é^^^^\ " ^ ^ ^ ^
WORLD H E A L T H ORGANIZATION ORGANISATION M O N D I A L E D E L A S A N T É
VIR/RC/74.25 E N G L I S H ONLY
(Arbo)
MEETING O F DIRECTORS OF WHO REFERENCE CENTRES FOR ARBOVIRUSES, CHLAMYDIAE AND RICKETTSIAE Geneva, 1-5 A p r i l 1974
JNDEX.;'
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3
^ ^
WHO COLLABORATIVE STUDIES - YELLOW FEVER VACCINE POTENCY TEST by Dr Y. Robin D i r e c t o r , WHO R e g i o n a l R e f e r e n c e C e n t r e f o r A r b o v i r u s e s , I n s t i t u t P a s t e u r , Dakar, S e n e g a l
1.
PURPOSE The purpose o f t h i s t e s t i s t o e s t i m a t e the amount o f a c t i v e v i r u s c o n t a i n e d i n the
vaccine. There a r e s e v e r a l methods o f c o n d u c t i n g v i r u s p a r t i c l e c o u n t s . two a r e c u r r e n t l y used: i n o c u l a t i o n o f s u s c e p t i b l e h o s t s w i t h v a r i e d d i l u t i o n s o f the t e s t s u s p e n s i o n and r e c o r d i n g f o r each d i l u t i o n o f t h e number o f p o s i t i v e r e a c t i o n s . The LD^Q i s d e t e r m i n e d statistically ; the p l a q u e method, which has the advantage o f b e i n g e x t r e m e l y s i m p l e and h i g h l y reproducible. The average number o f p l a q u e s formed p e r d i l u t i o n g i v e s an i n d i c a t i o n o f the v i r u l e n c e o f the s u s p e n s i o n t o be t i t r a t e d . T h i s i s expressed i n plaque u n i t s per u n i t volume o r PFU/ml. Up t i l l now the potency of the 17D v a c c i n e has been d e f i n e d as the number o f mouse volume o f the recommended human dose. LD^QS
Among b i o l o g i c a l
methods
per
For the reasons mentioned above and a l s o because i t i s easy t o s t a n d a r d i z e a c e l l system, the use o f the p l a q u e method f o r t i t r a t i n g 17D seems h i g h l y recommendable. I t i s necessary however t o conduct t r i a l s b e f o r e h a n d i n o r d e r t o d e t e r m i n e whether t h e method can be used i n a l l the c o n t r o l l a b o r a t o r i e s and t o d e f i n e t h e new potency s t a n d a r d s i n r e l a t i o n t o the new method. That i s why a c o l l a b o r a t i v e s t u d y has been c o n d u c t e d by WHO on the b a s i s o f a p r o t o c o l drawn up by Dr F. T. P e r k i n s . The p r o t o c o l e n v i s a g e s t i t r a t i o n o f f o u r v a c c i n e p r e p a r a t i o n s d i s t r i b u t e d t o a l l the p a r t i c i p a t i n g l a b o r a t o r i e s and a v a c c i n e p r e p a r e d l o c a l l y w h i c h w i l l s u b s e q u e n t l y be used as a secondary r e f e r e n c e b a t c h . Each p r e p a r a t i o n w i l l be t i t r a t e d f o u r days i n s u c c e s s i o n b o t h i n mice and i n c e l l c u l t u r e . To b e g i n w i t h i t i s w i s h e d t o r e c a l l b r i e f l y the method o f t i t r a t i o n i n mice; method as l a i d down f o r t h e c o l l a b o r a t i v e s t u d y w i l l then be d i s c u s s e d . the p l a q u e
The
issue of this document does not constitute It should not be reviev\/ed, Authors alone in signed
Ce
document ne constitue pas une publication.
formal publication.
