Effects of monoclonal antibodies on alpha-staphylotoxin action against erythrocytes and model phospholipid membranes.

It was found that one of twenty tested monoclonal antibodies (MABs) existed which drastically enhanced ability of Staphylococcus aureus alpha-toxin (ST) to both lysis of human erythrocytes and increase of planar phospholipid bilayer conductance more than 10 and 1,000 times respectively. Other 19 MABs possessed only neutralized effect. The activation could only be observed if the activating MAB (AMAB) interacted with ST in solution but not in membrane. The one molecule of AMAB was able to activate approximately 2-4 molecules of ST. It was assumed that this activation was a result of the AMAB-induced transition of ST from a hydrophilic to an amphiphilic form. The activation could not be observed when the activity of AMAB/ST mixtures was tested on highly sensitive rabbit erythrocytes. All the tested MABs (including AMAB) were able to inhibit the ST-induced lysis of rabbit erythrocytes. The activating effects of AMAB on ST action in BLM and in human erythrocytes as well as their inhibiting influence on the ability of toxin to cause a lysis of rabbit erythrocytes indicate the presence of an ST-specific receptor on the membrane of rabbit erythrocytes.

Immunopathogenesis of atherosclerosis: endotoxin accelerates atherosclerosis in rabbits on hypercholesterolemic diet.

BACKGROUND: On the basis of our concept that atherosclerosis has an immunopathological background, we tested whether activation of the innate immune system influences its progression. METHODS AND RESULTS: Hypercholesterolemic (0.5% wt/wt diet) rabbits received either repeated intravenous injections of endotoxin (Escherichia coli lipopolysaccharide 1.25 to 2.5 microg, once per week) or a self-limiting cutaneous Staphylococcus aureus infection with or without a quinolone antibiotic. Measured laboratory parameters, including LDL and HDL cholesterols, were similar in the different groups of hypercholesterolemic animals. All endotoxin-treated animals developed transient episodes of fever after endotoxin administration. The extent of atherosclerosis was evaluated by computer-assisted morphometry in the aortas en face (Sudan IV) and by histology at 8 weeks after start of the experiments. Endotoxin-treated animals exhibited significantly accelerated atherosclerosis compared with control animals (141+/-38 versus 45+/-16 mm(3) total lesion volume, n=7 to 9 rabbits each, P<0.001). CONCLUSIONS: Nonspecific stimulation of the innate immune system accelerates cholesterol-induced atherosclerosis. These data support the concept that atherosclerosis has an immunopathological component and render it improbable that a single infectious agent should assume particular importance in its initiation or progression.

Humoral antibody responses in periodontal disease.

Several forms of periodontal disease are considered to be infectious diseases with associated specific bacteria. This study examined the humoral antibody levels as assayed by ELISA to Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in adult periodontitis (AP), localized juvenile periodontitis (LJP), rapidly progressive periodontitis (RPP), and in periodontally healthy subjects (HS). Sixty-two of the 64 (96.9%) patients had significantly elevated antibody levels to at least one of the three organisms. Elevated levels of antibodies to P. gingivalis occurred in 82.8% of the RPP, LJP, and AP patients with all 3 disease groups showing greater responses than HS controls. Antibodies to A. actinomycetemcomitans were found in 59.4% of the RPP, LJP, and AP patients and were significantly higher in both LJP and RPP patients. Only 21.9% of the RPP, LJP, and AP patients showed elevated levels to P. intermedia with only significantly higher levels in the RPP and LJP groups. Antibodies to A. actinomycetemcomitans and P. intermedia were rarely found alone (only 5.1% and 2.6% of the patients respectively) but were usually accompanied by P. gingivalis. These results suggest that one or more combinations of these 3 bacteria may play a role in the pathogenesis of these forms of periodontal disease.

Characterization of neutralizing monoclonal antibodies directed against Staphylococcus aureus alpha-toxin.

