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PubMed1991

Selection for immune response in goats: the antibody response to diphtheria toxoid after 12 years of selection.

A herd of Norwegian dairy goats was subject to selection for high and low antibody response to diphtheria toxoid for 12 yr, or approximately 5.5 generations because sires were used for one mating season, whereas dams were used several years. The herd comprised approximately 100 milking goats. Only sires were tested and selected, five to seven sires were used in each line each year, and the percentage of male kids used for breeding varied between 15 and 50%. The mean phenotypic values of the lines diverged until the 4th yr, but the lines did not diverge any further. The means of both lines decreased during the experimental period. The restricted maximum likelihood (REML) estimate of the heritability of the trait in the base population was .19, whereas the realized heritability approached zero in the last six cycles of selection. Using the REML-estimate of heritability in an individual animal model, the mean breeding values (BLUP) of the lines were significantly different. The difference in mean BLUP values between the lines increased throughout the study. Yet, this increase was only 40% from the 2nd to the 12th yr. No effect of a major gene was observed in a test based on individual BLUP.

Analysis of Variance↗
PubMed1993

Responses of children to booster immunization with their primary conjugate Haemophilus influenzae type B vaccine or with polyribosylribitol phosphate conjugated with diphtheria toxoid.

Children primed with one of four conjugate Haemophilus influenzae type b vaccines received booster immunization with their primary vaccine or with polyribosylribitol phosphate conjugated with diphtheria toxoid. The latter vaccine produced postbooster antibody levels that equaled or exceeded those produced by boosting with the original vaccine, and thus may be used as a booster irrespective of the original vaccine.

Antibodies↗
PubMed1984

Safety and immunogenicity of Haemophilus influenzae type B polysaccharide-diphtheria toxoid conjugate vaccine in adults.

Sixty-one adults received either Haemophilus influenzae type b capsular polyribosephosphate (PRP) vaccine or H. influenzae type b polysaccharide-diphtheria toxoid conjugate (PRP-D) vaccine in two doses, one month apart. Both vaccines were well tolerated with no fever or systemic reactions. There were no significant local reactions. Mild local tenderness was reported by about half of the subjects in both groups. There was no observed effect of the vaccines on hematologic parameters, including reticulocyte and platelet counts, evaluated before immunization, one month after the first dose, and seven to 10 days after the second dose. A single dose of PRP-D induced levels of IgG antibody to PRP three times that observed with PRP alone one month after the first inoculation.

Adult↗
PubMed2000

Quantification of free polysaccharide in meningococcal polysaccharide-diphtheria toxoid conjugate vaccines.

A precipitation method using deoxycholate/HCI has been applied successfully to separate unconjugated free polysaccharide from carrier protein-bound material in meningococcal polysaccharide-diphtheria toxoid conjugate vaccines. The method effectively separated free and bound polysaccharide in conjugate vaccines prepared from Neisseria meningitidis serotypes A, C, W135 and Y. Free polysaccharide remained in the supernatant after deoxycholate treatment while protein-bound polysaccharide was fully precipitated. Testing by both colorimetric assay and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has confirmed the selective loss of protein-bound polysaccharide in samples of conjugate vaccine or conjugate vaccine mixed with known amounts of free polysaccharide. This rapid separation method requires minimum sample handling and is specific, reproducible, and allows assessment of free polysaccharide levels in vaccines at final container concentration.

Diphtheria Toxoid↗
PubMed1954

Immunochemical studies of antitoxin produced in normal and allergic individuals hyperimmunized with diphtheria toxoid. IV. Differences between human precipitating and non-precipitating skin-sensitizing diphtheria antitoxin as shown by electrophoresis.

Electrophoresis on a starch-supporting medium was used to fractionate sera containing human diphtheria antitoxin of the following varieties (a) precipitating antitoxin, (b) non-precipitating skin-sensitizing antitoxin, and (c) mixtures containing precipitating and skin-sensitizing antitoxins. Aliquots of the protein fractions thus separated were tested for activity using the rabbit toxin neutralization test, precipitin techniques, and passive transfer tests in human skin. Non-precipitating, skin-sensitizing diphtheria antitoxin migrated largely as a fast moving gamma (gamma(1)) globulin. Passive transfer studies of isolated antitoxic fractions showed that they were as potent as whole serum in the ability to cause immediate wheal reactions. These fractions were not precipitable using appropriate quantities of purified toxoid. Precipitating diphtheria antitoxin migrated largely as a slow moving gamma (gamma(2)) globulin. Isolated antitoxic fractions of appropriate strength obtained from representative sera were precipitable by toxin and were unable to cause immediate wheal reactions upon toxoid challenge in human recipients. Mixtures of skin-sensitizing and precipitating antitoxins were separable by the technique of starch electrophoresis. The individual components removed from mixtures by this method retained the properties by which they could be characterized in whole serum.

