5.Produccion de La Vacuna vs Pertusis

8/12/2019 5.Produccion de La Vacuna vs Pertusis

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Antonie van Leeuwenhoek29 1963 ) 183-201 183

D ev e lop m en t o f Pe r tuss i s Vacc ine Produc t ion and C on t ro lin the N at iona l Ins t i tu te of Publ ic H ea l th in the N ethe r land s

d u r in g th e y ea rs 1 9 5 0 1 9 6 2

H H C O H EN

N a t i o n a l h l st i t u t e o f P u b l i c H e a l t h U t r e ch t T h e N e t h e r l a n d s

CO HE N , H . H . 1 96 3. D e v e lo p m en t o f p e r tu s s is v acc in e p r o d u c t io n an dc o n t r o l i n th e N a t i o n a l I n s t i tu t e o f P u b l ic H e a l t h i n t h e N e t h e r l a n d sd u r i n g t h e y e a r s 1 9 5 0- 1 96 2 . A n t o n i e v a n L e e u w e n h o e k 2 9 : 1 8 3 -2 0 1 .

T h e d e v e l o p m e n t o f t h e p e r tu s s is v a c c i n e p r o d u c t i o n i n th e N a t i o n a lI n s t i t u t e o f P u b l i c H e a l th i n t h e N e th e r l an d s s in ce 19 53 , an d th er e s u l ts w i th t h e co n s ecu t iv e l o ts o f v acc in e i n t h e m o u s e p r o t e c t i o n

t e s t an d th e U . S . A . t o x i c i t y t e s t a re d e s c r ib ed . T h e r e s u l ts i n t h el a t t e r te s t a r e co m p a r ed w i th t h e r e s u lt s o f a lo ca l ly d ev e lo p ed g u in eap ig tox ic i ty tes t. S pec ia l a t ten t ion is g iven to the d i ff icu l t ie s en co un te r edw h e n t h e U . S . A . t o x i c it y t es t is u s ed f o r a d s o r b e d D P T v a c c i ne s .T h e p o t e n c y d a t a o f a ll l o ts o f D P T v a c c in e s p r o d u c e d s in c e 19 58f a ll w i th in t h e l im i ts o f t h e p o t en cy t e s t a s p r e s c r ib ed in t h e U . S . A .M i n i m u m R e q u i r e m e n t s . T h e r e a r e i n d ic a t io n s th a t t h e i n c r e a s e dp o t e n c y o f th e v a c c i n e m a y h a v e l ed t o a l o w e r m o r t a l i t y r a t e o fper tuss is .

I N T R O D U C T I O N

S i n c e 195 0 a s o li d m e d i u m s e e M a t e r i a ls a n d M e t h o d s ) w a s u s ed f o r t h ep r o d u c t i o n o f p e rt u ss i s v a c c in e i n th e N a t i o n a l I n s t it u t e o f P u b l i c H e a l t h .F r e s h ly i s o l a t ed p h as e I s t r a in s , s t o r ed in t h e l y o p h i l i z ed s ta t e , w e r e u s ed .A f t e r h a r v es t i n g , t h e b ac t e r i a w e r e k i ll ed an d d e to x i f i ed b y k eep in g th e v acc in e ,i n a co n c en t r a t ed s u s p en s io n 8 0 o p ac i ty u n i t s p e r m l ) , f o r 7 2 h o u r s a t 3 7 C

i n t h e p r e s e n c e o f 0 .0 1 ~o m e r t h i o l a t e . F o r D P T d i p h t h e r ia - p e r t u s s is - t e ta n u s )v a c c i n e p r o d u c t i o n b a c t e r ia l c o n c e n t r a t e s w e r e m i x e d w i t h p u r i f ie d d i p h t h e r i aa n d t e ta n u s t o x o id s a n d a d s o r b e d o n A I P Q . T h e f in a l D P T v a c c in e c o n t a i n e d2 0 o p a c i ty u n i t s i n 0 .5 m l . It s t o x i c i t y w as m eas u r ed b y in j ec t in g th e v acc in ei n 2 m l a m o u n t s i n t r a p e r i t o n e a l t y i n e a c h o f 1 2 g u i n e a p i g s . T h e a n i m a l ss h o u ld s u r v iv e f o r 3 5 d ay s , b u t a 2 5 ~o lo s s b y in t e r cu r r en t i n f ec t i o n s w asa c c e p t e d .

Ta s m a n 1 9 59 ) c a r r i e d o u t a f ie ld t ri a l w i t h a n u m b e r o f D P T a n d D T



184 H . H . COHEN

va c c ine s p r e pa r e d in th i s wa y in o r de r t o e xa mine loc a l a nd ge ne r a l un f a vour-

ab le s ide e ffec ts . The la t te r were mos t f r equent ly obse rved wi th vacc ines con-t a in ing the pe r tu s s i s c ompone n t . Af t e r subc u ta ne ous in j e c t ions c ons ide r a b lym o r e l oc a l r e a c ti o n s, o f l o n g e r d u r a t i o n , w e r e f o u n d t h a n a f t e r i n tr a m u s c u l a rin jec tion . But on the w hole , vacc ines prep a red in th i s w ay caused on ly a f ewun towa r d r e a c t ions a nd se e me d a c c e p ta b le f o r ge ne r a l u se .

I n 195 5 the in tr a c e r e b r a l m ouse p r o te c t ion t e s t wa s in t r odu c e d in ou rl a bo r a to r ie s . W i th the a id o f a r e f e re nc e p r e p a r a t ion ga uge d by c om pa r i so nwi th the U . S . A . s t a nd a r d va c c ine s no . 4 a nd 5 ( Cohe n , 1958) i t be c a m e poss ib leto de t e r m ine the po te nc y o f t he va c c ine s a va i la b le a t t he t ime .

The a ve r a ge po te nc y o f 13 c onse c u t ive lo t s o f D P T va c c ines p r e pa r e dbe f o r e 955 wa s c a l c u la t e d a s 4 . 5 p r o te c t ive un i t s pe r t o t a l immuniz ing dose .The r a nge wa s 1 . 5 - 15 . 2 un i ts . O n ly 3 lo ts m e t the U . S . A . po te nc y r e qu ir e -me n t s o f min im a l 8 un i t s in t he to t a l imm uniz ing dose .

I n a g r e e m e n t w i th the r e su l ts o f po te n c y de te r m ina t ions , D P T va c c ine sf r om th is s e rie s c on f e r r e d on ly pa r t i a l im m un i ty to the va c c ina te d c h i ld r e n .Vos (1961) was ab le to e s t im a te the imm un iz ing e ffec t in ch i ld ren . He obse rv eda n e p ide mic in a sma l l v il la ge , whe r e a m ino r i ty o f t he popu la t ion ha d c on -sc i en t ious ob je c t ions to the u se o f va cc ine . The ma jo r i ty, howe ve r, no t be long ingto th i s pa r t icu la r r e l ig ious group , a ccep ted v acc in a t ion of the i r ch i ld ren . InTab le 1 a su m m ar y of Vos re su lt s , is g iven . To a ce r ta in ex ten t the y agreewi th the da ta o f Spro nk (195l ) , Sm een k (1952) a nd Bos (1960).

In gene ra l , these re su l ts agree wi th the re su l ts o f the M .R .C . f ie ld t r ia l sin G rea t Br i ta in (1956). The resu l ts o f po ten cy de te rm ina t io ns cor re la te wi th

the e p ide mio logy ; t he y r e f l e c t t he r e l a t ive ly low immuniz ing p r ope r t i e s o fthe pe r tuss i s vacc ines produced in these yea rs .

