Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies.

Two glutathione S-transferase fusion proteins containing 44 (p1503) and 75 (p1509) amino acid residues of the rubella virus E1 glycoprotein were expressed in Escherichia coli with the aim of producing a recombinant rubella virus antigen for use in serological assays. p1503 contained three neutralizing and hemagglutinating epitopes (G. M. Terry, L. M. Ho-Terry, P. Londesborough, and K. R. Rees, Arch. Virol. 98:189-197, 1988); p1509 contained the putative neutralization domain described by Mitchell et al. (L. A. Mitchell, T. Zhang, M. Ho, D. Decarie, A. Tingle, M. Zrein, and M. Lacroix, J. Clin. Microbiol. 30:1841-1847, 1992) in addition to the three epitopes present in p1503. Both fusion proteins were soluble and affinity purified on glutathione-Sepharose 4B. In Western blots (immunoblots), p1503 and p1509 reacted with human sera containing rubella virus-specific immunoglobulin G. When used as antigens in indirect enzyme immunoassays to detect rubella virus-specific immunoglobulin G, p1503 correctly identified the rubella virus antibody status of 43 (76.8%) and p1509 correctly identified that of 48 (85.7%) of 56 serum samples received for routine rubella virus antibody screening. The results obtained with p1509 compare well with those obtained with a latex agglutination assay.     

Three T-cell epitopes within the C-terminal 265 amino acids of the matrix protein pp65 of human cytomegalovirus recognized by human lymphocytes

Although a T-cell response in human cytomegalovirus (HCMV)-immune individuals exists against the most abundantly expressed protein pp65 of the virus matrix, less is known about the determinants that evoke this response. The aim of the study was to identify regions within HCMV pp65 (ppUL83) that contain sequences for the cellular immune response by the use of three recombinant overlapping β-galactosidase pp65 fusion proteins (C74, C35, and C47), covering the C-terminal 265 amino acids of the entire pp65 sequence. Two T-cell epitope determinants were recognized by human lymphocytes of healthy, HCMV-seropositive, human leukocyte antigen (HLA)-typed individuals. One T-cell determinant (amino acids [aa] 303–388) was localized in the mid-region of the entire pp65 sequence and a second T-cell determinant (aa 477–561) within the C-terminal region. By fine mapping with synthetic hexadecamer peptides three T-cell epitopes were identified within these two regions: P10-I (aa 361–376) in the mid-region, P3-II (aa) 485–499), and P6-II (aa 509–524) in the C-terminal region. Inhibition studies with monoclonal antibodies to HLA class I or class II revealed a class I restricted response to peptides P10-I or P6-II, respectively. P10-I responders shared the HLA-DR11 allele and P6-II responders the -DR3 allele. Therefore, these T-cell epitopes of HCMV pp65 might be presented in association with particular HLA class II alleles. J. Med. Virol. 52:68–76, 1997. © 1997 Wiley-Liss, Inc.     

用点突变的方法组建巨细胞病毒抗原决定簇基因的表达克隆

研究资料表明，人巨细胞病毒（ＨＣＭｖ）单一蛋白的单一抗原决定簇只能被部分患者阳性血清识别。组建在血清学诊断中能够替代全病毒抗原的基因工程抗原，需要含有病毒多种主要抗原蛋白的抗原决定簇。为搞清在表达载体中重复插入某一抗原决定簇基因是否能表达出更高抗原效价的融会蛋白，我们用点突变的方法，在表达载体中分别插入了人ＨＣＭｖ的ｐｐＵＬ３２蛋白羧基端一个抗原决定簇基因的１个、２个和３个拷贝。在免疫转印检测中，这些克隆表达的融合蛋白与特异性阳性血清的反应性差别不明显。这表明，插入表达载体中目的基因的多寡对表达蛋白的抗原效价没有显著影响。     

Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus

We have previously shown that avian reovirus (ARV) sigmaA and sigmaNS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on sigmaA (I and II) and three epitopes (A, B, and C) on sigmaNS. To further define the location of epitopes on sigmaA and sigmaNS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of sigmaA and sigmaNS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on sigmaA was located at amino acid residues 340QWVMAGLVSAA350 and epitope B on sigmaNS at amino acid residues 180MLDMVDGRP188. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of sigmaA and sigmaNS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete sigmaNS but not with truncated sigmaNS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on sigmaNS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on sigmaA was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the sigmaNS with MAb 1F9. The sigmaNS of ARV with ssRNA-binding activity could be blocked by monoclonal antibody 1F9. The epitope B on sigmaNS is required for ssRNA binding because its deletion fully abolished the ssRNA binding activity of sigmaNS.     