Antibodies↗
PubMed1983

Isoelectric focusing of human anti-diphtheria toxoid antibodies: identical spectrotypes of anti-fragment A antibodies with the same IgG subclass and light chain constant regions are expressed in multiple individuals.

The serum antibodies from humans booster-immunized with diphtheria toxoid were analyzed by isoelectric focusing. To restrict the antibody response, we visualized those antibodies that reacted to the Fragment A moiety of diphtheria toxin. Of the 100 normal human donors examined, 20 were estimated to be nonresponders to Fragment A. Sixty-three of the 80 donors who responded to Fragment A had restricted IgG-anti-Fragment A spectrotypes that consisted of three to eight distinct bands. Six series of repeat or shared spectrotypes were delineated among our donor population with repeat frequencies ranging from 0.025 to 0.312. We determined that the repeat spectrotypes were IgG1-kappa anti-Fragment A antibodies. Three percent of our donors had complex IgG-anti Fragment A spectrotypes consisting of greater than 10 bands. In some instances, the complex spectrotypes were composed of simple spectrotypes found among other donors. The complex IgG-anti Fragment A spectrotypes of these donors consisted of both IgG1 and IgG4-anti-Fragment A antibodies. From a statistical analysis, we calculate that only a limited number of IgG-anti-Fragment A spectrotypes are expressed in vivo after booster immunization. Our analysis suggests two groups of IgG-anti-Fragment A spectrotypes exist in our donor population. One IgG-anti-Fragment A spectrotype group is expressed randomly, and a second group is preferentially expressed among individuals in our donor population.

Antibodies, Bacterial↗
PubMed1978

A practical method for quantitation of antibody responses to tetanus and diphtheria toxoids in normal young children.

Antibodies to diphtheria and tetanus toxoids were assayed in 46 children from 16 to 33 months of age following vaccination. A reversed rocket immunoelectrophoretic technique was used for antibody determination. This method is simple and consistent, and although the limit of detection for antibodies of both types (about 1 U/ml) is above the normal range for primary immune responses, antibody levels in immunized children five weeks after a second injection of vaccine were within measureable range in all but two of the children studied. Levels more than one standard deviation above the mean were found in one child for both antitetanus and anti-diphtheria and in four children for anti-tetanus toxoid antibody only. All four of the high responders to tetanus toxoid were Black, carrying both Black and Caucasian Gm haplotypes. The two apparent nonresponders were Caucasian. All children had normal immunoglobulin levels, except for one with low IgA, who showed normal antibody responses to both toxoids. The results suggest that measurements of precipitating antibodies to diphtheria and tetanus toxoids provide a practical index of humoral immune function in small children.

Antibodies, Viral↗
PubMed1993

[Granule-accumulation in VERO cells used for determination of antitoxin titration by micro cell culture method: II. Nature of granules and influence on the potency testing of diphtheria toxoid].

Accumulation of granules occurs in VERO cells when mouse serum was added in high concentration. The nature of the granule and influence of the granule-accumulation on the potency testing of diphtheria antitoxin were studied. The concentration of triglyceride in VERO cells increased 15 folds after 24 hours of incubation by the addition of undiluted mouse serum. Kinetical study of the increase of triglyceride and electron microscope observation suggested that the accumulated granules might be oil droplets with an average diameter of 0.27 micron. The end point reaction of neutralization of diphtheria toxin by the standard antitoxin was analyzed by the addition of mouse serum to the micro cell culture using VERO cells. The mean values of the end point reactions in the cell cultures added with high concentrations of mouse serum were -2.324, -2.338 and -2.346, while that of the control group was -2.343. The variance of these mean values in the experiment and control groups were 0.0017, 0.0024, 0.0017 and 0.0021, respectively. A test of homogeneity of variance for these data showed no significant difference between them. Furthermore, antitoxin titer of the serum specimens of mouse immunized with diphtheria toxoids determined by the micro cell culture method were compared with that obtained by rabbit skin test; a high correlation between the antitoxin titers by the two techniques was recognized. The results were comparable with those of guinea-pig sera. From these results it is evident that little influence, if any, was observed in the potency testing of diphtheria antitoxin using VERO cells, in spite of the remarkable accumulation of granules.

Animals↗
PubMed1987

Safety and immunogenicity of Haemophilus influenzae type b polysaccharide and polysaccharide diphtheria toxoid conjugate vaccines in children 15 to 24 months of age.