MATERI ALS AN D METHODS

Preparation o f m edia

Solid medium(G) . To I kg of g ro un d m ea t , 4 li te r s o f d i s ti ll ed wa te r and 12

g o f p a p a i n s u s p e n d e d in a s m a l l a m o u n t o f w a t e r w e r e a d d e d . T h e m i x t u r ewa s ke p t a t 6 5 - 70 C f o r 3 h r a n d the r e a f t e r fi lt er e d th r ou gh pa pe r a nd the pHa d jus t e d to 8 - 8 . 2 . The b r o th wa s s t e ri li z ed by h e a t ing i t on two suc ce s siveda ys f o r two h r i n the Ko c h a t 100 C . P h osph a te s we r e r e mov e d by f i lt r a t iont h r o u g h p a p e r a n d t h e b r o t h w a s d i lu t e d w i th t h e s a m e v o l u m e o f s a li ne .T h e p H w a s a d j u s t e d t o 7 . 4 ; 3 ~ a g a r a n d 1 ~ p o t a t o s t a r c h w e r e a d d e d t othe b r o th . T he m e d ium wa s subd iv ide d in 1500 ml qua n t i t i e s in e r l e nme y e rf la sks and s te ri li zed for 30 ra in a t 120 C. W he n the f la sks ha d cooled d ow n



P ERTU SS IS VA C C IN E I N T H E N E T H E R L A N D S 1 9 5 0 - 1 9 6 2

TA B L E 1

E p i d e m i o l o g i c a l r e s u lt s in a g r o u p o f c h i l d r e n ( 0 - 6 y e a r s o l d ) i m m u n i z e dw i t h D P T a d s o r b e d v a c c in e s n o t m e e t in g t h e U . S .A . M i n i m u mP o t e n c y R e q u i r e m e n t s a s c o m p a r e d w i t h a n u n v a c c i n a t e d c o n t r o l g r o u p( 0 - 6 y e a r s o l d )

185

C l i n i c a l a p p r a i s a l Va c c i n a t e d ( 2 06 ) U n v a c c i n a t e d ( 4 6)

1 56 ( 7 5 . 7 % ) 2 4 ( 5 2 . 2 % )ot i l lA s p ec i f i c co u r s e o f t h e d i s -

e a s e ; a v e r a g e d u r a t i o n o ft h e d i s ea s e l e s s t h an 2w e e k s

L e s s t h a n 3 a t t a c k s a d a y ;a v e r a g e d u r a t i o n o f t h ed i s ea s e l e s s t h an 4 w eek s

3 - 1 0 a t t a c k s a d a y ; a v e r a g ed u r a t i o n o f t h e d i s e a s e 6 - 8w e e k s

1 0 a t t a c k s a d a y ; a v e r a g e

d u r a t i o n o f t h e d i s e a s e >I0 w eek s

31 (50 = 24.3 )

11

1( 22 = 4 7 . 8 % )

912

This table was compi led from the data g iven by A. J . Vos (1961).

t o 4 8 C , 1 2 ½ ~ o f s t er il e d e f ib r in a t ed s h eep b lo o d w as ad d e d to each . T h em e d i u m w a s s u b d i v i d e d i n 8 5 m l a m o u n t s i n R o u x f l a s k s .

Fluid medium(V ) . T h i s w as p r ep a r ed a s d e s c r ib ed b y Ve r w e y e t a l. ( 19 4 9 ).

Tes ts fo r f r eedom o f t ox ic it y

U S A mo use toxici ty testT h i s t e s t w as ca r r i ed o u t a cco r d in g to t h e s p eci fi -c a t i o n s o f th e U . S . A . M i n i m u m R e q u i r e m e n t s P e r tu s s is Va c c i n e , 1st r e v is i o nOctober 31s t , 1952 ( sec t ion 4 .11 , 4 .12 ) as fo l lows :

A t l e a s t 2 h o u r s b e f o r e i n j e c t i o n 5 o r m o r e m i c e o f o n e s ex , w e i g h i n g 1 4 - 1 6 g r a m s e a c h ,a r e h e l d i n a s u it a b l e c a g e a n d g i v e n a d e q u a t e f o o d a n d f re e ac c e ss t o w a t e r. T h e y a r e w e i g h e da s a g r o u p i m m e d i a t e l y p r e c e d i n g t h e i n j e c t i o n w h i c h is m a d e i n t r a p e r i to n e a l l y. F o r a f i n alv a c c i n e , t h e d o s e f o r e a c h m o u s e i n a g r o u p s h a l l r e p r e s e n t o n e - t e n t h o f t h e t o t a l h u m a nd o s e o f a p l a i n v a c c i n e a n d o n e - f i f th o f t h e c o r r e s p o n d i n g d o s e o f v a c c i n e c o n t a i n i n g a p r e -c i p i t a t i n g o r a n a d s o r b i n g a g e n t . F o r a n u n f i n i s h e d b a c t e r i a l s u s p e n s i o n , t h e d o s e s h o u l drep re s e n t t h e eq u i v a l en t o f 1 0 o p a c i t y u n i t s . A v acc i n e i s co n s i d e red f r ee o f t o x i c i t y i f a t t h ee n d o f 7 2 h o u r s t h e g r o u p w e i g h t is n o l es s t h a n a t t h e t i m e o f i n j e c t io n a n d n o v a c c i n e - r e l a t e dd e a t h s o f m i c e h a v e o c c u r r e d .

R e c e n t l y ( A u g u s t 19 61), in a m e n d e n t n o . 5 , t hi s p a r a g r a p h w a s a m e n d e das f o l l o w s :



1 8 6 H . H . C OH EN

A group of no t less than I0 mice , each mouse w eigh ing 1 4- t6 g rams, sha l l be used . The

mice shal l have free access to food and water, for no less than 2 hou rs before in jection. Thegrou p weight of the mice shal l be determin ed im me diate ly before in ject ion. Each m ouse shal lbe injected intraperi tone al ly with a dose o f the bacter ia l suspension, consis t ing of n o less than10 opaci ty uni ts or a dose of the solut ion of bacter ia l f ract ion, consis t ing of no less thans ix- ten th o f the amount to be incorpora ted in a s ing le human dose o f the f ina l p roduc t .The grou p w eigh t o f the m ice sha l l be de te rmined a t the end of 72 hours and 7 days . The bu lkm ater ia ls shal l be considered free from toxici ty i f a t the end of 72 hou rs the gro up weight isno less than the weight preceding the injection and at the e nd of 7 days the average gain permo use is no less than 3 .0 grams a nd the vaccine death ra te does not exceed 5 percent . Resu l tsof a l l tes ts performed shal l be reported .

I n t h e w o r k r e p o r t e d i n t h is p a p e r t h e t o x i c i t y t e s t h a s b e e n c a r r i e d o u t

a c c o r d i n g t o t h e o r i g i n a l p r e s c r i p t i o n . T h e r e s u lt s g iv e n i n F ig . 1 s h o w, h o w -

e v e r , t h a t a p r o d u c e r , w h e n u s i n g t h is p r o c e d u r e , m a y r u n i n t o t ro u b l e . I n

t h is fi g u re th e g a i n i n w e i g h t a n d t h e d e a t h r a t e s o f t w o g r o u p s o f m i c e o n

d i f fe r e n t d i e t s, s e ve n d a y s a f t e r i n t r a p e r i t o n e a l i n j e ct i o n o f i n c r e a s i n g a m o u n t s

o f p l a i n p e r t u s s is v a c c i n e , a r e p r e s e n t e d .

4 V E R A G E W E I G H T G A I NI N , S EV E N D AY 3

80 (GRA , '4 ,$)

/2 0 I N T E R C U R R E N T D E AT H S

0120

o o

o o l . . . . =C O N T R O L ,¢ 00 2 0 0 0 1 0,0 00 5 0 ,0 0 0 X y O 6 B A C T E R I A

Fig. 1 . Av erage weight gain and death ra te in m ice injected with increasing amo unts

of p la in pertussis vaccine and supp lied ad l ib i tum with two different k inds o f food.

