Antonie van Leeuwenhoek 29 (1963) 183-201

183

Development of Pertussis Vaccine Production and Control in the National Institute of Public Health in the Netherlands during the years 1950--1962
H. H. COHEN
National hlstitute o f Public Health, Utrecht, The Netherlands

COHEN, H. H. 1963. Development of pertussis vaccine production and control in the National Institute of Public Health in the Netherlands during the years 1950-1962. Antonie van Leeuwenhoek 29: 183-201. The development of the pertussis vaccine production in the National Institute of Public Health in the Netherlands since 1953, and the results with the consecutive lots of vaccine in the mouse protection test and the U.S.A. toxicity test are described. The results in the latter test are compared with the results of a locally developed guinea pig toxicity test. Special attention is given to the difficulties encountered when the U.S.A. toxicity test is used for adsorbed DPT vaccines. The potency data of all lots of DPT vaccines produced since 1958 fall within the limits of the potency test as prescribed in the U.S.A. Minimum Requirements. There are indications that the increased potency of the vaccine may have led to a lower mortality rate of pertussis. INTRODUCTION Since 1950 a solid medium (see Materials and Methods) was used for the production of pertussis vaccine in the National Institute of Public Health. Freshly isolated phase I strains, stored in the lyophilized state, were used. After harvesting, the bacteria were killed and detoxified by keeping the vaccine, in a concentrated suspension (80 opacity units per ml), for 72 hours at 37 C in the presence of 0.01 ~o merthiolate. For DPT (diphtheria-pertussis-tetanus) vaccine production bacterial concentrates were mixed with purified diphtheria and tetanus toxoids and adsorbed on A I P Q . The final DPT vaccine contained 20 opacity units in 0.5 ml. Its toxicity was measured by injecting the vaccine in 2 ml amounts intraperitonealty in each of 12 guinea pigs. The animals should survive for 35 days, but a 25~o loss by intercurrent infections was accepted. Tasman (1959) carried out a field trial with a number of DPT and DT

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vaccines prepared in this way in order to examine local and general unfavourable side effects. The latter were most frequently observed with vaccines containing the pertussis component. After subcutaneous injections considerably more local reactions, of longer duration, were found than after intramuscular injection. But on the whole, vaccines prepared in this way caused only a few untoward reactions and seemed acceptable for general use. In 1955 the intracerebral mouse protection test was introduced in our laboratories. With the aid of a reference preparation gauged by comparison with the U.S.A. standard vaccines no. 4 and 5 (Cohen, 1958) it became possible to determine the potency of the vaccines available at the time. The average potency of 13 consecutive lots of DPT vaccines prepared before 1955 was calculated as 4.5 protective units per total immunizing dose. The range was 1.5-15.2 units. Only 3 lots met the U.S.A. potency requirements of minimal 8 units in the total immunizing dose. In agreement with the results of potency determinations, DPT vaccines from this series conferred only partial immunity to the vaccinated children. Vos (1961) was able to estimate the immunizing effect in children. He observed an epidemic in a small village, where a minority of the population had conscientious objections to the use of vaccine. The majority, however, not belonging to this particular religious group, accepted vaccination of their children. In Table 1 a summary of Vos' results, is given. To a certain extent they agree with the data of Spronk (195l), Smeenk (1952) and Bos (1960). In general, these results agree with the results of the M.R.C. field trials in Great Britain (1956). The results of potency determinations correlate with the epidemiology; they reflect the relatively low immunizing properties of the pertussis vaccines produced in these years. MATERIALS AND METHODS

Preparation of media Solid medium (G). To I kg of ground meat, 4 liters of distilled water and 12 g of papain suspended in a small amount of water were added. The mixture was kept at 65- 70 C for 3 hr and thereafter filtered through paper and the pH adjusted to 8-8.2. The broth was sterilized by heating it on two successive days for two hr in the Koch at 100 C. Phosphates were removed by filtration through paper and the broth was diluted with the same volume of saline. The pH was adjusted to 7.4; 3 ~ agar and 1 ~ potato starch were added to the broth. The medium was subdivided in 1500 ml quantities in erlenmeyer flasks and sterilized for 30 rain at 120 C. When the flasks had cooled down

PERTUSSIS VACCINE IN THE NETHERLANDS 1 9 5 0 - 1 9 6 2 TABLE 1 Epidemiological results in a group of children (0-6 years old) immunized with DPT adsorbed vaccines not meeting the U.S.A. Minimum Potency Requirements as compared with an unvaccinated control group (0-6 years old) Clinical appraisal Vaccinated (206) Unvaccinated (46) 24 (52.2%)

185

Not ill 156 (75.7%) Aspecific course of the disease; average duration of the disease less than 2 weeks Less than 3 attacks a day; average duration of the disease less than 4 weeks 31 (50 = 24.3%) 3-10 attacks a day; average duration of the disease 6-8 weeks 11 10 attacks a day; average duration of the disease > I0 weeks 1

01
9!
12

1 (22 = 47.8%)

This table was compiled from the data given by A. J. Vos (1961).

to 48 C, 12~ of sterile defibrinated sheep blood was added to each. The medium was subdivided in 85 ml amounts in Roux flasks.

