Development of an enzyme-linked immunosorbent assay for studying Vibrio cholerae cell surface antigens.

An enzyme-linked immunosorbent assay for the measurement of antibodies directed against cell surface antigens of Vibrio cholerae (CSA ELISA) was developed. NaN3-killed whole cells of V. cholerae, adsorbed to polystyrene tubes, were used as immobilized antigens. The assay was capable of detecting antibodies directed against lipopolysaccharide and non-lipopolysaccharide surface antigens. In addition, the CSA ELISA was capable of detecting non-vibriocidal antibody. An antiserum raised in rabbits by immunization with live V. cholerae 1418 (Ogawa, El Tor) was capable of reacting with various heterologous strains of V. cholerae used as immobilized antigens. Therefore, common antigens shared by V. cholerae strains could be detected by using the CSA ELISA.     

Protection against colibacillosis in neonatal piglets by immunization of dams with procholeragenoid.

Protection against colibacillosis in neonatal piglets was obtained by immunization of pregnant dams with procholeragenoid. Procholeragenoid is a stable high-molecular-weight aggregate of cholera toxin formed during the heating of cholera toxin. Procholeragenoid retained approximately 1% of the toxicity of native toxin as determined in the rabbit ileal loop and Y-1 adrenal cell assays and 5% of the activity in the rabbit skin assay. Immunization of pregnant dams with 50 micrograms of procholeragenoid 5 and 2 weeks before the expected delivery date elicited high titers of antitoxic immunoglobulin G and toxin-neutralizing antibody in both the colostrum and serum. In three independent field trials, immunization with procholeragenoid resulted in a substantial decrease in diarrhea (73% in controls versus 11% in immunized) and death (4.7% in controls versus 0.77% in immunized) in neonatal piglets. The protection rate in the immunized population was approximately 85% for both diarrhea and death. In the following gestation period, reimmunization of dams with a single dose of procholeragenoid (50 micrograms) 2 weeks before delivery elicited titers of antitoxic immunoglobulin G and toxin-neutralizing antibody comparable to those obtained during the primary immunization. The death rate in neonatal piglets (0.86%) was comparable to that seen after immunization during the first gestation period (0.77%). These results indicate that substantial protection of neonatal piglets against colibacillosis can be obtained by immunization of dams with procholeragenoid. Protection was found to be based solely on antitoxic immunity.     

Immunity in Experimental Salmonellosis I. Protection Induced by Rough Mutants of Salmonella typhimurium

Different rough mutants of Salmonella typhimurium were tested to determine their virulence and immunizing capacity when used as live vaccines for mice. All uridine diphosphate-galactose-4-epimeraseless mutants tested were much more potent immunizing agents than any other mutants. This capacity was not correlated with virulence or complexity of cell wall polysaccharide. For good protection, persistence of the rough strains in vivo was essential, but the protection lasted longer than the period during which bacteria were demonstrable in the liver and spleen of the mice. The outstanding immunizing capacity of the “epimeraseless” mutants is not dependent on the persistence of viable bacteria in the mouse.     

Experimental Klebsiella pneumoniae burn wound sepsis: role of capsular polysaccharide.

The role of Klebsiella pneumoniae capsular polysaccharide in relation to virulence in a murine burn wound sepsis model was investigated. Burn trauma markedly predisposed mice to lethal K. pneumoniae sepsis. A highly encapsulated variant (KP1-O) derived from K. pneumoniae KP1 was found to be extremely virulent for burned mice (50% lethal dose less than 10 organisms), whereas another variant (KP1-T), which possessed a much smaller capsule, was comparatively nonvirulent (50% lethal dose greater than 10(6) organisms). Production of large quantities of capsular material by KP1-O allowed for its rapid growth in vivo and persistence in the blood and liver. These traits were not demonstrated by KP1-T, which was effectively cleared after challenge.     

Immunity in Experimental Salmonellosis III. Comparative Immunization with Viable and Heat-Inactivated Cells of Salmonella typhimurium

Vaccination with viable cells of an avirulent Salmonella typhimurium galE mutant provides mice with solid specific immunity against subsequent infection with a virulent smooth strain. Such a live vaccine is markedly more potent than one prepared from inactivated cells of the virulent smooth strain. The superiority of the live vaccine is particularly well demonstrated when the oral route of application is used. The protective capacity of the galE mutant is based on its ability to synthesize complete smooth-like cell wall lipopolysaccharide in vivo.     

Protection against fatal Pseudomonas aeruginosa burn wound sepsis by immunization with lipopolysaccharide and high-molecular-weight polysaccharide.

