สารบัญ

Contents
MENU

15.3 VACCINES

15.3 VACCINES

15.3.1 Biological Assay of Adsorbed Diphtheria Vaccine

            The potency of Adsorbed Diphtheria Vaccine is determined by comparing the dose of the vaccine required to protect guinea-pigs from the effects of either an erythrogenic dose of diphtheria toxin administered intradermally or a lethal dose of diphtheria toxin administered subcutaneously with the dose of a reference preparation, calibrated in International Units, needed to give the same protection.

            The International Unit is the activity contained in a stated amount of the International Standard which consists of a quantity of diphtheria toxoid adsorbed on aluminium hydroxide.

            The design of the assay described below follows a parallel-line model with three dilutions for the test and reference preparations. Once the analyst has sufficient experience with this method for a given vaccine, it is possible to apply a simplified model using a single dilution for both test and reference preparations. Such a model enables the analyst to determine whether the potency of the test preparation is significantly higher than the minimum required but does not give information on linearity, parallelism and the dose-response curve.

METHOD OF INTRADERMAL CHALLENGE

            Selection and distribution of the test animals Use in the test, healthy, white guinea-pigs from the same stock and of a size suitable for the prescribed number of challenge sites, the difference in body weight between the heaviest and the lightest animal being not more than 100 g. Distribute the guinea-pigs in not less than six equal groups; use groups containing a number of animals sufficient to obtain results that fulfil the requirements for a valid assay prescribed below. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardized, include five guinea-pigs as unvaccinated controls. Use guinea-pigs of the same sex or with males and females equally distributed between the groups.

            Selection of the challenge toxin Select a preparation of diphtheria toxin containing 67 to 133 lr/100 in one Lf and 25,000 to 50,000 minimal reacting doses for guinea-pig skin in one Lf. If the challenge toxin preparation has been shown to be stable, it is not necessary to verify the activity for every assay.

            Preparation of the challenge toxin solution Immediately before use, dilute the challenge toxin with a suitable diluent to obtain a challenge toxin solution containing about 0.0512 Lf in 0.2 ml. Prepare from this a further series of five fourfold dilutions containing about 0.0128, 0.0032, 0.0008, 0.0002, and 0.00005 Lf in 0.2 ml.

            Determination of potency of the vaccine Using saline TS, prepare dilutions of the vaccine to be examined and of the reference preparation, such that for each, the dilutions from a series differing by not more than 2.5-fold steps and in which the intermediate dilutions, when injected subcutaneously at a dose of 1.0 ml per guinea-pig, will result in an intradermal score of approximately 3 when the animals are challenged. Allocate the dilutions one to each of the groups of guinea-pigs and inject subcutaneously 1.0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. After 28 days, shave both flanks of each guinea-pig and inject 0.2 ml of each of the six toxin dilutions intradermally into six separate sites on each of the vaccinated guinea-pigs in such a way as to minimize interference between adjacent sites.

            Determination of the activity of the challenge toxin If necessary, inject the unvaccinated control animals with dilutions containing 80, 40, 20, 10, and 5 millionths of an Lf of the challenge toxin.

            Reading and interpretation of results Examine all injection sites 48 hours after injection of the challenge toxin and record the incidence of specific diphtheria erythema. Record also the number of sites free from such reactions as the intradermal challenge score. Tabulate together the intradermal challenge scores for all the animals receiving the same dilution of vaccine and use those data with a suitable transformation, such as (score)2 or arcsin ((score/6)2 ), to obtain an estimate of the relative potency for each of the test preparations by parallel-line quantitative analysis.

            Requirements for a valid assay The test is not valid unless:

            – for both the vaccine to be examined and the reference preparation, the mean score obtained at the lowest dose level is less than three and the mean score at the highest dose level is more than three;

            – if applicable, the toxin dilution that contains 40 millionths of an Lf gives a positive erythema in at least 80 per cent of the control guinea-pigs and the dilution containing 20 millionths of an Lf gives a positive erythema in less than 80 per cent of the guinea-pigs (if these criteria are not met, a different toxin has to be selected);

– the confidence limits (P = 0.95) are not less than 50 per cent and not more than 200 per cent of the estimated potency;

– the statistical analysis shows no deviation from linearity and parallelism.

            The test may be repeated but when more than one test is performed, the results of all valid tests must be combined in the estimate of potency.

