Fisher Immunotype Research Articles

Mouse protection induced byPseudomonas aeruginosaPAC1R and its defective mutants,Salmonella minnesotaRe-mutant andEscherichia coliO14

Pseudomonas aeruginosa PAC1R and its defective mutants (acetone-killed bacteria), Salmonella minnesota Re mutant (acetone-killed bacteria and Re-LPS) and Escherichia coli O14 (acetone-killed bacteria and enterobacterial common antigen, ECA) were studied in a mouse active protection test. Immunized mice were challenged with wild-type P. aeruginosa strains. It was established that P. aeruginosa LPS-defective mutants induced cross-immunity against different Fisher immunotypes of P. aeruginosa. S. minnesota Re-LPS and ECA gave mice protection against P. aeruginosa.

Mouse protection induced by Pseudomonas aeruginosa PAC1R and its defective mutants, Salmonella minnesota Re-mutant and Escherichia coli O14

Pseudomonas aeruginosa PAC1R and its defective mutants (acetone-killed bacteria), Salmonella minnesota Re mutant (acetone-killed bacteria and Re-LPS) and Escherichia coli O14 (acetone-killed bacteria and enterobacterial common antigen, ECA) were studied in a mouse active protection test. Immunized mice were challenged with wild-type P. aeruginosa strains. It was established that P. aeruginosa LPS-defective mutants induced cross-immunity against different Fisher immunotypes of P. aeruginosa. S. minnesota Re-LPS and ECA gave mice protection against P. aeruginosa.

Specificity and function of murine monoclonal antibodies and immunization-induced human polyclonal antibodies to lipopolysaccharide subtypes of Pseudomonas aeruginosa serogroup 06.

Structural and antigenic heterogeneity has been noted among lipopolysaccharides (LPS) produced by Pseudomonas aeruginosa within serogroups previously considered to be serologically homogeneous. We characterized murine monoclonal antibodies (MAbs) and immunization-induced human polyclonal antibodies reactive with one or more of five structurally variant LPS subtypes belonging to serogroup 06 of the International Antigenic Typing System. Analyses of five different MAbs employing purified LPS or whole patterns of subtype specificity, ranging from recognition of a single subtype to reactivity with all five. MAb-mediated opsonophagocytic killing and in vivo protection against live challenge in mice correlated, in general, with differential binding to various LPS subtypes. In comparison, sera from human vaccinees immunized with LPS-derived high-molecular-weight polysaccharide from P. aeruginosa Fisher immunotype 1, one of five serogroup 06 subtypes, exhibited LPS binding and opsonic activity against all five subtypes. Antibodies in the human sera effectively inhibited binding to all five LPS subtype antigens of the cross-reactive MAb, LC3-2H2, suggesting the existence of a common serogroup-related epitope. These findings emphasize the importance of defining subtype-associated variations in LPS antigenicity and corresponding differences in antibody specificity and function as a basis for designing immunoprophylactic or therapeutic strategies which target P. aeruginosa LPS.

Therapeutic effects of a human antiflagella monoclonal antibody in a neutropenic murine model of Pseudomonas aeruginosa pneumonia

Human immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) reactive with type-specific Pseudomonas aeruginosa lipopolysaccharide (LPS) and flagella were compared for their protective activities against Fisher immunotype 2 P. aeruginosa pneumonia in neutropenic mice. The activity of the antiflagella MAb at a dose of 500 micrograms per mouse was comparable to that of the anti-LPS MAb at the same dose. In vivo protection was correlated with bacterial density in the lung tissue and blood of infected mice. In vitro data suggested that the protective activity of the antiflagella MAb was due more to inhibition of bacterial motility than to opsonophagocytosis of bacteria by alveolar macrophages. In contrast, the protective activity of the anti-LPS MAb was primarily related to alveolar macrophage-mediated opsonophagocytosis. Antiflagella MAb at a dose of 500 micrograms combined with oral sparfloxacin at a subtherapeutic dose of 62.5 micrograms produced a significant increase in survival (P < 0.05) compared with that produced by either agent alone or no treatment. The additive effects between the antiflagella MAb and sparfloxacin at sub-MICs on the inhibitory effects of bacterial motility supported the in vivo effect of the combination. These data suggest that human isotype-matched antiflagella and anti-LPS MAbs have similar protective activities against Pseudomonas pneumonia in neutropenic mice, despite discrete mechanisms of antibody-matched protection. In addition, in vivo synergy was demonstrated between antiflagella MAb and sparfloxacin in this model.

