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Immunogenicity of hepatitis B vaccine in guinea-pigs, mice and man
Journal of Biological Standardization (1985) 13, 2 2 1 - 2 2 7
Immunogenicity of hepatitis B vaccine in g u i n e a - p i g s , m i c e a n d m a n * Marie-Christine Mazert, t Liliane Rabier, t Marc Girardt and Alain Goudeau~
T h e i m m u n o g e n i c i t y o f the h e p a t i t i s B vaccine Hevac B Pasteur was assayed in g u i n e a - p i g s . T h e r e p r o d u c i b i l i t y o f the test was d e t e r m i n e d by c o m p a r i n g the ImDso oft-~o samples of vaccine in 12 and e i g h t g r o u p s o f 4 0 animals each. T h e variation coefficient between g r o u p s was a p p r o x i m a t e l y 3 0 % , s i m i l a r to t h a t found for other vaccines assayed in biological tests in vivo. The s e n s i t i v i t y o f the test was d e t e r m i n e d by c o m p a r i n g the dose response curves in mice, g u i n e a - p i g s and h u m a n s . T h e results s h o w e d t h a t one I m D s o in m a n (alter one injection) was a p p r o x i m a t e l y equivalent to 3 ImDso in g u i n e a pigs and to 100 l m D s 0 in Balb/C mice. S t a b i l i t y o f Hevac B at 37°C was assayed by d e t e r m i n i n g in guinea=pigs the ImDs0 of a series o f samples o f vaccine t h a t h a d been i n c u b a t e d at 37°C for varying a m o u n t s o f time. T h e i m m u n o g e n i c i t y o f t h e vaccine was f o u n d to decay w i t h a half-life o f t e n to 15 days at 37°C.
INTRODUCTION Following t he first e x p e r i m e n t a l H e p a t i t i s B vaccines c o n t a i n i n g purified H B s A g , 1.2 the Hevac B Pasteur Vaccine ( I n s t i t u t Pasteur P r o d u c t i o n , France) was the first vaccine available on the m a r k e t , c o n t a i n i n g a 9 5 - 9 9 % pure preparation o f H B s A g , treated u l t i m a t e l y w i t h formalin. 3.4 T h e i m m u n o g e n i c i t y o f H e v a c B has been tested in m u l t i p l e clinical trials in France, 5-7 Africa s a nd A s i a ? Usually, from 90 to 9 5 % o f the vaccinees developed a long-tasting a n t i - H B s antibodies response after three injections of 5 gLg H B s A g each, given at zero, one a nd t w o m o n t h s , respectively, a n d followed by a booster injection at 12 m o n t h s . H o w e v e r , the need to c o m p a r e the i m m u n o g e n i c i t y of successive vaccine * Received for p u b l i c a t i o n 28 A u g u s t 1984. t I n s t i t u t Pasteur P r o d u c t i o n , 3, I3
~ ) 1985 The International Associatioa of Biological gtandardization
221
/~f.-C. M A Z E R T ET AL. b a t c h p r e p a r a t i o n a n d to assess t h e b a t c h to b a t c h c o n s i s t e n c y o f t h e p r o d u c t i o n process, as well as t h e necessity to f o l l o w t h e effect o f a g e i n g o n t h e p o t e n c y o f t h e vaccine a n d ro be able to c o n t r o l t h e v a l i d i t y o f a g e d b a t c h e s o f vaccine, call for a s u i t a b l e l a b o r a t o r y test. F u r t h e r m o r e , t h e fact t h a t t h e r e c o m m e n d e d d o s e o f I q B s A g r e q u i r e d for v a c c i n a t i o n o f h u m a n s a g a i n s t h e p a t i t i s B varies f r o m a p p r o x i m a t e l y o n e to sixfold f r o m o n e m a n u f a c t u r e r to a n o t h e r , s u g g e s t s t h a t t h e s a m e a m o u n t o f a n t i g e n c o u l d e x h i b i t q u i t e d i f f e r e n t levels o f i m m u n o g e n i c i r y , p e r h a p s as a c o n s e q u e n c e o f tLe d i f f e r e n t e x t e n t o f d e n a t u r a t i o n o f t h e a n t i g e n in t h e d i f f e r e n t c o m m e r c i a l p r e p a r a t i o n s o f vaccine. T h i s in t u r n , w o u l d i m p l y