Standards for the manufacture and control of yellow fever vaccine: annex I: annex II: annex III

ANNEX I

STAÏŒIAKDS FOR THE MANUFACTURE AND CONTROL OF YiLLLQW FEVS:-: VACCINE

The I n t e r n a t i o n a l S a n i t a r y Convention f o r A e r i a l Navigation 1944, A r t i c l e XI(IO) provides t h a t UNRRA s h a l l l a y dc-vm standards w i t h which y e l l o w f e v a r vaccine s h a l l conform. A c c o r d i n g l y , the UNRRA Expert Commission on Quarantine prepared the . f o l l o w i n g memorandum s e t t i n g f o r t h standards f o r the manufacture and c o n t r o l of y e l l o w fever vaccine and methods of storage, t r a n s p o r t and a d m i n i s t r a t i o n . This memorandum represents a combination of the view of the Inter-Departmental Committee on Yellow Fever C o n t r o l i n London w i t h those set f o r t h by Dr. M i l t o n V. Veldae, C h i e f of the B i o l o g i e s C o n t r o l Laboratory of the U.S.Public Health S e r v i c e , a f t e r c o n s u l t a t i o n w i t h Dr. Hugh Smith and Dr. J.H.Bauer of the R o c k e f e l l e r Foundation. I t was subsequently adopted by the Standing Technical Committee on Health. • MINIMUM REQUIREMENTS 1. Type-of Vaccine : The vaccine s h a l l be of the aqueous-base (serum f r e e ) type. The production method employed s h a l l be e s s e n t i a l l y as described i n the r e p o r t s r e f e r r e d to below (references 1 and 2 ) . A r i g i d a s e p t i c technique must be employed during a l l stages of production. The f i n i s h e d v i r u s suspension contains only v i r u s - i n f e c t e d chick-embryo pulp suspended i n d i s t i l l e d water and must be f r e e of a l l ether v i a b l e micro-organisms as determined by s u i t a b l e t e s t s . The f i n i s h e d vaccine s h a l l be d r i e d i n the f i n a l c o n t a i n e r from the f r o z e n s t a t e under high vacuum by an acceptable method. The d r i e d product s h a l l c o n t a i n not more than 1.0 per cent of moisture ( p r e f e r a b l y not more than 0.5 per cent) as determined by the phosphorous pentoxid-vacuum'method. The f i n i s h e d vaccine s h a l l be flame-sealed i n an airpoule of sxùtable s i z e and prepared from glass c f not l e s s than U.S.P. type I q u a l i t y . P r i o r to s e a l i n g , the ampoule s h a l l be f i l l e d to atmospheric pressure w i t h pure, moisture-free nitrogen,2. I d e n t i t y and P u r i t y c f the Virus S t r a i n : The acceptable primary seed v i r u s i s known as the 17D s t r a i n of y e l l o w fever v i r u s ana s h a l l be of the •2OO-300th subculture (reference 3). I t shall be s a t i s f a c t o r y w i t h regard to the f o l l o w i n g c l i a r a c t e r i s t i c s i n the monkey. (These are discussed i n d e t a i l i n references 4 and 5)s (a) The degree of v i s c e r o t r o p i s m as i n d i c a t e d by the amount of c i r c u l a t i n g virus following intracerebral inoculation, (b) The onset and d u r a t i o n of a t y p i c a l f e b r i l e réaction. ( c ) The degree of neurotropism as i n d i c a t e d by the incidence of c l i n i c a l manif e s t a t i o n of e n c e p h a l i t i s and death. (d) Tha development of an immune response as measured by the a b i l i t y of the serum to prevent i n f e c t i o n i n the mouse, • • The v i r u s must be f r e e of a l l other v i a b l e micro-organisms as determined by s u i t a b l e r tests. The "seed l o t " system (reference 2) s h a l l be employed i n preparing and maintaining the stock of secondary seed v i r u s .

WCT/78.55

-23. Monkey S a f e t y Test on the Secondary Seed Virus i The o b j e c t of t h i s t e s t i s to evaluate the behavior c h a r a c t e r i s t i c s of t h e v i r u s i n a s u s c e p t i b l e animal as determined by t e s t s on tha secondary seed v i r u s using not l e s s than 6 rhesus monkeys. Tho t e s t dose i s i n j e c t e d i n t r a c e r e b r a l l y i n t o h e a l t h y rhesus monkeys (Macaca mulatta) which have proven to be non-immune t c yellov/ fever by v i r u s n e u t r a l i z a t i o n t e s t s of bluod specimens drawn from each monkey j u s t p r i o r tc i n j e c t i n g the seed v i r u s . The monkeys subsequently s h a l l be observed f o r a minimum p e r i o d c f 30 days. Not more than one (16 per c e n t ) o f t h e animals s h a l l f a i l to become immune to y e l l o w fever as determined by v i r u s n e u t r a l i z a t i o n t e s t s of blood (senun) drawn 14 or more days subsequent t o i n o c u l a t i o n of the v a c c i n e . Not more than 2 (33 per cent) of the monkeys under t e s t s h a l l develop e n c e p h a l i t i s , which may be manifested by p a r a l y s i s or i n a b i l i t y t o stand and vi^iich may or may not r e s u l t i n the death of the animal. Blood serum obtained from each of these t e s t monkeys on the 2nd, 4 t h , and 6th day, when i n j e c t e d i n t e n f o l d d i l u t i o n s i n t r a c e r e b r a l l y i n t o s u i t a b l e mice, s h a l l cause deaths i n lower d i l u t i o n s but not more than L e t h a l Dose 50 death r a t e i n d i l u t i o n 10-^ due t o a s p e c i f i c y e l l o w fever v i r u s e n c e p h a l i t i s during an observation p e r i o d of 21 d a y s . Not l e s s than 6 mice s h a l l be used at each d i l u t i o n . Each l o t " o f seed v i r u s must pass s a t i s f a c t o r y monkey s a f e t y and immunity t e s t s (references 1 and 5). • 4. Chicken Bnbryos : .. • ,

———^———

•: , ) J

Only l i v i n g , t y p i c a l chick embryos from v i r u s - i n o c u l a t e d eggs s h a l l be ^ harvested. The age of the embryo s h a l l be computed from the hour of i n i t i a l i n t r o d u c t i o n i n t o the i n c u b a t o r . Although the age of the harvested embryo i s not i n f l e x i b l y d e f i n e d , i t p r e f e r a b l y should be not mere than 11 days ( r e f e r e n c e 6). The i n c l u s i o n of the heads c f the embryos i n preparing the pulp i s o p t i o n a l , 5. Potency of the F i n i s h e d Vaccine : 'w • The f i n i s h e d , d r i e d , chick-embryo pulp vriaen rehydrated to i t s o r i g i n a l volume s h a l l c o n t a i n not l e s s than 150,000 MLD of 17D v i r u s per m i l l i l i t e r o f vaccine a t the time of p a s s i n g f i n a l potency t e s t . Present i n f o r m a t i o n i n d i c a t e s that f o r the s a t i s f a c t o r y immunization of man not l e s s than 500 MT.f) of a c t i v e v i r u s should be contained i n the immunizing dose i n j e c t e d , an excess b e i n g p r e f e r r e d . (One MLD i s d e f i n e d as that q u a n t i t y of y e l l o w fever v i r u s which -vrtien i n o c u l a t e d i n t r a c e r e b r a l l y i n t o each of an adequate number of yellov; f e v e r s u s c e p t i b l e a d u l t mice (16-20) grams w i l l k i l l 50 per cent of the animals due t o a s p e c i f i c y e l l o w fever v i r u s e n c e p h a l i t i s w i t h i n 21 days. See reference 7 f o r method of determining 50 per cent and p o i n t . ) 6. Guinea P i g Safety Test î '. /„

A safety t e s t i n the guinea p i g s h a l l be made on each l o t of f i n i s h e d v a c c i n e , u s i n g rehydrated chick-embryo pulp from ançcules s e l e c t e d a t random from the f i l l i n g . Four to f i v e ml. s h a l l be i n j e c t e d i n t r a p e r i t o n e a l l y i n t o each o f 2 or more normal guinea p i g s weighing 300 to 500 grams. The animals s h a l l be observed f o r 7 or more days d u r i n g vihich time m s i g n i f i c a n t c l i n i c a l m a n i f e s t a t i o n s s h a l l occur. I f both of the animals show s i g n i f i c a n t r e a c t i o n s the e n t i r e l o t s h a l l be regarded as u n s a t i s f a c t o r y . I f one animal shows s i g n i f i c a n t c l i n i c a l r e a c t i o n the t e s t s h a l l be repeated, u s i n g 3 t e s t animals. I f i n the r e p e a t e d t e s t more than one of the 3 animals show s i g n i f i c a n t r e a c t i o n s the product i s vmsatisfactory.

-37. S t e r i l i t y of the Vaccine : S t e r i l i t y of the product must be maintained a t a l l times w h i l e p r o c e s s i n g . As a f i n a l check the f i n i s h e d vaccine s h a l l be s t e r i l e as i n d i c a t e d by t e s t s on not l e s s than three f i n a l c o n t a i n e r s s e l e c t e d at random when the t o t a l f i l l e d i s 100 or l e s s , p l u s one a d d i t i o n a l c o n t a i n e r f o r each a d d i t i o n a l 50 c o n t a i n e r s f i l l e d . Not l e s s than 1 ml. from each c o n t a i n e r , or the e n t i r e contents i f l e s s than 1 ml. of the chick-embryo pulp when rehydrated as d i r e c t e d on the l a b e l s h a l l be t e s t e d . D u p l i c a t e c u l t u r e s s h a l l be made w i t h i n c u b a t i o n at 37°C and 22°G. I f contaminat i o n appears i n any of the tubes p l a n t e d the t e s t may be repeated vdth the same number of c o n t a i n e r s and a l o t s h a l l be d i s c a r d e d i f the same type of organism appears i n more than one t e s t , but no l o t s h a l l be passed u n t i l the f i n a l t e s t shows ho growth throughout. 8. I d e n t i t y Test : The potency t e s t on the f i n i s h e d vaccine s h a l l be considered a s a t i s f a c t o r y Identity test. 9. L a b e l i n g t The outside l a b e l on each c o n t a i n e r s h a l l shov; (a) the o f f i c i a l name of the product, namely, "Yellow Fever V a c c i n e " ; (b) the statement " L i v i n g d r i e d c u l t u r e of 17D v i r u s prepared from i n f e c t e d chicken embryo"5 (c) the volume and k i n d of d i l u e n t to add; (d) dose; (e) the l o t number and e x p i r a t i o n d a t e ; (f) the l i c e n s e number, name, and address of manufacturer; • ' (g) a statement of the c o n d i t i o n s of storage (see s e c t i o n 1 0 ) ; (h) a statement of the volume of chick-embryo pulp before d r y i n g i s o p t i o n a l ; (1) the statement "No U.S.Standard of Potency" s h a l l not appear; 10. E x p i r a t i o n Date : The dating p e r i o d of Yellow Fever v a c c i n e i s d i v i d e d i n t o three periods i a. P e r i o d of v e r y low temperature storage between h a r v e s t i n g and the : time of passing a s a t i s f a c t o r y potency t e s t (the l a t t e r date i s ; • designated the "date of manufacture" ). This i s a v a r i a b l e p e r i o d dependent upon "the manufacturing c o n d i t i o n s , demands f o r v a c c i n e , and ;j storage temperature. b. P e r i o d between date of manufacture and date of beginning d i s t r i b u t i o n • , (the l a t t e r i s designated the "date of i s s u e " ) . This p e r i o d s h a l l be not more than 1 year a f t e r date of manufacture and during t h i s time the , • : product must be kept c o n s t a n t l y a t a temperature below -5°C. (23°F), p r e f e r a b l y below minus 20°C. • c. P e r i o d of d i s t r i b u t i o n . This p e r i o d (ending w i t h the " e x p i r a t i o n date") s h a l l be not more than 1 year a f t e r the date of i s s u e , and d u r i n g t h i s time the product must be kept c o n s t a n t l y at a temperature below 5°C (41 F ) , and the l a b e l must bear^the statement f o l l o v d n g the e x p i r a t i o n date t " I f kept below 5 C. (41 F . ) . Keep p r e f e r a b l y below 0°C. (32°F.)."