A panel of neutralizing murine monoclonal antibodies (MAbs) against Staphylococcus aureus alpha-toxin has been established, using formaline-inactivated alpha-toxin as an immunogen. Five independent groups of neutralizing epitopes have been identified representing five functionally important structures in the toxin molecule. Because none of the antibodies binds to overlapping decapeptides representing the toxin sequence or to bromocyanogen cleavage products of alpha-toxin, they may all bind to conformational epitopes. Nevertheless, they all bind to monomeric alpha-toxin in a Western blot. Three of the antibodies bind to the toxin monomer in an enzyme-linked immunosorbent assay (ELISA) in the presence, but not in the absence, of detergent. These epitopes are not accessible in hexameric toxin; two of them may represent the contact sites of the toxin monomers upon hexamerization and one is related to a structurally important glycine-rich central hinge region. Two different antibodies bind to monomeric toxin in an ELISA in the presence and absence of detergent and their epitopes are present more than once on oligomeric toxin; they bind strongly to hexameric toxin in a Western blot. The binding properties of the antibodies to alpha-toxin in different assay systems are summarized in an epitope model, which describes the presence of neutralizing domains in the different conformational steps required for pore formation.

[In vitro and in vivo efficacy of a toxin-neutralizing human staphylococcal immunoglobulin]. / In vitro- und In vivo-Wirksamkeit eines Toxin neutralisierenden humanen Staphylokokken-Immunglobulins.

The pathogenic relevance of alpha-hemolysin of Staph. aureus in human infections is up to the present in discussion. Numerous therapeutic human trials to modify the outcome of a Staph. aureus infection by giving passively hyperimmune sera did not clarify the situation. First the work of Bhakdi22 has provided us with a rational approach for a systemic use of specific immunoglobulin preparations. A highly purified Staph. aureus alpha-hemolysin was carefully detoxified and injected into volunteers. From these sera were collected by plasmapheresis and a 5S immunoglobulin was prepared. In preclinical trials the antitoxin efficacy was evaluated. We examined in vitro cultures of human thrombocytes and monocytes, and of porcine pulmonary arterial endothelial cells. As in vivo models we used the intoxication of mice and monkeys and an intradermal test in rabbits. In all these models we were able to demonstrate the high antitoxic efficacy of a human anti-alpha-hemolysin-5S-immunoglobulin preparation.

Modern trends in the development of bacterial vaccines for human use.

The development of vaccines against infectious diseases is one of the great medical achievements of the 20th century. What other medical measure had such an influence on the statistical prolongation of life, as the use of vaccines did? The successful development of today's vaccines was from the beginning due to the intensive cooperation between the basic research sciences (e.g. bacteriology virology, chemistry, immunology) and the applied sciences (e.g. medicine-practiced). During the last few years newly developed techniques have awakened hopes for the development of new vaccines which are better tolerable and more effective. Molecular biologic methods have shown first successes in the development of epitope vaccines. Monoclonal anti-idiotype antibodies are being used in active protection experiments. Modern galenic preparations can perhaps one day replace the parenteral administration of antigens. In the following some of the new techniques are listed and their significance for the development of new vaccines discussed.

A new polyvalent pseudomonas vaccine.

A new polyvalent Pseudomonas aeruginosa vaccine consisting of extracts of the 24 serotypes of the Lanyi typing scheme was developed and evaluated in mouse protection studies. The vaccine, mainly consisting of lipopolysaccharides and outer membrane proteins was protective in passive and active protection studies. A rabbit antiserum against the vaccine showed high protective capacity in the IgG-fraction and was protective against 3 out of 6 challenge strains in a leucopenic mouse model.

Survey on experiences with latex-Chagas-test in various countries.

The principle of the latex agglutination technique was applied on the serodiagnosis of Chagas' disease. The latex particles coated with antigen from Trypanosoma cruzi are agglutinated by antibodies against T. cruzi in the serum of patients suffering thereof. In 11600 comparative determinations worked out mainly in a number of South American laboratories, the sensitivity of this test was compared with xenodiagnosis, CF, IFT and IHA. The latex Chagas test shows a mean coincidence of 90% with xenodiagnosis, 88% with CF, 75% with IFT, and 83% with IHA. The results indicate that the test is a useful new tool for the serodiagnosis of Chagas' disease. Also the data from CF, IFT and IHA were compared with each other. The latex Chagas test is sensitive even in the early stage of the disease. Also the specificity of the test was evaluated in other parasitic, bacterial and general diseases.

