共查询到20条相似文献,搜索用时 15 毫秒
1.
A predominant T-cell epitope of Escherichia coli outer membrane protein F (OmpF) that encompasses amino acids 295 to 314 was identified in H-2(d) mice. BALB/c-derived T-cell hybridomas generated against this region were CD3(+), CD4(+), CD8(-), and T-cell receptor alphabeta(+) and secreted TH-1-associated cytokines (interleukin-2 [IL-2] and gamma interferon), but not a TH-2-associated cytokine (IL-4), when restimulated with peptide 295-314. Class II(+) mouse lymphoma (A20) cells, but not class II(-) mouse mastocytoma (P815) cells, supported IL-2 secretion of hybridomas when substituted for syngeneic splenocytes as antigen-presenting cells (APCs). Antibodies specific for I-E(d) blocked IL-2 secretion by hybridomas, but I-A(d)-specific antiserum did not. When transfected L cells expressing I-A(d) (AalphaAbeta(d)), I-E(d) (EalphaEbeta(d)), or the hybrid molecule I-EalphaAbeta(d) were used as APCs, hybridomas recognized peptide only when presented by the I-E(d)-transfected cells. When peptide 295-314 truncated at either the C or the N terminus of the sequence was used, the minimal epitope was determined. Critical residues were determined by using alanine-substituted peptide analogues. T-cell hybridomas were only stimulated by peptides that encompassed amino acids 295 to 303 (9-mer), and the core sequence required a minimum of three additional amino acids at either the amino or the carboxy terminus to induce IL-2 secretion. Critical residues were determined to be phenylalanine at position 295, threonine at position 300, and tyrosines at positions 301 and 302. This study is the first to identify a minimal T-cell epitope and major histocompatibility complex restriction element of the OmpF protein and confirms previous observations that there is considerable degeneracy in the length of peptides that can bind I-E(d) and variability in the amino acid composition of the C and N termini of these peptides. 相似文献
2.
Neonatal Escherichia coli meningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics. Our earlier studies have shown that outer membrane protein A (OmpA) is one of the major factors responsible for Escherichia coli traversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC). In this study we showed that OmpA binds to a 95-kDa human BMEC (HBMEC) glycoprotein (Ecgp) for E. coli invasion. Ecgp was partially purified by wheat germ agglutinin and Maackia amurensis lectin (MAL) affinity chromatography. The MAL affinity-purified HBMEC proteins bound to OmpA(+) E. coli but not to OmpA(-) E. coli. In addition, the deglycosylated MAL-bound proteins still interact with OmpA(+) E. coli, indicating the role of protein backbone in mediating the OmpA binding to HBMEC. Interestingly, the MAL affinity-bound fraction showed one more protein, a 65-kDa protein that bound to OmpA(+) E. coli in addition to Ecgp. Further, the 65-kDa protein was shown to be a cleavage product of Ecgp. Immunocytochemistry of HBMEC infected with OmpA(+) E. coli by using anti-Ecgp antibody suggests that Ecgp clusters at the E. coli entry site. Anti-Ecgp antibody also reacted to microvascular endothelium on human brain tissue sections, indicating the biological relevance of Ecgp in E. coli meningitis. Partial N-terminal amino acid sequence of Ecgp suggested that it has 87% sequence homology to gp96, an endoplasmic reticulum-resident molecular chaperone that is often expressed on the cell surface. In contrast, the 65-kDa protein, which could be the internal portion of Ecgp, showed 70% sequence homology to an S-fimbria-binding sialoglycoprotein reported earlier. These results suggest that OmpA interacts with Ecgp via the carbohydrate epitope, as well as with the protein portion for invading HBMEC. 相似文献
3.
P6 outer membrane protein is one of the candidates for a vaccine formulation against non-typeable Haemophilus influenzae (NTHi) infection. However, otitis-prone children who have recurrent episodes of acute otitis media due to NTHi fail to respond adequately to P6. An innovative approach to vaccination is therefore required to augment such children's immune response. To develop an effective peptide vaccine, we established P6-specific CD4(+) T-cell lines (TCLs) restricted by the human histocompatibility leukocyte antigen (HLA)-DR9 molecule, and revealed a human T-cell epitope on P6 and its core peptide sequence (p77-85; EYNIALGQR). Furthermore, we found that 3 analog peptides, E77D (the substitution of E at position 77 with D), N79G, and R85K, induced high proliferative responses as well as marked cytokine production when compared to the T-cell epitope peptide. These peptides may be candidates for a peptide vaccine formulation effective against NTHi infections, even in otitis-prone children. 相似文献
4.