F r o m t h e r e s u l t s o f t h i s t e st , i t is a p p a r e n t t h a t m i c e f e d o n a p e l l e t d i e t

r e s p o n d e d w i t h l es s g r o w t h t h a n d i d m i c e f ed o n a s p e c i al p r o t e i n d i e t. I t is

c l e a r , a c c o r d i n g l y, t h a t t h e q u a l i t y o f t h e d i e t a f fe c ts th e r e s u l ts o f t h e t o x i c i t y

t e s t .

Guinea pig test T h i s t e s t w a s d e v e l o p e d a s f o l l o w s :

A s t h e D P T- v a c c i n e s , c o n t a i n i n g t he p e r tu s s is c o m p o n e n t p r e p a r e d o n th e



PERTUSSIS VACCINE IN THE NETHERLANDS 19 50 -1 96 2 187

G m e d i u m d i d n o t g i ve u n f a v o u r a b l e r e a c t io n s in c h i ld r e n d u r i n g m a s s v a c c i-

n a t i o n c a m p a i g n s , i t w a s d e c i d e d t o u s e a n u m b e r o f p l a i n p e r tu s s is c o n c e n t r a t e s ,p r e p a r e d o n t h e G m e d i u m a s r e f e r e n c e p r e p a r a t i o n s . T e n l o ts o f G v a c c i n esw e r e c o m p a r e d w i t h te n l o t s o f V v a cc i n e s f o r r e a c ti o n s o f V v a c c i n e s i nch i ld r en , s ee p ag e 1 96 ). G r o u p s o f 5 g u in ea p ig s w e r e i n j ec ted s u b cu tan e o u s lywi th 160 × 109 bac te r ia pe r an im al . T hr ee c r i t e r ia were used to assess d i ff e r-en ces i n t o x i c i t y o f t h e d i f f e r en t l o t s :

I ) t e m p e r a t u r e ri se ,

2 ) we igh t ga in ,3) d i a m e t e r o f i n f il tr a t io n m e a s u r e d d a i l y f r o m t h e t h i r d t o t h e f o u r t e e n t hd ay a f t e r i n j ec t i o n .

N o d i ff e re n c e s b e t w e e n G a n d V v a c c i n es w e r e f o u n d as f a r a s t e m p e r a t u r ea n d w e i g h t g a in w e r e c o n c e r n e d . H o w e v e r , t h e r e w a s a c l e a r d i ff e r e nc e inth e d i am e te r s o f s u b c u tan eo u s i n f i lt r a ti o n . F ig . 2 s h o w s th e av e r a g e v a lu e sf o u n d b e t w e e n t h e t h ir d a n d t h i r te e n t h d a y o f o b s e r v a t io n .

0,9

a 7

a s

0 3

O f

D I A M E T E R I N F I L T R A T I O NI N C M

0

3 7 9 f ' / f3> O B S E R VA T I O N p E R I O D I N DA Y S

Fig. 2. M ean diameter value in cm) of subcutaneous infiltrations caused by theinjection o f 160,000 × 106 g erm s of 10 lots of V an d 10 lots of G vaccine ov era period of I0 days . For each vaccine 5 guinea pigs we re used.

T h e d i f f e r e n c e b e t w e e n G a n d V v a c c i n e s is a lr e a d y o n t h e t h i r d d a y h i g h l ys ig n if i can t . T h e r e s u l ts a r e c a l cu l a t ed w i th t h e an a ly s i s o f t h e v a r i an ce t e ch -n iq u e . Wi th t h i s m e th o d i t i s p o s s ib l e t o e l im in a t e a l l d i f f e r en ceswit in t h e Va n d G g r o u p s . T h a t i n t e rg r o u p d i ff e r en c e s r e a ll y e x i st is s h o w n b y t h e f i g u re sin Tab le 2 :



188 H H C O H E N

TA B L E 2

Mean d iam e te r v a lu e in cm o f in f i l t r a t io n s 7 d ay s a f t e r su b cu tan eo u s in j ec t io n o f160,000 × 106 bac te r ia f rom 10 lo ts o f V vacc ine an d 10 lo ts o f G vacc ine , F or eachv acc in e , 5 g u in ea p ig s were u sed .

Av e r a g e d i a m e t e r v a l u e in c m

G v ac c in e s 0 .6 5 ; 0 .5 ; 0 .5 ; 0 .6 ; 0 . I ; 0 . 5 5 ; 0 .9 5 ; 0 .6 ; 0 .3 5 ; 0 .3V vacc ines 0 .95 ; 0 .7 ; 0 .85 ; 0 .3 ; 0 .6 ; 0 .65 ; 1 .7 ; 0 .75 ; 0 .75 ; 0 .55

I n th i s Ta b le f o r e a c h lo t o f va c c ine the me a n d i a m e te r va lue on the se ve n thda y i s g ive n . I t t u r ns ou t t ha t t he va lue s in bo th g r oups do ove r l a p .

Th i s is c on f i r me d b y a na lys ing the r e sul ts w i th the W i lc oxon m e thod . I nTa b le 3 the r e su l ts o f t he a na lyse s f r om the th i r d to the th i r t e e n th d a y a r e g ive n .

TA B L E 3

E x p e c t a t io n o f t h e o b s e r v e d r a n k i n g o r d e r s i n t h eWi lco x o n t e s t o f t h e d i am e te r o f i n f i l t r a t io n v a lu es o f

1 0 V an d I0 G v acc in es f ro m th e th i rd to th e th i r t een thday af te r in jec t ion

D a y E x p e c t a t i o n o f t h e D a y E x p e c t a t i o n o f t h eo b s e r v e d r a n k in g o b s e r v e d r a n k i n go r d e r o r d e r

3 63 8 3 14 43 10 1 J)5 33 11 2 1

6 22 12 1 l)7 1 l) 13 2 l)

0 Values excee ding 95 confidence limits,

The e xpe c ted va lue e xce e ds the 95 70 c on f ide nc e l imi ts on ly f r o m the se v e n thda y onwa r ds . Th i s s t r e s se s a ga in tha t l oc a l r e a c t ions w i th in e a c h g r oup a r es t r ong ly d i f f e r e n t . On ly f r om th i s da y on , t he d i f f e r e nc e s be twe e n bo th type so f va c c ine s ar e su ffi c ie n tly p r o no un c e d to m a ke the obse r ve d r a nk in g o r de r

h igh ly impr oba b le .Af t e r t he e va lua t ion o f t he se r e su l ts i t c ou ld be c on c lude d tha t :a V vacc ines were mo re tox ic fo r gu inea p igs than G vacc ines ;z G rea t va r ia t ions ex is ted be tween severa l lo t s o f one type o f vacc ine .

pplicat ion o f sequential analysis to results obtained in the guinea pig tes t

I t wa s de c ide d to ba se f u tu r e tox ic i ty t e s ts on the loc a l r e a c t ions in g u ine ap igs a f t e r subc u ta n e ou s in j e c tion o f va c c ine . The r e su lt s w i th G va c c ine s



PERTUSSIS VACCINE INTH N ET H ER LA N DS 1 9 5 0 1 9 6 2 1 8 9

w e r e a c c e p t e d a s a r e f e r e n c e . T h i s s e e m e d a l o g i c a l p o l i c y s i n ce o n l y a f e w

c o m p l a i n t s w e r e r e c ei v e d f r o m g e n e r a l p r a c t i t i o n e r s a f te r i n je c t io n o f D P Tv a c c i n e a d s o r b e d ) p r e p a r e d w it h G v ac c i n e .

