Identification of Acinetobacter baumannii strains with monoclonal antibodies against the O antigens of their lipopolysaccharides.
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Despite the emergence of Acinetobacter baumannii strains as nosocomial pathogens, simple methods for their phenotypic identification are still unavailable. Murine monoclonal antibodies specific for the O-polysaccharide moiety of the lipopolysaccharide (LPS) of two A. baumannii strains were obtained after immunization with heat-killed bacteria. The monoclonal antibodies were characterized by enzyme immunoassay and by Western and dot blot analyses and were investigated for their potential use for the identification of A. baumannii strains. The antibodies reacted with 46 of the 80 A. baumannii clinical isolates that were investigated, and reactivity was observed with 11 of 14 strains which were isolated during outbreaks in different northwestern European cities; no reactivity was observed with Acinetobacter strains of other genomic species, including the closely related genomic species 1 (Acinetobacter calcoaceticus), 3, and 13 sensu Tjernberg and Ursing, or with other gram-negative bacterial strains. The results show that O-antigen-specific monoclonal antibodies such as the ones described are convenient reagents which can be used to identify Acinetobacter strains in clinical and research laboratories. (+info)
Immunoglobulin G (IgG) subclass distribution and IgG1 avidity of antibodies in human immunodeficiency virus-infected individuals after revaccination with tetanus toxoid.
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In human immunodeficiency virus (HIV)-infected individuals the amount of antibodies formed after vaccination with T-cell-dependent recall antigens such as tetanus toxoid is proportional to the peripheral blood CD4(+) T-lymphocyte counts. To investigate whether the immunoglobulin G (IgG) subclass distribution and avidity of the antibodies produced after vaccination are affected as well, we gave 13 HIV-infected adults with low CD4(+) T-lymphocyte counts (<200 x 10(6)/liter; group I), 11 HIV-infected adults with intermediate CD4(+) T-lymphocyte counts (>/=200 x 10(6)/liter; group II), and 5 healthy controls booster immunizations with tetanus toxoid. The prevaccination antibody concentrations against tetanus toxoid were similar in the HIV-infected and healthy adults. After vaccination the total IgG and the IgG1 anti-tetanus toxoid antibody concentrations were significantly lower in group I than in group II and the controls. The avidity of the IgG1 anti-tetanus toxoid antibodies formed by HIV-infected adults was within the range for healthy controls, irrespective of their CD4(+) T-lymphocyte counts. (+info)
Immunization with a peptide corresponding to chlamydial heat shock protein 60 increases the humoral immune response in C3H mice to a peptide representing variable domain 4 of the major outer membrane protein of Chlamydia trachomatis.
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C3H (H-2(k)) mice are susceptible to a vaginal challenge with human strains of Chlamydia trachomatis and thus are a useful strain for testing potential Chlamydia vaccine candidates. However, C3H mice are fairly poor responders in terms of the level of antibody resulting from immunization with potential protective peptides representing variable domains (VDs) of the major outer membrane protein (MOMP). C57BL/6 (H-2(b)) mice, on the other hand, are moderately resistant to a vaginal challenge but are good responders to the chlamydial MOMP VDs. Peptides representing universal T-cell helper epitopes were employed to determine whether the antibody response to a peptide representing VD4 of the MOMP, which has been shown to contain neutralizing epitopes, could be enhanced in C3H and C57 mice. Universal T-cell helper peptides from tetanus toxin, the pre-S2 region of hepatitis B virus, and the mouse heat shock protein 60, as well as the corresponding segment of the Chlamydia heat shock protein 60 (hspct), were coadministered with the VD4 peptide. Peptides were coencapsulated in liposomes containing the adjuvant monophosphoryl lipid A and administered by using a combination of mucosal and intramuscular injection. The only T-cell helper peptide that improved the immune response as judged by antibody level, in vitro neutralization assays, and T-cell proliferation was hspct. The response in the C57BL/6 strain was not significantly enhanced with hspct over levels achieved with VD4 alone; however, in C3H mice the levels of serum antibody to C. trachomatis increased to that seen in C57 mice. However, the molecular specificity and immunoglobulin subclass distribution differed from those of the C57 response, and the neutralizing titers and T-cell proliferation responses were lower. In both strains of mice, titers of vaginal antibody to C. trachomatis were low. In summary, of the T-helper peptides used, only hspct significantly enhanced the immune response of C3H mice to the VD4 peptide, but it had only a modest effect on the immune response of C57 mice. (+info)
Helicobacter pylori heat shock protein A: serologic responses and genetic diversity.
