Biologicals (1992) 20, 267-275

Swedish Inactivated Polio Vaccine: Laboratory Standardization and Clinical Experience over a 30-year Period Margareta BSttiger and Berit Larsson* Department of Epidemiology and *Production Department, National Bacteriological Laboratory, S- 105 21 Stockholm, Sweden Abstract. Swedish inactivated polio vaccines have contained per single human dose a mean amount of viral antigen equivalent to 1 x 1075CCIDs0 of type 1, 1 x 1074of type 2 and 1 x 1078 of type 3 produced on primary monkey kidney cells. Potency tests were made in comparison with an equivalent amount of live virus suspension of all three types. Validation of tests has been based on the response to type 1 only. Based on clinical experience with vaccine lots from 1957 and the establishment of the second live reference virus suspension in 1966, the minimum limit of immune response in guineapigs--expressed in extinction values--was decided as 1.5 for type 1 and type 3, and 1.0 for type 2. The potency test method used since 1959 in Sweden was two subcutaneous injections 2 weeks apart using 10 guinea-pigs per dilution and blood collected 1 week thereafter. Potency tests made according to European Pharmacopoeia revealed a somewhat lower value for type 2. D-antigen content in Swedish vaccines was low, however, the Swedish vaccine has protected against many episodes or outbreaks of wild virus in Sweden and immunized individuals elsewhere in the world. For the Swedish population a clear-cut clinical motivation for requiring a higher potency for type 2 as required in the European Pharmacopoeia or increased levels of D-antigen in the final product has not been presented. It was concluded that the European Pharmacopoeia method did not distinguish between doses of 0.5-1.0 ml. The minimum limit extinction value for type 2, i.e. 2.0 seemed too high. There was no direct relationship between the D-antigen content and the guinea-pig potency values. It is useful only for 'inhouse testing'.

Introduction For more than 30 years, i.e. from 1957 to 1989, inactivated polio vaccine (IPV) produced on primary monkey kidney cells has been used in Sweden. Several technical changes were made in the process during this period, but the principles were the same.' During the first 15 years, the potency of the vaccine batches was tested regularly, both in guinea-pigs and in young, previously unvaccinated children. Later, testing in children was carried out only sporadically. So far, no one who has received three doses of this vaccine has contracted paralytic poliomyelitis. Apparently, the immunity levels obtained by the use of this vaccine according to the recommended vaccination schedule of four doses have been protective during t h a t period.'-' Challenges to this protection have occurred. A large number of Swedes travel to tropical countries each year. Foreigners are known to bring poliovirus into the country. No poliovirus has 1045-1056/92/040267 +09 $08.00/0

been coincidentally isolated from any of the thousands of cell-culture isolation tests carried out annually throughout the years on Swedes with meningo-encephalitis or diarrhoea2 However, when attempts were made to isolate poliovirus from the sewage, positive results were obtained from time to time 4 and in 1976 poliovirus spread within an unvaccinated community. 8In 1984, it became apparent that wild poliovirus type 3 had become widely disseminated in Finland, a neighbouring country with which Sweden has a frequent large interchange of people. 6 In the winter of 1984-5, poliovirus similar to the Finnish one was also isolated from sewage in Stockholm. 7 In 1987, it was decided to abandon the use of primary monkey kidney cells for the traditional polio vaccine, in order to prepare for the next generation of inactivated polio vaccine. This development involved cultivation of cells on beads in fermentors, purification and standardization, s O 1992 The International Associationof BiologicalStandardization

268

M. B~ttiger and B. Larsson

The aim of this article is to record the experience of the potency testings of the vaccines successfully used throughout the years, and to use this as a basis for standardization of the newer vaccines. Material and M e t h o d s

Vaccine The inactivated vaccine used in Sweden was produced by the National Bacteriological Laboratory (NBL). The manufacturing procedure has been described in detail in earlier publications.~ The seed viruses used for the types 1, 2 and 3 poliovirus components in the vaccine were the type 1 Stockholm 1423/53 (up to 1961), the type 1, Brunenders (1961 and later), the type 2, MEF-1 and the type 3, Saukett strains, respectively. The virus seed lots used since 1974 were passage 3 from 1963. Vaccine preparations were also obtained from the Rijks Institute Voor Volksgezondheid, Utrecht, Holland.

