Antibacterial properties of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide-specific monoclonal antibody (MAb) in a murine thigh infection model: combined effects of MAb and ceftazidime

A murine monoclonal antibody (MAb) specific for the Pseudomonas aeruginosa immunotype 1 (It-1) lipopolysaccharide (LPS) O-side chain was evaluated in terms of its in vitro bactericidal opsonophagocytic activity and in vivo bacterial killing in a mouse thigh infection model. An immunoglobulin (Ig) G2a MAb Ld3-2F2, specific for It-1 LPS, mediated in vitro complement-dependent opsonophagocytic killing at a concentration of 10 microg/ml. MAb-mediated, complement-dependent killing also occurred in the absence of neutrophils at serum concentrations in excess of 20%. A remarkable synergy was observed in opsonophagocytic assays between MAb Ld3-2F2 (0.5 microg/ml) and ceftazidime (1/4 MIC). The administration of MAb Ld3-2F2 at a level of 1 microg resulted in a significant decrease in the number of bacteria in the thigh muscles of normal mice, while 100 microg of the same MAb was required for one log of reduction in the number of bacteria at the same site in neutropenic mice. The combined therapy with MAb Ld3-2F2 and ceftazidime provided a significant reduction in the density of bacteria in the thigh muscle at 9 hr post-infection in normal and neutropenic mice as compared with those after treatment alone or with no treatment (P< 0.01). These favorable in vitro and in vivo interactions of an LPS-specific IgG MAb and ceftazidime strongly support their potential for use in therapy, combined with an LPS-reactive MAb and parenteral antipseudomonas beta-lactam antibiotics in the therapy of systemic Pseudomonas infections in normal and neutropenic hosts.     

Isolation and characterization of a lipopolysaccharide-specific bacteriophage of Pseudomonas aeruginosa

Phage H22 was isolated from sewage using Pseudomonas aeruginosa NCTC 8505 (serotype 0:3) as the host. Although not O-specific, this phage was found to have lipopolysaccharide (LPS) as a receptor. The broad host-range and lack of O-specificity of the phage suggested that its receptor site was in the core region of the LPS. Phage H22 had a Bradley type A structure. It was unaffected by chloroform and diethyl ether, and was stable between pH 5 and 8 and in the temperature range 0 to 60 degrees C. The adsorption rate constant was 14.6 X 10(-9) ml min-1. The phage had a latent period of 43 min, with a rise time of 18 min and a burst size of 6. The adsorption of phage to whole cells and LPS occurred over a broad pH range. Maximum adsorption occurred at 50 degrees C and pH 7.5 in the presence of 0.001 M Ca2+.     

Anti-idiotype-induced, lipopolysaccharide-specific antibody response to Pseudomonas aeruginosa. II. Isotype and functional activity of the anti-idiotype-induced antibodies

We recently developed a murine anti-idiotypic mAb that functioned as a molecular mimic of the O-specific polysaccharide side chain (Ps) of Pseudomonas aeruginosa LPS in vitro, and which induced Ps-specific antibodies in syngeneic BALB/c ByJ mice. In the current studies, we demonstrate that these anti-Id-induced, Ps-specific antibodies fix complement to the bacterial cell surface, and protect neutropenic mice from fatal P. aeruginosa sepsis. The isotypic distribution of the anti-Id-induced antibodies, however, resembles the restricted pattern (IgM and IgG3) seen after administration of purified Ps to mice. The immunogenicity and number of isotypes of Ps-specific antibody produced could be enhanced by conjugating the anti-Id to keyhole limpet hemocyanin. Finally, the anti-Id administered before immunization with purified Ps, primed BALB/c ByJ mice for production of other Ps-specific antibody isotypes (IgG1). These studies show that this anti-Id induces functional anti-Ps antibodies in syngeneic mice, and when used in conjugate form or as a priming agent before Ps immunization, yields an antibody response consistent with "T cell dependence." These immunization strategies may be useful for the induction of polysaccharide-specific antibodies in man.     