The efficacy of combination immunotherapy in experimental Pseudomonas sepsis.

Immunotherapy has been shown to be an effective adjuvant in the management of septic shock. A neutropenic rat model of septic shock induced by infection with Pseudomonas aeruginosa 12.4.4 (Fisher immunotype 6) was used to determine the relative efficacy of single, double, and triple combination immunotherapy. A Pseudomonas O serotype-specific, opsonophagocytic monoclonal antibody (MAb), polyclonal J5 antiserum, and a MAb directed against tumor necrosis factor-alpha (TNF) were studied as single therapy and in combination. The combination of all three immunotherapeutic agents resulted in a 77% survival rate (33/43 animals). This level of protection was superior to that achieved with any combination of two antibody treatments (50%-60% survival; P = .029) or single antibody therapy (25%-43% survival; P < .001) or compared with a control group (0/25 survivors; P < .0001). Immunotherapy directed against multiple steps of the septic process is more active than single or double antibody regimens and may offer an improved approach to the adjunctive treatment of septic shock.

Cloning and surface expression of Pseudomonas aeruginosa O antigen in Escherichia coli.

As a step toward developing recombinant oral vaccines, we have explored the feasibility of expression of O polysaccharide antigens from Pseudomonas aeruginosa by Escherichia coli. We cloned in E. coli HB101 a 26.2-kilobase DNA fragment from P. aeruginosa strain PA103 that specifies the production of the O polysaccharide of Fisher immunotype 2 (IT-2) strains. The recombinant organism incorporated the P. aeruginosa IT-2 O polysaccharide onto the core of the E. coli lipopolysaccharide (LPS). Transfer of the recombinant plasmid to three LPS-rough strains of P. aeruginosa resulted in synthesis of IT-2 O antigen, and two of these transconjugant strains also synthesized a second O polysaccharide, presumably representing expression of a repressed, or an incomplete, set of genes for an endogenous O polysaccharide. Rabbits injected with the purified recombinant LPS made antibody specific for P. aeruginosa IT-2 O side chains, as did mice fed the recombinant E. coli strain. Expression of P. aeruginosa O antigens by enteric bacteria makes it possible to study these recombinant strains as oral vaccines to prevent P. aeruginosa infections.

Sn-chlorin e6 antibacterial immunoconjugates: An in vitro and in vivo analysis

Monoclonal antibody-Sn-chlorin e6 immunoconjugates were prepared by the site-selective covalent modification of the monoclonal oligosaccharide moiety. By carefully controlling the reaction conditions and introducing triethanolamine groups as axial ligands of the Sn moiety, conjugates with in vivo biodistribution properties similar to underivatized IgG were prepared. By varying the reaction conditions, conjugates were reproducibly with p range of photosensitizer to mAb molar ratios from 1.6 to 10. Based on a competitive inhibition radioimmunoassay, conjugates prepared by this method showed selectivity and binding affinity comparable to the unmodified antibody. The immunoconjugates had only slightly lower singlet oxygen yields than that observed with the Sn-chlorin e6 precursor indicating that negligible aggregation or structural modification of the chromophores occurred during the synthesis process. In vitro cell killing experimments demonstrated that all conjugates possessed significant cytotoxic activity. Biodistribution studies in mice showed that conjugates prepared with axial ligands had significant serum retention 24 h after injection while conjugates prepared without the triethanolamine ligand were much more rapidly cleared. In vivo specificity was demonstrated using rats infected with Fisher immunotype I P. aeruginosa at a site in the left posterior thigh muscle. Target to background ratios exceeded 60 at 120 h after conjugate injection of the specific immunoconjugate, compared to a ratio of only 6 for a non-specific mouse IgG conjugate. Biodistribution patterns at 120 h post injection indicate that the conjugates were both biologically active and structurally intact.

Effects of the combination of lipopolysaccharide-specific monoclonal antibodies and sparfloxacin against Pseudomonas aeruginosa pneumonia in neutropenic mice.

