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Quantitative estimation of diphtheria and tetanus toxoids. 4. Toxoids as international reference materials defining Lf-units for diphtheria and tetanus toxoids.

The Lf-unit, which is used in the control of diphtheria and tetanus toxoid production and in some countries also to follow immunization of horses for production of antitoxins, has hitherto been defined by means of antitoxin preparations. A diphtheria toxoid and a tetanus toxoid preparation, both freeze-dried, were examined in an international collaborative study for their suitability to serve as reference reagents in the flocculation tests and for defining the Lf-units. It was shown that flocculation tests using the reference toxoids are very reproducible and reliable and the WHO Expert Committee on Biological Standardization established: the toxoid called DIFT as the International Reference Reagent of Diphtheria Toxoid for Flocculation Test with a defined content of 900 Lf-units of diphtheria toxoid per ampoule; and the toxoid called TEFT as the International Reference Reagent of Tetanus Toxoid for Flocculation Test with a defined content of 1000 Lf-units of diphtheria toxoid per ampoule.

Animals↗

[The kinetics of vaccine antibodies against tetanus toxoid, diphtheria toxoid, measles virus, poliomyelitis virus and pneumococcus after allogeneic and autologous bone marrow transplantation and revaccination. 3: The kinetics of vaccine antibodies against tetanus toxoid and diphtheria toxoid after allogeneic and autologous bone marrow transplantation and combined revaccination against diphtheria and tetanus].

The 3rd part of the paper deals with the results of a combined revaccination against diphtheria and tetanus in a group of 25 children after allogeneic bone marrow transplantation (BMT) with and without graft versus host disease (GvHD) and after autologous transplantation. It can be shown that in the allogeneic transplanted groups with and without GvHD it is possible to build up a tetanus and diphtheria antitoxin titre in a safe protective cause by a 2nd basic immunisation consisting of 3 single vaccinations starting about 9 to 12 months later. For autologous transplanted children only 1 to 2 vaccinations at a later term than for the allogeneic transplanted children may possibly be sufficient.

Bone Marrow Transplantation↗

Can Haemophilus influenzae type b-tetanus toxoid conjugate vaccine be combined with diphtheria toxoid-pertussis vaccine-tetanus toxoid?

OBJECTIVE: To assess the side effects and immune responses after three serial doses of PRP-T vaccine (a Haemophilus influenzae type b [Hib]-tetanus toxoid conjugate vaccine) given concurrently or mixed with adsorbed DPT vaccine (diphtheria toxoid-pertussis vaccine-tetanus toxoid). DESIGN: Multicentre randomized controlled trial. SETTING: Four public health units in western Canada. PARTICIPANTS: Healthy infants 8 to 15 weeks old at entry who were able to receive routine primary vaccinations. Of 444 infants enrolled, 433 (98%) completed the study. INTERVENTIONS: All infants received PRP-T and DPT vaccines at 2, 4 and 6 months of age: half received them mixed in one injection and the others as separate, bilateral injections. MAIN OUTCOME MEASURES: Side-effects 24 and 48 hours after each dose and serologic responses to each vaccine component. RESULTS: Follow-up was obtained after all 1312 vaccinations. Fever was infrequent in the two treatment groups. Local adverse effects of the PRP-T vaccine were infrequent and mild (e.g., redness was noted in 5.9% of cases and the area of redness was more than 2.5 cm in diameter in 0.8%). The incidence rate of local effects of the DPT-containing vaccines was the same in the two groups except for tenderness, which was more frequent in the group given the mixed vaccine (26.6% v. 17.9%, p < 0.001). Serologic data were available for 97% of the subjects. After the three doses 98.1% of the subjects had a PRP antibody level of 0.15 micrograms/mL or more, and 87.9% had a level of 1.0 micrograms/mL or more, both levels compatible with protection against Hib. Responses to PRP-T were comparable between the treatment groups as were responses to the diphtheria and tetanus toxoids. Pertussis agglutinin titres were reduced after administration of one of two PRP-T lots mixed with DPT vaccine, but responses to four other pertussis antigens were not impaired. CONCLUSION: PRP-T vaccine is well tolerated and immunogenic. Combined PRP-T and DPT vaccines performed satisfactorily and may be the preferred method of administration.

Antibodies, Bacterial↗

Physicochemical analysis of purified diphtheria toxoids: is toxoided then purified the same as purified then toxoided?

