W O R L D H E /\ L T H O R G A N I Z M ^ I O N
EXPEET CŒvMITTEE ON YELLOW F F ; S E VACCINE
Geneva 8-13 A p r i l 1937 Provisiong.1 -^.p^enda item 7
THE MOUSE NEUTRALIZATION (EROTECTION) TEST FOR YELLOW FEVEE
by
Dr F . N . Macnamara
I. A standard Test
Standardization of the yellow fever neutralization test would have the great advantage that the results from d i f f e r e n t laboratories would be more nearly
comparable than they are now. Nevertheless, i t i s u n l i k e l y that early agreement on a standard technique can be reached. The introduction of a universal standard technique moreover might also bring with i t the disadvantage that laboratories might not seel: to improve on the t e s t and conduct research on i t s application,
It wxiiuld seem advisable therefore at the present to allow laboratcrries to conduct n e u t r a l i z a t i o n tests according to the methods i n which they are most suited and experienced. The most important consideration, however, i s that i t should be p o s s i b l j to evaluate the results of one laboratory i n terms of those of another.
For t h i s purpose i t i s essential that a laboratory should be able to compare the method of i t s choice with some "standard", and f o r t h i s reason, i f f o r no other, i t i s suggested that a standard n e u t r a l i z a t i o n t e s t should be evolved. The methods and materials of the standard test should be l a i d down i n the greatest d e t a i l and they should be those which produce the least experimental v a r i a t i o n . The controls used i n the test should be r i g i d , and a standard immune serum should be available, as w e l l as a standard "non-immune" serum and a standard v i r u s .
If about 200 sera, of which about one half should be positive and one half negative, were tested i n p a r a l l e l i n the two tests a good i n d i c a t i o n would be
given of the s e n s i t i v i t y f o r use i n surveys of the peculiar method of the laboratory compared with the standard t e s t .
ORGANISATION MONDIALE DE LA SANTÉ
V , T I0 /Y F V/ 1 3
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»
19 ï-ferch 1957
ORIGINAL; ENGLEH
WH0/ïFV/l5 page 2
I f , however, the a n t i b o d y t i t r e s of s e r a were t o be e v a l u a t e d , c o n s i d e r a b l y more work would need t o be done. F o r i n s t a n c e , i t would be necessary t o show t h a t i n
t e s t s u s i n g d i f f e r e n t methods the same an'.ibodies were i n f a c t b e i n g measured; s i n c e 2 5
t h e r e i s evidence t h a t t h i s may not always be the c a s e , ' F o r t h i s purpose i t would be necessary t o prepare standard sera c o n t a i n i n g d i f f e r i n g q u a n t i t i e s o f the
v a r i o u s a n t i b o d i e s ,
S i n c e i t i s d i f f i c u l t t o predetermine the exact c h a l l e n g e dose mixed w i t h the t e s t serum, even i n t e s t s conducted on e x a c t l y s i m i l a r l i n e s , i t i s necessary t o know the f u n c t i o n r e l a t i n g v i r u s dose and a n t i b o d y t i t r e . Therefore f o r each d i f f e r e n t method the c h a r a c t e r i s t i c s of the curve v i r u s dose-antibody t i t r e should be
deterrained.
The amount of v i r u s added t o the serum can o n l y be determined by t i t r a t i n g t h e amount of v i r u s w h i c h i s v i a b l e under the c o n d i t i o n s of the t e s t . F o r t h i s purpose I t i s necessary t o add the v i r u s t o a "non-imnune" serum and t i t r a t e the v i r u s a f t e r i t has been subjected t o the c o n d i t i o n s of the t e s t . Therefore a standard
"non-immune" serum must be used and be a v a i l a b l e t o a l l l a b o r a t o r i e s .