The effects of the combination of a murine monoclonal antibody (MAb) specific for the O side chain of Pseudomonas aeruginosa Fisher immunotype 1 lipopolysaccharide and sparfloxacin in a neutropenic mouse model of P. aeruginosa pneumonia were examined. Under the condition that neither MAb at a dose of 500 micrograms per mouse administered intravenously nor a suboptimal dose of oral sparfloxacin (5 mg/kg of body weight) protected mice from challenge with a fatal dose, the combination therapy with MAb and sparfloxacin caused a significant increase in the survival rate (P less than 0.001 compared with either treatment alone). The effect of the combination was closely correlated to bacterial killing in plasma and lung tissue of infected mice. In vitro, a significant MAb-dependent, complement-mediated killing of P. aeruginosa was documented in the presence of sparfloxacin at one-half the MIC, while the killing was not observed in the absence of sparfloxacin. These in vivo and in vitro data suggest the usefulness of combination therapy with a lipopolysaccharide-reactive immunoglobulin G MAb and sparfloxacin in neutropenic patients with P. aeruginosa pneumonia.

Characterization of Antibody Response to Pseudomonas aeruginosa in Patients with Wound Infections

ELISA was used to measure the amount and avidity of IgG antibodies to exotoxin A (ExA) and 7 Fisher's immunotypes of lipopolysaccharide (LPS) in the sera of 13 patients with mild or moderate Pseudomonas aeruginosa infections. Changes in the specificity of tested sera during the course of infection were demonstrated. A statistically significant increase was seen in the amount and avidity of the antibodies to ExA in a majority of the sera, and an increase was seen in amount of antibodies to LPS immunotype 4 in the sera of patients with moderate infections.

Isolation and characterization of a human monoclonal antibody that recognizes epitopes shared by Pseudomonas aeruginosa immunotype 1, 3, 4, and 6 lipopolysaccharides

A hybridoma line secreting a human monoclonal antibody (HMAb) capable of recognizing Fisher immunotype (IT) 1, 3, 4, and 6 lipopolysaccharide (LPS) in vitro was isolated. Peripheral blood lymphocytes (PBL) were obtained from volunteers immunized with an experimental Pseudomonas aeruginosa O polysaccharide-toxin A vaccine. PBL-expressing surface antibodies able to bind to P. aeruginosa LPS were isolated by adsorption onto LPS-coated plastic wells. Such PBL were transformed with Epstein-Barr virus. Lymphoblastoid cell lines secreting anti-P. aeruginosa LPS antibodies were identified by an enzyme-linked immunosorbent assay and fused to the F3B6 heteromyeloma line. A hybridoma line producing a HMAb (2-8AH79) able to bind IT-1, IT-3, IT-4, and IT-6 LPS was identified by an enzyme-linked immunosorbent assay. This HMAb was found to bind to the O-polysaccharide regions of IT-1, IT-3, IT-4, and IT-6 LPS, as determined by immunoblotting. By using an immunofluorescence microscopy assay, the cell surfaces of IT-3 and IT-4 bacteria were strongly stained by HMAb 2-8AH79, whereas those of IT-1 and IT-6 bacteria were weakly stained. This HMAb was found to promote the uptake and killing of IT-3 and IT-4 bacteria, but not IT-1 or IT-6 organisms, by human polymorphonuclear leukocytes. Similarly, the passive transfer of HMAb 2-8AH79 to mice afforded significant protection only against a challenge with IT-3 and IT-4 bacteria.

The Structure and Serologic Distribution of an Extracellular Neutral Polysaccharide from Pseudomonas aeruginosa Immunotype 3

Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.

Treatment of experimental Pseudomonas aeruginosa pneumonia with a human IgM monoclonal antibody.

A human IgM monoclonal antibody (MA-1C1) to Fisher immunotype 3 Pseudomonas aeruginosa lipopolysaccharide antigen was evaluated for in vivo activity in a guinea pig model of experimental pneumonia. Pharmacokinetics of MA-1C1 were compared in infected and noninfected animals. Intravenous bolus infusion of MA-1C1, 1 mg/kg, resulted in peak serum antibody concentrations of 3.8 +/- 0.08 and 3.7 +/- 0.05 micrograms/ml in infected and noninfected animals, respectively. Serum half-lives were 25 and 22 h in infected and noninfected groups. Treatment with a single intravenous infusion of MA-1C1 improved survival from pneumonia and was effective over a broad dose range (0.1-2.5 mg/kg). Cumulative survivals were 18 of 47 in the MA-1C1 group and 0 of 31 in controls (P less than .001). Treatment with MA-1C1 also resulted in fewer positive blood cultures 12 h after infection (P = .04). Although MA-1C1 penetrated into inflamed bronchial fluids, local concentrations were only 5% of the concentrations achieved in serum. Thus, MA-1C1 seems to provide significant therapeutic activity against experimental P. aeruginosa pneumonia by preventing dissemination of infection from the lung.

