Study of diphtheria anatoxins in immunochemical and tissue culture assays

Equine diphtheria antitoxins from different manufacturers were studied. Their immunochemical interaction with diphtheria toxin, toxoid, and antigens of Corynebacterium diphtheriae in ELISA and immunoblotting assays as well as biological activity in CHO cell assay were compared. The discovered differences between antitoxin samples with stated equal activity in IU/ml point to heterogeneity of antigen composition in preparations used for immunization. Mentioned methods allow to standardize antitoxins basing on their biological activity and immunochemical characteristics.     

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.     

Immune response in adult immunized with adsorbed DT-M anatoxin with reduced antigen content and Imovax-DT-adulte vaccine

The comparative study of immune response after immunization of adults with adsorbed DT toxoid with reduced antigen content and Imovax-DT-adulte vaccine, as well as the safety of these preparations, was made. The study revealed that immunization of adults with adsorbed DT toxoid having reduced antigen content, made in two injections, and the injection of Imovax-DT-dulte vaccine, as well as the successive injection of these preparations, produced the same the levels of antitetanus immunity. Antidiphtheria immunity, evaluated by the number of seroconverted to diphtheria persons following two injections immunization was similar for the two preparations, while the level of antidiphtheria antibodies was higher in persons immunized with adsorbed DT toxoid. The immune stratum index was rather high among persons aged 16-29 years. This age group exhibited the highest number of persons, seropositive to both diphtheria and tetanus. Both vaccine preparations, adsorbed DT toxoid with reduced antigen content and Imovax-DT-adulte vaccine, were found to be equally capable of inducing autoimmune reactions in the vaccinees, detected by laboratory methods.     

The specific prophylaxis of diphtheria in adults with adsorbed DT-m anatoxin]

The dependence of immune response on commonly observed immunity characteristics prior to immunization has been established on the basis of the study of the kinetics of immune response in adults receiving injections of adsorbed diphtheria-tetanus (DT) toxoid with reduced antigen content, both for routine immunization and on epidemiological indications. The necessity of the practical use of immunological screening for the differentiated approach to the choice of a suitable preparation (adsorbed diphtheria toxoid, adsorbed DT toxoid or adsorbed DT toxoid with reduced antigen content) and immunization schedule for adults, especially in epidemic foci, has been substantiated.     

A comparison of the indirect haemagglutination test with the toxin neutralization test for the estimation of diphtheria antitoxin in mouse sera

Serum samples from 42 groups of mice immunized for different immunization periods with various doses of Adsorbed Diphtheria-Tetanus Vaccine, Adsorbed Diphtheria-Tetanus and Pertussis Vaccine and a standard diphtheria toxoid were assayed for their diphtheria antitoxin content by indirect haemagglutination (IHA) and by toxin neutralization (TN) tests. A very good correlation of 0.91 was obtained between the results of the two methods. There was no statistically significant difference between the IHA and the TN titres obtained. Adsorption with sheep red cells and treatment of the sera with 2-mercaptoethanol had no effect on the IHA titres. The minimum level of antitoxin detectable by the IHA test was 0.00039 IU ml-1. IHA proved to be a sensitive, specific and reproducible method which can be used reliably for the assay of diphtheria antitoxin in mouse sera.     

Duration of immunity in children who received a primary complex of inoculations with ADT-M anatoxin and an assessment of postponed secondary revaccination against diphtheria and tetanus (the results of an epidemiologic trial)

The prolonged observations of the immunological effectiveness of adsorbed diphtheria-tetanus toxoid with reduced antigen content in children who had received the primary course of immunization with this preparation showed that the preparation induced the development of prolonged and intensive immunity to both infections. In 2-3 years after the first booster immunization the protective level of diphtheria antitoxin was registered in 89.9% and that of tetanus antitoxin, in 99% of children. 6 years later the level of immunity remained practically unchanged: the titers of diphtheria antitoxin above the protection level were determined in 92% and those of tetanus antitoxin, in 97% of children. These data made it possible to increase intervals between booster immunizations to 6-7 years in children of this category. The results of the epidemiological trial made to find out the possibility of a change in the timing of the second booster immunization confirmed the expediency of postponing booster immunization from 6 and 11 years to 9 and 16 years of age.     

