Production and experimental testing of the polyvalency of corpuscular vaccine for the prevention of Pseudomonas aeruginosa infections II. Experimental testing of the immunogenicity of polyvalent corpuscular Pseudomonas aeruginosa vaccine

Newly developed P. aeruginosa vaccine has been shown to be safe and apyrogenic for experimental animals. Immunization with the vaccine in a single injection of 0.5 ml has been found to ensure the protection of 80--98% of mice from lethal infection caused by virulent vaccine strains, with the exception of P. aeruginosa strain No. 1311, for 9 weeks. Immunity to P. aeruginosa strain No. 1311 develops only by day 56 after vaccination. No sharp correlation between the specific agglutinin level and the degree of protective effect induced by the immunization of animals with the polyvalent vaccine has been established. The vaccine has been shown to possess high immunogenicity in respect to clinical P. aeruginosa strains belonging to different serotypes (homo- and heterological vaccine strains).     

Antigenic complexes of Pseudomonas aeruginosa slime: their isolation and biological properties

The possibility of using antigenic complexes contained in the extracellular slime of P. aeruginosa clinical strains belonging to different serological groups as the components of a chemical vaccine has been revealed. Animal experiments have demonstrated a high immunogenicity of these preparations, as well as their low toxicity. The use of slime antigens stimulates the production of specific antibodies exerting a protective action against infection with homologous P. aeruginosa strains.     

Protective action of a corpuscular polyvalent Pseudomonas aeruginosa vaccine against representative enterobacteria

Animal experiments have demonstrated that P. aeruginosa vaccine is capable of protecting animals from experimental P. aeruginosa infection, as well as rendering a protective effect with respect to some representatives of the family Enterobacteriaceae. The comparative study of the antigenic spectra of the vaccine strains and some representatives of Enterobacteriaceae (Enterobacter, Serratia, Citrobacter and Klebsiella) has revealed no direct relationship between the degree of this protective effect and the presence of common antigenic determinants in them.     

Protective effects of affinity-purified antibody and truncated vaccines against Pseudomonas aeruginosa V-antigen in neutropenic mice

Virulent P. aeruginosa strains express PcrV, one of the translocational components of the type III secretion system. PcrV has been reported to be a protective antigen against lethal P. aeruginosa infection. The PcrV region, which contributes to protective immunity against P. aeruginosa infection, was investigated by using genetically engineered, truncated PcrV proteins and affinity-purified anti-PcrV antibodies against the truncated PcrV proteins. The efficacy of active and passive immunization against PcrV was tested in mice with cyclophosphamide-induced immunosuppression by intraabdominal challenge of P. aeruginosa . Active immunization with either full-length PcrV1-294 or PcrV139-294 significantly improved the survival of mice infected with P. aeruginosa , while PcrV139-258, PcrV139-234, PcrV197-294, and PcrV261-294 were not protective. These results suggest that an effective PcrV vaccine needs to contain not only the Mab166 epitope (PcrV144-257) but also the carboxyl terminal tail of PcrV. In the case of passive immunization, administration of affinity-purified anti-PcrV IgG against either PcrV1-294 or PcrV139-258 showed significantly higher efficacy against lethal P. aeruginosa infection than did original anti-PcrV IgG and Mab166. The increased efficacy of affinity-purified anti-PcrV IgG implies that more potent anti-PcrV strategies are possible. The results of this study are crucial to the development of an effective PcrV vaccine for active immunization and to an appropriate blocking anti-PcrV antibody against P. aeruginosa infection in humans.     

Immunologic study of the cellular components of bacillus pyocyaneus. III. Immunochemical analysis, toxicity and protective properties of water-soluble antigenic complexes]

Aqueous extracts of two Ps. aeruginosa strains killed with acetone were subjected to fractionation by preparative ultracentrifugation and gel-chromatography. Toxic activity of the extract was found to be connected with the high-molecular, possibly glycoprotein components reacting with the corresponding antiserum in the immunoprecipitation test, and protecting 30--40% of rats against the generalized infection with Pseudomonas aeruginosa. This protective activity is apparently connected with the protein components (molecular weight--20000--60000 dalton), nontoxic for mice, not reacting with the corresponding antiserum in the immunoprecipitation test, and protecting 60 to 80% of rats against Ps. aeruginosa infecsion. Thus, as a result of preparative ultracentrifugation and gel-chromatography it was postible to divide the toxic and the nontoxic protective components of Ps. aeruginosa.     

Effect of a corpuscular polyvalent Pseudomonas aeruginosa vaccine on the proliferation and differentiation of hematopoietic stem cells and erythropoiesis

P. aeruginosa corpuscular polyvalent vaccine stimulates hematopoiesis in sublethally stimulated mice. The stimulating effect is dose-dependent. The most effective method of immunization is the intravenous injection of the vaccine. The degree to which the stimulation of hematopoiesis is manifested varies in different strains of mice. The stimulation of hematopoiesis is not linked with an increase in the content of erythropoietin. P. aeruginosa polyvalent corpuscular vaccine and monovaccine, prepared from P. aeruginosa strain 1313 and incorporated into the polyvalent vaccine, protect lethally irradiated mice in the postradiation survival test.     