Fluid medium (V). This was prepared as described by Verwey et al. (1949). Tests for freedom of toxicity U.S.A. mouse toxicity test. This test was carried out according to the specifications of the U.S.A. Minimum Requirements Pertussis Vaccine, 1st revision October 31st, 1952 (section 4.11, 4.12) as follows:
" A t least 2 hours before injection 5 or more mice of one sex, weighing 14-16 grams each, are held in a suitable cage and given adequate food and free access to water. They are weighed as a group immediately preceding the injection which is made intraperitoneally. For a final vaccine, the dose for each mouse in a group shall represent one-tenth of the total human dose of a plain vaccine and one-fifth of the corresponding dose of vaccine containing a precipitating or an adsorbing agent. For an unfinished bacterial suspension, the dose should represent the equivalent of 10 opacity units. A vaccine is considered free of toxicity if at the end of 72 hours the group weight is no less than at the time of injection and no vaccine-related deaths of mice have occurred".

Recently (August 1961), in amendent no. 5, this paragraph was amended as follows:

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COHEN

"A group of not less than I0 mice, each mouse weighing 14-t6 grams, shall be used. The mice shall have free access to food and water, for no less than 2 hours before injection. The group weight of the mice shall be determined immediately before injection. Each mouse shall be injected intraperitoneally with a dose of the bacterial suspension, consisting of no less than 10 opacity units or a dose of the solution of bacterial fraction, consisting of no less than six-tenth of the amount to be incorporated in a single human dose of the final product. The group weight of the mice shall be determined at the end of 72 hours and 7 days. The bulk materials shall be considered free from toxicity if at the end of 72 hours the group weight is no less than the weight preceding the injection and at the end of 7 days the average gain per mouse is no less than 3.0 grams and the vaccine death rate does not exceed 5 percent. Results of all tests performed shall be reported". In the w o r k r e p o r t e d in this p a p e r the toxicity test has been carried o u t a c c o r d i n g to the original prescription. T h e results given in Fig. 1 show, h o w ever, that a p r o d u c e r , when using this p r o c e d u r e , m a y run into trouble. In this figure the gain in weight a n d the d e a t h rates o f two g r o u p s o f mice on different diets, seven days after i n t r a p e r i t o n e a l injection o f increasing a m o u n t s o f plain pertussis vaccine, are presented.
4VERAGE WEIGHT GAIN IN ,SEVEN DAY3 80 (GRA ,'4,$) ""/20 0120 INTERCURRENT DEATHS

,o

ool

CONTROL

,00

2000

10,000

50,000

,, =

XyO6BACTERIA

Fig. 1. Average weight gain and death rate in mice injected with increasing amounts of plain pertussis vaccine and supplied ad libitum with two different kinds of food. F r o m the results o f this test, it is a p p a r e n t that mice fed o n a pellet diet r e s p o n d e d with less g r o w t h than d i d mice fed on a special p r o t e i n diet. It is clear, accordingly, t h a t the quality o f the diet affects the results o f the toxicity test.

Guinea pig test. This test was d e v e l o p e d as follows:

As the DPT-vaccines, c o n t a i n i n g the pertussis c o m p o n e n t p r e p a r e d on the

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G medium did not give unfavourable reactions in children during mass vaccination campaigns, it was decided to use a number of plain pertussis concentrates, prepared on the G medium as reference preparations. Ten lots of G vaccines were compared with ten lots of V vaccines (for reactions of V vaccines in children, see page 196). Groups of 5 guinea pigs were injected subcutaneously with 160 109 bacteria per animal. Three criteria were used to assess differences in toxicity of the different lots: I) temperature rise, 2) weight gain, 3) diameter of infiltration measured daily from the third to the fourteenth day after injection. No differences between G and V vaccines were found as far as temperature and weight gain were concerned. However, there was a clear difference in the diameters of subcutaneous infiltration. Fig. 2 shows the average values found between the third and thirteenth day of observation.
DIAMETER I N F I L T R A T I O N I N CM 0,9 0

a7

a,s

03

Of

f'/ f3 > OBSERVATION pERIOD

I N DAYS

Fig. 2. Mean diameter value (in cm) of subcutaneous infiltrations caused by the injection of 160,000 106 germs of 10 lots of V and 10 lots of G vaccine over a period of I0 days. For each vaccine 5 guinea pigs were used. The difference between G and V vaccines is already on the third day highly significant. The results are calculated with the analysis of the variance technique. With this method it is possible to eliminate all differences within the V and G groups. That intergroup differences really exist is shown by the figures in Table 2:

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TABLE 2

Mean diameter value in cm of infiltrations 7 days after subcutaneous injection of 160,000 106 bacteria from 10 lots of V vaccine and 10 lots of G vaccine, For each vaccine, 5 guinea pigs were used. Average diameter value in cm Gvaccines 0.65; V vaccines 0.95; 0.5; 0.7; 0.5; 0.85; 0.6; 0.3; 0.I; 0.6; 0.55; 0.65; 0.95; 1.7; 0.6; 0.75; 0.35; 0.75; 0.3 0.55

In this Table for each lot of vaccine the mean diameter value on the seventh day is given. It turns out that the values in both groups do overlap. This is confirmed by analysing the results with the Wilcoxon method. In Table 3 the results of the analyses from the third to the thirteenth day are given.
TABLE 3 Expectation of the observed ranking orders in the Wilcoxon test of the diameter of infiltration values of 10 V and I0 G vaccines from the third to the thirteenth day after injection Day Expectation of the observed ranking order 63% 43% 33% 22% 1% l) Day Expectation of the observed ranking order 3% 1) 1% J) 2% 1) 1% l) 2% l)

3 4 5 6 7

8 10 11 12 13

0 Values exceeding 95% confidence limits,

The expected value exceeds the 95 70 confidence limits only from the seventh day onwards. This stresses again that local reactions within each group are strongly different. Only from this day on, the differences between both types of vaccines are sufficiently pronounced to make the observed ranking order highly improbable. After the evaluation of these results it could be concluded that: a) V vaccines were more toxic for guinea pigs than G vaccines; z) Great variations existed between several lots of one type of vaccine.

Application of sequential analysis to results obtained in the guinea pig test

It was decided to base future toxicity tests on the local reactions in guinea pigs after subcutaneous injection of vaccine. The results with G vaccines

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were accepted as a reference. This seemed a logical policy since only a few complaints were received from general practitioners after injection of D P T vaccine (adsorbed) prepared with G vaccine. Tentatively the following rules were drafted: 1) a plain vaccine without adsorbent is accepted if we are 99 ~ sure that the average diameter of infiltration on the seventh day after subcutaneous injection of 160,000 10~ germs is less than 0.6 cm, 2) a plain vaccine without adsorbent is rejected if we are 99 ~o sure that the average diameter of infiltration on the seventh day after subcutaneous injection of 160,000 10~ germs is larger than 0.8 cm, 3) as long as no decisive data are available the experiments are continued. As the observation period is comparatively long, groups of 5 animals should be injected simultaneously with the same vaccine. In Fig. 3 a graphical design of such a sequential test is given. As long as the value of the sum of the diameters remains between the lines the experiment must be continued. As soon as it passes the upper line the vaccine is rejected, while the crossing of the lower line means acceptance.
..RUM D I A M E T E R INFILTRATION.S

M=O.60 MJ080

/" ~ -~REJECTF.O

, NUMBER

OF

AI41MAL5

Fig. 3. Sequential test for acceptance or rejection of pertussis vaccknes on account

of their local toxicity for guinea pigs after subcutaneous injection. As long as the sum of the diameter values falls between the lines the experiment is continued. Passing the lower line means acceptance, passing the upper line means rejection. In Fig. 4 the relation between the theoretically expected mean value of the number of animals needed to reach a decision, and the mean diameter value is

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given. It is clear that at the value of 0.7 cm, i.e. the value just between 0.6 and 0.8 the curve reaches its maximum (33 animals, which means continuation of the experiments during 33/5 ----- 7 weeks).
NUMBER OF ANIMALS Mf = t~O M 2: oao ~( = o O f ~2

28

20

15

12

055

060

065

Q70 075 080 085 " ~ J . H E A N REAL OIAMETER iNFILTRATION

Fig. 4. The graph gives the relation between the real diameter value of infiltration of a vaccine and the average number of animals which must be used t? reach a decision. It is clear that when a real value of 0.7 is obtained, a long time is required to reach a decision; 33 animals have to be injected, which means continuation of the test during 33/5 = 7 weeks.

Mouse protection test

This test is performed according to the U.S.A. Minimum Requirements, 1st revision October 31st, 1952. As a reference vaccine the National Institute of Public Health, Utrechtreference vaccine C was used (Cohen, 1958). Results were calculated by probit analysis.

