Collaborative study for the calibration of a replacement International Standard for diphtheria toxoid for use in flocculation test.

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for diphtheria toxoid for use in flocculation test and its calibration in Lf units. Calibration was performed using Ramon flocculation method, standardized using the 2nd IS. The candidate standard was assigned a unitage of 1870 Lf/ampoule based on results from 25 laboratories in 15 different countries and was established as the 3rd IS for diphtheria toxoid for use in flocculation test by the WHO Expert Committee on Biological Standardization (ECBS) in October 2015. The study also assessed the use of alternative methods for measuring Lf. Participants were asked to determine the Lf value of the candidate standard using an Enzyme Linked Immunosorbent Assay (ELISA) established at NIBSC, or other suitable in-house method. 10 laboratories performed ELISA according to the NIBSC protocol, 1 laboratory performed flocculation using laser-light scattering according to an in-house protocol, and 1 laboratory performed another in-house ELISA. Results suggest these methods may provide suitable alternatives to the Ramon flocculation test, subject to validation, and that the new standard could act as a suitable reference preparation in these methods.

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.

Investigation of immunity level against diphtheria and reinforcement of immunity by booster vaccination for infection control staff in Okayama prefecture.

The prevalence of immunity against diphtheria among Okayama local government staff members involved in diphtheria infection control was measured. Diphtheria booster vaccination was administered to staff members with low antitoxin levels (<0.1 IU/ml) in order to reinforce of immunity. Ninety-one (36.7%) of 248 staff members, 20-69 years of age, had fully protective antitoxin levels (> or =0.1 IU/ml), and the remaining 157 (63.3%) showed levels of <0.1 IU/ml. The rate of full protection was higher in females (44.9%) than in males (22.8%) and was also higher in the diphtheria-pertussis mixed vaccine (born in 1958-1967) and diphtheria-pertussis-tetanus mixed vaccine (born in 1968-) (58.3-61.0%) groups than in diphtheria vaccine (born in 1948-1957) and non-vaccinated (born until 1947) (7.4-18.9%) groups. Though antitoxin levels of 13 (68.4%) out of 19 staff members given booster vaccinations increased to 0.1 IU/ml, 50% of these individuals then showed levels of <0.1 IU/ml after 3 years. Most of the staff members with antitoxin levels of > or =0.1 IU/ml in the non-booster vaccination group maintained their immunity levels for 2-4 years, independent of their history of vaccination. To ensure that staff members of the local government have fully protective antitoxin levels against diphtheria, periodical confirmation of antitoxin levels and booster vaccination should both be systematically carried out.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccine (part 2).

The study is a contribution to the EDQM's efforts to meet some of the expectations of the 3 Rs: Replacement, Reduction and Refinement of animal assays as proposed by Russell and Burch in 1959 and adopted by the European Union in 1986, and specifically to validate alternative assays to replace, for batch-release purposes, the European Pharmacopoeia (Ph. Eur.) in vivo direct challenge procedures for the potency determination of diphtheria toxoid vaccines. The study results may be used in support of the replacement of the multi-dilution direct challenge procedures in different animal models by a single dilution serology test, where appropriate, and to use sera from the same animals for potency testing of several components in combined vaccines. With regard to the latter, the present study explores the possibility of testing both diphtheria and tetanus toxoid potencies using serum from the same animals.

Consistency testing of diphtheria and tetanus to replace potency testing for lot release.

Diphtheria and tetanus vaccines are among the most effective and safe vaccines in the EPI programme. Their mechanism of toxicity and clinical protection is well documented and toxin neutralising antibodies induced by the vaccines are generally accepted as correlates of protection. Despite these positive aspects there are still no generally accepted methods to estimate their potency for routine lot release. Some of these tests use large numbers of animals and rely on lethal challenge tests. Consequently there are ethical and financial barriers to perform these tests. Test results expressed in IU, depend on the animal species or strain used and there is limited information about their predictive value for clinical protection. WHO, supported by the ECBS, has made a proposal to harmonise the current methods into simplified consistency tests, after clinical safety and efficacy, as well as consistency in manufacturing has been established to the satisfaction of the National Regulatory Authority. In principle these tests aim for a proof of consistency in biochemical and immunological characteristics in comparison with the lots shown to be clinically safe and effective. Given that many manufacturers have recently made, or are planning to make clinical trials with new combination vaccines in the near future, recent clinical data are already available, or will be soon, on the clinical safety and efficacy of the D and T components present in these combination vaccines. This will create a unique opportunity to compare the biochemical and immunological characteristics of routinely produced vaccine lots with the clinical lots and to use the consistency approach as suggested by WHO for lot release purpose. Background information and an update will be given about the proposed consistency approach of WHO for routine lot release of the D and T components in vaccines.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines-part 1.