t h a t t h e a m o u n t o f a n t i g e n in a g i v e n vaccine p r e p a r a t i o n c a n n o t be t a k e n as an a c c u r a t e m e a s u r e o f its efficacy. F o r t h e s e reasons, it was felt to be o f i n t e r e s t to set u p a n a n i m a l test to evaluate t h e i m m u n o g e n i c i t y o f t h e H e v a c lff vaccine. I n t h e p r e s e n t s t u d y we i n v e s t i g a t e d t h e r e p r o d u c i b i l i t y a n d s e n s i t i v i t y o f a p o t e n c y t e s t in g u i n e a - p i g s a n d t h e d o s e response c u r v e in m a n , m i c e a n d g u i n e a - p i g s . T h e g u i n e a - p i g s y s t e m w~s t h e n used to s t u d y t h e s t a b i l i t y o f t h e v a c c i n e d u r i n g accelerated a g e i n g at 370(2. MATERIALS
AND
METHODS
Immunogenicity tests in the animal Commercial samples o f t h e H e v a c B P a s t e u r vaccine c o n t a i n e d 5 / z g o f H B s A g a d s o r b e d o n t o 1 m g o f A I ( O H ) 3 in a v o l u m e o f 1 m l . P o t e n c y tests w e r e carried o u t a c c o r d i n g to t h e W H O r e q u i r e m e n t s for h e p a t i t i s B vaccine, lo L a b o r a t o r y a n i m a l s u s e d were 300-350 g, male guinea-pigs (HartIey strain, randomized, ten guinea-pigs per g r o u p ) . G u i n e a - p i g s w e r e i n j e c t e d by t h e s u b - c u t a n e o u s r o u t e w i t h u n d i l u t e d a n d serial d i l u t i o n s o f t h e v a c c i n e in 0 " 9 % N a C l . A n i m a l s w e r e b l e d after 28 days a n d their sera w e r e a n a l y s e d i n d i v i d u a l l y for a n t i - H B s A g a n t i b o d i e s . A n t i b o d y t i t r e s w e r e d e t e r m i n e d by r a d i o i m m u n o a s s a y ( R I A ) u s i n g t h e A u s a b k i t ( A b b o t t ) . M e a n t i t r e s w e r e e x p r e s s e d as g e o m e t r i c m e a n s o f t h e titres o f t h e a n i m a l s in each g r o u p . I m m u n o g e n i c doses 5 0 ( I m D s o ) w e r e t a k e n as t h e a m o u n t o f H B s A g r e q u i r e d for o b t a i n i n g a p o s i t i v e a n t i b o d y response in 5 0 % o f t h e a n i m a l s i n j e c t e d , as c a l c u l a t e d by l o g i t t r a n s f o r m a t i o n . 11 A p o s i t i v e r e s p o n s e was d e f i n e d as a titre o f at least 50 R I A units. Immunogenicity results wen compared with those obtained with one batch of H e v a c B v a c c i n e u s e d t h r o u g h o u t as a reference s t a n d a r d a n d t h e R e l a t i v e P o t e n c y ( R P ) was c a l c u l a t e d . Experimentalsamples w e r e p r e p a r e d c o n t a i n i n g e i t h e r 5, 1- 25 or 0 - 3 1 2 / ~ g o f H B s A g , all a d s o r b e d o n t o 1 m g o f A l ( O H ) s in a v o l u m e c~f I m l . G u i n e a - p i g s were i n j e c t e d by t h e s u b c u t a n e o u s r o u t e w i t h 1 m l o f each ~accine. M i c e w e r e i n j e c t e d by t h e i n t r a p e r i t o n e a l r o u t e , u s i n g serial d i l u t i o n s o f v a c c i n e in 1 m g / m l o f A I ( O H ) 3 . T h e mice used were six-week-old females (Balb/C strain, randomized, 20 mice per group). I m m u n o g e n i c i t y r e s u l t s w e r e c o m p a r e d w i t h t h o s e o b t a i n e d in m a n . A p o s i t i v e r e s p o n s e was d e f i n e d as a t i t r e o f at least 50 R I A u n i t s . Immunogenicity in man T h r e e g r o u p s o f 8 0 y o u n g a d u l t s w e r e i n j e c t e d by t h e s u b c u t a n e o u s r o u t e w i t h 1 m l o f v a c c i n e c o n t a i n i n g e i t h e r 5, 1-25 o r 0 - 3 1 2 / z g o f H B s A g . I n j e c t i o n s w e r e at days O, 30 a n d 6 0 . A n t i b o d y t i t r e s w e r e d e t e r m i n e d at days 3 0 , 6 0 a n d 9 0 by ILIA, u s i n g t h e A u s a b k i t ( A b b o t 0 . T i t r e s w e r e e x p r e s s e d i n I n t e r n a t i o n a l U n i t s (IU) by comparison with the International Reference Preparation of Hepatitis B Immunog l o b u l i n s . A p o s i t i v e a n t i b o d y r e s p o n s e was d e f i n e d as a t i t r e o f at least 3 m l U / m L 222