Test for H.S.F. (Histamine-Sensitizing-Factor) content

This factor whs determined in comparison with reference vaccine C and expressed in units according to the method of Preston (1959). The test was carried out by injecting decreasing doses of the vaccine being tested (6, 1.2 and 0.24 opacity units in 0.5 ml) in groups of 20 mice. Four days later, the mice were challenged with 4.4 mg of histamine-acid phosphate. The animals dying within 24 hr were recorded. Results were calculated by probit analysis.

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RESULTS Increase of potency of pertussis vaccines As early as in 1952 Lansberg in this laboratory made experiments on the production of pertussis vaccine in a liquid medium. For the development of such a method the V medium (Verwey et al., 1949) was chosen. A number of specially selected strains (Cohen and Leppink, 1956) were cultivated on solid medium G and fluid medium V. After centrifugation, the bacterial mass was resuspended in saline with 0.01 ~o merthiolate. Detoxification and killing took place by heating the suspensions for 3 days at 37 C. In Table 4 the results of potency tests are given as potency in units per 60 opacity units.
TABLE 4 Potency per total immunizing dose of a number of pertussis vaccines prepared from different strains and grown on fluid and on solid medium Strain Passage Medium solid (G) 512 534 3838 509 530 a b a
b

fluid (V)
12.0 20.4

1.8 2.4
18.0 13.2 13.2 1.8

6.0
d4,4 42.0 12.0 15.2 48.0

a
b

a
b

6.6
--

a b Average

1.2 4.8 7.2

3.6 7.8
21.0

Italics: Data meeting the U.S.A. Mimmum Requirement (8 Units)

From the results it can be inferred that the amount of protective antigen formed largely depends on the cultural conditions. The usefulness of fluid medium V for vaccine production is also apparent from the results in Table 5. This table gives among other things the potency, the H.S.F. content and the mouse toxicity of the pertussis component, produced in fluid medium in this laboratory since 1958, and used in different lots of adsorbed DPT vaccines. From the data presented, it can be concluded that the potency data of all vaccines produced, fall well within the limits 8 - 36 protective units/total immunizing dose, as stipulated in the U.S.A. Minimum Requirements. The

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COHEN

TABLE 5 Potency, H.S.F. content and mouse toxicity of a number of DPT vaccines, produced in the National Institute of Public Health, Utrecht, the Netherlands Lot. nr. Potency H,S.F. Ratio U.S.A. Toxicity test (I.U./total (U/total P/HSF Weight gain Intercurrent imm. dose) imm. dose) after 3 days after 7 days deaths

R 38

24.6 10.8

39 40 4t 42 43 44 45 46 47 48

18.9 74.4(!) 18.0 36.0 21.0 11.4 4.2-20.4 (av. 12.3) 20.4 10.8

24.6 6.6-6.015.6 (av. 9.4) 12.6 23.4 21.0 13.2 12.0 5.8 5.7 2.4 1.5-7.2 (av. 4.4) 2.4 10.8 15.2 11.4 16.2 13.8 24.6 10.8 25.2

1.0 1.1

1.5 3.2(!) 0.9 2.7 1.8 2.0 2.2 8.5 2.5 9.1 2.7 1.5 1.4 0.7 1.9 0.9 1.7 1.3

---0.3 +0.3 +0.2

+2.3 +2.8 +2.2

3/10 2/10 2/10

+1.0 ---0.2

+3.1 +0.9

0/10 0/I0

15.6-28.2 (av. 21.9) 49 28.8 50 22.8 51 15.6 52 11.4 53 26.6 54 22.8 55 18.0 56 33.0 57 27.0 58 58.8-11.4 (av. 35.1) 59 29.4 60 I3.8 63 20.4 64 25.2 65 21.0 66 35.4 67 14.4 68 16.8 Average 20.9

+2.6,+0.3 --t.7,--9.2 --0.9 --0.9 --9.6 --0.9 --0.1 --9.7,--0.9 --0.6,+0.0 --0.9 --0.5 --0.3 ---0.6 +0.5 +0.0 --0.1,+0.0 ---0.2, +0.7 --0.2

+2.7,+2.1 --0.7,+0.9 +0.2 +0.7 +2.2 +2.8 +0.8 --1.2,+1.1 +2.3,+1.0 + 1.3 + 1.2 +0.5 +0.5 +1.5 + 1.4 +t.8,+1.7 +1,1,+2.0 + 1.4

1/20 3/20 0/10 0/10 0/10 0/10 1/10 5/20 3/20 1/10 2/10 1/10 0/10 1/10 0/10 2/20 1/20 28/280 (10%)

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numbers 61 and 62 are omitted from these series as they were rejected for other reasons. The epidemiological situation does not contradict the potency data given in Table 5. As morbidity figures are not available, only mortality figures
PERTUSSIS MORTALITY IN THE NETHERLANDS
m o r to hty/i,oo o,o o o 30--

20

$ POTENCY (U,S,A. MIN. REQ.

