A murine monoclonal antibody against Klebsiella capsular polysaccharide is opsonic in vitro and protects against experimental Klebsiella pneumoniae infection

Murine monoclonal antibodies (mAb) of the immunoglobulin M class specific for the K2 capsular polysaccharide (CPS) of Klebsiella were isolated. One such mAb, termed III/5-1, was selected for further study. This mAb promoted the uptake and killing of Klebsiella pneumoniae K2 strains by human granulocytes and activated complement after contact with the bacteria. The efficiency of mAb-mediated phagocytosis and complement activation was inversely related to the amount of capsular material produced by the test strain. MAb III/5-1 was found to be effective at preventing fatal experimental K. pneumoniae K2 sepsis when administered prophylactically, the degree of protection being dependent upon the amount of CPS produced by the challenge strain.     

Mouse monoclonal antibodies to anthrax edema factor protect against infection

Bacillus anthracis is the causative agent of anthrax, and the tripartite anthrax toxin is an essential element of its pathogenesis. Edema factor (EF), a potent adenylyl cyclase, is one of the toxin components. In this work, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic C(B) domain. MAb 7F10 shows potent neutralization of edema toxin activity in vitro and in vivo; it targets the N-terminal protective antigen binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor in Western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore-induced foreleg edema model. A combination of three of the MAb also significantly delayed the time to death in a third subcutaneous spore challenge model. This appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression.     

Stx2-specific human monoclonal antibodies protect mice against lethal infection with Escherichia coli expressing Stx2 variants

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for causing hemolytic-uremic syndrome (HUS), and systemic administration of Shiga toxin (Stx)-specific human monoclonal antibodies (HuMAbs) is considered a promising approach for prevention or treatment of the disease in children. The goal of the present study was to investigate the ability of Stx2-specific HuMAbs to protect against infections with STEC strains that produce Stx2 variants. Dose-response studies on five HuMAbs, using the mouse toxicity model, revealed that only the three directed against the A subunit were protective against Stx2 variants, and 5C12 was the most effective among the three tested. Two HuMAbs directed against the B subunit, while highly effective against Stx2, were ineffective against Stx2 variants. In a streptomycin-treated mouse model, parenteral administration of 5C12 significantly protected mice up to 48 h after oral bacterial challenge. We conclude that 5C12, reactive against the Stx2 A subunit, is an excellent candidate for immunotherapy against HUS and that antibodies directed against the A subunit of Stx2 have broad-spectrum activity that includes Stx2 variants, compared with those directed against the B subunit.     

Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-mug dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.     

Immunization against fatal experimental Klebsiella pneumoniae pneumonia.

The ability of serospecific anti-capsular polysaccharide (CPS) antibody to prevent fatal Klebsiella pneumoniae pneumonia was evaluated in a rat lung model. Rats were immunized intramuscularly with 100 micrograms of purified serotype 2 CPS and challenged intrabronchially 14 days later with a serotype 2 strain of K. pneumoniae. Vaccination engendered high levels of serum anti-CPS antibody which afforded significant protection (P less than 0.01) against fatal pneumonia. Immunization promoted clearance of the challenge bacteria from the lungs and prevented bacteremia. Histological examination of lung tissue from infected control animals showed pronounced inflammatory cellular infiltrate in the alveolar spaces, intra- and peribronchial inflammation, and tissue necrosis. In contrast, pathological changes noted in lungs from immunized animals were restricted to infrequent intra- and peribronchial involvement.     

Recognition specificity of monoclonal antibodies which protect mice against Salmonella typhimurium infection.

We used enzyme-linked immunosorbent assay (ELISA), competitive inhibition ELISA, flow cytometry and western immunoblots to study the antigenic specificity of two monoclonal antibodies (mAbs) raised against the cell surface antigens of Salmonella typhimurium. These mAbs (SH6.11 and WB60.4) protect CAF1 (Ity(r)) mice against endotoxemia and mouse typhoid. We found that SH6.11 and WB60.4 recognize Salmonella serogroup B-specific lipopolysaccharide O4 and O5 factors, respectively. These mAbs did not bind to Salmonella serotypes that belong to serogroup A, D1, E4, G2, or R and did not cross-react with other enteric and nonenteric bacterial species.     

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.     

Murine anti-cytomegalovirus monoclonal antibodies with autoreactivity.

