Biologic activities of antibodies to the neutral-polysaccharide component of the Pseudomonas aeruginosa lipopolysaccharide are blocked by O side chains and mucoid exopolysaccharide (alginate).

Virulent strains of Pseudomonas aeruginosa are either of a nonmucoid, lipopolysaccharide (LPS)-smooth or mucoid, LPS-rough phenotype, and immunity to these different variants is efficiently mediated by antibodies specific to O antigens or mucoid exopolysaccharide (also called alginate), respectively. In addition to O side chains and core polysaccharide components, the LPS of P. aeruginosa also contains neutral-polysaccharide components that express antigenic determinants common to many clinical isolates. We evaluated antibodies specific to neutral polysaccharides for the ability to mediate opsonic killing and protective immunity. Antibodies to these antigens mediated opsonic killing of poorly virulent nonmucoid LPS-rough isolates but not of isogenic strains with either a LPS-smooth or a mucoid phenotype. Antibodies to neutral-polysaccharide antigens also failed to protect neutropenic mice from challenge with modest doses of LPS-smooth P. aeruginosa strains (< 10(3) CFU per mouse), whereas O-antigen-specific antibodies were highly protective. Antibodies to neutral polysaccharides deposited significantly (P = 0.002) more C3 onto LPS-rough strains than did antibodies to O side chains, but this situation was reversed when isogenic LPS-smooth strains were tested. Given that protective immunity against P. aeruginosa must be directed against either nonmucoid LPS-smooth strains or mucoid LPS-rough strains, it appears that antibodies specific to neutral-polysaccharide antigens do not protect against P. aeruginosa infection. Lack of protection is likely due to the ability of both O side chains and mucoid exopolysaccharide (alginate) to interfere with the opsonic killing activity of neutral-polysaccharide-specific antibodies.     

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.     

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.     

Mucosal vaccination with a multivalent, live-attenuated vaccine induces multifactorial immunity against Pseudomonas aeruginosa acute lung infection

Many animal studies investigating adaptive immune effectors important for protection against Pseudomonas aeruginosa have implicated opsonic antibody to the antigenically variable lipopolysaccharide (LPS) O antigens as a primary effector. However, active and passive vaccination of humans against these antigens has not shown clinical efficacy. We hypothesized that optimal immunity would require inducing multiple immune effectors targeting multiple bacterial antigens. Therefore, we evaluated a multivalent live-attenuated mucosal vaccination strategy in a murine model of acute P. aeruginosa pneumonia to assess the contributions to protective efficacy of various bacterial antigens and host immune effectors. Vaccines combining 3 or 4 attenuated strains having different LPS serogroups were associated with the highest protective efficacy compared to vaccines with fewer components. Levels of opsonophagocytic antibodies, which were directed not only to the LPS O antigens but also to the LPS core and surface proteins, correlated with protective immunity. The multivalent live-attenuated vaccines overcame prior problems involving immunologic interference in the development of O-antigen-specific antibody responses when closely related O antigens were combined in multivalent vaccines. Antibodies to the LPS core were associated with in vitro killing and in vivo protection against strains with O antigens not expressed by the vaccine strains, whereas antibodies to the LPS core and surface proteins augmented the contribution of O-antigen-specific antibodies elicited by vaccine strains containing a homologous O antigen. Local CD4 T cells in the lung also contributed to vaccine-based protection when opsonophagocytic antibodies to the challenge strain were absent. Thus, multivalent live-attenuated vaccines elicit multifactorial protective immunity to P. aeruginosa lung infections.     

Immunochemical characterization of high-molecular-weight polysaccharide from Fisher immunotype 3 Pseudomonas aeruginosa.