-411* A d n d n i s t r a t i o n of the Vaccine î Yellow fever vaccine processed i n accordance w i t h these minimum r e q u i r e m e n t s i s prepared f o r human i n j e c t i o n i n the f o l l o v d n g manner t Draw i n t o a s t e r i l e needle and syringe of s u i t a b l e s i z e the d i l u e n t ( s t e r i l e p h y s i o l o g i c a l sodium c h l o r i d e s o l u t i o n on the b a s i s of 10 ml. of d i l u e n t f o r each 1 ml* of d r i e d vaccine, as shown on l a b e l of ampoule), then open the vaccine c o n t a i n i n g ampoule and c a r e f u l l y mix the vaccine and the d i l u e n t u n t i l a l l has been drawn i n t o t h e syringe and i s i n a uniform suspension. Because of the volume i n case of t h e l a r g e r ampoules, the l i q u e f y i n g and d i l u t i n g v d l l need to be accomplished w i t h a separate b o t t l e of d i l u e n t and a s u i t a b l e s y r i n g e . Administer the s i n g l e immunizing dose of 0.5 ml. stibcutaneously at once. C h i l d r e n r o c e i v e the same d o s e as a d u l t s . 12. Shipping Requirements : Yellow f e v e r vaccine s h a l l be shipped i n a s u i t a b l e container adequately p a c k e d i n carbon d i o x i d e i c e , or provided w i t h other means of r e f r i g e r a t i o n , so as to ensure a teiiperature c o n s t a n t l y below 0 C. 13. General Rp;quirements : A l l other requirements as to methods of manufacture, and the -maintenance o f adequate processing and d i s t r i b u t i o n records -vAich are r e q u i r e d by governmental regulitie^ns s h a l l apply. 14. Requirements f o r Rglease : '

Yûiere r e q u i r e d by governmental r e g u l a t i o n s , as i n the United S t a t e s , d i s t r i b u t i o n of each l o t s h a l l be vdthhald pending o f f i c i a l r e l e a s e . For t h i s release there s h a l l be submitted the f o l l o w i n g : (a) A p r o t o c o l of the potency and s t e r i l i t y t e s t s . (b) No l e s s than 3 v i a l s , or not l e s s than the equivalent of 50 c c . of f i n i s h e d • a c c i n e , shipped as d i r e c t e d i n s e c t i o n 12. , . DIRECTIONS FOR PRESERVAHON OF SEED VIRUS The primary seed v i r u s s h a l l be preserved i n the d r i e d s t a t e under n i t r o g e n and s t o r e d continuously at minus 70 C or c o l d e r . The v i r u s c o n t a i n i n g m a t e r i a l i s d i s t r i b u t e d i n t o ampoules of U.S.P. type I g l a s s , or b e t t e r , s h e l l f r o z e n q u i c k l y , f o l l o w i n g which the m a t e r i a l i s d r i e d from the frozen s t a t e under h i g h vacuum u n t i l the moisture content of the v i r u s c o n t a i n i n g m a t e r i a l i s l a s s t h a n 0.5 per cent. The airpoules are then f i l l e d w i t h dry n i t r o g e n , flame-sealed, i n s p e c t e d , l a b e l e d , and immediately stored at minus 70 C. or c o l d e r . I f properly prepared and stored, the v i r u s w i l l remain v i a b l e f o r an i n d e f i n i t e niimber of y e a r s . The secondary seed v i r u s i s prepared as chick-embryo pulp by the method described f o r the p r e p a r a t i o n of the pulp f o r vaccine production. The pulp i s ampouled, d r i e d and stored i n the manner described f o r the p r e p a r a t i o n of the primary seed v i r u s .

-5REFERENCES 1. 2, HargQtt, M.v., Burruss, H.^., and Donovan, A.: Aquaous-base y e l l o w f e v e r v a c c i n e s . Pub. Health Rep., 58; 505-512 (1943). Fox, J.P., Kossobudzki, S.L., and Fonseca da Gunha, J . : F i e l d s t u d i e s on the immune response to 17D y e l l o w f e v e r v i r u s ; r e l a t i o n t o v i r u s s u b s t r a i n , dose, and route of i n o c u l a t i o n . Am. J . Hyg., 38; 113-138 (1943).

3* T h e i l e r , M., and Smith, H.H.: The e f f e c t c f prolonged c u l t i v a t i o n i n v i t r o upon the p a t h o g e n i c i t y of y e l l o w f e v e r v i r u s . J . Exp. Med., 65; 757-7^6 (1937). 4. Fox, J.P., and Penna, H.A.: Behavior of 17D y e l l o w f e v e r v i r u s i n rhesus monkeys: R e l a t i o n to s u b s t r a i n , dose,and n e u r a l or e x t r a n e u r a l i n o c u l a t i o n . Am. J . Hyg., 38; 152-172 (1943). Hargett, i .V., and Burruss, H.Vif.: The use of rhesus monkeys i n the t e s t i n g of aqueous-base y e l l o w f e v e r v a c c i n e . Am. J . Trop. Med., 25; 19-30 (1945). Berge, T.D., and Hargett, M.V.s Anaphylaxis i n guinea p i g s f o l l o w i n g s e n s i t i z a t i o n w i t h chick-embryo y e l l o w f a v e r vaccine and normal c h i c k embryo. Pub, Health Rep., 57; 652-667 (1942). Reed, L . J . , and Muench, H.* A simple method of e s t i m a t i n g 50 p e r cant end p o i n t s . Am. J . Hyg., 27: 493-497 (1938).

5. 6.

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ANNEX I I STANDARDS FOR THE MANUFACTURE AND CONTROL OF YELLOiV FEVER VACCINE (with amendments incorporated) MINIMUM REQUIREMENTS 1. Type of Vaccine t

The vaccine s h a l l be of the aqueous-base (serum f r e e ) type. The produotion method employed s h a l l be. e s s e n t i a l l y as d e s c r i b e d i n the reports r e f e r r e d to below (references 1, 2, and 2 a l . A r i g i d a s e p t i c technique must be employed during a l l stages of p r o d u c t i o n . The f i n i s h e d v i r u s suspension contains v i r u s - i n f e c t e d chickembryo j u i c e alone or d i l u t e d i n d i s t i l l e d water and must be free of a l l other v i a b l e micro-organisms as determined by s u i t a b l e t e s t s . (Embryo j u i c e i s here used f o r the supernatant f l u i d a f t e r c e n t r i f u g a t i o n of the pulp.) The f i n i s h e d vaccine s h a l l be d r i e d i n the f i n a l .container from the f r o z e n s t a t e under h i g h vacuum by an acceptable method. The did.ed product s h a l l c o n t a i n not more than 1.0 per cent of moisture ( p r e f e r a b l y not more than 0.5 per cent) as determined by the phosphorous pentoxid-vacuum method. (The contents of a t l e a s t two ampoules are d r i e d t o conPtant weight over P^O i n vacuo a t 50 C.) The f i n i s h e d vaccine s h a l l be flaraesealed i n an ampoule or Suitable s i z e and prepared from glass of not l e s s than U.S.P. type I q u a l i t y . P r i o r to s e a l i n g , the ampoule s h a l l be f i l l e d t o atmospheric pressure w i t h pure, moisture-free n i t r o g e n . 2. I d e n t i t y and P u r i t y of the Virus S t r a i n :

The acceptable primary seed v i r u s i s knovm as the 17D s t r a i n of y e l l o w fever v i r u s c u l t i v a t e d i n chick embryb t i s s u e c u l t u r e (reference 3 ) , or i n developing chick embryo, and s h a l l be of the 200-300th subculture. I t s h a l l be s a t i s f a c t o r y w i t h regard to the f o l l o w i n g c h a r a c t e r i s t i c s i n the monkey. (These are discussed i n d e t a i l i n references 4 and 5) t. • • (a) The degree of v i s c e r o t r o p i s m as i n d i c a t e d by the amount of c i r c u l a t i n g v i r u s following intracerebral inoculation. • •• (b) The onset and d u r a t i o n of a t y p i c a l f e b r i l e r e a c t i o n . ( c ) The degree of neurotropism as i n d i c a t e d by the 'incidenee «f c l i n i c a l manifestat i o n s of e n c e p h a l i t i s and death. " — • • . . (d) The development of an immune response as measured by the a b i l i t y of the serum to prevent i n f e c t i o n i n the mouse. The v i r u s must be f r e e of a l l other v i a b l e microf-organisms as determined by s u i t a b l e tests. The "seed l o t " system (reference 2 ) s h a l l be employed i n p r e p a r i n g and maintaining the stock of secondary seed v i r u s . 3. Monkey S a f e t y Test on the Secondary Seed V i r u s :

