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BIOASSAY of PERTUSSIS
VACCINE
Introduction
Whooping Cough (Pertussis)
•More than 200,000 children used to get whooping cough
each year. Thanks to vaccines, that number has dropped
significantly.
There are 2 vaccines that include protection against
whooping cough:
The DTaP vaccine protects young children from diphtheria,
tetanus, and whooping cough
The Tdap vaccine protects preteens, teens, and adults from
tetanus, diphtheria, and whooping cough
ANIMAL MODELS FOR THE STUDY
OF WHOOPING COUGH.
 Bordetella pertussis does not naturally cause disease in animals.
Nevertheless, experiments in animals have made important
contributions to the present, although incomplete,
understanding of pertussis.
 Mice, rats, rabbits, dogs, ferrets, and primates have been used.
The respiratory colonization of mice by B. pertussis mimics that
of humans, but mice do not cough, and so the infection is not
spread from mouse to mouse (Pittman et al., 1980).
 Among experimental animals, only primates have been found to
develop a paroxysmal cough and mucus production; they do
transmit the infection from one animal to another (Weiss and
Hewlett, 1986). However, adult primates can become resistant to
pertussis, so that newborn animals are needed for use in
experiments, and this is impractical. Rats are very hard to infect
with B. pertussis, and rabbits carry the organism for months
without showing signs of disease (Ashworth et al., 1982; Weiss
and Hewlett, 1986).
STANDARDIZED ANIMAL TESTS OF
VACCINE MATERIALS
 The intracerebral mouse protection test (Kendrick et al.,
1947, 1949) has served importantly in the progress in
vaccine development that has been made to date. The test
uses a standardized strain of bacteria (strain 18-323) stored
in liquid nitrogen (Cameron, 1988), standardized mice
(strain HSFS/ N) (Manclark et al., 1976), a freeze-dried
reference vaccine (Armitage and Perry, 1957), and an
interval between immunization and injection of 14 to 17
days (Cameron, 1988).
 The intranasal mouse protection test has been improved
by use of a standardized system for delivery of bacteria by
aerosol (Sato and Sato, 1988). This test has been used for
the study of the role in pathogenesis of bacterial adherence
proteins, for example, the 69-kilodalton outer membrane
protein (Shahin et al., 1990)
 The toxicities of vaccines have been studied by the mouse weight
gain test. This test depends on the observation that
intraperitoneal injection of vaccine into young mice leads to a
weight loss within hours, followed by total recovery of weight
within the next 7 days (Cameron, 1988). The causes of toxicity
(manifested as poor weight gain) in the test are not well
understood; the test is not very sensitive to endotoxin (Cameron,
1977). Results of the test have been shown to vary with the
adjuvant or absorbent used with the vaccine, mouse strain, diet,
size of cage, ambient temperature, and duration of exposure to
light (Cameron, 1988). These vagaries further illustrate the
difficulty of generalizing to humans the results obtained from
studies in animals.
 A sensitive assay for the particularly important toxin PT and for
anti-PT has been developed by using Chinese hamster ovary
(CHO) cells (Gillenius et al., 1985; Hewlett et al., 1983). In the
presence of PT, CHO cells undergo a characteristic clumping,
which can be blocked with antibody to PT. The test can detect PT
at levels one-fiftieth those of the next most sensitive assay
(Cameron, 1988).
Kendrick test
 Principles: The current pertussis potency test for whole-cell
vaccine is based on the intracerebral mouse protection test
as described by Kendrick (1947). The predictive value of
this test for the protective activity of the vaccine in man
was investigated by the Medical Research Council (1956).
The Kendrick test is an assay designed to estimate the
potency of pertussis containing vaccines on the basis of
their ability to protect mice against intra-cerebral challenge
with virulent Bordetella pertussis. The potency is expressed
in International Units (IU) calculated by comparing the
effective dose of the test vaccine to the reference vaccine.
Materials: Animals: Healthy male or female mice from a strain, all of the same sex,
3–4 weeks old and weighing not less than 10 g and not more than 18 g, but all
within a weight range of 4 g (this weight range should not vary from assay to
assay). If there is a need to use mice of both sexes and different weight described
above, they should be distributed equally throughout the test and the sexes
segregated. Validation needs to be carried out and should give appropriate results.
