Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.     

The evaluation of the vibriocidal antibody test in establishing a diagnosis of cholera or Vibrio carriage]

On the basis of the serological survey of cholera patients, vibrio carriers and persons having had contacts with the source or reservoir of Vibrio cholerae the conclusion has been made that the test for the presence of vibriocidal antibodies, together with the bacteriological study of the patient, is of diagnostic importance in the diagnosis of cholera or vibrio carriership. The detection of vibriocidal antibodies, especially in the study of paired sera, permits the detection of cholera cases which have not been bacteriologically confirmed due to various reasons; besides, it makes it possible to exclude the diagnosis of cholera made only on the basis of clinical data. Like bacteriological study, the determination of vibriocidal antibodies must be obligatory for persons hospitalized in a provisory hospital or an isolation ward; it will undoubtedly improve the quality of cholera diagnosis and permit taking timely antiepidemic measures in the focus of infection.     

A semi-automated vibriocidal assay for improved measurement of cholera vaccine-induced immune responses

Vibriocidal antibody assay has been a surrogate standard assay in the evaluation of cholera vaccine efficacy because it has a good correlation with protection. Although the optical density-based vibriocidal assay in a 96-well microtiter-plate format is widely used in clinical trials, it has limitations as vibriocidal titers are altered by incubation time and samples with the same end-point titers could have potentially different vibriocidal kinetics. In the present study, we developed an improved agar-plate assay coupled with an automated colony counting system. Through testing 30 pairs of human sera from vaccinees administered with a cholera vaccine or placebo, these two assays showed good correlations for the vibriocidal titers and fold increases in titers between pre- and post-vaccinated sera as determined by the Pearson correlation coefficient and the Regression coefficient. Notably, the newly-developed semi-automated assay demonstrated that serum samples with the same end-point titers turned out to have distinct vibriocidal kinetics that were not distinguishable by the microtiter-plate assay. The semi-automated assay responded specifically to Vibrio cholerae but not to irrelevant bacteria such as Salmonella typhi and Escherichia coli. These results demonstrate that the semi-automated assay provides better sensitivity, accuracy, and stability of the assay results with minimized efforts than conventional microtiter-plate assay and could provide a useful tool as an in vitro surrogate assay for the evaluation of cholera vaccine efficacy.     

Identification of vibriocidal compounds from medicinal plants using chromatographic fingerprinting

The aim of the present study was to investigate the vibriocidal activity of bark of Syzygium cumini, leaves of Lawsonia inermis, fruits of Terminalia bellerica and identify the bioactive compounds. The vibriocidal activity of plant extracts was determined in aqueous and organic solvents, and the minimum inhibitory concentration (MIC) against Vibrio spp. using the disk diffusion method was established. The chemical constituents of the plant extracts were analysed by thin layer chromatography (TLC), the vibriocidal compounds were determined by TLC-bioautography and were further confirmed by high performance liquid chromatography (HPLC). Significant inhibitory activity was observed with ethanol extract of plants against the test bacteria while less antibacterial activity was observed in acetone, methanol and aqueous extracts. The MIC of the plant extracts ranged between 2.5 and 20 mg/ml. The TLC, TLC-bioautography and HPLC analysis showed that gallic acid and tannin present in ethanol extracts of S. cumini, tannin present in L. inermis and gallic acid present in T. bellerica may be responsible for the vibriocidal activity. S. cumini, L. inermis and T. bellerica can be used for the treatment of gastroenteritis, diarrhoea and cholera diseases after detailed investigations. We also conclude that the plants rich in gallic acid and tannin can be used as an alternative to search for new vibriocidal drugs.     

Seroepidemiology of cholera in Gulf coastal Texas

Single serum samples from 559 volunteers from a Texas Gulf Coast area were examined for vibriocidal antibody to Vibrio cholerae O1 (biotype El Tor, serotype Inaba) by a microtiter method. Elevated levels of vibriocidal antibody were present in 14% of the subjects. Also, 6.8% of the subjects had elevated levels of antibody to the enterotoxin of V. cholerae O1 by the immunoglobulin G enzyme-linked immunosorbent assay. Recent infection, defined on the basis of elevations in both vibriocidal and antitoxin antibodies, had occurred in 1.3% of the subjects. When subjects who reported Brucella infection, travel to a cholera-endemic area, and/or cholera vaccination within a year of the study were removed from the analysis, a prevalence of recent infection of 0.89% was obtained. Significantly higher titers of vibriocidal antibody were found in those with exposure to seawater (fishermen, shrimpers, merchant marines, and dock workers) than in those without such exposure (P less than 0.005). Furthermore, titers of antitoxin antibody were significantly higher in those who consumed shellfish than in nonconsumers. Finally, titers of vibriocidal antibody were significantly higher in Vietnamese subjects than in non-Vietnamese subjects. The results of this study indicate that an endemic focus of infection with V. cholerae occurs in this area.     

