The effect of the synergistic action of enterotoxin on the specific protective complex of Shigella sonnei]

The study has first established that enterotoxin enhances the protective potency of S. sonnei specific protective complex. This effect has been revealed both in experiments of the oral immunization of mice and in experiments of the conjunctival immunization of guinea pigs and depends on the dose of enterotoxin used in the experiment. The increase of protection has a specific character. These observations open prospects for further enhancement for the protective properties of S. sonnei specific protective complex, which should be taken into consideration in developing the vaccinal preparation.     

The protective activity of the detoxified lipopolysaccharide of Neisseria meningitidis serogroup A in in vivo experiments

The immunogenic potency, toxicity, homologous and heterologous protective activity of lipopolysaccharide preparations obtained from serogroup A N. meningitidis (LPS A) were studied in animal experiments. These preparations were shown to possess very high protective activity. The alkaline treatment of native LPS A decreased the toxicity of the preparation almost 20 times and did not affect its immunogenic potency. Detoxified LPS A was capable of protecting mice from fatal meningococcemia resulting from infection with N. meningitidis strains, serogroups A, B and C; the adsorption of the preparation on aluminium hydroxide did not affect its protective activity. In view of the properties of detoxified LPS A revealed in this investigation, it may be regarded as a possible vaccinal preparation.     

Human monoclonal antibodies to Pseudomonas aeruginosa produced by EBV-transformed cells

Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD50) values were 0.5, 2.6, and 3.1 micrograms immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.     

Protective cross activity of the extracellular mucus of Pseudomonas aeruginosa

Extracellular slime was isolated from 15 P. aeruginosa typing strains of different O-serotypes (immunotypes). The isolated slime, partially purified by ethanol precipitation, was later referred to as crude slime. Glycolipoprotein was obtained from crude slime and lipopolysaccharide (LPS) was obtained from acetone-dried microbial cells by the method of aqueous-phenol extraction. All these antigenic preparations were studied in the active mouse cross-protection tests: immunized mice were challenged with 7 strains of different immunotypes, strain No. 170 019 or toxigenic strain PA-103. In experiments on mice the slime of different P. aeruginosa serotypes (immunotypes) was found to stimulate immunity to intraperitoneal infection with P. aeruginosa, both homologous or heterologous in respect to their immunotype, including toxigenic strains. Slime glycoprotein also stimulated active cross-immunity in mice, but the level of this immunity was higher than that of immunity stimulated by crude slime. LPS showed mostly weak protective activity in experiments on mice.     

The protective properties of the endotoxin protein

The isolation and properties of endotoxin protein, or lipid A-associated protein (LAP), from Shigella sonnei were described earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 11-17, and No. 7). In this report the data on its protective activity are presented. In experiments on mice one nanogram of LAP injected i. v. protected 50% of the animals against i. p. challenge with 40 LD50 of virulent S. sonnei. Guinea pigs injected s. c. with 10 micrograms of LAP were protected against local (keratoconjunctival) challenge with S. sonnei, the efficiency of immunization being 58%. LAP preparations containing no detectable amounts of O-antigen (less than 0.003%) were found to have a protective effect. Hyperimmune anti-LAP rabbit serum prevented local infection when incubated with S. sonnei challenge inoculum before injection into guinea pigs. Both active and passive protection induced by LAP was specific since no effect was observed in animals challenged with Shigella flexneri. In the homologous system the protective effect of anti-LAP serum was abolished by the addition of protein-free LPS. These results are compatible with the hypothesis that the protective activity of LAP depends on the presence of minute amounts of O-antigen whose immunogenic effect is greatly amplified by the protein component of the natural endotoxin complex.     

Heterogeneity and variation among Neisseria meningitidis lipopolysaccharides.