153 28412~;~ 143

[ POTENCY J/ )U.S,A, MIN. REQ.

f27 f 2 3

IO

24

28

32

f3

19

25

. . . . . . . .

.........
"49

1947

:'51

%3

"55

"57

"59

'61

Fig. 5. Mortality rate from pertussis since 1947. In 1953 mass vaccination with vaccines of relatively low potency (see Table 1) was introduced. These vaccines were gradually replaced by better vaccines from the end of 1958 onwards.

are given. In Fig. 5 the death rate/I,000,000 from pertussis is given since the year 1947. After the beginning of mass vaccination in 1953 this figure has dropped considerably to 1 - 3. Since the end of 1958, vaccines of higher potency were gradually introduced. In 1960 and 1961 the death rate was respectively 0.2 and 0.8 per 1,000,000.

Control of toxicity
Although the higher potency of fluid medium vaccines given in Table 4 was promising, this was not considered sufficient for introduction of this medium for mass production of the vaccine. It was decided that more data on toxicity were needed especially after it became apparent (Lansberg, 1953, personal communication) that many general reactions - especially fever which ran as high as 104 F (40 C) - and local reactions were observed after vaccination with vaccines produced in fluid medium and detoxified by keeping them for 3 days at 37 C.

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COHEN

Toxicity of plain vaccines prepared in fluid and in solid medium and submitted to different methods of detoxifieation Toxicity test in guinea pigs and mice, In T a b l e 6 the results o f a n u m b e r o f
toxicity tests in guinea pigs with 5 freshly p r e p a r e d G a n d V vaccines are given. TABLE 6 Results of guinea pig toxicity test of 5 G and 5 V vaccines Solid medium (G) vaccine Vaccine prepared from strain : 530 3838 509 512 534 Average Number of animals required for acceptance 4 7 5 4 5 5.0 Average diameter value of the infiltrate (cm) 0.35 0.40 0.48 0.30 0.35 0.38 Fluid medium (V) vaccine Number of animals required for acceptance 7 13 5 19 5 9.8 Average diameter value of the infiltrate (cm) 0.45 0.56 0.45 0.60 0.40 0.49

It is clear, t h a t a l t h o u g h all vaccines are finally accepted, the V vaccines are m o r e toxic than the G vaccines. This is clear from the greater n u m b e r o f a n i m a l s necessary to reach a decision a n d the larger average infiltration. In T a b l e 7 the results with the U.S.A, m o u s e toxicity test with the vaccines m e n t i o n e d in T a b l e 6 are given. TABLE 7 Toxicity of 5 G and 5 V vaccines (see Table 6) in the U.S.A. mouse toxicity test Solid medium (G) vaccine Vaccine prepared from strain : 530 3838 509 512 534 Average of values Average weight Intergain (10 mice) current after 3 after 7 deaths days days -t-0.6 --0.4 ~0.1 q0.2 ---0.4 +0.0 +2.8 +3.4 +2.6 +2.8 +1.9 +2.7 0/10 0/10 0/I0 1/10 0/10 1/50 Fluid medium (V) vaccine Average weight Intergain (10 mice) current after 3 after 7 deaths days days --I.0 --3.7 --2.0 --0.6 ---0.5 --1.6 +2.2 +1.0 --0.5 +0.9 +2.8 +1.3 0/10 5/10 2/10

2/10
0/10 9/50

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195

Although the results with the individual vaccines are not clearly correlated, on the whole the data of this Table confirm the results of Table 5: the V vaccines are more toxic than the G vaccines. Moreover only two G vaccines (strain 530 and 509) and not a single V vaccine really passed the test. This leads to the conclusion that the U.S.A. toxicity test is more sensitive in discovering residual traces of toxin. We then tried to improve the methods of vaccine detoxification, passing of the U.S.A. test being the criterion for freedom of toxicity. It was found that by heating the vaccines for 3 0 - 6 0 min at 56 C in the presence of 0.01 ~o merthiolate, vaccines meeting the U.S.A. Minimum Requirements for Toxicity could be produced routinely in the V medium. In Table 8 the results with four lots of V vaccines prepared in this way are given. All four vaccines passed the test. TABLE 8 Mouse toxicity of 4 V vaccines heated at 56 C (30 min) in the presence of merthiolate Vaccine prepared from strain : Averageweight gain of Inter10 mice current after 3 after 7 deaths days days +0.1 +0.2 +1.3 +0.8 + 1.6 +2.7 +2.1 +2.1 +2.6 +2.6 +2.4 +4.2 0/10 0/10 0/10 0/10 0/10