Structure of the genome of equine herpesvirus type 3

Restriction endonuclease mapping studies were performed to determine the molecular structure of the genome of equine herpesvirus type 3 (EHV-3). Purified EHV-3 DNA, either unlabeled or 32P-labeled, was analyzed using the restriction enzymes BamHI, BclI, BglII, EcoRI, and HindIII. The findings that four 0.5 M (molar) fragments were present, that two of these were terminal fragments, and that all 0.5 M fragments contained homologous DNA sequences as judged by DNA hybridization analyses indicated that DNA sequences located at one terminus are repeated within the molecule and that two populations of molecules exist with regard to the arrangement of this pair of shared sequences. Mapping of BamHI, BclI, BglII, EcoRI, and HindIII fragments by double digestion of intact EHV-3 DNA, reciprocal digestion of isolated restriction enzyme fragments, and blot hybridization experiments revealed that the EHV-3 genome is a linear, double-stranded DNA molecule with a molecular size of 96.2 +/- 0.48 MDa and is comprised of two covalently linked segments, designated L (long) and S (short). The S region is approximately 22.9 MDa in size and consists of a unique segment (Us) of approximately 5.8 MDa bracketed by 8.5 MDa inverted repeat sequences that allow the S region to invert relative to the fixed L region which is approximately 73.3 MDa in size and consists only of unique sequences. Thus, these data confirm that EHV-3 DNA exists in two isomeric forms and has a molecular structure similar to that of the genomes of EHV-1 (B. E. Henry, S. A. Robinson, S. A. Dauenhauer, S. S. Atherton, G. S. Hayward, and D. J. O'Callaghan, Virology 115, 97-114, 1981; D. J. O'Callaghan, G. A. Gentry, and C. C. Randall, "The Herpesvirus," Vol. 2, pp. 215-318, Plenum, New York, 1983; D. J. O'Callaghan, B. E. Henry, J. H. Wharton, S. A. Dauenhauer, R. B. Vance, J. Staczek, and R. A. Robinson, "Developments in Molecular Virology," Vol. 1, pp. 387-418, Nijhoff, The Hague, 1981; W. T. Ruyechan, S. A. Dauenhauer, and D. J. O'Callaghan, J. Virol., 42, 297-300, 1982), pseudorabies virus (W. Stevely, J. Virol., 22, 232-234, 1977; T. Ben-Porat, F. J. Rixon, and M. L. Blankenship, Virology, 95, 285-294, 1979), varicella-zoster virus (A. M. Dumas, J. L. Geelen, M. W. Weststrate, P. Wertheim, and J. Van Der Noordaa, J. Virol., 39, 390-400, 1981; S. E. Straus, H. S. Aulakh, W. T. Ruyechan, J. Hay, T. A. Casey, G. F. Vande Woude, J. Owens, and H. A. Smith, J. Virol., 40, 516-525, 1981.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fusion Proteins with Heterologous T Helper Epitopes. Recombinant E. coli Heat-Stable Enterotoxin Proteins

Fusion proteins containing specific B cell and T cell epitopes were used to examine how the intramolecular arrangement of T and B cell epitopes within a chimeric protein influences antigen-specific B cell antibody responses as well as specific T cell activation. Chimeric proteins, containing single or multiple copies of the T_h epitope ovalbumin 323-339 (ova) linked at different positions to STa, the heat-stable enterotoxin of E. coli, were compared with respect to their ability to induce STa-specific antibody production and to induce ova-specific T cell activation.

Chimeric proteins induced ova-dependent antibody production against STa at the amino terminal end, irrespective of the positioning of ova. Multiple tandem copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against a second B cell epitope at the carboxy terminus of the fusion proteins was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. A fusion protein, containing four copies of ova effectively elicited T cell help for antibody production against both examined B cell determinants, showing that activated T_h cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein.