Il ne doit faire l'objet d'aucun compte rendu ou résumé ni d'aucune citation sans l'autorisation de l'Organisation Mondiale de la S a n t é . Les opinions e x p r i m é e s dans les articles s i g n é s n'engagent que leurs auteurs.
abstracted or quoted without the agreement of the World Health Organization. articles. are responsible for views expressed
VIR,'RC,^7 1 . 2 5
(Arbo)
page 2 2. 2.1 MOUSE TEST M a t e r i a l and methods
2.1.1 Vaccine. Three f i n a l c o n t a i n e r s are s e l e c t e d at random and r e h y d r a t e d to the c o n c e n t r a t i o n i n d i c a t e d f o r use by means o f the d i l u e n t advocated by the m a n u f a c t u r e r . After 2 0 m i n u t e s ' d i f f u s i o n at 2 0 - 3 0 ° C , s e r i a l t e n f o l d d i l u t i o n s are made from each ampoule, usiner as d i l u e n t a s o l u t i o n c o n s i s t i n g of 0 . 7 5 " r b o v i n e albumin, f r a c t i o n V, i n i s o t o n i c sodium c h l o r i d e s o l u t i o n b u f f e r e d w i t h phosphate to a pH o f 7 . 4 , o r any o t h e r e q u i v a l e n t d i l u e n t (phosphate b u f f e r c o n t a i n i n g lO'c normal r a b b i t serum). The d i l u t i o n s are c a r r i e d out i n an i c e - w a t e r b a t h and s t o r e d t h e r e u n t i l the mouse i s i n o c u l a t e d . 2.1.2 Mouse. A h i g h l y s u s c e p t i b l e s t r a i n must be used. I n o c u l a t i o n s are g i v e n i n t r u c u t a n e o u s l y to a t l e a s t s i x 4 - 6 weeks o l d mice f o r each d i l u t i o n . The mice are kept under o b s e r v a t i o n f o r 2 1 days. 2.2 Results The r e s u l t s are expressed i n LDSQ-
C a l c u l a t i o n s should 1000 2.3 An the are mouse LD^QS
e s t a b l i s h that
the
ready-to-use vaccine
does not
contain
less
than
per
human dose.
Discussion important c r i t i c i s m the mice. that In can fact, be made o f t h i s method i s t h a t susceptibility i t depends e s s e n t i a l l y on i s used variations hot less during very
c o n d i t i o n of observed
even when a h i g h l y s u s c e p t i b l e s t r a i n being
i n r e s p o n s e to c l i m a t i c c o n d i t i o n s ,
weather. 3. 3.1 TEST IN CELL CULTURES M a t e r i a l and methods L15
3.1.1 Vaccine. The v a c c i n e i n the ampoules i s r e h y d r a t e d i n c u l t u r e medium ( L e i b o v i t z medium c o n t a i n i n g 3'"c f o e t a l c a l f serum). F o u r f o l d d i l u t i o n s are made (1 ml of v i r u s s u s p e n s i o n i n 3 ml of d i l u e n t ) a t the r a t e of f i v e d i l u t i o n s per v a c c i n e .
3.1.2 Cell cultures. For the t i t r a t i o n o f y e l l o w f e v e r v i r u s the PS c e l l l i n e i s the most s e n s i t i v e , a l t h o u g h i t i s c h r o n i c a l l y c o n t a m i n a t e d w i t h swine f e v e r v i r u s . VERO c e l l s can a l s o be u s e d . I f f o r e a c h l a b o r a t o r y a bank i s e s t a b l i s h e d c o n t a i n i n g c e l l s p r e s e r v e d i n l i q u i d n i t r o g e n , i t i s p o s s i b l e always to use c e l l s which have undergone the same number o f passages. The t e c h n i q u e s o f c e l l p r o p a g a t i o n used are the r o u t i n e ones. 3.1.3 Titration. The t e c h n i q u e used i s an a p p l i c a t i o n o f the method o f De Madrid t P o r t e r f i e l d , 1969. When c o n f l u e n t c e l l growth has been o b t a i n e d , the c e l l s are t r y p s i n i z e d and suspended i n L e i b o v i t z L15 medium i n such a way as to o b t a i n about 6 x 10 c e l l s per ml. E q u a l volumes (0.2 ml) o f c e l l L i n b r o FB 16 2 4 TC t r a y , f o u r w e l l s s u s p e n s i o n and v i r u s d i l u t i o n are b e i n g used f o r each d i l u t i o n . placed i n the w e l l s of a
The t r a y s a r e c o v e r e d and i n c u b a t e d f o r f o u r hours at 36°C. A f t e r t h i s period of i n c u b a t i o n 0 .4 ml o f o v e r l a y medium i s added to each w e l l . T h i s medium i s made up o f : L e i b o v i t z L15 medium F o e t a l c a l f serum Carboxymethyl c e l l u l o s e , 3.2% The t r a y s are covered and incubated at 36°C f o r f i v e 47% 3% 50% days.
