Inhibition of bacterial motility with human antiflagellar monoclonal antibodies attenuates Pseudomonas aeruginosa-induced pneumonia in the immunocompetent rat

Landsperger, William J.; Kelly-Wintenberg, K.; Montie, Thomas C.; Knight, L S; Hansen, Morten Bagge; Huntenburg, Carolyn C.; Schneidkraut, Marlowe J.

doi:10.1128/iai.62.11.4825-4830.1994

Cited by 46 publications

(11 citation statements)

References 23 publications

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“…d Apparent M r Ϫ adjusted molecular weight. (24,30). With at least one mouse MAb, i.e., that raised against the flagella of 170018, there was an association between M r and cross-reactivity (30).…”

Section: Discussionmentioning

confidence: 98%

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Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin of Pseudomonas aeruginosa a-Type Strains

1998

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Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of severalPseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.

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Section: Discussionmentioning

confidence: 98%

“…The membrane was removed and washed according to standard procedures. Primary antibody, either human anti-a flagellin MAb or human anti-b flagellin MAb (24), was added at a 1:1,000 dilution in Tris-buffered saline (TBS)-Tween and incubated overnight at 4°C with shaking. Washes were performed as outlined above with TBS-Tween.…”

Section: Methodsmentioning

confidence: 99%

Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin of Pseudomonas aeruginosa a-Type Strains

1998

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“…A number of studies have demonstrated the protective effects of antiflagellar antiserum or MAbs against lethal P. aeruginosa infections in a burn wound model (15,19,21,28,33,39) and in a pneumonia model (22,29). Landsperger et al (22) demonstrated that decreased bacterial motility by antiflagellar antibody is associated with a decreased pathogenicity of P. aeruginosa in rat model of pneumonia (22). As a mechanism of protective effects of antiflagellar antibodies, Anderson and Montie (3,4) reported that antiflagellar antibodies stimulate opsonophagocytosis of P. aeruginosa by polymorphonuclear leukocytes.…”

Section: Discussionmentioning

confidence: 99%

Effect of Antiflagellar Human Monoclonal Antibody on Gut-Derived Pseudomonas aeruginosa Sepsis in Mice

Matsumoto

Tateda

Miyazaki

et al. 1999

Clin Diagn Lab Immunol

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We evaluated the effect of antiflagellar human monoclonal antibody on gut-derived Pseudomonas aeruginosa sepsis. Mice were given a suspension of P. aeruginosa SP10052 in their drinking water and were simultaneously treated with ampicillin (200 mg/kg of body weight) to disrupt the normal bacterial flora. Cyclophosphamide was then administered to induce leukopenia and translocation of the P. aeruginosa that had colonized the gastrointestinal tract, thereby producing gut-derived generalized sepsis. In this model, intraperitoneal injection of 100 μg of antiflagellar human monoclonal antibody (SC-1225) per mouse for 5 consecutive days significantly (P < 0.01) increased the survival rate compared with that for mice treated with bovine serum albumin (BSA). Treatment with SC-1225 significantly reduced the average number of viable bacteria in portal blood, liver, and heart blood compared with the average number after treatment with BSA. Furthermore, the presence in serum of the inflammatory cytokines tumor necrosis factor alpha and interleukin 6 were evaluated as markers of severity of infection, and the results showed that the levels of these cytokines in mice treated with SC-1225 were significantly decreased in comparison with those in BSA-treated control mice. Although there was no significant difference in the number of bacteria that colonized the intestine, SC-1225 treatment significantly increased bacterial opsonophagocytosis by cultured peritoneal macrophages from mice with or without cyclophosphamide pretreatment. Our results indicate that antiflagellar human monoclonal antibody SC-1225 protects mice against gut-derived sepsis caused by P. aeruginosa and suggest that such an effect is due to its opsonophagocytic activity and the reduced motility of the translocated bacteria once the bacteria move from the intestine into the bloodstream.

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“…Thus, strategies evaluating either different monovalent antigens from serogroup O2 strains in humans or monoclonal antibodies to this epitope may identify broadly reactive and protective antibodies with potential as immunotherapeutic agents. Alternative antigenic targets to LPS O antigens have been intensely investigated, with outer membrane proteins (OMPs) (8,11,16,40) and flagella (21) receiving the most attention. However, levels of antibodies to O antigens are clearly correlated with resistance to lethal outcomes during human infection (38).…”

Section: Discussionmentioning

confidence: 99%

Complex Serology and Immune Response of Mice to Variant High-Molecular-Weight O Polysaccharides Isolated from Pseudomonas aeruginosa Serogroup O2 Strains

Hatano

Pier

1998

Infect Immun

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The O antigen of the Pseudomonas aeruginosalipopolysaccharide is the optimal target for protective antibodies, but the unusual and complex nature of their sugar substituents has made it difficult to define the range of these structures needed in an effective vaccine. Most clinical isolates of P. aeruginosacan be classified into 10 O-antigen serogroups, but slight chemical differences among O polysaccharides within a serogroup give rise to subtype epitopes. These epitopes could impact the reactivity of O-antigen-specific antibodies, as well as the susceptibility of a target strain to protective, opsonic antibodies. To define parameters of serogroup and subtype-epitope immunogenicity, antigenicity, and surface expression on P. aeruginosa cells, we prepared high-molecular-weight O-polysaccharide vaccines from strains ofP. aeruginosa serogroup O2, for which eight structurally variant O antigens expressing six defined subtype epitopes (O2a to O2f) have been identified. A complex pattern of immune responses to these antigens was observed following vaccination of mice. The high-molecular-weight O polysaccharides were generally more immunogenic at low doses (1 and 10 μg) than at a high dose (50 μg) and usually elicited antibodies that opsonized the homologous strain for phagocytic killing. Some of the individual polysaccharides elicited cross-opsonic antibodies to a variable number of strains that express all of the defined serogroup O2 subtype epitopes. Combination into one vaccine of two antigens that individually elicited cross-reactive opsonic antibodies to most members of the O2 serogroup inhibited, instead of enhanced, the production of antibodies broadly reactive with most serogroup O2 subtype strains. Thus, immune responses to P. aeruginosa O antigens may be restricted to a limited range of epitopes on structurally complex O antigens, and combining multiple related antigens into a single vaccine formulation may inhibit the production of those antibodies best able to protect against mostP. aeruginosa strains within a given O-antigen serogroup.

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Inhibition of bacterial motility with human antiflagellar monoclonal antibodies attenuates Pseudomonas aeruginosa-induced pneumonia in the immunocompetent rat

Cited by 46 publications

References 23 publications

Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin of Pseudomonas aeruginosa a-Type Strains

Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin of Pseudomonas aeruginosa a-Type Strains

Effect of Antiflagellar Human Monoclonal Antibody on Gut-Derived Pseudomonas aeruginosa Sepsis in Mice

Complex Serology and Immune Response of Mice to Variant High-Molecular-Weight O Polysaccharides Isolated from Pseudomonas aeruginosa Serogroup O2 Strains

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