Histopathology of experimental Chagas disease in NMRI-mice. A long term study following paw infection.

NMRI-mice were infected subcutaneously with T. cruzi Brazil and the pathohistological findings in 17 organs at different times after infection are described. The paw was chosen as injection site, for, due to the great variety of tissues found in a single histological section, the first reactions between host and parasite are best studied here. For this purpose the complete paw was embedded in plastic and sectioned longitudinally. After infection with 5 X 10(4) parasites a shift of parasitism from the initial host cells (macrophages) to the neighbouring muscle cells was observed. This occurred on the 9th day after infection. On the 18th day a generalized parasitism of organs containing muscle had developed, especially of myocardium and alimentary tract. Destruction and inflammation of heart muscle was the most important consequence with respect to mortality. The autonomous nervous system of heart and alimentary tract was involved, although not selectively. Liver and spleen played no role as host tissues. After infection with 10(2) or 10(3) parasites specific alterations in the animals surviving the acute phase were not observed from the 60th to 201st day. A perivenous encephalitis without cerebral parasitism was found in about one half of the experimental animals. It does not correspond to the known 'Chagas encephalitis' and is for the present defined as parainfectious.

Tetanus toxoid loaded nanoparticles from sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide): evaluation of antibody response after oral and nasal application in mice.

PURPOSE: Aim of the study was the evaluation of the potential of novel tetanus toxoid (TT) loaded nanoparticles (NP) for electing an immune response in mice against TT. METHODS: Six week-old female Balb/c mice were immunized by oral (p.o.), nasal (i.n.) and intraperitoneal (i.p.) application of TT NP loaded by adsorption. As polymer a novel polyester, sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide), SB(43)-PVAL-g-PLGA was used. Blood samples were collected 4 and 6 weeks after immunization and assayed for serum IgG- as well as IgA antibody titers by ELISA. NP formulations varying in size and loading were compared to alum adsorbates as well as to TT solutions. RESULTS: Both, p.o. and i.n. administration of TT associated NP increased serum titers up to 3 x 10(3) (IgG) and 2 x 10(3) (IgA). While small NP induced significantly higher titers then larger ones after oral administration, intermediate NP induced antibodies after nasal application. Of the mucosal routes investigated, i.n. seems to be more promising compared to p.o. immunization. CONCLUSIONS: Antigen loaded NP prepared from surface modified polyesters combined with CT show considerable potential as a vaccine delivery system for mucosal immunization. The results warrant further experiments to explore in more detail the potential use of NP as mucosal vaccine delivery system.

Microencapsulation of antigens using biodegradable polyesters: facts and phantasies.

New vaccination approaches and new delivery systems have been subject of intensive research activities recently. Controlled release vaccine delivery systems depend on the microencapsulation of antigens into biodegradable polymers, yielding small spherical polymeric particles, in the size range of 1-100 microns. By manipulating the micromorphology of the microparticles and degradation properties of the polymer either continuous or pulsatile release patterns can be adjusted. As biodegradable polymers mainly copolymers of lactic- and glycolic acid have been utilized, since these materials are known to be biocompatible and non-toxic. Apart from modulation of antigen release, an improvement of the adjuvant effect and an increase of in vitro (shelf-life) and in vivo stability of the antigen are issues of general interest with respect to parenteral vaccine delivery systems. Using different microparticles that release antigens in a pulsatile pattern at predetermined timepoints one hopes to induce protective immunity by a single administration of the vaccine delivery system. Using tetanus toxoid (TT) as a model antigen we have examined the stability during preparation, in vitro release and storage of TT microparticles. TT is a complex protein mixture sensitive to changes in pH conditions (pH < 5) and to thermal stress. TT microparticles can be prepared by a W/O/W double emulsion technique with satisfactory encapsulation efficiencies in good yields. In accordance with other investigators we observe an adjuvant effect of TT microspheres in mice upon sc administration leading to a long-lasting antibody response. In challenge experiments we could demonstrate a protective effect. The issue of an ideal release pattern remains open, since a boosting of the antibody titers during the bioerosion of the TT microspheres was not observed, possibly due to desactivation of TT in the degrading microspheres.

