Differences In Epitope Recognition Research Articles

IgE and IgG 4 antibodies from patients with mite allergy recognize different epitopes of Dermatophagoides pteronyssinusgroup II antigen (Der p 2)

BACKGROUND: Der p 2, composed of 129 amino acids, is one of the major allergens of Dermatophagoides pteronyssinus . Several groups reported epitope mapping of IgE antibody but not of IgG or IgG subclass antibodies in Der p 2. OBJECTIVE: The purpose of this study was to compare the epitopes recognized by Der p 2–specific IgE antibodies with those recognized by IgG 4 antibodies. METHODS: Recombinant fusion proteins containing a full-length peptide or overlapping five fragments of Der p 2 were prepared and analyzed for their reactivity with IgE and IgG subclass antibodies in sera from patients with mite allergy by means of Western blot procedure. RESULTS: IgE antibodies in 12 of 13 sera tested bound to full-length Der p 2 (1-129). In 11 of these 12 sera, IgE antibodies bound to one or more of fragments 41-80, 64-105, or 81-129 but not to those of 1-40 or 20-63. Of the 11 sera in which the IgE antibodies to the fragments were detected, only four contained detectable levels of IgG 4 antibodies reactive to the fragments. The binding spectra of the IgG 4 antibodies against the fragments were rather wide and differed from those of IgE antibodies. CONCLUSION: Results revealed that there are differences in epitope recognition between IgE and IgG 4 antibodies; IgE antibodies recognize restricted parts of Der p 2 antigen compared with IgG 4. (J A LLERGY C LIN I MMUNOL 1996;97:638-45.)

Glutamic acid decarboxylase autoantibodies in stiff-man syndrome and insulin-dependent diabetes mellitus exhibit similarities and differences in epitope recognition.

Glutamic acid decarboxylase (GAD) is an autoantigen in two autoimmune diseases, insulin-dependent diabetes mellitus (IDDM) and stiff-man syndrome (SMS). However, most individuals with one of these diseases do not have the other disease. Prior studies have suggested that the natures of the GAD Abs associated with each of these diseases are different, which may have implications for the autoimmune pathogenesis. We have compared the GAD autoantibody profile and have mapped GAD protein epitope regions in the two diseases using an immunoprecipitation assay with recombinant GAD 65 and GAD 67 proteins, GAD protein fragments, and synthetic GAD peptides, as well as chimeric GAD proteins. Our results indicate that individuals with SMS have GAD Abs in 100- to 500-fold higher titer than individuals with IDDM. The population of GAD Abs in SMS sera is quite complex and includes those that recognize at least three GAD 65 epitope regions located between amino acids 1-16, 188-442, and 442-563. These types of GAD Abs are not found in IDDM sera. All SMS sera also had Ab specificity that binds GAD 67 in a region highly homologous to amino acids 188-442 of GAD 65. In contrast to prior studies that used immunoblotting to measure GAD Abs, we find GAD Abs in SMS sera also target two conformation-dependent regions of GAD 65, one located in the middle and one near the C-terminus of the protein. These two regions of the GAD 65 protein are similar to regions targeted by GAD 65-specific Abs found in individuals with IDDM. These results indicate that although disease-specific epitopes may exist, there is also overlap in the humoral response between the two diseases.

Characterization of the IgA and subclass IgG responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus.

In this study, we have investigated the characteristics of secreted IgA and other classes of Ig induced after vaccination of sows with transmissible gastroenteritis virus (TGEV) or the antigenically related porcine respiratory coronavirus (PRCV). Both viruses induced the secretion of neutralizing antibodies of different classes in the sows' milk, but these protected suckling piglets against TGEV to different degrees. Quantitative differences in the induction of IgA by both viruses were found among the different viral antigenic sites and subsites of glycoprotein S. In TGEV-vaccinated sows, antigenic subsite A was the best inducer of IgA, followed by antigenic site D. After vaccination with PRCV, lower levels of IgA were detected on colostrum and milk, antigenic site D and subsite Ab being the immunodominant sites. This quantitative difference in epitope recognition could explain the differences in newborn piglet protection found using Ig classes purified from the milk of sows immunized with both viruses. Apparently only IgA recognizing at least antigenic sites A and D confers good protection in vivo, whereas any Ig class recognizing only one antigenic site may neutralize the virus in cell culture. These results indicate that the formulation of a subunit vaccine against TGEV has to consider the inclusion of more than one antigenic site involved in virus neutralization.

