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.     

Glycolipoprotein from Pseudomonas aeruginosa as a protective antigen against P. aeruginosa infection in mice.

After primary subcutaneous immunization of rabbits with glycolipoprotein from Pseudomonas aeruginosa BI, indirect hemagglutinating and bacterial agglutinating activities appeared in the antiserum 6 days after immunization and reached a peak between 15 and 20 days. Both these in vitro activities paralleled in vivo antipseudomonas-induced leukopenia and mouse passive-protection activities. Further experiments indicated that a functional association exists between the hemagglutinating and passive-protection activities, and that passive protection depends on activity levels in the plasma rather than in the peritoneum. After intraperitoneal injection in mice, in vitro and in vivo activities of antiglycolipoprotein serum declined in the peritoneal cavity as the plasma levels increased. After intravenous injection of the antiglycolipoprotein serum, initially high levels of in vitro and in vivo activity declined at approximately equal rates. Immunoglobulin G (IgG) and immunoglobulin M (IgM) fractions from 15-day antiglycolipoprotein serum were assayed for biological activity. Most of the hemagglutinating and bacterial agglutinating activity and all of the mouse passive-protection activity were found in the IgM fraction. Assay of antiglycolipoprotein serum after 2-mercaptoethanol inactivation of IgM showed that most of the in vitro and all of the passive-protection activities had been destroyed, again locating these activities principally in the IgM fraction of the original antiserum.     

Production of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa.

A panel of 22 monoclonal antibodies against 8 of the 17 International Antigenic Typing Scheme (IATS) serotypes of Pseudomonas aeruginosa was produced. The antibodies were characterized for cross-reactivities, isotypes, titers, and epitope specificities. The results complemented those of our previous study and marked the completion of a set of monoclonal antibodies for serotyping P. aeruginosa.     

Generation and characterization of murine antiflagellum monoclonal antibodies that are protective against lethal challenge with Pseudomonas aeruginosa.

Two murine monoclonal antibodies, IIG5 (IgG3) and IVE8 (IgG2a), that bind to Pseudomonas aeruginosa type a flagella and type b flagella, respectively, were prepared by conventional hybridoma methodology. Specificity of each monoclonal antibody for type a or type b flagella was demonstrated by enzyme-linked immunosorbent assay, indirect immunofluorescence, and immunoblotting. The percentage of P. aeruginosa isolates recognized by each monoclonal antibody was analyzed by enzyme-linked immunosorbent assay. Among a panel of 257 flagellated P. aeruginosa clinical isolates, IIG5 bound to 67.7% of the isolates and IVE8 bound to another 30.7%, for a combined coverage of 98.4%. Inhibition of motility of P. aeruginosa by the monoclonal antibodies was observed in vitro in a soft agar assay and was dose dependent. The protective efficacy of IIG5 and IVE8 was examined in a mouse burn wound sepsis model. The antiflagellum monoclonal antibodies provided specific and significant prophylactic and therapeutic protection against lethal challenge with P. aeruginosa strains.     

Isolation and characterization of monoclonal antibodies against alkaline phosphatase of Pseudomonas aeruginosa.

Monoclonal antibodies against the alkaline phosphatase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of P. aeruginosa ATCC 10145 and SP20/Ag-14 myeloma cells. The eight stable clones established produced antibodies that reacted by enzyme-linked immunosorbent and indirect immunofluorescence assays with all bacterial strains of P. aeruginosa, including the 17 serotypes and two nontypable strains. Three of the clones cross-reacted only with some Pseudomonas species of the rRNA homology group I defined by N. J. Palleroni (in N. R. Krieg and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, 8th ed., p. 140-218, 1984). The other clones also interacted with other species, including Pseudomonas acidovorans and Xanthomonas maltophilia. Because other species of the genera Aeromonas and Acinetobacter and species of the family Enterobacteriaceae were not detected by these monoclonal antibodies, the antibodies could be used as reagents for routine detection of P. aeruginosa in clinical specimens. Interactions of the antibodies with other Pseudomonas species such as P. fluorescens and P. stutzeri are not important, since these species are susceptible to the same antipseudomonal agents.     

Protection of immunosuppressed mice against infection with Pseudomonas aeruginosa by recombinant P. aeruginosa lipoprotein I and lipoprotein I-specific monoclonal antibodies.

Outer membrane protein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. The protective effect of OprI vaccination and that of three OprI-specific monoclonal antibodies (MAbs) against infection with P. aeruginosa were tested in immunosuppressed mice. The combination of Oprl and MAb 2A1 protected the mice against a challenge with a 96-fold 50% lethal dose. The binding site of MAb 2A1 was mapped, resulting in the identification of a protective epitope (amino acids 7 to 20).     