A collaborative study on the evaluation of an alternative functional assay, the Vero cell method, to the Ph. Eur. in vivo challenge procedures for potency determination of diphtheria toxoid in 6 different combined vaccines was initiated in January 2001. The study was an extension of a previous study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. To allow interim evaluation of test results and to monitor study progress, the project was divided into three consecutive phases. The results of Phase I and II studies are presented in this report. Pre-validation (Phase I) study, performed in two laboratories, indicated that comparable diphtheria potency estimates were obtained in the Ph. Eur. direct intradermal challenge assay in guinea pigs, in Vero cell assay and in indirect ELISA for five vaccines of different potencies (range of estimates: ca. 20-200 IU/ml). The correlation coefficients between the challenge assay and the Vero cell assay corresponded to those between the challenge assay and ELISA, confirming that the antibodies play an important role in protection and that predominantly protective/neutralising antibodies are present in guinea pigs, at the time point investigated. It was observed, for Vero cell assays, that about 16-35 (9-28 in Phase II study) fold lower titre of individual serum samples were obtained when using equine, rather than guinea pig reference serum. The study also provided preliminary information that sera from the same guinea pigs may be used for potency determination of both diphtheria and tetanus toxoid components of vaccines. In Phase II, another five laboratories analysed a subset of the vaccines included in Phase I study plus an additional vaccine. Four laboratories performed the lethal challenge assay and one laboratory carried out the intradermal challenge assay. All laboratories also performed the Vero cell assay and both ELISA for diphtheria antitoxin and ELISA for tetanus antitoxin. One laboratory also performed the tetanus ToBI assay. The correlation coefficient (r) between Vero cell assay and ELISA for diphtheria antitoxin ranged from 0.76 to 0.91 in the different laboratories. The correlation between diphtheria serological assays and challenge assays were confirmed satisfactory as ca. 90 per cent of serum-estimates lead to correct prediction of mortality. All laboratories had identical rankings of the vaccines in all serological assays and in the valid challenge assays. The ranking order was identical to assumed/provided potency for the highest and the lowest vaccine. Two of the vaccines had an inversion in some assays and laboratories. As these two vaccines have almost identical potencies in all assays, these inversions are not significant. As the vaccine doses were optimised for the diphtheria component, serum anti-tetanus toxoid/toxin activities varied widely between the vaccines, making it questionable to apply a parallel line model to calculate exact potencies. However, the dose levels used showed a clear regression and good linearity in general. DTaP vaccines containing the IPV component did not always meet the present Ph. Eur. requirements in the serological assays. It should be further investigated in the Phase III study if this is a general feature of such combined vaccines. Preliminary investigations on samples from two laboratories indicate that the neutralising activity of type 1, 2 and 3 polioviruses can also be detected, in a dose-dependent way. Further studies are in progress with serum samples from other laboratories. In the light of results obtained in the first two phases, it is recommended to proceed with Phase III study to investigate reliability of the in vitro assays. In Phase III it will also be further investigated whether the serological assays for D and T components are suitable for the control of the multi-component vaccines currently marketed in Europe.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines - extended study: correlation of serology with in vivo toxin neutralisation.

Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.

[The determination of the protective potency of the diphtheria component in combined vaccines by the indirect hemagglutination (IHA) method]. / Kombine asilarda difteri komponentinin koruyucu gücünün indirekt hemaglutinasyon (IHA) yöntemiyle belirlenmesi.

The sheep erythrocytes which were coated with diphtheria toxoid by formaldehyde-tannic acid method are used to identify the specific antibody response in guinea-pigs that were immunized by adsorbed DPT and DT. It has been demonstrated that this antibody response detected by IHA is highly correlative with protective antibody response detected by Lethal Challenge Test. It has also been determined by IHA method that the immunization potency of diphtheria component can be detected by testing only one immunized guinea-pig.

An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

[Pilot study of the safety and immonogenicity of the diphtheria adsorbed toxoid "d"]. / Badanie pilotowe bezpieczenstwa i immunogennosci anatoksyny bloniczej "d" o zmniejszonej zawartosci antygenu.

The absorbed vaccine d recommended as the booster dose contained in 0.5 ml 2 Lf diphtheria toxoid and was safety and stable, the potency was 30 JO/ml. Eight adults volunteers 25-65 years received toxoid d. The tolerance and the serological response was very good.

[A case of diphtheria in a young man]. / Przypadek blonicy gardla u mlodego mezczyzny.

A case of pharyngeal diphtheria with mild course was observed in a man aged 21 years who had had received four vaccinations. This was the first case of this disease in Poland since 1983, and the possibility is discussed of diphtheria spread in a country with widespread Di-Per-Te vaccinations, as pointed out in the present literature.

Experimental study on estimating the immunogenic potency of diphtheria and/or tetanus toxoids in monovaccines and associated vaccines.

The results obtained using the "classical" active immunoprotection tests on guinea pigs, biometrically interpreted by the regression analysis in "PROBIT" systems revealed that the biopreparations of the Cantacuzino Institute meet the validity conditions related to the parallelism of the "slopes" enabling the calculation of ED50 and of the relative potencies against the reference preparations. The results also showed that the relative potencies of the biopreparations tested by the two methodologies were very close. The geometrical means and the geometrical standard deviations of the titrers obtained in mice pointed to the similarity of the values following inoculation of the Cantacuzino Institute preparations on the one hand and of the international reference preparation on the other.

[Experience of the People's Republic of Bulgaria in controlling diphtheria, tetanus and whooping cough]. / Opyt Narodnoi Respubliki Bolgarii v bor'be s difteriei, stolbniakom i kokliushem.

The analysis of the morbidity and mortality rates in diphtheria, tetanus and pertussis in Bulgaria after the introduction of the compulsory mass immunization of children with combined DPT vaccine is presented. These data indicate that morbidity in diphtheria, tetanus and pertussis has sharply decreased. Favorable results with respect to these three diseases are the consequence of the complete coverage of the child population by immunization with the vaccine whose quality has been steadily improving for the last 20 years. A higher purity of toxoids has been achieved, and at present it exceeds the latest WHO requirements. Pertussis vaccine is produced with the use of strains whose serological characteristics correspond to those of the pertussis strains circulating in the country. The study of the reactogenicity of DPT vaccine, carried out over the period of 20 years, has shown that the vaccine has low reactogenicity.