Diphtheria toxin can be converted into a highly immunogenic and irreversibly detoxified vaccine either using the conventional process in which the crude toxin is formalinised and subsequently purified (DTxd(conv)) or by detoxification of the highly purified toxin (DTxd(new)). In this study, both DTxd(new) and DTxd(conv) were evaluated by use of physico-chemical methods. For some methods, results were also compared to those obtained for cross-reacting material (CRM197), which is a non-toxic mutant of diphtheria toxin. DTxd(new) was assayed to have a specific purity of at least 2300 LF/mg protein N, thereby exceeding Pharm. Eur. requirements by up to 35%. Superior purity of DTxd(new) could also be demonstrated by size exclusion HPLC experiments and by amino acid composition studies. Far-UV circular dichroism spectroscopy revealed that the secondary structure of DTxd(new) almost resembled that of CRM197, suggesting only minor molecular changes during detoxification. This study worked out differences between purified diphtheria toxoids. Physico-chemical means revealed the advantages of DTxd(new) being the purer and more defined product, thus making it highly efficient for its use as a vaccine carrier as well as a component of vaccine combinations.

Amino Acids↗

Reduced immunogenicity of diphtheria and tetanus toxoids when combined with pertussis toxoid.

The effect of pertussis toxoid on the immunogenicity of diphtheria and tetanus toxoids (DT) was studied during a double blind efficacy trial of an acellular pertussis vaccine. Infants received DT with or without pertussis toxoid at 3, 5 and 12 months of age. Geometric mean concentrations were higher in the DT than in the DT-pertussis toxoid group 1 month (diphtheria toxoid 4.76 versus 3.58 IU/mL, P = 0.009; tetanus toxoid 4.42 versus 2.66 IU/mL, P < 0.0001) and 2 years after the third injection (diphtheria toxoid 0.15 versus 0.10 IU/mL, P < 0.0001; tetanus toxoid 0.38 versus 0.18 IU/mL, P < 0.0001). Pertussis toxoid causes a small but significant reduction of the immunogenicity of diphtheria toxoid and tetanus toxoid.

Animals↗

[Kinetics of vaccination antibodies against tetanus toxoid, diphtheria toxoid, measles virus, poliomyelitis virus and pneumococcus after allogenic and autologous bone marrow transplantation and booster vaccination. 2: Kinetics of vaccination antibodies against diphtheria toxoid after allogenic and autologous bone marrow transplantation].

In the second part of the paper we report on the results of the diphtheria antitoxin valuation of 8 children after allogeneic bone marrow transplantation (BMT) with and without graft versus host disease (GvHD) as well as on the kinetics of the diphtheria antitoxin of 5 children after allogeneic BMT with and without GvHD and of 5 children after autologous transplantation. The antibody valuation was done by a cell-culture assay. Whereas the inspection of the isolated data gives the impression of a swift antibody decrease up to the non-protective level from the 7th month after BMT, the kinetic tests are more highly differentiated. Besides rapidly decreasing values below the accepted protection rate of 0.01 IU/ml in the allogeneic transplanted groups, there are patients with positive antibody titres within an observation period of up to 24 months after BMT in the allogeneic as well as in the autologous transplantation group.

Bone Marrow Transplantation↗

[Kinetics of vaccine antibodies to tetanus toxoid, diphtheria toxoid, measles virus, poliomyelitis virus and pneumococci after allogenic and autologous bone marrow transplantation and booster immunization. 1: The kinetics of vaccine antibodies to tetanus toxoid after allogenic and autologous bone marrow transplantation].

Today BMT belongs to the established methods of treatment in haematology and oncology. Because of the constant increase of healthy long-term survivors after BMT the problem of immunological reconstitution and eventual possible late effects gets more and more importance. One problem, which til now has been few attention paid to, is that of the protection by vaccination after BMT. We report on the kinetics of the tetanus-antitoxin in 20 patients after allogeneic or autologous BMT and demonstrate the influence of a graft-versus-host disease and its therapy on the antibody kinetics. In the group of allogeneic transplanted children without a GvHD the tetanus-antitoxin titers felt below their detection range after a time of about 8 months whereas in the group with GvHD this effect already occurred after nearly 4 months. The autologous transplanted patients have a positive antibody level til the time of 20 months after BMT. As a consequence of the lost protection by vaccination after BMT follows the necessity of revaccinations respectively of boostering after immunological reconstitution.