Eight immunotype lipopolysaccharides (LPSs) of Neisseria meningitidis were prepared by the phenol-water procedure and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sugar analyses. By SDS-PAGE and a highly sensitive silver strain. N. meningitidis LPSs from cells grown in tryptic soy broth were shown to contain one or two predominant components and a few minor, somewhat higher-molecular-weight components. The molecular sizes of the two predominant components were approximately the same as those of two E. coli rough-type LPSs, one with a complete core and the other with an incomplete core. The molecular weight of the major LPS component varied somewhat among different immunotypes but was estimated to be in the range of 4,200 to 5,000. By sugar analyses, the eight immunotype LPSs were different in their monosaccharide compositions. All contained glucose, galactose, heptose, glucosamine, and 2-keto-3-deoxyoctonate, but in different molar ratios. The growth of N. meningitidis in tryptic soy broth under different levels of aeration resulted in a change in the two major LPS components seen on the SDS-PAGE gel. High aeration increased the amount of the smaller component, whereas low aeration increased the amount of the larger component. Sugar analyses of LPSs from high and low aeration indicated that the larger LPS component contained more galactose residues per molecule. Use of different media for cell growth may also result in small, but noticeable, variations in the LPS components and in the galactose content of the LPS. The observed heterogeneity of N. meningitidis LPS may explain why many strains of N. meningitidis appear to possess more than one immunotype.     

Protective activity of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae

Protective efficacy of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae cultivated on cardiocerebral broth and semisynthetic growth medium respectively was studied in vivo. Fraction with molecular weight 30 - 50 kDa obtained by the method of membrane fractionation had high protective efficacy. Two-dose immunization of mice with this fraction provided 80 - 100% protection from infection by homologous strains of S. pneumoniae and K. pneumoniae. Cross-protective activity of the fraction was revealed when infecting immunized mice by different K-types of K. pneumoniae. Blood sera of mice immunized with 30 - 50 kDa fraction possessed preventive features protecting from infection 90% of animals while 100% of death in the control group. It was determined that protective efficacy of the mentioned fraction was determined by protein-containing antigens because proteolytic disruption of the protein component resulted in loss of protective properties of the preparation.     

Evaluation of TcpF-A2-CTB Chimera and Evidence of Additive Protective Efficacy of Immunizing with TcpF and CTB in the Suckling Mouse Model of Cholera

The secreted colonization factor, TcpF, which is produced by Vibrio cholerae 01 and 0139, has generated interest as a potential protective antigen in the development of a subunit vaccine against cholera. This study evaluated immunogenicity/protective efficacy of a TcpF holotoxin-like chimera (TcpF-A2-CTB) following intraperitoneal immunization compared to TcpF alone, a TcpF+CTB mixture, or CTB alone. Immunization with the TcpF-A2-CTB chimera elicited significantly greater amounts of anti-TcpF IgG than immunization with the other antigens (P<0.05). Protective efficacy was measured using 6-day-old pups reared from immunized dams and orogastrically challenged with a lethal dose of El Tor V. cholerae 01 Inaba strain N16961. Protection from death, and weight loss analysis at 24 and 48 hours post-infection demonstrated that immunization with TcpF alone was poorly protective. However, immunization with TcpF+CTB was highly protective and showed a trend toward greater protection than immunization with CTB alone (82% vs 64% survival). Immunization with the TcpF-A2-CTB chimera demonstrated less protection (50% survival) than immunization with the TcpF+CTB mixture. The TcpF-A2-CTB chimera used for this study contained the heterologous classical CTB variant whereas the El Tor CTB variant (expressed by the challenge strain) was used in the other immunization groups. For all immunization groups that received CTB, quantitative ELISA data demonstrated that the amounts of serum IgG directed against the homologous immunizing CTB antigen was statistically greater than the amount to the heterologous CTB antigen (P≤0.003). This finding provides a likely explanation for the poorer protection observed following immunization with the TcpF-A2-CTB chimera and the relatively high level of protection seen after immunization with homologous CTB alone. Though immunization with TcpF alone provided no protection, the additive protective effect when TcpF was combined with CTB demonstrates its possible value as a component of a multivalent subunit vaccine against Vibrio cholerae 01 and 0139.     