Potency testing in guinea-pigs Guinea-pigs were used for checking potency. 9.~°,15.~6 The vaccine was tested for antibody response in five groups o f l 0 guinea-pigs each in 10-fold dilutions. The starting dilution (undiluted or 10-1) depended on the expected potency in the vaccine. In order to get a safe endpoint five dilutions were injected. Two doses were administered at 2 weeks' interval and the guinea-pigs were bled 1 week after the second injection (for the first vaccines in the 1950s, this interval was 3 weeks). For neutralization studies virus of the type 1, Stockholm 1423/53, the type 2, MEF-1, and the type 3, Saukett strain, were used at a concentration of 100 CCIDso and mixed with sera diluted 1:5 and incubated for 18 h at 37°C. The mixture was then used to inoculate primary monkey kidney cell cultures. The vaccine dilution, expressed in -log~o, which evoked neutralizing antibody response in 50% of the guineapig serum samples tested was given as the potency value, extinction limit. The potency values were corrected by comparison with the result of a reference preparation tested at the same time. The standard deviation of this test amounted to +_0.4. The method described later in the European Pharmacopoeia was also used for comparison.17

National Standard Reference Preparation A mixture of live virus of all three types (Brunenders, MEF-1 and Saukett) corresponding to the antigen amount in the vaccines was established in 1966 as the second National Reference Preparation.

Since that time the preparation distributed into aliquot samples was maintained at -20°C. Five groups of 10 guinea-pigs were injected (dilution 10-~-10 5) at the same time as the vaccines to be tested.

D-antigen test A certain number of vaccine lots were analyzed for D-antigen contents by Dr J Beale in the U.K. and by Dr van Wezel in Holland, using their D-antigen test and ELISA technique. H Test sera were for the type 1 strain anti-Mahoney, for type 2 anti-MEF-1 and type 3 anti-Saukett. Potency tests in children Vaccination comprised of two doses of 1 ml, given 1-2 months apart, a third dose 6-18 months later and a fourth dose about 5 years after the primary course. The vaccination was not initiated until the age of 9 months when interference of maternal antibodies has ceased. When the potency of vaccines in guinea-pigs became higher, new trials of vaccination of children at earlier age than 9 months were carried out. 1'~ The potency testing in children was described in detail.1 Blood was collected 1 month after the two primary doses by finger puncture and immediately diluted 1:8 in heparinized medium. For a number of years, a further test was carried out on a second sample collected 1 month after the third injection (administered about 7 months later). The antibody content of the sera was measured by neutralization tests in cell cultures. As challenge virus for the tests, between 30-100 CCID~o of the type 1, Stockholm 1423/53 strain, the type 2, MEF-1, and the type 3, Saukett strain, were used. Serum was tested in fivefold (earlier tests) or fourfold dilutions. The dilution, expressed in -loglo, that gave 50% neutralization of the cell-culture tubes was used as the antibody-response value. The same laboratory carried out all the tests throughout the years. Potency testing in children comparing the high D-antigen containing Dutch vaccines and the Swedish vaccines was carried out in a similar manner. Results

Second National Reference Preparation (B) A second reference preparation (Ref. B) was established in 1966. It consisted of a mixture of a live virus suspension of all three types equivalent in concentration to the Swedish IPV. Based on the results of 13 potency tests and 42 individual titrations; the extinction limit value for type 1 was established as 3.0 (Table 1). This reference preparation distributed in tubes for each test has been stored at -20°C since

Swedish inactivated polio vaccine

T a b l e 1. Standardization of the Second Reference P r ep ar atio n (B) Date injected

Extinction limit*

1969-04-28

1969-08-25

1969-09-08

1969-10-06

1969-11-03

1969-11-06

1969-12-23

1970-01-12

1970-02-09

1970-03-16

1970-04-13

1970-05-11

1970-06-08

Mean (42 titrations): Mean (13 tests): Assigned Potency:

Mean

3-55

3.50 3.81 3.45

3.59

2.51 3.08 2.06

t h a t time and has kept its antigenicity. Inactivated virus can not be stored for such a long period without losing the antigenicity.

Antigen content in vaccines

3.21 3.63 3.55 3.81

2.20 2.98 2.42 3.42

269

The antigen concentration of Swedish IPV per single h u m a n dose has been equivalent to or more than 107.0 CCID~o of all three types since its introduction in 1960. The antigen concentration in IPV lots during 1985-89, based on titre values obtained before inactivation, is shown in Table 2. Production lots T a b l e 2. Antigen content in polio vaccines during 1985-9

2.76 Virus titre/CCID~o) before inactivation 2.73

2.10 1.91 1-62 2.43

2.02

2.23 1.86 2.50

2.20

2.32 2.91 3.00

2.74

3.61 2.60 3-44

3.22

2.82 3.65 3.53

3.33

3.31 3.58 3.24

3.38

3.62 3.12 3.43

3.39

3.26 3.39 2.72

3.12

2.50 3.35 3.35

3.07

2.99 3.01 3.0

* Antibody titrations carried out against type 1.