Systemic immunity of experimental animals immunized with cholera vaccines

The levels of antitoxic and vibriocidal antibodies in the sera of suckling rabbits after their parenteral immunization with cholera vaccine, cholera toxoid and a combination of cholera vaccine and toxoid were examined. Cholera vaccine induces intensive production of vibriocidal antibodies, and cholera toxoid, of antitoxic antibodies. The parenteral administration of the serum of rabbits immunized with cholera toxoid neutralized the action of cholera toxin in the small intestine of suckling rabbits. The complex preparation combines the properties of the corpuscular vaccine and the toxoid, inducing the production of both vibriocidal and antitoxic antibodies.     

Seroepidemiology of cholera in Gulf coastal Texas.

Single serum samples from 559 volunteers from a Texas Gulf Coast area were examined for vibriocidal antibody to Vibrio cholerae O1 (biotype El Tor, serotype Inaba) by a microtiter method. Elevated levels of vibriocidal antibody were present in 14% of the subjects. Also, 6.8% of the subjects had elevated levels of antibody to the enterotoxin of V. cholerae O1 by the immunoglobulin G enzyme-linked immunosorbent assay. Recent infection, defined on the basis of elevations in both vibriocidal and antitoxin antibodies, had occurred in 1.3% of the subjects. When subjects who reported Brucella infection, travel to a cholera-endemic area, and/or cholera vaccination within a year of the study were removed from the analysis, a prevalence of recent infection of 0.89% was obtained. Significantly higher titers of vibriocidal antibody were found in those with exposure to seawater (fishermen, shrimpers, merchant marines, and dock workers) than in those without such exposure (P less than 0.005). Furthermore, titers of antitoxin antibody were significantly higher in those who consumed shellfish than in nonconsumers. Finally, titers of vibriocidal antibody were significantly higher in Vietnamese subjects than in non-Vietnamese subjects. The results of this study indicate that an endemic focus of infection with V. cholerae occurs in this area.     

A duplex vibriocidal assay to simultaneously measure bactericidal antibody titers against Vibrio cholerae O1 Inaba and Ogawa serotypes

Currently available cholera vaccines are formulated with killed-whole cells of Vibrio cholerae O1 Inaba and Ogawa serotypes. A serum vibriocidal assay has been widely used to evaluate the immunogenicity of cholera vaccines in clinical trials. In this study, we developed a duplex vibriocidal assay to obtain vibriocidal antibody titers against both serotypes simultaneously. Initially, serial dilutions of serum from vaccinees were incubated with guinea pig complements along with both streptomycin-resistant Inaba and ampicillin-resistant Ogawa strains for 1 h. The mixture was then inoculated on separate agar plates containing each antibiotic to selectively culture each corresponding serotype and incubated at 37 °C for 16 to 20 h. Bacterial colonies were enumerated using an automated colony counting system to obtain the vibriocidal antibody titers defined by the reciprocal of serum dilution inhibiting bacterial growth by 50%. Performance of the duplex vibriocidal assay was examined by comparison with a single serotype vibriocidal assay using 20 clinical sera consisting of ten-paired sera prepared at pre- and post-vaccination. Both assays showed a good correlation for vibriocidal titers against the two serotypes, respectively, as determined by Pearson correlation coefficient (r) and regression coefficient (β) analyses; r = 0.998, β = 1.003 for Inaba and r = 0.997, β = 0.999 for Ogawa, respectively. The duplex vibriocidal assay can diminish the amount of sera required for the assay and enhance assay efficiency in terms of time, labor intensity, and expenditure.     

O1群霍乱Ogawa血清型结合疫苗免疫学效果研究

霍乱是经粪口传播的烈性传染病,相应的疫苗研究已逾百年,但目前还没有理想的疫苗。实验中以霍乱O1群小川血清型的脂多糖为目标抗原,用不同方法制备了其四种霍乱结合疫苗,通过小鼠模型验证了各结合物的免疫学效果。结果显示,不同结合物免疫学效果不一,其中增大多糖分子量后制备的结合物免疫效果较好,氨还原法制备的结合物多针免疫后也可诱导特异性抗体产生,而且具有针对小川和稻叶两种血清型的杀弧菌活性。     

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.     