The object of t h i s t e s t i s t o evaluate the behavior c h a r a c t e r i s t i c s of the J • v i r u s i n a s u s c e p t i b l e animal as determined by t e s t s on the secondary seed v i r u s , u s i n g not l a s s than 6 rhesus monkeys. The t e s t dose i s i n j e c t e d i n t r a c e r e b r a l l y i n t o healthy rhesus monkeys (Macaca mulatta) which have proven t o be non-imraune t o y e l l o w f e v e r by v i r u s n e u t r a l i z a t i o n t e s t s of blood specimens drawn from each monkey j u s t p r i o r t o i n j e c U n g the seed v i r u s . The monkeys subsequently s h a l l be 2a. Smith, H.H. 1951. N.Y., p.612. MH/CT/78,55^/ Y e l l o w Fever. ~ E d i t . G.H.Strode, McGraw H i l l Book Co., ^

-2observed f o r a miniraum period of 30 days. Not more than one (16 per cent) of the animals s h a l l f a i l to become immune to yellov; fever as determined by v i r u s neutralization tests of blood (serum) drawi 14 or more days subsequent to i n o c u l a tion cf the vaccine. Not more than 2 (33 per cent) of the monkeys under test s h a l l develop encephalitis, which may be manifested by paralysis or i n a b i l i t y to stand and which may or may not result i n the death of the animal. Blood serum obtained from each of these test monkeys on the 2nd, 4th, and 6th day, when injected i n tenfold dilutions intracerebrally intc suitable mice, s h a l l cause deaths i n lower dilutions but not more than Lethal Dose 50 death rate i n d i l u t i o n 10- due to a specific yellow fever virus encephalitis during an observation period of 21 days. Not less than 6 mice shall be used at each d i l u t i o n . Each lot of seed virus must pass satisfactory monkey safety and immunity tests (references 1 and 5), 4. Chicken Embryos :

Developing eggs s h a l l be obtained from poultry free from disease. Only l i v i n g , t y p i c a l chick embryos from virus-inoculated eggs s h a l l be harvested. The ageof the embryo s h a l l be computed from the hour of i n i t i a l introduction into the incubator. Although the age of the harvested embryo i s not i n f l e x i b l y defined, i t preferably should be not more than 11 days (reference 6). The i n c l u s i o n of the heads of the anbryos i n preparing the pulp i s optional. 5. Potency test of Vaccine « (a) Three ampoules •

are taken out at random from a vaccine l o t consisting of a

number of ampoules containing freeze-dried chick embryo j u i c e . (b) The anyoules are stored f o r one week at room temperature (18°Q). (c) The juice i s rehydrated by using d i s t i l l e d water to i t s o r i g i n a l volume. (d) A tenfold d i l u t i o n series from IC to 10 ^ i s made up from the content of each ampoule using 0.2$^ bovine albumin,"fraction V, i n saline as d i l u e n t . (e) Three series of Swiss albino mice of the Rockefeller s t r a i n weighing 16-20 grams are inoculated intracerebrally under ether anaesthesia. The dose s h a l l be 0.03 ml. using s i x mice f o r each d i l u t i o n , together 150 mice. The time between d i l u t i o n and inoculation s h a l l be exactly 30 minutes during vrtiich time the d i l u t i o n i s kept at room temperature. (f) The mice are observed for 21 days. A l l deaths occurring within 7 days a f t e r inoculation are ignored. Deaths caused by t y p i c a l yellow fever v i r u s i n f e c t i o n are recorded. ' ' (g) After the period of observation, the LD50 {50% mortality end point) f o r the three series i s calculated according to Reed and Munch (reference 7 ) . " (h) The potency of the vaccine s h a l l be expressed i n mouse LD50 per ml. and s h a l l constitute the mean value of results received by testing three amooulp.; from the same vaccine l o t ^ '

The term "vaccine l o t " refers to material that i s processed together as a u n i t and that i s of uniform composition^ ~ ~ ' ~~~

6.

Potency Requirements

of the F i n i s h e d Vaccine Î

The f i n i s h e d , d r i e d , c h i c k embryo j u i c e when rehydrated to i t s o r i g i n a l volume s h a l l c o n t a i n not l e s s than 150,000 mouse LD50 of 17D v i r u s per ml. at the time of p a s s i n g f i n a l potency t e s t according to the method given above. The potency of the dose used f o r human v a c c i n a t i o n s h a l l be expressed i n mouse LD50 and be of the order of 500-5000 mouse ID50» 0.5 ml. of d i l u t e d vaccine s h a l l be the dose of v a c c i n a t i o n and the content of chick embryo j u i c e of t h a t dose must not exceed 8%. I t would be a d v i s a b l e to t e s t the potency of the dose of v a c c i n a t i o n on the a b i l i t y to produce n e u t r a l i z i n g a n t i b o d i e s i n non-immune humans i n , say, every tenth vaccine l o t . 7. Guinea P i g Safety Test i

A s a f e t y t e s t i n the guinea p i g s h a l l be made on each l o t of f i n i s h e d v a c c i n e , u s i n g rehydrated chick-embryo pulp from ampoules s e l e c t e d at random from the filling. Four to f i v e ml. s h a l l be i n j e c t e d i n t r a p e r i t o n e a l l y i n t o each of 2 or more normal guinea p i g s weighing 300 to 500 grams. The animals s h a l l be observed f o r 14 or more days during which no s i g n i f i c a n t c l i n i c a l m a n i f e s t a t i o n s s h a l l occur. I f both of the animals show s i g n i f i c a n t r e a c t i o n s the e n t i r e l o t s h a l l be regarded as u n s a t i s f a c t o r y . I f one animal shows s i g n i f i c a n t c l i n i c a l r e a c t i o n the t e s t s h a l l be repeated, u s i n g 3 t e s t animals. I f i n the repeated t e s t more than one of the 3 animals show s i g n i f i c a n t r e a c t i o n s the product i s u n s a t i s f a c t o r y . 8. S t e r i l i t y of the Vaccine :

S t e r i l i t y df the product must be maintained at a l l times w h i l e p r o c e s s i n g . As a f i n a l check the f i n i s h e d vaccine s h a l l be s t e r i l e as i n d i c a t e d by t e s t s on not l e s s than three f i n a l containers s e l e c t e d at. random when the t o t a l f i l l e d i s 100 or l e s s , plus one a d d i t i o n a l c o n t a i n e r f o r each a d d i t i o n a l 50 c o n t a i n e r s f i l l e d . Not l e s s than 1 ml. from each c o n t a i n e r , or the e n t i r e contents i f l e s s than 1 ml.' of the chick-embryo j u i c e when rehydrated as d i r e c t e d on the l a b e l s h a l l be tested-o D u p l i c a t e c u l t u r e s s h a l l be made w i t h i n c u b a t i o n a t 37 C and 22 C u s i n g dextrose b r o t h , both aerobic and anaerobic tubes,'and Brewer's or Linden's t h i o g l y c o l l a t e media. I t might a l s o b"e"advisable to usé chocolate agar s l a n t s to detect contaminants. The c u l t u r e s are observed f o r 10 days and i f contamination appears i n any of the tubes p l a n t e d tho t e s t may be repeated w i t h the same number of containers. I f the contamination i s caused by l a c k of s t e r i l i t y of the vaccine tho l o t s h a l l be d i s c a r d e d . ^. I d e n t i t y Test i • • . . . ~: ,

The potency t e s t on the f i n i s h e d vaccine s h a l l be considered a s a t i s f a c t o r y identity test. 10. L a b e l i n g : The outside l a b e l on each container s h a l l show (a) the o f f i c i a l name of the product, namely, "Yellow Fever Vaccine"; (b) the statement " L i v i n g d r i e d c u l t u r e of 17D v i r u s prepared from i n f e c t e d chicken embryo"; (c) the volume and k i n d of d i l u e n t to add;

-4(d) dose and mouse LD50 per dose; (e) the l o t number and e x p i r a t i o n date; ( f ) the l i c e n s e nuntier, name, and address of manufacturer; (g) a statement of the c o n d i t i o n s of storage (see s e c t i o n 1 1 ) ; 11» E x p i r a t i o n date : The d a t i n g period of Yellow Fever vaccine i s d i v i d e d i n t o three p e r i o d s t (a) Period of v e r y lov; temperature storage between h a r v e s t i n g and the time o f passing a s a t i s f a c t o r y potency t e s t (the l a t t e r date i s designated the "date of manufacture"). This i s a v a r i a b l e p e r i o d dependent upon the manufacturing c o n d i t i o n s , demands f o r vaccine, and storage temperature. (b) Period between date of manufacture and date of beginning d i s t r i b u t i o n ( t h e l a t t e r i s designated the "date of i s s u e " ) . This p e r i o d s h a l l be not more than 1 year a f t e r date of manufacture and^during t h i s time the product must be kept c o n s t a n t l y a t a temperature below -5 C. (23°F.), p r e f e r a b l y below minus 20*^0. (c) Period of d i s t r i b u t i o n . This neriod (ending w i t h the " e x p i r a t i o n date") s h a l l be not more than 1 year a f t e r the date of i s s u e , and during t h i s time t h e product must be kept c o n s t a n t l y at a temperature below 5 C. (41 "^F.), and the l a b e l must bear the statement f o l l o w i n g the e x p i r a t i o n date t " I f kept below 0°C. (32 F . ) " . . ' • ' ^ 12. A d m i n i s t r a t i o n of the Vaccine : • •

Yellow f e v e r vaccine processed i n accordance w i t h these minimum requirements i s prepared f o r human i n j e c t i o n i n the f o l l o w i n g manner : Draw i n t o a s t e r i l e needle and syringe of s u i t a b l e s i z e the d i l u e n t ( s t e r i l e p h y s i o l o g i c a l sodium c h l o r i d e s o l u t i o n on the b a s i s of 10 ml, of d i l u e n t f o r each 1 ml. of d r i e d v a c c i n e , as shown on l a b e l of ampoule), then open the vaccine c o n t a i n i n g ampoule and c a r e f u l l y mix the vaccine and the d i l u e n t u n t i l a l l has been drawn i n t o the syringe and i s i n a uniform suspension. Because of the volume i n case of the l a r g e r ampoules, the l i q u e f y i n g and d i l u t i n g w i l l need to be accomplishedvith a separate b o t t l e of d i l u e n t and a s u i t a b l e s y r i n g e . Administer the s i n g l e immunizing dose of 0.5 ml. subcutaneously at once. I f vaccine i s intended t o be used i n a f i n a l d i l u t i o n of more than I t l O a second d i l u t i o n " l . s prepared"i The f i r s t d i l u t i o n ma/ be kept a t ice-box temperature (5 0.) or lower f o r not more than three hours. The f i n a l d i l u t i o n must be administered a t once. C h i l d r e n ' r e c e i v e the same dose as a d u l t s . . , , 13. Shipping Requirements : . - - r

Yellow f e v e r vaccine s h a l l be shipped i n a s u i t a b l e c o n t a i n e r adequately packed i n carbon d i o x i d e i c e , or provided w i t ^ other means of r e f r i g e r a t i o n , so as to ensiire a tenperature c o n s t a n t l y below 0 C.