An antiserum (WA-SAA) that agglutinates specifically with mouse virulent but not avirulent strains of Yersinia enterocolitica was used to identify virulence-associated factors by Western blot techniques. Several outer membrane polypeptides were identified only in the virulent strains, which included serotypes O:8, O:3, O:9, O:4,32, O:5,27, and O:21. These included three, and possibly four, major outer membrane polypeptides. The prominent high-molecular-weight species was demonstrated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot, whereas the others were only revealed by the Western blot technique. Expression of these polypeptides correlated with antiserum agglutination reaction and the presence of a 42- and/or 82-megadalton plasmid. These polypeptides were highly temperature dependent and only slightly affected by the inclusion of 10 mM Ca2+ in the growth medium. These polypeptides were produced during both the logarithmic and stationary phases of growth at 37 degrees C. We suggest that the production of these specific polypeptides and calcium dependency may be coded for by the plasmid(s) but are regulated by independent mechanisms. These polypeptides appear to be novel markers specific for virulent strains of Y. enterocolitica and may be important to the pathogenicity of this organism. 相似文献
5.
The P2 protein of Haemophilus influenzae type b has a porin activity and is the most abundant protein in the outer membrane. We have employed fusion protein constructs and synthetic peptides along with monoclonal antibodies to map B-cell epitopes in this protein. A linear, surface-exposed epitope was identified between residues 158 and 174. A second surface-exposed epitope was identified near the carboxy-terminal end of the protein (residues 319 to 341). Two additional B-cell epitopes were identified. One was localized between residues 28 and 55, whereas the other was located between residues 148 and 174. These epitopes were not present on the surface of intact H. influenzae cells. Thus, four distinct immunogenic and antigenic regions on the P2 protein have been identified. 相似文献
6.
CD8 T cells play an important role in controlling Ebola infection and in mediating vaccine-induced protective immunity, yet little is known about antigenic targets in Ebola that are recognized by CD8 T cells. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding Ebola nucleoprotein (NP). CD8 T-cell responses were mapped to a H-2(d)-restricted epitope (NP279-288) and two H-2(b)-restricted epitopes (NP44-52 and NP288-296). The identification of these epitopes will facilitate studies of immune correlates of protection and the evaluation of vaccine strategies in murine models of Ebola infection. 相似文献
7.
The outer membrane protein (OMP) prepared from Escherichia coli was found to be a potent mitogen for murine B cells and to be capable of inducing polyclonal antibody formation as well as a proliferative response. Spleen cells from nude mice responded equally as well to OMP as those from their normal litter-mates, whereas nylon-wool-purified T cells or thymocytes failed to respond. The proliferative response was dependent on the presence of macrophages. The macrophage dependency of the polyclonal antibody response seemed to be less than that of the proliferation. OMP was mitogenic for lipopolysaccharide (LPS)-resistant C3H/HeJ spleen cells, further indicating that OMP is an unique B-cell mitogen distinct from LPS. OMP also enhanced the specific antibody response sixty-seven-fold to an optimal dose of sheep red blood cells (SRBC) in vitro. The kinetics of the response, however, was not altered from that of cultures without OMP. The anti-SRBC response of spleen cells from C3H/HeJ mice was also enhanced by the addition of OMP, suggesting that the adjuvant effects were not due to the LPS in the preparation. Antibody responses in vitro to TI-1 antigens, trinitrophenyl-LPS (Boivin) (TNP-LPSB) and TNP-Brucella abortus, were not enhanced in the presence of OMP. In contrast OMP enhanced the response to TI-2 antigens, TNP-LPSW (Westphal) and dinitrophenyl-Ficoll and T cells were shown to be required for these augmented antibody responses. Enhancement was not seen in nude mouse spleen cell cultures but was seen when nylon-wool-purified T cells were added to the cultures. 相似文献
8.
