Mapping of B-cell epitopes in a Trypanosoma cruzi immunodominant antigen expressed in natural infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.     

Peptide-based analysis of the amino acid sequence important to the immunoregulatory function of Trypanosoma cruzi Tc52 virulence factor

The intracellular protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas' disease. We have previously identified a T. cruzi-released protein called Tc52, which is crucial for parasite survival and virulence. In the present study, we attempted to define the Tc52 epitope(s) responsible for its immunoregulatory function. A naturally occurring major peptide fragment of molecular mass 28 kDa (Tc28k) was identified, which was localized in the C-terminal portion of Tc52 and was inhibitory for T-cell activation. Synthetic peptides corresponding to amino acid sequences in Tc52 were evaluated for their ability to modulate T-cell proliferation and cytokine production. Results obtained using five peptides spanning the N-terminal or C-terminal domain of the Tc52 protein indicated that the activity mapped to Tc52 residues 432-445. Moreover, it was found that the peptide, when coupled to a carrier protein (ovalbumin), exhibited increased inhibitory activity on T-lymphocyte activation. Incubation with 8 nm ovalbumin-coupled peptide 432-445 resulted in approximately the same levels (>75%) of inhibition of T-cell proliferation as 5 micro g/ml Tc28k. Furthermore, we showed that the coupled peptide significantly down-regulated the secretion of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Likewise, in immunized mice, the coupled peptide 432-445 was a very poor B- and T-cell antigen compared with the other Tc52-derived peptides. These results suggest that the immunomodulatory portion of the T. cruzi Tc52 virulent factor may reside, at least in part, in a conserved sequence within its C-terminal domain, which could minimize its antigenicity.     

B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

Trypanosoma cruzi-Induced Host Immune System Dysfunction: A Rationale for Parasite Immunosuppressive Factor(s) Encoding Gene Targeting

An intense suppression of T cell proliferation to mitogens and toantigens is observed in a large number of parasitic infections.The impairment of T cell proliferation also occurred during theacute phase of Chagas' disease, caused by the intracellularprotozoan parasite Trypanosoma cruzi. A wealth ofevidence has accumulated that illustrates the ability ofT. cruzi released molecules to influence directly avariety of diverse immunological functions. In this paper, wereview the data concerning the immunoregulatory effects ofT. cruzi Tc24 (a B cell activator antigen) and Tc52 (animmunosuppressive protein) released molecules on the host immunesystem. The gene targeting approach developed to further explorethe biological function(s) of Tc52 molecule, revealed interestingunexpected functional properties. Indeed, in addition to itsimmunusuppressive activity a direct or indirect involvement ofTc52 gene product alone or in combination with othercellular components in T. cruzi differentiation controlmechanisms have been evidenced. Moreover, targeted Tc52replacement allowed the obtention of parasite mutants exhibitinglow virulence in vitro and in vivo. Thus, thegeneration of a complete deficiency state of virulence factors bygene targeting should provide a means to assess the importance ofthese factors in the pathophysiological processes and diseaseprogression. It is hoped that such approaches might allowrational design of tools to control T. cruziinfections.     

Evidence of Trypanosoma cruzi II infection in Colombian chagasic patients

Trypanosoma cruzi is genetically classified into at least two major lineages named T. cruzi I (also named Tc I) and T. cruzi II (also named Tc IIb). T. cruzi II is associated with Chagas’ disease in the southern cone of South America, while T. cruzi I is the only one so far identified in chagasic patients of Central America and in the northern part of South America. Herein we identified T. cruzi IIb directly in 9.9% of blood of chronic chagasic patients of Colombia. This finding establishes that in this region, the two T. cruzi lineages are associated with the pathology of Chagas’ disease and have implications in the morbidity and epidemiology of the disease.     