Cell-free Pseudomonas vaccine. III. Immunochemical analysis of purified Pseudomonas aeruginosa protein antigens and protective properties of antisera against these antigens

The immunochemical analysis of isolated and purified antigens A and B obtained from P. aeruginosa, strains 868 (serogroup O3 according to Lányi or immunotype 3/7 according to Fisher) and 170015 (serogroup O7 or immunotype 2), was carried out. Rabbit antisera to proteins A and B, as well as to the initial aqueous extracts and partially purified aqueous extracts, were obtained. Cross activity between the protein antigens of different strains was established by the methods of immunodiffusion and two-dimensional immunoelectrophoresis. Isolated proteins A and B contained both common and specific antigenic determinants detected by the method of two-dimensional immunoelectrophoresis. The immunization of rabbits with proteins A and B was found to stimulate the synthesis of protective, probably species-specific, antibodies.     

In vitro T cell-mediated killing of Pseudomonas aeruginosa. IV. Nonresponsiveness in polysaccharide-immunized BALB/c mice is attributable to vinblastine-sensitive suppressor T cells

We reported previously that BALB/c mice immunized with a polysaccharide (PS) antigen isolated from immunotype 1 Pseudomonas aeruginosa and vinblastine sulfate develop T cell-mediated protective immunity, despite their failure to produce specific antibody. In vitro, Lyt-1-,2+, I-J+ T cells from vinblastine- and PS-immunized mice kill P. aeruginosa by secretion of a bactericidal lymphokine. BALB/c mice immunized with PS alone generate neither protective antibodies nor a protective T cell response. The current studies indicate that T cells from mice immunized with PS alone significantly suppress the bactericidal activity of T cells from mice immunized with vinblastine and PS. The suppressor T cells are of the same Lyt-1-,2+, I-J+ phenotype as the bactericidal T cells. Suppression is mediated by a soluble product of these suppressor T cells which both inhibits T cell proliferation and interferes with the production or release of the bactericidal lymphokine. Cyclophosphamide, used in other systems to remove suppressor T cells, fails to enhance bacterial killing and does not inhibit suppressor cell activity. These studies indicate that immunization with PS elicits responses in two functionally distinct subgroups of Lyt-1-,2+, I-J+ T cells, and that these cells are distinguishable by their sensitivity to vinblastine sulfate.     

Single immunization with MF59-adjuvanted inactivated whole-virion H7N9 influenza vaccine provides early protection against H7N9 virus challenge in mice

H7N9 influenza infection in humans would result in severe respiratory illness. Vaccination is the best way to prevent influenza virus. In this paper, we investigated the effect of early protection provided by inactivated whole-virion H7N9 influenza vaccine in a mouse model.Mice were immunized intramuscularly once with different doses of inactivated whole-virion H7N9 influenza vaccine alone or in combination with MF59 adjuvant. Specific IgM and IgG antibody titers in sera of mice were detected by ELISA 3, 5 and 7days after immunization. To evaluate the early protection provided by the vaccine, mice were challenged with lethal dose (40LD₅₀) of homologous virus 3, 5 and 7 days after immunization respectively. The survival rate and body weight change of mice during 21 days after challenge and the residual lung virus titer on 3rd day after challenge were determined. The results demonstrated that mice could obtain effective protection 3 days after immunization with the vaccine at a high dose, and 5–7 days after immunization even at a low dose. Thus early immune responses induced by inactivated whole-virion H7N9 vaccine could provide effective protection.     

Protective properties of anatoxin obtained from a homogeneous preparation of Pseudomonas aeruginosa exotoxin A

The protective properties of formulated toxoid obtained from the highly purified preparation of P. aeruginosa exotoxin A have been studied in the test of the active immunization of mice. The study has revealed that the preparation when introduced in 1 or 2 injections in a dose of 15 micrograms, shows faint protective potency with respect to P. aeruginosa strains differing in virulence. Immunization with this toxoid in 3 and 4 injections has been found to ensure 60-100% and 50-60% protection of mice infected with P. aeruginosa toxigenic and proteolytic strains respectively. Immunization with toxoid has been found to induce the appearance of short-term antibacterial immunity which loses its capacity to protect the immunized animals, challenged with both toxigenic and proteolytic P. aeruginosa strains, as early as on day 28. The immunization of mice with toxoid in 4 injections has been shown to induce the development of antitoxic immunity capable of neutralizing up to 150 LD50 of purified exotoxin A.     