Adolescent↗

Tetanus toxoid stimulation of the hypothalamic-pituitary-adrenal axis correlates inversely with the increase in tetanus toxoid antibody titers.

In humans, endotoxin activates the hypothalamic-pituitary-adrenal (HPA) axis, and the resulting increase in cortisol modulates the immune response. There is little information on the HPA axis response to other antigens. We examined the effect of the protein antigen tetanus toxoid on HPA axis activity in 10 healthy, premenopausal women (aged 28.6 +/- 2.6 yr). Subjects received im injections of placebo and tetanus toxoid at 1600 h on consecutive days. Blood samples for ACTH and cortisol were obtained every half-hour from--1 to 6 h and at 8, 12, and 16 h after each injection. Compared to placebo, tetanus toxoid administration stimulated significant increases in plasma ACTH and serum cortisol, with the maximum cortisol increase of 1.6-fold occurring 4.5 h after drug administration. Urinary free cortisol increased 1.8-fold in the 8 h after tetanus toxoid administration compared to that after placebo administration. Additionally, there was a significant inverse correlation (r = 0.87; P < 0.005) between the tetanus toxoid-induced increase in serum cortisol and the increase in tetanus antibody levels measured 1 month postvaccination. Thus, administration of the protein antigen tetanus toxoid activated the HPA axis in healthy, premenopausal women. This activation of the HPA axis correlated inversely with the antibody response to tetanus toxoid.

Adrenal Glands↗

In vitro induction of a diphtheria toxoid specific antibody response in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid.

In comparison with the presently used potency test for diphtheria vaccine, in vitro examination of the immunogenicity of the vaccine would have great advantages. For this reason in vitro induction of diphtheria toxoid specific antibody synthesis in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid was investigated. The results showed that a dose dependent synthesis of diphtheria antibody was induced by adsorbed diphtheria toxoid and combined vaccines containing the diphtheria toxoid component. Plain diphtheria toxoid appeared to be less immunogenic in comparison with adsorbed toxoid. There is some indication that the pertussis component had a stimulating effect on the diphtheria antibody synthesis. In conclusion, these results are promising for in vitro examination of the immunogenicity of diphtheria vaccines. The model will be validated for the routine control of diphtheria vaccine.

ABO Blood-Group System↗

Experience with diphtheria toxoid-tetanus toxoid-acellular pertussis vaccine in Japan.

In Japan, the morbidity rate for pertussis per 100,000 population was 147.6 in 1950 when whole cell pertussis vaccine was introduced but dropped to 0.2 in 1972 when routine immunization with a combined vaccine consisting of diphtheria toxoid, tetanus toxoid, and whole cell pertussis had been widely accepted. Thereafter, adverse reactions to the whole cell pertussis vaccine became a social problem and lowered the acceptance of the vaccine. As a result, the morbidity rate increased to 11.3 in 1979. Introduction of the safer yet efficacious acellular pertussis vaccine, consisting of mainly pertussis toxoid and filamentous hemagglutinin, into the routine childhood vaccination in combination with diphtheria and tetanus toxoids in 1981 increased the acceptance rate. The lowest morbidity rate, 0.1, was achieved in 1993. During the next 16 years, almost all cases were in unvaccinated or incompletely vaccinated persons. Regardless of whether whole cell or acellular pertussis vaccine was used, > 90% of the reported pertussis cases were in children < 10 years of age until 1990. However, since 1991, the rate of pertussis in young adults 20-44 years of age has been clearly increasing. To control pertussis, booster vaccination with diphtheria toxoid-tetanus toxoid-acellular pertussis vaccine in adults should be considered.

Adult↗

Development of a purified cholera toxoid. II. Preparation of a stable, antigenic toxoid by reaction of purified toxin with glutaraldehyde.