Development of an equine herpesvirus type 4-specific enzyme-linked immunosorbent assay using a B-cell epitope as an antigen

The equine herpesvirus type 4 (EHV-4)-specific region of glycoprotein G has served as an antigen for serodiagnosis and seroepizootic studies of EHV-4 infection (B. S. Crabb and M. J. Studdert, J. Virol. 67:6332-6338, 1993; S. Yasunaga, K. Maeda, T. Matsumura, K. Kai, H. Iwata, and T. Inoue, J. Vet. Med. Sci. 60:1133-137, 1998; S. Yasunaga, K. Maeda, T. Matsumura, T. Kondo, and K. Kai, J. Vet. Med. Sci. 62:687-691, 2000). Here we identified a major B-cell epitope in the type-specific region of EHV-4 and applied it as an antigen in enzyme-linked immunosorbent assays (ELISAs). A 24-amino-acid repeat sequence expressed as a glutathione S-transferase fusion protein specifically reacted as well as the type-specific region with sera from foals infected with EHV-4. Five synthetic peptides (12-mer peptides) in the repeat sequence were included as ELISA antigens. The results indicated that the 12-mer peptide MKNNPIYSEGSL contained a major B-cell epitope specific for EHV-4 infection. Inclusion of this 12-mer peptide in ELISAs for an epidemiological study specifically detected EHV-4 infection in the field. These results indicated that the 12-mer epitope was responsible for the type-specific antibody response and therefore is useful for seroepizootic studies and serodiagnosis of EHV-4 infection.     

Signal peptides and trans-membrane regions are broadly immunogenic and have high CD8+ T cell epitope densities: Implications for vaccine development

Cell mediated immune response has a major role in controlling the elimination of infectious agents. The rational design of sub-unit peptide vaccines against intracellular pathogens or cancer requires the use of antigenic sequence/s that can induce highly potent, long lasting and antigen-specific responses in the majority of the population. A promising peptide selection strategy is the detection of multi-epitope peptide sequences with an ability to bind multiple MHC alleles. While past research sought the best epitopes based on their specific antigenicity, we ask whether specific defined domains have high epitope densities. Signal peptides and trans-membrane domains were found to have exceptionally high epitope densities. The improved MHC binding of these domains relies on their hydrophobic nature and, in signal peptides, also on their specific sequence. The high epitope density of SP was computed using in-silico methods and corroborated by the high percentage of identified SP epitope in the IEDB (immune epitope database). The enhanced immunogenicity of SP was then experimentally confirmed using a panel of nine peptides derived from Mycobacterium tuberculosis (MTb) proteins used in human PBMC proliferation assays and T cell lines functional assays. Our results show the exceptionally high antigen specific response rates and population coverage to SP sequences compared with non-SP peptide antigens derived from the same proteins. The results suggest a novel scheme for the rational design of T cell vaccines using a domain based rather than an epitope based approach.     

Identification of novel helper epitope peptides of Survivin cancer-associated antigen applicable to developing helper/killer-hybrid epitope long peptide cancer vaccine

We identified novel helper epitope peptides of Survivin cancer antigen, which are presented to both HLA-DRB1*01:01 and DQB1*06:01. The helper epitope also contained three distinct Survivin-killer epitopes presented to HLA-A*02:01 and A*24:02. This 19 amino-acids epitope peptide (SU18) induced weak responses of Survivin-specific CD4⁺ and CD8⁺ T cells though it contained both helper and killer epitopes. To enhance the vaccine efficacy, we synthesized a long peptide by conjugating SU18 peptide and another DR53-restricted helper epitope peptide (SU22; 12 amino-acids) using glycine-linker. We designated this artificial 40 amino-acids long peptide containing two helper and three killer epitopes as Survivin-helper/killer-hybrid epitope long peptide (Survivin-H/K-HELP). Survivin-H/K-HELP allowed superior activation of IFN-γ-producing CD4⁺ Th1 cells and CD8⁺ Tc1 cells compared with the mixture of its component peptides (SU18 and SU22) in the presence of OK-432-treated monocyte-derived DC (Mo-DC). Survivin-H/K-HELP-pulsed Mo-DC pretreated with OK-432 also exhibited sustained antigen-presentation capability of stimulating Survivin-specific Th1 cells compared with Mo-DC pulsed with a mixture of SU18 and SU22 short peptides. Moreover, we demonstrated that Survivin-H/K-HELP induced a complete response in a breast cancer patient with the induction of cellular and humoral immune responses. Thus, we believe that an artificially synthesized Survivin-H/K-HELP will become an innovative cancer vaccine.     