Amount, avidity, and specificity of antibodies to Pseudomonas aeruginosa in normal human sera

Seventy-two normal human sera from healthy blood donors were tested by an enzyme-linked immunosorbent assay in order to determine the amounts and avidities of immunoglobulins M and G antibodies to lipopolysaccharides of seven Fisher's immunotypes of Pseudomonas aeruginosa and to exotoxin A. The patterns of specificity for seven immunotypes in all individual sera were determined. These data show a predominance of antibodies directed to Fisher's immunotypes 7 and 4 in the human population tested and may reflect frequency of occurrence of immunotypes outside the hospital environment.

Saccharides of sevenPseudomonas aeruginosaimmunotypes

The structures of O-specific polysaccharides obtained by mild acid degradation of lipopolysaccharides (LPS) from seven Pseudomonas aeruginosa Fisher's immunotypes have been studied. The polysaccharides consist mainly of monoamino and diamino sugars, frequently also carrying acidic functions. Some of the sugars were detected in nature for the first time in these organisms. The structures of the O-specific polysaccharides of the immunotypes 2, 3, 4, 5 and 6 are identical to those of the polysaccharides of the 011; 0(2a)2c; 01; 010a, 10b and 07a, 7d Lányi-Bergan serological subgroups respectively, whereas no analogues have been found for the immunotypes 1 and 7. Some cross-reactions between the LPS of different immunotypes were observed in passive haemagglutination tests; the results of inhibition of passive haemagglutination and agar gel immunoprecipitation point, however, to a specificity of the LPS. Many of the LPS of the seven Pseudomonas aeruginosa immunotypes manifest rather a high cross-protective activity in active immunization tests in mice. The nature of the cross-protective activity of the LPS is discussed.

Isolation, structure, and immunogenicity of Pseudomonas aeruginosa immunotype 4 high-molecular-weight polysaccharide

A high-molecular-weight, immunogenic form of the lipopolysaccharide O side chain of Pseudomonas aeruginosa Fisher immunotype 4 (type 4, International Antigenic Typing System 1, Lanyi O:6) was isolated and characterized. Analysis by nuclear magnetic resonance spectroscopy confirmed the structural similarity of this high-molecular-weight polysaccharide and the type 4 O side chain. The polysaccharide was immunogenic in rabbits and mice, eliciting opsonophagocytic killing antibodies. Immunization with the polysaccharide produced significant protection against homologous challenge in both burned and granulocytopenic mice. Naturally acquired opsonic killing antibodies to type 4 polysaccharide were present in sera from unimmunized normal adults at levels comparable to postimmunization levels achieved after immunization with other type-specific polysaccharides. The specificity of the naturally occurring antibodies for the O side chain was documented by immunoblot analysis and inhibition studies. Naturally occurring polysaccharide-specific antibodies were comparable in their protective activity against live challenge in neutropenic animals to immunization-induced murine antibodies with similar specificity. These data suggest that naturally occurring serum antibody to P. aeruginosa type 4 lipopolysaccharide O side chains in most adults is not distinguishable in quantity or quality from immunization-induced antibodies in mice; evaluation of type 4-specific vaccines in humans may be complicated by this finding.

Human monoclonal antibodies that protect mice against challenge with Pseudomonas aeruginosa.

Lymphocytes from healthy volunteers and from cystic fibrosis patients were transformed with Epstein-Barr virus and cultured at a limiting dilution to generate lymphoblastoid cell lines that secreted human monoclonal antibodies specific for lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Three cell lines (RM5, FDD7, and 11F9) produced immunoglobulin M (IgM) antibody species that reacted specifically with P. aeruginosa Fisher immunotypes 2, 4, and 5, respectively, and with LPS extracted from these immunotypes. A fourth cell line (9H10) produced a single IgM antibody species that recognized P. aeruginosa immunotypes 3, 6, and 7 and LPS extracted from them. Monoclonal antibodies secreted by cell lines RM5, FDD7, and 11F9 protected neutropenic mice prophylactically against challenge with P. aeruginosa immunotypes 2, 4, and 5, and those secreted by 9H10 protected against P. aeruginosa immunotypes 3 and 6 but did not protect against immunotype 7. In vivo experiments indicated that antibodies protected mice against infection by increasing the rate of bacterial clearance.

X-linked immunodeficient mice as a model for testing the protective efficacy of monoclonal antibodies against Pseudomonas aeruginosa.