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.     

Radioimmunodiffusion technique for determining diphtheria antitoxin.

A radioimmunodiffusion technique for detecting low levels of diphtheria antitoxin was developed. Diphtheria toxoid was labelled with 125I to facilitate detection of lines of precipitation by the use of X-ray film, the lower limit of detection being 0.001 unit per millilitre of diphtheria antitoxin.     

Characteristics of immune response to diphtheria anatoxin in children with different immune status during the second age-scheduled revaccination

The dynamics of the intensity of specific antidiphtheria immunity after the second age-scheduled revaccination was studied in 129 practically healthy children. The study revealed that the formation of immunity depended on the initial functional state of the immune system before the injection of diphtheria toxoid. Three variants of immune response were determined and the immune status corresponding to each of these variants was characterized. As shown in this study, children with the hyperergic character of immune response were characterized by relatively high initial titers of antitoxin, and the injection of an additional dose of the antigen led to the prolonged state of hyperimmunization with the subsequent decrease of the intensity of immunity by half, registered in the catamnestic observation for 4 years. Children with the hypo- and normoergic variants of immune response were characterized by the most stable immune response to diphtheria toxoid, and during the catamnestic observation they formed the levels of antibody titers 2.5- to 3-fold higher than before immunization. But the protection characteristics in children with the third variant were the lowest among the children under study.     

Variants in the immune response to revaccination with diphtheria anatoxin in adults

The immunological effectiveness of the revaccination (made in two injections) of 488 adults aged 18-67 years with diphtheria-tetanus toxoid is discussed; the parallel study of the results of this revaccination was carried out in the diphtheria toxin neutralization test on Vero cells and in the passive hemagglutination (PHA) test. The specific features of the dynamics of the increase of diphtheria antitoxic antibodies, depending on the initial immunity level, the age and the sex of revaccinated persons, were determined. Among persons with the low level of circulating antibodies before revaccination four variants of immune response to the injection of diphtheria toxoid were registered: variant 1--rapid reaction like in secondary immune response (53.6%); variant 2--delayed but effective reaction like in primary immune response (27.3%); variant 3--slow weak response (6.5%); and variant 4--the absence of effective immune response to immunization made in 2-3 injections (12.6%). The immunological and neutralizing properties of diphtheria antitoxic antibodies in the process of immunization made in 2 injections were evaluated. Persons with abnormal immune response (variants 3 and 4) produced defective antibodies, displaying immunological activity in the PHA test, but in most cases unable to neutralize diphtheria toxin in vitro when tested on Vero cells.     

Collaborative study for the calibration of a replacement international standard for diphtheria toxoid adsorbed

We present the results of a collaborative study for the characterization of a preparation of diphtheria toxoid adsorbed, and its calibration in terms of the 3rd International Standard (IS) for Diphtheria Toxoid Adsorbed. Calibration was performed using established World Health Organization (WHO) and European Pharmacopoeia (Ph. Eur.) protection models. Two candidate toxoid preparations were included in the study, one of which was adopted as a replacement Ph. Eur. Biological Reference Preparation (BRP, batch 4) in February 2009. The second candidate preparation was found to have a unitage of 213 IU/ampoule based on the calibration by in vivo bioassay in 19 laboratories in 16 countries, and was established as the 4th IS for Diphtheria Toxoid Adsorbed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2009.The study also assessed performance of the replacement standard in mouse and guinea pig serological assays which are used as alternative procedures for diphtheria potency testing. Participants tested both candidate preparations and potency was expressed in relative terms only. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays and that the Vero cell assay may be suitable for calibration of future replacement standards.     

The characteristics of the protective and antigenic activities of purified staphylococcal anatoxin in trials with mice of different genotypes

The protective potency of purified staphylococcal toxoid by the survival rate of immunized mice challenged with the culture of Staphylococcus aureus strain L-1726 and the antigenic properties of the toxoid, determined by the level of antitoxin in the blood of mice and by the intensity of cell-mediated immunity in the spleen-cell migration inhibition test, were studied. The experiments were made on CBA and C57BL/6 mice. Purified staphylococcal toxoid was shown to possess antigenic and protective properties in a wide range of doses between 0.15 and 15 binding units per mouse. The protective effect of the toxoid in CBA mice was manifested in the presence of circulating antibodies and cell-mediated reaction or only in the presence of the toxoid. In C57BL/6 mice the protective effect of the toxoid was less pronounced and appeared in combination with the induction of cell-mediated immunity in the presence of an extremely low antibody level (0.062 I.U.).     