Identification of a MHC I-restricted epitope of DsRed in C57BL/6 mice

Fluorescent proteins can be used to visualize cells and their constituents by various imaging techniques. Adoptive transfer of T cells from C57Bl/6 (B6) mice that expressed DsRed.T3 under the actin promoter lead to frequent rejection of transferred cells. In short term in vivo cytotoxicity assays these mice showed detectable, but weak lysis of DsRed spleen cells but their responses could be boosted by re-challenge with DsRed spleen cells. To determine whether DsRed protein may contain an H-2^b MHC I-restricted T cell epitope, B6 mice immune to DsRed spleen cells were examined for in vivo lysis of target cells coated with various DsRed-derived peptides selected by the SYFPEITHI epitope prediction program. This analysis identified one D^b-restricted peptide sequence within DsRed (SSLQDGCFI) that acted as an epitope for B6 target lysis. Knowledge of this epitope could allow DsRed to be used as a model antigen in B6 mice and cautions against using this fluorochrome, as well as several others containing the immunogenic sequence, in adoptive transfer studies where rejection is not desirable.     

Trypanosoma cruzi-infected macrophages are defective in major histocompatibility complex class II antigen presentation

Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagas' disease, interferes with the host immune response to establish a persistent infection. In this report, we demonstrate that macrophages infected with T. cruzi are unable to effectively present antigens to CD4 T cells. The interference is due to defective antigen-presenting cell (APC) function, as antigen-independent stimulation of the T cell in the presence of infected macrophages is not affected. The defect is distal to antigen processing and is not due to decreased major histocompatibility complex (MHC) class II expression, decreased viability, defective peptide loading in the infected macrophages, nor absence of CD28 co-stimulation. There was a role for gp39:CD40 co-stimulation during antigen presentation to the T cells we studied, but the expression of CD40 on T. cruzi-infected macrophages was not decreased. Antigen-specific adhesion between macrophages and T cells was reduced by infection. Equivalent levels of the adhesion molecules lymphocyte function-associated antigen-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or very late antigen-4 are found on infected and uninfected APC, suggesting that reduced expression of these adhesion molecules was not responsible for the defect in antigen-specific adhesion. The defective T cell:macrophage adhesion may be due to the reduced expression of other adhesion molecules or other changes in the cell induced by infection. Interfering with MHC class II antigen presentation in infected macrophages may help T. cruzi to blunt the immune response by the host.     

The reactivity of naturally sensitized human CD4 cells and IgG antibodies to synthetic peptides derived from the amino terminal sequences of a 3800 MW Streptococcus mutans antigen.

Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 MW streptococcal antigen (SA) was found in human subjects. Significant serum IgG antibodies were detected both to the native SA and to peptides consisting of residues 3-13, 1-15 and 1-20. Inhibition studies confirmed cross-reactivity between the native SA and SP. A series of short peptides with deletions at the amino and carboxy termini were then tested to determine the sequence of B-cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies, but residues 8-13 were more effective and consistent in inhibiting human antibodies than residues 1-6. These results suggest that residues 8-13 constitute a major B-cell epitope but that residues 1-6 may represent a minor B-cell epitope. The human CD4 subset of T cells was then examined by stimulating the cells with SA or SP and measuring the uptake of [3H]thymidine [( 3H]TdR). The cells were found to be sensitized in vivo to both the native SA and the SP and cross-reactivity between the SA and SP was shown by enrichment and depletion experiments on antigen-coated monocytes. As with the B-cell epitope, the series of short peptides was used to stimulate CD4 cells, in order to determine the T-cell epitope. Residues 6-15 were the shortest SP which stimulated significant [3H]TdR uptake and this peptide was designated as a T-cell epitope. The results suggest that natural oral immunization with Streptococcus mutans induces serum antibodies and T-cell sensitization to a peptide in which a T-cell epitope (residues 6-15) overlaps with a B-cell epitope (residues 8-13). Furthermore, a comparison between linear and cycled peptides suggests that unlike immunogenicity which is commonly enhanced by the more rigid cyclized peptides, antigenicity is favoured by linear peptides. This was evident not only for antibodies but also for T-cell proliferative responses.     

Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.     

Cross-recognition by T cells of an epitope shared by two unrelated mycobacterial antigens

The molecular mimicry represented by cross-recognition of determinants shared by unrelated antigens by antibodies or T cells is of broad immunological interest. In this study, we analyzed the cross-recognition by CD4⁺ T cells of a peptide epitope shared by two mycobacterial proteins of diverse sequence, represented by the 19-kDa antigen of Mycobacterium tuberculosis and the 28-kDa antigen of Mycobacterium leprae. This epitope was immunodominant with respect to the 19-kDa antigen, but cryptic in relation to the 28-kDa antigen. The cross-reactive epitope cores were identified by Pepscan window analysis and found to be eight residues long in both antigens (residues 69–76 and 127–134). Alignment of these octameric sequences revealed two identical and five conservatively related amino acids. Within the epitope core, two residues (⁷³Asn and ⁷⁶Ile) were identified as critical for recognition on the basis of inhibition of the cross-reactive T cell proliferative response using singly substituted analog peptides. These results suggest that T cell cross-reactive epitopes can exist in proteins with apparently not more than random levels of sequence homology. Their potential for unsuspected cross-sensitization may play a role in the maintenance of T cell memory, in the pathogenesis of autoimmune diseases and possibly in a wide range of host immune responses to infectious pathogens.     

Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

In Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruzi-infected animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected dogs were used. The study''s findings suggest that these three peptides can constitute a potential tool for more sensitive and specific serodiagnosis of L. infantum infection in dogs.     

Trypanosoma cruzi infection does not impair major histocompatibility complex class I presentation of antigen to cytotoxic T lymphocytes

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas' disease, lives free within the cytoplasm of infected host cells. This intracellular niche suggests that parasite antigens may be processed and presented on major histocompatibility complex (MHC) class I molecules for recognition by CD8⁺ T cells. However, the parasite persists indefinitely in the mammalian host, indicating its success at evading immune clearance. It has been shown that T. cruzi interferes with processing and presentation of antigenic peptides in the MHC class II pathway. This investigation sought to determine whether interference in MHC class I processing and presentation occurs with T. cruzi infection. Surface expression of MHC class I molecules was found to be unaffected or up-regulated by T. cruzi infection in vitro. A model system employing a β-galactosidase (β-gal)-specific murine cytotoxic T lymphocyte (CTL) line (0805B) showed: (i) in vitro infection of mouse peritoneal macrophages or J774 cells with T. cruzi did not inhibit MHC class I presentation of exogenous peptide (a nine-amino acid epitope of β-gal) to the CTL line, (ii) in vitro infection of a β-gal-expressing 3T3 cell line (LZEJ) with T. cruzi did not inhibit MHC class I presentation of the endogenous protein to the CTL line and (iii) mouse renal adenocarcinoma cells infected with T. cruzi and subsequently infected with adenovirus expressing β-gal were able to present antigen to the β-gal-specific CTL line. These findings indicate that the failure of the immune response to clear T. cruzi does not result from global interference by the parasite with MHC class I processing and presentation. Parasites engineered to express β-gal were unable to sensitize infected antigen-presenting cells in vitro to lysis by the CTL 0805B line. This was probably due to the intracellular localization of the β-gal within the parasite and its inaccessibility to the host cell cytoplasm.     

