W O R L D H E A L T H O R G A N I Z A T I O N
O R G A N I S A T I O N M O N D I A L E DE LA SANTÉ
EXPERT COMMITTEE ON YELLOW PEVER
VACCINE
Geneva, 8-13 A p r i l 1957 P r o v i s i o n a l agenda itam 7
'.VHOAFVA
8 March 1957
ORIGINAL; FRENCH
THE MOUSE PROTECTION TEST POR YELLOW PEVER by
Dr R. P a n t h i e r
R e c e n t l y ( c f . B u l l . Soc. Path. Exot. I956, 4^, 759) we hoped, by u s i n g the SVEN GARD v i r u l e n c e curve, t o f a c i l i t a t e the t i t r a t i o n of antibody by txie mouse p r o t e c t i o n t e s t .
As soon as we were i n a p o s i t i o n t o continue t h i s study u s i n g a s u i t a b l e b a t c h of v i r u s (Dakar s t r a i n s t o r e d u n d r i e d a t -60°C, and a batch chosen from amonn, batches obtained on v a r y i n g the d i l u t i o n of the i n o c u l a t i o n and the day of h a r v e s t i n g the b r a i n s ) i t r a p i d l y became e v i d e n t t h a t the v i r u l e n c e curve o b t a i n e d w i t h a mixture of v i r u s and normal serum was d i f f e r e n t from t h a t g i v e n by t h e same v i r u s w i t n p r o t e c t i v e serum, although the d i l u t i o n s of the l a t t e r were c a l c u l a t e d so as t o g i v e , i n t n e o r y , an i d e n t i c a l curve.
I n c o l l a b o r a t i o n w i t h J . P. CARTEAUD and R. A. HUSSON, we undertook a
s y s t e m a t i c i n v e s t i g a t i o n of the e f f e c t s on the mouse of d i f f e r e n t c o n c e n t r a t i o n s of v i r u s and a n t i b o d y . The d i f f e r e n t t i t r a t i o n s were c a r r i e d out under
c o n d i t i o n s as i d e n t i c a l as p o s s i b l e ( v i r u s and serum l e f t i n c o n t a c t f o r 20 minutes a t the ambient temperature).
I n o i ^ e r t o r e p r e s e n t t h i s actic^n we s e l e c t e d a t h r e e - d i m e n s i o n a l graph, as a l r e a d y used by D. SEGRE e t a l . (Amer. J . V e t . Res. 1955^ 1^, 517) i n a study on the v i r u s o f bovine p a p i l l o m a .
The r e s p e c t i v e viirus and antibody d i l u t i o n s a r e shown on the h o r i z o n t a l plane ( l o g a r i t h m i c s c a l e ) . F o r the t h i r d dimension, one of the f o l l o w i n g may be chosen o r a comparison made between them: the 3VEN GARD v i r u l e n c e curve
( r e c i p r o c a l o f the s u r v i v a l time o r time f o r development of p a r a l y s i s ) , the percentage s u r v i v a l r a t e of the mice (the PR, o r p r o t e o t i a n r a t e , of American workers) o r t h e r e s u l t s c a l c u l a t e d a c c o r d i n g t o BUGHER's method.
WHO/yPV/V page 2
To make the r e s u l t o b t a i n e d e a s i e r t o understand as compared w i t h the c o n t r o l , we show i n d o t t e d l i n e s a second volume formed by the v i r u l e n c e curves which would t h e o r e t i c a l l y be obtained i f the d i l u t e d v i r u s - a n t i b o d y r e l a t i o n s h i p were the same, i . e . i f X PD^„ + X LD^^ always gave the same r e s u l t , whatever the value of "x".
50 50
To complete the p i c t u r e g i v e n by the t h r e e - d i m e n s i o n a l g r a p h i c a l r e p r e s e n t a t i o n , we have p l o t t e d on the h o r i z o n t a l plane of the volume the most important p o i n t s
(ID ID LD ) o b t a i n e d by the v i r u s d i l u t i o n s corresponding to ID V, ID V and ID^QQV, ard by the a c t i o n of the antiserum on the v i r u s (ID^SV, LD^^SV, ID^^^SV).