I M M U N O G E N I C I T Y OF H E V A C B RESULTS
Reproducibility of poteucy test in guinea-pigs T h e I m D s o o f t w o b a t c h e s o f H e v a c B v a c c i n e w a s d e t e r m i n e d b y 12 a n d e i g h t s u c c e s s i v e assays, u s i n g 4 0 g u i n e a p i g s p e r assay ( T a b l e 1). T h e I m D s o w a s c o m p u t e d for e a c h a s s a y as t h e a m o u n t o f H b s A g ( / z g ) s u f f i c i e n t t o i n d u c e s e r o c o n v e r s i o n in 5 0 % o f t h e i n j e c t e d a n i m a l s . S t a n d a r d d e v i a t i o n s o f m e a n I m D s 0 w e r e 0 " 9 0 a n d 0 - 7 7 /~g f o r batch A and batch B, respectively. Coefficients of variation between individual assays w e r e c o m p u t e d . T h e f i g u r e s o b t a i n e : l w e r e 3 0 % for b a t c h A a n d 3 5 % for b a t c h B . These figures are quite comparable to those obtained in the laboratory when performing r o u t i n e p o t e n c y t e s t o n o t h e r v a c c i n e s s u c h as r a b i e s or t e t a n u s v a c c i n e s . T h e R P o f b a t c h B , in t e r m s o f b a t c h A t a k e n as a r e f e r e n c e , w a s 1 - 2 5 ( a v e r a g e o f e i g h t assays) w i t h an a v e r a g e S D o f 0 " 3 2 . T h e c o n f i d e n c e l i m i t s ( P = 0 " 9 5 ) o f t h e R P a r e i n d i c a t e d in T a b l e 1. T h e c o e f f i c i e n t o f v a r i a t i o n b e t w e e n t h e i n d i v i d u a l d e t e r m i n a t i o n s o f t h e R P o f batch B was 26%. This observation implies that ImDsso should always be determined in a c o m p a r a t i v e f a s h i o n . Batch to batch consistency and the RPs of successive vaccine batches were determ i n e d o n s a m p l e s f r o m 12 s u c c e s s i v e p r o d u c t i o n b a t c h e s ( T a b l e 2). T h e a v e r a g e R P o f t h e 12 b a t c h e s w a s 1-3 in r e l a t i o n t o a r e f e r e n c e ~-accine p r e p a r a t i o n , t h e S D w a s 0 - 3 6 and the variation coefficient 27%. Analysis of the data revealed that most of the v a r i a t i o n s o b s e r v e d w e r e d u e to s t a t i s t i c a l f l u c t u a t i o n in t h e a s s a y s ( p e r h a p s i n r e l a t i o n to the animals) and not to lack of batch-to-batch consistency. A great part of the v a r i a t i o n s in t h e I m D s o v a l u e s w a s n o t r e f l e c t e d in v a r i a t i o n o f t h e R P - T h i s f u r t h e r strengthens the conclusion that ImDsso should always be determined comparatively. TABLE I. R e p r o d u c i b i l i t y o f p o t e n c y test in guinea-pigs: the ImD~0 o f two batches o f H e v a c B vaccine (A and B) was assayed as described in Materials and M e t h o d s using ten g u i n e a - p i g s p e r dilution I m D s o (/zg) Assay no. 1
Batch A 4-2
Relative p o t e n c y o f b a t c h B ~*
Batch B --
2 3 4 5 6 7 8 9 10 11 12
3"5 2"5 4-3 1-8 3-8 3" I 3-5 2-4 1-6 2-2 2-9
-a . . 2-5 3-8 2-4 2-2 1-9 1-3 1-6 1-7
Average
3"0
2- 2
N .
. 0-7 1-0 1-3 1-6 1-2 1-2 1-4 1-7
(0-4-1-3) (0-5-2.0) (0-8-2-0) (0-9-3-1) (0-5--3"7) (0-5--3-9) (0-7--3-(3) (1-0-3-0) l- 25
*~Figures in parentheses indicate the confidence limits (P -----0-95) o f the RP data. 223
I~i.-C. MAZERT ET AL,. TABLE 2. Batch-to-batch consistency of potenty test: the ImDso of 12 successive vaccine batches was measured and compared to that of a reference vaccine as described in the legend o f Table 1
Batch no.
