residue (K) of this epitope can be replaced by isoleucine without loss of autoantibody binding. Here we have investigated whether other variants of this epitope are present on the p68 autoantigen and if these are recognized by anti-p68 autoantibodies. We identified four related motifs in the carboxy-terminal half of the p68-protein, and three of these (all containing glutamic acid instead of lysine (ERERR] mapped to the previously characterized autoantigenic domains C and D. Immunoreaction of anti-ERKRR autoantibodies, affinity-purified from domain A with recombinant fusion proteins containing either domain C or domain D of p68, revealed that anti-ERKRR autoantibodies cross-react with the ERERR-motifs. This finding, which was confirmed by competitive inhibition-ELISA with solid-phase coupled domain A-, C- and D-fusion proteins and ERKRR-containing synthetic peptides as competitors, suggests that a subset of patient autoantibodies is directed against repetitive structures on a single snRNP component.     

Antigenic domains on the U1 small nuclear ribonucleoprotein-associated 70K polypeptide: a comparison of regions selectively recognized by human and mouse autoantibodies and by monoclonal antibodies.

Antigenic regions on the U1 small nuclear ribonucleoprotein (snRNP)-associated 70K polypeptide recognized by human and mouse autoantibodies or by monoclonal antibodies were identified and compared. Using a set of 70K fusion proteins as antigen in enzyme-linked immunosorbent assay and immunoblotting revealed that serum autoantibodies of human and of MRL/Mp mouse origin recognized a common region of the 70K polypeptide. Monoclonal anti-70K antibodies derived from a patient with mixed connective tissue disease, from an autoimmune MRL/Mp mouse, and from a BALB/c mouse immunized with purified U1 snRNP were all shown to bind to a part of the 70K polypeptide rich in charged residues and different from the region recognized by most human and MRL/Mp mouse serum autoantibodies.     

Biochemical and functional characterization of the cis-spliceosomal U1 small nuclear RNP from Trypanosoma brucei

Recent studies in the trypanosome system have revealed that in addition to trans splicing of a short spliced leader (SL) exon, there is also cis splicing of internal introns. It has been suggested that cis splicing requires base-pairing of U1 small nuclear RNA (snRNA) and the 5' splice site. We have cloned the gene for U1 snRNA from Trypanosoma brucei and characterized the U1 snRNP. Based on immunoprecipitation and direct mass-spectrometric protein analysis the U1 snRNP contains the common Sm core found also in the known trans-spliceosomal snRNPs U2, U4/U6, and U5. The 5' end of U1 snRNA in the U1 snRNP is accessible for and functional in specific recognition of the 5' splice site of the poly(A) polymerase intron.     

Genetic analysis of the role of human U1 snRNA in mRNA splicing: I. Effect of mutations in the highly conserved stem-loop I of U1

The 5' splice site mutation known as hr440 can be suppressed efficiently in vivo by a compensatory base change in U1 small nuclear RNA (snRNA). We have now begun a second-site reversion analysis of this suppressor U1-4u snRNA (which has a C----U change at position 4) to identify U1 nucleotides that are essential for mRNA splicing. Point mutations in U1-4u that disrupt the structure of stem-loop I or alter phylogenetically conserved nucleotides within the loop cause loss of suppression. The level of suppressor activity observed for most mutants correlated with the abundance of the corresponding suppressor RNA, suggesting that mutations in stem-loop I cause loss of suppression by destabilizing U1 snRNA or the U1 snRNP (small nuclear ribonucleoprotein particle). We favor the interpretation that incompletely or improperly assembled U1 snRNPs are unstable, because two severe point mutations in stem-loop I were found to decrease the binding of U1 snRNP-specific proteins in vitro. In a separate set of experiments, we found that increasing the distance between stem-loop I and the 5' end of U1 snRNA also inhibited suppression but did not affect assembly or stability of the U1 snRNP. This suggests that the relationship between the 5' splice site and the body of the U1 snRNP is important for mRNA splicing.     

Thirteen anti-Sm monoclonal antibodies immunoprecipitate the three cytoplasmic snRNP core protein precursors in six distinct subsets

The small nuclear ribonucleoprotein particle (snRNP) common core proteins are the lupus-associated Sm autoantigens. In mouse fibroblasts the seven snRNP core proteins form a particle with a suggested stoichiometry of B2[D1,D2(E,F,G)2] D3. Core particle assembly occurs in the cytoplasm where newly synthesized snRNAs assemble with core proteins stored in three RNA-free complexes of (1) a 6S complex of [D1,D2(E,F,G)2] (2) a 20S complex of (B,D3 and an unidentified 70 kDa protein) and (3) a 2S-6S complex that minimally contains the B protein. In this report a panel of 13 anti-Sm monoclonal antibodies is shown to immunoprecipitate six different subsets of the cytoplasmic snRNP proteins. Four epitopes are shared by the three aforementioned complexes and five other epitopes are shared by two of the complexes. In addition, the 6S or 20S complexes are apparently disrupted by five of the antibodies. Kinetic studies show that the three cytoplasmic snRNP protein complexes have independent half-lives. These studies provide another approach for characterizing the Sm epitopes. They also complement previous in vitro snRNP assembly studies and suggest that snRNP core assembly occurs by the initial binding of snRNA to the 6S particle followed by addition of the B and D3 proteins.     

Antigenic mimicry and autoimmune diseases.

The finding of cross-reactive autoantibodies or sequence homology does not necessarily mean that this molecular mimicry is biologically meaningful or associated with disease pathogenesis. For example, relatives of persons with putative autoimmune insulin-dependent diabetes [123], and elderly humans [124] have a high incidence of autoantibodies which are generally not associated with autoimmune disease. In addition, natural antibodies to cell constituents [125] may be present in normal sera. These antibodies need to be directed against biologically important domains of host cell proteins in order to mediate autoimmune disease [27]. In spite of extensive homology between two sequences, a cross-reactive immune response may not be generated. The dissimilar amino acids should not be radical substitutions or affect the binding properties of the molecule. For instance, antibodies to synthetic peptides with only one substitution in a 19 amino acid sequence may not bind the whole protein [126]. Despite an identical six amino acid sequence shared by HLA-B27 and an EBV protein, no cross-reactive antibodies to EBV peptides were found in HLA-B27 positive patients with AS or RS. Unless the homology and subsequent crossreactive immune response can recognize a host protein intimately involved in disease pathogenesis, autoimmune disease is unlikely to occur.