T cell recognition of ova in all chimeric peptides, independently of its position, followed the same pattern of genetic restriction (i.e. immunodominant in H-2^d and nonimmunogenic in H-2^k) as in the native ovalbumin molecule. At high doses, the level of ova-specific T cell activation in LNC of immunized mice was similar, irrespective of the number of ova copies in the fusion proteins. In contrast, at low antigen doses chimeric proteins containing multiple copies of ova effectively primed ova-specific T cells at 20-fold lower concentrations than did fusion proteins containing a single copy of ova. Furthermore, when the ova-containing constructs were presented to a ova-specific T cell line by A20 B lymphoma cells, an increased level of IL-2 production by the clonal T cells in response to multiple copies of ova in the chimeric antigens was observed.     

Characterization of the neutralizing epitopes of VP7 of the Gottfried strain of porcine rotavirus.

The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.     

Molecular and evolutionary analysis of Borrelia burgdorferi 297 circular plasmid-encoded lipoproteins with OspE- and OspF-like leader peptides

We previously described two OspE and three OspF homologs in Borrelia burgdorferi 297 (D. R. Akins, S. F. Porcella, T. G. Popova, D. Shevchenko, S. I. Baker, M. Li, M. V. Norgard, and J. D. Radolf, Mol. Microbiol. 18:507-520, 1995; D. R. Akins, K. W. Bourell, M. J. Caimano, M. V. Norgard, and J. D. Radolf, J. Clin. Investig. 101:2240-2250, 1998). In this study, we characterized four additional lipoproteins with OspE/F-like leader peptides (Elps) and demonstrated that all are encoded on plasmids homologous to cp32 and cp18 from the B31 and N40 strains, respectively. Statistical analysis of sequence similarities using the binary comparison algorithm revealed that the nine lipoproteins from strain 297, as well as the OspE, OspF, and Erp proteins from the N40 and B31 strains, fall into three distinct families. Based upon the observation that these lipoproteins all contain highly conserved leader peptides, we now propose that the ancestors of each of the three families arose from gene fusion events which joined a common N terminus to unrelated proteins. Additionally, further sequence analysis of the strain 297 circular plasmids revealed that rearrangements appear to have played an important role in generating sequence diversity among the members of these three families and that recombinational events in the downstream flanking regions appear to have occurred independently of those within the lipoprotein-encoding genes. The association of hypervariable regions with genes which are differentially expressed and/or subject to immunological pressures suggests that the Lyme disease spirochete has exploited recombinatorial processes to foster its parasitic strategy and enhance its immunoevasiveness.     

幽门螺杆菌黏附素A有效T和B联合抗原表位噬菌体展示和抗原性测定

目的 分析并确定幽门螺杆菌黏附素A(HpaA)分子中有效T细胞和B细胞联合(T/B)抗原表位.方法 重组表达HpaA(rHpaA)并免疫家兔制备抗血清.采用生物信息学技术预测和分析HpaA分子中T细胞和B细胞表位,PCB扩增T/B联合表位肽片段并构建其噬菌体展示系统.采用PEG/NaCl沉淀法提纯展示了T/B联合表位的重组噬菌体PⅢ蛋白(rPⅢ).分别以商品化幽门螺杆菌全菌IgG抗体和rHpaA抗血清为一抗,采用Western blot和ELISA对rPⅢ蛋白中展示的T/B联合表位进行筛选和鉴定.采用MTT检测rHpaA免疫小鼠脾细胞在不同重组噬菌体蛋白刺激下的增殖情况.结果 HpaA分子中共有5个T/B联合表位:HpaA10、HpaA37、HpaA79、HpaA116和HpaA143.所有T/B联合表位均成功地展示于M13噬菌体PⅢ蛋白表面.Western blot、ELISA和淋巴细胞增殖试验结果 均显示,HpaA116是优势抗原表位,HpaA37和HpaA79为有效抗原表位,HpaA10和HpaA143为无效抗原表位.结论 本研究成功地构建了幽门螺杆菌HpaA的T/B联合表位肽噬菌体展示系统.HpaA37和HpaA79,尤其是HpaA116是HpaA有效T/B联合抗原表位.     