Certain murine monoclonal antibodies (mAb) raised against structural proteins of mouse cytomegalovirus (MCMV) display distinct patterns of multiple organ-autoreactivity in addition to their viral specificities. We analysed the autoreactivity of five such mAb by immunoperoxidase histochemistry, western immunoblot and enzyme-linked immunosorbent assay (ELISA). Four mAb recognized cellular autoantigens in the salivary gland, lung, heart, liver, kidney, ileum, striated muscle and brain, as detected by immunoperoxidase histochemistry. However, the mAb showed different specificities for nuclear, cytoplasmic and surface membrane antigens on various cell types in addition to common autoreactivities. Immunoblot analyses showed that some of the mAb recognized polypeptides of various molecular weights obtained from 100,000 g supernatants of normal BALB/c liver, brain, striated and cardiac muscle homogenates. Reactivity of the mAb with a 200,000 molecular weight (MW) polypeptide was similar to our previous finding of the reaction of late immune polyclonal sera with a 200,000 MW polypeptide, the heavy chain of myosin. The mAb reacted to the cardiac isoform of myosin as determined by ELISA and immunoblot. Reactivity of mAb with cardiac myosin, as detected by immunoblot, was removed by absorption with cardiac myosin and recovered in the eluate. However, cardiac myosin used in a competitive inhibition ELISA did not abrogate the reactivity of the mAb with MCMV antigens. These anti-MCMV mAb appear to be multispecific for both virus and self-antigens, including cardiac myosin, and possibly recognize these different antigens through partly similar or distinct antigen-binding sites.     

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.     

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.     

Binding to and opsonophagocytic activity of O-antigen-specific monoclonal antibodies against encapsulated and nonencapsulated Klebsiella pneumoniae serotype O1 strains

The high mortality of nosocomial infections caused by Klebsiella spp. has acted as a stimulus to develop immunotherapeutic approaches targeted against surface molecules of these bacteria. Since O-antigen-specific antibodies may add to the protective effect of K antisera, we tested the functional and binding capacity of O-antigen-specific monoclonal antibodies (MAbs) raised against different Klebsiella O antigens. The MAbs tested were specific for the O-polysaccharide partial antigens D-galactan II (MAb Ru-O1), D-galactan I (MAb IV/4-5), or core oligosaccharide (MAb V/9-5) of the Klebsiella serogroup O1 antigen. In enzyme-linked immunosorbent assay binding experiments, we found that all MAbs recognized their epitopes on intact capsule-free bacteria; however, binding to encapsulated wild-type strains belonging to different K-antigen serotypes was significantly reduced. The K2 antigen acted as the strongest penetration barrier, while the K7 and K21 antigens allowed some, though diminished, antibody binding. In vitro phagocytic killing experiments showed that MAb Ru-O1 possessed significant opsonizing activity for nonencapsulated O1 serogroup strains and also, to a much lesser extent, for encapsulated strains belonging to the O1:K7 and O1:K21 serotypes. MAbs or antisera specific for the D-galactan II antigen may thus be the most promising agents for further efforts to develop a second-generation Klebsiella hyperimmune globulin comprising both K- and O-antigen specificities.     

Lactoferrin can protect mice against a lethal dose of Escherichia coli in experimental infection in vivo.

Experiments were undertaken to demonstrate and partially explain the protective effect of bovine lactoferrin (LB) when administered intravenously to mice 24 h before a challenge with a lethal dose of Escherichia coli. About 70% of mice pretreated with LB survived challenge. The survival rates in control mice treated with E. coli alone and pretreated with bovine serum albumin (BSA), were 4 and 8%, respectively. Human lactoferrin (LH) had almost the same protective effect as LB. Sufficient amounts of ferric ions were given to mice, in single and multiple doses, for full serum transferrin saturation 30 min before or after E. coli administration. The multiple dose of ferric ions did not change considerably the survival rate of mice pretreated with LB. In contrast, a single dose of ferric ions gradually decreased the survival rate of the mice after the first week of experiment. From day 14 this decrease was statistically significant in all groups of mice treated with a single dose of ferric ions when compared with mice pretreated only with LB, and the difference ranged from 25 to 35% on day 30. The possible mechanism(s) of protective effect of LB and role of iron ions are discussed.     

Rabbit antibodies to the cell wall polysaccharide of Streptococcus pneumoniae fail to protect mice from lethal challenge with encapsulated pneumococci.

