Quality control of routine, experimental and real-time aged diphtheria toxoids by in vitro analytical techniques.

Physicochemical and immunochemical techniques can be used to assess the quality of diphtheria toxoid vaccines. In a previous paper [Metz B, Jiskoot W, Hennink WE, Crommelin DJA, Kersten GFA. Physicochemical and immunochemical techniques predict the quality of diphtheria toxoid vaccines. Vaccine 2003;22(2):156-67], techniques were introduced which indicated toxoid quality with respect to safety and potency: SDS-PAGE, primary amino group determination, fluorescence/denaturation, circular dichroism and biosensor analyses. These analyses were performed with experimental toxoids from one toxin batch. In the present study, the quality of regular vaccine batches of different manufacturers, the properties of real-time aged products and a number of experimental toxoids were investigated, using the above-mentioned analytical techniques. We had the unique opportunity to analyse toxoids that were up to 40 years old. The real-time aged diphtheria toxoids showed hardly any structural differences as compared to the recently prepared products in both the analytical chemical techniques and the conventional potency and safety tests. The analytical assays discriminated between regular diphtheria toxoids and experimental toxoids prepared by methylation, acetylation or glutaraldehyde treatment. The analytical data showed a clear correlation with potency and safety of these toxoids. Based on the results, we refined the described physicochemical and immunochemical criteria that a standard diphtheria toxoid has to meet. We recommend further validation of these techniques for quality control of diphtheria toxoid vaccine because of their high precision and easy performance as compared to conventional in vivo procedures.

Ionic interfaces and diphtheria toxoid interactions

We present here a systematic study on the purification of the diphtheria toxoid (Dtxd) produced at the Instituto Butantan, byadding only one step on the entire process of its production. Aliquots of 1.0 ml of Dtxd were added to an equal amount of QSepharosepreviously equilibrated with 500mM Tris, pH 5.0–9.0 (increments of 0.5 pH units). The best condition for the Dtxdmonomer adsorption was achieved at pH 9.0. The best condition for desorption was obtained with 300mM NaCl. After studyingthe gel binding capacity for Dtxd, a column (C20/20) equilibrated with 500mM Tris, pH 9.0, was prepared. The purification factorfor Dtxd was 1.5. The final recovery of Dtxd was 68.75%, with 90.31% purity. The process methodology presented here is a veryrealistic sequence of separation steps, which is perfectly compatible with the production requirements. Vaccination with ‘‘toxoidhighly purified toxin’’ is known to confer a strong immunity on people in the absence of undesirable reactions, which led experts ofEuropean Pharmacopoeia to recommend its use both for children and adult vaccination.

Disturbance of cardiovascular circadian rhythms by pertussis vaccine in freely-moving rats.

Vaccination of young children with diphtheria, tetanus, poliomyelitis and pertussis (DTPoP) vaccine is effective in preventing outbreaks of whooping cough but adverse events sometimes occur. This pilot study shows that in freely-moving rats, multiple treatment with DTPoP (at day 0 and day 5, 6 ml/kg i.v.) increased heart rate (HR) for 5 days after the first treatment and decreased diastolic blood pressure (DBP) for at least 26 days after the first treatment and inhibited the circadian rhythm of HR and DBP for at least 10 days. DTPo vaccine, containing no pertussis vaccine, was free of such effects. Thus, in rats, the pertussis component of DTPoP acts on the cardiovascular system and disturbs its circadian rhythm. The contribution of these findings to clinical adverse effects is as yet unknown and needs further research.

Statistical evaluation of numbers of animals to be used in vaccine potency testing: a practical approach.

Some of the guidelines for potency testing of vaccines issued by regulatory bodies such as the European Pharmacopoeia (EP) and WHO are detailed and stringent (e.g. EP monograph for Newcastle Disease (ND) Vaccine (inactivated)), whereas others only stipulate that the number of animals used should be sufficient to meet the required accuracy (e.g. EP monograph for Hepatitis A vaccine (inactivated)). Simulation studies in our laboratory using historical ND potency test data indicated that the number of animals specified in the monograph is too high; a considerable reduction from 10 to seven animals per group does not substantially influence the precision of the results. Multipoint models (e.g. EP monograph for Tetanus Vaccine (adsorbed)) require at least three dilutions per vaccine for testing for response linearity. However, when historical data clearly show that in the range used the response curves are linear, it is superfluous to verify this in every test. Furthermore, linearity has little priority for a valid parallel line assay calculation. A simulation study using historical Diphtheria potency test data showed that calculations using two dilutions per vaccine in relatively small groups of animals produced results comparable to those obtained from the full assay. This procedure still enables calculation of the relative potency, in contrast to the 1 + 1 method, which gives only a pass or fail result, while the number of animals required is only slightly higher. This method could be applied in cases where the 1 + 1 method fails. In conclusion, by providing guidelines on methods in which proven consistency in production and testing may be taken into account, manufacturers are more stimulated to look for other (cheaper) ways to test the potency of a vaccine using less animals.