-514. General Requirements :

A l l other requirements as t o methods of manufacture, and the maintenance of adequate processing and d i s t r i b u t i o n records which are r e q u i r e d by governmental regulations s h a l l apply. 1$. Requirements f o r Release :

Where r e q u i r e d by governmental r e g u l a t i o n s , as i n the U n i t e d S t a t e s , d i s t r i b u t i o n o f each l o t s h a l l be w i t h h e l d pending o f f i c i a l r e l e a s e . For t h i s r e l e a s e there s h a l l be submitted the f o l l o w i n g : (a) A p r o t o c o l of the potency and s t e r i l i t y t e s t s . (b) No l e s s than 3 v i a l s , or not l e s s than the equivalent of 50 c c . o f f i n i s h e d v a c c i n e , shipped as d i r e c t e d i n s e c t i o n 13. DIRECTIONS FOR PRESERVATION OF SEED VIRUS Not to be amended a t the present stage.

REFERENCES As i n o r i g i n a l Standards, w i t h the a d d i t i o n of 2a. the t e x t o f which i s t o be found on page 1 of t h i s Annex.

ANNEX I I I Comments on Standards f o r the Manufacture and C o n t r o l of Yellow Fever Vaccine (UNRRA E p i d e m i o l o g i c a l Information B u l l e t i n , 1945, V o l . I , No.9)

1.

Type of Vaccine

• ^ ^ D^' A. Cast G a l v i s ( D i r e c t o r , I n s t i t u t e de Estudios E s p e c i a l e s "Carlos F i n d l a y " , M i n i s t e r i o d i Higiene, Bogota) The very wide experience of the R o c k e f e l l e r Foundation workers and of v a r i o u s South American o r g a n i z a t i o n s attached t o i t has shewn t h a t e n t i r e l y s a t i s f a c t o r y r e s u l t s have been obtained w i t h vaccine prepared according to the technique described by Smith ( l o c o c i t . ) i . e . i n the f i r s t p l a c e , 25 ml. of water i s added per 100 ml. by volume of embryoj a f t e r m i l l i n g and c e . i i r i f u g i n g , the same amount of water i s added t o the supernatant f l u i d . In t h i s v a c c i n e , the embryo j u i c e represents .40/È by volume of the f i n a l d r i e d product. With the l / l O d i l u t i o n f o r human use, each s u b j e c t v a c c i n a t e d r e c e i v e s 4% by volume of embryo j u i c e . To t h i s vaccine, which contains more water than that prepared according to the technique of Hargett et c o l . , the UNRRA standards whereby the f i n a l d r i e d product rehydrated to i t s o r i g i n a l volume must c o n t a i n a t l e a s t 150,000 mouse LD5n per ml. have been and s t i l l are a p p l i e d . Smith a l s o mentions t h a t the supernatant f l u i d a f t e r m i l l i n g and c e n t r i f u g i n g of the embryos can be used as vaccine without the a d d i t i o n of the equal volume of water; t h i s means that the vaccine thus prepared w i l l have art 80;^ (by volume) c o n c e n t r a t i o n of embryo j u i c e i n the d r i e d product and, t h e r e f o r e , that each I n d i v i d u a l v a c c i n a t e d w i t h t h i s product d i l u t e d to l / l O w i l l receive 8% of embryo j u i c e . I t has also been adequately demonstrated that vaccines prepared according t o the technique r e f e r r e d to by Smith can, once the l / l O d i l u t i o n of the d r i e d product has been made, be administered to man up to three hours from the moment when the d i l u t i o n i s prepared. These r e s u l t s prove that the d r i e d product can be d i l u t e d f o r humaji use i n such a way t h a t the d i l u t i o n contains a miiiimum of 15,000 mouse LD50 per ml. (on the b a s i s of the f i n a l t i t r e of the d r i e d p r o d u c t ) , and 4^ to 8jS (by volume) of embryo j u i c e , and that t h i s d i l u t i o n can be administered to man up to 180 minutes from the time of p r e p a r a t i o n . I t i s important t o note t h a t we have considered the e f f e c t of the a d d i t i o n of water to the embryo e x c l u s i v e l y i n i t s r e l a t i o n t o the f a c t o r s of d i l u t i o n and p r e s e r v a t i o n of the v i r u s , without reference to other techniques i n # i i c h the a d d i t i o n of water a f f e c t s the product during c e n t r i f u g i n g ^ and e s p e c i a l l y during milling. In view of the above, i t would be d e s i r a b l e to c o n s i d e r modifying the e x i s t i n g standards. I n the l i g h t of our expe-r^onc^' and that of the R o c k e f e l l e r Foundation workers, the f o l l o w i n g are some of the m o d i f i c a t i o n s we would suggest :

LMH/CT/78.55

\I

-2Type of Vaccine Î U n i f i c a t i o n of method f o r the production of vaccine Here we would include ; 1.1 M i l l i n g of embryos i n a "Waring Blender" f o r 10 minutes w i t h the a d d i t i o n of s t e r i l e d i s t i l l e d water i n the r a t i o of 0.25 g. of water to 1 g. of embryos. In p r a c t i c e i t i s much e a s i e r to obtain t h i s p r o p o r t i o n by weight than by volume. The m i l l e d product t o be put i n t o s u i t a b l e f l a s k s , f r o z e n , and stored at -75 C. while awaiting separation i n t o ampoules. The m i l l e d product to be t e s t e d f o r s t e r i l i t y . The t i t r a t i o n of the product i n containers to be o p t i o n a l ; i t makes i t p o s s i b l e t o r e j e c t , before d e s s i c a t i o n , any low t i t r e m i l l e d l o t s . Unfreezing of the s t e r i l e m i l l e d pulp and c e n t r i f u g i n g i n 250 m l . f l a s k s at 2,000 r.p.m. f o r 40 minutes. The supernatant f l u i d to be f i l t e r e d . V

- " C

1.2

S ' ..a ' •

1.3 1.4 1.5 1.6 1.7 2.

Separation of the supernatant f l u i d into appropriate ampoules (USP type 1 ) . .

F r e e z i n g of the ampoule contents and d e s i c c a t i o n i n accordance w i t h c u r r e n t methods. S e a l i n g of the ampoules i n accordance w i t h the standard p r a c t i c e . '

Dr. F.N.Macnamara (Yfest A f r i c a n C o u n c i l f o r Research L a b o r a t o r i e s , Lagos) b e l i e v e s t h a t some of the techniques recommended by UNRRA could be improved c o n s i d e r a b l y and that p r o v i s i o n should be made to i n c l u d e i n the standards reference t o a 17D vaccine to be administered by s c a r i f i c a t i o n . He submits the f o l l o w i n g comments • 1. Type of vaccine : I t i s h i g h l y probable t h a t a s a t i s f a c t o r y mouse b r a i n 17D vaccine a d m i n i s t e r e d by s c a r i f i c a t i o n can be developed. Such m a t e r i a l might be d e s i c c a t e d by o t h e r means than l y o p h i l i s a t i o n .

3.

Dr. R.A.Penna ( I n s t i t u t e Oswaldo Cruz, Rio de J a n e i r o ) declares t h a t amendments should be made to the standards e s t a b l i s h e d by UNRRA and submits the f o l l o w i n g m o d i f i c a t i o n s to make allowance f o r the methods of manufacture adopted by the Oswaldo Cruz I n s t i t u t e , Rio de J a n e i r o : 1. Type of vaccine : The vaccine s h a l l be of the serum f r e e type. Ihe f i n i s h e d v i r u s suspension contains v i r u s - i n f e c t e d chick-embryo j u i c e alone or d i l u t e d i n d i s t i l l e d water e t c .

-34. Dr. R.A.Penna : Tho f i n i s h e d v i r u s suspension contains v i r u s - i n f e c t e d chicken embryo j u i c e alone or d i l u t e d i n d i s t i l l e d water, e t c . Dr. F.G.Morgan ( D i r e c t o r , Commonwealt Serum L a b o r a t o r i e s , P a r k v i l l e , A u s t r a l i a ) : I t might be considered d e s i r a b l e , p a r t i c u l a r l y w i t h such a hygroscopic substance as y e l l o w f e v e r v a c c i n e , to s p e c i f y e i t h e r by a reference to a p u b l i s h e d paper or i n d e t a i l , the acceptable method f o r determining the moisture content. Dr. D.G.Edward (Virus Laboratory, The lyellcome Research L a b o r a t o r i e s , Beckenham) : The r e s i d u a l moisture content i s estimated on the contents of at l e a s t two ampoules b^ determining the l o s s i n weight i n c u r r e d a f t e r t r e a t i n g i n a vacuum oven at 50 C. f o r 30 minutes, u s i n g an anhydrous desiccant t r a p . • Dr. F.N.Macnamara : Ampoules would be e i t h e r f i l l e d w i t h n i t r o g e n or sealed i n vacuo u (vacuum s e a l i n g a l l o w s e a s i e r d e t e c t i o n of f a u l t y ampoules a f t e r storage.) Health L.aboratory Methods S e c t i o n ; The r i s k of contamination cannot be excluded i n connection vi'ith opening of a vacuum sealed ampoule.

5.

6.

7.

8.

2. 1.

I d e n t i t y and p u r i t y of the V i r u s S t r a i n The seed v i r u s should always be 17D s t r a i n c u l t i v a t e d i n

Dr. F.N.Iliiacnamara s the c h i c k embryo.

3 - Monkey S a f e t y Test on the Secondary Seed V i r u s Cr. A. Gast Galvi-s : No change, but i t would be d e s i r a b l e , before adopting a secondary seed v i r u s , to i n o c u l a t e tvro s u f f i c i e n t l y l a r g e batches of embryos w i t h the s a i d v i r u s so that two p i l o t batches of vaccine can be prepared. At l e a s t one of these tiro batches should be i n j e c t e d i n t o a rhesus monkey. Dr. F.N.Macnamara : The t e s t dose to be i n j e c t e d i n t r a c e r e b r a l l y i n t o the monkeys should be d e f i n e d i n terms of LD50 f o r mice of the v a c c i n e . I suggest 500 - 5000 LD50. A l l rehydrated m a t e r i a l should be alloî/ed t o stand f o r a defined time before t i t r a t i o n (10 *'.nutes)< The nature of the d i l u e n t f o r a l l v a c c i n e and blood serum t i t r a t i o n s should be defined ( l suggest 0^2% bovine aloumiu F r a c t i o n V ) . 12 mice should be used f o r each t e n - f o l d dilution<. The monkey t e s t i s not s a t i s f a c t o r y f o r the d e t e c t i o n of neurotropism. I t a l l depends on the observation of e n c e p h a l i t i c symptoms i n one monkey t o r a i s e the e n c e p h a l i t i c r a t e from the pass of 33jSl t o the h i g h l y dangerous s t a t e of S0%.