S t a n d a r d i z a t i o n of the v i r u s w i t h r e ^ r d t o s t r a i n , passage l e v e l , and potency i n t e r n s of mouse LD^^ would be r e l a t i v e l y easy. What, however, would be extremely d i f f i c u l t t o determine i s the amount of n o n - v i a b l e v i r u s p r e s e n t ; and t h e r e i s a t present v e r y l i t t l e known o f the e f f e c t of t h i s n o n - v i a b l e v i r u s on the behaviour o f a n e u t r a l i z a t i o n t e s t . C o n s i d e r a t i o n of the presence of n o n - v i a b l e v i r u s ,
however, shoxald not be ignored when i t i s r e a l i z e d t h a t any standard v i r u s
p r e p a r a t i o n wovild almost of n e c e s s i t y have t o be a f r e e z e - d r i e d p r o d u c t , and t h a t i n f r e e z e - d r y i n g about 90 p e r c e n t , of the l i v e v i r u s i s d e s t r o y e d .
With standard immune s e r a , "non-immune" s e r a , and v i r u s , i t would be p o s s i b l e t o e v a l u a t e o t h e r v a r i a b l e s such as those o f the w h i t e n i c e and the s k i l l of the
12 o p e r a t o r s as have heen d e s c r i b e d by Smithburn,
In making s u g g e s t i o n s f o r standard t e s t s , sera., and v i r u s , i t i s not t h e r e b y recommended t h a t standards should n e c e s s a r i l y be developed i n t h a t o r d e r , Use of
V7H0/YFV/15 page 5
any s t a n d a r d m a t e r i a l o r method i s l i k e l y t o r e s u l t i n t h e s u b s e q u e n t s t a n d a r d s b e i n g o f a h i g h e r q u a l i t y t h a n i f t h e y t h e m s e l v e s had b e e n u s e d f i r s t . T h e r e f o r e i t i s l a r g e l y a m a t t e r o f c h o i c e v h i c h s t a n d a r d i s f i r s t d e v e l o p e d h a v i n g due r e g a r d t o t h i s c o n s i d e r a t i o n .
I n d e s c r i b i n g t h e d e t a i l s o f a n e u t r a l i z a t i o n t e s t t h e f o l l o w i n g p o i n t s a r e l i s t e d as w o r t h y o f a t t e n t i o n :
1. I n a c t i v a t i o n o f t h e serum samples P o r i o d . T e m p e r a t u r e . 2. N a t u r e o f d i l u t i o n
10^ non-immune monkey serum 105^ " " human 105& " " r a b b i t "
105^ " " h o r s e 10^ o t h e r serum
I s 10^ t h e c o r r e c t c o n c e n t r a t i o n ? 0.2^/ 0.75^ and ?^ b o v i n e a l b u m i n
E x c i p i e n t - Sodium c h l o r i d e 0.8^, 0.85^, 0.9^
B u f f e r e d s a l i n e
A d d i t i o n o f f r e s h non-immune serum c o n t a i n i n g complement o r c o m p l e m e n t - l i k e s u b s t a n c e s
5» V i r u s S t r a i n , Passage l e v e l .
P r e p a r e d f r o m : mouse b r a i n , c h i c k embryo
M e t h o d o f t r i t u r a t i o n , n a t u r e and q u a n t i t y o f e x c i p i e n t C e n t r i f u g a t i o n : p e r i o d and g r a v i t a t i o n c o n s t a n t
F i l t r a t i o n
D e t a i l s o f d e s i c c a t i o n S t o r a g e
M e t h o d o f r e h y d r a t i n g d e s i c c a t e d v i r u s D u r a t i o n b e t w e e n r e h y d r a t i o n and use
WHO/YFV/15
page k
h. S e r u m - v i r u a m i x t u r e s Mouse LD50 o f v i r u s added
P r o p o r t i o n o f v o l u m e s o f serum and v i r u s - p r e p a r a t i o n
Time and t e m p e r a t u r e o f i n c u b a t i o n o f t h e s e r u m - v i r u s m i x t u r e s b e f o r e i n o c u l a t i o n
5« M i c e S t r a i n , a g e , r a n d o m i z a t i o n 6. I n o c u l a t i o n
A n a e s t h e t i c
Dose o f i n o c u l u m R o u t e o f i n o c u l a t i o n
S i z e o f n e e d l e
I n t r a c e r e b r a l s t a r c h i n g ; n a t u r e , d o s e , and t i m e i n t e r v a l b e f o r e / a f t e r i n t r a - p e r i t o n e a l i n o c u l a t i o n
7. C o n t r o l s
T i t r e o f t h e v i r u s i n t h e t e s t d e t e r m i n e d
(a) b y d i l u t i n g t h e t e s t v i r u s i n t h e "non-imniune" serum b e f o r e i n c u b a t i o n and i n o c u l a t i n g a f t e r i n c u b a t i o n ;
(b) b y t i t r a t i n g v i r u s r e m a i n i n g i n t h e "non-immune" s e r u m - v i r u s m i x t u r e a f t e r i n c u b a t i o n ;
( c ) b y t i t r a t i n g i m m i x e d v i r u s w h i c h h a s s t o o d w i t h t h e t e s t Number o f non>-immune c o n t r o l s
T i t r e o f immune serum c o n t r o l
(a) d i l u t i o n o f serum w i t h v i r u s h e l d c o n s t a n t ; (b) d i l u t i o n o f v i r u s w i t h serum h e l d c o n s t a n t .