Te n t a t i v e l y t h e f o l l o w i n g r u l e s w e r e d r a f t e d :

1) a p l a i n v a c c i n e w i t h o u t a d s o r b e n t is a c c e p t e d i f w e a r e 9 9 ~ s u r e t h a tt h e a v e r a g e d i a m e t e r o f i n f il tr a t io n o n t h e s e v e n t h d a y a f t e r s u b c u t a n e o u sin jec t ion o f 160 ,000 × 10~ g e r m s i s le s s t h a n 0 .6 c m ,

2 ) a p l a i n v a c c i n e w i t h o u t a d s o r b e n t i s r e j e c te d i f w e a r e 9 9 ~o s u r e t h a t

t h e a v e r a g e d i a m e t e r o f i n f il t ra t io n o n t h e s e v e n t h d a y a f t e r s u b c u t a n e o u sin je ct ion o f 160 ,000 × 10~ g e r m s i s l a rg e r t h a n 0 .8 c m ,

3 ) a s lo n g a s n o d e c i s i v e d a t a a r e a v a i l a b l e t h e e x p e r i m e n t s a r e c o n t i n u e d .A s t h e o b s e r v a t i o n p e r i o d is c o m p a r a t i v e l y lo n g , g r o u p s o f 5 a n i m a l ss h o u l d b e i n j e c t e d s i m u l t a n e o u s l y w i t h t h e s a m e v a c c i n e .

I n F i g . 3 a g r a p h i c a l d e s i g n o f s u c h a s e q u e n t i a l t e s t i s g i v e n . A s l o n g a st h e v a l u e o f t h e s u m o f th e d i a m e t e r s r e m a i n s b e t w e e n th e l in e s t h e e x p e r i m e n tm u s t b e c o n t i n u e d . A s s o o n a s i t p a s s e s t h e u p p e r l i n e t h e v a c c i n e i s r e j e c t e d ,

w h i l e t h e c r o s s i n g o f t h e l o w e r li n e m e a n s a c c e p t a n c e .

. . R U M D I A M E T E RI N F I LT R A T I O N . S

M = O . 6 0 /M J 0 8 0 ~ - ~ R E J E C T F . O

x

x

• , N U M B E R O F A I 4 1 M A L 5

Fig . 3 . Sequent ia l t es t fo r acceptance or re jec t ion of per tuss i s vaccknes on accountof their local toxicity for guinea pi gs after subcutaneous injection. As long asthe sum of the diameter values fallsetween the lines the experiment is continued.Passing the lower lin e me ans acceptance, passing the upper line m eans rejection.

I n F i g. 4 th e r e l a t io n b e t w e e n t h e t h e o r e t ic a l l y e x p e c t e d m e a n v a l u e o f th en u m b e r o f a n i m a l s n e e d e d t o r e a c h a d e c is io n , a n d t h e m e a n d i a m e t e r v a l u e is



1 90 H . H . C OH EN

g i v e n . I t is c l e a r t h a t a t t h e v a l u e o f 0 .7 c m , i .e . th e v a l u e j u s t b e t w e e n 0 .6 a n d

0 .8 t he c u r v e r e a ch e s it s m a x i m u m 3 3 a n i m a l s , w h i c h m e a n s c o n t i n u a t i o no f the ex pe r im en t s du r ing 33 /5 ----- 7 week s ) .

NUMBER OF ANIMALS

M f = t ~

M 2: o a o

~( = o O f

~2

28

20

15

12

55 6 6 5 Q 7 75 8 8 5

~J .H EA N REAL OIAMETER iN FILTRAT ION

Fig. 4. The grap h g ives the relation betw een the real diameter value of infiltrationof a vaccine and the average number of animals which must be used t? reach a de-cision. It is clear that when a real value of 0.7 is obtained, a long time is requiredto reach a decision; 33 animals have to be injected, wh ich means continuation of thetest during 33/5 = 7 weeks.

M o u s e p r o te c t io n t es t

T h i s te s t is p e r f o r m e d a c c o r d i n g to t h e U . S .A . M i n i m u m R e q u i r e m e n t s ,1s t rev is ion O ct o be r 31s t , 1952 .

A s a r e f er e n c e v a c c i n e t h e N a t i o n a l I n s t it u t e o f P u b l i c H e a l t h , U t r e c h t -r e f e r e n c e v a c c i n e C w a s u s e d C o h e n , 1 95 8). R e s u l t s w e r e c a l c u l a t e d b y p r o b i tana ly s i s .

Te s t f o r H . S . F. H i s t a m i n e - S e n s i ti z in g - F a c t o r ) c o n t e n t

T h i s f a c t o r w h s d e t e r m i n e d i n c o m p a r i s o n w i t h r e f e r e n c e v a c c i n e C a n de x p r e s s e d i n u n i ts a c c o r d i n g t o t h e m e t h o d o f P r e s t o n 1 9 59 ). T h e t e s t w a sc a r r i e d o u t b y in j e c t i n g d e c r e a s i n g d o s e s o f t h e v a c c i n e b e i n g t e s t e d 6 , 1 .2a n d 0 .2 4 o p a c i t y u n i t s i n 0 .5 m l ) i n g r o u p s o f 20 m i c e . F o u r d a y s l a t e r, t h em i c e w e re c h a ll e n ge d w i th 4 .4 m g o f h i s ta m i n e - a c i d p h o s p h a t e . T h e a n i m a l sd y i n g w i t h in 2 4 h r w e r e re c o r d e d . R e s u l t s w e r e c a l c u la t e d b y p r o b i t a n a l y s i s.



P E RT U S SI S VA C C IN E I N T H E N E T H E R L A N D S 1 9 5 0 - 1 9 6 2 1 9 1

RESULTS

ncrease of potency of per tussis vacc ines

As ear ly as in 1952 Lansberg in th i s l abora tory made exper iments on thep rod uc t ion o f pe r tus s is vacc ine i n a l iqu id med ium. Fo r t he dev e lopm en t o fsuch a metho d the V m ed ium Verwey e t a l., 1949) was chosen . A nu m ber o fspecia lly se lec ted s t ra ins Co hen and Lepp ink , 1956) were cu l tiva ted on so l idm edium G and f lu id m edium V. A f ter cen tr i fugat ion , the bacter ia l m ass was

resu spen ded in sal ine with 0.01 ~o m erthiolate . D etoxif icat ion an d ki ll ingtook p lace by heat ing the suspens ions for 3 days a t 37 C.In T able 4 the resu l ts of po tency test s a re g iven as potency in uni ts p er 60

opaci ty uni ts .

TA B L E 4

P o t e n c y p e r t o t al i m m u n i z i n g d o s e o f a n u m b e ro f p e r t u s s i s v a cc i n e s p r e p a r e d f r o m d i f fe r e n t s t r a i n sa n d g r o w n o n f l u id a n d o n s o li d m e d i u m

S t r a in P a s s a g e M e d i u m

s o l i d G ) f l u id V )

512 a 1.8 1 2 . 0

b 2 .4 2 0 . 4

534 a 1 8 . 0 6.0b 1 3 . 2 d 4 4

3838 a 1 3 . 2 4 2 . 0

b 1 . 8 1 2 . 0

509 a 6 .6 1 5 . 2b - - 4 8 . 0

530 a 1 .2 3 .6b 4 .8 7 .8Av e r a g e 7 . 2 2 1 . 0

I t a l i c s : D at a m ee t in g t h e U .S .A . M i mm u m Re-q u i r emen t 8 U n i t s )

Fr om the resu lt s it can be infer red tha t the a m ou nt of pro tec t ive an tigen

form ed largely depen ds o n the cu l tura l condi t ions . Th e usefu lness of f lu idmedium V for vaccine product ion i s a l so apparent f rom the resu l t s in Table 5 .