METHOD OF LETHAL CHALLENGE

            Selection and distribution of the test animals Use in the test healthy guinea-pigs from the same stock, each weighing 250 to 350 g. Distribute the guinea-pigs in not less than six equal groups; use groups containing a number of animals sufficient to obtain results that fulfil the requirements for a valid assay prescribed below. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardized, include four further groups of five guinea-pigs as unvaccinated controls. Use guinea-pigs of the same sex or with males and females equally distributed between the groups.

            Selection of the challenge toxin Select a preparation of diphtheria toxin containing not less than 100 LD50 per millilitre. If the challenge toxin preparation has been shown to be stable, it is not necessary to verify the lethal dose for every assay.

            Preparation of the challenge toxin solution Immediately before use, dilute the challenge toxin with a suitable diluent to obtain a challenge toxin solution containing approximately 100 LD50 per millilitre. If necessary, dilute portions of the challenge toxin solution 1 in 32, 1 in 100 and 1 in 320 with the same diluent.

            Determination of potency of the vaccine Using saline TS, prepare dilutions of the vaccine to be examined and of the reference preparation, such that for each, the dilutions form a series differing by not more than 2.5-fold steps and in which the intermediate dilutions, when injected subcutaneously at a dose of 1.0 ml per guinea-pig, protect approximately 50 per cent of the animals from the lethal effects of the subcutaneous injection of the quantity of diphtheria toxin prescribed for this test. Allocate the dilutions one to each of the groups of guinea-pigs and inject subcutaneously 1.0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. After 28 days, inject subcutaneously into each animal 1.0 ml of the challenge toxin solution (100 LD50).

            Determination of the activity of the challenge toxin If necessary, allocate the challenge toxin solution and the three dilutions made from it, one to each of the four groups of five guinea-pigs and inject subcutaneously 1.0 ml of each solution into each guinea-pig in the group to which that solution is allocated.

            Reading and interpretation of results Count the number of surviving guinea-pigs 4 days after injection of the challenge toxin. Calculate the potency of the vaccine to be examined relative to the potency of the reference preparation on the basis of the proportion of animals surviving in each of the groups of vaccinated guinea-pigs, using the “Statistical Analysis of Results of Biological Assay and Tests” (Appendix 9).

Requirements for a valid assay The test is not valid unless:

– for the vaccine to be examined and the reference preparation the 50 per cent protective dose lies between the largest and smallest doses of the preparations given to the guinea-pigs;

– if applicable, the number of animals that die in the four groups of five injected with the challenge toxin solution and its dilutions indicates that the challenge dose was approximately 100 LD50;

– the confidence limits (P = 0.95) are not less than 50 per cent and not more than 200 per cent of the estimated potency;

– the statistical analysis shows no deviation from linearity and parallelism.

            The test may be repeated but when more than one test is performed the results of all valid tests must be combined in the estimate of potency.

            15.3.2 Biological Assay of Adsorbed Pertussis Vaccine

            The potency of Adsorbed Pertussis Vaccine is determined by comparing the dose necessary to protect mice against the effects of a lethal dose of Bordetella pertussis, administered intracerebrally, with the quantity of a reference preparation, calibrated in International Units, needed to give the same protection.

            The International Unit is the activity contained in a stated amount of the International Standard which consists of a quantity of dried pertussis vaccine.

            Selection and distribution of the test animals Use in the test, healthy mice less than 5 weeks old of a suitable strain from the same stock, the difference in weight between the heaviest and the lightest being not more than 5 g. Distribute the mice in six groups of not less than 16 and four groups of 10. The mice must all be of the same sex or the males and females should be distributed equally between the groups.

            Selection of the challenge strain and preparation of the challenge suspension Select a suitable strain of B. pertussis capable of causing the death of mice within 14 days of intracerebral injection. If more than 20 per cent of the mice die within 48 hours of the injection the strain is not suitable. Make one subculture from the strain and suspend the harvested B. pertussis in a solution containing 1 per cent w/v of casein hydrolysate and 0.6 per cent w/v of sodium chloride and having a pH of 7.0 to 7.2 or in another suitable solution. Determine the opacity of the suspension. Prepare a series of dilutions in the same solution and allocate each dilution to a group of 10 mice. Inject intracerebrally into each mouse a dose (0.02 ml or 0.03 ml) of the dilution allocated to its group. After 14 days, count the number of mice surviving in each group. From the results, calculate the expected opacity of a suspension containing 100 LD50 in each challenge dose. For the test of the vaccine to be examined make a fresh subculture from the same strain of B. pertussis and prepare a suspension of the harvested organisms with an opacity corresponding to about 100 LD50 in each challenge dose. Prepare three dilutions of the challenge suspension.

