Identification of the epitope for a highly cross-reactive monoclonal antibody on the major sigma factor of bacterial RNA polymerase

A highly cross-reactive monoclonal antibody (MAb), 2G10, was found to react in a conserved region of Escherichia coli RNA polymerase sigma70. The epitope was localized to amino acids 470 to 486, which included part of conserved region 3.1. The epitope for MAb 3D3, a MAb which maps close to the 2G10 epitope, was also determined.     

alpha-GlcNAc-1-->2-alpha-glc, the Salmonella homologue of a conserved lipopolysaccharide motif in the Enterobacteriaceae, elicits broadly cross-reactive antibodies

To define cross-reactive epitopes in Salmonella lipopolysaccharide (LPS), antisera designated anti-S, anti-Ra, and anti-Re were generated against smooth (S), complete-core (Ra), and deep-core mutant (Re) strains, respectively, and characterized immunochemically. The reactivities of anti-Ra and anti-S with rough LPS (rLPS) chemotypes in enzyme-linked immunosorbent assays (ELISA) decreased progressively with increasing truncation of the complete-core oligosaccharide (e.g., Ra > Rb1 >.Re), while that of anti-Re increased (Ra < Rb1 <.Re). Anti-Ra was relatively more reactive with nonhomologous smooth LPS (sLPS) than anti-S, which in turn was more reactive than anti-Re. This order reflected the relative reactivities of these sera with outer-core rLPS but not those with inner-core rLPS, which suggests that the cross-reactivities of all three sera with sLPS were mediated by antibodies which bind outer-core determinants. Anti-Ra, but not anti-S or anti-Re, reacted with molecules substituted by O chains in immunoblots and revealed ladder-like patterns in sLPSs of various serospecificities. Anti-Ra, however, did not react with O-antigen-specific neoglycoconjugates in ELISA, thus demonstrating specificity for core epitopes. Ra and Rb1 but not other Salmonella core chemotypes inhibited the reactivity of anti-Ra with sLPS in ELISA, which showed that the terminal outer-core disaccharide, alpha-GlcNAc-1-->2-alpha-Glc (GlcNAc-->Glc), was the major epitope of cross-reactive antibodies in the serum. GlcNAc-->Glc represents the conserved motif alpha-hexose-1-->2-alpha-hexose in cores of the Enterobacteriaceae, other homologues of which should likewise be cross-reactive. These results demonstrate that S or Re strains do not elicit cross-reactive antibodies and indicate that immunization with Ra strains may represent a general strategy for eliciting cross-reactive antibodies against LPSs from enteric bacteria.     

1H nuclear magnetic resonance study on equilibrium between two four-stranded solution conformations of short d(CnT)

NMR analysis of d(C4T) showed the slow exchange between two distinct tetramers (each fully symmetric) in solution. For one tetramer, NOE cross-peak patterns characteristic of an i-motif structure (H1'-H1' and H6-H1'/H1'-H6) were observed between C1 and T5, indicating that this tetramer takes a completely intercalated conformation where the T5 residue is stacked on the C1.C1(+) pair of the other duplex (S-form). The other was found to be a tetramer in which one of the duplexes is shifted by one nucleotide unit (R-form), resulting in nonstacking 3' end thymidine residues and an equal number of stacked C.C+ pairs to that of the S-form. The same spectral features were observed for d(C3T) but neither for d(TC3) nor d(TC4), indicative of the critical role of the position of the thymidine residue in the tetrad isomerization. From NMR denaturation profiles, the S-forms were found to be more stable than the R-forms, and the linear relationship between the logarithm of the equilibrium constant (K = [tetramer]/[single]4) and the inverse of temperature (1/T) was confirmed for both forms, indicating conformity to the two-state transition model. Both enthalpy and entropy values of the formation of the S-form from four single strands were more negative than those of the R-form. The enthalpy term should contribute to the stabilization of the S-forms at low temperatures. The difference of the free energy values [DeltaG degrees(S-form) - DeltaG degrees(R-form)] was found to be -2.1 and -2.7 kJ.mol-1 at 20 degreesC for d(C4T) and d(C3T), respectively, explaining the higher stability of the S-forms. With increasing temperature, these two topologies were found to comparably exist at equilibrium in solution with slow exchange via dissociation to the single strands. A biological role of this topological isomerization is also suggested.     

Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria

The binding of IgG in antiserum to Escherichia coli J5 to the surface of Enterobacteriaceae and to cell wall fragments released from serum-exposed bacteria was studied in a search for potentially protective epitopes other than lipopolysaccharide (LPS). IgG titers to multiple heterologous gram-negative smooth bacteria increased following incubation of the bacteria in serum and decreased following absorption with serum-exposed heterologous bacteria. IgG eluted from absorbing bacteria bound to at least three conserved bacterial outer membrane proteins (OMPs), but not LPS, as assessed by immunoblotting. The same OMPs were present in LPS-containing macromolecular cell wall fragments released by incubation of heterologous gram-negative bacteria in human serum. Part of the protection offered by J5 antiserum could be from binding of IgG to conserved OMPs at the bacterial surface or to OMPs in cell-wall fragments released from dying bacteria.     

Preferential formation and decreased removal of cisplatin-DNA adducts in Chinese hamster ovary cell mitochondrial DNA as compared to nuclear DNA

We report here evaluation of a competitive enzyme-linked immunosorbent assay (c-ELISA) for detection of Salmonella spp. in chicken organs and faeces. The c-ELISA used a monoclonal antibody (MAb), specific for a genus-specific epitope of the outer core oligosaccharide of salmonellae. Salmonella lipopolysaccharide (LPS) in samples competed with Salmonella LPS coated on microtitre plates, for binding to the MAb. Competition reduced binding of the MAb to the LPS on the plate and of the secondary antibody to the MAb hence reducing the chromogenic signal. Stable coating and minimal false positive were achieved by conjugating LPS to poly-L-lysine. The c-ELISA was compared with motility enrichment culture using modified semisolid Rappaport Vassiliadis (MSRV) medium, which detected less than 10(2) CFU/g, and did not allow migration of non-salmonella species. The c-ELISA detected 10(6) CFU of enriched culture or 10(2)-10(3) CFU of Salmonella/g of faeces. Its limit of detection was thus higher than that of MSRV culture and it had a sensitivity of 92.9% and a specificity of 96.7%.     

Participation of CD14 in the phagocytosis of smooth-type Salmonella typhimurium by the macrophage-like cell line, J774.1

The role of CD14 in the phagocytosis and killing of microorganisms was investigated using macrophage-like cell lines, CD14-positive J774.1 cells and CD14-negative mutant J7.DEF.3 cells derived from J744.1 cells. The cells were infected with Salmonella typhimurium organisms of the smooth (S)-form LT2, mutant rough (R)-form TV148 or Staphylococcus aureus 248betaH. At 30 or 180 min incubation, the cells were washed and disrupted. Colony-forming units (CFUs) liberated from the disrupted cells were determined by quantitative cultivation, and the phagocytic index and killing rate were calculated. Both the phagocytic index and killing rate of J774.1 cells against LT2 organisms were greater than those of J7.DEF.3 cells. However, the index and rate of J774.1 cells against TV148 and 248betaH organisms were similar to those of the J7.DEF.3 cells. The phagocytosis of LT2 organisms by J774.1 cells was partially inhibited by S-form LPS (S-LPS) and anti-CD14 antibody, but not by R-chemotype LPS (R-LPS). These results suggest that CD14 participates in the phagocytosis of S-form Salmonella.     