Lot no. 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 Mean: NBL minimum requirement WHO FDA

Type 1

Type 2

Type 3

7.1 7.7 7.7 7.6 7.8 7.8 7.2 7.1 7.1 7.2 7.2 7.0 7.8 7.7 7-7 7.2 7.1 7-6 7.1 7.2 7.7 7.2 7-6 7.5 7.4 8.0 7.7 7.6 7.6 7-5 7.8 7.6 7.5

7-3 7-6 7.1 7-4 7.4 7.4 7.4 7-5 7.5 7-5 7-5 7.1 7.1 7.2 7.3 7-3 7.3 7.3 7.3 7-3 7.3 7.7 7.7 7.7 7-6 7.5 7.5 7.5 7.4 7.3 7.3 7-5 7.4

7.9 7.9 7.9 7.6 7.6 7.6 7.6 8-0 8.0 8.0 8.0 7.9 7.6 7.2 7.2 7.2 7.5 7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.8 7-9 7.9 7.9 7.8 7.8

7.0 7-0 6.5

7. 0 7-0 6.5

7.0 7.0 6.5

270

M. BSttiger and B. Larsson

have varied, ranging from 7.0 to 8.0 for type 1, from 7.1 to 7.7 for type 2 and 7.2 to 8.0 for type 3. The minimum requirement of 7-0 for all three types, set in the early1960s, was maintained and met with the later World Health Organization and Food and Drug Administration requirements. ~.~..,o

Potency test in guinea-pigs The results of guinea-pig potency tests, carried out according to the Swedish method, on vaccine lots delivered since the year 1960 had the minimum requirement set at t hat time, viz., extinction limit values of 1.5 for type 1 and type 3, and 1.0 for type 2. As an example, the potency values for IPV lots

T a b l e 3. Guinea-pig potency values for polio vaccines during 1985-9 Lot no. 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 Mean NBL minimum requirement

Type 1

Type 2

Type 3

2.4 2.9 3.8 3-3 3.6 3.7 4-1 2.8 2.1 2.7 2-5 2.3 2-8 3.1 3.4 2.5 2-6 3.4 3.1 3.5 3.1 2.7 2.9 3.9 3.1 3-4 3.4 3.0 3-5 3.4 3-4 2.8 3.1

2.1 3-3 3.5 3.6 2.5 2-2 3.2 3.0 2-2 2.3 2.6 2-8 2.8 2.2 2.9 2.1 3.0 2.8 2.3 3.1 3.4 3.0 2.4 2.9 2.4 2.7 2.4 2-3 3.0 2.9 2.6 3.0 2.7

3.6 3.9 4.0 3.9 3.9 2.7 3-9 2.9 3.3 3.3 2-9 3.0 3.7 3-2 4.0 2.9 2-7 3.5 3.3 3.6 4.1 3.6 3.0 3.9 3.4 3.8 3.3 2.9 3.7 4.1 4.1 3.4 3.5

1.5

1.0

1-5

released during 1985-89 are shown in Table 3. The extinction limit values for these vaccines were considerably higher than the minimum requirement set in the past. The mean values obtained for these 32 lots were: for type 1, 3-1 (range 2.4-4.1); for type 2, 2.7 (range 2.1-3.6) and for type 3, 3.5 (range 2.7-4.1}. Tests were done according to the European Pharmacopoeia (EP) method ~7 on two vaccine lots (Table 4). Vaccine lot 254 was tested using both 0.5 and 1.0 ml per guinea-pig according to the EP. The extinction limit values when using 0.5 ml were 2.6, 1.7 and 3.5, and with 1.0 ml 2-7, 1.7 and 4.0, respectively, for types 1, 2 and 3. Thus, no quantitative increase in antibody response in relation to doubling the immunizing dose could be obselwed. The Swedish IPV preparations fell below the requirement set by Em'opean Phmnnacopoeia for type 2 antigen.

D-antigen content and guinea-pig potency values Values for D-antigen content and guinea-pig potency are given in Table 5. Also included are the cotresponding values for a num ber of Dutch vaccines. The content of D-antigen in Swedish IPV was considerably lower than those in the extra-high potency one- to twodose Dutch vaccines. Guinea-pig potency values were colTespondingly higher for the Dutch vaccines.

Potency test in children The results of potency tests in children with IPV vaccines are shown in Tables 6, 7 and 8. During the years 1957-8, part of the vaccines used were imported from the U.S.A. The mean neutralizing antibody titre values in sera of children vaccinated during 1957-9 were found to be low, especially for type 1. In Table 7, antibody levels after vaccination are shown. During 1957-9, the non-reactors to type 1 were high in proportion (27-46%), whereas it was 1-13c~ for type 2 and 3-23c~ for type 3. With the improvements in vaccine quality, the percentage of non-reactors decreased during this development period, 1960-4.