            Determination of potency of the vaccine Prepare three serial dilutions of the vaccine to be examined and three similar dilutions of the reference preparation such that in each the intermediate dilution may be expected to protect about 50 per cent of the mice from the lethal effects of the challenge dose of B. pertussis. Suggested doses are 1/8, 1/40 and 1/200 of the human dose of the vaccine to be examined and 0.5 IU, 0.1 IU and 0.02 IU of the reference preparation, each dose being contained in a volume not exceeding 0.5 ml. Allocate six dilutions one to each of the groups of not less than 16 mice and inject intraperitoneally into each mouse one dose of the dilution allocated to its group. After 14 to 17 days inject intracerebrally into each animal in the groups of not less than 16, one dose of the challenge suspension. Allocate the challenge suspension and the three dilutions made from it one to each of the groups of 10 mice and inject intracerebrally one dose of each suspension into each mouse in the group to which that suspension is allocated. Exclude from consideration any mice that die within 48 hours of challenge. Count the number of mice surviving in each of the groups after 14 days. Calculate the potency of the vaccine to be examined relative to the potency of the reference preparation on the basis of the numbers of animals surviving in each of the groups of not less than 16.

            Requirements for a valid assay The test is not valid unless:

– for both the vaccine to be examined and the reference preparation, the 50 per cent protective dose lies between the largest and the smallest doses given to the mice;

– the number of animals which die in the four groups of 10 injected with the challenge suspension and its dilutions indicates that the challenge dose is approximately 100 LD50;

– the statistical analysis shows no deviation from linearity or parallelism.

            The test may be repeated but when more than one test is performed, the results of all valid tests must be combined.

15.3.3 Biological Assay of Adsorbed Tetanus Vaccine

            The potency of Adsorbed Tetanus Vaccine is determined by administration of the vaccine to animals (guinea-pigs or mice) followed either by challenge with tetanus toxin (method A or B) or by determination of the titre of antibodies against tetanus toxoid in the serum of the guinea-pigs (method C). In both cases, the potency of the vaccine is calculated by comparison with a reference vaccine, calibrated in International Units.

            Adsorbed Tetanus Vaccine Reference Preparation is calibrated in International Units with reference to the National or International Standard.

SUGGESTED METHOD

            The method chosen for assay of tetanus vaccine (adsorbed) depends on the intended purpose.

            Method A or B may be used for routine assay of batches of vaccine but in the interests of animal welfare, method C is used wherever possible.

            Method C may be used after verification of the suitability of the method for the product. For this purpose, a suitable number of batches (usually 3) are assayed by method C and method A or B. Where different vaccines (monovalent or combinations) are prepared from tetanus toxoid of the same origin, suitability demonstrated for the combination with the highest number of components can be assumed to be valid for combinations with less components and for monovalent vaccine.

            For combinations with a whole-cell pertussis component, a separate demonstration of equivalence must be made for the highest combination.

            The design of the assays described below uses multiple dilutions for the test and reference preparations. Based on the potency data obtained in multidilution assays, it may be possible to decrease the number of animals needed to obtain a statistically significant result by applying a simplified model using a single dilution of both test and reference preparations. Such a model enables the analyst to determine whether the potency of the test preparation is significantly higher than the minimum required but does not give information on the dose-response curves and their linearity, parallelism and significant slope. The simplified model may lead to a considerable reduction in the number of animals required and its use must be considered in accordance with the provisions of the National Authority.

            Where a single-dilution assay is used, production and test consistency over time are monitored via suitable indicators and by carrying out a full multipledilution assay periodically, for example every 2 years. For serological assays, suitable indicators to monitor test consistency are:

– mean and standard deviation of relative antitoxin titres or scores of the serum samples obtained after administration of a fixed dose of the vaccine reference preparation,

– antitoxin titres or scores of run controls (positive and negative serum samples),

– ratio of antitoxin titres or scores for the positive serum control and the serum samples corresponding to the reference vaccine.

Method A. Challenge Test in Guinea-pigs

            Selection and distribution of the test animals Use in the test healthy guinea-pigs from the same stock, each weighing 250 to 350 g. Distribute the guinea-pigs in not less than six equal groups; use groups containing a number of animals sufficient to obtain results that fulfil the requirements for a valid assay prescribed below. If the activity of the challenge toxin has to be determined, include three further groups of five guineapigs as unvaccinated controls. Use guinea-pigs of the same sex or with the males and females equally distributed between the groups.