Microtiter assay for detecting Campylobacter spp. and Helicobacter pylori with surface gangliosides which bind cholera toxin

Campylobacter jejuni with Gm1 ganglioside in the core of its lipopolysaccharide has been associated with Guillain-Barré syndrome. Since this epitope may be of considerable pathophysiologic importance and since this ganglioside binds cholera toxin, a rapid screening assay to detect bacteria that bind cholera toxin as an indication of Gm1 on their surfaces was developed. In the assay, bacterial lawns were grown on agar plates, harvested with phosphate-buffered saline, boiled, and incubated with a standard concentration of cholera B subunit. Preparations from strains with Gm1 were observed to inhibit the binding of cholera B subunit to Gm1 in a microtiter enzyme-linked immunosorbent assay. By using this assay with two groups of strains, 37 positive strains were detected among the 197 tested. Species with positive isolates included C. jejuni, Campylobacter coli, and Helicobacter pylori. The assay is capable of testing large numbers of isolates and should prove useful in future clinical and epidemiological studies of bacteria with this epitope.     

Use of an isogenic mutant constructed in Moraxella catarrhalis To identify a protective epitope of outer membrane protein B1 defined by monoclonal antibody 11C6

Moraxella catarrhalis-induced otitis media continues to be a significant cause of infection in young children, prompting increased efforts at identifying effective vaccine antigens. We have previously demonstrated that M. catarrhalis expresses specific outer membrane proteins (OMPs) in response to iron limitation and that this organism can utilize transferrin and lactoferrin for in vitro growth. One of these proteins, which binds human transferrin, is OMP B1. As the human host presents a naturally iron-limited environment, proteins, like OMP B1, which are expressed in response to this nutritional stress are potential vaccine antigens. In this study, we have developed monoclonal antibody (MAb) 11C6, which reacts to a surface-exposed epitope of OMP B1 expressed by M. catarrhalis 7169. This antibody was used to clone ompB1, and sequence analysis suggested that OMP B1 is the M. catarrhalis homologue to the transferrin binding protein B described for pathogenic Neisseriaceae, Haemophilus influenzae, Actinobacillus pleuropneumoniae, and M. catarrhalis. Expression of recombinant OMP B1 on the surface of Escherichia coli confers transferrin binding activity, confirming that this protein is likely involved in iron acquisition. In addition, ompB1 was used to construct an isogenic mutant in M. catarrhalis 7169. This mutant, termed 7169b12, was used as the control in bactericidal assays designed to determine if OMP B1 elicits protective antibodies. In the presence of MAb 11C6 and human complement, wild-type 7169 demonstrated a 99% decline in viability, whereas the ompB1 isogenic mutant was resistant to this bactericidal activity. Further analysis with MAb 11C6 revealed the presence of this OMP B1 epitope on 31% of the clinical isolates tested. These data suggest that OMP B1 is a potential vaccine antigen against M. catarrhalis infections.     

All accessible epitopes in the Salmonella lipopolysaccharide core are associated with branch residues

Antisera generated against each of the nine known chemotypes of Salmonella lipopolysaccharide (LPS) core were characterized in order to delineate cross-reactive epitopes and define the bases for their accessibility. Strongly cross-reactive epitopes were associated with three chemotypes: Ra and Rb4, which recognized alpha-GlcNAc-1-->2-alpha-Glc, and Rd1, which recognized L-alpha-D-heptose-1-->7-L-alpha-D-heptose. Both these disaccharides and the more weakly cross-reactive alpha-Gal-1-->6-alpha-Glc terminal in Rb3 LPS represent branch points along the core oligosaccharide. Therefore, branch points in endotoxin core oligosaccharides may generally be cross-reactive.     