Media and reagents: Currently two types of agar media are used to grow Bordetella
pertussis
Bordet Gengou Agar (Copenhagen
Ingredients
Distilled water 4000 ml
NaCl 24 g
Glycerol 160 ml
Potatoes 2000 g
Beef broth 6000 ml
Bacto Agar 210 g
Norit (Carbon) 40 g
Fresh sheep blood (or other animal blood e.g.
horse) 120 ml
Charcoal Agar (CA)
CA Base 12.75 g
Distilled Water 250 ml
Defibrinated horse blood 25 ml
Experimental Procedures: Mice should be either all of the same sex or
equally distributed between male and female into groups by using the
computer program for randomization of animals over cages as following
[1]:
1) At least three groups of not less than 16 mice each for immunization with
reference preparation
2) At least three groups of not less than 16 mice each for immunization with
test vaccine sample
3) Four groups of 10 mice each are unimmunized control
Immunisation: The choice of appropriate doses for immunisation is critical
for the test and it should be cautiously adjusted to keep the median dose of
both test vaccine and reference preparations as close to the ED50 as
possible under each laboratory conditions. Since the response to the
vaccine differs between mouse strains, it is recommended that each
laboratory establish the optimal dose range during the validation stage ,
therefore the following immunisation doses (dilutions) serves only as an
example
1) Reconstitute an ampoule or ampoules of reference vaccine in
sterile PBS or 0.9% sodium chloride to give a stock suspension
containing 2.5 IU/ml.
2) Two further five-fold serial dilutions are made, also in sterile
saline or PBS, to generate suspensions containing 0.5 IU/ml and
0.1 IU/ml. All dilutions are of sufficient volume to inject the
appropriate group of mice with 0.5 ml each. The dilutions are
made in, and kept in, sterile labeled glass containers.
3) The test vaccine is initially diluted one in four, in sterile saline or
PBS, and then two further five-fold serial dilutions are made, all
to final volumes sufficient to inject the appropriate groups of
mice with 0.5 ml each. All dilutions are made in sterile labeled
glass containers. Any unused PBS remaining is discarded.
For Immunization
4) The stock suspension and dilutions are then kept at +2 to +8°C
and must be used within 4 hours.
5) Inject 0.5 ml of the appropriate dilution of the standard or
test vaccines intraperitoneally into each animal.
6) The four control groups remain untreated.
Care should be taken to ensure that cage position and
injection sequences are randomly allocated, since failure to do so
can result in invalid or misleading estimates of potency. This can
be achieved by allocating vaccine dilutions to cages of mice
according to a random number sequence by using the computer
program for randomization of dilutions over cages [1].
Animals are observed
for 14 days post-
immunization.
Preparation of challenge
suspension
 A challenge suspension can be prepared directly from a freeze-
dried ampoule of B. pertussis strain 18.323 or a B. pertussis strain
18.323 suspension previously prepared, aliquot and stored in
liquid nitrogen or -70ºC
 Five days before the intracerebral challenge, prepare the
challenge suspension by re-suspension of one ampoule of freeze-
dried B. pertussis strain 18.323 into 0.5 ml sterile distilled water.
If a pre-frozen suspension is used, allowed to thaw at room
temperature.
 Two to four pre-prepared BG agar plates are removed from the
refrigerator and labelled with assay number, culture name and
date. Any condensation is removed by leaving the plates open in
the cabinet until all the condensation has evaporated.
•Using a sterile glass Pasteur pipette, the bacterial suspension is
dropped equally onto 2 to 4 BG plates.
•The plates are incubated for 2-3 days at +33o C to +39o C (the exact
time needed to obtain a well grown culture under local conditions
should be determined by each laboratory).
•These plates are examined for contamination and only non-
contaminated cultures are used for the next step.
•The culture is then inoculated by spread/or streak on to 3 to 8 fresh
BG plates. These plates are then incubated at +33ºC to +39o C for 18
to 24 hours before they are used to prepare the challenge
suspension. In some laboratories, a frozen challenge suspension
made from a 18 hours culture, (e.g. 5% Glycerol in 1% sterile Casein
as described in Appendix I), and pre-calibrated in terms of c.f.u/ml
and LD50 was used to give better consistency of the challenge
suspension (see Appendix I)
Challenge administration:
Fourteen to seventeen days after the immunization, the
three groups of mice receiving the dilutions of each test
vaccine and the three groups of mice receiving the
dilutions of the reference vaccine
challenged intracerebrally with 10-30 µl of the suspension
A through the frontal bone of the cranium, 2 mm behind
the eye and 2 mm from the mid-line
In some laboratories, animals are under light halothane
narcosis (a mixture of halothane (1.8%), nitrous oxide (9
L/min) and oxygen (3 L/min)) before the challenge.