Anti-idiotype-induced, lipopolysaccharide-specific antibody response to Pseudomonas aeruginosa

Antibody directed to the O-specific polysaccharide (Ps) side chain of Pseudomonas aeruginosa LPS provides immunotype-specific protection against infection by virtue of enhancing opsonophagocytosis. We have developed a syngeneic anti-idiotypic antibody (mAb2) directed to a functionally active monoclonal immunotype 1 Ps-antibody (mAb1). The mAb2 performed as a molecular mimic of Ps as evidenced by 1) blocking of mAb1/mAb2 interaction by Ps, 2) blocking of mAb1/Ps binding by mAb2, 3) cross-species binding of mAb2 to human Ps antibodies from individuals immunized with the same immunotype 1 Ps, and 4) induction of anti-LPS antibody by immunization with mAb2 in syngeneic mice. Our studies thus show that an anti-idiotypic antibody may functionally mimic the O-polysaccharide of P. aeruginosa LPS, and bind to cross-reactive Id present in human Ps antibodies. We have further shown that this anti-idiotypic antibody induces anti-LPS antibody when used as an Ag in syngeneic mice, suggesting that this approach may eventually be used to successfully immunize humans.     

Characterization of the specificity of lipopolysaccharide antigens from seven Fisher's immunotypes of Pseudomonas aeruginosa, using ELISA technique

Lipopolysaccharides of seven Fisher's immunotypes of P. aeruginosa, extracted by water-phenol method, were fractionated on Sepharose 2B column. On the basis of molecular weight sugar and KDO content, eluents were separated into 4 fractions. An analysis of the antigenic specificity of the chromatographic fractions of seven immunotypes of LPS was carried out, using sera of mice vaccinated with several crude LPS preparations or whole-cell suspensions each of P. aeruginosa immunotypes, by ELISA. The antigenic specificity of fraction 1 and 2 of several immunotypes (with the exception of LPS from immunotype 2) in reaction with mice antisera for crude LPS was shown. Not quite full specificity of fractions 1-3 of all LPS preparations during analysis of these fractions reactivity with antisera to whole P. aeruginosa cells were observed, but specific reactions predominated in all test systems except LPS 2.     

Structure of the O-specific polysaccharides and the protective activity of the lipopolysaccharides in Pseudomonas aeruginosa of 7 immunotypes (after Fisher)

The structure of O-specific polysaccharides and the protective activity of lipopolysaccharides (LPS) obtained from seven P. aeruginosa immunotypes (according to Fisher's classification) have been studied. The structure of O-specific polysaccharides, immunotypes 2, 3, 4, 5 and 6, is identical to that of polysaccharides of serotypes 011; 0(2a), 2c; 01; 010a, 10b; 07a, 7d respectively. No structural analogs of O-polysaccharide characteristic of immunotypes 1 and 7 have been detected among serotypes classified according to the scheme of Lányi-Bergan-Akatova-Smirnova. The specific character of O-polysaccharides is confirmed by the results of the passive hemagglutination inhibition test, but the data of the passive hemagglutination test indicate that LPS of different immunotypes are antigenically related. As revealed in experiments on the active immunization of mice, LPS of seven immunotypes possess more or less pronounced cross protective properties. The causes of the cross protective activity observed in experiments with these LPS are discussed.     

Protective protein antigens of Pseudomonas aeruginosa

Partially purified water extract was obtained from the initial water extract by ultracentrifugation. Nine protein fractions differing in molecular weight, homogeneity and the content of lipopolysaccharide (LPS) were obtained by stepwise precipitation with ammonium sulfate and subsequent fractionation in columns packed with Sephadex G-100 and DEAE cellulose. Two protein fractions with a molecular weight of 30000 and 40000 daltons were practically free of LPS. These fractions were homogeneous as shown by analytical centrifugation and formed a single precipitation line with P. aeruginosa antiserum; both fractions were found to be antigenically identical. In the enzyme immunoassay these two fractions proved to be least active in comparison with the other protein fractions, but when used for the immunization of rabbits, they induced the formation of specific protective (for mice) antibodies. Both antisera were equally active in the experiments of the passive protection of mice. The isolated LPS-free proteins are supposed to be the proteins of the outer membrane of P. aeruginosa cell wall and have the properties of protective antigens.     