Abnormal sex development and impotence

OBJECTIVE: Cross-reactivity of anti-Sm autoantibodies with a certain ribosomal protein has been reported previously. The present study was undertaken to identify the anti-Sm-reactive ribosomal protein, and to characterize the cross-reactive epitope. METHODS: Two-dimensional gel electrophoresis followed by immunoblotting was used to identify the ribosomal protein (S10) which was reactive with the Y12 anti-Sm monoclonal antibody (MAb). Human anti-Sm antibodies were also tested for cross-reactivity with the Sm-B/B', Sm-D, and isolated S10 proteins by immunoblotting. Epitope analysis was performed by immunoprecipitation of in vitro-translated products of the recombinant S10 and its various mutants. RESULTS: The Y12 MAb and the affinity-purified human anti-Sm autoantibodies cross-reacted with ribosomal S10 protein. Reactivity of the Y12 MAb with S10 protein was abolished by deletion of 19 amino acids at the carboxyl-terminus of S10, containing the Gly-Arg-Gly sequence motif shared by Sm-B/B' and Sm-D (D1 and D3). Replacements of Arg-158 with Gly and of Arg-158/Arg-160 with Gly/Gly at the carboxyl-terminal 157-Gly-Arg-Gly-Arg-Gly region disrupted the Y12 MAb recognition. CONCLUSION: At least a part of human anti-Sm antibodies and Y12 MAb show cross-reactivity among Sm-B/B', Sm-D, and ribosomal protein S10. The carboxyl-terminal Gly-Arg-Gly region of S10 protein is involved in constructing the cross-reactive epitope. This demonstrates that a common structural feature is shared by the ribosomal protein and the small nuclear RNP proteins.     

Protection against lethal endotoxemia by anti-lipid A murine monoclonal antibodies: comparison of efficacy with that of human anti-lipid A monoclonal antibody HA-1A

The protective capacities of murine anti-lipid A monoclonal antibodies (MAbs) 8-2 and 26-20 were examined and compared with those of the human MAb HA-1A with respect to inhibition of lipopolysaccharide (LPS) priming of human polymorphonuclear leukocytes (PMNL) in vitro and protection against lethal endotoxemia in mice. HA-1A did not prevent the priming effect of either rough or smooth LPS, while MAb 26-20 effectively inhibited LPS priming of human PMNL. Also, both murine MAbs protected mice against an otherwise lethal challenge with rough Re LPS of S. minnesota R595 as well as with smooth LPS of E. coli O111:B4. HA-1A exerted no protection against the lethal effects of Re LPS in this in vivo model. The enhanced survival in mice by treatment with MAbs 8-2 and 26-20 was associated with decreased levels of LPS-induced tumor necrosis factor. Neutralization of lipid A as a mechanism of protection was strongly suggested by efficacious inhibition of LPS priming of human PMNL by MAbs 8-2 and 26-20 in vitro.     

Binding of monoclonal antibody 4B1 to homologs of the lactose permease of Escherichia coli

The conformationally sensitive epitope for monoclonal antibody (mAb) 4B1, which uncouples lactose from H+ translocation in the lactose permease of Escherichia coli, is localized in the periplasmic loop between helices VII and VIII (loop VII/VIII) on one face of a short helical segment (Sun J, et al., 1996, Biochemistry 35;990-998). Comparison of sequences in the region corresponding to loop VII/VIII in members of Cluster 5 of the Major Facilitator Superfamily (MFS), which includes five homologous oligosaccharide/H+ symporters, reveals interesting variations. 4B1 binds to the Citrobacter freundii lactose permease or E. coli raffinose permease with resultant inhibition of transport activity. Because E. coli raffinose permease contains a Pro residue at position 254 rather than Gly, it is unlikely that the mAb recognizes the peptide backbone at this position. Consistently, E. coli lactose permease with Pro in place of Gly254 also binds 4B1. In contrast, 4B1 binding is not observed with either Klebsiella pneumoniae lactose permease or E. coli sucrose permease. When the epitope is transferred from E. coli lactose permease (residues 245-259) to the sucrose permease, the modified protein binds 4B1, but the mAb has no significant effect on sucrose transport. The studies provide further evidence that the 4B1 epitope is restricted to loop VII/VIII, and that 4B1 binding induces a highly specific conformational change that uncouples substrate and H+ translocation.     