Functionally inactive S. aureus alpha-toxin containing a single amino acid substitution: potential usefulness as a vaccine.

Substitution of His-35 with arginine in staphylococcal alpha-toxin leads to loss of its membrane perturbating properties in vitro. In this study, we report that the inactive mutant toxin is also devoid of toxic properties when injected intravenously into rabbits. Whereas a single application of native toxin at a dose of 20 micrograms/kg proved uniformly lethal within 3 hours (n = 3), all animals receiving 20, 100 or 200 micrograms/kg mutant toxin (n = 3 for each group) remained in unaltered, healthy states over a 15 day period of observation. Furthermore, all animals receiving inactive toxin developed antibody titers on day 10-15. No dose dependency was noted in the tested range. Thus, the site-directed alpha-toxin mutant (His-35:Arg) is an excellent vaccine candidate.

A human monoclonal antibody with the capacity to neutralize Staphylococcus aureus alpha-toxin.

A human monoclonal antibody, CB STL-1, against staphylococcal alpha-toxin has been established by hybridoma technology. It is of IgG1 subclass with lambda light chain and possesses a dissociation constant of 8 x 10(-10) mol/l. 1 mg of purified antibody neutralizes the hemolytic activity of 800 micrograms/ml alpha-toxin in an in vitro hemolysis assay using rabbit erythrocytes. The antibody does not bind to overlapping (7 residues) decapeptides spanning the sequence of alpha toxin, thus it might bind to a conformational epitope. The epitope recognized by the antibody is not accessible in oligomeric toxin. The antibody binds both to the hydrophilic and amphipathic forms of the monomeric toxin Fab fragments of the antibody are stable and show no significant loss of activity. CB STL-1 was able to protect mice in vivo from i.p. challenge with alpha toxin. Thus, the antibody is a candidate for passive immunotherapy. The variable regions of the antibody secreted by CB STL-1 were sequenced and found to be encoded by a VH gene segment belonging to the VH1 family, and a Vlambda segment most likely belonging to the VlambdaIII subgroup. Further analysis concerning the third complementarity determining region (CDR3) of the heavy chain is presented.

Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo.

Alpha-toxin, the major cytolysin of Staphylococcus aureus, preferentially attacks human platelets and cultured monocytes, thereby promoting coagulation and the release of interleukin-1 and tumor necrosis factor. Titers of naturally occurring antibodies in human blood are not high enough to substantially inhibit these pathological reactions. In the present study, F(ab')2 fragment preparations from hyperimmune globulin obtained from immunized volunteers were tested for their capacity to inhibit the cytotoxic action of alpha-toxin in vitro and in vivo. These antibody preparations exhibited neutralizing anti-alpha-toxin titers of 80 to 120 IU/ml, whereas titers in commercial immunoglobulin preparations were 1 to 4 IU/ml. In vitro, the presence of 2 to 4 mg of hyperimmune globulin per ml protected human platelets against the action of 1 to 2 micrograms of alpha-toxin per ml. Similarly, these antibodies fully protected human monocytes against the ATP-depleting and cytokine-liberating effects of 0.1 to 1 microgram of alpha-toxin per ml. Intravenous application of 0.5 mg (85 to 120 micrograms/kg of body weight) of alpha-toxin in cynomolgus monkeys elicited acute pathophysiological reactions which were heralded by a selective drop in blood platelet counts. Toxin doses of 1 to 2 mg (170 to 425 micrograms/kg) had a rapid lethal effect, the animals presenting with signs of cardiovascular collapse and pulmonary edema. Prior intravenous application of 4 ml of hyperimmune globulins per kg inhibited the systemic toxic and lethal effects of 1 mg (200 micrograms/kg) of alpha-toxin. In contrast, normal human immunoglobulins exhibited no substantial protective efficacy in vitro and only marginal effects in vivo. It is concluded that high-titered anti-alpha-toxin antibodies effectively protect against the cytotoxic actions of alpha-toxin.