A high-molecular-weight polysaccharide (PS) was isolated from the culture supernatant of a Fisher immunotype 3 (IT-3) strain of Pseudomonas aeruginosa. Consistent with previously reported findings for IT-1 and IT-2 PS, the preparation of IT-3 PS was found to be an immunogenic, nontoxic form of the O polysaccharide side chain on the lipopolysaccharide (LPS). The IT-3 PS was mainly carbohydrate in composition. It was serologically and chemically identical to LPS O side chain, but distinct from that structure in molecular size and immunogenicity. The IT-3 PS was nontoxic in mice and guinea pigs, nonpyrogenic in rabbits, and greater than 1,000-fold less reactive than IT-3 LPS in gelation of the Limulus amoebocyte lysate. Preliminary analyses by gas-liquid chromatography and 13C nuclear magnetic resonance have established the structural identity of IT-3 high-molecular-weight PS and the IT-3 O side chain. IT-3 PS was immunogenic in rabbits and mice. After active immunization, mice were protected against P. aeruginosa IT-3 intraperitoneal infection and burn wound sepsis. IT-3 PS also elicited protection against challenge with an IT-5 strain of P. aeruginosa, indicating that low-level contamination of the IT-3 PS with IT-3 LPS was not responsible for the immunogenic activity. These findings demonstrate the feasibility of preparing nontoxic immunogenic IT-3 PS capable of eliciting serotype-specific protective antibodies, employing methods similar to those previously described for the isolation of PS from other P. aeruginosa immunotypes.     

Protective effect of antilipopolysaccharide monoclonal antibody in experimental Klebsiella infection.

An O-antigen-specific murine monoclonal antibody (MAb) directed against an immunodominant epitope expressed on Klebsiella O1, O6, and O8 lipopolysaccharides (LPS) was examined with respect to its binding to nonencapsulated and encapsulated bacterial cells and its ability to protect against lethal murine Klebsiella sepsis. While the MAb (clone Ru-O1, mouse immunoglobulin G2b) bound well to nonencapsulated organisms of the O1 serogroup, binding was significantly, but not completely, abolished by the presence of the K2 capsule. In a model of experimental Klebsiella peritonitis and sepsis induced by a virulent O1:K2 serogroup strain, higher doses of anti-LPS MAb Ru-O1 than of a previously described anticapsular MAb specific for the K2 capsular polysaccharide were needed to provide protection. However, high-dose (40 microg/g of body weight) pretreatment with anti-LPS MAb Ru-O1 significantly reduced bacterial dissemination to various organs as well as macroscopic and histologic pulmonary alterations. Thus, since the number of Klebsiella capsular antigens occurring in clinical material is too large to be completely "covered" by a K-antigen-specific hyperimmunoglobulin preparation, O-antigen-specific antibodies may supplement K-antigen-specific immunoprophylaxis and -therapy of clinical Klebsiella infection.     

Phage display and bacterial expression of a recombinant Fab specific for Pseudomonas aeruginosa serotype O6 lipopolysaccharide.

Immunotherapy with antibodies (Abs) against the lipopolysaccharide (LPS) of Pseudomonas aeruginosa remains an alternative to serotype-specific LPS-based vaccines due to their limited use and to antibiotics due to the intrinsic resistance to antimicrobials observed in P. aeruginosa. We have chosen a monoclonal Ab (MAb), MF23-1, that binds to the O antigen of the most clinically relevant serotype, IATS O6, for producing a recombinant antibody. Heavy (H) and light (L) chain genes were isolated from MF23-1 to form a functional Fab molecule in the periplasm of Escherichia coli and on the surface of phage by using phagemid vector pComb3. The entire kappa L chain gene was used, but the H chain gene was amplified to 2 amino acids past cysteine 128 which is involved in interchain disulfide bond formation with the L chain. The truncated H chain associated with the L chain in the periplasm of E. coli to form a functional Fab molecule that bound in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay to O6 LPS. Therefore, the remainder of the CH1 past cysteine 128 is not essential for stable formation of the Fab portion of MF23-1. This recombinant Fab (r-Fab) was shown to be specific for the LPS of the most predominant clinical isolate, serotype O6, while no cross-reactivity was detected to the LPS of the other 19 remaining serotypes. This r-Fab was also expressed on the surface of filamentous phage upon addition of helper phage to recombinant E. coli containing phagemid. Recombinant phage from clones MT13 and MT24 bound specifically to O6 LPS in ELISA. These results represent an important step toward the design of therapeutic Abs to be used against P. aeruginosa infections.     

Characterization of anti-core glycolipid monoclonal antibodies with chemically defined lipopolysaccharides.