Human monoclonal antibodies against Pseudomonas aeruginosa lipopolysaccharide derived from transgenic mice containing megabase human immunoglobulin loci are opsonic and protective against fatal pseudomonas sepsis

Pseudomonas aeruginosa is a significant human pathogen, and no vaccine is commercially available. Passive antibody prophylaxis using monoclonal antibodies (MAb) against protective P. aeruginosa epitopes is an alternative strategy for preventing P. aeruginosa infection, but mouse MAb are not suitable for use in humans. Polyclonal human antibodies from multiple donors have variable antibody titers, and human MAb are difficult to make. We used immunoglobulin-inactivated transgenic mice reconstituted with megabase-size human immunoglobulin loci to generate a human MAb against the polysaccharide (PS) portion of the lipopolysaccharide O side chain of a common pathogenic serogroup of P. aeruginosa, 06ad. The anti-PS human immunoglobulin G2 MAb made from mice immunized with heat-killed P. aeruginosa was specific for serogroup 06ad pseudomonas. The MAb was highly opsonic for the uptake and killing of P. aeruginosa by human polymorphonuclear leukocytes in the presence of human complement. In addition, 25 microg of the MAb protected 100% of neutropenic mice from fatal P. aeruginosa sepsis. DNA sequence analysis of the genes encoding the MAb revealed V(H)3 and Vkappa2/A2 variable-region genes, similar to variable-region genes in humans immunized with bacterial PS and associated with high-avidity anti-PS antibodies. We conclude that human MAb to P. aeruginosa made in these transgenic mice are highly protective and that these mice mimic the antibody response seen in humans immunized with T-cell-independent antigens such as bacterial PS.     

Inhibitory activity on bacterial motility and in vivo protective activity of human monoclonal antibodies against flagella of Pseudomonas aeruginosa.

Three stable hybridoma cell lines, IN-2A8, IN-5D6, and ZI-3A8, that secrete human monoclonal antibodies (MAbs) specific for b-type flagella of Pseudomonas aeruginosa were established by fusing peripheral blood lymphocytes from healthy volunteers with murine myeloma P3X63-Ag8.653 cells. The immunoglobulin M MAbs reacted specifically with flagellin (Mr, 52,000) by Western blotting (immunoblotting) analysis and bound specifically to clinical isolates belonging to Homma serotypes A, B, H, I, and M at frequencies of 58, 50, 46, 30, and 35%, respectively, but did not bind to any serotype E or G isolates. Overall, the MAbs bound to 31% of the clinical isolates. MAb IN-2A8 strongly protected burned mice challenged with P. aeruginosa bearing b-type flagella from death following parenteral administration of 0.1 microgram per mouse. This MAb also inhibited P. aeruginosa colony spreading in soft agar at a concentration of more than 1 microgram/ml but only slightly enhanced opsonophagocytosis by human polymorphonuclear leukocytes. A line of evidence suggests that the potent in vivo activity of MAb IN-2A8 in the burned-mouse model is likely to be caused by its inhibition of bacterial motility after binding to flagella.     

Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa

Monoclonal antibodies against 12 of the 17 IATS serotype strains of Pseudomonas aeruginosa were produced. Eighty-seven hybridoma clones were isolated, and the antibodies secreted were found to be reactive with both Formalin-fixed whole cells and purified lipopolysaccharide of homologous strains in enzyme-linked immunosorbent assays. Among these monoclonal antibodies, the predominant antibody class was immunoglobulin M (IgM) (76%), although antibodies of the IgG2a and IgG3 isotypes were also produced. The monoclonal antibodies could further be divided into two groups based on their ability to agglutinate whole cells of homologous strains. The agglutinating monoclonal antibodies were found to immunoblot with the O side chains of homologous lipopolysaccharide, while the nonagglutinating monoclonal antibodies were found to be reactive with outer membrane protein-associated lipopolysaccharide. The applicability of monoclonal antibodies for serotyping was examined, and several antibodies were found to agglutinate whole cells and immunoblot with the O antigen of corresponding serotypes of clinical isolates from cystic fibrosis patients. In conclusion, a set of monoclonal antibodies against the IATS serotype strains of P. aeruginosa have been produced. These antibodies represent a bank of invaluable immunological reagents which may have application in serotyping, epitope mapping, lipopolysaccharide structural determination, and studies of protection against P. aeruginosa.     

In vivo protective effect of lipopolysaccharide against Pseudomonas aeruginosa exotoxin A in mice.