A conjugate, composed of the cell wall polysaccharide (C polysaccharide) of Streptococcus pneumoniae and bovine serum albumin (BSA), was prepared with the bifunctional agent N-succinimidyl-3-(2-pyridyldithio)-propionate. Analysis with monoclonal antibodies provided evidence that the phosphocholine (PC) moiety of the C polysaccharide was retained during the conjugation procedure. The C polysaccharide-BSA conjugate elicited antibodies to C polysaccharide in rabbits; no PC-specific antibodies were detected in globulins prepared from these hyperimmune sera obtained early and late after a second immunization. Rabbit hyperimmune sera were taken after multiple intravenous injections of the pneumococcus strain SRC-2, which has a capsulelike structure composed of the C polysaccharide. Globulin prepared from these antisera had both C polysaccharide- and PC-specific antibodies. Antibodies to C polysaccharide elicited by the C polysaccharide-BSA conjugate failed to protect mice against intraperitoneal challenge with a strain of type 3 or type 6A pneumococci. The anti-SRC-2 globulin conferred protection against both of these pneumococcal strains. Absorption of the SRC-2 globulin with C polysaccharide, however, failed to change its protective activity. These data provide evidence that antibodies to the C polysaccharide do not confer immunity against infection of mice with encapsulated pneumococci inoculated by the intraperitoneal route.     

Protection of neonatal macaques against experimental SHIV infection by human neutralizing monoclonal antibodies.

Neonatal macaques were completely protected against oral challenge with SHIV-vpu+, a simian-human immunodeficiency virus that encodes the envelope gene of a laboratory-adapted HIV strain, by pre- and post-natal treatment with a triple combination of human neutralizing monoclonal antibodies (mAbs). The mAbs were directed either against the CD4 binding site, a glycosylation-dependent gp120 epitope, or against a linear epitope on gp41. This triple combination was highly synergistic in vitro and neutralized primary HIV completely. Subsequently, oral challenge was performed with pathogenic SHIV89.6P, an animal-passaged variant of a chimeric virus that encodes the envelope gene of the primary, dual-tropic HIV89.6. Only post-natal treatment with a similar triple mAb combination was used. One out of 4 mAb-treated infants was completely protected from infection. In the other 3 treated animals, there was a tendency towards lower peak viral RNA loads compared with untreated controls. Two out of 4 mAb-treated infants maintained normal CD4+ T-cell numbers, in contrast to all controls that had steep declines at 2 weeks post-challenge. We conclude that the triple mAb combination significantly protected the neonates, even against mucosal challenge with pathogenic SHIV89.6P. Passively administered synergistic human mAbs may play a role in preventing mother-infant transmission of HIV, both against intrapartum transmission as well as against infection through breast milk. As passive immunization is a tool to assess correlates of immune protection, we conclude that the epitopes recognized by the mAbs in our combinations are important for AIDS vaccine development. Future passive immunization studies may reveal other important conserved epitopes.     

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.     

Interleukin-17 and lung host defense against Klebsiella pneumoniae infection

Bacterial pneumonia remains an important cause of morbidity and mortality worldwide, especially in immune-compromised patients. Cytokines and chemokines are critical molecules expressed in response to invading pathogens and are necessary for normal lung bacterial host defenses. Here we show that interleukin (IL)-17, a novel cytokine produced largely by CD4+ T cells, is produced in a compartmentalized fashion in the lung after challenge with Klebsiella pneumoniae. Moreover, overexpression of IL-17 in the pulmonary compartment using a recombinant adenovirus encoding murine IL-17 (AdIL-17) resulted in the local induction of tumor necrosis factor-alpha, IL-1beta, macrophage inflammatory protein-2, and granulocyte colony-stimulating factor (G-CSF); augmented polymorphonuclear leukocyte recruitment; and enhanced bacterial clearance and survival after challenge with K. pneumoniae. However, simultaneous treatment with AdIL-17 provided no survival benefit after intranasal K. pneumoniae challenge. These data show that IL-17 may have a role in priming for enhanced chemokine and G-CSF production in the context of lung infection and that optimally timed gene therapy with IL-17 may augment host defense against bacterial pneumonia.     

Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin.