This t ab le g ives among o ther th ings the potency, the H.S .F. conten t andthe m ouse t ox i c i ty o f t he pe r tus s is co mp onen t , p rodu ced in f lu id med iumin th is l abo ra tory s ince 1958, and used in d i fferen t lo t s of adso rbed D P Tvacc ines . F rom the da t a p resen t ed , i t can be conc luded tha t t he po t ency da t ao f a ll vaccines prod uce d , fa ll wel l wi th in the l imi t s 8 - 36 pro tec tive uni t s / to ta limmuniz ing dose , as s t ipu la ted in the U.S .A. Minimum Requi rements . The



1 9 2 H . H . C O H E N

TA B L E 5

P o t e n cy, H . S . F. c o n t e n t a n d m o u s e t o x i c it y o f a n u m b e r o f D P T v a c c in e s , p r o d u c e di n th e N a t i o n a l I n s ti tu t e o f P u b l i c H e a l t h , U t r e c h t , t h e N e t h e r l a n d s

Lo t . n r. Po ten cy H ,S .F. Ra t io U .S .A . To x ic i ty t e st

( I . U . / to t a l ( U / t o t a l P / H S F W e i g h t g a i n I n t e r c u r r e n tim m . d o se ) im m . d o se ) a f t e r 3 d ay s a f t e r 7 d ay s d ea th s

R 24.6 24.6 1 .03 8 1 0 . 8 6 . 6 - 6 . 0 - 1.1

15.6(av. 9.4)

39 18.9 12.6 1.540 74.4( ) 23 .4 3.2( )4t 18.0 21.0 0 .942 36.0 13.2 2.743 21.0 12.0 1.844 11.4 5.8 2.0

45 4 .2-2 0.4 5 .7 2 .2(av. 12.3)

46 20.4 2 .4 8 .547 10.8 1 .5-7.2 2 .5

(av. 4 .4)48 15.6-2 8.2 2.4 9.1

(av. 21.9)49 28.850 22.851 15.652 11.453 26.654 22.855 18.056 33.057 27.058 58.8-11.4

(av. 35.1)59 29 .460 I3 .863 20.464 25.265 21.066 35.467 14.468 16.8Av erag e 2 0 .9

- --0 .3 +2 .3 3 /10

+ 0 . 3 + 2 . 8 2 / 10+ 0 . 2 + 2 . 2 2 /1 0

1 . 0 3 . 1 0/10- --0 .2 +0 .9 0 / I0

10.8 2.71 5.2 1 .5 +2 .6 ,+ 0 . 3 +2 .7 , +2 .1 1 /2 011.4 1 .4 - - t . 7 , - - 9 . 2 - - 0 . 7 , + 0 . 9 3 /2 01 6.2 0 .7 - -0 .9 +0 .2 0 /1 01 3.8 1 .9 - -0 .9 +0 .7 0 /1 02 4 .6 0 . 9 - - 9 . 6 + 2 . 2 0 / 1010.8 1 .7 - -0 .9 +2 .8 0 /1025 .2 1 .3 - -0 .1 +0 .8 1 /10

- - 9 . 7 , - - 0 . 9 - - 1 . 2 , + 1 . 1 5 /2 0- - 0 . 6 , + 0 . 0 + 2 . 3 , + 1 . 0 3 /2 0

-- 0 .9 + 1 .3 1/10- -0 .5 + 1 .2 2 /1 0- -0 .3 +0 .5 1 /1 0- - -0 .6 +0 .5 0 /10+0 .5 +1 .5 1 /1 0+0 .0 + 1 .4 0 /1 0- - 0 . 1 , + 0 . 0 + t . 8 , + 1 . 7 2 /2 0- -- 0. 2, + 0 . 7 + 1 , 1 , + 2 . 0 1 /2 0- -0 .2 + 1 .4 2 8 /2 80 (1 0 %)



1 9 4 H . H . C OH EN

To xic i ty o f p la in vaccines prepared in f lu id and in so lid med ium and su bm it tedto different methods of detoxifieat ion

Toxicity test in guinea pigs and miceI n Ta b l e 6 t h e r e su l ts o f a n u m b e r o f

t o x i c i t y t e st s i n g u i n e a p i g s w i t h 5 f r e s h l y p r e p a r e d G a n d V v a c c i n e s a r e g iv e n .

TA B L E 6

Results of guinea pig toxici ty tes t of 5 G and 5 V vaccines

Vaccinepreparedf romstrain :

So l id medium G ) vaccine F lu id medium V) vacc ine

N um b er Av e rage d i a - N um ber Ave rage d i a -of an imals mete r va lue o f an imals mete r va luerequired for of the inf il - required for of the inf il -acceptance t ra te cm) acceptance t ra te cm)

530 4 0.35 7 0.453838 7 0.40 13 0.56

509 5 0.48 5 0.45512 4 0.30 19 0.60534 5 0.35 5 0.40

Av erag e 5.0 0.38 9.8 0.49

I t i s c le a r , t h a t a l t h o u g h a l l v a c c i n e s a r e f in a l l y a c c e p t e d , t h e V v a c c i n e s a r e

m o r e t o x i c t h a n t h e G v a c c in e s . T h i s is c l e a r f r o m t h e g r e a t e r n u m b e r o f

a n i m a l s n e c e s s a r y t o r e a c h a d e c i s i o n a n d t h e l a r g e r a v e r a g e i n f i l t r a ti o n .

I n Ta b l e 7 t h e r e s u l ts w i t h t h e U . S . A , m o u s e t o x i c i t y t e s t w i t h t h e v a c c i n e s

m e n t i o n e d in Ta b l e 6 a r e g iv e n .

TA B L E 7Tox ici ty of 5 G and 5 V vaccines see Tab le 6) in theU.S.A. mo use toxici ty tes t

S o l id med ium G ) F lu id med ium V )vaccine vaccine

530 - t-0.6 +2 .8 0/10 -- I .0 +2 .2 0/103838 - -0 .4 +3 .4 0 /10 - -3 .7 +1 .0 5 /10

509 ~0 .1 +2 .6 0 / I0 - -2 .0 - -0 .5 2 /10512 q 0 . 2 + 2 . 8 1 / 1 0 - - 0 . 6 + 0 . 9 2/10534 ---0.4 +1 .9 0/10 ---0.5 +2 .8 0/10

Averageo f v a lu e s + 0 . 0 + 2 . 7 1 / 5 0 - -1 .6 + 1 .3 9 /50

Vaccine Av erage weight Inter- Av erage weight Inter-prepa red gain 10 mice) cu rrent gain 10 mice) curren tfrom after 3 af ter 7 deaths af ter 3 af ter 7 d eathsstra in : days days days days



PERTUSSIS VACCINE ]N THE NETHERLANDS 19 50 -1 96 2 195

A l th o u g h th e r e s u l ts w i th t h e i n d iv id u a l v acc in e s a r e n o t c l e a r ly co r r e l a t ed ,

o n t h e w h o l e t h e d a t a o f t hi s Ta b l e c o n f i rm t h e r e s u lt s o f Ta b l e 5 : t h e V v a c c in e sa r e m o r e t o x i c t h a n t h e G v a c ci ne s . M o r e o v e r o n l y tw o G v a c c in e s s t r a in5 3 0 an d 5 0 9) an d n o t a s in g le V v ac c in e rea l l y p a s s ed th e t e s t. T h i s l e ad s t oth e co n c lu s io n th a t t h e U . S . A . t o x i c i t y t e s t is m o r e s en s it iv e in d i s co v e r in gr e s id u a l t ra c e s o f t o x in . W e t h e n t r i e d t o i m p r o v e t h e m e t h o d s o f v a c c i n ed e t o x i f ic a t i o n , p a s s in g o f th e U . S . A . t e s t b e i n g t h e c r it e r io n f o r f r e e d o mo f to x i c it y. I t w a s f o u n d t h a t b y h e a t i n g t h e v a cc i n e s f o r 3 0 - 6 0 m i n a t 5 6 Cin t h e p r e s en ce o f 0 .0 1 ~o m er th io l a t e , v acc in e s m e e t in g th e U . S . A . M in im u mR e q u i r e m e n t s f o r To x i c i t y c o u l d b e p r o d u c e d r o u t i n e l y i n th e V m e d i u m . I nTa b le 8 t h e r e s u l ts w i th f o u r l o t s o f V v acc in e s p r ep a r e d in t h i s w ay a r e g iv en .A l l f o u r v acc in e s p a s s ed th e t e s t .

