Immunoprotective extracellular polysaccharides of Klebsiella aerogenes capsular type K1, expressed in Escherichia coli.

A gene library of genomic DNA Klebsiella aerogenes of capsular serotype K1 was constructed in E. coli LE392 using the cosmid pMMB33. Culture filtrates of E. coli recombinants were screened by ELISA for extracellular polysaccharides specific for K. aerogenes K1. Extracellular polysaccharide extracts from K. aerogenes K1 and 3% of the E. coli recombinants contained immunoprotective extracellular polysaccharides (IEP) with similar chemical and immunological properties as shown by gel filtration through Sephacryl 1000, double immunodiffusion and mouse protection tests. IEPs contained no detectable protein, had molecular weights of several hundred million and protected mice against lethal autologous K. aerogenes K1 challenge at a dosage of 10 nanograms per mouse.

Passive immunization of mice against Klebsiella aerogenes.

Klebsiella vaccines were isolated by mild diafiltration techniques from culture filtrates of nine capsular types of K. aerogenes (K1, K2, K3, K15, K20, K35, K36, K44 & K63). The bacteria were grown in a chemically defined medium in standardized conditions in a fermenter. The vaccines had a molecular weight of more than 20 000, a carbohydrate content of 40-89%, a protein content of between less than 1 and 16% and small amounts (0.6-1.2%) of lipopolysaccharide. Antisera raised in rabbits to the nine klebsiella vaccines were standardized by passive haemagglutination, immunoglobulin G enzyme-linked immunosorbent assay and by autologous passive protection tests. Each rabbit antiserum when passively transferred to mice showed a variable capacity to passively protect mice against lethal infections by a panel of ten capsular types of K. aerogenes (K1-K10). Seven of the rabbit antisera protected mice against more than half of the challenge strains. A pool of six rabbit antisera (anti-K1, K2, K3, K20, K35 & K44) passively protected mice against lethal infections from strains of bacteria representing each of 77 capsular types of K. aerogenes.

Characterisation of mouse monoclonal antibodies produced by immunisation with a single serotype component of a polyvalent Pseudomonas aeruginosa vaccine.

Mouse monoclonal antibodies raised by immunisation with a protective antigen extract from Pseudomonas aeruginosa serotype 1 varied in immunoglobulin isotype, in passive protective properties against infection by homologous P. aeruginosa serotype 1, and in cross-reactions in ELISA against antigen preparations from 15 other P. aeruginosa serotypes. All monoclonal antibodies with specificity in ELISA for the immunising antigen gave some degree of protection to mice against lethal infection by the homologous P. aeruginosa serotype. The IgG antibodies were more protective than the IgM antibodies.

Enzyme-linked immunosorbent assay (ELISA) and a passive haemagglutination test (PHT) for the detection of pseudomonas antibodies in burned patients.

Pseudomonas antibodies were measured in serial plasma samples from patients with burns using an enzyme-linked immunosorbent assay (ELISA), passive haemagglutination test (PHT) and a passive protection test (PPT). ELISA and PHT showed that from 7 days after burning, 15 patients immunized with a 16-part polyvalent vaccine had higher titres of antibody to the 16 antigens in the vaccine than 15 unvaccinated patients. There was evidence that ELISA and PHT detected different pseudomonas antibodies in the plasma samples. When a single antigen (type 6) from the polyvalent vaccine was used in ELISA it failed to monitor antibodies in the plasma from vaccinated and unvaccinated burned patients shown by a PPT to protect mice against Pseudomonas aeruginosa serotype 6. PHT gave a closer correlation of protective antibodies against type 6 antigen than ELISA.

Vaccination against 77 capsular types of Klebsiella aerogenes with polyvalent Klebsiella vaccines.

A method is described for producing monovalent and polyvalent vaccines from culture filtrates of Klebsiella aerogenes. With a single injection, each monovalent vaccine protected mice against lethal intraperitoneal challenge by more than 30 capsular types; and polyvalent vaccines containing 2-12 monovalent components protected against 46-61 of the 77 capsular types of K. aerogenes. One vaccine with 12 components, administered in two doses, induced full protection against 71 types and protected half of the mice challenged with the other six types.

Vaccination against Klebsiella aerogenes.

Klebsiella vaccine was prepared from strains of Klebsiella aerogenes with capsular types K1, K36, K44 and K Cross (a type which cross-reacts in vitro with sera from many klebsiella capsular types). The vaccine was extracted by dialysis and ultrafiltration from capsular material released during growth of the bacteria in a five-day batch culture. Mice given one dose of vaccine from K1a (1.0 microgram/mouse) survived lethal intraperitoneal challenge of 11/11 homologous klebsiella strains four days after vaccination; 14 days after vaccination protection against the same challenge strains had declined to 5/11 strains. Vaccines from K1a, b, c, K36, K44 and K Cross induced homologous protection and protected mice against different ranges of heterologous klebsiella capsular types. The protective response of the mice was greatly enhanced by administering three doses of the vaccines. Vaccines from K1, K36, K44 and K Cross protected mice against 14/20, 11/20, 10/20 and 9/20 homologous and heterologous klebsiella challenge strains respectively. None of the klebsiella vaccines was toxic for mice at the immunizing dose (1.0 microgram/mouse). Vaccine from K36 was the most lethal, killing mice at 10(3) immunizing doses. The least toxic vaccine was from K44, which killed mice at 10(4) immunizing doses.