V I R / R C / 7 1 .25 page After for 3.2 3.2.1 this incubation the preparation i s washed w i t h dried. saline, stained 3
(Arbo)
i n naphthalene
black
30 m i n u t e s Results
t h o n r i n s e d i n t a p w a t e r and
Qualitative. plaques, 2,3
Under the mm in
conditions described
the
yellow
fever
v i r u s produced
easily
countable
diameter,
3.2.2 Quanti t a t i v e . The p u r p o s e o f t h e m e t h o d i s o f c o u r s e t i t r a t i o n o f t h e i n f e c t i v e particles. The u s e o f f o u r f o l d i n s t e a d o f t e n f o l d d i l u t i o n s m a k e s i t p o s s i b l e t o a r r i v e more e a s i l y a t t h o e x a c t t i t r e by u s i n g d i l u t i o n s i n w h i c h t h o p l a q u e s a r e c l e a r l y c o u n t a b l e . C a l c u l a t i o n o f t h e t i t r e , i , e . o f t h e number o f p l a q u e u n i t s p e r u n i t v o l u m e ( P F u / m l ) , i s c a r r i e d out t a k i n g i n t o account: the the the The d i l u t i o n of the v i r u s suspension n u m b e r o f c e l l s c o u n t e d n; t o t a l number o f p l a q u e s o b t a i n e d C; N, to the square root of the total number of
relative
error i s inversely proportionate i.e. •
plaques counted 3.3 Discussion
There a r e numerous a d v a n t a g e s i n u s i n g a c e l l c u l t u r e s y s t e m f o r the t i t r a t i o n o f v i r u s particles. Among them i s t h e f a c t t h a t a l l l a b o r a t o r i e s c a n u s e t h e same c e l l c u l t u r e w i t h t h e same n u m b e r o f p a s s a g e s i n t h e same medium a n d t h i s e l i m i n a t e s v a r i a t i o n s r e s u l t i n g f r o m the use o f d i f f e r e n t s t r a i n s o f m i c e . The u s e o f PS c e l l s , w h i c h h a v e b e e n s h o w n t o be r e g u l a r l y s e n s i t i v e t o y e l l o w f e v e r v i r u s , a n d t h e i r r e a d y g r o w t h i n L e i b o v i t z L15 m e d i u m w i t h o u t t h e u s e o f a CO2 i n c u b a t o r , together w i t h s u b s t i t u t i o n of carboxymethyl c e l l u l o s e f o r agar, are a l l s i m p l i f i c a t i o n s which s h o u l d make t h e p l a q u e - c o u n t i n g t e c h n i q u e the method of c h o i c e f o r t i t r a t i n g the p o t e n c y o f yellow fever virus. REFERENCES 1. 2. 3. 4. 5. De Madrid, A, K. T. & Porterfield, J . S. (1969) B u l l . Wld H l t h Org., 40, 113-121
I n o u e , Y. Wld Wld
k O g u r a , R. techn. Rep. Rep.
( 1 9 6 2 ) V i r o l o g y , 16, Ser. Ser. roy. ( 1 9 5 7 ) No. ( 1 9 5 9 ) No. Soc. 136 179
205-207
H l t h Org,
H l t h Org, techn. R.
S a l i m , A.
(1967) T r a n s ,
t r o p . Med.
Hyg.,
61,
259-264