Human anti-Pseudomonas aeruginosa outer membrane proteins IgG cross-protective against infection with heterologous immunotype strains of P. aeruginosa.

In order to develop an effective means to treat and prevent Pseudomonas aeruginosa infections, we have purified P. aeruginosa outer membrane protein (Oprs)-specific human IgG antibody using a large-scale affinity column. In this study, we investigated the cross-protective activity of the purified anti-Oprs IgG against various immunotype strains of P. aeruginosa. The anti-Oprs IgG reacted with Oprs isolated from seven Fisher-Devlin immunotype strains of P. aeruginosa and was able to promote opsonophagocytic killing of all seven immunotype strains by human phagocytic cells. Administration of 500 microg anti-Oprs IgG to mice raised the LD50 of the P. aeruginosa strains by 8-250-fold, indicating the protective capacity against heterologous P. aeruginosa strains as well as homologous strains. In contrast, despite high titers against P. (aeruginosa Oprs, total serum IgG isolated from burn patient sera was no better than normal serum IgG in protecting mice from infection with P. aeruginosa. These data demonstrate that the affinity-purified human anti-Oprs IgG could afford protection against heterologous immunotype P. aeruginosa strains and provide a rationale to use anti-Oprs IgG as an adjunct for treatment of P. aeruginosa infections in humans.     

The protective activity of polyclonal and monoclonal antibodies to the lipopolysaccharide of Neisseria meningitidis serogroup A in in vivo experiments

The protective activity of the sera of mice immunized with the preparations of native and detoxified N. meningitidis lipopolysaccharide (LPS), group A, as well as with monoclonal antibodies to N. meningitidis antigens, groups A and B, was studied on the mucin model of meningococcal infection. The study showed that the maximum level of anti-LPS antibodies in mice was observed on day 7 after the injection of LPS. Immune sera obtained from mice were capable of protecting the animals from fetal meningococcemia induced by N. meningitidis strains of homologous and heterologous groups. As shown by the results of this study, the alkaline treatment of N. meningitidis native LPS did not decrease the protective properties of antibodies. The monoclonal antibodies under study were found to possess high preventive activity in mice challenged with N. meningitidis, groups A and B. Anti-LPS monoclonal antibodies showed greater protective activity than antipolysaccharide monoclonal antibodies.     

Immunocycte stimulation in vitro by nontoxic bacterial lipopolysaccharide derivatives.

Intact lipopolysaccharides (LPS), considered nonspecific enhancers of B cell responses, as well as nontoxic derivatives from Serratia marcescens LPS, were studied with regard to their ability to stimulate in vitro immune responses to a T-dependent antigen, sheep erythrocytes. Intact LPS, at a dose of 10 to 50 microgram, consistently enhanced the in vitro anti-SRBC immune response by normal splenocytes. The LPS also increased the background PFC response to SRBC in nonimmunized cultures. A chemically detoxified preparation derived from LPS (Mex B) had no stimulatory activity in vitro. A completely nontoxic, relatively small m.w., polysaccharide-rich preparation (PS), free of detectable lipid and protein, was stimulatory in vitro and at a dose of 10 microgram resulted in a 40 to 70% enhancement of the anti-SRBC response. The PS also stimulated an enhanced background response to SRBC as well as several other RBC species in nonimmunized cultures. PS had no mitogenic effect in vitro since addition of this bacterial derivative failed to stimulate thymidine incorporation into mouse splenocytes, as occurred with the intact LPS. The use of nontoxic preparations from gram-negative bacterial LPS for dissecting the stimulatory vs antigenic properties of bacterial products provides a model system for determining the role of a mitogenic stimulus in B cell activation.     

The structure and serologic distribution of an extracellular neutral polysaccharide from Pseudomonas aeruginosa immunotype 3

Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.