Five anti-core glycolipid monoclonal antibodies (MAb) (four against Escherichia coli J5 lipopolysaccharide [LPS] and one against the Re core glycolipid of Salmonella typhimurium) were characterized using LPS from several rough and smooth strains and derivatives of E. coli J5 LPS, obtained by N acetylation and hydrolysis. The MAb against E. coli J5 were not only weakly cross-reactive with clinical isolates, whereas the anti-Re MAb was highly cross-reactive. The MAb differed in their reaction pattern with E. coli J5 LPS. MAb 4-7B5 (immunoglobulin M) and MAb 4-6A1 (immunoglobulin G1) cross-reacted with LPS of Salmonella minnesota R5 and S. typhimurium Ra and Rc and little with Re and lipid A. The dominant binding site of these MAb was located in the glucose-heptose-heptose region and was independent of phosphate substitution. The MAb 4-9A1 reacted with the terminal part of the core region (glucose-heptose) and was dependent on phosphate substitution of the LPS. The MAb BA7 (immunoglobulin G3) was E. coli J5 LPS specific and reacted with the glucosaminyl-heptose disaccharide. Antibody 8-2C1 was directed against the common parts of LPS, 3-deoxy-D-manno-octulosonic acid, and lipid A, which are not (or only weakly) recognized by the four anti-J5 LPS MAb. Thus, MAb that are not cross-reactive can be directed against at least three different antigenic determinants present on the core oligosaccharide of E. coli J5 LPS.     

Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia.

A human immunoglobulin G preparation, enriched in antibodies to lipopolysaccharide (LPS) Pseudomonas aeruginosa antigens (PA-IGIV) and murine monoclonal antibodies (MAb) to P. aeruginosa Fisher immunotype-1 (IT-1) LPS antigen and outer membrane protein F (porin), were evaluated for therapeutic efficacy in a guinea pig model of P. aeruginosa pneumonia. The concentration of antibodies to IT-1 LPS was 7.6 micrograms/ml in PA-IGIV and 478 micrograms/ml in the IT-1 MAb preparation. No antibody to IT-1 was detected in MAb to porin. For study, animals were infected by intratracheal instillation of IT-1 P. aeruginosa and then treated 2 h later with intravenous infusions of PA-IGIV, IT-1 MAb, or porin MAb. Control groups received intravenous albumin, and routinely died from pneumonia. Both PA-IGIV (500 mg/kg) and IT-1 MAb (greater than or equal to 2.5 mg/kg) treatment resulted in increased survival (P less than 0.01 to 0.001), and also improved intrapulmonary killing of bacteria. Porin MAb failed to protect from fatal pneumonia. IT-1 MAb treatment produced more survivals than did PA-IGIV treatment but only at dosages of MAb resulting in serum antibody concentrations greater than those achieved with PA-IGIV. PA-IGIV and IT-1 MAb demonstrated in vitro and in vivo (posttreatment guinea pig serum) opsonophagocytic activity for the IT-1 challenge strain. However, the polyclonal preparation required complement, whereas the MAb did not. We conclude that passive immunization with polyclonal hyperimmune P. aeruginosa globulin or with MAb to LPS antigens may be useful in the treatment of acute P. aeruginosa pneumonia. The relative efficacies of such preparations may be limited, however, by their type-specific LPS antibody concentrations.     

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).     

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.     

Murine monoclonal antibodies to Klebsiella pneumoniae protect against lethal endotoxemia and experimental infection with capsulated K. pneumoniae.

To prepare monoclonal antibodies (MAbs) directed against the core-lipid A fractions of smooth lipopoly-saccharide (LPS) from Klebsiella pneumoniae O1:K2, we immunized BALB/c mice with the LPS-associated proteins plus LPS. This preparation exposed the core-lipid A moiety, which is normally hidden in the micellar structure of classical LPS preparations. Among 10 MAbs selected for their reactivity with LPS-associated proteins plus LPS from K. pneumoniae O1:K2, 6 (3A3, 3C2, 3C4, 7D2, 11C3, and 12B6) were directed against the core fraction and 2 (6C5 and 10A5) were directed against the lipid A fraction. Only one (2A4) recognized the O antigen, and one (6D5) had an undefined specificity. When injected before challenge with K. pneumoniae O1:K2 LPS in galactosamine-sensitized mice, five of the MAbs (3C4, 6D5, 7D2, 11C3, and 12B6) provided protection in this model of lethal endotoxemia. MAb 7D2 was also protective in an experimental infection with capsulated K. pneumoniae O1:K2.     