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays.     

A protective epitope of Moraxella catarrhalis is encoded by two different genes

The high-molecular-weight UspA protein of Moraxella catarrhalis has been described as being both present on the surface of all M. catarrhalis disease isolates examined to date and a target for a monoclonal antibody (MAb 17C7) which enhanced pulmonary clearance of this organism in a mouse model system (M. E. Helminen et al., J. Infect. Dis. 170:867-872, 1994). A recombinant bacteriophage that formed plaques which bound MAb 17C7 was shown to contain a M. catarrhalis gene, designated uspA1, that encoded a protein with a calculated molecular weight of 88,271. Characterization of an isogenic uspA1 mutant revealed that elimination of expression of UspA1 did not eliminate the reactivity of M. catarrhalis with MAb 17C7. In addition, N-terminal amino acid analysis of internal peptides derived from native UspA protein and Southern blot analysis of M. catarrhalis chromosomal DNA suggested the existence of a second UspA-like protein. A combination of epitope mapping and ligation-based PCR methods identified a second M. catarrhalis gene, designated uspA2, which also encoded the MAb 17C7-reactive epitope. The UspA2 protein had a calculated molecular weight of 62,483. Both the isogenic uspA1 mutant and an isogenic uspA2 mutant possessed the ability to express a very-high-molecular-weight antigen that bound MAb 17C7. Southern blot analysis indicated that disease isolates of M. catarrhalis likely possess both uspA1 and uspA2 genes. Both UspA1 and UspA2 most closely resembled adhesins produced by other bacterial pathogens.     

Epitope mapping of capsid proteins VP2 and VP3 of infectious bursal disease virus

Twenty hybridoma cell lines producing monoclonal antibodies (MAbs) against serotype 1 infectious bursal disease virus (IBDV) of GBF-1 and the attenuated GBF-1E strains were produced. The MAbs recognized major structural proteins VP2 and VP3. MAb recognition sites were mapped using recombinant Escherichia coli clones which expressed N-terminal and (or) C-terminal truncated virus antigens, and competitive-binding assays. At least 3 conformation-dependent serotype 1 specific virus neutralizing antigenic sites and a linear antigenic site were defined on VP2 and VP3, respectively. Two of the conformational virus neutralizing antigenic sites were localized in the central area of VP2 consisting of 156 amino acid residues, and the linear epitope was localized in C-terminal 105 amino acid residues of VP3. Another conformational virus neutralizing antigenic site recognized with the virus neutralizing MAb GK-5 was not defined because GK-5 did not react with virus antigen expressed in recombinant E. coli. The conformational antigenic site was supposed to be composed of tertiary or quaternary protein structure, which may not be constructed in recombinant E. coli.     

Detection of an Actinobacillus pleuropneumoniae serotype 2 lipopolysaccharide (LPS) variant

Until now 12 serotypes of Actinobacillus pleuropneumoniae have been recognized. The specificity of the serotypes reside in the carbohydrate composition of the capsular polysaccharides and lipopolysaccharides (LPS). The LPS of A. pleuropneumoniae serotype 2 is a smooth type LPS with O-chains of linear repeating pentasaccharide units with an O-acetyl group linked to a glucose unit. A monoclonal antibody (MAb 102-G02) directed against A. pleuropneumoniae serotype 2 was characterized in enzyme linked immunosorbent assay (ELISA) and in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The MAb 102-G02 was directed against an epitope on the O-chain of the LPS and was used to define a new. LPS variant of A. pleuropneumoniae serotype 2 (referred to as A. pleuropneumoniae serotype 2X). Investigation of the reactivity of the MAb 102-G02 against an A. pleuropneumoniae serotype 2X, field isolate (9008) and the Danish App-2 strain 4226 in electron microscopy, confirmed the different binding patterns.     