Estrogen aggravates inflammation in Pseudomonas aeruginosa pneumonia in cystic fibrosis mice

Background

Among patients with cystic fibrosis (CF), females have worse pulmonary function and survival than males, primarily due to chronic lung inflammation and infection with Pseudomonas aeruginosa (P. aeruginosa). A role for gender hormones in the causation of the CF "gender gap" has been proposed. The female gender hormone 17β-estradiol (E2) plays a complex immunomodulatory role in humans and in animal models of disease, suppressing inflammation in some situations while enhancing it in others. Helper T-cells were long thought to belong exclusively to either T helper type 1 (Th1) or type 2 (Th2) lineages. However, a distinct lineage named Th17 is now recognized that is induced by interleukin (IL)-23 to produce IL-17 and other pro-inflammatory Th17 effector molecules. Recent evidence suggests a central role for the IL-23/IL-17 pathway in the pathogenesis of CF lung inflammation. We used a mouse model to test the hypothesis that E2 aggravates the CF lung inflammation that occurs in response to airway infection with P. aeruginosa by a Th17-mediated mechanism.

Results

Exogenous E2 caused adult male CF mice with pneumonia due to a mucoid CF clinical isolate, the P. aeruginosa strain PA508 (PA508), to develop more severe manifestations of inflammation in both lung tissue and in bronchial alveolar lavage (BAL) fluid, with increased total white blood cell counts and differential and absolute cell counts of polymorphonuclear leukocytes (neutrophils). Inflammatory infiltrates and mucin production were increased on histology. Increased lung tissue mRNA levels for IL-23 and IL-17 were accompanied by elevated protein levels of Th17-associated pro-inflammatory mediators in BAL fluid. The burden of PA508 bacteria was increased in lung tissue homogenate and in BAL fluid, and there was a virtual elimination in lung tissue of mRNA for lactoferrin, an antimicrobial peptide active against P. aeruginosa in vitro.

Conclusions

Our data show that E2 increases the severity of PA508 pneumonia in adult CF male mice, and suggest two potential mechanisms: enhancement of Th17-regulated inflammation and suppression of innate antibacterial defences. Although this animal model does not recapitulate all aspects of human CF lung disease, our present findings argue for further investigation of the effects of E2 on inflammation and infection with P. aeruginosa in the CF lung.     

Exposure of mice to live Pseudomonas aeruginosa generates protective cell-mediated immunity in the absence of an antibody response

In previous studies we have elicited T cell-mediated protective immunity to the extra-cellular Gram-negative bacterium Pseudomonas aeruginosa by administering P. aeruginosa polysaccharide Ag and the anti-mitotic agent vinblastine sulfate to BALB/c mice. The current studies indicate that T cells which inhibit the growth of P. aeruginosa in vitro and protect granulocytopenic mice from P. aeruginosa infection can be generated by exposure of BALB/c mice to as few as 10(2) live bacteria without simultaneous administration of vinblastine. The in vitro inhibition of bacterial growth and mouse protection are P. aeruginosa immunotype specific. Exposure to 10(6) live bacteria is required to elicit a detectable antibody response. These findings indicate a potential role for T cells in resistance to P. aeruginosa infection in the large majority of individuals who lack anti-P. aeruginosa antibody.     

Antigenic properties of Pseudomonas aeruginosa anatoxin and the protective action of antitoxic anti-Pseudomonas aeruginosa serum

The antigenic properties of P. aeruginosa toxoid, prepared with the use of casein culture medium, were not inferior to those of the toxoid obtained in Martin broth. In experiments on white mice antisera obtained by the immunization of rabbits with the toxoid prepared on the basis of casein culture medium showed sharply pronounced protective properties against P. aeruginosa homologous and heterologous strains, as well as toxigenic reference strain PA-103.     

N-acetyl-L-galactosaminuronic acid as an epitope common to the O-polysaccharides of Pseudomonas aeruginosa serotype A and H (Homma) recognized by a protective human monoclonal antibody.

