WORLD H E /\ L T H O RGA N IZ ^ I O N M
ORGANISATION MONDIALE DE LA SANTÉ V ,TI 0/ YFV /13
EXPEET CŒvMITTEE ON YELLOW F F ; S E VACCINE
'^2 M Geneva 8-13 A p r i l 1937 Provisiong.1 -^.p^enda item 7
19 ï-ferch 1957 »
ORIGINAL;
ENGLEH
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 unlikely that early agreement The introduction of a universal standard on a standard technique can be reached.
technique moreover might also bring with i t the disadvantage that laboratories might not seel: to improve on the test and conduct research on i t s application, It wxiiuld seem advisable therefore at the present to allow laboratcrries to conduct neutralization 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 possiblj 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 neutralization test should be evolved. they should be those which produce the least experimental v a r i a t i o n . 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 indication 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 . The methods The controls and materials of the standard test should be l a i d down i n the greatest d e t a i l and used i n the test should be r i g i d , and a standard immune serum should be available,
WH0/ïFV/l5 page 2
I f , however, the antibody t i t r e s of sera were t o be evaluated, c o n s i d e r a b l y more work would need t o be done. For instance, i t would be necessary t o show t h a t i n since
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 being measured; 2 5 there i s evidence t h a t t h i s may not always be the case, ' 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 of the various antibodies, Since i t i s d i f f i c u l t t o predetermine the exact challenge 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 Therefore f o r each d i f f e r e n t
the f u n c t i o n r e l a t i n g v i r u s dose and antibody t i t r e .
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 only be determined by t i t r a t i n g the amount of v i r u s which 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 . For t h i s purpose
I t i s necessary t o add the v i r u s to 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 i n terns of mouse LD^^ would be r e l a t i v e l y easy. d i f f i c u l t t o determine What, however, would be potency extremely
i s the amount of non-viable v i r u s present;
and there i s a t
present v e r y l i t t l e known of the e f f e c t of t h i s non-viable v i r u s on the behaviour of a neutralization test. C o n s i d e r a t i o n of the presence of non-viable 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 product, and t h a t i n f r e e z e - d r y i n g about 90 per c e n t , of the l i v e v i r u s i s destroyed. 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 evaluate other v a r i a b l e s such as those of the white n i c e and the s k i l l of the 12 operators as have heen described by Smithburn, In making suggestions f o r standard t e s t s , sera., and v i r u s , i t i s not thereby recommended t h a t standards should n e c e s s a r i l y be developed i n that order, Use of
V7H0/YFV/15 page 5
any s t a n d a r d m a t e r i a l or method i s l i k e l y t o r e s u l t
i n the
subsequent s t a n d a r d s b e i n g Therefore i t is this
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 been u s e d f i r s t . l a r g e l y a m a t t e r of c h o i c e v h i c h s t a n d a r d consideration.
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
I n d e s c r i b i n g the 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 l i s t e d as w o r t h y o f 1. 2. attention: serum samples Poriod. Temperature.
are
I n a c t i v a t i o n o f the Nature of d i l u t i o n
10^ non-immune monkey serum 1 0 5 ^ " 1 0 5 & " 1 0 5 ^ " 10^ o t h e r " " " serum concentration? human rabbit horse "
I s 10^ the c o r r e c t
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 Buffered saline 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 complement-like 5» Virus Strain, Prepared f r o m : substances level.
0.8^, 0.85^, 0.9^
Passage
mouse b r a i n , c h i c k embryo
Method 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 Centrifugation: Filtration D e t a i l s of d e s i c c a t i o n Storage Method 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 between r e h y d r a t i o n and use p e r i o d and g r a v i t a t i o n constant
WHO/YFV /15
page k
h.
Serum-virua mixtures Mouse LD50 o f v i r u s added Proportion o f volumes of serum and virus-preparation of the serum-virus mixtures
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 before inoculation 5« 6. Mice S t r a i n , age, r a n d o m i z a t i o n
Inoculation Anaesthetic Dose o f i n o c u l u m Route of i n o c u l a t i o n Size of needle
Intracerebral starching; n a t u r e , dose, and time i n t e r v a l b e f o r e / a f t e r intra-peritoneal inoculation 7. Controls T i t r e of the v i r u s i n the t e s t determined (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 and i n o c u l a t i n g a f t e r incubation;
before incubation (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 incubation;
mixture a f t e r (c)
b y t i t r a t i n g immixed v i r u s w h i c h has 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) (b) d i l u t i o n o f serum w i t h v i r u s h e l d d i l u t i o n of v i r u s w i t h serum h e l d constant; constant.