Molecular manipulation with the arthritogenic epitopes of the G1 domain of human cartilage proteoglycan aggrecan

Systemic immunization of BALB/c mice with human cartilage proteoglycan (PG) aggrecan induces progressive polyarthritis. The G1 domain of the PG aggrecan molecule contains most of the T cell epitopes, including three immunodominant ('arthritogenic') and at least six subdominant T cell epitopes. The three dominant T cell epitopes (P49, P70 and P155) were deleted individually or in combination by site directed mutagenesis, and the recombinant human G1 (rhG1) domain (wild type and mutated) proteins were used for immunization. Close to 100% of BALB/c mice immunized with the wild-type (nonmutated) rhG1 domain developed severe arthritis, which was 75% in the absence of P70 (5/4E8) epitope, and very low (< 10% incidence) when all three dominant T cell epitopes were deleted. The onset was delayed and the severity of arthritis reduced in animals when dominant T cell epitopes were missing from the immunizing rhG1 domain. The lack of T cell response to the deleted epitope(s) was specific, but the overall immune response against the wild-type rhG1 domain of human PG was not significantly affected. This study helped us to understand the dynamics and immune-regulatory mechanisms of arthritis, and supported the hypothesis that the development of autoimmune arthritis requires a concerted T cell response to multiple epitopes, rather than the immune response to a single arthritogenic structure.     

T and B cell responses to chimeric proteins containing heterologous T helper epitopes inserted at different positions.

The ability of a T helper (Th) epitope to induce help for B cells recognizing different determinants within a multideterminant antigen was investigated. Chimeric fusion proteins, containing inserts of single or multiple copies of the Th epitope ovalbumin 323-339 (ova) at two different positions, were compared with respect to their ability to induce specific antibody production and ova-specific T cell activation. The antibody responses against B cell determinants at the amino and carboxy terminus, respectively was differently influenced by the molecular positioning of the inserted Th determinant. All ova-containing fusion proteins induced antibody production against the B cell determinant at the amino terminal end irrespective of the positioning of ova. In addition, multiple copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against the determinant at the carboxy terminus was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. Furthermore a fusion protein, containing four copies of ova effectively elicited T cell help for high levels of antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. Immunodominant T cell recognition of ova in all chimeric peptides, independently of its position, was demonstrated by lymph node cell (LNC) proliferation of primed BALB/c mice. The level of ova-specific T cell proliferation was similar, irrespective of which chimeric peptide that had been used for priming, and thus did not reveal any differences in T cell priming efficiencies related to the number of ova copies in the fusion proteins. However, when the peptides were presented to a ova-specific T cell line by A20 B lymphoma cells, a close correlation between IL-2 production by the clonal T cells and the number of ova epitopes in the chimeric peptides was observed. Thus, increased cytokine production by ova-specific T cells may be important for the increased level of in vivo antibody production observed in response to multiple copies of ova in the chimeric antigens.     

Humoral immune response to functional regions of human cytomegalovirus glycoprotein B

Sera from patients with primary human cytomegalovirus (HCMV) infections, both acute and convalescent phase, and from HCMV-seropositive healthy subjects were analyzed to determine whether the sera would recognize antigenic domains on HCMV glycoprotein B (gB) that function in virion infectivity and spread of virus from cell to cell. The intact gB molecule, amino-terminal derivatives of different lengths, and internal deletion derivatives were expressed in eukaryotic cells and reacted by immunofluorescence with the sera. All convalescent-phase sera and most sera from healthy seropositive individuals reacted with full-length gB and with an amino-terminal derivative containing 687 amino acids (aa), gB-(1–687); approximately half of the sera recognized an amino-terminal derivative of 447 aa, gB-(1–447), and one-third reacted with the shortest deletion derivative of 258 aa, gB-(1–258). Of the acute-phase sera, 77% recognized intact gB and gB-(1–687), 32% recognized gB-(1–447), and 14% recognized gB-(1–258). Deletion of aa 548 to 618 dramatically reduced the percentage of reactive sera, whereas deletion of aa 411 to 447 had a minor impact on reactivity of sera. To investigate the epitope specificity of human antibodies to gB, we carried out competition experiments between human sera with neutralizing activity and selected monoclonal antibodies (mAbs) to conformational epitopes on gB. We found that antibodies in human sera preclude syncytium formation in UB15-11 glioblastoma cells constitutively expressing gB and compete with certain murine mAbs that block virus entry into cells and transmission of infection from cell to cell. Our results show that HCMV-immune human sera contain antibodies to functional regions on gB, and the abundance of these antibodies in convalescent-phase sera suggests that they may play a central role in limiting dissemination of virus in the host. J. Med. Virol. 52:451–459, 1997. © 1997 Wiley-Liss, Inc.     

Comparative analysis of Brucella serotype A and M and Yersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats.