„ . 2.

-43, Health Laboratory . ethods S e c t i o n : The s t a n d a r d i z a t i o n of the vaccine potencyt e s t , which w i l l be proposed at 5, w i l l l e a d to some improvements a l s o f o r the c o n t r o l of the seed v i r u s . The question w i l l be taken up i n HLM S e c t i o n comments on the l a s t paragraph bf the present UNRRA Standards which deal w i t h " D i r e c t i o n s f o r P r e s e r v a t i o n of Seed V i r u s . "

4. 1. 2» 3.

Chicken Embryos Eggs should be obtained from f l o c k s f r e e from Newcastle disease.

Dr. F.N.Macnamara : Dr. A. Gast G a l v i s : of e x t r a c t i o n .

The embryos must not be more than 12 days o l d at the moment

Health Laboratory Methods S e c t i o n : The i n d i c a t i o n of the age of the embryo when harvested i s so vague that i t s u r e l y permits the 12 days' e x t r a c t i o n .

(Here note i n t r o d u c t i o n of new paragraph 5. 1. Potency Test of Vaccine

5.)

P r . Gh. Courtois ( L a b o r a t o i r e Médicale, S t a n l e y v i l l e ) : The l a b o r a t o r i e s p r o d u c i n g yellow f e v e r vaccines t o be employed f o r the issue of i n t e r n a t i o n a l c e r t i f i c a t e s of v a c c i n a t i o n should inform the World Health Organization of the methods they u s e d f o r the t e s t i n g of t h e i r v a c c i n e . 'mO would then decide whether or not a p p r o v a l should be given. Dr. C. Durieux ( D i r e c t e u r de l ' I n s t i t u t Pasteur, Dakar) : The technique t o be adopted c o n s i s t s of the i n o c u l a t i o n of batches of mice w i t h successive t e n - f o l d d i l u t i o n s of the vaccine to be t e s t e d , and i n the determination of the potency o f t h i s vaccine by u s i n g Reed and Muench's method f o r the e v a l u a t i o n of the 50 p e r cent end-point. This technique i s employed by the Pasteur I n s t i t u t e , Dakar, and by most l a b o r a t o r i e s . I f e e l , however, that i t i s important to define the u n i t to be employed as s t a r t i n g p o i n t f o r preparing the vaccine d i l u t i o n s . For 17D~ vaccine, t h i s u n i t i s obtained by making up the volume of the embryonic pulp contained i n the ampoule to i t s o r i g i n a l volume before d e s i c c a t i o n . The volume should consequently be s p e c i f i e d on the l a b e l . As regards the Dakar v a c c i n e , one 100-dose ampoule,diluted to tha t i t r e of the suspension employed f o r v a c c i n a t i o n i s considered as a u n i t . Dr. D.G.Edward Î i s as f o l l o w s : The method used i n the Wellcome Research L a b o r a t o r i e s , Beckenham '

2.

3.

The potency of the vaccine i s estimated by i n o c u l a t i n g s e r i a l d i l u t i o n s of the contents of an ampoule i n t r a c e r e b r a l l y i n t o mice. The number of mouse u n i t s p e r ml. of vaccine i s then estimated, and the human dosage assessed by a l l o w i n g l,00o mouse u n i t s per human dose. (One mouso-imit i s d e f i n e d as that q u a n t i t y of y e l l o w f e v e r v i r u s which, when i n o c u l a t e d i n t r a c e r e b r a l l y i n t o each of an adequate number

-5• of adult mice (16-20 grams) susceptible to yellow fever, w i l l k i l l 50% of the animals with a specific yellow-fever virus encephalitis within 21 days - Lethal Death Rate 50, or LD-50, The potency test on the finished vaccine i s considered a satisfactory identity t e s t . A safety test on the guinea-pig i s made on each l o t 01' finished vaccine i n order to determine the possible presence of organisms which did not grow i n culture, Rehydrated chick-embryo pulp from ampoules selected at random from the f i l l i n g i s used and a f t e r d i l u t i n g 1 i n 10 with d i s t i l l e d water 4-5 ml, are injected intraperitoneally into each of 2 or more normal guineapigs v;eighing 300-500 grams. The animals are observed f o r 7 or more days. If both of the animals show s i g n i f i c a n t reactions, i . e . elevation of temperature and/or c l i n i c a l illness^ the entire l o t i s regarded as unsatisfactory. I f one animal only shows s i g n i f i c a n t c l i n i c a l reaction, the test i s repeated, using three test animals. I f i n the repeated test more than one of the three animals show s i g n i f i c a n t reactions, the product i s unsatisfactory. The residual moisture 1 - content i s estimated on the contents ui at least x.vro ampoules by determining the loss i n weight incurred a f t e r heating -n a vacuum oven at 50 C for 30 minutes, using an anhydrone desiccant t r a p j i t i s expressed as a percentage. The moisture content o l the vaccine mvist bo less than 1%. A uniform technique of testing by a l l laboratories would be desirable, In my opinion, i t i s p a r t i c u l a r l y desirable that the medium used for suspending the virus should be standardized.., I use 105 normal orse serum i n s s l i n o , 4. Dr.._J. W.Field_( Institute _for_Medix:£l Jî_e ' ThFToiloivïng te"chnique"'is used at ijie""institute f o r Medical"Researc'h, Kuala Lampiir ; Two 20"-dose ampoules are rehydrated each i n one ml. of s t e r i l e d i s t i l l e d water. These are pooled and this i s considered undiluted vaccine. Tenfold dilutions are made i n 10 por cent, inactivated, normil rabbit serum i n 0.85 per c e j t saljne., ^ ^ i x 3--4.vreek old mice are inoculated each vàth 0-03 m l . of dilutions 10 , 10 , 10 ' > lo" , lo" , The mice are observed f o r 21 days^ deaths up to the fourth day being considered non-specific. Mice paralysed at the 21st day are considered as deaths. The 50 per cent end-point i s calculated by the method of Reed and Muench and the number of ID50 i n one dose of vaccine made accorï^ing to the instructions on the ampoule i s calculated^ This number of W50 per standard dose i s reported to the centres administering the vaccine. Less than 500 LD5Û per dose would be considered unsatisfactory f o r use. The adoption of a uniform technique i s favoured. 5. Dr. A . Gast Galvis : At f i r s t i t is advisable to secure the following inform'^tion s

(a) a l i s t of the e n t i t i e s er.gaged i n yellow-fever work and their addresses; (b) which of the above e n t i t i e s rvr.v'r''tv.re yellow-fever vaccine, which types of virus they use, and vitiich techniques they f o l l o w . Ir. The technique used at the "Carlos Finlay" Institute of Bogota f o r testing the a c t i v i t y of yello\\~fever vaccine consists i n t i t r a t i n g each batch and effecting sero-protection tests on a small percentage of the persons vaccinated vdth the batch.

-61.1 Titration. Determination of the number of mouse LD50 per unit of volume i n accordance with the Reed and Muench method, inoculating i n t r a c e r e b r a l l y groups of at least six mice with each d i l u t i o n . Each mouse i s given 0.03 m l . Ten i s used as the d i l u t i o n f a c t o r . Vgry often (vrtien the supply of mice permits) 12 mice are inoculated with each d i l u t i o n . The t i t r e s as mentioned i n 1.1.1 are obtained. V/hite, Swiss, Albino and Rockefeller strain mice are used, 40-100 days o l d . The mice are inoctilated as soon as the dilutions are ready. The diluent employed i s 0.9 per cent NaCl solution, with 10% normal human serum. T i t r a t i o n of chick embryo pulp immediately a f t e r extraction of tha embryos The pulp from each m i l l i s t i t r a t e d , T i t r a t i o n of the pool of tho various milling which are to form a batch of vaccine, immediately before sealing into containers and drying. As a r u l e , only one t i t r a t i o n io carried out, usually inoculating 12 mice w i t h each d i l u t i o n . T i t r a t i o n of the dried vaccine, rehydrated to i t s o r i g i n a l volume. A t least three ampoules from each batch are t i t r a t e d separately. When the supply of mice permits, each d i l u t i o n i s injected into a group of 12 m i c e . The f i n a l t i t r e i s considered to be the geometrical mean of the various t i t r e s obtained, the number of the doses being rounded o f f to the nearest ten when i t i s between 51 and 100; to the nearest hundred when i t i s between 51 and lOOj to the nearest thousand when i t i s between 1,001 and 10,000, and so on. Or - i n terms of logarithms : the logarithm of the doses i s rounded o f f to one place of decimals. Subseouent t i t r a t i o n of the vaccine a f t e r desiccation. This has not been carried out as a routine procedure on a l l batches, but a second t i t r a t i o n of the dried product of at least some of them has been made a month o r two months after désignation, T i t r a t i o n of the vaccine diluted f o r human use and maintained at 28°C. One out of each 50 batches i s selected at random; two ampoules are diluted to l / l O (as f o r human use). This l/lO d i l u t i o n i s t i t r a t e d and immediately afterwards divided into two parts, one of which i s stored a t 0 C. and the other at 28 C. They are then both t i t r a t e d simultaneously every 20 minutes f o r three hours, that i s , u n t i l nine t i t r e s have been obtained. Sero-protection tests i n vaccinated persons. Up to the present time blood samples f o r the purpose of making post-^vaccination sero-protection tests have been taken from only a very small nuiber of persons vaccinated with a few l o t s of vaccine. A register i s kept very c a r e f u l l y giving the name and address of each vaccinated person and indicating the batch of vaccine used. W e are i n favour of the adoption of a uniform technique f o r the t e s t i n e of dried vaccine. The following factors i n such uniformity may be considered as being i n l i n e with our own c r i t e r i a :

1.1.1 1.1.2

1.1.3

1.1.4

1.1.5

1.2

1.3 2*

-72.1 2o2 2.3 Use of mice of the same s t r a i n , age. and weight (Swiss, A l b i n o , R o c k e f e l l e r mice of 50-60 days o l d , average weight 16 g. each), Use of the same type of d i l u e n t ( s a l i n e s o l u t i o n w i t h 10 per cent normal human serum). Use of the same d i l u t i o n s and a f i x e d number of mice per d i l u t i o n i n titration. For example ; d i l u t i o n f a c t o r 4 and i n o c u l a t i o n of each d i l u t i o n i n s i x mice, o r , accordance w i t h our own method; d i l u t i o n f a c t o r 10, i n o c u l a t i o n of each d i l u t i o n i n 12 mice. T i t r a t i o n should be i n accordance w i t h paragraph 1.1 «3 • > P r e p a r a t i o n of the d i l u t i o n s f o r i n o c u l a t i o n to be completed w i t h i n a f i x e d i n t e r v a l a f t e r r e h y d r a t i o n of the product up to i t s o r i g i n a l volume, e i t h e r immediately afterwards or 20 minutes l a t e r . One batch to be s e l e c t e d at random from an e s t a b l i s h e d number of batches, the product to be d i l u t e d as f o r human use and the d i l u t i o n to be maintained at 28 C f o r throe hours ( t i t r a t e d every 20 minutes). I n d i c a t i o n s i n connection vdth other t e s t s of a c t i v i t y of vaccines s A r e g i s t e r of a l l v a c c i n a t e d persons to be kept, i n d i c a t i n g the name, age; address,and number of the batr^h used, f o r purposes of comparison w i t h cases of yelloiv f e v e r observed » I n zones where v a c c i n a t i o n of the p o p u l a t i o n i s to be c a r r i e d out, systematic taking of b l o o d samples before and a f t e r v a c c i n a t i o n f o r the purpose of s e r o - p r o t e c t i o n t e s t s so t h a t a t l e a s t 0,.2 per cent of the persons v a c c i n a t e d w i t h each batch of vaccine are subjected t o the s a i d test.