3838 512a 512b 509 Average weight gain Control

Absence of toxic reactions in children with V vaccines which passed the U.S.A. mouse toxicity test. A plain vaccine, detoxified by heating, which had passed the U.S.A. toxicity test was compared with a G vaccine. In Table 9 the reactions in children are given. It must be stressed that virtually no unfavourable local or general reactions were met with after subcutaneous injection of either of these types of vaccine. However, the percentage children with temperatures higher than 37.5 C suggest that the V vaccines still have a somewhat higher residual toxicity than the G vaccines. Toxicity of adsorbed DPT-vaccines prepared with V vaccines detoxified by heating for 60 rain at 56 C Toxicity reactions in animals. Establishment of a unit H.S.F. D P T vaccines prepared in our laboratory contain 16 opacity units of pertussis vaccine per

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single dose, adsorbed in AIPO 4 (1.5 mg per dose). Originally, to measure toxicity, only the intraperitoneal guinea pig test was carried out as described under Materials and Methods. In Table 5 the results are given of the potency tests and the toxicity tests of all D P T adsorbed vaccines prepared since 1957 with the V pertussis component. TABLE 9 Toxic reactions in children after injection of 20 opacity units plain pertussis vaccines (G vaccines detoxified at 37 C, V vaccines detoxified 60 min at 56 C) Vaccine Number of children Average temperature after 4 hr G V 48 49 after 24 hr Numberofchildren Whining with temperature > 37.5 C after after 4 hr 24 hr 15/48 (31%) 21/49 (42.8%) 7/48 (14.6%) 12/49 (24.5%) t4/48 15/49

37.2 C 37.0 C 37.4 C 37.1 C

The mouse toxicity test was carried out routinely beginning with lot 50. Moreover, a number of vaccines were compared with our standard preparation to determine their relative H.S.F. content. The ratio of potency/H.S.F. content seems to be somewhat larger than 1, with the exception of lots 4 4 - 48, in which much higher values were found. (Average vatues lot 3 8 - 4 3 , 4 9 - 5 6 = 1.66, lots 4 4 - 4 8 = 4.17). For the preparation of lots 4 4 - 4 8 a plain vaccine was used with a comparatively low protective value (1.0 unit per 8.000 106 germs). After adsorption an explicit adjuvant effect of the AIPO4 on the potency was observed. The mechanism probably does not affect the H.S.F. factor. It is interesting that the adjuvant effect is less marked with plain vaccines of higher potencies ( 2 - 3 units per 8.000 106 bacteria), as are currently produced in our laboratory. F r o m the results obtained after the adoption of the U.S.A. mouse toxicity test it becomes obvious that most of the vaccines did not pass this test (see also Materials and Methods, mouse toxicity test).
Reactions years about them could part of the in children vaccinated with D P T vaccine. In the course of two 15 complaints of untoward reactions were received. Many of be ascribed to faulty injection techniques in which the greater vaccine was deposited in the subcutaneous tissue.

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Some cases of convulsions occurring either within some hours, or one or two days after vaccination, were reported. We received information about four cases of serious encephalopathies. One of these cases was published (Bos, 1960). It has been possible to carry out two small trials in which two D P T adsorbed vaccines (lots 52 and 53) were tested. These lots (52 and 53) were chosen because a number of complaints about reactions with these lots were received. The results are given in Table I0.
T A B L E 10 Toxic reactions in babies after injection o f 0.5 ml D P T vaccines (adsorbed) (lots 52 and 53) *) Vaccine no. N u m b e r o f children observed DPT52 DPT53 31 96 127 Categories B

19(61.3% ) 11(35.5% ) 1(3.2% ) 57(59.4% ) 35(36.4% ) 4(4.2% ) 76(59.8% ) 46(36.2% ) 5(3.9% )

A = no toxic reactions observed. B = small complaints- whining, disappearing within 24 hr. temperature not exceeding 38.5 C. C = ill, local reactions, fever higher than 38.5 C. duration longer than 24 hr. ') For results of potency determinations and mouse toxicity test see Table 5.

It is obvious that the percentage of more serious toxic reactions is 3 - 4 . By comparing the results in Table 10 with the results with plain vaccine (Table 9) it may be inferred that toxic reactions occur more frequently in adsorbed vaccines. In this respect the results in the mouse toxicity test reflect the situation in children. The relatively small number of complaints has led us to the conclusion, that vaccines prepared in this way can be used in mass vaccination campaigns. DISCUSSION In this survey of the results of 10 years pertussis vaccine production in the National Institute of Public Health in the Netherlands, there are three points of special interest. The first is, that the epidemiological situation seems to reflect the quality of the vaccine, as determined in the mouse protection test. This is apparent from the data of Vos (1961) and Bos (1960); it can also be inferred from the death rate from 1947 onwards as given in Fig. 5. There are two important features in this graph. The first is, the mortality drop since