Long-term high immune response to diphtheria toxoid in rodents with diphtheria toxoid conjugated to dextran as a single contact point delivery system.

Cross-linked dextran beads were used as a carrier for development of a 'single-contact' vaccine delivery system. Diphtheria toxoid (DT) was covalently coupled to dextran beads (DEX-DT conjugate). The conjugate was immunoreactive with antibodies raised against native DT. Immunization of rats with DEX-DT conjugate generated on average 24 times higher antibody titres against diphtheria toxoid than immunization with the conventional DT absorbed on alum. The immune response was sustained for 9 months, with gradual decline of antibody titres thereafter. Significant antibody titres were measurable in rats after 1 year of immunization. DEX-DT had neither acute nor long-term adverse effects on the health of animals, as evidenced by local reactions, general behaviour, food intake, body weight gain and survival compared with controls.

The pyrogenicity of pertussis vaccine in mice and the factors in the vaccine responsible for this effect.

The injection of whole cell pertussis vaccine into mice produced a biphasic fever reaction with two peaks appearing after about one and four hours, respectively. A method for the quantitative determination of each peak fever activity was developed and the factor responsible for each activity was investigated. The first and the second peak fever activities did not parallel each other in individual vaccines. The earlier fever activity appeared to correlate with endotoxin activity in individual vaccines while the later appeared to correlate with histamine-sensitizing factor (HSF) activity. The later peak fever activity was greatly reduced by heating the vaccine at 100 degrees C for 30 min while the first was little affected by such treatment. It was concluded that the fever activity of pertussis vaccine in mice may be ascribed to the combined actions of endotoxin and a heat-labile substance, possibly HSF.

Determination of the epinephrine-refractory hypoglycaemic activity of whole-cell pertussis vaccine in mice.

A new assay method has been developed for the quantitative estimation of the inhibitory effect of pertussis vaccine on epinephrine-induced hyperglycaemia in mice. The statistical analysis of the assay was based on logarithm-transformed estimates of the blood glucose levels. The method was sufficiently sensitive to detect the activity of 0.004 millilitre of commercial combined diphtheria-tetanus-whole cell pertussis vaccine. The estimated common variance was as small as 0.0034 and the assay was highly reproducible. Among commercial vaccines there was a significant difference in activity. The activity of a stock pertussis vaccine was inactivated by 5 mM glutaraldehyde at 37 degrees C for 30 min, but resisted treatment with 40 mM formaldehyde at 37 degrees C for 5 days. The extent of inactivation with the chemicals was calculated by a parallel line assay as the activity relative to that of untreated control pertussis vaccine.

Local reactions of the saponin Quil A and a Quil A containing iscom measles vaccine after intramuscular injection of rats: a comparison with the effect of DPT-polio vaccine.

A short-term toxicity study was performed to investigate local reactions and hematological changes after im injection of Quillaia A (Quil A; 50 or 600 micrograms/ml) an essential component of an immunostimulating complex (iscom), a novel form of a subunit vaccine, and of iscom measles vaccine containing 360 micrograms Quil A/ml. The effects were compared with those caused by a sterile phosphate-buffered saline solution and by diphtheria-pertussis-tetanus-polio (DPT-polio) vaccine. Groups of six male rats were injected im with 0.25 ml of the test solution: on Day 0 in the left and on Day 7 in the right musculus gastrocnemius. On Day 14 the animals were killed, and the left and right inguinal lymph nodes were weighed. These organs and the left and right musculus gastrocnemius were investigated microscopically. The only hematological changes observed occurred in the group injected with DPT-polio vaccine: a decrease in the Hb value and in the number of erythrocytes and an increase in mean corpuscular hemoglobin content and in the number of leucocytes. The weights of the left and right inguinal lymph nodes were significantly increased in rats injected with DPT-polio vaccine, iscom measles vaccine, and the high dose of Quil A. The most intense granulomatous inflammatory reaction, mainly consisting of activated macrophages, was observed at the injection sites of all animals of the DPT-polio group. Only one animal injected with the iscom measles vaccine showed a moderate inflammatory reaction of the same type.(ABSTRACT TRUNCATED AT 250 WORDS)

Acellular pertussis vaccines: evaluation of reversion in a nude mouse model.

An animal model has been developed to assess the safety of acellular pertussis vaccines in terms of reversion to toxicity. Adsorbed pertussis toxoid preparations, alone or combined in a DTP formulation, were administered to nude mice intraperitoneally. In parallel, groups of positive and negative control mice received pertussis toxin and buffer, respectively. The circulating white blood cells of the animals were monitored for 28 days. Mice immunized with glutaraldehyde toxoid preparations did not develop a lymphocytosis during the observation period, whereas mice immunized with an experimental formalin pertussis toxoid vaccine exhibited a high lymphocytosis six days after vaccine administration, demonstrating, in this model, a reversion of the toxoid. The nude mouse model thus appears to reveal the in-vivo reversion of pertussis toxoids and could be included in the quality control panel for the assessment of the safety of acellular pertussis vaccine.