2.4

2.5

2.6 2.6.1

2.6.2

6.

Dr. J.H.S.Gear, The South A f r i c a n I n s t i t u t e f o r Medical Research, Johannesburg : li/e favour the adoption of a uniform technique so that r e s u l t s obtained by d i f f e r e n t I n s t i t u t e s may be s t r i c t l y comparable. The o n l y d e t a i l s not given i n the above B u l l e t i n (UNRRA) i s the method of t i t r a t i o n of v i r u s i n mice. This, of course^ has to bo done by i n t r a c e r e b r a l i n c u l a t i o n so that there i s not much v a r i a t i o n possible i n the dose v h i c h i s g e n e r a l l y 0«03 ml. per mouse. I t may be of value to l a y down that the d i l u t i o n s should be made i n 10;^ serum s a l i n e and that the d i l u t i o n s should be kept c o l d at say 5 C ^ . u n t i l immediately before inoculation. Dr. A.J.Haddow? as f o l l o w s f The technique used at the V i r u s Research I n s t i t u t e , Entebbe, i s

The vaccine i s rehydrated i n the ampoule w i t h s t e r i l e d i s t i l l e d water to the o r i g i n a l volume p r i o r to d e s i c c a t i o n , This suspension i s w e l l shaken and c a l l e d 10 or neat. g e r i a l 1 0 - f o l d d i l u t i o n s are now made i n 0«5^ bovine albumin i n s a l i n e up to l O " . The d i l u t i o n s are then immediately i n j e c t e d i n t r a c e r e b r a l l y i n t o mice, one group of 6 mice (28 days o l d on the day of i n o c u l a t i o n ) b e i n g used per d i l u t i o n . The inoculum being 0.03 ml. per mouse. The mice used hare are descended from a stock of Swiss mice o r i g i n a t i n g from the Carworth Farm, New York, The p e r i o d of observation of a l l i n o c u l a t e d mice i s 21 days. The i n f e c t i v i t y of the inoculum i s c a l c u l a t e d by the method of Reed and Muench (1938). Paralysed mice are counted as dead at the end of the p e r i o d of observation.

.-8I would favour the adoption of a uniform technique. I have no s t r o n g f e e l i n g s about vrtiich, so long as an adequate t i t r a t i o n i s i n v o l v e d . 8. Dr. F.N.Macnamara* I b e l i e v e t h a t , as f a r as p o s s i b l e , a uniform technique s h o u l d be followed by a l l approved y e l l o w - f e v e r t e s t i n g s t a t i o n s . There should, however be d i f f e r e n t techniques f o r mouse b r a i n m a t e r i a l and egg m a t e r i a l . Dr. A.F,Mahaffy, V i c t o r i a , B.C., Canada : Standard methods f o r assaying the potency of vaccines are most c e r t a i n l y d e s i r a b l e — perhaps i m p e r a t i v e , I suggest an i n f o r m a l meeting of a few s p e c i a l i s t s t o hammer out a set of methods vrtiich would be acceptable to a l l - or n e a r l y a l l . Dr. R.A.Penna : I am i n favour of the adoption of a uniform technique. The f o l l o w i n g t e s t s should be performed : (a) s t e r i l i t y t e s t ; (b) r e s i d u a l m o i s t u r e determination; ( c ) v i r u s potency t-^ct; (d) monkey t e s t ; (e) guinea p i g t e s t e The techniques f o r the execution of these t e s t s might be those d e s c r i b e d i n the UNRRA Standards. Dr. M.R.Pinto (Professeur de v i r o l o g i e . I n s t i t u t de Médecine t r o p i c a l e , L i s b o n n e ) : The d i r e c t o r s of the i n s t i t u t e s manufacturing yellow-fever vaccines s h o u l d f i r s t " " be consulted^ then a study should be made of the answers r e c e i v e d . Dr. K.C.Smithburn, The R o c k e f e l l e r Foundation, Johannesburg) : I am i n s t r o n g agreement w i t h the d e s i r a b i l i t y of a s t a n d a r d i z a t i o n of methods f o r the assay o f vaccines to be employed f o r the issue of i n t e r n a t i o n a l c e r t i f i c a t e s of v a c c i n a t i o n against yellov; f e v e r . I b e l i e v e very s t r o n g l y t h a t the method of assay s h o u l d be s p e c i f i e d i n considerable d e t a i l and high standards of potency r e q u i r e d . i would suggest, f u r t h e r , that any statement of methods to be employed should i n c l u d e a requirement that the t e s t s of potency be made a f t e r a p r e s c r i b e d p e r i o d , s a y , one week, during which the vaccine i s stored, without r e f r i g e r a t i o n - a procedure which would e l i m i n a t e i i r p r o p e r l y d r i e d vaccines but would not s e r i o u s l y a f f e c t the t i t r e of w e l l desiccated m a t e r i a l . I t might a l s o be advisable t o r e q u i r e submission of c e r t i f i e d p r o t o c o l s of t i t r a t i o n r e s u l t s f o r vaccines •which are t o be used f o r the i s s u e of i n t e r n a t i o n a l c e r t i f i c a t e s . Dr. F.l.Soper, D i r e c t o r , \7H0 Regional O f f i c e f o r the Americas : This matter merits considerable a t t e n t i o n . Th- standards e s t a b l i s h e d i n the past are v e r y l e n i e n t regarding the amount of moisture content of the vaccine, but are r a t h e r severe as to the p e r i o d of time during vnhich that vaccine can be considered satisfactory following i t s preparation. I s h a l l not undertake t o d i s c u s s t h i s p o i n t more than to c a l l a t t e n t i o n to the p o s s i b i l i t y of a v e r y dry vaccine l o s i n e a much smaller percentage of i t s potency during storage than one w i t h a l a r g e r amount of moisture, thus not only the length of time during ï r t i i c h a Taccine can be stored may be subject to i n c r e a s e , but a l s o may be a corresponding degree of d i l u t i o n a t the time the vaccine i s f i n a l l y a p p l i e d . Dr. N.H.Swellengrebel, K o n i n k l i j k I n s t i t u u t voor de Tropen, Amsterdam : The methods used i n the I n s t i t u t e of T r o p i c a l Hygiene and Geographical Pathology, Amsterdam are as f o l l o w s : , *

9.

10.

Hi

12.

13.

14.

-9Each f r e s h l y prepared batch of v a c c i n e i s d i v i d e d i n t o l o t s of 400 ampoules, which have been d r i e d simultaneously. The t i t r e of each l o t i s determined separately, u s i n g "Swiss white" mice aged from 35 t o 42 days. F i v e groups of s i x mice receive .an i n t r a c e r e b r a l i n j e c t i o n of 0.02 ml. of a d i l u t i o n of 1 i n 1024, 1 i n 4096, 1 i n 16,384, 1 i n 65,536, and 1 i n 262,144 r e s p e c t i v e l y . The mice are kept under observation f o r 21 days. Those which succumb before the 10th day are discarded. The t i t r e i s determined by Reed and Muench's method according to the number of mice dying between the 10th and 20th days w i t h t y p i c a l symptoms of paralysis. A uniform technique to be employed a l l over the w o r l d i s the i d e a l at which to aim. Of course, everyone p r e f e r s h i s own way, but I assure you that t h i s I n s t i t u t e i s prepared to observe any r u l e w i t h i n reason which WHO might l a y dowi. 15• Dr. R.N.Taylor, Department of V i r o l o g y , U.S.Naval Medical Research U n i t No. 3, c/g American Embassy, C a i r o : I t would seem advisable that each l a b o r a t o r y , which has been designated as a " c o n t r o l l a b o r a t o r y " , submits the exact procedures which i t employs f o r assaying potency of vaccines. lïith t h i s i n f o r m a t i o n at hand, the members of the Expert Comnlttee on Yellow Fever, or p r e f e r a b l y the d i r e c t o r s of the r e p r e s e n t a t i v e l a b o r a t o r i e s , would be i n a b e t t e r p o s i t i o n t o decide upon the standard method which i t would be agreeable, as w e l l as e f f e c t i v e , f o r a l l l a b o r a t o r i e s engaged i n c o n t r o l l i n g vaccines to f o l l o w ,

16. Dr. P. iifagle. D i r e c t o r , Haffkine I n s t i t u t e , Bombay : The techniqtie of the . , . potency t e s t of y e l l o w - f e v e r vaccine on mice i s described below : ( i ) Take a s e r i e s of t e s t tubes, and put i n t o each 1.8 c c . of 10^ normal horse serum (pooled) i n s a l i n e . ( i i ) Take one ampoule of y e l l o w - f e v e r vaccine c o n t a i n i n g e i t h e r 1 c c . or 5 c c . of d r i e d m a t e r i a l and rehydrate i t w i t h 1 c c . or 5 c c . p h y s i o l o g i c a l s a l t s o l u t i o n , as the case may be. ( i i i ) Take 0.2 c c . of the rehydrated vaccine and add i t t o the f i r s t t e s t tube, drawing the contents i n and out of the p i p e t t e 3 times. Then thoroughly mix by shaking the tube f o r 60 seconds. ( i v ) Transfer 0,2 c c . from the f i r s t t e s t tube to the second t e s t tube, and mix as above. (v) Thus make t e n - f o l d d i l u t i o n s lO"''', t o lo''* or l O " ^ . ( v i ) Inoculate i n t r a c e r e b r a l l y under ether anaesthesia 0.03 ml. i n t o each mouse, using 8 mice f o r each d i l u t i o n . Mice i n o c u l a t i o n should be • • completed as q u i c k l y as p o s s i b l e , and i n any case w i t h i n 30 minutes of .\ , , „ making d i l u t i o n s . • : A t u b e r c u l i n syringe w i t h 0.03 c c , graduations i s used f o r t h i s piu»pose. Needle used i s 27 gauge, 1/4" l o n g . Swiss mice, 3-4 weeks o l d , 16-18 g. i n weight, are to be used i n t h i s test. At the end of the l a s t d i l u t i o n , a l l mice i n o c u l a t e d are examined, . • and i f deaths due to trauma are n o t i c e d , the dead mice are replaced by f r e s h l y i n o c u l a t e d ones.