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cγ1

B cells contribute to the immune system in many ways such as antigen presentation to CD4⁺ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas’ disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cγ1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.     

The composition of upstream open reading frames (uORF) in four genes from Trypanosoma cruzi typical strains

Upstream open reading frames (uORF) are small open reading frames located in the 5′ untranslated region (5′ utr) of a mature mRNA. We analysed in four strains representing the Trypanosoma cruzi groups Tc I, Tc II, Tc IV and Tc VI the uORF present in 5′ utr sequences of four genes: P-type H+-ATPase 1, DEAD/H RNA helicase, casein kinase 1.1 and ferredoxin–NADP+ reductase. A segment in the 5′ utr at each of these genes encompassing one or more uORF was PCR amplified and sequenced. An analysis of these sequences reveals that the uORF in T. cruzi show minor variations; however, these nucleotide substitutions mirror the divergence of T. cruzi strains into major groups.     

The relationship between colonization and haemagglutination inhibiting and B cell epitopes of Porphyromonas gingivalis

Passive immunization with the monoclonal antibody 61BG1.3 selectively prevents colonization by Porphyromonas gingivalis in humans (Booth V, Ashley FP, Lehner T. Infect Immun 1996; 64:422-7). The protective MoAb recognizes the j3 component of the RI protease of P. gingivalis which is formed by proteolytic processing of a polyprotein precursor termed PrpRl. This subunit is both a haemagglutinin and an antigen which is recognized by sera from patients with periodontitis. In this study the relationship was investigated between a colonization epitope which is recognized by the MoAb 61BG1.3, a haemagglutinating and B cell epitope which are recognized by sera from patients with periodontitis. B cell epitopes were mapped by Western blotting with a series of truncated recombinant polypeptides spanning the adhesion domain within residues 784–1130 of PrpRl and by ELISA using a panel of synthetic peptides spanning the same sequence. The epitope which is recognized by the protective MoAb was mapped within residues 907–931 of PrpRl, while serum responses of patients were directed predominantly to the adjacent carboxy-terminal sequence within residues 934–1042. The haemagglutinating epitope was mapped to residues 1073–1112. In view of our previous findings that the MoAb 61BG1.3 prevents colonization of P. gingivalis in vivo and inhibits haemagglutination, these two epitopes may be in proximity in the native protein. Active or passive immunization strategies which target the protective or haemagglutinating epitopes of the adhesion domain of PrpRl may provide a means of preventing infection with P. gingivalis.     

Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.     

Major histocompatibility complex and T cell receptor interaction of the P91A tum− peptide

The P91A antigen was identified following mutation of P1 mastocytoma cells. The peptide epitope is encoded by a mutant form of the S3 subunit of the PA 700 proteasome regulatory complex. P91A stimulates a strong CD8⁺ T cell response when expressed on tumor cells or normal tissue and P91A-specific T cells express a restricted range of T cell receptors. Although it is a strong L^d-binding peptide, P91A does not conform to the established motif for this major histocompatibility complex (MHC) molecule and this has hampered elucidation of the precise epitope. L^d predominantly associates with nonamer peptides; however, using a variety of complementary approaches, the P91A epitope is identified as the octamer QNHRALDL. In the absence of the L^d motif residue proline at position 2, residues 5–7 are primarily involved in MHC interaction. P91A is thus atypical in its interaction with L^d. Residues 1, 3, and 4 are found to influence T cell recognition of P91A. Definition of the P91A peptide will allow studies on P91A processing and interactions of the P91A peptide/MHC complex with T cell receptors of differing avidity to establish the basis for restricted T cell receptor usage. The basis for the failure of the P91A tum⁺ peptide (QNRRALDL) to bind to L^d is addressed by molecular modeling.