To evaluate the safety and immunogenicity of the Haemophilus influenzae type b polysaccharide vaccine, PRP, and a new polysaccharide-diphtheria toxoid conjugate vaccine, PRP-D, a collaborative study was carried out in six centers in five states. Subjects were 585 infants 15 to 24 months of age. They were randomly assigned to receive a single dose of PRP or PRP-D vaccine. There were no significant differences in the rate of adverse reactions between the two vaccine groups. Minor local reactions occurred in 10.3% of PRP and 12.5% of PRP-D recipients, and fever in 27.4% of PRP and 23.8% of PRP-D recipients. All reactions resolved within 48 hours. Serum samples were obtained just before vaccination and after 1 month. Prevaccination antibody levels were similar for the PRP (0.035 micrograms/mL) and PRP-D (0.027 micrograms/mL) groups, with no differences in levels by age, sex, race, vaccine lot, or study site. Both groups had significant rises in geometric mean levels, but this difference was significantly greater for PRP-D (2.166 micrograms/mL) than for PRP (0.154 micrograms/mL). In addition, the percentage of responders as determined by three definitions (twofold titer rise, greater than 0.15 micrograms/mL, and greater than 1.0 micrograms/mL) was also significantly greater for PRP-D than PRP. In contrast to a marked age-related immunogenicity to PRP (P less than 0.001), there was no significant variation in immune response to PRP-D by age. PRP-D conjugate vaccine appears to be as safe and significantly more immunogenic than PRP vaccine for children vaccinated at 15 to 24 months of age.

Age Factors↗
PubMed2006

Diphtheria toxoid loaded poly-(epsilon-caprolactone) nanoparticles as mucosal vaccine delivery systems.

Poly-(epsilon-caprolactone) (PCL), a poly(lactide-co-glycolide) (PLGA)-PCL blend and co-polymer nanoparticles encapsulating diphtheria toxoid (DT) were investigated for their potential as a mucosal vaccine delivery system. The nanoparticles, prepared using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method, demonstrated release profiles which were dependent on the properties of the polymers. An in vitro experiment using Caco-2 cells showed significantly higher uptake of PCL nanoparticles in comparison to polymeric PLGA, the PLGA-PCL blend and co-polymer nanoparticles. The highest uptake mediated by the most hydrophobic nanoparticles using Caco-2 cells was mirrored in the in vivo studies following nasal administration. PCL nanoparticles induced DT serum specific IgG antibody responses significantly higher than PLGA. A significant positive correlation between hydrophobicity of the nanoparticles and the immune response was observed following intramuscular administration. The positive correlation between hydrophobicity of the nanoparticles and serum DT specific IgG antibody response was also observed after intranasal administration of the nanoparticles. The cytokine assays showed that the serum IgG antibody response induced is different according to the route of administration, indicated by the differential levels of IL-6 and IFN-gamma. The nanoparticles eliciting the highest IgG antibody response did not necessarily elicit the highest levels of the cytokines IL-6 and IFN-gamma.

Administration, Intranasal↗
PubMed1984

Immunogenicity of Haemophilus influenzae type b polysaccharide--diphtheria toxoid conjugate vaccine in adults.

The capsular polysaccharide of Haemophilus influenzae type b is a poor immunogen in human infants. In an attempt to enhance immunogenicity, this polysaccharide was covalently coupled to diphtheria toxoid and the conjugate tested as a vaccine in adult volunteers. Two injections of PRP-D vaccine were given, separated by one month. The anti-PRP antibody responses in this group were compared with those in a group receiving a comparable dose (20 micrograms) of conventional PRP vaccine. Both vaccines were well tolerated. A single injection of PRP-D was significantly more immunogenic than PRP, eliciting higher serum concentrations of total anti-PRP antibody 1 month later (geo means of 248 and 62 micrograms/ml, respectively; P less than 0.001). In addition, higher concentrations of IgG anti-PRP antibody were observed in the PRP-D group (P less than 0.001). One month after reinjection of vaccine, subjects receiving PRP-D showed a small but significant decline in total antibody (P = 0.03), whereas the serum antibody concentrations in the group that received PRP remained unchanged. At 12 months, the antibody concentrations of the two groups were not significantly different. Bactericidal activity and passive protection activity (infant rat model) were tested in pooled sera from the three highest and three lowest responders in each vaccine group; both PRP and PRP-D vaccines induced biologically active anti-PRP antibody. Thus PRP-D was found to elicit biologically active serum antibody and to be more immunogenic in adults than PRP vaccine; however, the duration of higher concentrations of antibody was transient.