Bacterial lipopolysaccharide (LPS)-specific antibodies in commercial human immunoglobulin preparations: superior antibody content of an IgM-enriched product

The anti-LPS antibody content of commercial intravenous immunoglobulins was examined by quantitative ELISA using LPS preparations from Escherichia coli, Klebsiella and Pseudomonas aeruginosa O serotypes occurring most frequently in Gram-negative septicaemia. Three IgG products from different manufacturers and one IgM-enriched product were tested. Mean antibody levels were significantly higher in the IgM fraction of the IgM-enriched product compared with ‘pure’ IgG products, indicating that natural antibodies against bacterial LPS belong primarily to the IgM class. Immunoblotting studies showed that antibody specificities were directed mainly against O side chain epitopes. Antibodies against rough mutant LPS representing various chemotypes were detected in IgG but not in IgM products. The virtual absence of antibodies against Vibrio cholerae LPS indicated that human anti-LPS antibodies result from continuous environmental exposure to Gram-negative pathogens. These data support the further development of IgM-enriched preparations for prophylaxis and treatment of Gram-negative nosocomial infections.     

Evaluation of murine monoclonal antibodies targeting different epitopes of the hepatitis B virus surface antigen by using immunological as well as molecular biology and biochemical approaches

The hepatitis B virus surface protein (HBsAg) displays the major B cells antigenic determinants that can induce protective immunity and prevent the hepatitis B virus (HBV) infection, a major health problem. A panel of murine monoclonal antibodies against the HBsAg (MAb anti-HBs), raised after mice immunization with a pool of plasma of hepatitis chronic carriers, has been established. Mainly using simple immunological tools such as enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, we could trace the location of the epitopes on the HBsAg determinants. We also report the use of two specific methodology approaches based on molecular biology and biochemical techniques such as, respectively, cloning and expression of preS1 major neutralizing epitope of the HBsAg in Escherichia coli and ELISA accomplished to chemical reduction with dithiothreitol (DTT), which were able to complete the MAb anti-HBs characterization. Our results showed that the majority of the MAbs anti-HBs were directed to the HBV common determinant a. One MAb recognizes a discontinuous epitope present in all forms of the HBsAg when evaluated by Western blot.     

Influence of Immunopotentiators on the Antiporin Immunoglobulin G Subclass: Distribution and Protective Immunity Against Murine Salmonellosis

To improve the immune potential of porin (a pore-forming protein of Salmonella sp.), different immunopotentiators such as Freund's complete adjuvant (FCA), lipopolysaccharide (LPS) and polyoxydonium (PO) were evaluated by studying the nature of the protective immune response induced against murine Salmonellosis. The nontoxic, synthetic heteropolymer polyoxydonium was as good as LPS at inducing antiporin immunoglobulin G (IgG) antibodies and protective immunity. Analysis of the antiporin IgG subclass pattern revealed a preferential increase in a particular subclass based on the immunopotentiator used. Porin, alone or emulsified in FCA, elicited predominantly antiporin IgG1 antibodies, whereas LPS preferentially evoked antiporin IgG2a, IgG2b and IgG3 antibodies. Polyoxydonium induced a clear shift towards antiporin IgG2b antibodies. The significance of these antiporin IgG subclass antibodies in protection against murine Salmonellosis was studied by passive immunization and by analysing the infected mouse sera.     

Monoclonal antibodies reactive with K1-encapsulated Escherichia coli lipopolysaccharide are opsonic and protect mice against lethal challenge

Seven murine monoclonal antibodies (MAbs) directed against O-side-chain determinants of the K1-encapsulated Bortolussi strain of Escherichia coli (O18:K1:H7) were evaluated for their in vitro and in vivo activities. All the MAbs reacted well in Western blots against E. coli O18 lipopolysaccharide antigens. Two MAbs of the immunoglobulin G (IgG) class promoted in vitro opsonophagocytosis and protected mice lethally challenged with bacteria. Two IgM MAbs showed partial protection, although they had no in vitro opsonic activity, and the remaining three IgM MAbs showed no apparent functional activities. Monoclonal IgG antibodies against bacterial lipopolysaccharide can be opsonic and protective in spite of the presence of the K1 capsule on the bacterium.     