The whole procedure from the moment of preparing the
challenge culture to the injection of the last mouse shall
not last more than 4 hours
. The four unimmunized control groups are used for the titration
of the challenge dose (LD50). Each animal is inoculated
intracerebrally with 10-30µl of the challenge suspension (either
dilution A, B, C or D). After finishing the challenge procedure,
inoculate again 0.1 ml of suspension D on 2 BG plates
Observation: Observe the animals daily post challenge for
fourteen days and record the number of dead animals.
Mice in which pertussis infection has progressed to the
point where they no longer can reach food and water
should be killed and recorded as died a day later. The
deaths are recorded for 14 days after challenge. Mice dying
within 72 hours should be excluded from the results as
considered to be caused by the technical inoculation
error.
Calculations of Result:
Mice dying in the first three days after
challenge are not included in the calculation
of vaccine potency.
The data from the control groups are used to
estimate the LD50 of the challenge
suspension using probit analysis or any
other suitable statistical method.
The potency of vaccine test sample should be
calculated by comparing the ED50 of the
standard and vaccine sample using parallel
probit analysis program
Assay validity criteria:
1) The ED50 of each vaccine is between the largest and smallest
immunizing doses.
2) The challenge dose has been shown to contain between 100-1000
LD50.
3) The LD50 should contain no more than 300 colony forming units
(CFU).
4) The dose-response curves of the test and reference vaccine do not
deviate significantly (p < 0.05) from parallelism and linearity. This
means that the assay should meet the criteria set in the probit
analysis programme for linearity and parallelism of the dose–
response relationships
Pass criteria: The vaccine passes the test if it has a potency of not
less than 4.0 International Units (IU) per single human dose and the
lower fiducial limit (P = 0.95) of the estimated potency is not less
than 2.0 IU.
Pre clinical evaluation of pertussis vaccine (1)

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Pre clinical evaluation of pertussis vaccine (1)

  • 2. Introduction Whooping Cough (Pertussis) •More than 200,000 children used to get whooping cough each year. Thanks to vaccines, that number has dropped significantly. There are 2 vaccines that include protection against whooping cough: The DTaP vaccine protects young children from diphtheria, tetanus, and whooping cough The Tdap vaccine protects preteens, teens, and adults from tetanus, diphtheria, and whooping cough
  • 3.
  • 4. ANIMAL MODELS FOR THE STUDY OF WHOOPING COUGH.  Bordetella pertussis does not naturally cause disease in animals. Nevertheless, experiments in animals have made important contributions to the present, although incomplete, understanding of pertussis.  Mice, rats, rabbits, dogs, ferrets, and primates have been used. The respiratory colonization of mice by B. pertussis mimics that of humans, but mice do not cough, and so the infection is not spread from mouse to mouse (Pittman et al., 1980).  Among experimental animals, only primates have been found to develop a paroxysmal cough and mucus production; they do transmit the infection from one animal to another (Weiss and Hewlett, 1986). However, adult primates can become resistant to pertussis, so that newborn animals are needed for use in experiments, and this is impractical. Rats are very hard to infect with B. pertussis, and rabbits carry the organism for months without showing signs of disease (Ashworth et al., 1982; Weiss and Hewlett, 1986).