The lethal toxin of Bacillus anthracis consists of two components, protective antigen and lethal factor. Protective antigen is cleaved after binding to cell receptors, yielding a receptor-bound fragment that binds lethal factor. Sixty-one monoclonal antibodies to the lethal factor protein have been characterized for specificity, antibody subtype, and ability to neutralize lethal toxin. Three monoclonal antibodies (10G3, 2E7, and 3F6) neutralized lethal toxin in Fisher 344 rats. However, in a macrophage cytolysis assay, monoclonal antibodies 10G3, 2E7, 10G4, 10D4, 13D10, and 1D8, but not 3F6, were found to neutralize lethal toxin. Binding studies showed that five of the monoclonal antibodies that neutralized lethal toxin in the macrophage assay (10G3, 2E7, 10G4, 10D4, and 13D10) did so by inhibiting the binding of lethal factor to the protective antigen fragment bound to cells. Monoclonal antibody 1D8, which was also able to neutralize lethal toxin activity after lethal factor was prebound to cell-bound protective antigen, only partially inhibited binding of lethal factor to protective antigen. Monoclonal antibody 3F6 did not inhibit the binding of lethal factor to protective antigen. A competitive-binding enzyme-linked immunosorbent assay showed that at least four different antigenic regions on lethal factor were recognized by these seven neutralizing hybridomas. The anomalous behavior of 3F6 suggests that it may induce a conformational change in lethal factor. Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.     

Monoclonal antibody against Klebsiella capsular polysaccharide reduces severity and hematogenic spread of experimental Klebsiella pneumoniae pneumonia.

Klebsiella pneumoniae is an important nosocomial pathogen causing severe pulmonary infections. The majority of clinical Klebsiella isolates produce a high-molecular-weight capsular polysaccharide (CPS) which is one of the dominant virulence factors. In the present study, we examined the potency of a murine immunoglobulin M monoclonal antibody (MAb) with specificity to Klebsiella type 2 CPS to protect rats against experimental Klebsiella pneumonia. The MAb did not prevent the invasion of virulent bacteria into the interalveolar space. However, the resolution of infection was accelerated in MAb-treated animals. This was demonstrated by (i) less severe weight loss and (ii) markedly reduced inflammatory reactions in the lung. The elimination of bacteria was significantly increased not only in the lungs but also in the livers of antibody-treated rats. This was reflected by reduced levels of circulating, soluble CPS and MAb-bound CPS. A mixture of human MAbs with specificity to CPS of clinically important Klebsiella serotypes may prove to be a useful tool for the prevention or supportive treatment of Klebsiella pneumonia.     

Lactoferrin can protect mice against a lethal dose of Escherichia coli in experimental infection in vivo

Experiments were undertaken to demonstrate and partially explain the protective effect of bovine lactoferrin (LB) when administered intravenously to mice 24 h before a challenge with a lethal dose of Escherichia coli. About 70% of mice pretreated with LB survived challenge. The survival rates in control mice treated with E. coli alone and pretreated with bovine serum albumin (BSA), were 4 and 8%, respectively. Human lactoferrin (LH) had almost the same protective effect as LB. Sufficient amounts of ferric ions were given to mice, in single and multiple doses, for full serum transferrin saturation 30 min before or after E. coli administration. The multiple dose of ferric ions did not change considerably the survival rate of mice pretreated with LB. In contrast, a single dose of ferric ions gradually decreased the survival rate of the mice after the first week of experiment. From day 14 this decrease was statistically significant in all groups of mice treated with a single dose of ferric ions when compared with mice pretreated only with LB, and the difference ranged from 25 to 35% on day 30. The possible mechanism(s) of protective effect of LB and role of iron ions are discussed.     

Enhanced protection by use of a combination of anticapsule and antilipopolysaccharide monoclonal antibodies against lethal Escherichia coli O18K5 infection of mice.

To study antibody-mediated protection against Escherichia coli peritonitis in BALB/c mice, monoclonal antibodies (MAbs) were generated against the capsule (K5) and the lipopolysaccharide (O18) of E. coli. Flow cytometric analysis with two selected immunoglobulin M MAbs revealed that bacteria were antigenically heterogeneous. Arbitrarily, three subpopulations in E. coli O18K5 cultures could be distinguished by double immunofluorescence. A subpopulation bound only the anti-K5 MAb, and another subpopulation bound only the anti-O18 MAb. An intermediate subpopulation, however, bound both MAbs. In agreement with this result, combinations of both MAbs enhanced phagocytosis of fluorescein isothiocyanate-labeled bacteria by human polymorphonuclear leukocytes and mouse macrophage J774 cells as well. In protection experiments, combinations of both MAbs, preincubated with 3 50% lethal doses of E. coli O18K5, protected all mice upon intraperitoneal challenge. Relatively high doses of either MAb alone proved to be not fully protective in this infection model. Protection of mice by the combination of MAbs was associated with significantly lower (P < 0.02) tumor necrosis factor levels in serum 90 min after challenge compared with any other treatment group. Similarly, prophylactic administration of MAbs yielded significantly lower (P < 0.01) tumor necrosis factor levels in mice that received the combination of MAbs than in any other treatment group.