The ehrlichiae express variable outer membrane proteins (OMPs) that play important roles in both pathogenesis and host defense. Previous studies revealed that OMPs are immunodominant B-cell antigens and that passive transfer of anti-OMP antibodies can protect SCID mice from fatal ehrlichial infection. In this study, we used a model of fatal monocytotropic ehrlichiosis caused by Ehrlichia bacteria from Ixodes ovatus (IOE) to determine whether OMP immunization could generate protective immunity in immunocompetent mice. Immunization of C57BL/6 mice with a purified recombinant OMP expressed by IOE omp19 generated protection from fatal IOE infection and elicited robust humoral and CD4 T-cell responses. To identify CD4 T-cell epitopes within OMPs, we performed enzyme-linked immunospot analyses for gamma interferon (IFN-gamma) production using a panel of overlapping 16-mer peptides from IOE OMP-19. Five immunoreactive peptides comprising residues 30 to 45, 77 to 92, 107 to 122, 197 to 212, and 247 to 264 were identified; the strongest response was generated against OMP-19(107-122). Most of the peptides are conserved between E. muris and E. chaffeensis OMP-19, and they elicited IFN-gamma production in CD4 T cells from E. muris-infected mice, indicating that T-cell epitope cross-reactivity likely contributes to heterologous immunity. Accordingly, CD4 T-cell responses to both OMP-19 and OMP-19(107-122) were of greater magnitude following high-dose IOE challenge of mice that had been immunized by prior infection with E. muris. Our studies cumulatively identify B- and T-cell epitopes that are associated with protective homologous and heterologous immunity during ehrlichial infection. 相似文献
9.
The OmpC outer membrane protein of Escherichia coli was used as a carrier molecule for the nonimmunogenic heat-stable enterotoxin STa. Two fragments of different lengths of the gene encoding STa were fused in vitro to the 3' terminus of the truncated ompC gene. The resulting OmpC-STa hybrid proteins could be detected by L-[35S]cysteine labeling, and they were processed and thus exported. All synthesized hybrid protein remained cell bound and was found by fractionation mainly in the periplasm. Immunoblot analysis showed that the hybrid proteins reacted in vitro both with anti-OmpC and anti-STa antibodies, and immunization of rabbits evoked an antibody response to either of these proteins. 相似文献
10.
The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein. 相似文献
11.
Enteroaggregative Escherichia coli (EAggEC) is a distinct category of diarrheal pathogen implicated as the cause of persistent diarrhea. The pathogen exhibits a characteristic "stacked-brick" pattern of aggregation when incubated with HEp-2 cells. The outer membrane protein (OMP) profile of a prototype EAggEC strain (F03) reflected the presence of one major 30-kDa protein. The OMP is expressed in the presence of the 60-MDa plasmid that the strain harbors. Antibodies were raised against the OMP by injecting the protein into a rabbit. The manifestation of an adherence phenotype on HEp-2 cells was observed for F03 and other strains that express OMP in the presence and absence of anti-OMP serum. Clumps of bacteria forming an aggregative pattern were observed in the HEp-2 cell assay in the absence of OMP antibodies, whereas a few bacteria attached to the cells in the presence of OMP antibodies. Mannose-resistant hemagglutination of human erythrocytes observed in the presence of EAggEC strains was inhibited in the presence of anti-OMP serum. Sequence analysis of a peptide generated by protease digestion of OMP exhibited 90% homology to a peptide of flagellin protein encoded by the hag gene of Serratia marcescens. Immunolabeling of the outer membrane by colloidal gold confirmed the protein to be an OMP. Our results suggest that the OMP of EAggEC have common antigenic properties. Antibodies raised against the protein can prevent adherence in vitro and could potentially interrupt the natural disease. 相似文献
12.
The antigenically variant Chlamydia trachomatis major outer membrane protein (MOMP) is a target of antibody-mediated neutralization in vitro, and it is an important protein for designing a subunit vaccine. Knowledge of MOMP T-cell determinants will be essential to elicit rapid and strong immune responses following an encounter with infectious organisms. C. trachomatis-specific T-cell lines were derived from MOMP-immunized BALB/c mice and selected with intact organisms. We used these short-term T-cell lines to identify determinants of MOMP that could be recognized by T cells following processing of the intact organism. T-cell line proliferation in response to overlapping MOMP peptides showed that only a peptide encompassing the third variable segment (VS3) elicited a strong proliferative response. We further mapped determinants within the VS3 peptide and found that a sequence-conserved portion of the VS3 peptide elicited T-cell proliferation of T-cell lines from BALB/c mice. Thus, unlike the response to several MOMP peptides with unselected T cells, development of short-term T-cell lines with intact organisms restricted the repertoire of antigens capable of being recognized by MOMP-specific T cells. 相似文献
13.