TABLE 8

M ouse toxicity of 4 V vaccines heated at 56 C 30 min)in the presence of merthiolate

Vaccine prepared A ve ra geweight gain of Inter-from strain : 10 mice current

after 3 after 7 deathsdays days

3838 +0.1 +2.1 0/10512a +0 .2 +2.1 0/10512b +1.3 +2 .6 0/10509 +0 .8 +2 .6 0/10

Average weight gain + 1.6 +2 .4 0/10

Control +2 .7 +4 .2

Ab sence o f toxic reactions in children with V vaccines which pa sse d the U .S.A.mouse toxicity test .A p la in v acc in e , d e to x i f ied b y h ea t in g , w h ich h ad p a s s edth e U . S . A . t o x i c i t y t e s t w as co m p a r ed w i th a G v acc in e . I n Tab le 9 t h e r eac t io n sin ch i ld ren a re g iven .

I t m u s t b e s tr e s s ed th a t v i r t u a l l y n o u n f av o u r a b le l o ca l o r g en e r a l r e ac t io n sw e r e m e t w i th a f t e r s u b c u t a n e o u s i n je c t io n o f e it h e r o f t h e se t y p e s o f v a c c in e .

H o w e v e r , t h e p e r c e n t a g e c h il d r e n w i t h t e m p e r a t u r e s h i g h e r th a n 3 7 .5 C s u g g e s tth a t t h e V v ac c in e s st il l h av e a s o m ew h a t h ig h e r r e s id u a l t o x i c i t y t h an th e Gv acc in e s .

Toxici ty o f adsorbed DPT vaccines prepa red with V vaccines d etoxif ied byheating for 60 rain at 56 C

To xic ity reactions in animals. Esta blishm ent o f a unit H .S.F .D P T v a c c i n e sp r e p a r e d in o u r l a b o r a t o r y c o n t a i n 16 o p a c i t y u n i ts o f p er tu s s is v a c c i n e p e r



1 96 H . H . C O HE N

s in g l e d o se , a d s o r b e d i n A I P O 4 ( 1.5 m g p e r d o s e ) . O r i g i n a l l y, t o m e a s u r e t o x -

i ci ty, o n l y t h e i n t r a p e r i t o n e a l g u i n e a p i g t e s t w a s c a r r i e d o u t a s d e s c r i b e du n d e r M a t e r i a l s a n d M e t h o d s . I n Ta b l e 5 t h e re s u l ts a r e g iv e n o f th e p o t e n c yt e st s a n d t h e t o x ic i t y t e st s o f a ll D P T a d s o r b e d v a c c i n e s p r e p a r e d s in c e 1 95 7w i t h t h e V p e r t u s s i s c o m p o n e n t .

TABLE 9

Toxic reactions in children afte r injection of 20 opa city units plainpertussis vaccines (G vaccines detoxified at 37 C, V vaccines detoxified60 min at 56 C)

Vac cine Num ber of Average tem- Num berofchi ldren Whiningchildren perature with tempe rature

> 37.5 Cafter after after after4 hr 24 hr 4 hr 24 hr

G 48 37.2 C 37 .0 C 15/48 7/48 t4/48(31 ) (14.6 )

V 49 37.4 C 37.1 C 2 1 /4 9 12/49 15/49(42.8 ) (24.5 )

T h e m o u s e t o x i c i ty te s t w a s c a r r i e d o u t r o u t i n e l y b e g i n n i n g w i t h l o t 5 0.M o r e o v e r , a n u m b e r o f v a cc in e s w e r e c o m p a r e d w i th o u r s t a n d a r d p r e p a r a -t i o n t o d e t e r m i n e t h e i r r e l a ti v e H . S . F. c o n t e n t . T h e r a t i o o f p o t e n c y / H . S . F.c o n t e n t s e e m s t o b e s o m e w h a t l a rg e r t h a n 1, w i t h t h e e x c e p t i o n o f l o t s 4 4 - 4 8,

in w h i c h m u c h h i g h e r v a lu e s w e r e f o u n d . ( Av e r a g e v a t u e s l o t 3 8 - 4 3 , 4 9 - 5 6 =1.66, l o ts 4 4 - 4 8 = 4 .1 7) . F o r t h e p r e p a r a t i o n o f lo t s 4 4 - 4 8 a p l a i nv a c c i n e w a s u s e d w i t h a c o m p a r a t i v e l y l o w p r o t e c t i v e v a l u e ( 1.0 u n i t p e r8 .0 0 0 × 106 g e r m s ) . A f t e r a d s o r p t i o n a n e x p l ic i t a d j u v a n t e f fe c t o f t h e A I P O 4o n t he p o t e n c y w a s o b s e r v e d . T h e m e c h a n i s m p r o b a b l y d o e s n o t a f f e ct t h eH . S . F. f a c t o r. I t is i n t e r e s t in g t h a t t h e a d j u v a n t e f f e ct i s l e ss m a r k e d w i t h p l a i nv a c c i n e s o f h i g h e r p o t e n c i e s ( 2 - 3 u n i t s p e r 8 . 0 0 0 × 106 b a c t e r i a ) , a s a r ec u r r e n t l y p r o d u c e d i n o u r l a b o r a t o r y.

F r o m t h e r e s u lt s o b t a i n e d a f t e r t h e a d o p t i o n o f t h e U . S . A . m o u s e t o x i c i tyt e s t i t b e c o m e s o b v i o u s t h a t m o s t o f t h e v a c c i n e s d i d n o t p a s s t h i s t e s t ( se ea l so M a t e r i a ls a n d M e t h o d s , m o u s e t o x i c it y t es t) .

Reactions in children vaccinated with DPT vaccine I n t h e c o u r s e o f t w oy e a r s a b o u t 15 c o m p l a i n t s o f u n t o w a r d r e a c ti o n s w e r e r e ce iv e d . M a n y o ft h e m c o u l d b e a s c r ib e d t o f a u l t y i n j e c ti o n t e c h n i q u e s i n w h i c h t h e g r e a t e rp a r t o f t h e v a c c i n e w a s d e p o s i t e d i n t h e s u b c u t a n e o u s t is su e .



198 H . H . COHEN

1953, the ye a r in wh ic h a p r o g r a m o f ma ss va c c ina t ion wa s s t a r t e d . F r o m

this yea r on , u n t i l 1960 , the m or ta l i ty r emains m ore or le ss on the sam e leve l,w h e r e a f t e r a s e c o n d d r o p b e c o m e s a p p a r e n t . T h i s se c o n d d r o p r o u g h l y c o i n -c ide s w i th the in t r odu c t ion in the e nd o f 1958 , o f a v a c c ine o f imp r ove dqu a l i ty which , in the course of tha t yea r, r ep laced the supp l ie s s to red in thec oun t r y. We r e a l i z e tha t t he se f a c t s c a nno t be a c c e p te d a s f ina l p r oo f o f t hep r o te c tive a c t iv i ty o f t he va c c ine ; e spe c ia lly no t , s inc e we do no t know e xa c t lythe pe r c e n ta ge o f t he popu la t ion va c c ina te d . H ow e ve r, t he qua n t i ti e s o f t heva c c ine s sh ippe d f r om th is l a b o r a to r y in the l a st f e w ye a r s ha ve be e n ne a r lyc ons t a n t . A t a n y r a t e t hese da t a do no t d i sa g re e w i th the r e su l ts o f t he Br i ti shFie ld Tria l 1956).

T h e p o t e n c y li m it s o f th e U . S .A . M i n i m u m R e q u i r e m e n t s a r e a p p a r e n t l ywel l chosen . The resu l t s in Table 5 show c lea r ly tha t a l l lo t s p roduced exceptth ree lo ts 40 , 45 an d 58), have a po tenc y fa l l ing we l l be tween the l imi t s o f8 - 3 6 p r o te c tive un i ts pe r to t a l imm uniz ing dose . Exc lud ing a bn or m a l va lue s ,the a r i t hme t i c me a n o f t he po te nc y va lue is 20 .9 w i th a v a r i a t ion be twe e n10.8 and 36.0.