Cell-free Pseudomonas vaccine: its immunochemical characteristics and immunogenicity

Two variants of cell-free protein vaccine have been prepared from the mixture of 4 P. aeruginosa strains, serovars 02, 06, 07 and 011, and from a single P. aeruginosa strain, serovar 02. The preparation contains proteins with molecular weight ranging from 20,000 to 100,000 and the admixture of lipopolysaccharide in negligible amounts (not exceeding 0.08% of dry weight). The vaccine produces no signs of toxicosis in laboratory animals. The vaccine effectively protects mice challenged with P. aeruginosa of different O-serotypes and stimulates the formation of specific protective antibodies in rabbits.     

Increase of resistance to Pseudomonas aeruginosa infection in mice caused by Staphylococcus aureus]

In our study of opportunistic pathogens, we have some indication that Staphylococcus aureus can increase resistance in mice against Pseudomonas aeruginosa. Intraperitoneal injections of sublethal doses of S. aureus had a protective effect in mice against lethal doses of P. aeruginosa, more so if living and coagulase-positive S. aureus strains were injected. This protective effect was obtained both with laboratory and freshly isolated hospital strains. The interval between these infections can be extended from 2 h up to 1 week and it is still possible to observe the resistance phenomenon. The increased resistance was accompanied by a decrease in viable units of P. aeruginosa in the peritoneal cavity of mice 6 h after the injection of this species. There was no protection by S. aureus against Candida albicans in similar experimental conditions. These observations indicate that intermicrobial ecology, understood here as the previous presence of another species in a host, may be a significant factor in the resistance to infection with opportunistic pathogens such as P. aeruginosa.     

Immunological study of the cellular components of Pseudomonas aeruginosa. VI. The toxicity and protective properties of its mucus and its fractions]

Capsule-like mucus was obtained from two newly isolated mucoid strains of P. aeruginosa and then treated with ethanol. The mucus was fractionated by the method of differential centrifugation (at 15, 000 g for 1 h, at 105, 000 or 170, 000 g for 3 h) and gel chromatography in columns packed with Sepharose 4B. The sediment fractions contained 30--80% of high molecular polysaccharide protein (peptide) mucus components which were toxic for mice and protected 25--77% of rats against experimental P. aeruginosa infection. The supernatant fluid fractions contained 60--80% of predominantly protein components with molecular mass between 20,000 and 60,000 daltons. These mucus components were slightly toxic for mice and rats and protected 80--100% of rats against P. aeruginosa infection.     

Comparative study of the antibody concentration and protective activity against Pseudomonas aeruginosa of gamma-globulin and IgM-enriched immunoglobulin preparations

The protective effect of IgM-enriched immunoglobulin isolated by the caprylate-alcohol technique has been found to be more pronounced in comparison with that of the commercial preparation of gamma globulin in experiments on mice infected with P. aeruginosa live culture. The effectiveness of this protective action correlates with a higher content of antibodies to P. aeruginosa O-antigen in IgM-enriched immunoglobulin. In mice injected with P. aeruginosa toxin both preparations have shown equal intensity of their protective action.     

Immunologic study of the cellular components of bacillus pyocyaneus. V. Antigenic analysis of slime and its fractions]

Various slime fractions were obtained from newly isolated mucoid strains of P. aeruginosa by the method of ultrafiltration or differential centrifugation with subsequent gel chromatography. Purified slime was found to react with a broader spectrum of typing O sera than the corresponding cell wall lipopolysaccharides. Erythrocytic diagnostic preparations produced on the basis of slime antigens allowed to reveale the presence of circulating antibodies against P. aeruginosa. The slime components with molecular weight of 30,000--100,000 daltons and greater contained common antigenic determinants, and the slime components with molecular weight of 10,000--30,000 daltons contained both specific antigenic determinants and those also common to the high molecular components.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

Experimentally determined safety and immunological activity of vaccine based on antigens isolated from Pseudomonas aeruginosa in medium K-4

The safety and immunological activity of P. aeruginosa vaccine were experimentally evaluated. The vaccine was prepared on the basis of the antigens of P. aeruginosa extracellular slime which was accumulated in medium K-4, obtained with the use of original technology. The immunization of animals with P. aeruginosa vaccine induced the synthesis of antibodies. The introduction of the vaccine in 2 or 3 injections resulted in a high level of antibody formation, differing with the use of various strains. Hyperimmune sera, obtained by the multiple immunization of rabbits with P. aeruginosa vaccine, ensured high protection of mice from P. aeruginosa infection. The vaccine proved to be safe when evaluated in experiments of acute and chronic toxicity, made on laboratory animals.     

Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia coli.

Lipoprotein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. Like porin protein F (OprF), it is a vaccine candidate because it antigenically cross-reacts with all serotype strains of the International Antigenic Typing Scheme. Since lipoprotein I was expressed in Escherichia coli under the control of its own promoter, we were able to isolate the gene by screening a lambda EMBL3 phage library with a mouse monoclonal antibody directed against lipoprotein I. The monocistronic OprI mRNA encodes a precursor protein of 83 amino acid residues including a signal peptide of 19 residues. The mature protein has a molecular weight of 6,950, not including bound glycerol and lipid. Although the amino acid sequences of protein I of P. aeruginosa and Braun's lipoprotein of E. coli differ considerably (only 30.1% identical amino acid residues), peptidoglycan in E. coli, are identical. Using lipoprotein I expressed in E. coli, it can now be tested whether this protein alone, without P. aeruginosa lipopolysaccharide contaminations, has a protective effect against P. aeruginosa infections.     

Isolation and study of the properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine. An evaluation of the biological properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine

P. aeruginosa killed polyvalent corpuscular vaccine, tested in a trial on 42 volunteer donors, is safe, low reactogenic and possesses pronounced immunogenicity, as the injection of the vaccine induced a rise in the titer of antibodies to P. aeruginosa up to 1:1280 in 95% of the immunized donors. To obtain specific antibodies in the blood plasma of the donors in an amount sufficient for the protective activity of the plasma becoming manifest, it is expedient to use the vaccination schedule providing for subcutaneous injections of 0.5, 0.5 and 1.0 ml at intervals of 7 days. Intense immunity thus induced in the donors lasts for 3-4 months. The hyperimmune plasma obtained from the donors has an antibody titer of at least 1:320 and shows a 90-100% protective effect in mice infected intraperitoneally with P. aeruginosa. The preliminary results of the clinical trial of anti-P. aeruginosa plasma have demonstrated its efficiency as a part of the complex treatment of patients with purulent septic complications of P. aeruginosa etiology.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Results of using polyvalent Pseudomonas aeruginosa vaccine in children with burns by various medical centers

Polyvalent Pseudomonas aeruginosa vaccine, prepared at the Institute of Hematology from 10 hospital strains isolated from burn wounds, was administered to 32 children with extensive and deep burns. The vaccine was well tolerated. The vaccine produced a high degree of the immunity against Pseudomonas aeruginosa infection. Agglutinin serum titre increased significantly. Vaccination either prevented or inhibited the infection of burn wounds with Pseudomonas aeruginosa in all immunized children. The symptoms of Pseudomonas aeruginosa infection usually disappeared following one or two vaccinations. Bacteriemia caused by P. aeruginosa was not observed in 31 out of 32 children. In the remaining child transient bacteriemia was noted. No septicemia caused by P. aeruginosa was seen. Due to the high efficiency of the polyvalent P. aeruginosa vaccine all burned children with burns exceeding 10% of the total body surface should by vaccinated to prevent the life-threatening infections with Pseudomonas aeruginosa.     

Vaccines against Pseudomonas aeruginosa results and perspectives of investigations (survey)

At the present time, there are many preparations for active immunization against P. aeruginosa infection (pseudomonas infection), but none of the proposed preparations has so far been widely used in medical practice. Development of P. aeruginosa vaccine (PV) should obviously be based on findings concerning the pathogenesis of infection, the mechanisms of immunogenesis and the factors of virulence of the causative agent. On the basis of results of their studies the authors believe that PV should include a non-toxic low-molecular component (or components) of the extracellular slime and water-soluble protein antigens of the cell wall, isolated from one or three selected strains of P. aeruginosa. Adoption of these components onto aluminium hydroxide can obviously increase the efficiency of PV.     

Combination effect with some aminoglycoside antibiotics

Strains of resistant species -- Staphylococcus aureus and Streptococcus faecalis -- isolated from material of nosocomial infections in the surgical department of a Prague hospital were tested for combined effect with further chemotherapeutics. The same concerned Ps, aeruginosa noted, at present, by a high degree of resistance. In Ps. aeruginosa it was the combination sisomicin-carfecillin that proved a success in 50 p. c. of cases as well as that of sisomicin-doxycyclin. At the same time the authors draw attention to the possibility of antagonistic action of the combination sisomicin-chloramphenicol in a third part of the strains tested. Potentiation of the effect in Staphylococcus aureus strains was observed also in the combination sisomicin-carfecillin in more than one half of the strains tested and in case of sisomicin-doxycyclin in one third of the strains. In Streptococcus faecalis strains sisomicin was combined with amoxycillin, carbenicillin, carfecillin and doxycyclin; synergistic action being observed with all those combinations in more than one half of strains tested. No antagonism was registered in those cases. (Ta.).