            Selection of the challenge toxin Select a preparation of tetanus toxin containing not less than 50 times the 50 per cent paralytic dose per millilitre. If the challenge toxin preparation has been shown to be stable, it is not necessary to verify the paralytic dose for every assay.

            Preparation of the challenge toxin solution Immediately before use, dilute the challenge toxin with a suitable diluent (for example, peptone buffered saline solution pH 7.4) to obtain a stable challenge toxin solution containing approximately 50 times the 50 per cent paralytic dose per millilitre. If necessary, use portions of the challenge toxin solution diluted 1 to 16, 1 to 50 and 1 to 160 with the same diluent to determine the activity of the toxin.

            Dilution of the test and reference preparations Using saline TS, prepare dilutions of the vaccine to be examined and of the reference preparation, such that for each, the dilutions form a series differing by not more than 2.5-fold steps and in which the intermediate dilutions, when injected subcutaneously at a dose of 1.0 ml per guinea-pig, protect approximately 50 per cent of the animals from the paralytic effects of the subcutaneous injection of the quantity of tetanus toxin prescribed for this test.

            Immunization and challenge Allocate the dilutions, one to each of the groups of guinea-pigs and inject subcutaneously, 1.0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. After 28 days, inject subcutaneously into each animal 1.0 ml of the challenge toxin solution (containing 50 times the 50 per cent paralytic dose).

            Determination of the activity of the challenge toxin If necessary, allocate the three dilutions made from the challenge toxin solution, one to each of the three groups of five guinea-pigs, and inject subcutaneously 1.0 ml of each solution into each guinea-pig in the group to which that solution is allocated. The activity and stability of the challenge toxin are determined by carrying out a suitable number of determinations of the 50 per cent paralytic dose. It is then not necessary to repeat the determination for each assay.

            Reading and interpretation of results Examine the guinea-pigs twice daily. Remove and humanely kill all animals showing definite signs of tetanus paralysis. Count the number of guinea-pigs without paralysis 5 days after injection of the challenge toxin. Calculate the potency of the vaccine to be examined relative to the potency of the reference preparation on the basis of the proportion of challenged animals without paralysis in each of the groups of vaccinated guinea-pigs, using the “Statistical Analysis of Results of Biological Assay and Tests” (Appendix 9).

            Requirements for a valid assay The test is not valid unless:

            – for both the vaccine to be examined and the reference preparation the 50 per cent protective dose lies between the largest and smallest doses of the preparations given to the guinea-pigs,

            – if applicable, the number of paralyzed animals in the three groups of five injected with the dilutions of the challenge toxin solution indicates that the challenge was approximately 50 times the 50 per cent paralytic dose, – the confidence limits (P = 0.95) are not less than 50 per cent and not more than 200 per cent of the estimated potency,

            – the statistical analysis shows significant slope and no deviation from linearity and parallelism of the doseresponse lines (Appendix 9).

            The test may be repeated but when more than one test is performed, the results of all valid tests must be combined in the estimate of potency.

Method B. Challenge Test in Mice

            Selection and distribution of the test animals Use in the test healthy mice from the same stock, about 5 weeks old and from a strain shown to be suitable. Distribute the mice in not less than six equal groups; use groups containing a number of animals sufficient to obtain results that fulfil the requirements for a valid assay prescribed below. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardized, include three groups of not less than five mice to serve as unvaccinated controls. Use mice of the same sex or with males and females equally distributed between the groups.

            Selection of the challenge toxin Select a preparation of tetanus toxin containing not less than 100 times the 50 per cent paralytic dose per millilitre. If the challenge toxin preparation has been shown to be stable, it is not necessary to verify the paralytic dose for every assay.

            Preparation of the challenge toxin solution Immediately before use, dilute the challenge toxin with a suitable diluent (for example, peptone buffered saline solution pH 7.4) to obtain a stable challenge toxin solution containing approximately 50 times the 50 per cent paralytic dose in 0.5 ml. If necessary, use portions of the challenge toxin solution diluted 1 to 16, 1 to 50 and 1 to 160 with the same diluent to determine the activity of the toxin.