The LD^Q p o i n t s were c a l c u l a t e d by the REED and MUENCH method, the LD^ p o i n t s by the p o i n t of i n t e r s e c t i o n of the two SVEN GARD curves p l o t t e d w i t h the s u r v i v a l and p a r a l y s i s r a t e s , v/hile the ID-j^^^ p o i n t s , which were i n e v i t a b l y more i n e x a c t , r e p r e s e n t e d the lowest v i r u s c o n c e n t r a t i o n s i n the experiments c a u s i n g the death of 18 mice out of l 8 i n o c u l a t e d .
The serum used was from a horse immunized by repeated i n j e c t i o n s of the b r a i n s of mice i n f e c t e d w i t h Dakar v i r u s . Examination of these volumes and curves
suggests the f o l l o w i n g comments:
(1) S i n c e the serum used i s not human serum, i t may c o n t a i n two a n t i b o d i e s w i t h r e s p e c t to the a n t i g e n used (mouse b r a i n ) , namely, a b r a i n antibody and a v i r u s a n t i b o d y . We do not b e l i e v e t h a t t h i s e x p l a i n s the d i s t o r t i o n of the t h r e e - d i m e n s i o n a l f i g u r e , s i n c e :
(a) a very p o s i t i v e human serum had been p r e v i o u s l y t i t r a t e d and gave r e s u l t s which were d i f f i c u l t t o i n t e r p r e t . We then v a r i e d the v i r u s d i l u t i o n s . I n t h i s way we o b t a i n e d 11 p o i n t s ; however, a p a r t from a s i n g l e s t r a y p o i n t , a l l of them f i t p e r f e c t l y i n t o the volume which we o b t a i n e d ;
(b) the r e s u l t s p u b l i s h e d i n F i g u r e 3 of the a r t i c l e by SEGRE et a l . , f o r horse 37, when r e p l o t t e d u s i n g a l o g a r i t h m i c s c a l e f o r the d i l u t i o n s of the inoculum, g i v e a t h r e e - d i m e n s i o n a l r^raph very c l o s e t o t h a t which we o b t a i n e d . I n t h i s case, however, t h e r e i s not a double a n t i b o d y , and t h e r e i s not even n e u t r a l i z a t i o n i n v i t r o .
LD = 5 0 per c e n t , l e t h a l dose
5 0
PD = 5 0 per c e n t , p r o t e c t i v e dose
5 0
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I t i s , i n f a c t , probable t h a t i n t h i s experiment the authors measured the v a r i o u s v i r u s - a n t i b o d y r e l a t i o n s h i p s i n r e l a t i o n to time, which i s i n accordance w i t h our own experiment.
(2) The importance of the breed of mice seems here t o be s t i l l g r e a t e r than i n o r d i n a r y t i t r a t i o n by v i r u s d i l u t i o n .
We know t h a t on u s i n g mice of the same o r i g i n , but bred i n a d i f f e r e n t p l a c e , t h e r e i s a s l i g h t v a r i a t i o n i n the v i r u l e n c e curve o b t a i n e d by v i r u s d i l u t i o n .
I t would seem t h a t the t h r e e - d i m e n s i o n a l f i g u r e g i v e n by the v i r u s - a n t i b o d y mixture shows more a p p r e c i a b l e d i f f e r e n c e s (experiments under way but not y e t completed).
T h i s i s i n l i n e w i t h the f i n d i n g s cf SEGRE e t a l . , f o r horse 3 8 as compared w i t h horse 3 7 (experiment d u r i n g which the q u a n t i t y of antibody may p l a y a p a r t i n a d d i t i o n to the s u s c e p t i b i l i t y of the h o r s e ) .
(3) Thus, we are only a t the commencement of t h i s experiment, and i f we communicate these i n i t i a l r e s u l t s , i t i s because i t appears c e r t a i n t h a t these f a c t s must be
*
borne i n mind i n a t t e m p t i n g to d e f i n e the F D ^ ^ . How should the PD^^ be d e f i n e d
5 0 5 0
w i t h r e s p e c t t o the ID^^?