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1953, the year in which a program of mass vaccination was started. From this year on, until 1960, the mortality remains more or less on the same level, whereafter a second drop becomes apparent. This second drop roughly coincides with the introduction in the end of 1958, of a vaccine of improved quality which, in the course of that year, replaced the supplies stored in the country. We realize that these facts cannot be accepted as final proof of the protective activity of the vaccine; especially not, since we do not know exactly the percentage of the population vaccinated. However, the quantities of the vaccines shipped from this laboratory in the last few years have been nearly constant. At any rate these data do not disagree with the results of the British Field Trial (1956). The potency limits of the U.S.A. Minimum Requirements are apparently well chosen. The results in Table 5 show clearly that all lots produced except three (lots 40, 45 and 58), have a potency falling well between the limits of 8-36 protective units per total immunizing dose. Excluding abnormal values, the arithmetic mean of the potency value is 20.9 with a variation between 10.8 and 36.0. A second point of interest is, that the H.S.F. content of the vaccines does not run parallel with the protective antigen. Theoretically the ratio between both values should be unity, as all vaccines are gauged on the same reference preparation. On the whole (see Tables) the ratio is larger, especially in lots 44-48. These lots were prepared from one batch of plain vaccine with comparatively low potency. On page 196 this was explained by assuming a difference in adjuvant effect of A1PO 4 on both factors. However, there may be an alternative explanation. Munoz et al. (1959) have shown that both factors are closely connected within the bacterial cell wall. In continuous cultures their optimal synthesis takes place at the same dilution rate (Van Hemert and Cohen, 1962). Joo et al. (1961a, I961b) even suggested to use the H.S.F.test in mice as an indicator of vaccine potency as this test is less time consuming than the mouse protection test. There are, however, also indications (Pittman, 1951; Cohen, 1962; Sutherland, 1962) that the H.S.F. can be specifically destroyed, while protective antigen remains intact. The evaluation of the pharmacological activity of this factor in the human being is very difficult especially with regard to the rare cases of serious encephalopathies, which sporadically occur directly after pertussis vaccination. Theoretically this factor could be responsible for this complication and in attempts for purification full attention should be paid to this fact. The thh'd point of interest is, that DPT vaccines adsorbed on AIPO4, which apparently do not pass the U.S.A. toxicity test as carried out in our laboratories, give few untoward reactions in children. This is the more re-

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markable, as the test seems to be quite sensitive for the detection of traces of toxins in plain vaccines (see Table 8 and Table 9). However, some factors must be kept in mind lest conclusions are drawn too early. The fact that many of our DPT vaccines did not pass the test may be ascribed to at least 3 factors: 1) The use o f an adsorbent in the vaccine. It is fairly well known, that adsorbed vaccines are more toxic for mice, especially if A1POa is used as an adsorbant, than plain ones. In this respect the results of the mouse test correlate with the results in children. However, the quantity of AIPO 4 used by us (1.5 mg per dose) is not exceptionally high and, in our opinion, does not explain our divergent results. 2) The relatively large number o f bacteria in our vaccines - 16 opacity units in one dose, i.e. the maximum permitted in the U.S.A., Minimum Requirements. The favourable results after the introduction of a vaccine with a high average potency has kept us, so far, from lowering the amount of pertussis to 10 or 12 opacity units per dose, the amount used in many vaccines prepared by American producers. Assuming that our production is consistent as regards potency, this would lower the average value to the otherwise acceptable level of 13-16 protective units. It is clear that the number of bacteria present can play a decisive rote in the outcome of the toxicity test, but, on the other hand, may influence the epidemiological situation. 3) The breed and condition of the mice. In this respect the paper given by Piersma (1962) in the "Prague Meeting on Pertussis" is interesting. This author gave the results of a number of control laboratories of American companies all working with the same lots of pertussis vaccine but with different inbred strains of mice. He pointed out that the results, which diverged very much from each other, could be caused by the strain of animals used. The results of a feeding experiment (Fig. 1) show that the composition of the food also may play a role in these experiments. During our testing program, early in 1961, we had a period in which the number of intercurrent deaths in our breeding stock of Swiss mice rose to some extent, especially with animals subjected to stress. The total intercurrent death rate exceeded the 5 ~ level permitted in the 5th amendment U.S.A. Minimum Requirements for Pertussis Vaccine. Out of 280 animals 28 died (10~o). The average gain of weight of the surviving mice was 1.3 g. Noninjected mice generally do not show a weight gain of more than 3 - 4 g. We are inclined to express as our opinion that the National Institute of Public Health-mouse test has served a useful purpose, but should be modified. This has been done recently (see Materials and Methods). However, it seems