Evidence is presented which confirms that cholera toxoids obtained by reaction of purified toxin with Formalin possess the ability to partially reactivate both in vivo and in vitro. At the same time, conditions are presented for the preparation of stable, antigenic cholera toxoids by reaction of purified toxin with glutaraldehyde. Treatment of purified cholera toxin with approximately 200 mol of glutaraldehyde per mol of toxin at pH 7.8 reproducibly resulted in the preparation of toxoids which: (i) possessed less than 20 bluing doses per 100 mug; (ii) did not reactivate in vivo or in vitro; (iii) precipitated with, and neutralized antitoxin; (iv) elevated prolonged serum antitoxin in immunized rabbits; (v) protected immunized guinea pigs against toxin skin challenge; and (vi) lent themselves to enhanced antigenicity by means of an in situ adjuvant system which may be suitable for man. Acrylamide gel electrophoresis and molecular sieve chromatography of a series of glutaraldehyde-derived toxoids suggested that the reaction products consisted of monomeric and polymeric species and that the proportion of higher-molecular-weight species was determined by the relative concentrations of toxin and glutaraldehyde. The results suggested a relationship between complete and irreversible elimination of toxicity and the formation of higher-molecular-weight toxoids.

Adsorption↗

The early primary immune response to absorbed tetanus toxoid in man: A study of the influence of antigen concentration, carrier concentration, and sequence of dosage on the rate, extent, and persistence of the immune response to one and to two doses of toxoid.

A quantitative study was performed to determine the effect of toxoid concentration and aluminium salt concentration on the primary immune response (PIR) and the secondary response induced by tetanus toxoid in human volunteers. Four toxoid preparations having 5-fold differences in toxoid concentration, aluminium salt concentration, or both, were administered to four comparable groups of human volunteers. Antitoxin titres in the serum of each volunteer were determined at intervals. The PIR was found to be a function of the antigen concentration, the mineral concentration, and the interaction of both. The secondary response was a function of the antigen concentration; increase in mineral adjuvant concentration had no significant effect. The data suggested that the higher the post-secondary response, the slower the rate of decline over the ensuing 10 months. The distribution of primary responses at day 28 tended to be bimodal. The response to the best preparation suggested that a single-dose toxoid might be developed to immunize populations that may be difficult to retrieve for multiple injections.

Adsorption↗

Preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccines recommendations of the Advisory Committee on Immunization Practices (ACIP).

During spring 2005, two tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine (Tdap) products formulated for use in adolescents (and, for one product, use in adults) were licensed in the United States (BOOSTRIX, GlaxoSmithKline Biologicals, Rixensart, Belgium [licensed May 3, 2005, for use in persons aged 10-18 years], and ADACEL, sanofi pasteur, Toronto, Ontario, Canada [licensed June 10, 2005, for use in persons aged 11-64 years]). Prelicensure studies demonstrated safety and efficacy against tetanus, diphtheria, and pertussis when Tdap was administered as a single booster dose to adolescents. To reduce pertussis morbidity in adolescents and maintain the standard of care for tetanus and diphtheria protection, the Advisory Committee on Immunization Practices (ACIP) recommends that: 1) adolescents aged 11-18 years should receive a single dose of Tdap instead of tetanus and diphtheria toxoids vaccine (Td) for booster immunization against tetanus, diphtheria, and pertussis if they have completed the recommended childhood diphtheria and tetanus toxoids and whole cell pertussis vaccine (DTP)/ diphtheria and tetanus toxoids and acellular pertussis vaccine (DTaP) vaccination series (five doses of pediatric DTP/DTaP before the seventh birthday; if the fourth dose was administered on or after the fourth birthday, the fifth dose is not needed) and have not received Td or Tdap. The preferred age for Tdap vaccination is 11-12 years; 2) adolescents aged 11-18 years who received Td, but not Tdap, are encouraged to receive a single dose of Tdap to provide protection against pertussis if they have completed the recommended childhood DTP/DTaP vaccination series. An interval of at least 5 years between Td and Tdap is encouraged to reduce the risk for local and systemic reactions after Tdap vaccination. However, an interval less than 5 years between Td and Tdap can be used; and 3) vaccine providers should administer Tdap and tetravalent meningococcal conjugate vaccine (Menactra, sanofi pasteur, Swiftwater, Pennsylvania) to adolescents aged 11-18 years during the same visit if both vaccines are indicated and available. This statement 1) reviews tetanus, diphtheria and pertussis vaccination policy in the United States, with emphasis on adolescents; 2) describes the clinical features and epidemiology of pertussis among adolescents; 3) summarizes the immunogenicity, efficacy, and safety data of the two Tdap vaccines licensed for use among adolescents; and 4) presents recommendations for tetanus, diphtheria, and pertussis vaccination among adolescents aged 11-18 years.