We r e g a r d i t as more l o g i c a l t o c o n s i d e r t h a t the a c t i o n c f x P^^Q ^ I D ^ ^ g i v e s a f i n a l r e s u l t of 1 I D ^ ^ , r a t h e r than a f i n a l r e s u l t of 1 L D ^ . Whatever the d e f i n i t i o n chosen, the t a b l e showing the v a r i a t i o n s of L D ^ Q V and I ^ ^ Q S V makes the d i f f i c u l t y of t h i s d e f i n i t i o n c l e a r . I t seems indeed, t h a t the v i r u l e n t dose of 100 ID^Q g e n e r a l l y s e l e c t e d makes i t p o s s i b l e to work i n the range where r e s u l t s are most c o n s t a n t . However, i t must be p o i n t e d out t h a t although the ID^^ p o i n t may be e x a c t , the s l o p e of the curve o b t a i n e d by v i r u s d i l u t i o n i s very d i f f e r e n t from the s l o p e of the curve o b t a i n e d by the a c t i o n of antibody on the 10 L D ^ Q, ^ of v i r u s , so t h a t o n l y the s a i d I D ^ ^ p o i n t w i l l be exact.
5 0
Even i n t h i s p a r t i c u l a r case, i t i s consequently necessary t o give a t h e o r e t i c a l d e f i n i t i o n i n advance.
*
J . P. Pox e t a l . (Amer._J;;_Hyg. 1 9 ^ 3 , 3 8 , 113) have a l r e a d y r e p o r t e d the a p p r e c i a b l e d i f f e r e n c e s observed i n the r e s u l t s a c c o r d i n g as serum and concentrated v i r u s or v i r u s d i l u t i o n and d i l u t e d serum are used.
WHOAFVA
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(4) T h i s t h r e e - d i m e n s i o n a l r e p r e s e n t a t i o n i s v a l i d o n l y f o r a g i v e n l a b o r a t o r y
w o r k i n g w i t h a g i v e n v i r u s and a known aerurri.
We f e e l t h a t , i f i t i s d e s i r e d t o make t h e r e s u l t s o b t a i n e d by d i f f e r e n t w o r k e r s w i t h d i f f e r e n t t e c h n i q u e s c o m p a r a b l e , t h e n a s i m i l a r e x p e r i m e n t s h o u l d be c a r r i e d o u t u s i n g t h e o t h e r s e r o - p r o t e c t i o n methods and n a t u r a l l y t h i s s h o u l d be done w i t h one and t h e same serum. We u s e d a h o r s e sei-um, s i n c e we d i d n o t h a v e human serum w i t h v e r y p r o t e c t i v e p r o p e r t i e s a v a i l a b l e i n s u f f i c i e n t q u a n t i t y .
F o r o u r p a r t :
(a) '.;e w o u l d w i l l i n g l y r e p e a t t h i s e x p e r i m e n t w i t h any human serum s u p p l i e d t o u s j
(b) we c o u l d f u r n i s h a r e a s o n a b l e q u a n t i t y o f t h i s h o r s e serum t o a n o t h e r l a b o r a t o r y w h i c h m i g h t d e s i r e , e i t h e r t o r e p e a t t h i s e x p e r i m e n t u n d e r t h e same c o n d i t i o n s , o r t o v e r i f y i t s r e s u l t s by a n o t h e r method.
(5) I f c o m p a r i s o n o f t h e r e s u l t s o b t a i n e d w i t h d i f f e r e n t methods u s i n g t h e same serum shovjs a p o i n t i n common, t h e n i t w i l l be p o s s i b l e t o d e f i n e t h e PD^^.
50
I f no common p o i n t were f o u n d , a t l e a s t t h e same serum c o u l d be u s e d t o c o r r e l a t e t h e r e s u l t s o b t a i n e d by e a c h l a b o r a t o r y . I n b r i e f , a l l t h e r e s u l t s p u b l i s h e d w o u l d be c o m p a r a b l e .