T and B cell responses to myelin basic protein and encephalitogenic epitopes

The major encephalitogenic epitope of myelin basic protein (MBP) for the Lewis rat includes residues 68–84, although a minor epitope has been localized to MBP residues 87–99. We synthesized MBP68–84 and MBP87–99, and immunized rats with these peptides or with MBP in complete Freund's adjuvant (CFA). MBP and MBP68–84 induced paralytic experimental autoimmune encephalomyelitis (EAE) at equimolar concentrations, whereas significantly higher dosages of MBP87–99 were required to elicit paralytic disease. Spleen cells (SpC) from MBP- or MBP68–84-immunized rats could be activated with either MBP or MBP68–84 to transfer EAE to recipients. Anti-MBP antibodies were detected by ELISA in rats immunized with MBP-CFA, and anti-MBP68–84 specific antibodies were present in serum obtained from MBP68–84-immunized animals. However, these antibodies were non-cross reactive. MBP87–99 elicited only a meager antibody response to the immunizing peptide, and cross reactivity with MBP was not observed. Thus, although MBP and each peptide exhibited encephalitogenic activity, and MBP and MBP68–84 were cross reactive at the T cell level, the absence of cross reactivity at the humoral level indicates that significant immunological differences exist between MBP and the synthetic determinants, which may reflect differences in epitope recognition by T and B lymphocytes.

T cell responses to myelin basic protein in experimental autoimmune encephalomyelitis-resistant BALB/c mice

In strains of mice that are susceptible to experimental autoimmune encephalomyelitis (EAE), cloned CD4 + T cells reactive with autologous myelin basic protein (MBP) have been shown to cause disease when transferred to naive syngeneic recipients. Recent reports indicate that under particular experimental conditions, ‘resistant’ strains of mice can also develop EAE, although cloned cells have not been isolated and characterized. An analyis of the characteristics of a panel of MBP-specific T cells and the antigen presenting capability of CNS-derived cells obtained from the resistant strain BALB/c is presented here. The data demonnstrate that immunization of EAE-resistant BALB/c mice results in the activation of a heterogeneous group of T cells reactive with autologous MBP. Both peripheral antigen presenting cells, as well as microglia isolated from brains of BALB/c mice, are capable of stimulating these cloned MBP-specific T cells to proliferate. When optimally activated in vitro and then injected in vivo into syngeneic BALB/c recipients, three clones studied induced severe cachexia, resulting in loss of up to 35% of body weight before death. Two of the clones also induced clinical and histological EAE, while the third induced only occasional histological evidence of disease. Differences in epitope recognition, T cell receptor usage, cytokine profiles or regulatory mechanisms of self tolerance, may play important roles in preventing potentially destructive autoimmune reactions by these T cells capable of recognizing autologous myelin in the central nervous system.

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.

Silencing of immunodominant epitopes by contiguous sequences in complex synthetic peptides

We have previously shown that the T cell response to the synthetic peptide cI 12–26:NP 365–380 (covalently linked epitopes of λ repressor (cI) and influenza A nucleoprotein (NP) polypeptides) requires amino acid sequences located in the junctional region between the cI 12–26 and NP 365–380 epitopes in the H-2 d and H-2 k haplotypes. In this study, we show that the dominant epitope of cI 12–26:NP 365–380 in H-2 b mice is also located within the junctional region of the peptide, indicating that the same amino acid sequence is immunodominant in three different H-2 haplotypes. Based on results using fixed APC, there was no qualitative difference in epitope recognition due to antigen processing. In addition, antigen presentation by APC expressing mutant I-A molecules constructed by hemiexon shuffling of regions of the molecule containing primarily β sheet or α helix showed that many different substitutions were permissive for at least one of the T hybridomas. More importantly, however, when the junctional sequences are covalently linked in composite synthetic peptides containing additional previously defined T cell epitopes, antigenicity of the immunodominant junctional region was silenced and a new epitope assumed immunodominance. Thus, immunodominance does not correlate with the primary amino acid sequence of the potential epitope. Instead, the immunodominant epitope is determined by complex interactions among the epitopes, which most likely depend on the structural conformation of the composite peptide.