            Dilution of the test and reference preparations Using saline TS, prepare dilutions of the vaccine to be examined and of the reference preparation, such that for each, the dilutions form a series differing by not more than 2.5-fold steps and in which the intermediate dilutions, when injected subcutaneously at a dose of 0.5 ml per mouse, protect approximately 50 per cent of the animals from the paralytic effects of the subcutaneous injection of the quantity of tetanus toxin prescribed for this test.

            Immunization and challenge Allocate the dilutions, one to each of the groups of mice and inject subcutaneously 0.5 ml of each dilution into each mouse in the group to which that dilution is allocated. After 28 days, inject subcutaneously into each animal 0.5 ml of the challenge toxin solution (containing 50 times the 50 per cent paralytic dose).

            Determination of the activity of the challenge toxin If necessary, allocate the three dilutions made from the challenge toxin solution, one to each of the three groups of not less than five mice and inject subcutaneously 0.5 ml of each solution into each mouse in the group to which that solution is allocated.

            Reading and interpretation of results Examine the mice twice daily. Remove and humanely kill all animals showing definite signs of tetanus paralysis. Count the number of mice without paralysis 4 days after injection of the challenge toxin. Calculate the potency of the vaccine to be examined relative to the potency of the reference preparation on the basis of the proportion of challenged animals without paralysis in each group of vaccinated mice, using the “Statistical Analysis of Results of Biological Assay and Tests” (Appendix 9)

            Requirements for a valid assay The test is not valid unless:

– for both the vaccine to be examined and the reference preparation the 50 per cent protective dose lies between the largest and smallest doses of the preparations given to the mice,

– if applicable, the number of paralyzed animals in the three groups of not less than five injected with the dilutions of the challenge toxin solution indicates that the challenge dose was approximately 50 times the 50 per cent paralytic dose,

– the confidence limits (P = 0.95) are not less than 50 per cent and not more than 200 per cent of the estimated potency,

– the statistical analysis shows a significant slope and no deviation from linearity and parallelism of the dose-response lines (Appendix 9).

The test may be repeated but when more than one test is performed the results of all valid tests must be combined in the estimate of potency.

Method C. Determination of Antibodies in Guinea-pigs

            Selection and distribution of the test animals Use in the test healthy guinea-pigs from the same stock,each weighing 250 to 350 g. Use guinea-pigs of the same sex or with males and females equally distributed between groups. Distribute the guinea-pigs in not less than six equal groups; use groups containing a number of animals sufficient to obtain results that fulfil the requirements for a valid assay. Use a further group of non-vaccinated guinea-pigs of the same origin to provide a negative serum control. If test consistency has been demonstrated, a reference negative serum control may be used.

            Dilution of the test and reference preparation Using saline TS, as diluent, prepare serial dilutions of the vaccine to be examined and the reference preparation; series differing by 2.5- to fivefold steps have been found suitable. Use not less than three dilutions within the range for example 0.5 to 16 IU/ml for each series. Use dilutions for immunization preferably within 1 hour of preparation. Allocate one dilution to each group of guinea-pigs.

            Immunization Inject subcutaneously in the nape of each guinea-pig 1.0 ml of the dilution allocated to its group.

            Blood sampling Thirty-five to 42 days after immunization, take a blood sample from each vaccinated and control guinea-pig using a suitable method.

            Preparation of serum samples Avoid frequent freezing and thawing of serum samples. To avoid microbial contamination, it is preferable to carry out manipulations in a laminar-flow cabinet.

            Determination of antibody titre Determine the relative antibody titre of each serum sample by a suitable immunochemical method (Appendix 14.5). Enzyme-linked immunosorbent assay (ELISA); either indirect ELISA or competitive ELISA (Toxin-Binding Inhibition; ToBI) has been found suitable.

            Calculation of potency Calculate the potency of the vaccine to be examined in International Units relative to the reference preparation, using the “Statistical Analysis of Results of Biological Assay and Tests” (Appendix 9).

(Note International Units of potency refer to the reference vaccine and not to the International Units of antitoxin of the reference guinea-pig serum.)

            Requirements for a valid assay The test is not valid unless:

– the confidence limits (P = 0.95) are not less than 50 per cent and not more than 200 per cent of the estimated potency,

– the statistical analysis shows significant slope and no deviation from linearity and parallelism of the doseresponse lines (Appendix 9).

The test may be repeated but when more than one test is performed, the results of all valid tests must be combined in the estimate of potency.

APPENDICES • 15.3 VACCINES
view 1,257 ผู้เข้าชม / View
หมายเหตุ / Note : TP II 2011 PAGE 708 - 712