  • 5. STANDARDIZED ANIMAL TESTS OF VACCINE MATERIALS  The intracerebral mouse protection test (Kendrick et al., 1947, 1949) has served importantly in the progress in vaccine development that has been made to date. The test uses a standardized strain of bacteria (strain 18-323) stored in liquid nitrogen (Cameron, 1988), standardized mice (strain HSFS/ N) (Manclark et al., 1976), a freeze-dried reference vaccine (Armitage and Perry, 1957), and an interval between immunization and injection of 14 to 17 days (Cameron, 1988).  The intranasal mouse protection test has been improved by use of a standardized system for delivery of bacteria by aerosol (Sato and Sato, 1988). This test has been used for the study of the role in pathogenesis of bacterial adherence proteins, for example, the 69-kilodalton outer membrane protein (Shahin et al., 1990)
  • 6.  The toxicities of vaccines have been studied by the mouse weight gain test. This test depends on the observation that intraperitoneal injection of vaccine into young mice leads to a weight loss within hours, followed by total recovery of weight within the next 7 days (Cameron, 1988). The causes of toxicity (manifested as poor weight gain) in the test are not well understood; the test is not very sensitive to endotoxin (Cameron, 1977). Results of the test have been shown to vary with the adjuvant or absorbent used with the vaccine, mouse strain, diet, size of cage, ambient temperature, and duration of exposure to light (Cameron, 1988). These vagaries further illustrate the difficulty of generalizing to humans the results obtained from studies in animals.  A sensitive assay for the particularly important toxin PT and for anti-PT has been developed by using Chinese hamster ovary (CHO) cells (Gillenius et al., 1985; Hewlett et al., 1983). In the presence of PT, CHO cells undergo a characteristic clumping, which can be blocked with antibody to PT. The test can detect PT at levels one-fiftieth those of the next most sensitive assay (Cameron, 1988).
  • 7. Kendrick test  Principles: The current pertussis potency test for whole-cell vaccine is based on the intracerebral mouse protection test as described by Kendrick (1947). The predictive value of this test for the protective activity of the vaccine in man was investigated by the Medical Research Council (1956). The Kendrick test is an assay designed to estimate the potency of pertussis containing vaccines on the basis of their ability to protect mice against intra-cerebral challenge with virulent Bordetella pertussis. The potency is expressed in International Units (IU) calculated by comparing the effective dose of the test vaccine to the reference vaccine.
  • 8. Materials: Animals: Healthy male or female mice from a strain, all of the same sex, 3–4 weeks old and weighing not less than 10 g and not more than 18 g, but all within a weight range of 4 g (this weight range should not vary from assay to assay). If there is a need to use mice of both sexes and different weight described above, they should be distributed equally throughout the test and the sexes segregated. Validation needs to be carried out and should give appropriate results. Media and reagents: Currently two types of agar media are used to grow Bordetella pertussis Bordet Gengou Agar (Copenhagen Ingredients Distilled water 4000 ml NaCl 24 g Glycerol 160 ml Potatoes 2000 g Beef broth 6000 ml Bacto Agar 210 g Norit (Carbon) 40 g Fresh sheep blood (or other animal blood e.g. horse) 120 ml Charcoal Agar (CA) CA Base 12.75 g Distilled Water 250 ml Defibrinated horse blood 25 ml
  • 9. Experimental Procedures: Mice should be either all of the same sex or equally distributed between male and female into groups by using the computer program for randomization of animals over cages as following [1]: 1) At least three groups of not less than 16 mice each for immunization with reference preparation 2) At least three groups of not less than 16 mice each for immunization with test vaccine sample 3) Four groups of 10 mice each are unimmunized control Immunisation: The choice of appropriate doses for immunisation is critical for the test and it should be cautiously adjusted to keep the median dose of both test vaccine and reference preparations as close to the ED50 as possible under each laboratory conditions. Since the response to the vaccine differs between mouse strains, it is recommended that each laboratory establish the optimal dose range during the validation stage , therefore the following immunisation doses (dilutions) serves only as an example
  • 10. 1) Reconstitute an ampoule or ampoules of reference vaccine in sterile PBS or 0.9% sodium chloride to give a stock suspension containing 2.5 IU/ml. 2) Two further five-fold serial dilutions are made, also in sterile saline or PBS, to generate suspensions containing 0.5 IU/ml and 0.1 IU/ml. All dilutions are of sufficient volume to inject the appropriate group of mice with 0.5 ml each. The dilutions are made in, and kept in, sterile labeled glass containers. 3) The test vaccine is initially diluted one in four, in sterile saline or PBS, and then two further five-fold serial dilutions are made, all to final volumes sufficient to inject the appropriate groups of mice with 0.5 ml each. All dilutions are made in sterile labeled glass containers. Any unused PBS remaining is discarded. For Immunization
  • 11. 4) The stock suspension and dilutions are then kept at +2 to +8°C and must be used within 4 hours. 5) Inject 0.5 ml of the appropriate dilution of the standard or test vaccines intraperitoneally into each animal. 6) The four control groups remain untreated. Care should be taken to ensure that cage position and injection sequences are randomly allocated, since failure to do so can result in invalid or misleading estimates of potency. This can be achieved by allocating vaccine dilutions to cages of mice according to a random number sequence by using the computer program for randomization of dilutions over cages [1]. Animals are observed for 14 days post- immunization.