A s e c o n d po in t o f i n te r e s t is , t ha t t he H . S . F. c on te n t o f the va c c ine s doe sno t r un pa r a ll e l w i th the p r o te c t ive a n t ige n . The o r e t i c a l ly the r a t io be twe e nboth va lues should be un i ty, a s a l l vacc ines a re gauged on the same re fe renceprep ara t ion . On the whole see Tables ) the ra t io is l a rge r, e spec ia l ly in lo t s44 - 4 8 . The se lo t s we r e p r e pa r e d f r om o ne ba t c h o f p l a in va c c ine w i th c om-pa r a t ive ly low po te nc y . On pa ge 196 th i s wa s e xp la ine d by a s sum ing a d i f f e re nc ein a d juva n t e f fe c t o f A1P O 4 on bo th f a c to r s .

How e ve r, the r e ma y be a n a l t e r na tive e xp la na t ion . M un oz e t a l. 1959) ha veshow n tha t bo th fac tor s a re c lose ly con nec ted w i th in the bac te r ia l ce ll wa l l. Inc on t inuou s c u l tu r e s the ir o p t ima l syn thes i s t a ke s p l a c e a t the s a me d i lu t ion r a t eVa n He m e r t a nd Coh e n , 1962) . Joo e t a l. 1961a , I 961b) e ve n sugge s t e d

to u se the H . S . F. t e s t i n mic e a s a n ind ic a to r o f va c c ine po te n c y a s th i s t e st isle ss t ime c ons um ing tha n the mouse p r o te c t ion t es t. The r e a r e , how e ve r, a l soind ica t ions P i t tm an , 1951; Co hen , 1962; Su the r lan d , 1962) tha t the H .S .F .can be spec i fica lly des t ro yed , whi le p ro tec t ive an t igen rem ains in tac t .

The e va lua t ion o f t he pha r m a c o log ic a l a c tiv i ty o f th i s f a c to r in the hu m a nbe ing i s ve ry d i ff icu l t e spec ia lly wi th rega rd to the ra re cases o f se r ious en ce -pha lopa th ies , which sporad ica l ly occur d i rec t ly a f te r pe r tuss i s vacc ina t ion .The or e t i c a l ly th is f a c to r c ou ld be r e spons ib l e f o r t h i s c omp l i c a tion a nd ina t t e mpt s f o r pu r i f i c a t ion f u l l a t t e n t ion shou ld be pa id to th i s f a c t .

T h e t h h d po in t o f i n te r e s t is , t ha t D P T va c c ines a dso r be d on AI P O4 ,wh ic h a pp a r e n t ly do no t pa s s the U . S . A . t ox ic i ty t e s t a s c a rr i e d o u t in o u rlabora tor ie s , g ive few untoward reac t ions in ch i ld ren . This i s the more re -



PERTUSSIS VACCINE IN THE NETHERLANDS 19 50 19 62 199

m ark ab le , a s the te s t seems to be qu i te sens i tive for the de tec t ion of t r aces

o f tox ins in p la in va cc ines ( see Table 8 and Tab le 9).H o w e v e r, s o m e f a c t o r s m u s t b e k e p t i n m i n d l e s t c o n c l u s i o n s a r e d r a w n

t o o e a rl y. T h e f a c t t h a t m a n y o f o u r D P T v a c ci ne s d i d n o t p as s t h e t e s tma y be a sc r ibe d to a t l e a s t 3 f a c to r s :

1) T h e u s e o f a n a d s o r b e n t i n t h e v a c ci n e.I t i s fa i r ly we l l kno w n , t ha tads orb ed vacc ines a re mo re tox ic fo r m ice , e spec ia l ly i f A1POa is used as anad so rba nt , than p la in ones . In th i s r e spec t the re su l ts o f the m ou se te s t cor re -l a te w i th the r e su l ts in c h i ld r e n . How e ve r, t he qua n t i ty o f AI P O 4 use d byus (1 .5 m g pe r dose ) is no t exce pt iona l ly h igh and , in ou r op in ion , does no texpla in our d ive rgent r e su l t s .

2) T h e r e l a t i v e l y l a rg e n u m b e r o f b a c t e r i a i n o u r v a c c in e s16 opac i ty un i t sin one dose , i.e . t he ma x imu m pe r m i t te d in the U . S . A . , M in im um Re qu i r e -me n t s .

The f a vou r a b le r e su lt s a f t e r t he in t r od uc t ion o f a va c c ine w i th a h igh a ve r a gepo te nc y ha s ke p t u s , so f a r, f r om lowe r ing the a m ou n t o f pe r tu s s i s to 10 o r12 opa c i ty un i t s pe r dose , t he a moun t u se d in ma ny va c c ine s p r e pa r e d byA m e r i c a n p r o d u c e r s . A s s u m i n g t h a t o u r p r o d u c t i o n i s c o n s i s t e n t a s r e g a r d spo te nc y, t h i s wou ld lowe r the a ve r a ge va lue to the o the r w ise a c c e p ta b le l e ve lo f 13 - 16 p r o te c t ive un i ts . I t is cl e a r t ha t t he nu m be r o f ba c t e r ia p r e se n t c a nplay a dec is ive ro te in the ou tcom e o f the tox ic i ty te st , bu t , on the o the r ha nd ,may in f luence the ep idemiolog ica l s i tua t ion .

3) T h e b r e e d a n d c o n d i t i o no t he mice . In th i s r e spec t the paper g iven byPie r sm a (1962) in the P r ag ue Me e t ing on Per tu ss i s i s in te res t ing . This au tho r

ga ve the r e su lt s o f a n um be r o f c on t r o l l a bo r a to r ie s o f Am e r ic a n c om pa n ie sa ll w or k ing w i th the s a me lo t s o f pe r tu s s is va c c ine bu t w i th d i f f e re n t i nb r e ds t r a in s o f mice . He po in t e d ou t t ha t t he r e su l ts , wh ic h d ive rge d ve r y m uc hf r om e a c h o the r, c ou ld be c a use d by the s t r a in o f a n ima l s u se d . The r e su lt so f a f e e d ing e xpe r im e n t ( F ig . 1) show th a t t he c om pos i t i on o f the f o od a l som a y p la y a r o l e i n the se e xpe r ime n t s .

D ur ing ou r t e s ting p r og r a m , e a r ly in 1961, we ha d a pe r iod in wh ic h thenu m be r o f i n te r c u r r e n t de a ths in o u r b r e e d ing s toc k o f Swiss mice ro se to

som e e x te n t , e spe c ia l ly w i th a n im a l s sub je c t e d to s tr e ss . Th e to t a l i n t e r c u r r e n tde a th r a t e e xc e e de d the 5 ~ le vel pe r m i t t e d in the 5 th a m e n dm e n t U . S . A .M in im um Re q u i r e me n t s f o r P e r tu s s is Va c c ine. O u t o f 280 a n ima l s 28 d i e d(10~o). Th e ave rage g a in o f we igh t o f the su rv iv ing mice was 1 .3 g. No n-in je c t ed mic e ge ne ra l ly do no t sho w a we igh t ga in o f mo r e tha n 3 - 4 g .