ImDso (/~g)
2 3 4 5 6 7 8 9 10 11 12 13
1-4 1"7 3"5 2"9 3"5 3"6 1"9 2-2 3-8 2-6 1-6 3-3
Average
2"7
Relative porency*'~ 1-8 (1-2--2-5) ND~: 1-2 (0-7-2.2) 1-2 (0-6-2"5) 1-25 ( 0 - 9 - t - 8 ) 1-2 ( 0 - 7 - 1 . 8 ) 1-0 (0-6-1-7) 1-25 ( 1 - 0 - t - 5 ) 1-0 (0-6-1-7) 1-2 (0-7-2-0) 2-2 (1-3--4-5) 1-2 (0-7-2-0) 1- 3
"* The SD of average relative potency was 0-36. ~" Figures in parentheses indicate the confidence limits (P -----0-95) of the RP data. :[: 1MD -----Not detezmined.
Comparisou of dose-response effects iu guinea-pigs, mice a n d m~o T h r e e e x p e r i m e n t a l v a c c i n e p r e p a r a t i o n s c o n t a i n i n g 0 " 3 1 2 , 1-25 a n d 5 / z g H B s A g were injected into three groups of ten guinea-pigs, and into three groups of 80 a n t i - H B s A g - n e g a t i v e y o u n g m e n . T h e i n j e c t i o n s w e r e r e p e a t e d t w i c e at m o n t h l y i n t e r v a / s i n t h e m e n . P a r t o f t h e v a c c i n e was d i l u t e d w i t h a s u s p e n s i o n o f I m g / m l o f Al(OH)3 and samples containing 0-109, 0-078 and 0-312/a.g HBsAg, were injected into three groups of 20 mice each. A n t i - l - I B s A g a n t i b o d i e s w e r e m e a s u r e d 2 8 d a y s l a t e r in all t h e a n i m a l s , a n d o n e m o n t h after e a c h o f t h e successive i n j e c t i o n s in t h e m e n . R e s u l t s w e r e e x p r e s s e d as p e r c e n t a g e s o f s e r o c o n v e r s i o n (Fig. 1) o r a s a v e r a g e a n t i - H B s A g a n t i b o d y t i t r e s (Fig. 2). I n t h e case o f t h e m i c e , s o m e a n t i b o d y t i t r e s w e r e h i g h e r t h a n t h e c u t - o f f for s e r o c o n v e r s i o n b u t c o u l d n o t b e d e t e r m i n e d a c c u r a t e l y in v i e w o f i n s u f f i c i e n t s e r u m r e c o v e r y . T h e real level o f a n t i - H B s A g a n t i b o d i e s in m i c e m u s t , t h e r e f o r e , be h i g h e r t h a n i n d i c a t e d . A c u t - o f f f o r s e r o c o n v e r s i o n o f 3 m l U was u s e d i n m a n , as c o m p a r e d to 5 0 R I A u n i t s ( a p p r o x i m a t e l y 8 m I U ) ' i n g u i n e a - p i g s a n d m i c e , as is u s u a l in clinical s t u d i e s . 6-8 T h e r e s u l t s in F i g . 1 s h o w t h a t t h e i m m u n o g e n i c i t y o f H B s A g i n g u i n e a - p i g s was a p p r o x i m a t e l y t h r e e t i m e s h i g h e r t h a n in m a n (after o n e i n j e c t i o n ) a n d 100 t i m e s l o w e r t h a n i n m i c e . T h i s is i n c o n t r a d i c t i o n w i t h t h e r e p o r t t h a t g u i n e a - p i g s r e s p o n d e d r e a d i l y t o l o w d o s e s o f H B s A g a n d c o u l d n o t b e u s e d f o r . p o t e n c y tests. 12
Stability of H B s A g immuuogenicity Samples~ o f o n e v a c c i n e b a t c h w e r e s t o r e d at 3 7 ° C f o r s e v e n , 14 a n d 2 8 d a y s , after w h i c h t h e y w e r e assayed for r e l a t i v e p o t e n c y in g u i n e a p i g s by c o m p a r i s o n w i t h s a m p l e s
224
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Fi 8 . 1. P e r c e n t a g e s e r o c o n v e r s i o n in g u i n e a - p i g s a n d m i c e after one i n j e c t i o n o f w c c i n e a n d in m a n after one, t w o o r t h r e e i n j e c t i o n s . × , M i c e , o n e i n j e c t i o n ; A , g u i n e a - p i g s , o n e i n j e c t i o n ; e , m a n , one injection; o , m a n , ~wo i n j e c t i o n s ; • m a n , t h r e e i n j e c t i o n s .