Utilization of serological methods in the retrospective diagnosis of cholera and in detecting vibrion carriers]

Sensitivity and specificity of the three serological methods were studied comparatively: the vibriocidal test, the reaction of bacterial agglutination and of indirect hemagglutination, with the use of erythrocytes sensitized with the vibrio lyzate, cholera species O-antigen and cholerogen. Investigations were conducted with the blood sera of cholera patients, vibrio carriers and contacts. Vibriocidal test proved to be the most sensitive; its data correlated with the results of bacterial agglutination and indirect hemagglutination with erythrocytes, sensitized with the lysate of the vibrios and the cholera O-antigen. None of the used serological methods provided a 100% coincidence with the results of bacteriological analysis. The frequency of detection of anticholera antibodies decreased in the following order: cholera patients, vibrio carriers, contacts.     

The characteristics of the reactogenicity and immunological activity of a new cholera bivalent chemical vaccine based on the results of controlled trials]

The reactogenic properties and immunological potency of modified cholera chemical vaccine (choleragen-toxoid + O-antigens Inaba and Ogawa) were tested in 278 volunteers aged 18 years and over in comparison with those of a commercial batch of monovalent cholera vaccine (choleragen-toxoid + O-antigen Inaba). The cholera vaccine, enriched with O-antigen Ogawa, was found to be safe; vaccination with this vaccine was not accompanied by the development of systemic and local reactions whose frequency and intensity met the requirements for the reactogenic properties of commercial cholera vaccine. The immunological potency of the bivalent vaccine with respect to strain Inaba was not inferior to that of the commercial vaccine; at the same time in persons immunized with the new preparation the titers of vibriocidal antibodies to strain of serovar Inaba were five-fold higher. The conclusion on the expediency of using cholera chemical vaccine enriched with O-antigen Ogawa was made.     

Titration of Cholera Antitoxin in Human Sera by Microhemagglutination with Formalinized Erythrocytes

Microtiter hemagglutination tests employing formalinized sheep erythrocytes sensitized with either crude or purified cholera toxin were used to assay the cholera antitoxin content of human sera. Comparable results were obtained with either crude or purified toxin-sensitized cells with the exception of two sera that gave unusually high hemagglutination titers with the crude toxin. Sera from 13 convalescent cholera patients showed a high degree of correlation between antitoxin levels as determined in vitro by the hemagglutination test and in vivo by the skin permeability factor neutralization test. Fourfold or greater rises in antitoxin levels between acute and convalescent sera were detected in 9 of 15 patients with bacteriologically proven cholera. No significant increases in titer were observed in 14 cases of noncholera diarrhea. Cholera antitoxin was detected by hemagglutination in only 1 of 33 sera, obtained from eight countries, containing vibriocidal antibodies. Formalinized sheep erythrocytes sensitized with toxin and stored at 4 C in the presence of 1:10,000 thimerosal were stable and sensitive for at least 6 months (the longest time tested).     

Agglutinating and Bactericidal Properties of Fractions of Rabbit Anti-Vibrio cholerae Serum

The major portion of the agglutinating and bactericidal activity of the sera of rabbits immunized with live Vibrio cholerae or with cholera vaccine was found in the gammaM fractions during the early stages of immunization. After 5 weeks or more, gammaG fractions accounted for more than half of the agglutinating activity. When late antibody was measured as the amount of protein precipitated by somatic antigens, nearly 3 times as much gammaG as gammaM was required for agglutination, and about 30 times as much gammaG as gammaM was required to kill 50% of a standard inoculum in the presence of complement. The ratio of vibriocidal to agglutinin titer of gammaG fractions at different stages of immunization was more variable than that of gammaM fractions. More complement was required for a vibriocidal effect by gammaG than by gammaM. Increasing the amount of complement decreased the amount of both gammaG and gammaM required to kill, but smaller amounts of gammaM required disproportionately larger amounts of complement. Less time was required by gammaM than by gammaG to kill 50% of the inoculum. Removal of the group-reactive antibody from anti-Ogawa serum and serum fractions by absorption with Inaba reduced the vibriocidal titer by more than one-half.     

A Cholera Conjugate Vaccine Containing O-specific Polysaccharide (OSP) of V. cholerae O1 Inaba and Recombinant Fragment of Tetanus Toxin Heavy Chain (OSP:rTTHc) Induces Serum,Memory and Lamina Proprial Responses against OSP and Is Protective in Mice

Background

Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS).

Methodology

Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization.

Principle Findings

Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model.

Conclusion

We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens.     

Preparation and characterization of monoclonal antibodies to the cholera toxin

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.     