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Helicobacter pylori synthesizes an unusual GroES homolog, heat shock protein A (HspA). The present study was aimed at an assessment of the serological response to HspA in a group of Chinese patients with defined gastroduodenal pathologies and determination of whether diversity is present in the nucleotide sequences encoding HspA in isolates from these patients. Serum samples collected from 154 patients who had an upper gastrointestinal pathology and the presence of H. pylori defined by biopsy were tested for an immunoglobulin G (IgG) serologic response to H. pylori HspA by an enzyme linked immunosorbant assay. HspA-encoding nucleotide sequences in H. pylori isolates from 14 patients (7 seropositive and 7 seronegative for HspA) were analyzed by PCR and direct sequencing of the PCR products. The sequencing results were compared to those of 48 isolates from other parts of the world. Of the 154 known H. pylori-positive patients, 54 (35.1%) were seropositive for HspA. The A domain (GroES homology) of HspA was highly conserved in the 14 isolates tested. Although the B domain (metal-binding site unique to H. pylori) resembled that in the known major variant, particular amino acid substitutions allowed definition of an HspA variant associated with isolates from East Asia. There were no associations between patient characteristics and HspA seropositivity or amino acid sequences. We confirmed in this study that the clinical outcomes of H. pylori infection are not related to HspA antigenicity or to sequence variation. However, B-domain sequence variation may be a marker for the study of the genetic diversity of H. pylori strains of different geographic origins. (+info)
Molecular cloning of the gene for a conserved major immunoreactive 28-kilodalton protein of Ehrlichia canis: a potential serodiagnostic antigen.
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A gene encoding a 28-kDa protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes of E. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with the E. chaffeensis outer membrane protein-1 (omp-1) gene family, with the Cowdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologous p28 gene copies in the E. canis genome, confirming that p28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed that E. canis P28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28. The p28 genes from seven different E. canis isolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen. (+info)
Intranasal immunization against dental caries with a Streptococcus mutans-enriched fimbrial preparation.
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Streptococcus mutans has been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination of S. mutans surface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected with S. mutans at 18 to 20 days of age, were intranasally immunized with a mixture of S. mutans surface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly (P < 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recovered S. mutans cells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture of S. mutans surface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries. (+info)
Effect of early antibiotic treatment on the antibody response to cytoplasmic proteins of Brucella melitensis in mice.
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To test whether antibiotic therapy hampers the antibody response to Brucella antigens, 30 BALB/c mice were infected with Brucella melitensis H38 and randomized for treatment with doxycycline administered intraperitoneally for 42 days starting at 7 or 28 days postinfection (p.i.) (groups DOX7 and DOX28, respectively) or for no treatment (control group). Antibodies to smooth lipopolysaccharide (LPS) reached peak levels (mean optical density [OD] = 2.618) between days 56 and 70 p.i. in the control group, and similar peak levels (mean OD = 2.486) were observed in the DOX28 group, but significantly lower peak levels (mean OD = 0.821) were observed at 28 days p.i. in the DOX7 group. The antibody response against cytoplasmic proteins depleted of LPS (CPs) reached maximal levels (mean OD = 2.402) between days 56 and 70 p.i. in the control group, but no response was detected in the DOX7 group. Anti-CP antibodies were detected in only three animals from the DOX28 group, at levels significantly lower than those in the control group (mean maximal OD = 0.791). The pattern of antibody response to an 18-kDa cytoplasmic protein of Brucella spp. was similar to that against the CP antigen. This study shows that early antibiotic treatment affects the antibody response of mice to cytoplasmic proteins of Brucella and, to a lesser extent, to LPS. (+info)
Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin.
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Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor. (+info)