(DBA/N[female] X CBA/2[male])F1 males have been reported to be deficient in producing antibodies against a number of antigens, including carbohydrates (I. Scher, Adv. Immunol. 35:1-71, 1982). We show that F1 male mice, in contrast to females, made less lipopolysaccharide (LPS)-specific antibodies after immunization with heat-inactivated Pseudomonas aeruginosa and had significantly less naturally occurring LPS-specific antibodies. Furthermore, neutropenic males were 50 to 1,000 times more sensitive to challenge with representative isolates belonging to the seven Fisher immunotypes. Administration to neutropenic F1 males of a human monoclonal antibody specific for the O carbohydrates of P. aeruginosa immunotype 2 LPS or administration of serum from rabbits immunized with heat-inactivated P. aeruginosa immunotype 1 raised the level of resistance to bacterial challenge close to that of females. The results show that the X-linked immunodeficient mouse is an excellent model with which to test the protective efficacy of P. aeruginosa-specific monoclonal antibodies.

Human monoclonal antibodies to Pseudomonas aeruginosa produced by EBV-transformed cells.

Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD50) values were 0.5, 2.6, and 3.1 micrograms immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of lipopolysaccharides of P. aeruginosa O3 (Lányi), O25 (Wokatsch) and Fisher immunotypes 3 and 7.

O-specific polysaccharides, obtained on mild acid degradation of lipopolysacchrides of the serologically related strains Pseudomonas aeruginosa O3 (Lányi classification), O25 (Wokatsch classification) and immunotypes 3 and 7 (Fisher classification), are built up of trisaccharide repeating units involving 2-acetamido-2,6-dideoxy-D-galactose (N-acetyl-D-fucosamine), 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid or 2,3-diacetamido-2,3-dideoxy-L-guluronic acid and 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid or 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic acid. Lányi O3(a),3d,3f and Wokatsch O25 polysaccharides contain also O-acetyl groups. On the basis of solvolysis with anhydrous hydrogen fluoride, resulting in trisaccharide fragments with N-acetylfucosamine residue at the reducing terminus, chemical modifications of the acetamidino group (alkaline hydrolysis to the acetamido group or reductive deamination to the ethylamino group), as well as analysis by 1H-NMR (including nuclear Overhauser effect experiments) and 13C-NMR spectroscopy, and fast-atom bombardment mass spectrometry, it was concluded that the repeating units of the polysaccharides have the following structures: (Formula: see text) where HexNAcAmA = alpha-L-GulNAcAmA (approximately 70%) or beta-D-ManNacAMA (approximately 30%). Lányi O3(a),3d,3f polysaccharide involves two types of repeating units, which differ from each other only in the configuration at C-5 of the 3-acetamidino-2-acetamido-2,3-dideoxyuronic acid residue. Lányi O3(a),3c,O3a,3d,3e and Fisher immunotypes 3 and 7 polysaccharides contain, together with the major repeating units shown above, a small proportion of units in which the derivative of alpha-L-guluronic acid is replaced by the corresponding beta-D-manno isomer. The data obtained provide the opportunity to substantiate the serological interrelations between these strains of P. aeruginosa by the presence in the O-specific polysaccharides of common monosaccharides or disaccharide fragments. The distinctions between them stem from the presence or absence of the O-acetyl group, a different configuration of the glycosidic linkage of the N-acetylfucosamine residue and/or a different configuration at C-5 of one or both derivatives of diaminouronic acids.

Production of cytotoxin by clinical strains of Pseudomonas aeruginosa.

Presence of cytotoxin was studied in extracts of 57 strains of Pseudomonas aeruginosa (46 bacteremia, 4 environmental, and 7 Fisher immunotype), 10 Pseudomonas species, and 7 nonpseudomonas isolates. Cytotoxin was identified by Western immunoblot in extracts of all P. aeruginosa isolates. None of the Pseudomonas species or nonpseudomonas isolates were shown to produce this protein. No immunologic cross-reactivity was observed between cytotoxin antibody and P. aeruginosa alkaline protease, toxin A, or elastase. In partially purified extracts of two bacteremia strains and PA 158 (parent strain for cytotoxin production), detection of cytotoxin by Western immunoblot was correlated with biological activity, as measured by the cell swelling assay. Cytotoxin appears to be produced by all strains of P. aeruginosa and biological activity can be demonstrated in extracts of the strains tested. This biological activity is neutralized by specific antibody. Because of its known marked cytotoxic effect on most eukaryotic cells, P. aeruginosa cytotoxin might be an important factor in the pathogenesis of P. aeruginosa infections.