吸附霍乱菌苗免疫后人群血清杀弧菌抗体观察

本文介绍了用霍乱杀弧菌抗体微量法,检测经Ｏ１型吸附霍乱菌苗免疫前后人群的Ｏ１型霍乱和Ｏ１３９型霍乱的血清杀弧菌抗体。结果表明,免前人群的Ｉｎａｂａ、Ｏｇａｗａ、Ｏ１３９型三种自然杀弧菌抗体很低（ＧＭＴ在５．４９以下）,免后Ｉｎａｂａ和Ｏｇａｗａ型杀弧菌抗体大幅度增加,ＧＭＴ分别达到７８２和５９２、Ｏ１３９型霍乱杀弧菌抗体免疫前后无变化     

Immunity to Cholera: Relation of Fraction II of Type 2 Cholera Toxin to Vibriocidal Antibody

The nontoxic protein component in supernatant fluids of young cultures of the cholera vibrio in peptone dialysate broth contains an antigen identical in specificity to vibrio lipopolysaccharide. This material was heterogeneous after elution from diethylaminoethyl A50 Sephadex, and it contained at least five additional minor antigens. Identity was demonstrated by immunodiffusion methods, by the induction of specific vibriocidal antibody formation, and by specific interference in the vibriocidal reaction. The minor antigens appeared to be unrelated to the vibriocidal reaction. The major antigen was more highly immunogenic than lipopolysaccharide, giving higher and longer-persisting antibody titers in the rabbit, but lipopolysaccharide was the more effective interfering antigen per unit weight in the vibriocidal reaction. The nontoxicity and high immunogenic potency of the protein antigen suggest that it may be useful as an immunizing agent for the production of the antibacterial component of an effective immunity.     

Lifestyle factors and 5-year abdominal fat accumulation in a minority cohort: the IRAS Family Study

The objective of this study was to examine whether lifestyle factors were associated with 5‐year change in abdominal fat measured by computed tomography (CT) in the Insulin Resistance and Atherosclerosis (IRAS) Family Study. We obtained abdominal CT scans at baseline and at 5 years, from African Americans (AA) (N = 339) and Hispanic Americans (N = 775), aged 18–81 years. Visceral (VAT) and subcutaneous (SAT) adipose tissue was measured at the L4/L5 vertebral level. Physical activity was documented by self‐report of vigorous activity and a 1‐year recall instrument. Dietary intake was assessed at follow‐up using a semi‐quantitative food frequency questionnaire referencing the previous year. Generalized linear models, accounting for family structure, were used to assess the associations between percent change in fat accumulation and smoking, physical activity, total calories, polyunsaturated, monounsaturated, protein, and saturated fat intake, percent of calories from sweets, and soluble and insoluble fiber. Soluble fiber intake and participation in vigorous activity were inversely related to change in VAT, independent of change in BMI. For each 10 g increase in soluble fiber, rate of VAT accumulation decreased by 3.7% (P = 0.01). Soluble fiber was not associated with change in SAT (0.2%, P = 0.82). Moderately active participants had a 7.4% decrease in rate of VAT accumulation and a 3.6% decrease in rate of SAT accumulation versus less active participants (P = 0.003 and P = 0.01, respectively). Total energy expenditure was also inversely associated with accumulation of VAT. Soluble fiber intake and increased physical activity were related to decreased VAT accumulation over 5 years.     

Development of peptide mimics of a protective epitope of Vibrio cholerae Ogawa O-antigen and investigation of the structural basis of peptide mimicry

As an alternative approach toward the development of a cholera vaccine, the potential of peptide mimics of Vibrio cholerae lipopolysaccharide (LPS) to elicit cross-reactive immune responses against LPS was investigated. Two closely related protective monoclonal antibodies, S-20-4 and A-20-6, which are specific for Ogawa O-antigen (O-specific polysaccharide; O-SP) of V. cholerae O1, were used as the target antibodies (Abs) to pan phage display libraries under different elution conditions. Six phage clones identified from S-20-4 panning showed significant binding to both S-20-4 and A-20-6. Thus, it is likely that these phage-displayed peptides mimic an important conformational epitope of Ogawa antigens and are not simply functionally recognized by S-20-4. Each of the six phage clones that could bind to both monoclonal antibodies also competed with LPS for binding to S-20-4, suggesting that the peptides bind close to the paratope of the Ab. In order to predict how these peptide mimics interact with S-20-4 compared with its carbohydrate counterpart, one peptide mimic, 4P-8, which is one of the highest affinity binders and shares motifs with several other peptide mimics, was selected for further studies using computer modeling methods and site-directed mutagenesis. These studies suggest that 4P-8 is recognized as a hairpin structure that mimics some O-SP interactions with S-20-4 and also makes unique ligand interactions with S-20-4. In addition, 4P-8-KLH was able to elicit anti-LPS Abs in mice, but the immune response was not vibriocidal or protective. However, boosting with 4P-8-KLH after immunizing with LPS prolonged the LPS-reactive IgG and IgM Ab responses as well as vibriocidal titers and provided a much greater degree of protection than priming with LPS alone.