The purpose of this study was to screen and identify the linear B-cell epitopes of Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2). The secondary structure and surface properties of EBV LMP2A protein were analyzed. In combination with hydrophilicity, accessibility, flexibility, and antigenicity analysis, and average antigenicity index (AI) of epitope peptide investigation, three peptides were selected as potential candidates of linear B-cell epitopes. The peptides were 199-209 (RIEDPPFNSLL), 318-322 (TLNLT), and 381-391 (KSLSSTEFIPN). The fragments encoding potential B-cell epitopes were cloned and overexpressed in an E. coli system. The immune sera of these fusion proteins were collected from BALB/c mice by subcutaneously immunizing them three times. Western blotting results showed that these epitope recombinant proteins could be recognized by the serum antibodies against the whole LMP2 from nasopharyngeal carcinoma (NPC). Indirect ELISA measuring individual sera from 196 NPC patients, 44 infectious mononucleosis (IM) patients, 253 healthy adults, and 61 healthy children, indicated that NPC patients had significantly higher reactivity to these epitope-fused proteins compared with IM and healthy individuals (p?0.05). In addition, all the immune sera of peptide-fused proteins responded to native LMP2A antigen obtained from the EBV prototype strain, B95-8?cells. IFA results confirmed that specific antibodies induced by epitope peptide-fused proteins recognized intracellular regions of LMP2A. These results demonstrated that these three predictive epitopes not only were immunodominant B-cell epitopes of LMP2A, but also may be potential targets for applications in the design of diagnostic tools. 相似文献
14.
In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis, and Mycobacterium leprae, recombinants were frequently encountered that expressed an immunodominant 65-kilodalton (kDa) protein antigen that was shown to react with a high proportion of mycobacterium-reactive human and murine T cells and murine monoclonal antibodies. In this study, recombinant antigens were used to map T-cell and B-cell epitopes on the M. bovis BCG 65-kDa protein that was previously designated MbaA. Four different T-cell-epitope-containing regions (amino acid residues 1 through 16, 17 through 61, 85 through 108, and 235 through 279) were defined that were recognized by seven T-cell clones from patients with tuberculoid leprosy. These regions are distinct from two previously described T-cell epitopes recognized by T cells from a tuberculosis patient. As T-cell clones restricted by different class II determinants were shown to be specific for different regions on the 65-kDa protein, the presented data suggested that the products of different human leukocyte antigen class II loci and alleles present different parts of MbaA to the immune system. B-cell epitopes recognized by 20 monoclonal antibodies were assigned to eight different regions of MbaA. Using 15 of these antibodies, we previously showed that MbaA was antigenically related to a common antigen present in many bacterial species. The dispersed localization of the involved epitopes defined here shows that various different parts of MbaA are indeed conserved. These results show that well-defined recombinant antigens are useful tools for the localization of both B- and T-cell-epitope-containing regions of a protein. Peptides synthesized from the sequences of such regions may then exactly define the epitopes relevant for the development of specific diagnostic tests or of vaccines against mycobacteria. 相似文献
16.
We are interested in antigen processing mechanisms of antigen-presenting cells, and to what extent the susceptibility of protein antigens to degradation contributes to immunogenicity. Understanding the biochemistry of antigen processing may be essential for reliable prediction of T-cell epitopes and for the design of vaccines that are optimized for T-cell priming. To examine possible effects of protein structural context on antigen presentation, we used genetic engineering techniques to insert helper T-cell epitopes derived from sperm whale myoglobin into surface loops of the highly stable Escherichia coli alkaline phosphatase, with the expectation that presentation of the myoglobin guest epitopes might vary with their position in the carrier protein. Levels of recombinant protein expression in E. coli cells and residual enzyme activity depended on the location of the guest peptides in the alkaline phosphatase carrier. Mutants with insertions between residues 189-190 of the carrier were recovered with yields and activities similar to the wild type protein; however, insertion of the same peptides at a second site, between residues 165-166, led to low recoveries and diminished phosphatase activities. Subcutaneous injection of mice with one of the purified recombinant proteins in complete Freund's adjuvant induced T cells that responded to in vitro challenge with myoglobin. The potential use of this system to dissect processing mechanisms is discussed. 相似文献
17.