A murine burn wound model was employed to evaluate the relative efficacy of purified Pseudomonas aeruginosa lipopolysaccharide (LPS) and high-molecular-weight polysaccharide as protective immunogens. LPS was found to be both highly immunogenic and protective. As little as three 0.001-microgram doses elicited good immunoglobulin M and G titers and increased the mean lethal dose more than 1,000-fold. The level of protection against a live challenge correlated with antibody titers and was found to be serotype specific. An immunizing regimen which evoked only an immunoglobulin M response was still found to offer substantial protection. Immunization with a high-molecular-weight polysaccharide was also found to be protective. However, approximately 1,000-fold more high-molecular-weight polysaccharide, as compared with LPS, was needed to protect mice to an equivalent degree. Immunization with LPS was found to promote bacterial clearance and prevent establishment of bacteremia. A multivalent LPS vaccine conferred high levels of protection (110- to 53,000-fold) against eight different challenge strains of various serotypes.     

Purification and vaccine potential of Klebsiella capsular polysaccharides.

Capsular polysaccharide (CPS) from 18 Klebsiella strains of different capsular types was isolated and characterized. Purified CPSs were composed primarily of carbohydrate with trace quantities of protein, nucleic acids, and lipopolysaccharide. All CPSs were of a high molecular weight, possessing a Kd of 0.01 to 0.11 as determined by gel filtration over Sepharose CL-4B. Low levels of lipopolysaccharide present in all preparations were responsible for the highly pyrogenic nature of one-half of the CPS preparations. Treatment of capsular material with dilute NaOH in 95% ethanol markedly reduced the pyrogenicity of all preparations and had a negligible effect on their molecular weight. The immunogenicity of the various native CPSs for mice varied considerably from serotype to serotype, but all evoked an anticapsular immunoglobulin G response. Five of 18 NaOH-treated polysaccharides were significantly (P less than 0.05) less immunogenic than their native counterparts. Human immunoglobulin G prepared from volunteers immunized with either native or NaOH-treated KP1-0 capsular polysaccharide was equally effective at preventing experimental fatal Klebsiella pneumoniae burn wound sepsis in mice.     

Protection against Pseudomonas aeruginosa infection in a murine burn wound sepsis model by passive transfer of antitoxin A, antielastase, and antilipopolysaccharide.

The protective capacity of passively transferred immunoglobulin G (IgG) fractions from antitoxin (AT-IgG), antielastase (AE-IgG), and antilipopolysaccharide (ALPS-IgG) against Pseudomonas aeruginosa infection was evaluated in a murine burn wound sepsis model. Complete protection was afforded by homologous ALPS-IgG against intermediate challenge doses (10 50% lethal doses) of P. aeruginosa PA220, whereas AT-IgG and AE-IgG offered no significant protection (P less than 0.5). The simultaneous transfer of AT-IgG or AE-IgG with ALPS-IgG gave no additional protection above that seen with ALPS-IgG alone. The transfer of ALPS-IgG did not dramatically alter bacterial multiplication in the skin at the site of infection. However, bacteremia and infection of the liver were prevented. In parallel experiments, AT-IgG or AE-IgG did not significantly alter either the course of the infection or the number of bacteria seen in the blood, liver, or skin when compared with controls. ALPS-IgG administered 24 h before infection, at the time of infection, or 4 h postinfection provided complete protection. Even when ALPS-IgG was transferred at a time when the infection was well established locally in the skin (8 h postinfection), highly significant protection (P greater than 0.999) was obtained. Protection afforded by ALPS-IgG was serotype specific. These results indicate that antibody to lipopolysaccharide is of critical importance for protection against P. aeruginosa challenge in a relevant animal model.     

Cell-associated hemagglutinin-deficient mutant of Vibrio cholerae.

Cell-associated hemagglutinin-negative mutants were derived from cholera enterotoxin-negative Vibrio cholerae JBK70 by Tn5 mutagenesis. One of the mutants identified, SB001, was characterized in greater detail. Its ability to colonize ilea of adult rabbits was determined by feeding approximately 10(8) V. cholerae to each animal. At 17 h after feeding, the numbers of viable vibrios in the ilea were determined. There was a significant, 4 log, decrease in the ability of the hemagglutinin-negative mutant to colonize ileal tissue compared with the parent strain JBK70. In addition, the higher levels of colonization attained by JBK70 and the wild-type parent of JBK70, N16961, were associated with intestinal fluid accumulation and death. Rabbits immunized orally with approximately 10(8) SB001, when challenged 3 weeks later with either homologous biotype and serotype El Tor Inaba N16961 or heterologous Classical Ogawa 395, were protected to the same extent as those animals immunized with either the challenge strain or JBK70. This was evidenced by decreases in both the number of animals showing detectable colonization and the level of colonization achieved. A hemagglutinin-negative mutant of V. cholerae may therefore be of potential use as a live oral vaccine against cholera.