'

.• . - -

-lO( v i i ) Observe mice f o r 21 days. Ignore a i l deaths o c c u r r i n g w i t h i n 7 days atfter i n o c u l a t i o n . Observe mice at a p a r t i c u l a r time every day, and maintain records showing P f o r p a r a l y s i s and D f o r death. ( v i i i ) A f t e r the p e r i o d of observation i s over, determine potency by c a l c u l a t i n g 50% m o r t a l i t y end-point by the method of need and Muench. I am i n favour of the general adoption of the above technique. 17. D r . F.G.Morgan : With regard to the method of t e s t i n g the a c t i v i t y of Y e l l o w Fever Vaccine, wo desire to suggest that i t might be d e s i r a b l e to s p e c i f y i n more d e t a i l the method of t e s t i n g potency of the f i n i s h e d v a c c i n e , p a r t i c u l a r l y i n regard to the d i l u e n t used i n preparing s t e r i l e s o l u t i o n s of the v a c c i n e f o r i n o c u l a t i o n i n t o s u s c e p t i b l e mice. Many l a b o r a t o r i e s , i t i s understood, use normal human serum a t a strength of 10$ i n normal s a l i n e f o r t h i s p u r p o s e . A t these l a b o r a t o r i e s we are commonly unable to check on the o r i g i n of serum u s e d , and i t i s suspected that some specimens may c o n t a i n of serum used, and i t i s suspected that some specimens may c o n t a i n antibodies to Dengue Fever and p o s s i b l y to Yellow Fever, as a r e s u l t i n the l a t t e r case of war-time i n o c \ i l a t i o n . Our experience of other mammalian sera i s l i m i t e d , but we would suggest c o n s i d e r a t i o n of the d e s i r a b i l i t y f o r u s i n g albumin as the p r o t e i n present i n the diluent. Thd albumin might p r e f e r a b l y be c r y s t a l l i n e bovine albiunin. 18. D r . Gast G a l v i s : No change. I t should be c l e a r , however, that t h i s f i n a l t i t r e must be the geometrical average of the various t i t r e s o b t a i r e d , the dose f i g u r e to be e s t a b l i s h e d as f o l l o w s : T i t r a t i o n of the d r i e d v a c c i n e , rehydrated t o i t s o r i g i n a l volume. A t l e a s t three ampoul3s from each batch are t i t r a t e d separately. «ftien the supply of mice permits, each d i l u t i o n i s i n j e c t e d i n t o a group of 12 mice. The f i n a l t i t r e i s considered to be the geometrical mean o f the various t i t r e s obtained, the number of the doses being rounded o f f t o the nearest ten vrtien i t i s between 51 and 100; to the nearest 100 when i t i s between 101 and 1000, e t c . D r . F.N.Macnamara : The o b j e c t of the f i r s t t i t r a t i o n i s presumably t o show t h a t the vaccine grows adequately i n the embryo and does not l o s e i t s potency on desiccation. The standard i s meaningless unless the amount of d i s t i l l e d water added to each embryo b e f o r e homogenisation and the average weight of the embryos are s t a t e d No standard i s given as to the number of ampoules of each batch or l o t t o be t e s t e d . I suggest that 4 ampoules of each batch should be t i t r a t e d i n d i v i d u ally. I f one or more of the f o u r f a i l s to reach the standard, s i x more ampoules should be t e s t e d . I f three or more of the 10 ampoules have t i t r e s of 500-5000 LD50 per human dose or two or more ampoules have t i t r e s l e s s than 500 LD50 p e r human dose, the whole batch should be condemned.

19.

^Reed, L . H . Sc Muench H . "A s i n ç l e method of e s t i m a t i n g 50$ e n d - p o i n t s " . J . H y g . , 1938, 27, 493-

Amer

-11(Hore note i n t r o d u c t i o n of-new paragraph No. 6) 6« 1. Potency Requirements of the F i n i s h e d Vaccine . .

Health Laboratory Methods S a c t i o n s I f the potency t e s t i s given i t s own paragraph;' the potency requirements of vaccine used i n human v a c c i n a t i o n w i l l s t i l l need to be s e t out. I t i s t h e r e f o r e suggested t h a t a new paragraph 6 be made and that t h i s be e n t i t l e d '"Potency Requirements of the F i n i s h e d Vaccine" Dr. Durieux b e l i e v e s t h a t UNRRA standards f o r the manufacture and c o n t r o l of y e l l o w - f e v e r vaccines a p p l i e d subcutaneously are s a t i s f a c t o r y , but t h a t the minim^tJi dose of 500 LD50 l a i d down f o r vaccines when a p p l i e d by s c a r i f i c a t i o n i s insufficient. As only p a r t of the dose i s absorbed by s c a r i f i c a t i o n , i t i s e s s e n t i a l t h a t i t should c o n t a i n more v i r u s . The minimum i n d i v i d u a l dose should be 5000 LD50 i n t h i s case. Dr. J .li¥.Field : The standards are g e n e r a l l y s a t i s f a c t o r y although an a d d i t i o n a l t e s t of potency based on tlie a b i l i t y of 500 LD50 of the vaccine to produce n e u t i c l l z i n g a n t i b o d i e s i n non-immune humans i n say every tenth batch would be an a d d i t i o n a l safeguard. Dr._F. N.Mac namara • : The immunization dose of 500 LD50 does not, i n my o p i n i o n , give a~sûfficTênt margin of s a f e t y y&i^.u the standard e r r o r of t i t r a t i o n s and d e t e r i o r a t i o n on storage are considered. Not l e s s than.5000 LD50 should he the standard . . . c. •J.parate standards w i l l have to be devised f o r vaccine administered by s c a r i f i c a t i o n « Since the d e s i c c a t e d m a t e r i a l i s u s u a l l y rehydrated i n l / l O the v o l m e of s a l i n e used f o r subcutaneous injection^, i t i s suggested that the r e c o n s t i t u t e d vaccine should c o n t a i n 10 times as much as the former. I t i s recommended that a l l manufacturers r e f e r t o the potency of t h e i r vaccines i n forms of mouse LD50 p e r "raman dose» I f other t i t r e s are given i t should be e x p r e s s l y s t a t e d t h a t such t i t r e s do not r e f e r t o the f i n i s h e d product.

2.

3.

4.

5*

Dr. H.A.Penna : Proposed amendment ; Replace the f u l l - s t o p a f t e r " t e s t " by a comma, and continue "or i f to be used i n a f i n a l d i l u t i o n of 1.100 s h a l l not c o n t a i n l e s s than 200,000 ID50 a t the moment of being used." Dr. F.G.Morgan s I t has been the p r a c t i c e t o assay samples from s t o c k s of vaccine and when there i s evidence of d e t e r i o r a t i o n of potency t o amend the d i l u t i o n or h y d r a t i o n of the f i n i s h e d d r i e d product t o ensure that the s p e c i f i e d number of mouse LD50 i s injected.Such a p r a c t i c e r e s u l t s i n a greater amount of chick p r o t e i n being i n j e c t e d . Advice i s asked as to vAiether t h i s i s a d e s i r a b l e p r a c t i c e and whether i t i s d e s i r a b l e to f i x a r a t i o of the amount of t o t a l n i t r o g e n p e r LD50 of vaccine. Health Laboratory Methods S e c t i o n 5 I f the vaccine d e t e r i o r a t e s by storage i n the l a b o r a t o r y of tSe manufacturer,"with the r e s u l t that the l a s t potency t e s t made by him gives l e s s than 150,000 Mouse LD50 of rehydrated o r i g i n a l volume, the vaccine s h a l l be discarded. That must be tha c o n c l u s i o n which i s drawn from the i n s t r u c t i o n s of the present UNRRA standard-.

6.

7.

-12(Note new number of next and subsequent paragraphs) 7. 1. Guinea P i g Safety Test

Dr. F.N.Macnamara : The o b j e c t of t h i s t e s t i s presumably to detect v i r a l or b a c t e r i o l o g i c a l contaminants not demonstrable i n other t e s t s . For egg v a c c i n e i t s use appears to be of doubtful value and I doubt that i t i s adequate f o r mouse b r a i n vaccine. To be of any value the animals should be observed f o r 14 days a t least. The s u s c e p t i b i l i t y of guinea p i g s to yellow fever i s so v a r i a b l e t h a t the t e s t can give l i t t l e information on the quîility of the vaccine per se. 8. S t e r i l i t y of the Vaccine

1,

Dr. F.N.Macnamara : Recommendations f o r the type of c u l t u r e media to be used should be made. Incubation at 37 C should be both aerobic and anaerobic. The period of i n c u b a t i o n of the c u l t u r e s should be s t a t e d . 9. I d e n t i t y Test • ...

No comments. 10. 1. Labeling

Dr. F.N.Macnamara : A d d i t i o n a l i n s t r u c t i o n s may be necessary i f the vaccine i s to be given by s c a r i f i c a t i o n . I t i s recommended t h a t a l l manufacturers r e f e r t o the potency of t h e i r vaccines i n terms of mouse LD50 per human dose. I f other t i t r e s are given i t should be e x p r e s s l y s t a t e d t h a t such t i t r e s do not r e f e r to the f i n i s h e d and r e c o n s t i t u t e d product.