Safety and immunogenicity of a Pseudomonas aeruginosa outer membrane protein I vaccine in human volunteers.

The outer membrane protein I (OprI) of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni2+ chelate-affinity chromatography. After safety and pyrogenicity evaluation in animals, four groups of seven adult human volunteers were vaccinated three times at four week intervals with either 500 micrograms, 200 micrograms, 50 micrograms or 20 micrograms of OprI adsorbed onto Al(OH)3. All vaccinations were well tolerated and no systemic side effects were detected. A significant rise of antibody titers against OprI could be measured in the serum of all volunteers who had received the 500 micrograms, 200 micrograms or 50 micrograms doses. Elevated antibody titers against OprI could still be measured 30 weeks after the final vaccination. Binding of the complement component C1q to the elicited antibodies could be demonstrated, showing the ability of the latter to promote antibody-mediated complement-dependent opsonization.

The Pseudomonas aeruginosa outer membrane protein I vaccine: immunogenicity and safe administration in man.

After expression in Escherichia coli and purification by Ni++ chelate-affinity chromatography, the outer membrane protein I (OprI) of Pseudomonas aeruginosa was tested in experimental animals for its safety and pyrogenicity. Four groups of 7 adult human volunteers were then vaccinated 3 times at four-weekly intervals with either 500 micrograms, 200 micrograms, 50 micrograms or 20 micrograms of OprI adsorbed onto aluminum hydroxide. The vaccinations were well tolerated and without systemic side effects, but a significant rise of antibody titers against OprI was measured in the serum of those who had received the 500 micrograms, 200 micrograms or 50 micrograms doses. Raised antibody titers against OprI were still present 30 weeks after the final vaccination. It was possible to demonstrate binding of the complement component C1q to the elicited antibodies, and this confirms their ability to promote antibody-mediated complement-dependent opsonization.

In vivo effects of intravascularly applied Escherichia coli hemolysin: dissociation between induction of granulocytopenia and lethality in monkeys.

The effects of intravascular application of endotoxin-depleted Escherichia coli hemolysin (HlyA) was studied in rabbits and monkeys. In rabbits, bolus application of HlyA calculated to effect final blood levels of approximately 2-3 HU/ml (200-300 ng/ml) caused an acute fall of polymorphonuclear blood leukocytes to less than 20% of starting levels within 5 min. Additionally, platelet counts dropped to approximately 30% of starting levels, whereas lymphocyte counts varied considerably and seldom fell to less than 50%. Nine out ten animals that received 2-4 HU/ml toxin died within 90 min post application. These animals presented with signs of acute respiratory failure and post mortem inspection of the internal organs revealed hemorrhagic pulmonary edema. Other internal organs appeared unaffected. Application of less than 1 HU/ml HlyA was never fatal (n = 9), and only transient leukopenia was noted. Monkeys presented with a remarkable and different response. Two animals were repeatedly given HlyA at high doses ranging from 3 to 10 HU/ml. Both animals developed selective granulocytopenia, but following a short, transient drop in blood pressure they showed no severe clinical signs of cardiovascular or pulmonary malfunction. Histological examinations revealed accumulation of polymorphonuclear granulocytes in both animals in liver, lung and spleen. Very high leukocyte elastase levels were measured in one animal over a period of 1.5 h. The present results demonstrate a remarkable tolerance of monkeys towards the leukocidal effects of E. coli hemolysin. Lethality in rabbits must be due to additional effects of the toxin, possibly on cells in the pulmonary vasculature. Neither pulmonary sequestration of granulocytes nor massive release of elastase from these cells is in itself sufficient to provoke pulmonary dysfunction in monkeys.