We have established a human--mouse heterohybridoma cell line producing a human monoclonal antibody TS-3G2 (IgG gamma 1, K). This monoclonal antibody specifically bound to O-polysaccharides belonging to plural Pseudomonas aeruginosa Homma serotypes, A and H, in contrast to serotype-specific monoclonal antibody which exclusively bound to strains belonging to a single specific serotype. The binding affinity for serotype A strains was higher than that for serotype H strains. Competitive enzyme immunoassay experiments with O-polysaccharide preparations derived from IID 1001, NCTC 8505 (serotype A) and IID 1009 (serotype H) and their derivatives demonstrated that the N-acetyl-L-galactosaminuronic acid residue in O-polysaccharide was essentially involved in the epitope for TS-3G2. Furthermore, a 6-deoxy-hexosamine residue neighboring the reducing terminal of N-acetyl-L-galactosaminuronic acid residues was also concerned with the epitope to some extent. In the experimental infection model of normal mice, the monoclonal antibody TS-3G2 showed a protective activity against both strains of serotype A and H.     

Pseudomonas aeruginosa antibody detection in cystic fibrosis patients.

Chronic respiratory infection due to Pseudomonas aeruginosa remains the most important prognostic factor in cystic fibrosis patients. One method to lengthen the patient's life is to extend the initial state of the illness with an early diagnosis, before Ps. aeruginosa infection becomes chronic. Often this is difficult because of the young age of the patients. This study tested an immunoenzymatic system to evaluate antibody response against three Ps. aeruginosa purified antigens, alkaline protease, elastase and exotoxin A. We studied 40 patients with cystic fibrosis, 20 affected and 20 unaffected by apparent Ps. aeruginosa infection, also from the bacteriological point of view. Serological and bacteriological results were compared for each patient and showed that serological screening can be useful in young subjects, who often have no bacteriological evidence of Ps. aeruginosa colonization.     

The protective effect of preparations made from plant seedlings in experimental Pseudomonas aeruginosa infection

The influence of preparations obtained from oat and wheat seedlings (immunostimulating factors IF-1 and IF-2, respectively) on the natural resistance of mice to P. aeruginosa infection was studied. IF-1 and IF-2 were introduced intraperitoneally in a single injection in doses of 100 micrograms and 1000 micrograms per mouse 2 and 7 days prior to the inoculation of P. aeruginosa strain 8 in doses of 1 and 10 LD50. The presence of substances capable of stimulating the immunobiological reserves of the body in actively growing plants (seedlings) was shown.     

Cell-free Pseudomonas vaccine. III. Immunochemical analysis of purified Pseudomonas aeruginosa protein antigens and protective properties of antisera against these antigens

The immunochemical analysis of isolated and purified antigens A and B obtained from P. aeruginosa, strains 868 (serogroup O3 according to Lányi or immunotype 3/7 according to Fisher) and 170015 (serogroup O7 or immunotype 2), was carried out. Rabbit antisera to proteins A and B, as well as to the initial aqueous extracts and partially purified aqueous extracts, were obtained. Cross activity between the protein antigens of different strains was established by the methods of immunodiffusion and two-dimensional immunoelectrophoresis. Isolated proteins A and B contained both common and specific antigenic determinants detected by the method of two-dimensional immunoelectrophoresis. The immunization of rabbits with proteins A and B was found to stimulate the synthesis of protective, probably species-specific, antibodies.     