Peptide 9 (TDAYNQKLSERRAN) and peptide 10 (NATAEGRAINRRVE) represent surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa. Rats immunized with four intramuscular inoculations on days 0, 14, 28, and 42 with either peptide 9 or peptide 10 conjugated to keyhole limpet hemocyanin were afforded protection against pulmonary lesions when examined 7 days subsequent to challenge (day 56) via intratracheal inoculation of P. aeruginosa-containing agar beads. Peptide 9 shares considerable homology with other OmpA-related outer membrane proteins in various bacteria, whereas peptide 10 displays little homology with these other proteins. Antisera directed to peptide 9 reacted weakly with cell envelope proteins from the various other OmpA-associated bacteria upon immunoblot analysis. However, antisera directed to peptide 10 reacted only with Neisseria gonorrhoeae cell envelope proteins upon immunoblot analysis. Antisera to both peptides 9 and 10 reacted at minimal titers with whole cells of the various other bacteria in a whole-cell enzyme-linked immunosorbent assay (ELISA) but antisera to each of the peptides reacted at high titers when various strains of P. aeruginosa were used as the ELISA antigen. Antibodies to peptides 9 and 10 were protective, reactive to all strain of P. aeruginosa tested except for a protein F-deficient mutant, and functionally specific against pseudomonads. 相似文献
18.
Inadequate knowledge of pathogenesis and pathophysiology has contributed to the high mortality and morbidity associated with neonatal Escherichia coli meningitis. We have shown previously that outer membrane protein A (OmpA) contributes to E. coli K1 membrane invasion of brain microvascular endothelial cells. In this study we report that this OmpA+ K1 E. coli invasion of brain microvascular endothelial cells was inhibited by wheat germ agglutinin and chitooligomers prepared from the polymer of 1,4-linked GlcNAc, chitin. The specificity of the interaction between OmpA and GlcNAc beta 1-4GlcNAc epitopes was verified by the demonstration that chitotriose-bound OmpA and wheat germ agglutinin-bound brain microvascular endothelial cell membrane proteins inhibit E. coli K1 invasion. Of interest, OmpA+ E. coli invasion into systemic endothelial cells did not occur, but invasion similar to that of brain microvascular endothelial cells was observed when systemic cells were treated with alpha-fucosidase, suggesting that the GlcNAc beta 1-4GlcNAc moieties might be substituted with L-fucose on these cells. More importantly, the chitooligomers prevented entry of E. coli K1 into the cerebrospinal fluid of newborn rats with experimental hematogenous E. coli meningitis, suggesting that the GlcNAc beta 1-4GlcNAc epitope of brain microvascular endothelial cells indeed mediates the traversal of E. coli K1 across the blood-brain barrier. A novel strategy with the use of soluble receptor analog(s) may be feasible in the prevention of devastating neonatal E. coli meningitis. 相似文献
19.
We previously identified 18 stimulatory Chlamydia trachomatis major outer membrane protein (MOMP) peptides containing at least 23 epitopes presented with various HLA class II allotypes. Only one peptide contained an epitope localized in a variable segment (VS2). Continued studies reported here identified a total of five VS peptides containing T-cell epitopes that are distributed among MOMPs VS1, VS2, and VS4. Only MOMP-primed T-cell cultures from subjects infected with serovar E responded to the serovar E VS peptides, while the response of such cultures to constant-segment peptides was independent of the infecting serovar. Furthermore, MOMP-primed T cells proliferated in response only to the VS peptides encoded in serovar E but not to the corresponding peptides derived from serovar F, I, or J, confirming that these responses were serovar specific. 相似文献
20.
DNA obtained from Chlamydia trachomatis (serovar L2) was partially digested with DNase I and inserted into the beta-galactosidase gene of bacteriophage lambda gt11. Seven recombinants were selected that produced immunoreactive fusion proteins which were detected with anti-C. trachomatis rabbit serum. One recombinant, designated lambda gt11/L2/33, reacted with various monoclonal antibodies that recognize species-, subspecies-, and type-specific determinants on the chlamydial major outer membrane protein (MOMP). Immunoblot analysis of a lambda gt11/L2/33 lysogen revealed a fusion protein that expressed a approximately 15,000-dalton carboxyl-terminal peptide of the chlamydial MOMP. This moiety of the MOMP possesses epitopes responsible for each of the unique reactivities demonstrated by anti-MOMP monoclonal antibodies. The lambda gt11/L2/33 recombinant contained a 1.1-kilobase DNA insert which hybridized to DNA isolated from each of the 15 C. trachomatis serovars. 相似文献
|