Numbers o f mice i n o c u l a t e d 8.
p e r d i l u t i o n , and d i l u t i o n I n t e r v a l s
P e r i o d ^f._obsg.^X^AiOP—Q^L jPAee.
vrao/ïFv/i5 page 5
9,
Evaluation
of r e s u l t s deaths/illnesses average s u r v i v a l times; Sven Gard's 1; T
Decision on non-specific
Sickness r a t e ; m o r t a l i t y r a t e j n e u t r a l i z a t i o n index
II. o
Use of the mouse n e u t r a l i z a t i o n t e s t i n diafyiosis
Sabin'' showed by complement f i x a t i o n t e s t s that y e l l o v fever v i r u s was 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 .
sero.-
P r e v i o u s l y Smithburn^^
had shown that West N i l e , Japanese B and St Louis 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 related i n protection tests. 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
haemagglutination i n h i b i t i o n ^ t h a t y e l l o w fever 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 relationships
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 fever 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 while 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 against y e l l o w fever 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 . weak and one-sided. The The r e l a t i o n s h i p s vere f o r the most part
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 sera c o l l e c t e d of sera g i v i n g a
from widespread regions of the globe i n d i c a t e that the proportion
p o s i t i v e y e l l o v fever 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 very s m a l l . Nevertheless i n West A f r i c a , where there i s probably as h i g h an B arthropod-borne v i r u s e s as anywhere, i t has been suggested that or more v i r u s e s of the group may r e s u l t i n the sera
incidence of Group
repeated i n f e c t i o n s w i t h one
becoming p o s i t i v e t o other v i r u s e s of the group although a c t i o n i n f e c t i o n may not . have occurred. That t h i s can occur w i t h haemagglutination t e s t s i s already 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 Porterfield has produced some c i r c u m s t a n t i a l though not conclusive grounds; evidence on
epidemiological
and a n a l y s i s of a survey of the sera 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 sera 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 serious problem and one more d i f f i c u l t t o r e s o l v e i s that of the r i s e i n y e l l o w fever antibody t i t r e i n a person already immune t o y e l l o w fever 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 , Bearcroft has shown a
r i s e i n t i t r e of over 400 timfôîfollowing i n f e c t i o n v i t h Zika v i r u s , and Schlesinger 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 a rise
now no longer advisable t o diagnose a case of yellow fever by demonstrating i n antibody t i t r e unless the i n i t i a l or acute phase serum i s completely
negative.
The r i s e i n t i t r e of the serum samples against the 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 that against y e l l o w f e v e r , so that n e u t r a l i z a t i o n t e s t s using other v i r u s e s may not neces-rarily i n d i c a t e the r e a l cause of the 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 yellow fever v i r u s i n man has been shown t o r a i s e considerably i n a n e u t r a l i z a t i o n t e s t the 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 not, 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 three 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 that 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 the acute phase serum contains evidence of antibody, should not be used as c o n c l u s i v e evidence of i n f e c t i o n f o r yellow f e v e r , or perhaps f o r any other of the group B Infectlor.;^,
page 7
EEFEEENCES 1, 2. 5. U, 5. 6. 7. 8. 9. 10. 11. 12. 13. Bearcroft, W. G. C. (l956) Bugher, J . C. (l95l) Trans, roy. Soc, trop. Med. Hyg. 22; hh2 G, K. Strode, New York, p. 373 J . exp. Med. 22; 1 ^ 2 9
Yellov Fever, (195^)
Casais, J . & Brown, L , V . C, Macnamara, F. N . (1953) " " " " (1955)
B r i t . J . exp. Path. ^ , 592 Thesis for M.D. Cambridge, Eng. i n preparation.
i't a l . (1957)
P o r t e r f i e l d , J . S. (1955) Proceedings V l i t h International Congress of Comparative Pathology, Lausanne, 1955» P o r t e r f i e l d , J . S. (195^) Sabin, A. B. (19^9) Tr ans, rov. Soc, trop. Med. Hyg. 5^+
Fed. Proc. 8, UlO J . Immunol, ZL, 352
Schlesinger, K. W. et a l . (195^) Smithburn, K, C, (l9^2) " " " " (19^+5) (195^)
J , Immunol, ij^, 25 J . Immunol. 173
Immunol, J2, 376