WORLD HEALTH O R G A N I Z A T I O N EXPERT COMMITTEE ON YELLOW PEVER VACCINE Geneva, 8-13 A p r i l 1957
O R G A N I S A T I O N MONDIALE DE LA SANTÉ
' . V H O A F V A 8 March 1957
ORIGINAL; FRENCH P r o v i s i o n a l agenda itam 7
THE MOUSE PROTECTION TEST POR YELLOW PEVER by Dr R. P a n t h i e r Recently ( c f . B u l l . Soc. Path. Exot. I956, 4^, 759) we hoped, by using 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 protection test.
As soon as we were i n a p o s i t i o n to continue t h i s study u s i n g a s u i t a b l e batch of v i r u s (Dakar s t r a i n stored undried a t -60°C, and a batch chosen from amonn, batches obtained on varying 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 brains) i t r a p i d l y became evident that the v i r u l e n c e curve
obtained 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 that given by the same v i r u s witn 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 to g i v e , i n tneory, 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 systematic 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 concentrations of v i r u s and antibody. conditions 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 ( v i r u s and serum l e f t i n contact f o r
as i d e n t i c a l as p o s s i b l e
20 minutes a t the ambient temperature). In o i ^ e r to represent t h i s actic^n we selected a three-dimensional graph, as already used by D. SEGRE e t a l . (Amer. J . Vet. Res. 1955^ 1^, 517) the v i r u s of bovine papilloma. The respective viirus and antibody d i l u t i o n s are shown on the h o r i z o n t a l scale). For the t h i r d dimension, one of the f o l l o w i n g may the 3VEN GARD v i r u l e n c e curve i n a study on
plane ( l o g a r i t h m i c
be chosen o r a comparison made between them:
( r e c i p r o c a l of 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 rate of the mice (the PR, or p r o t e o t i a n r a t e , of American workers) o r the r e s u l t s c a l c u l a t e d according t o BUGHER's method.
WHO/yPV/V page 2
To make the r e s u l t obtained e a s i e r to understand
as compared w i t h the c o n t r o l ,
we show i n dotted 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 given by the three-dimensional 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 points (ID ID LD ) obtained 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 points 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 , represented the lowest v i r u s concentrations i n the experiments 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 . suggests the f o l l o w i n g comments: (1) Since 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 Examination of these volumes and curves causing the death
respect 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 antibody. 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 three-
dimensional 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 We then v a r i e d the v i r u s however, apart from a
r e s u l t s which were d i f f i c u l t to i n t e r p r e t . dilutions.
I n t h i s way we obtained 11 p o i n t s ;
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 obtained; (b) the r e s u l t s published i n Figure 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, give a three-dimensional r^raph very c l o s e to that which we obtained.
I n t h i s case, however, there i s not a double antibody, and there i s not even neutralization i n vitro.
LD 50
= 5 0 per cent, l e t h a l dose = 50
PD
50 per cent, p r o t e c t i v e dose
W H O A F V / 7 page 3
I t i s , i n f a c t , probable that i n t h i s experiment the authors measured the various 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 (2) experiment.
The importance of the breed of mice seems here to be s t i l l greater than i n
ordinary 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 using 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 , there 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 obtained by v i r u s d i l u t i o n . I t would seem that the three-dimensional f i g u r e given by the v i r u s - a n t i b o d y mixture shows more appreciable d i f f e r e n c e s (experiments under way but not yet completed).
This i s i n l i n e w i t h the f i n d i n g s cf SEGRE et a l . , f o r horse 38 as compared w i t h horse 37 (experiment during which the q u a n t i t y of antibody may play a part 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 at 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 that these f a c t s must be borne i n mind i n attempting to define the F D ^ ^ . 50
How
should the PD^^ be d e f i n e d 50
w i t h respect t o the ID^^? We regard i t as more l o g i c a l to consider t h a t the a c t i o n c f x P^^Q gives 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 ^ . 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 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 . LD^QV
^ ID^^ Whatever
and
I^^QSV
makes
I t seems indeed, that 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 constant. However, i t must be pointed out t h a t although the ID^^ point
may be exact, the slope of the curve obtained 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 obtained by the a c t i o n of antibody on the v i r u s , so t h a t only the s a i d ID^^ point w i l l be exact. 50 10 LD^Q,^
of
Even i n t h i s p a r t i c u l a r case, i t i s consequently t h e o r e t i c a l d e f i n i t i o n i n advance.
necessary t o give a
* J . P. Pox et a l . (Amer._J;;_Hyg. 19^3, 3 8 , 113) have already reported the appreciable d i f f e r e n c e s observed i n the r e s u l t s according 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 page 4
(4)
This three-dimensional
representation i s v a l i d only f o r a given laboratory a known aerurri. different be be
w o r k i n g w i t h a g i v e n v i r u s and 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 the r e s u l t s o b t a i n e d by
workers w i t h d i f f e r e n t techniques
comparable, 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 n a t u r a l l y t h i s should
c a r r i e d out u s i n g the o t h e r s e r o - p r o t e c t i o n methods and done w i t h one and the same serum. We
used a h o r s e sei-um, s i n c e we d i d not have
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 our p a r t : (a) to usj (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 ' . ; e would 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
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 under the 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 the 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 the 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 the PD^^.
50 I f no common p o i n t were f o u n d , a t l e a s t the same serum c o u l d be used t o c o r r e l a t e the r e s u l t s o b t a i n e d by each l a b o r a t o r y . I n b r i e f , a l l the r e s u l t s p u b l i s h e d w o u l d
be comparable. (6) A c e r t a i n r e g i o n of the t h r e e - d i m e n s i o n a l corresponding t o 10 FD^„ flO""^ serum d i l u t i o n ) .
graph seems r e m a r k a b l e , namely t h a t A p a r t f r o m the v e r y weak v i r u s the v i r u l e n c e of tlie m i x t u r e seems
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 o be i n c r e a s e d . technique,
I f t h e same phenomenon were o b s e r v e d w i t h the SAWYER and LLOYD by the
t h i s would perhaps e x p l a i n the 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 t r o p . Med. Hyg.
PORTERFIELD ( T r a n s , r o y . Soc. G o l d C o a s t , where one
1956, 50, 344) i n the n o r t h o f
f i n d s a mixture
o f y e l l o w f e v e r and
other v i r u s diseases the least,
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 the mouse p r o t e c t i o n t e s t and haemagglutination inhibition test. Une: r our e x p e r i m e n t a l
conditions at
i f a t t e n t i o n i s not drawn t o t h i s phenorr. non, 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
a v e r y w e a k l y p o s i t i v e serum (about serum.
with a negative
W H O A P V / 7 page 5
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 usual r e s e r v a t i o n s applying 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 to allow f o r the phenomenon observed, i f i t i s wished t o standardize not the methods used f o r the 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 of c a l c u l a t i n g the r e s u l t s obtained w i t h the d i f f e r e n t techniques. We are ready t o c o l l a b o r a t e w i t h any other l a b o r a t o r y as regards more thorough research i n t h i s connexion. We could communicate more p r e c i s e d e t a i l s on the work,
and the serum employed, and could a l s o repeat, w i t h any other serum which may be suggested, t h i s experiment and communicate the r e s u l t s obtained.
0,24-1 0.23 -1 0,22 - i 0,21 - i 0,20 -Tk,
Volumes derived from calculations based on the method of Sven Gard T h i n l i n e s ; value j- calculated from the periods before appearance of paralysis Thick lines : " " ^M^h
F u l l l i n e s : values actually 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
LD..
and L D . . .
LD,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.
W H O 709t