Development of a mouse model to estimate the potency of the diphtheria toxoid component of diphtheria-tetanus and diphtheria-tetanus-pertussis vaccines

A mouse model to estimate the potency of the diphtheria toxoid component in diphtheria-tetanus vaccines and diphtheria-tetanus-pertussis vaccines has been developed as an alternative to the conventional method of testing in guinea-pigs. Optimal conditions with regard to dose, route and period of immunization have been standardized. The maximum levels of antitoxin were detected five weeks after vaccination and the s.c. route was found to be optimal. Potency data have been compared with other studies in mouse models and with those obtained by the conventional method in guinea-pigs.     

The immunomodulating properties of a diphtheria bacterial vaccine in in vitro experiments

In this investigation lymphocytes sensitized with Corynebacterium diphtheriae antigens obtained from carriers and convalescents were used. The new diphtheria bacterial vaccine Codivac, in contrast to other comparable preparations (diphtheria toxoid, staphylococcal toxoid, staphylococcal vaccine, levamisole), was found to produce a more direct effect by modulating the levels of T-lymphocytes, depending on their initial levels in the patient. Codivac, together with other preparations, can be used for the study of the problems of immunostimulation and immunocorrective therapy.     

Comparative study of the immunoglobulins and specific antibodies in the blood of rabbits immunized with ADTP vaccine and ADT-anatoxin with normal and reduced antigen contents

Adsorbed DPT vaccine and adsorbed DT toxoids with normal and reduced antigen content were used for the immunization of rabbits. The levels of IgM and IgG and the dynamics of antibodies to diphtheria and tetanus toxins and to Bordetella pertussis in the blood sera of the animals were studied in the postvaccinal period (on days 15 and 34). This study revealed that the reduction of the antigen content of adsorbed DT toxoid to 5 Lf of diphtheria toxoid and 5 binding units of tetanus toxoid did not decrease the capacity of the preparation for increasing the levels of IgG and IgM, antibodies to diphtheria and tetanus toxins in the sera of the rabbits. The reduced content of these toxoids in adsorbed DPT vaccine did not affect its capacity for inducing the enhanced synthesis of IgG, antibodies to diphtheria and tetanus toxins, while the production of IgM and IgA remained unchanged. At the same time an increase in the titers of antibodies to B. pertussis in the animals was less pronounced than that observed after the injection of commercial adsorbed DPT vaccine. Additional investigations are necessary in order to establish the protective potency of the pertussis component in adsorbed DPT vaccine with the reduced content of toxoids and to find out the optimum antigenic composition for this preparation.     

The immunization of children with combined diphtheria and tetanus vaccine in Sweden

Two groups derived from 97 children three-four months of age were vaccinated with diphtheria and tetanus vaccines containing either a routinely prepared diphtheria toxoid or a more purified preparation. Two injections were given with an interval of one month and a third injection was given one year after the first. Prior to the third injection no child was without protection against diphtheria, i.e. had an antitoxin titre less than 0.01 IU ml-1. After the third injection 95 and 94% of the children vaccinated with the routinely and more purified diphtheria toxoids, respectively, had diphtheria antitoxin titres greater than 1 IU ml-1 (estimated to provide protection for at least ten years). Systemic reactions such as fever and malaise occurred in five children. Local reactions greater than 10 cm were observed in three children and reactions greater than 5 but less than or equal to 10 cm were seen in 14% of the children. The routinely prepared combined diphtheria and tetanus vaccine, DT, produced very good immunity against diphtheria with moderate side effects. The use of a more purified diphtheria toxoid in the combined vaccine produced the same immunity and side effects.     

IgA and IgA diphtheria antitoxin responses from human tonsil lymphocytes.