Numbers o f m i c e i n o c u l a t e d p e r d i l u t i o n , a n d d i l u t i o n I n t e r v a l s 8. P e r i o d ^f._obsg.^X^AiOP—Q^L jPAee.
vrao/ïFv/i5 page 5
9, E v a l u a t i o n of r e s u l t s
D e c i s i o n on n o n - s p e c i f i c d e a t h s / i l l n e s s e s
S i c k n e s s r a t e ; m o r t a l i t y r a t e j average s u r v i v a l t i m e s ; Sven Gard's 1;
n e u t r a l i z a t i o n index T
I I . Use of the mouse n e u t r a l i z a t i o n t e s t i n d i a f y i o s i s o
Sabin'' showed by complement f i x a t i o n t e s t s t h a t y e l l o v f e v e r v i r u s was sero.-
L o g i c a l l y r e l a t e d t o dengue, West N i l e , and Japanese B v i r u s . P r e v i o u s l y Smithburn^^
had shown t h a t West N i l e , Japanese B and St L o u i s e n c e p h a l i t i s were s e r o l o g i c a l l y
r e l a t e d i n p r o t e c t i o n t e s t s . More r e c e n t l y i t has been shown by techniques i n v o l v i n g 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 h a t y e l l o w f e v e r v i r u s i s a member of a group of
v i r u s e s now u s u a l l y known as the Group B arthropod-borne v i r u s e s . The r e l a t i o n s h i p s have been demonstrated on the whole by t e s t s other than the p r o t e c t i o n t e s t which i s the most s p e c i f i c of them a l l . Macnamara showed by n e u t r a l i z a t i o n t e s t s the
15 s e r o l o g i c a l r e l a t i o n s h i p between y e l l o w f e v e r and Uganda 3 v i r u s e s , and Smithburn
7
showed s i m i l a r r e l a t i o n s h i p s w i t h other v i r u s e s w h i l e P o r t e r f i e l d was able t o show a s l i g h t p r o t e c t i o n a g a i n s t y e l l o w f e v e r v i r u s of the serxun of a rhosus monkey which had been i n j e c t e d w i t h West N i l e v i r u s . The r e l a t i o n s h i p s vere f o r the most p a r t weak and one-sided. The r e s u l t s , moreover, of n e u t r a l i z a t i o n t e s t s on s e r a c o l l e c t e d from widespread r e g i o n s of the globe i n d i c a t e t h a t the p r o p o r t i o n of s e r a g i v i n g a p o s i t i v e y e l l o v f e v e r n e u t r a l i z a t i o n t e s t r e s u l t i n g from s e r o l o g i c a l c r o s s - r e l a t i o n s h i p i s v e r y s m a l l . N e v e r t h e l e s s i n West A f r i c a , where t h e r e i s p r o b a b l y as h i g h an
i n c i d e n c e of Group B arthropod-borne v i r u s e s as anywhere, i t has been suggested t h a t repeated i n f e c t i o n s w i t h one or more v i r u s e s of the group may r e s u l t i n the s e r a becoming p o s i t i v e t o other v i r u s e s of the group a l t h o u g h a c t i o n i n f e c t i o n may not . have o c c u r r e d . That t h i s can occur w i t h h a e m a g g l u t i n a t i o n t e s t s i s a l r e a d y known.