Adolescent↗

Quantitative estimation of diphtheria and tetanus toxoids. 5. Comparative assays in mice and in guinea-pigs of two diphtheria toxoid preparations.

Two freeze-dried international reference diphtheria toxoids of different origin were compared in biological assays in guinea-pigs and mice under different adjuvant conditions. When the antigenic content in the two toxoids was used as denominator for determination of relative potency, that is to say quantitation of immunogenic power per unit amount of antigen, the design of the animal assay proved to have a major influence. Similar observations have been made previously also for tetanus vaccines. It is concluded that diphtheria vaccines as well as tetanus vaccines can hardly be quantitated unambiguously using the currently recommended potency assays in animals. A new scheme for control of toxoid vaccine production is suggested, with more emphasis on the control of the bulk purified toxoid, which would make the release of final products more simple and rapid.

Animals↗

Studies on the adequate composition of diphtheria and tetanus toxoids-with reference to the amounts of toxoids and aluminum adjuvant.

Immune response of man to tetanus and diphtheria-tetanus combined toxoids with different compositions was investigated, with special reference to the long-term immunity. Adsorbed tetanus toxoid with a potency of about 25 IU per human dose endowed a long-lasting immunity sufficient to prevent tetanus to the vaccines of various age groups, when administered in two doses, followed by a booster injection. Two doses of adsorbed diphtheria-tetanus combined toxoid with potencies of about 20 IU per dose for both components were sufficient to give immunity in infants against diphtheria and tetanus. However, a booster injection should be given within a year to endow a long-lasting immunity. In the case of booster immunization of children with a complete history of immunization against diphtheria and tetanus, a potent long-term immunity was endowed by a single injection of diphtheria-tetanus combined toxoid (plain) with 1 and 3.5 IU per dose, for diphtheria and tetanus components, respectively.

Adjuvants, Immunologic↗

IgG1 is the predominant subclass of in vivo- and in vitro- produced anti-tetanus toxoid antibodies and also serves as the membrane IgG molecule for delivering inhibitory signals to anti-tetanus toxoid antibody-producing B cells.

Peripheral blood leukocytes from individuals immunized with tetanus toxoid can be stimulated by pokeweed mitogen to produce IgG anti-tetanus toxoid antibody (IgG-Tet) in vitro. Previous studies have shown that treatment of these cells with tetanus toxoid or anti-human IgG reagents can inhibit this in vitro antibody synthesis. We have examined the four IgG subclasses on the surface of B cells for their relative contributions in the anti-IgG antibody-induced inhibition of IgG-Tet production. With all donors, the inclusion of anti-IgG1, but not anti-IgG2, -IgG3, or -IgG4, antiserum resulted in the in vitro inhibition of IgG-Tet synthesis. The magnitude of this inhibition was similar to that induced by treatment of the B cells with tetanus toxoid antigen. When the supernatants from normal in vitro cultures were assayed for IgG-Tet of the various IgG subclasses, it was observed that the IgG-Tet were almost exclusively IgG1. Similar results were obtained when serum IgG-Tet were measured. Thus, IgG1 appears to be the major subclass for (1) the in vivo-produced IgG-Tet, (2) the in vitro-produced IgG-Tet, and (3) the membrane receptor which can selectively convey an inhibitory signal to the IgG-Tet B cell.

Antibody Formation↗

Immunochemical studies of antitoxin produced in normal and allergic individuals hyperimmunized with diphtheria toxoid. I. Relationship of skin sensitivity to purified diphtheria toxoid to the presence of circulating, non-precipitating antitoxin.

Among a group of 131 young adults tested, there was a high degree of correlation between the occurrence of immediate skin reactions of the wheal and erythema type produced by purified diphtheria toxoid and personal or familial history of allergy of the hay-fever type. Of 39 Schick-negative subjects who received a "booster" dose of purified diphtheria toxoid, 19 showed no immediate skin reactions before immunization. The development of skin sensitivity in these subjects was associated with the production of non-precipitating circulating antitoxin. Three subjects produced 20 units or more antitoxin per cc. serum following immunization, without demonstrable precipitins. Despite the fact that none showed immediate skin reactions to Schick toxoid prior to immunization, all 3 possessed a high degree of skin sensitivity to toxoid following immunization.

Adult↗