Relationship between D-antigen content of vaccines and guinea-pig potency values Guinea-pig potency values were plotted against D-antigen content for both Swedish and Dutch vaccines (Fig. 1). For type 1 virus, a higher D-antigen content with Dutch vaccines was needed to give the same degree of sero-conversion as Swedish vaccines. For type 2 and type 3 viruses, the relationship was similar. The curves did not run parallel indicating t hat the D-antigen test could not be used to compare the two products.

Swedish inactivated polio vaccine

271

T a b l e 4. Comparison of potency testing in guinea-pigs between NBL and E P methods Extinction limit Vaccine no.

Dose (ml)

Method

Type 1

Type 2

Type 3

Po 254 Po 254

1.0 0.5

NBL EP

Po 254

1

Mean EP

Po 256

0.5

Mean EP

Po 256

1-0

Mean NBL

3.2 2.62 2.50 2.6 2.68 2.65 2.7 2.72 2.82 2.8 2.41 3.75 3.1

2.0 1.48 1.99 1.7 1.50 1.82 1.7 1.48 1.76 1.6 2-04 2.36 2.2

3.9 3.44 3.47 3.5 4-08 3.96 4.0 3.56 3.91 3.7 4.00 4.94 4.5

Mean

T a b l e 5. Guinea-pig potency values and D-antigen content of Swedish and Dutch vaccines Guinea-pig potency Product Batch NBL NBL NBL NBL NBL NBL NBL NBL NBL NBL NBL NBL NBL RIV RIV RIV RIV RIVM RIVM RIVM RIVM RIVM

202 203 208 229* 233 236 243 248 249 253 255 258 260 20 40 40 80 319 320 321 322 323

D-antigen content

1

2

3

1

2

3

2.7 2.4 2.4 3.2 2.9 2.8 2.3 3-3 2.2 2.4 3.4 3.6 2.2 3.5 3.1 3.0 4.3 3.6 4.1 3.3 3.9 4.4

1.6 1.8 1.2 3.1 1.9 1.9 2.1 3.0 2.6 2.5 2.2 3.3 2.2 2.3 2.6 2.8 3.2 3.6 4.1 3.3 4.1 4.3

3.0 3.1 2.6 3.7 2.9 3.1 3.0 3.3 3.3 3.3 3.4 4.0 3-4 3.6 3.9 3.2 3.8 3.6 4.4 3.4 4.2 4.2

4 3 5 6 8 4 6 7 5 7 6 9 2 20 40 40 80 51 46 41 51 47

1 1 1 2 2 1 1 1 1 1 1 1 1 2 4 4 8 10 9 8 11 9

2 2 2 3 3 2 2 2 2 1 1 3 2 8 16 16 32 32 32 30 42 35

* Tested by gel diffusion technique.

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M. B6ttiger and B. Larsson

T a b l e 6. The antibody response in children after two doses administered 1 month apart and the potency levels of the poliovaccines used Mean values of antibody titres Year

Mean values of extinction limits in guinea-pigs

No. tested

1

2

3

1

2

3

69 152 336 516 367 193 30

0.7 0.7 0.9 1.7 1.6 1.4 1.5

1.3 1.4 1.8 2.1 1-9 1.6 3.0

1.0 2.1 2.0 2.1 1.8 1.6 2.0

0.6 1.4 1.7 1.9 2.0 2.1 2.3

1.4 1.1 1.8 1.3 1.5 1.8 2.1

1.6 2-1 3.1 2.0 2.4 2.6 3.0

1957" 1958 1959 1960-4 1965-9 1970-4 1982

* Vaccines imported fi'om U.S.A. Swedish vaccines started usage as from 1958.

Relationship between guinea-pig potency values and estimated total live virus content of vaccines

T a b l e 7. P e r c e n t a g e of children with no demonstrable antibody titres after the 2 first doses of vaccine

When the guinea-pig extinction values were plotted against the estimated viral content of Swedish vaccines (102 lots between the years 1973-89 based on values obtained prior to inactivation with formaldehyde) no trend towards a dose-response relationship could be observed. It was possible t h a t viral content above 107.o CCIDso already had reached a peak and t h a t such a dose-response relationship occurred with viral antigen content below t h a t level.

Poliovirus type Year of vaccination 1957 1958 1959 1960 1961 1962 1963 1964 and later

1

2

3

42 46 27

Swedish inactivated polio vaccine: laboratory standardization and clinical experience over a 30-year period.

Swedish inactivated polio vaccines have contained per single human dose a mean amount of viral antigen equivalent to 1 x 10(7.5) CCID50 of type 1, 1 x...
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