Lipoprotein release by bacteria: potential factor in bacterial pathogenesis

Lipoprotein (LP) is a major component of the outer membrane of bacteria in the family Enterobacteriaceae. LP induces proinflammatory cytokine production in macrophages and lethal shock in LPS-responsive and -nonresponsive mice. In this study, the release of LP from growing bacteria was investigated by immuno-dot blot analysis. An immuno-dot blot assay that could detect LP at levels as low as 100 ng/ml was developed. By using this assay, significant levels of LP were detected in culture supernatants of growing Escherichia coli cells. During mid-logarithmic growth, approximately 1 to 1.5 microgram of LP per ml was detected in culture supernatants from E. coli. In contrast, these culture supernatants contained 5 to 6 microgram/ml of lipopolysaccharide (LPS). LP release was not unique to E. coli. Salmonella typhimurium, Yersinia enterocolitica, and two pathogenic E. coli strains also released LP during in vitro growth. Treatment of bacteria with the antibiotic ceftazidime significantly enhanced LP release. Culture supernatants from 5-h cultures of E. coli were shown to induce in vitro production of interleukin-6 (IL-6) by macrophages obtained from LPS-nonresponsive C3H/HeJ mice. In contrast, culture supernatants from an E. coli LP-deletion mutant were significantly less efficient at inducing IL-6 production in C3H/HeJ macrophages. These results suggest, for the first time, that LP is released from growing bacteria and that this released LP may play an important role in the induction of cytokine production and pathologic changes associated with gram-negative bacterial infections.     

Structure of a neutralizing antibody bound monovalently to human rhinovirus 2

The structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-A resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45 degrees to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2-Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.     

Contrasting effects of lipopolysaccharides (endotoxins) from oral black-pigmented bacteria and Enterobacteriaceae on platelets, a major source of serotonin, and on histamine-forming enzyme in mice

By measurement of serotonin levels, the translocation of platelets to various tissues was examined following intravenous injection of a lipopolysaccharide (LPS) into C3H/HeN mice. There was a rapid platelet accumulation (within 5 min and particularly in the lung), followed by a slower accumulation in the liver, which reached its plateau 3-5 h later. The severity of the anaphylactoid shock corresponded well with the magnitude of the rapid response. LPSs from the oral black-pigmented bacteria, Porphyromonas gingivalis and Prevotella intermedia, were much more potent in inducing the rapid platelet response than were those from the Enterobacteriaceae Escherichia coli and Salmonella typhimurium. However, LPSs from these Enterobacteriaceae were significantly more potent than those from black-pigmented bacteria in inducing the slow platelet response. There was also a contrast between their abilities to induce histidine decarboxylase, which forms histamine from histidine: LPSs from the Enterobacteriaceae were much more potent than those from black-pigmented bacteria.     

Agonists and antagonists for lipopolysaccharide-induced cytokines

Agonistic and antagonistic properties of LPS and partial structures in the induction of cytokines are reviewed. Studies on structure-activity relationships of LPS and lipid A with human mononuclear cells reveal that S- and notably R-form LPS are very potent cytokine inducers. Synthetic E. coli lipid A is somewhat less active, whereas synthetic S. minnesota-type lipid A is significantly less active. Pentaacylated forms of lipid A are less potent than hexaacylated forms, and tetraacylated synthetic precursor Ia and bisacylated disaccharides and monosaccharides are completely inactive, indicating that a structure-dependent hierarchy of LPS and lipid A partial structures determines the monokine-inducing capacity in human mononuclear cells. Precursor Ia is a potent LPS antagonist. The mechanism of its inhibitory activity is shown to be due to competitive binding to cellular binding sites (receptors). Proinflammatory and antiinflammatory cytokines, receptor antagonists, and soluble cytokine receptors influence the cytokine-inducing activity of LPS, suggesting a complex regulatory network.