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imperative that a "standard o f toxicity" preparation, adsorbed on mineral carrier, should be established, which does not give " t o o m a n y unfavourable" reactions in children. With the aid o f this standard preparation the influence o f strain- and feeding-differences can be excluded. The hope is justified that in the future pertussis vaccine can be purified so that toxic and H.S,F. properties are specifically destroyed, or separated from the protective antigen. W h e n such vaccines can be prepared on a large scale, our present problems will be solved completely. F r o m the comparative assays in guinea pigs (Table 6) and mice (Table 7) it is clear that the U.S.A. mouse test is more sensitive than the guinea pig test as devised in our laboratories. It must be emphasized that the reference points in the guinea pig were chosen on the basis o f some preliminary experiments in the laboratory (Table 2). It is, however, possible to increase the sensitivity o f this test by decreasing the parameters. The results o f such a test might parallel more accurately the local reaction in h u m a n beings. The a u t h o r is indebted to: Dr. M. Pittman, National Institutes o f Health, Bethesda, U.S.A., for stimulation to compile the data o f this paper and for critical evaluation o f the first concept, Dr. H. D. Piersma, Lederle Laboratories, Pearl River, U.S.A., for critical advise and correction o f the manuscript, and Dr. C. Smeenk, pediatrician, Doetinchem, The Netherlands, for the field work with the vaccines mentioned in this paper.

Received 16 January 1963

REFERENCES Bos, G. J. 1960. Een epidemie van kinkhoest in een redelijk gevaccineerde huisartsenpraktijk. Huisarts en Wetenscbap 3: 293-297. COHEN, H. H., and LEPPINKG. J. 1956. Selection of Hemophih~spertussis strains for vaccine production in the mouse protection test in a balanced design. J. lmmunol. 77: 299-304. COHEN, H. H. 1958. Establishment of a dried standard pertussis vaccine. Antonie van Leeuwenhoek 24: 33-48. COHEN, H. H. 1962. Discussion on the preparation of a semipurified protective antigen of Bordetella pertussis in connection with side effects and the serological response after vaccination and disease. Round Table Conference on Pertussis Immunization, Prague, Vol. 1, 97-103. VAN HEMERT, P. and COHEN, H. H. 1962. Development of agglutinogens, protective antigens and histamine sensitizing factor during continuous culture. Round Table Conference on Pertussis Immunization, Prague, Vol. 1, 24-29. Joo, 1., PUSZTAI,Z. and JUH~.SZ,V. P. 1961a. Histamine-sensitizing activity of various pertussis vaccines. Immun. Forsch. 121: 143-158. Joo, I., PUSZTAI,Z. and JUHASZ, V. P. 1961b. Comparative investigations into the mouseprotective potency, histamine-sensitizing activity and agglutinin-producing ability of pertussis vaccines. Immun. Forsch. 121: 250-266.

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MUNOZ, J., Rtal, E. and LARSON, C. L. 1959. Antigens of Bordetella pertussis. I. Activities of cell walls and protoplasm. J. lmmunol. 83: 496-501. PIERSMA, H. D. 1962. Recent laboratory experience in the U.S.A. with the pertussis toxicity test. Round Table Conference on Pertussis Immunization, Prague, Vol. 1, 111-119. PITTMAN, M., 1951. Comparison of the histamine-sensitizing property with the protective activity of pertussis vaccines for mice. J. infect. Dis. 89: 300-304. PRESTON, N. W. 1959. Factors influencing the assay of the histamine-sensitising factor of Haemophilus pertussis. J. Path. Bact. 78: 217-224. Report to the Whooping-cough Immunization Committee of the Medical Research Council, 1956. Vaccination against whooping-cough. Brit. Med. J. II: 454-462. SMEENK, C. S. 1952. Resultaat van de kinkhoest-immunisatie bij zuigelingen. Ned. T. Geneesk. 96: 1812-1814. SPRONK, M. G. 195l. Actieve immunisatie tegen kinkhoest. Thesis. Amsterdam. SUTHERLAND,1. W. 1962. The cell wall of Bordetetla pertussis as a protective antigen. Round Table Conference on Immunization, Prague, Vol. 1, 72-80. TASMAN, A. 1959. Het verband tussen de aard van de gebruikte entstof, de wijze van inspuiten en de ent-reacties na immunisatie tegen kinkhoest, difterie en tetanus. Ned. T. Geneesk. 103: 1049-1057. VERWEY, W. F., THIELE,E. H., SAGE,D. N. and SCHUCHAROT,L. F. 1949. A simplified liquid culture medium for the growth of Hemophilus pertussis. J. Bacteriol. 58: 127-134. Vos, A. J. 1961. Een beschouwing over de preventieve waarde van het kinkhoestvaccin naar aanleiding van een onderzoek in een algemene praktijk. Huisarts en Wetenschap 4:114-117.