Human tonsil lymphocytes were stimulated with diphtheria toxoid and then cultured in a Marbrook culture system so that antibodies could be measured in the culture supernatant. Specific antibodies were measured with excess radiolabeled antigen and antisera specific for each immunoglobulin class. Good IgG and IgA diphtheria antitoxin responses have been obtained and responding culture supernatants were shown to neutralize toxin. The relationship between antitoxin response in vitro and immunization of donors with toxoid was investigated. It was found that at least two immunizations after the age of 6 months were necessary to prime the tonsils for an in vitro antibody response. The IgG and IgA in culture supernatants were demonstrated by immunodiffusion and were measured by radioimmunoassay. By sucrose density gradient ultracentrifugation, it was shown that 40% of the IgA produced in the cultures was greater than 7S. Evidence was obtained that neither the IgA nor the specific IgA antitoxin bears secretory piece. It appears that human lymphocytes from tonsils produce polymer IgA in vitro without secretory piece.     

Immunisation of adults during an outbreak of diphtheria.

In an outbreak of infection due to Corynebacterium diphtheriae in a hospital for mentally subnormal adults sera from 211 members of staff were screened for diphtheria antitoxin titres. Of these, 79 (37%) required immunisation, and a low dose preparation (1 LfU of diphtheria and 10 LfU tetanus toxoids) was offered. Of the 64 subjects who accepted a single immunisation and were subsequently retested, seroconversion to diphtheria toxoid occurred in 45 (70%), the rate being highest in younger adults. Seroconversion to tetanus toxoid occurred in 59% of subjects. Local reactions to the single dose were reported by 29 (43%) subjects, and nine (13%) experienced moderately severe local reactions and systemic symptoms. We conclude that adults should not be vaccinated without previous screening for susceptibility to diphtheria; that neither previous immunisation nor age is reliable in predicting the need for vaccination; and that though a single booster dose of diphtheria toxoid is probably effective in adults under 45, two doses should be given to those in the older age group.     

不同粒径氢氧化铝佐剂对白喉类毒素免疫效果的影响

研究不同粒径的氢氧化铝[Al(OH)3]对白喉类毒素的吸附效果,用于指导疫苗生产,提高疫苗质量。用透射电镜测得的不同粒径的Al(OH)3分别吸附白喉类毒素及用絮状单位测定法和疫苗效价测定法对其吸附效果作比较,并经统计学处理。试验结果显示,粒径为210nm的Al(OH)3对白喉类毒素吸附效果及对白喉疫苗效力的免疫增强作用明显好于粒径600nm的Al(OH)3。实验证实,Al(OH)3佐剂的粒径大小与白喉类毒素的吸附效果及疫苗的免疫原性密切相关。     

The immunization of adults against diphtheria in Sweden

Two hundred and three women who disclaimed vaccination against diphtheria were divided into four groups and injected with either 2.0 or 6.25 Lf of a routine diphtheria toxoid or of a more purified preparation. One hundred and twenty-six of these women who did not show a secondary antibody response were given a second and a third injection one month and one year, respectively, after the first injection. Prebooster (third injection) antitoxin titres of greater than or equal to 0.01 IU ml-1 (the minimum level for protection) were found in 22 and 37% of those who received 2.0 and 6.25 Lf, respectively. Postbooster titres of greater than or equal to 1.0 IU ml-1 (calculated to give a protection of at least ten years of duration) were found in 23 and 58% of those who received 2.0 and 6.25 Lf, respectively. The rate of untoward reactions was low. Fever of short duration occurred in five women. Four out of the five women received 6.25 Lf of the more purified diphtheria toxoid and one 2 Lf of the routine toxoid. Local reactions greater than 10 cm were observed in three women. All received the higher dose, 6.25 Lf of diphtheria toxoid. Local reactions greater than 5 but less than or equal to 10 cm occurred in up to 13% (6.25 Lf of diphtheria toxoid). No significant difference between the groups of women vaccinated with routine or more purified toxoid was found. It was concluded that the diphtheria toxoids in the two doses of 2 Lf and 6.25 Lf did not induce a satisfactory immune response. To induce adequate protection the dose of diphtheria vaccine needs to be the same for adults and children, i.e. 12.5 Lf.