Lipopolysaccharide (LPS) treatment of mice 1 to 5 days prior to administration of Pseudomonas aeruginosa exotoxin A (PA) induced full or partial protection against PA intoxication. The optimal LPS dose that induced resistance was 50 to 100 micrograms per mouse. Simultaneous administration of LPS and PA to mice, however, increased their sensitivity to PA two- to fourfold. Mice pretreated with LPS demonstrated a markedly enhanced clearance rate of 125I-labeled PA from peripheral blood, livers, and kidneys. In mice exposed to LPS and PA simultaneously, the rate of elimination of labeled PA was lower than that in control mice. While protein synthesis was inhibited significantly in livers and other organs of PA-exposed mice, in LPS-pretreated mice, PA-induced inhibition of protein synthesis was either diminished or totally prevented and elongation factor 2 (EF2) levels were normal. In mice treated only with LPS, enhanced protein synthesis and increased levels of EF2 were observed, suggesting that LPS protection against PA intoxication was perhaps a consequence of excessive amounts of EF2 induced by LPS.     

Cross-reactivity of Pseudomonas aeruginosa antipilin monoclonal antibodies with heterogeneous strains of P. aeruginosa and Pseudomonas cepacia.

Much of the morbidity and mortality in patients with cystic fibrosis (CF) is secondary to pulmonary infections with Pseudomonas aeruginosa and, more recently, with Pseudomonas cepacia. Prevention of colonization and subsequent infection would be a useful therapeutic strategy. The pili (fimbriae) of P. aeruginosa are a potential vaccine antigen, as they have been implicated in binding to respiratory epithelium and appear to have limited antigenic diversity. Monoclonal antibodies (MAbs) raised to P. aeruginosa pilin demonstrated significant cross-reactivity, as four of five P. aeruginosa strains with known pilin sequences and 10 of 15 P. aeruginosa clinical isolates hybridized by immunoblot with at least one of the three MAbs tested. The P. cepacia strains demonstrated minimal cross-reactivity with these MAbs, as only 2 of 16 strains hybridized immunologically. The three MAbs decreased the adherence of 35S-labeled P. aeruginosa PA1244 to bovine tracheal cells by 56, 45, and 31%. One of these MAbs decreased the adherence of strains P. aeruginosa PAO1 and P. cepacia 249 to CF epithelial cells by 46 and 25%, respectively. While antibodies to Pseudomonas pili must be shown to be protective in patients with CF, these studies give support for a multivalent vaccine strategy using P. aeruginosa pilin as the immunogen.     

Subdivision of O serotypes of Pseudomonas aeruginosa with monoclonal antibodies.

Sixteen murine monoclonal antibodies (MAbs) against serotypes O2, O5, and O16 (serogroup II) and subtypes O2b and O5d of Pseudomonas aeruginosa were evaluated by agglutination and enzyme-linked immunosorbent assay. Six MAbs that exhibited different specificities were compared with absorbed rabbit O-type antisera for the serotyping of 55 clinical isolates of serogroup II. There was good agreement between the antibodies for strains of serotypes O2 and O2b, but MAbs revealed reproducible differences between strains that were indistinguishable with rabbit antisera. The greater serotype specificity of MAbs was illustrated by the fact that only 5 of 20 strains which were agglutinated by rabbit antiserum O16 reacted with the MAb to that serotype. One antibody, M89, that reacted with 27 of 55 serogroup II strains, apparently bound to core lipopolysaccharide epitopes. Three MAbs to the frequent serotype O6 identified six subtypes, one of which accounted for over half of the clinical strains, while two subtypes were represented by single strains only. Overall, MAbs provided a greater discrimination between strains of P. aeruginosa of the same serotype than did absorbed polyclonal antisera.     

Four epitopes of Pseudomonas aeruginosa elastase defined by monoclonal antibodies.

Monoclonal antibodies against the elastase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified elastase of P. aeruginosa and P3-X63-Ag8-U1 myeloma cells. The six clones established generated antibodies which reacted with a 33,000-Da peptide and recognized four different elastase epitopes by a competitive binding enzyme-linked immunosorbent assay. The monoclonal antibodies designated as ELA-17 and ELA-42 that recognize two different epitopes reacted by dot-enzyme immunoassay and by Western immunoblotting with all clinical and International Antigen Typing Scheme strains of P. aeruginosa positive for elastase.     

Use of monoclonal antibodies to protein F of Pseudomonas aeruginosa as opsonins for phagocytosis by macrophages.

Five protein F-specific monoclonal antibodies were found to opsonize Pseudomonas aeruginosa for complement-independent phagocytosis by unelicited mouse peritoneal macrophages, mouse macrophage cell line P388D1, and human monocyte-derived macrophages. Immunoglobulin G1 antibodies seemed to be a preferred isotype.     