Immunization of burned patients against Pseudomonas aeruginosa infection at Safdarjang Hospital, New Delhi.

Patients (746) aged one to 60 years admitted to Safdarjang Hospital with full skin-thickness burns over 5%-70% of their body surface were randomly allocated to a group in a controlled clinical trial in which polyvalent pseudomonas vaccine (PEV-01), antipseudomonas human immunoglobulin, pseudomonas vaccine and immunoglobulin, or no immunologic prophylaxis was administered. The mortality rate for 297 patients immunized with PEV-01 was reduced fourfold in adults and more than twofold in children as compared with that for the 263 patients who received no immunoprophylaxis. Children particularly benefited from prophylaxis with immunoglobulin; the mortality rate for children who received PEV-01 was more than twofold less than that for a control group of unimmunized children. In both children and adults, immunoprophylaxis with PEV-01 plus immunoglobulin was less effective in reducing mortality than was PEV-01 or immunoglobulin administered alone. Antibodies measured by passive hemagglutination and by passive protection tests failed to correlate with one another, but all three immunologic regimens enhanced the bactericidal capacity of whole blood against Pseudomonas aeruginosa. Klebsiella pneumoniae emerged as the dominant gram-negative bacterial species causing bacteremia in patients receiving immunoprophylaxis against P. aeruginosa.

Active and passive immunization against Pseudomonas aeruginosa infection of burned patients.

A 16-part polyvalent pseudomonas vaccine, PEV-01 and an immunoglobulin prepared from plasma from human volunteers vaccinated with PEV-01 were tested in a controlled clinical trial in burned patients at Safdarjang hospital, New Delhi. The mortality of the burned patients was reduced four-fold in adults and three-fold in children by the immunological treatments. Both immunoglobulin and vaccine increased the concentration of protective antibody. Patients responded to all 16 components in PEV-01. The vaccine caused no toxic side-effects and enhanced the bactericidal activity of polymorphonuclear leucocytes against Pseudomonas aeruginosa.

Controlled trial of Pseudomonas immunoglobulin and vaccine in burn patients.

In a controlled trial burn patients at risk of Pseudomonas aeruginosa septicaemia were passively immunised with an immunoglobulin prepared from plasma from healthy human volunteers vaccinated with a polyvalent pseudomonas vaccine; passively immunised and vaccinated; or only vaccinated. In children the mortality was lowest in those passively immunised (0%, 0/18); it was 21% (9/42) in controls. In adults the mortality rate of those receiving immunoglobulin or vaccine was 10% (3/30) or 8.3% (5/60), respectively, compared with 36% (22/61) in controls. Combined vaccine and immunoglobulin treatment gave rather less protection (mortality 13.6%, 3/22) than vaccine alone. Pseudomonas infection of burns was less common in patients who received immunoglobulin than in vaccinated or control patients.

Controlled trials of a polyvalent pseudomonas vaccine in burns.

A polyvalent pseudomonas vaccine has been tested in controlled clinical trials at two burns units, in Birmingham and New Delhi, in children and adults with burns more than 15% full skin thickness. None of the vaccinated patients in either trial showed blood cultures containing Pseudomonas aeruginosa, and vaccinees showed raised titres of protective antibody and increased phagocytic activity against Ps. aeruginosa. In the New Delhi unit, where death from Ps. aeruginosa infection is common, the mortality in adults was reduced from 40.6% (13/32) in the unvaccinated group to 6.6% (2/30) in the vaccinated group, and in children from 20.8% (5/24) in the unvaccinated group to 4.8% (1/21) in the vaccinated group.

Measurement of endotoxins with the limulus test in burned patients.

High titres of endotoxin as measured by the Limulus test were usually found in burned patients who had raised body temperatures, and were colonized with gram-negative bacteria; also some infected patients showed raised endotoxin without a raised temperature. Patients vaccinated with an antipseudomonas vaccine rarely showed endotoxin in their plasma but occasional plasma samples from vaccinated patients had a high titre of endotoxin which appeared unrelated to infection or to a raised temperature in the patient.

The NBT test in burned patients.

The number of polymorphs which stained with the dye nitro-blue tetrazolium (NBT "Positive") increased sharply during the first week after burning, reaching levels 4--5 times above values for healthy volunteers. In burns of more than 20% of the body surface a second, smaller increase in the number of NBT "positives" occurred 4 to 6 weeks after burning. The high levels of NBT "positive" polymorphs occurred independently of infection on the burns. A burned patient who died from septicaemia had very low numbers of NBT "positive" polymorphs for 3 weeks before death.

Low mortality in burned patients in a Pseudomonas vaccine trial.

In a controlled clinical trial of a polyvalent pseudomonas vaccine in burned patients infected with Pseudomonas aeruginosa all 18 vaccinated patients survived, whereas 8 of 20 unvaccinated patients died.