Hapten polysaccharides of Brucella smooth M and A serotypes were prepared from Brucella sp. and Yersinia enterocolitica O:9 by previously described hydrolytic (O chain) or nonhydrolytic (native hapten [NH]) procedures. The purified polysaccharides differed only in the presence (O chain) or absence (NH) of lipopolysaccharide core sugars. The polysaccharides were compared by reverse radial immunodiffusion for the diagnosis of brucellosis in cattle (Brucella abortus biotype 1 [A serotype] and Brucella melitensis biotype 3 [AM serotype]), sheep (B. melitensis biotypes 1 [M serotype] and 3), and goats (B. melitensis biotype 1). The reverse radial immunodiffusion test with the NH from B. melitensis 16 M (serotype M) showed the highest sensitivity (89.6 to 97.3%), regardless of the host species and the serotype of the infecting Brucella sp. Y. enterocolitica O:9 NH (A serotype) was useful for diagnosing disease in cattle infected with B. abortus biotype 1, but not in cattle infected with B. melitensis biotype 3, sheep, or goats. The different results obtained with the serotype M and A polysaccharides and the sera from animals infected with M, A, and AM serotypes of Brucella spp. showed that in naturally infected animals, a large proportion of the antibodies are directed to or react with a previously defined common epitope(s) (J. T. Douglas and D. A. Palmer, J. Clin. Microbiol. 26:1353-1356, 1988) different from the A or M epitopes. By using the radial immunodiffusion test with B. melitensis 16M NH, it was possible to differentiate infected from vaccinated cattle, sheep, and goats with a sensitivity and specificity similar to that of the complement fixation test.     

Pattern and persistence of the epitope-specific IgM response against human cytomegalovirus in renal transplant patients.

The humoral immune response against human cytomegalovirus (HCMV) was evaluated in immunocompromised patients by Western blotting (WB) based on recombinant viral envelope (gB and gH) and tegument (pp150 and pp65) proteins. Three groups of patients were investigated: (a) 74 renal transplant recipients; (b) 24 hemodialysis patients, both groups without clinical evidence of viral infections; and (c) 19 renal transplant patients with manifest HCMV infections. The results obtained suggest that (i) the WB is considerably more sensitive, recognizing the HCMV-specific IgM response rather than the enzyme-linked immunosorbent assays. An IgM response was detected in one-third of all clinically asymptomatic renal patients. (ii) The virus-specific IgM response is primarily directed against the pp150 epitope. (iii) In patients with clinically manifest HCMV disease, additional IgM reactivities are most frequently directed against the glycoprotein B epitope. (iv) The severity of HCMV infections correlates with the extent of the IgM antibody response, i.e. with the number of specific epitopes involved. (v) After transplantation, IgM reactivity and its epitope-specific pattern persist for years.     

Different antibody response to a neutralizing epitope of human cytomegalovirus glycoprotein B among seropositive individuals

The amino-terminal portion of human cytomeg-alovirus glycoprotein B (HCMV-gB) was expressed as a fusion protein to analyze the neutralizing epitope recognized by human monoclonal antibody C23 and the humoral immune response to this epitope. The linear neutralizing epitope was further localized to the pep-tide within 17 amino acids (position 68-84) which were conserved between two HCMV laboratory strains. Ten out of 17 HCMV-seropositive human sera contained the antibody against this epitope. Although seven sera were negative for reacting with the fusion protein, the viruses isolated from the same patients retained the epitope. The immunogenicity of the epitope and the possible application of C23 human monoclonal antibody for passive immunization against HCMV infections are discussed. © 1994 Wiley-Liss, Inc.     

Development, characterization, and biological properties of meningococcal immunotype L3,7,(8),9-specific monoclonal antibodies.