Cytokine-deficient mice as a model for generation of autologous anti-cytokine monoclonal antibodies

The possibility of inducing immune responses to murine interleukin-4 (IL-4) and IL-13 and generating anti-cytokine monoclonal antibodies (mAbs) was studied in IL-4- and IL-13-knockout mice. The minimal doses of IL-4 or IL-13 that could induce significant anti-cytokine responses with titers of 5000-10,000 were 20 microg per injection with the total doses of 100 microg. The highest titers in a range of 20,480-40,960 were achieved by triple immunization of IL-13-knockout mice with 30 microg of IL-13 per injection. Anti-IL-4 mAbs were generated at an antibody serum titer about 400; however, only 0.5% of primary hybridoma clones were anti-IL-4-positive. Anti-IL-13 cell fusions were successful at titers of 20,480 and 40,960 with 50% of the primary clones positive. Both hybridomas secreted low-affinity IgMkappa mAbs and were IL-6-dependent. These data demonstrate that IL-4- and IL-13-deficient mice may develop high polyclonal immune responses to "syngeneic" murine cytokines but fail to generate high-affinity mAbs at the tested conditions.     

Human monoclonal antibodies with a protective activity against tetanus toxin

The antibodies and their protective activity in response to tetanus toxoid in man were studied by producing human antitetanus monoclonal antibodies after transformation of peripheral blood lymphocytes with Epstein Barr virus. Two human monoclonal IgG that reacted with the heavy chain of the toxin were obtained. One of them binds the COOH-terminal moiety and the other the NH2-terminal moiety. Only the NH2-terminal specific monoclonal antibody neutralized toxin in mouse, but in doses approximately 100-fold higher than those of a polyclonal antiserum. However, the association of these 2 antibodies was protective with doses lower than necessary for the monoclonal antibodies alone. To replace the polyclonal antibodies used, a good protection could be achieved by mixed human monoclonal antibodies against different epitopes of tetanus toxin.     

Comparison of polyclonal rabbit antisera with monoclonal antibodies for serological typing of Pseudomonas aeruginosa.

A panel of 219 distinct strains of Pseudomonas aeruginosa were serotyped with a set of monoclonal antibodies prepared against the serotype strains of the Homma scheme (J. Y. Homma, Jpn. J. Exp. Med. 46:329-336, 1976). A total of 87.6% were typable, and there was very good agreement with the corresponding polyclonal serotype. A high proportion of strains that were polyagglutinating or nontypable with polyclonal antisera were agglutinated by antibody towards Homma group M.     

Inhibition of pilus-mediated adhesion of Pseudomonas aeruginosa to human buccal epithelial cells by monoclonal antibodies directed against pili.

The Pseudomonas aeruginosa PAK pilus is capable of mediating the binding of this strain to human respiratory epithelial cells. We have produced monoclonal antibodies (MAbs) to the PAK pilus in order to elucidate the location of the binding domain of the pilus for human buccal epithelial cells (BECs). Four MAbs are described. MAbs PK41C and PK34C were found to react with P. aeruginosa pilins produced by a large number of strains. The epitope recognized by PK41C was determined to lie within the N-terminal region of the pilin and is likely constituted by amino acid residues 22 through 33. The epitope for PK34C was located in the C-terminal region of the pilin and was partially dependent on an intact intrachain disulfide bridge between cysteine residues 129 and 142. PK99H and PK3B were found to react specifically with PAK pilin. The epitope for PK99H was also localized in the C-terminal region of the pilin protein and appears to reside between amino acid residues 130 and 138. The epitope for PK3B was not localized by using the methods of this study, but it is likely dependent on the three-dimensional structure of the pilin. Fab fragments of PK99H inhibited adhesion of strains PAK and 492c to BECs, but the adherence of five other strains was not affected. Fab fragments of PK34C inhibited adhesion of all piliated strains examined. Fab fragments from both of these antibodies inhibited PAK pilus binding to BECs. Fab fragments of PK41C and PK3B had no effect on P. aeruginosa binding to BECs. These results confirm that the C-terminal region of the pilin has adhesin qualities and that a conserved epitope lies within this region.     

Simple model for the study of Pseudomonas aeruginosa infections in leukopenic mice.

A simple, reproducible model of fatal Pseudomonas aeruginosa sepsis in mice during immunosuppression was developed. Mice were rendered leukopenic (less than or equal to 800 leukocytes per mm3 of blood) for a period of 5 days by multiple injections of cyclophosphamide. Mice were challenged at the onset of leukopenia by instilling the bacteria onto a 0.5-mm incision made into the back. The mean lethal dose (LD50) for P. aeruginosa PA220 and M-2 was less than 20 bacteria. The mean time to death for these strains ranged from 46 to 59 h. Leukopenic mice were comparatively resistant when challenged with Klebsiella pneumoniae (LD50 = 1.5 x 10(6)) or Staphylococcus aureus (LD50 greater than 10(6)). Infection with P. aeruginosa was characterized by rapid bacterial multiplication in the skin at the site of infection, producing ecthyma gangrenosum. Bacteremia and colonization of the liver were pronounced 21 h postinfection. This model should prove to be a useful tool for studying the pathogenesis of P. aeruginosa infections under immunosuppressed conditions.