A new polyvalent Pseudomonas vaccine.

This paper describes the preparation, chemical characterisation and immunogenic properties of a new polyvalent pseudomonas vaccine. New cultural methods were devised which allowed a build-up of the immunogen in the cell wall of the bacteria, and mild extraction techniques were used to remove the immunogens from the cell wall of living bacteria without apparent physical or chemical change which might affect immunogenicity. The polyvalent vaccine comprised 16 component vaccines, each a lipid-protein-carbohydrate complex extracted from one of the 16 different serotypes of Pseudomonas aeruginosa. Single injections into mice of each of the 16 component vaccines induced protection against several strains of homologous serotypes within 3 days of vaccination. The polyvalent vaccine induced similar protection against several strains of each of the 16 serotypes. We would like to thank R. E. Dyster, K. Digby and J. Simmonds for their technical assistance.

Protection against Gram-negative infection by 'super-active' antigen.

'Super-active' antigens modified antigens released from bacteria which had been phagocytosed and killed by human leucocytes, were found to induce protective responses in mice within 24 h of immunization. At the earliest time (24 h) when immunized mice were protected against lethal intrapertoneal (i.p.) challenge by the bacteria from which which the 'super-active' antigens were made (Proteus mirabilis) the leucocytes of peripheral blood from immunized mice showed enhanced phagocytosis and killing of autologous bacteria and there was an increase in the number of lymphocytes producing anti-proteus antibody. Another mouse protective factor inducing transient protection lasting 1-2 days against lethal i.p. challenge by P. mirabilis was found in preparations of lysed heman leucocytes not engaged in phagocytosis. Burned mice, immunized with 'super-active' antigen preparations were protected against lethal invasive proteus infection, inoculated on to the burn surface, 2 h after burning and immunization.

A new Pseudomonas vaccine: preliminary trial on human volunteers.

Fifteen healthy volunteers were given three weekly subcutaneous injections of a new polyvalent Pseudomonas aeruginosa vaccine (PEV-01). Four doses - 1-0 RHD (manufacturer's recommended human dose), 0-75 RHD, 0-5 RHD and 0-1 RHD - were used in separate groups of volunteers. Blood samples taken before each of the injections and one taken 7 days after the last injection were examined for immune response to the vaccine and for possible adverse clinical, biochemical and haematological effects. Raised titres of antibody in serum of volunteers given 0-5-1-0 RHD vaccine were shown, often by the seventh day, in passive haemagglutination tests against all of the 16 serotypes of Ps. aeruginosa represented in the vaccine; serum from volunteers who received 0-1 RHD usually showed a reduced antibody titre. Tests of mouse protection by serum against intraperitoneal challenge with Ps. aeruginosa P14 showed increased titres of mouse protective antibody in the blood of volunteers given 1-0, 0-75 or 0-5 RHD of vaccine but a reduced mouse-protective titre in two out of three sera from volunteers given 0-1 RHD vaccine. There was a suggestion of enhanced phagocytic ingestion and intracellular killing of two strains of Ps. aeruginosa by the blood of vaccinated volunteers, and more definite enhancement of ingestion of inert latex particles, which were less well ingested than were the bacterial cells by phagocytes from unvaccinated volunteers. Apart from slight or moderate local reactions and a transient rise of temperature in some volunteers, there were no clinical, biochemical or haematological abnormalities in the vaccinated volunteers.

Detection of endotoxins with the Limulus test in burned and unburned mice infected with different species of gram-negative bacteria.

The Limulus test detected endotoxins in the plasma of burned and unburned mice infected with different species of gram-negative bacteria produced different amounts of endotoxin in the plasma of infected mice. Plasma from mice given lethal infections showed very high concentrations of endotoxin. Low concentrations of endotoxin in the plasma were tolerated by mice but high concentrations were invariably fatal. A polyvalent pseudomonas vaccine reduced endotoxin in the plasma of mice given lethal infections of Pseudomonas aeruginosa.

Protective properties and haemagglutinins in serum from humans and in serum from mice injected with a new polyvalent Pseudomonas vaccine.

Mice given single injections of a polyvalent pseudomonas vaccine produced anti-pseudomonas haemagglutinins against the 16 component immunogens of the multivalent vaccine. Mice passively immunized with sera from vaccinated mice were protected against lethal challenge by 8/10 strains of Ps. aeruginosa of homologous serotype. Protection by the serum was inversely proportional to the virulence of the challenge strains. Anti-pseudomonas haemagglutinins were always present in sera which passively protected mice against pseudomonas infection. Low levels of anti-pseudomonas haemagglutinins were present in some sera which failed to passively immunize mice against pseudomonas infection. Anti-pseudomonas haemagglutinins and antibodies involved in passive protection were mainly in the IgM fractions of mouse serum. Control human sera contained anti-pseudomonas haemagglutinins against most serotypes of Ps. aeruginosa. Sera from patients with burns contained high levels of anti-pseudomonas haemagglutinins against some but not all serotypes of Ps. aeruginosa. Sera from both controls and patients with burns passively protected mice against pseudomonas infection.