2»

Dr. P. Wagle : The standards are adequate except that the date of manufacture as w e l l as the e x p i r a t i o n date should be shown on the l a b e l of the y e l l o w - f e v e r vaccine c o n t a i n e r . Date of manufacture has considerable importance f o r a centre l i k e ours, where we have s t o r e d a bulk supply of y e l l o w - f e v e r vaccine and i r r e s p e c t i v e of the e x p i r a t i o n date, have been using i t f o r years and years, t i l l the potency f e l l below the r e q u i r e d l e v e l , as found by the mouse potency t e s t c a r r i e d out on e v e r y l o t from time to time. The vaccine has maintained i t s potency remarkably w e l l ,

34. Health Laboratory Methods S e c t i o n : According to the UNRRA Standard the date of i s s u e precedes the date of e x p i r a t i o n by one year and the date of manufacture must f a l l w i t h i n the year foregoing the date of i s s u e . From the v a c c i n a t o r ' s p o i n t of view the l o t nimiber and the e x p i r a t i o n date seem t o give s u f f i c i e n t information. I t i s proposed above t h a t the concentration of chick embryo j\iice of the human dose must not exceed B% and t h a t the number of mouse 1D50 per human dose s h a l l be s t a t e d on the l a b e l . I f these amendments are agreed upon there w i l l be no need f o r (h) and ( i ) .

-1311. 1. E x p i r a t i o n Date

Dr. Swellengrebel b e l i e v e s t h a t the r u l e s t a t i n g that the vaccine may be stored f o r a year at 5 C. i n an o r d i n a r y r e f r i g e r a t o r o f f e r s unwarrantable r i s k s of deterioration. A vaccine w i t h a comparatively low i n i t i a l t i t r e may even become inactivated. He i n c l u d e s w i t h a l l ampoules d i r e c t i o n s t h a t the vaccine r e t a i n s i t s a n t i g e n i c powers for- s e v e r a l months i f s t o r e d i n the f r e e z i n g compartment of a r e f r i g e r a t o r , i n which the temperature remains w e l l below 0°C. 12. A d m i n i s t r a t i o n of the Vaccine

l"

Dr. Gast G a l v i s : In other words, the UNRRA standards demand that the 1/10 d i l u t i o n of the d r i e d product s h a l l c o n t a i n at l e a s t 15,000 mouse Ln50 per ml., and that the person vaccinated s h a l l r e c e i v e at l e a s t 7,500 mouse LD50 c a l c u l a t e d on the b a s i s of the f i n a l t i t r e of t h a d r i e d product and without t a k i n g i n t o account the l o s s of v i r u s vhich occurs i n the i n t e r v a l between f i n a l t i t r a t i o n and a d m i n i s t r a t i o n of the vaccine. I n view of the f a c t t h a t experience so f a r has shovm t h a t imnunity i s inducod by i n j e c t i o n of 500 mouse LD50, t h i s ample margin of s e c u r i t y - 15 times the necessary minimum dose - v/as e s t a b l i s h e d p r e c i s e l y i n .order to compensate the l o s s of v i r u s which occurs i n the i n t e r v a l betv/een f i n a l t i t r a t i o n and a d m i n i s t r a t i o n of the vaccine. Moreover, i t i s considered t h a t the vaccine when i t i s administered to man must be d i l u t e d to l / l O (UNRRA) because t h i s d i l u t i o n g i v e s , f o r human use, such a concentration of p r o t e i n s t h a t the p r e s e r v a t i o n of the v i r u s during the i n o c u l a t i o n operations i s guaranteed. As we understand i t , t h i s l/lO d i l u t i o n i s made i n view of the f a c t t h a t the d r i e d product has bean prepared w i t h embryos to which - according t o the UNRRA technique (Hargett- e t c o l . , l o c . c i t o ) 1 ml. of water has beon added per 3 g. of embryo. Therefore, the embryo j u i c e represents 75^ by weight of the f i n a l d r i e d product. Hargett e t c o l . , however, i n the a r t i c l e r e f e r r e d t o , state t h a t w i t h high t i t r e extracts' (200,000 or more but without mentioning the volume) i t i s s u f f i c i e n t to put h a l f of the s p e c i f i e d q u a n t i t i e s i n the ampoules. I f the UNRRA s t i p u l a t i o n s are a p p l i e d to the l e t t e r , the h a l f q u a n t i t i e s of vaccine i n the ampoules must be d i l u t e d , f o r human use, i n the r a t i o of 10 ml, of d i l u e n t t o 1 ml,, of d r i e d vaccine. I t has a l s o been adequately demonstrated that vaccines prepared according to the technique r e f e r r e d t o by Smith can, once the l/lQ d i l u t i o n of the d r i e d product has been made, be administered to man up to three hours from the moment when the d i l u t i o n i s prsparud. These r e s u l t s prove t h a t the di'xed product can be d i l u t e d f o r human use i n such a way that the d i l u t i o n contains a minimum of 15,000 mouse LD50 per ml. (on the b a s i s of the f i n a l t i t r e of the d r i e d product), and A% to 3% (by volume) of embryo j u i c e , and that t h i s d i l u t i o n can be administ e r e d to man up to 180 minuses from the time of p r e p a r a t i o n . On the b a s i s of -he f i n a l t i t r e of the product, the d i l u t i o n of the d r i e d vaccine f o r human use must be such as to c o n t a i n a minimum of 15,000 mouse LD50 per ml. and a f i n a l 4% to 8^ concentration of embryo j u i c e , embryo j u i c e t o be understood as the supern-itant f l u i d r e s u l t i n g from c e n t r i f u g i n g o f the m i l l e d embryos without any a d d i t i o n of water, and t a k i n g i n t o account the d i l u t i o n factor only. As mentioned i n an e a r l i e r p a r t of t h i s manuscript, the d r i e d vaccine prepared according t o t h a proposed technique, v d t h the a d d i t i o n of 0.25 g. of water per 1 g. of embryoa gives an 80^ concentration of embryo j u i c e ;

-14consequently the d i l u t i o n of the d r i e d product, f o r use i n man, v d l l be from 1/lQ to 1/20 according t o the t i t r e . The d i l u e n t used f o r t h i s d i l u t i o n v d l l be s t e r i l e normal s a l i n e and the immunizing dose i n man w i l l be 0.5 ml. a d m i n i s t e r e d subcutaneously and w i t h i n three hours from the moment o f p r e p a r a t i o n of the dilution. 2. Dr. F.N.Macnamara : I f the manufacturer places s u f f i c i e n t vaccine i n the ampoules to give on r e c o n s t i t u t i o n an adequate dosage, I do not t h i n k t h a t i t matters whether i t i s i n the p r o p o r t i o n of 1 m l , of d r i e d vaccine to 10 m l . d i l u e n t or not provided t h a t the p r o p o r t i o n i s not greater than one t o t e n ,

3. Dr. H.A.Penna : I f vaccine i s intended t o be used i n a f i n a l d i l u t i o n of 1:100, a second 1:10 d i l u t i o n i s prepared by adding 1 ml. of the f i r s t d i l u t i o n t o 9 ml. of d i l u e n t . The f i r s t d i l u t i o n may be kept a t ice-box temperature (5 C.) or lower f o r not more than three hours. The f i n a l d i l u t i o n s h a l l be administered at once. , , ; . ., , , , 4. Health Laboratory Methods S e c t i o n : The f i n a l potency t e s t of the f r e e z e - d r i e d vaccine gives i n f o r m a t i o n on the amount of chick embryo j u i c e vidiich w i l l c o n s t i t u t e one human dose. Each manufacturer can e a s i l y c o n t r o l how great a margin of potency t h i s product needs i n order f o r i t t o have a s u f f i c i e n t potency on the day of e x p i r a t i o n . I t i s reasonable, t h e r e f o r e , t o give t h e manufacturer the p o s s i b i l i t y to v a r y the amount of d i l u e n t needed from v a c c i n e l o t t o vaccine l o t . He s h a l l s t a t e on the l a b e l the amount o f mouse LD50 p e r human dose which i s obtained when a c e r t a i n amount o f d i l u e n t i s added. I t h i n k t h a t the proposed amendment gives a good s o l u t i o n of t h a t question. 13» Shipping Requirements •-

;v t v e •

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-

"ic "^ j^'"''"" *

No comments.

14,

General Requirements , •-:

' '. , ' i': c' jf , rj '' -^i^.^.-. -

.;

,. .

1:- No comments, 15.

Requirements f o r Release

No comments.

•

' DIRECTIONS FOR PRESERVATION OF SESD VIRUS 1, P r o f e s s o r J . Tréfouë'l, D i r e c t o r , Pasteur I n s t i t u t e , P a r i s : As the N a t i o n a l I n s t i t u t e s of Health, Bethesda, d e p o s i t o r y of the 17D s t r a i n , keep and d i s t r i b u t e secondary seed v i r u s i n the non-dried f r o z e n s t a t e , i t might be opportune t o amend t h i s p r o v i s i o n i n the UNRRA standards vitiich reads "The p u l p i s anpouled, d r i e d and s t o r e d " . Health Laboratory Methods S e c t i o n : The question of the s t o r i n g and d i s t r i b u t i of secondary seed v i r u s must be very c a r e f u l l y s t u d i e d . The main problem i s t o maintain o r improve the immunizing c a p a c i t y of the seed v i r u s , which means t h a t a f u r t h e r l o s s of v i s c e r o t r o p i c a f f i n i t y and increase i n neuro-tropism must be avoided. There are a l s o other problems i n t h i s f i e l d which need f u r t h e r

2.

-15investigation. I t has been mentioned b y many o f the Panel members t h a t there i s a great d e a l of u n c e r t a i n t y concerning the l e v e l of potency of the f r e s h l y produced vaccine u^iich i s needed i n order to give a s u f f i c i e n t immunizing e f f e c t at the moment of i n j e c t i o n . The r e l a t i o n between t h e o r i g i n a l potency of the vaccine and the potency a t the moment of i n j e c t i n g depends c h i e f l y on the e f f e c t i v i t y of the f r e e z e - d r y i n g process and upon the temperature a t which the vaccine i s s t o r e d . At i t s 9 t h meeting h e l d 10 - 15 October 1955 the Expert Committee on B i o l o g i c a l S t a n d a r d i z a t i o n d i s c u s s e d a report by P r o f e s s o r P. Lépine of the I n s t i t u t Pasteur, P a r i s , on the need f o r c h a r a c t e r i z a t i o n of seed l o t s of the 17D s t r a i n of y e l l o w f e v e r v i r u s , used i n the production of y e l l o w f e v e r v a c c i n e , and agreed t h a t the matter should be r e f e r r e d t o the Expert Committee on Yellow Fever. The Committee f u r t h e r decided t o c a l l the a t t e n t i o n of the World Health O r g a n i z a t i o n t o the urgency of the problem and t o recommend t h a t a c t i o n should be taken as soon as p o s s i b l e . The members of the Yellow Fever Advisory Panel are asked t o consider the questions r a i s e d and other questions which might be r e l e v a n t . The S e c t i o n of Health Laboratory Methods would v e r y much appreciate r e c e i v i n g suggestions on the best moans of t a c k l i n g these problems.