(6) A c e r t a i n r e g i o n o f t h e t h r e e - d i m e n s i o n a l g r a p h seems r e m a r k a b l e , namely t h a t c o r r e s p o n d i n g t o 10 FD^„ flO""^ serum d i l u t i o n ) . A p a r t f r o m t h e v e r y weak v i r u s
50
d i l u t i o n s , a p a r a d o x i c a l phenomenon i s o b s e r v e d : t h e v i r u l e n c e o f t l i e m i x t u r e seems t o be i n c r e a s e d . I f t h e same phenomenon were o b s e r v e d w i t h t h e SAWYER and LLOYD t e c h n i q u e , t h i s w o u l d p e r h a p s e x p l a i n t h e p a r a d o x i c a l r e s u l t s o b s e r v e d by
PORTERFIELD ( T r a n s , r o y . S o c . t r o p . Med. Hyg. 1956, 50, 344) i n t h e n o r t h o f t h e G o l d C o a s t , where one f i n d s a m i x t u r e o f y e l l o w f e v e r and o t h e r v i r u s d i s e a s e s d u r i n g a t t e m p t s t o compare t h e r e s u l t s o f t h e mouse p r o t e c t i o n t e s t and t h e h a e m a g g l u t i n a t i o n i n h i b i t i o n t e s t . Une: r o u r e x p e r i m e n t a l c o n d i t i o n s a t l e a s t ,
i f a t t e n t i o n i s n o t d r a w n t o t h i s phenorr. non, a v e r y w e a k l y p o s i t i v e serum ( a b o u t 10 PD ) m i g l i t v e r y e a s i l y be c o n f u s e d w i t h a n e g a t i v e s e r u m .
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I n c o n c l u s i o n , our reason f o r communicating these r e s u l t s d e s p i t e a l l the u s u a l r e s e r v a t i o n s a p p l y i n g t o an experiment s t i l l under way, i s t h a t we f e l t i t important t o a l l o w f o r t h e phenomenon observed, i f i t i s wished t o s t a n d a r d i z e not the methods used f o r t h e mouse p r o t e c t i o n t e s t (which i s u s e l e s s ) but the way o f c a l c u l a t i n g the r e s u l t s o b t a i n e d w i t h the d i f f e r e n t t e c h n i q u e s .
We a r e ready t o c o l l a b o r a t e w i t h any o t h e r l a b o r a t o r y as regards more thorough r e s e a r c h i n t h i s connexion. We c o u l d communicate more p r e c i s e d e t a i l s on the work, and the serum employed, and c o u l d a l s o r e p e a t , w i t h any o t h e r serum which may be suggested, t h i s experiment and communicate the r e s u l t s o b t a i n e d .
0 , 2 4 - 1 0.23 -1 0,22 - i 0,21 - i 0,20 -Tk,
Volumes derived from c a l c u l a t i o n s based on the method of Sven Gard
T h i n l i n e s ; value j- c a l c u l a t e d from the periods before appearance of p a r a l y s i s T h i c k l i n e s : " " ^ M ^ h
F u l l l i n e s : v a l u e s a c t u a l l y given by the serum-virus mixture
Broken l i n e s ; theoretical volues of these points if x P D + x L D „ always gave comparable r e s u l t s
WHO 7092
18 mice
Volume derived from number of mice surviving out of 18 mice for each point Thin lines : th» control volume which would be obtained if the results were constant for all mixtures of x PD^^ + x LDjp whatever the value of x
Thick lines : volume actually obtained
Full lines : Values for which one of the two volumes is greater than the other (thick lines as compared with thin)
Broken lines : Values for which one of the two volumes is smaller than the other
(Edifor'i nota : the lost two paragrophi indicate that where one of the two polyhedfa dellnooted by the thick or thin line» standi outside the other, the lines are " ( u H " , and whore it stands ins ide .the other the lines are "broken")
WHO 7093
Sérum horse 62
I I \ I I 10-8
Projection on the horizontal plane of points L D L D . . and L D . . .
L D , v
Curves of the different L D values which would be obtained if x PD5Q+ ^ L D j ^ gave the same results
whatever the value of x
The different points of the L D values actually obtained with the virus antiserum mixture The L D j ^ s v points were calculated by the Reed
and Muench method.
WHO 709t