Conjugation of metallothionein to a murine monoclonal antibody

A method of conjugation of the metal-binding protein, metallothionein, to an anticarcinoma murine monoclonal antibody, B72.3, and its F(ab')2 fragment has been developed utilizing the heterobifunctional crosslinking reagent, succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate. This crosslinking reagent is first reacted with the free amines on the immunoglobulin. After removal of unreacted crosslinker, conjugation is affected through a sulfhydryl group on metallothionein. Under the conditions employed all immunoglobulin aggregates contained metallothionein. The degree of undesired aggregation is directly proportional to the number of metallothioneins attached to the immunoglobulin. This aggregation can be controlled by the amount of crosslinker and metallothionein presented to the immunoglobulin. The immunoglobulin conjugate retains full immunoreactivity and can be readily purified from the unreacted metallothionein and high molecular weight aggregates. The metallothionein-B72.3 conjugate functions as an efficient and stable chelator of radiometals. Thus metallothionein-monoclonal antibody conjugates have potential utility in cancer diagnosis and therapy.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

Abstract International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately vn80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunolgobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studies in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD₅₀ of five strains of P. aeuruginosa was increased ≥ 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MA0bs in mice challenged with approximately 10 LD₅₀ of the homologous LPS serotype ranged from < 0.01 mg/kg to 1.00 mg/kg.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately 80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunoglobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studied in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD50 of five strains of P. aeruginosa was increased greater than or equal to 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MAbs in mice challenged with approximately 10 LD50 of the homologous LPS serotype ranged from less than 0.01 mg/kg to 1.00 mg/kg.     

Generation and characterization of a lipopolysaccharide-specific murine monoclonal antibody to <Emphasis Type="Italic">Proteus vulgaris</Emphasis> OX19

The three strains of non-pathogenic Proteus species namely, Proteus vulgaris OX2, P. vulgaris OX19 and Proteus mirabilis OXK used in the Weil–Felix test are the group-specific cross-reactive antigens for Rickettsia and Orientia species. Earlier studies have revealed that the group specific and cross-reactive antigens responsible for the Weil–Felix test lie mostly in the lipopolysaccharide (LPS) moiety of the bacterial cell wall [Amano et al. (1993a) Infect Immun 61:4350–4355, (1993b) Microbiol Immunol 37:927–933, (1998) Infect Immun 66:923–926]. The three Proteus strains (OX2, OX19 and OXK) were used to raise murine monoclonal antibodies (MAbs) by hybridoma technology. Several MAb-producing hybridomas could be stabilized following limiting dilution. Affinity and specificity of these MAbs were checked by indirect ELISA using a battery of homologous and heterologous antigens including LPS. Amongst these, one MAb was found to be specific for P. vulgaris OX19 LPS. Since the Weil–Felix reaction is based on the cross-reactivity between the LPS based epitopes, this MAb could be of potential use in mapping of epitopes on the cross-reactive LPS and may also be useful as a potential diagnostic reagent.     

Protective effects of affinity-purified antibody and truncated vaccines against Pseudomonas aeruginosa V-antigen in neutropenic mice

Virulent P. aeruginosa strains express PcrV, one of the translocational components of the type III secretion system. PcrV has been reported to be a protective antigen against lethal P. aeruginosa infection. The PcrV region, which contributes to protective immunity against P. aeruginosa infection, was investigated by using genetically engineered, truncated PcrV proteins and affinity-purified anti-PcrV antibodies against the truncated PcrV proteins. The efficacy of active and passive immunization against PcrV was tested in mice with cyclophosphamide-induced immunosuppression by intraabdominal challenge of P. aeruginosa . Active immunization with either full-length PcrV1-294 or PcrV139-294 significantly improved the survival of mice infected with P. aeruginosa , while PcrV139-258, PcrV139-234, PcrV197-294, and PcrV261-294 were not protective. These results suggest that an effective PcrV vaccine needs to contain not only the Mab166 epitope (PcrV144-257) but also the carboxyl terminal tail of PcrV. In the case of passive immunization, administration of affinity-purified anti-PcrV IgG against either PcrV1-294 or PcrV139-258 showed significantly higher efficacy against lethal P. aeruginosa infection than did original anti-PcrV IgG and Mab166. The increased efficacy of affinity-purified anti-PcrV IgG implies that more potent anti-PcrV strategies are possible. The results of this study are crucial to the development of an effective PcrV vaccine for active immunization and to an appropriate blocking anti-PcrV antibody against P. aeruginosa infection in humans.