In this study, we characterize the properties of nine monoclonal antibodies (MAbs) that recognize meningococcal lipopolysaccharides (LPS). The following three specific MAbs that had not been described previously were elicited in BALB/c mice by using an immunotype L3,7,9 oligosaccharide-tetanus toxoid conjugate in combination with Quil A: 4D1-B3, 3A12-E1, and 4A8-B2. These MAbs reacted with L3,7,9 LPS on immunoblots and in the LPS enzyme-linked immunosorbent assay (ELISA) and recognised strains containing L3, L3,7, L8 (except 3A12-E1), or L9 LPS in the whole-cell ELISA. The six other MAbs have been described in previous studies (K. Saukkonen, M. Leinonen, H. Abdillahi, and J.T. Poolman, Vaccine 7:325-328, 1989; R.J.P.M. Scholten, B. Kuipers, H.A. Valkenburg, J. Danjert, W.D. Zollinger, and J.T. Poolman, J. Med. Microbiol., in press) and were obtained after immunization with outer membrane protein complexes containing LPS: MN15A11, MN15A8-1, MN15A17-1, MN11A11G, MN14F20-11, and MN14F21-11. MN15A11 was specific for L3,7,9 LPS and displayed properties similar to those of 3A12-E1. MN15A17-1, MN14F20-1, and MN11A11G were cross-reactive, and MN14F21-11 was specific for the L1,8 immunotype. Epitope specificities of MAbs reacting with L3,7,(8),9 strains were analyzed. MAbs 4D1-B3, 3A12-E1, and 4A8-B2 recognized phosphoethanolamine group-containing oligosaccharide-specific epitopes. MN15A11 and MN15A17-1 were probably directed against a conformational epitope, although for MN5A11 recognition of an unknown L3,7,9-specific epitope in the 2-keto-3-deoxyoctulosonic acid (KDO)-lipid A region cannot be excluded. MN15A8-1, a strongly cross-reactive MAb, recognized a determinant which included the KDO-lipid A region and the more terminal saccharides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assignment of the genome segment coding for the neutralizing epitope(s) of orbiviruses in the Great Island subgroup (Kemerovo serogroup)

Reassortant viruses were produced from high-frequency recombination events between temperature-sensitive (ts) mutants of Broadhaven (BRD) (S. R. Moss and P. A. Nuttall, Virus Res. 4, 331-336, 1986) and Wexford (WEX) viruses, two serotypes in the Kemerovo serogroup of orbiviruses. The parental origin of each of the 10 genomic segments comprising each reassortant was determined by polyacrylamide gel electrophoresis. Comparison of neutralization titers with the results of genomic dsRNA analyses revealed that genome segment 5 codes for the neutralizing epitope(s). Kemerovo group viruses therefore differ from orbiviruses of the bluetongue serogroup in which segment 2 codes for the neutralizing epitopes (M. J. Grubman, J. A. Appleton, and G. J. Letchworth, Virology 131, 355-366, 1983; J. Kahlon, K. Sugiyama, and P. Roy, J. Virol. 48, 627-632, 1983). Results also indicated that the ts lesions in mutants of recombination groups II, III, and VI were in segments 1, 5, and 4, respectively. Reassortment of segments 2 and 10 appeared to be nonrandom, and evidence of possible "linkage" was obtained for segments 3 and 9.     

Soybean glycinin G1 acidic chain shares IgE epitopes with peanut allergen Ara h 3

BACKGROUND: The identification of IgE epitopes for proteins is the first step in understanding the interaction of allergens with the immune system. Proteins from the legume family have shown in vitro cross-reactivity in IgE-binding assays, but this cross-reactivity is rarely clinically significant. Resolution of this discrepancy requires IgE epitope mapping of legume family protein allergens. METHODS: We constructed six fusion proteins representing overlapping regions of soybean glycinin G1 acidic chain. These fusion proteins were used in immunoblotting and a novel sandwich ELISA with pooled sera from soy-allergic individuals to reveal a common IgE-binding region. This region was the focus for IgE epitope mapping using overlapping synthetic peptides. RESULTS: Data from the fusion protein experiments revealed an IgE-binding region consisting of residues F192-I265. Analysis of the overlapping synthetic peptides to this region indicated that IgE epitopes to glycinin G1 acidic chain consist of residues G217-V235 and G253-I265. The epitopes identified for glycinin G1 acidic chain are homologous to IgE epitopes previously identified for the peanut allergen Ara h 3 [1]. However, residues identified by alanine scanning in the peanut epitopes as being important for IgE binding are different in the natural soybean epitopes. CONCLUSIONS: The IgE epitopes identified for glycinin G1 acidic chain apparently represent an allergenic region of several legume family seed storage proteins. Our findings indicate that the identification of IgE epitopes and structural analysis of legume family proteins will provide valuable information to the study of food allergies.     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.     

Epitope mapping of the Brucella melitensis BP26 immunogenic protein: usefulness for diagnosis of sheep brucellosis

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others (O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis- or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.