  • 12. Preparation of challenge suspension  A challenge suspension can be prepared directly from a freeze- dried ampoule of B. pertussis strain 18.323 or a B. pertussis strain 18.323 suspension previously prepared, aliquot and stored in liquid nitrogen or -70ºC  Five days before the intracerebral challenge, prepare the challenge suspension by re-suspension of one ampoule of freeze- dried B. pertussis strain 18.323 into 0.5 ml sterile distilled water. If a pre-frozen suspension is used, allowed to thaw at room temperature.  Two to four pre-prepared BG agar plates are removed from the refrigerator and labelled with assay number, culture name and date. Any condensation is removed by leaving the plates open in the cabinet until all the condensation has evaporated.
  • 13. •Using a sterile glass Pasteur pipette, the bacterial suspension is dropped equally onto 2 to 4 BG plates. •The plates are incubated for 2-3 days at +33o C to +39o C (the exact time needed to obtain a well grown culture under local conditions should be determined by each laboratory). •These plates are examined for contamination and only non- contaminated cultures are used for the next step. •The culture is then inoculated by spread/or streak on to 3 to 8 fresh BG plates. These plates are then incubated at +33ºC to +39o C for 18 to 24 hours before they are used to prepare the challenge suspension. In some laboratories, a frozen challenge suspension made from a 18 hours culture, (e.g. 5% Glycerol in 1% sterile Casein as described in Appendix I), and pre-calibrated in terms of c.f.u/ml and LD50 was used to give better consistency of the challenge suspension (see Appendix I)
  • 14. Challenge administration: Fourteen to seventeen days after the immunization, the three groups of mice receiving the dilutions of each test vaccine and the three groups of mice receiving the dilutions of the reference vaccine challenged intracerebrally with 10-30 µl of the suspension A through the frontal bone of the cranium, 2 mm behind the eye and 2 mm from the mid-line In some laboratories, animals are under light halothane narcosis (a mixture of halothane (1.8%), nitrous oxide (9 L/min) and oxygen (3 L/min)) before the challenge. The whole procedure from the moment of preparing the challenge culture to the injection of the last mouse shall not last more than 4 hours
  • 15. . The four unimmunized control groups are used for the titration of the challenge dose (LD50). Each animal is inoculated intracerebrally with 10-30µl of the challenge suspension (either dilution A, B, C or D). After finishing the challenge procedure, inoculate again 0.1 ml of suspension D on 2 BG plates Observation: Observe the animals daily post challenge for fourteen days and record the number of dead animals. Mice in which pertussis infection has progressed to the point where they no longer can reach food and water should be killed and recorded as died a day later. The deaths are recorded for 14 days after challenge. Mice dying within 72 hours should be excluded from the results as considered to be caused by the technical inoculation error.
  • 16. Calculations of Result: Mice dying in the first three days after challenge are not included in the calculation of vaccine potency. The data from the control groups are used to estimate the LD50 of the challenge suspension using probit analysis or any other suitable statistical method. The potency of vaccine test sample should be calculated by comparing the ED50 of the standard and vaccine sample using parallel probit analysis program
  • 17. Assay validity criteria: 1) The ED50 of each vaccine is between the largest and smallest immunizing doses. 2) The challenge dose has been shown to contain between 100-1000 LD50. 3) The LD50 should contain no more than 300 colony forming units (CFU). 4) The dose-response curves of the test and reference vaccine do not deviate significantly (p < 0.05) from parallelism and linearity. This means that the assay should meet the criteria set in the probit analysis programme for linearity and parallelism of the dose– response relationships Pass criteria: The vaccine passes the test if it has a potency of not less than 4.0 International Units (IU) per single human dose and the lower fiducial limit (P = 0.95) of the estimated potency is not less than 2.0 IU.