Staithburn"'"^ has demonstrated a s i m i l a r phenomenon u s i n g the n e u t r a l i z a t i o n t e s t . o
P o r t e r f i e l d has produced some c i r c u m s t a n t i a l though not c o n c l u s i v e evidence on e p i d e m i o l o g i c a l grounds; and a n a l y s i s of a survey of the s e r a from a v i l l a g e i n N i g e r i a has shown s l i g h t c o r r e l a t i o n between s e r a g i v i n g p o s i t i v e r e s u l t s i n
6 n e u t r a l i z a t i o n t e s t s employed on Group B v i r u s e s .
vmoAFv/13 page 6
l'Tiat i s probably a more s e r i o u s problem and one more d i f f i c u l t t o r e s o l v e i s t h a t of the r i s e i n y e l l o w f e v e r antibody t i t r e i n a person a l r e a d y immune t o y e l l o w f e v e r as a r e s u l t of i n f e c t i o n w i t h another group B v i r u s , B e a r c r o f t has shown a r i s e i n t i t r e o f over 400 timfôîfollowing i n f e c t i o n v i t h Z i k a v i r u s , and S c h l e s i n g e r et a l , " ^ ^ have shown a r i s e f o l l o w i n g dengue v a c c i n a t i o n , VJith t h i s knowledge i t i s now no longer a d v i s a b l e t o diagnose a case of y e l l o w f e v e r by demonstrating a r i s e i n antibody t i t r e u n l e s s the i n i t i a l or acute phase serum i s completely n e g a t i v e .
The r i s e i n t i t r e of the serum samples a g a i n s t t h e homologous v i r u s may be c o n s i d e r a b l y l e s s than t h a t a g a i n s t y e l l o w f e v e r , so t h a t n e u t r a l i z a t i o n t e s t s u s i n g other v i r u s e s may not n e c e s - r a r i l y i n d i c a t e the r e a l cause of t h e i n f e c t i o n .
As a c o r o l l a r y i t should be noted t h a t i n f e c t i o n w i t h y e l l o w f e v e r v i r u s i n man has been shown t o r a i s e c o n s i d e r a b l y i n a n e u t r a l i z a t i o n t e s t t h e serum t i t r e a g a i n s t Uganda S v i r u s , ^ Such a r i s e does n o t , however, p e r s i s t , and the t i t r e f a l l s
g r a d u a l l y from a peak about t h r e e weeks t o one .-nonth a f t e r i n f e c t i o n n e a r l y t o i t s o r i g i n a l l e v e l which i s reached i n l e s s than 18 months.
I n c o n c l u s i o n i t should be emphasized t h a t a r i s e i n serum antibodj^ t i t r e , e s p e c i a l l y when t h e acute phase serum c o n t a i n s evidence o f a n t i b o d y , s h o u l d not be used as c o n c l u s i v e evidence o f i n f e c t i o n f o r y e l l o w f e v e r , or perhaps f o r any other of t h e group B Infectlor.;^,
page 7
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1, Bearcroft, W. G. C. (l956) Trans, roy. Soc, trop. Med. Hyg. 22; hh2 2. Bugher, J . C. (l95l) Yellov Fever, G, K. Strode, New York, p. 373 5. Casais, J . & Brown, L , V . C, (195^) J . exp. Med. 22; 1^29
U, Macnamara, F . N . (1953) B r i t . J . exp. Path. ^ , 592 5. " " (1955) Thesis f o r M.D. Cambridge, Eng.
6. " " i't a l . (1957) i n preparation.
7. P o r t e r f i e l d , J . S. (1955) Proceedings V l i t h International Congress of