Adult↗
PubMed1990

Persistence of antibody and booster responses to reimmunization with Haemophilus influenzae type b polysaccharide and polysaccharide diphtheria toxoid conjugate vaccines in children initially immunized at 15 to 24 months of age.

To evaluate the persistence of antibody after Haemophilus influenzae type b polysaccharide vaccine (PRP) and H influenzae type b polysaccharide diphtheria toxoid conjugate vaccine (PRP-D), a group of 141 infants initially immunized between 15 and 24 months of age were studied 1 year later. One month after immunization with PRP, the man anti-PRP antibody level was 0.27 microgram/mL and 1 year later was 0.29 microgram/mL (not significant). In the group immunized with PRP-D, the levels were 1.34 micrograms/mL and 1.20 micrograms/mL (not significant), respectively. To evaluate immunogenicity and safety of a booster immunization 1 year after initial vaccination, subjects were randomly assigned to receive saline, PRP, or PRP-D. In addition, 73 age-matched previously unimmunized subjects were vaccinated with PRP or PRP-D. In all groups, adverse reactions were minor and resolved by 48 hours. Subjects receiving booster immunization with PRP or PRP-D had significantly greater antibody responses than children of the same age receiving their first dose of vaccine. The highest antibody levels were achieved in children initially immunized with PRP-D, regardless of whether the booster vaccine was PRP (112.8 micrograms/mL) or PRP-D (122.0 micrograms/mL) (not significant). Antibody levels after booster vaccine were significantly lower in those initially given PRP compared with those initially given PRP-D but significantly higher than in age-matched previously unimmunized control subjects (PRP booster 3.16 micrograms/mL vs control of 0.62 microgram/mL [P less than .05]; PRP-D booster 12.31 micrograms/mL vs control 2.31 micrograms/mL [P less than .01]).

Antibody Formation↗
PubMed2000

Capillary electrophoretic analysis of meningococcal polysaccharide-diphtheria toxoid conjugate vaccines.

Protein-polysaccharide conjugate vaccines are large, complex molecules that present challenges in terms of characterization. Free solution capillary electrophoresis using alkaline (pH 9-10) borate or glycine/NaOH buffers permitted electrophoresis of diphtheria toxoid (Dt) or meningococcal polysaccharide-Dt conjugates as well-formed, relatively broad peaks. Resolution was observed between the conjugate and the Dt peaks for some serotypes; however, overlap between Dt and one or more conjugate peaks was observed with both buffers. Inclusion of SDS in the separation buffer at a level above the critical micelle concentration allowed separation of Dt and Dt-conjugates of meningococcal serotypes A, C, Y and W135. Using borate/SDS, a linear relationship between peak area and Dt concentration was observed between approximately 2 mg/ml and 20 microg/ml. A linear relationship between Dt peak area and injection times from 10 to 50 seconds was also observed. Capillary electrophoresis may therefore be a useful method for quantifying free protein level in meningococcal-Dt conjugate vaccines.

Carrier Proteins↗
PubMed1985

Immunogens consisting of oligosaccharides from the capsule of Haemophilus influenzae type b coupled to diphtheria toxoid or the toxin protein CRM197.

Haemophilus influenzae type b (Hib) capsular polysaccharide (PRP) was selectively hydrolyzed to reducing oligosaccharides, and the fraction containing 3-10 ribosylribitolphosphate repeating units (VS) was conjugated by reductive amination to diphtheria toxin (DTx), its nontoxic derivative CRM197 (Dcr), or diphtheria toxoid (DTd). Conjugate DTx-VS retained approximately 1% of native toxicity, which was eliminated by treatment with formalin. Immunization of rabbits with the conjugates elicited antibody (Ab) to PRP and to DTx but not to a model for the linkage determinant. Human adults given single subcutaneous injections had rises in serum Ab to PRP and in bactericidal activity in vitro; the Ab protected infant rats challenged with Hib. Adults had rises also in Ab to DTd, and these Ab protected rabbits against DTx. A series of two injections of the conjugates Dcr-VS and DTd-VS was tested in infants beginning at 19-23 mo of age. Rises in anti-PRP Ab after the primary resembled the rises after PRP vaccine. In contrast to PRP, the conjugates elicited large rises after the secondary vaccinations and a substantial IgG component. Development of bactericidal activity paralleled the rises in anti-PRP Ab. Secondary rises after Dcr-VS were higher than after DTd-VS. In infants 12-16 mo of age, Dcr-VS (but not DTd-VS) elicited strong primary and secondary Ab responses that included IgG and bactericidal activity. Both conjugates produced consistent rises in Ab to DTd.

Adult↗