Development of an anti-idiotype monoclonal antibody mimicking the structure of lipopolysaccharide (LPS) inner-core determinants

An anti-idiotype antibody has been developed which is specific for idiotypic determinants of a BALB/c mouse IgG₃ monoclonal antibody (MAbY1-4A6) directed against the inner-core Kdo region of lipopolysaccharide (LPS). Armenian hamsters were immunized with MAbY1-4A6 and splenocytes from immunized animals fused with Sp2/0 myeloma cells. Eight clones secreting antibodies that bound to MAbY1-4A6, but not control IgG₃, were identified and subcloned. Culture supernatants from one hybridoma, termed MAb4G2, contain monoclonal antibody that binds to the variable region of MAbY1-4A6 and dose-dependently inhibits binding of MAbY1-4A6 to Re chemotype rough mutant LPS (Re-LPS). This antibody also inhibits binding of three additional mouse monoclonal antibodies specific for the inner-core of Re-LPS. MAb4G2 also recognizes a significant proportion of antibodies present in polyclonal R-chemotype antisera generated in mice (Re-LPS) and rabbits (J5 Rc-LPS). Mice and hamsters immunized with MAb4G2 or Re-LPS generate antibodies which cross-react with both immunogens. Cumulatively, these data suggest that MAb4G2 can function as an internal image of the Kdo-specific monoclonal antibody, MAbY1-4A6, mimicking the antigenic structure and immunogenicity of a portion of the LPS inner-core Kdo region.     

Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis.

Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 10(3) organisms, whereas FT2F11 was able to detect only 10(5) organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified O side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the O side chain and that FT2F11 is directed against the core.     

Properties determining the potential of naturally occurring and vaccine-induced human antibodies to protect against lethal infection of Pseudomonas aeruginosa.

In order to characterize antibodies responsible for the protection against fatal infection with Pseudomonas aeruginosa we analysed the fine specificity, avidity and protective capacities of naturally occurring anti-lipopolysaccharide (LPS) antibodies in two standard human Ig preparations and of vaccine-induced anti-LPS antibodies in a hyperimmune Ig preparation. Applying competitive binding assays, immunoblotting and an in vivo protection assay, we provide evidence that only preparations from immunized volunteers contain significant amounts of antibodies which confer detectable protection in a murine burn-wound model. Supported by the parallel analysis of monoclonal antibodies, our data suggest that protection by passive immunization with anti-LPS antibodies is mediated by antibodies specific for the LPS O-chain moiety of the corresponding virulent bacterium. Furthermore, our results indicate that protectiveness is restricted to a small population of antibodies with high affinity for particular O-chain epitopes.     

Influence of surface modulations by enzymes and monoclonal antibodies on alternative complement pathway activation by Yersinia enterocolitica.

Effector mechanisms resulting from alternative complement pathway (ACP) activation cannot act efficiently against Yersinia enterocolitica serotype O3, as indicated by poor C3 to C9 consumption and by survival in EGTA (ethyleneglycoldiaminetetraacetic acid) Mg-serum. These results were not influenced by the lack or presence of plasmid-encoded outer membrane proteins or lipopolysaccharides (LPS) with different amounts of side chains or by treatment of the bacteria with pronase or neuraminidase. Surface modulation of Y. enterocolitica with polyclonal immunoglobulin G or the immunoglobulin G fragments F(ab')2 and Fab always converted Y. enterocolitica to a high ACP activator, with strong C3 to C9 consumption and surface deposition of activated C3. Killing of Y. enterocolitica as a result of antibody-mediated ACP activation was observed only with bacteria grown at 22 degrees C but not with bacteria from 37 degrees C cultures. The expression of complement resistance in Y. enterocolitica grown at 37 degrees C was not influenced by the presence or absence of plasmids. Using different monoclonal antibodies (MAb), we found that MAb with LPS specificity mediated ACP activation, whereas MAb specific for different plasmid-encoded outer membrane proteins were ineffective, despite surface binding. These results suggest a major inhibitory role of LPS on ACP activation which was neutralized by LPS-specific antibodies.