The effects of reductions in the numbers of animals used for the potency assay of the diphtheria and tetanus components of adsorbed vaccines by the methods of the European pharmacopoeia.

Forty eight assays of adsorbed diphtheria vaccine and seven assays of adsorbed tetanus vaccine using either a lethal challenge (38 assays) or a serum neutralization test (17 assays) were evaluated for the effects of reductions in the number of animals used at each dilution on the potency values and 95% confidence intervals. The results were assessed in the light of the requirements of the European Pharmacopoeia and the WHO. In the majority of assays, 50% of the number of animals presently required would have sufficed for the determination of a potency within the limits of confidence stipulated by the European Pharmacopoeia and the WHO. Therefore it is concluded that a simplified assay with a reduced number of animals is suitable for the routine potency testing of the diphtheria and tetanus components of the combined vaccines and the monovalent that were examined. Flexibility in the national and international requirements in respect of the numbers of animals used at each dilution is suggested for the routine potency assay of the diphtheria and tetanus components of adsorbed vaccines.

Safety and immunogenicity of a diphtheria-tetanus-pertussis vaccine containing an acellular pertussis component.

In an open-label study, we evaluated the safety and immunogenicity of a diphtheria-tetanus-pertussis (DTP) vaccine containing an acellular pertussis component. Thirty-one normal, healthy children 4 to 6 years old received a single dose. All subjects had received their primary DTP series and DTP booster at age 18 months. All but three of the subjects were followed up for one month. Two subjects developed fever (100 F [37.8 C]) during the 24 hours after dosing. Eleven subjects had mild local reactions reported by parents at the 24-hour visit. One subject had a small "sterile abscess." Paired antibody assays revealed enhancement of titers in 19 of 21 for pertussis, 20 of 21 for diphtheria, and 19 of 21 for tetanus. Although the sample is small, the experimental vaccine appears to have been safe and effective in the subjects studied.

Comparative study of rat-paw oedema tests for assessing reactivity of B. pertussis-containing combined vaccines.

Two volumetric (electrocapacity plethysmography and mercury volumetry) and two linear methods (a new type of scale and a calliper rule) for rat-paw oedema measurement used in local reactivity of combined vaccines containing a B. pertussis component were compared. The reproducibility and sensitivity of the methods was tested on a diphtheria-tetanus-pertussis vaccine and a toxic pertussis suspension. The linear methods proved more adequate for measuring small and chiefly unidirectionally (vertically) progressing oedemas.

Swelling of the brain in mice caused by pertussis vaccine--its quantitative determination and the responsible factors in the vaccine.

Intracerebral injection of vaccine into the mouse induced swelling of the brain. The swelling reached the maximum in the intensity by day 1 and persisted for several days. A method for quantitative determination of the brain-swelling activity of the vaccine was developed. A positive regression coefficient was found only between the brain-swelling and the lymphocytosis-promoting activities. Such activity was no longer shown with the vaccine heat-treated for 30 min at 80 C, but it was restored upon addition of the lymphocytosis-promoting factor (LPF) that caused no brain swelling by itself. The activity, therefore, was ascribed to cooperation of LPF and a certain heat-stable component other than endotoxin contained by pertussis vaccine.

[Evaluation of the toxic action of prophylactic and therapeutic preparations on cell cultures of different types and origin. II. The cytotoxic action of adsorbed DPT vaccine and its components on cells of the continuous L132 line]. / Otsenka toksicheskogo deistviia profilakticheskikh i lechebnykh preparatov na kulturakh kletok raznogo vida i proiskhozhdeniia. Soobshchenie II. Tsitotoksi-cheskoe deistvie vaktsiny AKDS i ee komponentov na kletki perevivaemoi linii L132.

Different batches of the same preparation manufactured at the same enterprise, or at different enterprises, in accordance with the same manufacturing regulations have been found to be capable of producing a damaging effect of different intensity on the continuous cell culture L132. The titers vary, according to their cytotoxic effect, from 1 : 32 to 1 :2048. The components of B. pertussis antigens and thimerosal solutions have been found to produce the most pronounced cytotoxic effect on the cells. The comparison of the results of the titration of adsorbed DPT vaccine in cell cultures with clinical manifestations has shown correlation between a greater degree of cell damage in vitro and severe local reaction. Therefore, in the process of the quality control of preparations cell cultures provide more sensitive tests than laboratory animals, which is confirmed by our data obtained in revealing the toxic properties of adsorbed DPT vaccine and its components.