We a r e inc l ine d to e xp r e s s a s ou r op in ion tha t t he Na t iona l I n s t i t u t e o fP ub l i c He a l th - m ouse t e s t ha s s er ve d a u sef u l pu r pose , bu t shou ld be mod i f ie d .Th i s ha s be e n don e r e c e n t ly (se e Ma te r i a l s a nd M e thods ) . How e ve r, it se e ms



2 0 0 H . H . C OH EN

i m p e r a t i v e t h a t a s t a n d a r d o f t o x i c i ty p r e p a r a t i o n , a d s o r b e d o n m i n e r a l

c a rr ie r, s h o u l d b e e s ta b li sh e d , w h i c h d o e s n o t g iv e t o o m a n y u n f a v o u r a b l er e a c t i o n s in c h il d r en . W i t h t h e a i d o f t hi s s t a n d a r d p r e p a r a t i o n t h e i n fl u e n c e

o f st r a in - a n d f e e d i n g - d i f f e r e n c e s c a n b e e x c l u d e d . T h e h o p e i s j u s t if i ed t h a tin t h e f u t u r e p er tu s s is v a c c i n e c an b e p u ri f ie d s o t h a t t o x i c a n d H . S , F. p r o p e r -

t ie s a r e s p ec i fi c a ll y d e s t r o y e d , o r s e p a r a t e d f r o m t h e p r o t e c t i v e a n t i g e n . W h e ns u c h v a c c i n e s c a n b e p r e p a r e d o n a l ar g e s ca le , o u r p r e s e n t p r o b l e m s w i ll b e

s o l v e d c o m p l e t e l y.F r o m t h e c o m p a r a t i v e a s s a y s i n g u i n e a p i g s ( Ta b l e 6 ) a n d m i c e ( Ta b l e 7 )

i t is c l e a r t h a t t h e U . S . A . m o u s e t e s t is m o r e s e n s it iv e t h a n t h e g u i n e a p i g

t e s t a s d e v i s e d i n o u r l a b o r a t o r i e s . I t m u s t b e e m p h a s i z e d t h a t t h e r e f e r e n c ep o i n t s in t h e g u i n e a p i g w e r e c h o s e n o n t h e b a si s o f s o m e p r e l i m i n a r y e x p e r i-m e n t s i n t h e l a b o r a t o r y ( Ta b l e 2 ) . I t is , h o w e v e r , p o s s i b l e t o i n c r e a s e t h e s e n -

s i ti v it y o f t h i s te s t b y d e c r e a s i n g t h e p a r a m e t e r s . T h e r e s u lt s o f s u c h a t es tm i g h t p a r a ll e l m o r e a c c u r a t e l y th e l o c al re a c t i o n i n h u m a n b e in g s .

T h e a u t h o r is i n d e b t ed t o :

D r. M . P i t t m a n , N a t i o n a l I n s t i tu t e s o f H e a l t h , B e t h e s d a , U . S . A . , f o rs t i m u l a t i o n t o c o m p i l e t h e d a t a o f t h is p a p e r a n d f o r c r it ic a l e v a l u a t i o n o ft h e f i r s t c o n c e p t ,

D r. H . D . P i e r s m a , L e d e r l e L a b o r a t o r i e s , P e a r l R i v e r , U . S . A . , f o r c r i ti c a la d v is e a n d c o r r e c t i o n o f t h e m a n u s c r i p t , a n d

D r. C . S m e e n k , p e d i a t ri c i a n , D o e t i n c h e m , T h e N e t h e r l a n d s , f o r th e f ie ldw o r k w i t h t h e v a c c i n e s m e n t i o n e d i n t h i s p a p e r .

Rece iv ed 6 J an u a r y1963

R E F E R E N C E S

Bos, G . J. 1960. Een epidemie van k inkh oest in een redelijk gevaccineerde huisartsenpraktijk.Huisarts en Wetenscbap 3: 293-297.

COHEN, H . H ., an d LEPPINKG . J. 1956. Selection o fHemophih~s pertuss isstrains for vaccineproduction in the mouse protection test in a balanced design. J. lmmunol. 77: 299-304.

COHEN, H. H. 19 58 . Establishment o f a dried stan dar d pertussis vaccine. A nto nie vanLeeuwenhoek 24: 33-48.

COHEN, H. H. 1962. Discussion on the pre par atio n o f a semipurified protective antigen o fBordetella p ertussisin connection with side effects and the serological response aftervaccination and disease. Round Table Conference on Pertussis Immunization, Prague,Vol. 1, 97-103.

VAN HEMERT P. and COHEN H. H . 1962. Development o f agglutinogens, protective antigensand histamine sensitizing factor during continu ous culture. Ro un d Table Co nference onPertussis Immunization, Prague, Vol. 1, 24-29.

Jo o , 1., PUSZTAI,Z. an d JUH~.SZ, V. P. 1961a. Histamine-sensitizing activity of various pertussisvaccines. Immun. Forsch. 121: 143-158.

Jo o, I. , PUSZTAI, Z. an d JUHASZ, V. P . 196 1b. Co m para tive investigations into the m ous e-protective potency, histamine-sensitizing activity an d agglutinin-producing ability o fpertussis vaccines. Immun. Forsch. 121: 250-266.



P ERTU SS IS VA C C IN E I N T HE N E T H E R L A N D S 1 9 5 0 - 1 9 6 2 2 0 1

MU N O Z J . R t a l E . an d LA RSON C . L . 1 95 9. A n t i g en s o fBordetella pertussisI . A c t i v i t i e s

o f ce ll w a l l s a n d p r o t o p l a s m . J . l m m u n o l . 8 3 : 4 9 6 -5 0 1 .P IERSMA H . D . 1 96 2. Re cen t l a b o ra t o ry ex p e r i en ce i n t h e U .S .A . w i t h t h e p e r t u s s i s t o x i c i tyt e st . R o u n d Ta b l e C o n f e r e n c e o n P e r t u s s is I m m u n i z a t i o n P r a g u e Vo l . 1 111 -119 .

PIT TM A N M . 1 95 1. C o m p a r i s o n o f t h e h i s t a m i n e - s e n s i t i z i n g p r o p e r t y w i t h th e p r o t e c t i v eac t i v i t y o f p e r t u s s i s v acc i n e s fo r mi ce . J . i n f ec t. D i s . 8 9 : 3 0 0 -30 4 .

PRESTON N . W . 1 95 9. F ac t o r s i n f l u en c i n g t h e a s s ay o f t h e h i s t am i n e - s en s i t i s i n g f ac t o r o fHaem ophilus pertussisJ . Pa t h . Bac t . 7 8 : 2 1 7 -2 2 4 .

R e p o r t t o t h e W h o o p i n g - c o u g h I m m u n i z a t i o n C o m m i t te e o f t h e M e d i c a l R e s e a r c h C o u n c i l1 9 5 6 . Vacc i n a t i o n ag a i n s t w h o o p i n g -co u g h . Br i t . Med . J . I I : 4 5 4 -4 6 2 .

SMEENK C . S . 1 95 2. Res u l t aa t v a n d e k i n k h o e s t - i m m u n i s a t i e b i j zu i g e l i n g en . N ed . T . G e -neesk. 96: 1812-1814.

S PR ON K M . G . 1 9 5 l . A c t i e v e i m m u n i s a t i e te g e n k i n k h o e s t . T h e s i s . A m s t e r d a m .

SUTHERLAND 1 . W. 1962 . The ce l l wal l o fBordetetla pertussisa s a p r o t e c t i v e a n t i g e n . R o u n dTa b l e C o n f e r e n c e o n I m m u n i z a t i o n P r a g u e Vo l . 1 7 2 -8 0 .TA SM AN A . 1 95 9. H e t v e r b a n d t u s s e n d e a a r d v a n d e g e b r u i k t e e n t s t o f d e w i jz e v a n i n s p u i t e n

e n d e e n t- r e a c t i e s n a i m m u n i s a t i e t e g e n k i n k h o e s t d i f te r i e e n t e t a n u s . N e d . T. G e n e e s k .103: 1049-1057.

VERWEY W. F. THIELE E. H . SAGE D . N . a n dSCHUCHAROT L. F. 1949 . A s imp l i f i ed l iqu idc u l t u r e m e d i u m f o r t h e g r o w t h o fHemophilus pertussisJ . Bac te r io l . 58 : 127-134 .

Vo s A . J . 19 61 . E e n b e s c h o u w i n g o v e r d e p r e v e n t i e v e w a a r d e v a n h e t k i n k h o e s t v a c c i n n a a ra a n l e i d in g v a n e e n o n d e r z o e k i n e e n a l g e m e n e p r a k ti jk . H u i s a r ts e n We t e n s c h a p 4 : 11 4 - 11 7 .

5.Produccion de La Vacuna vs Pertusis

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