o f t h e s a m e b a t c h o f v a c c i n e s t o r e d at 2 - 8 ° C . T h e results s h o w e d t h a t t h e immunogenicity of the samples decreased regularly with time. The slope of the curve relating l o g o f R P t o t i m e (in days) at 3 7 ° C was - - 0 - 0 2 , c o r r e s p o n d i n g to a half-life o f a b o u t 15 d a y s (Fig. 3). T h e e x p e r i m e n t was r e p e a t e d w i t h t w o o t h e r b a t c h e s o f vaccine: t h e slopes o f t h e curves o f i m m u n o g e n i c i t y d e c a y at 3 7 ° C w e r e - - 0 - 0 3 a n d - - 0 - 0 2 , c o r r e s p o n d i n g to half-lives o f t e n a n d 15 days, r e s p e c t i v e l y (results n o t s h o w n ) . T h e a v e r a g e R P o f t h e three b a t c h e s o f v a c c i n e w e r e 0 - 5 + 0- 12, 0 - 3 4- 0 - 2 a n d 0 - 2 + 0 - 1 6 after seven, 14 t0oo
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F i g . 2. M e a n a n t i - H B s A g a n t i b o d y t i t r e s in g u i n e a - p i g s a n d m i c e after one i n j e c t i o n o f vaccine a n d in m a n a f t e r o n e , t w o a n d t h r e e i n j e c t i o n s . X , M i c e , o n e i n j e c t i o n ; Ak, g u i n e a - p i g s , one inject/on; e , m a n , o n e i n j e c t i o n ; o , m a n , t w o i n j e c t i o n s ; A , m a n , t h r e e i n j e c t i o n s . M e a n a n t i - H B s A g t i t r e s w e r e expressed as m / U / m l for m a n o r r a d i o i m m u n o a s s a y u n i t s / m l for g u i n e a - p i g s a n d m i c e .
225
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10 20 Length o f storage at 37¢'C (days)
~50
Fig. 3. Decay ofimmunogenicity at 37°C. A batch o f H e v a c B vaccine was i n c u b a t e d at 37°C a n d the I m D s 0 was d e t e r m i n e d i n guinea-pigs at each o f the i n d i c a t e d ' t i m e s a n d c o m p a r e d to that of the same vaccine k e p t at zi°C.
and 28 days at 37°(=, respectively. F u r t h e r data is presently being collected in order to obtain better statistical precision. W e have also followed the t i m e course of i m m u n o g e n i c i t y decay o f the vaccine at 20°C. Pr e l i m i na r y results indicate that the Hevac 13 vaccine is very stable d u r i n g prolonged storage at this t e m p e r a t u r e (non-observable loss o f potency after four m o n t h s storage).
DISCUSSION Comparisons o f close-response effects of Hevac B in guinea-pigs, mice and m e n show that gui ne a - pi gs are a suitable m o d e l to evaluate t h e i m m u n o g e n i c i t y for hepatitis B vaccine. T h u s , the close o f H e v a c B required for 50% seroconversion in guinea-pigs, at 28 days after injection, was approximately one-third o f that required in man. T h e same dose, w h e n injected three times, at m o n t h l y intervais, in m a n , achieved m o r e than 9 5 % seroconversion. R e p r o d u c i b i l i t y o f the pot e nc y test in g u in ea-p ig s was fairly good, as judged from the fact t h a t the coefficient o f variation b etween e i g h t successive determinations of the relative potency o f t w o vaccine batches was 269~, using ten guinea-pigs per d i l u t i o n of each vaccine. T h e relative pot e n cy o f 12 successive product!on batches o f vaccine showed a variation coefficient of 2 7 % , indicative o f good batch to batch consistency. T h e p o t e n c y test in guinea-pigs was used to measure the stability o f the vaccine under conditions of accelerated a g e i n g at 37°(=. I m m u n o g e n i c i t y o f the vaccine was found to decay exponentially w i t h a half-life o f t e n to 15 days at 37°(=. T h e vaccine appears to be very stable at 20°C, as j u d g e d from p r e l i m i n a r y results obtained in guinea-pigs. Such a h i g h stability is c o n f i r m e d by the fact that vaccine samples-that were kept for up to four years at 4°(: are still fully i m m u n o g e n i c in guinea-pigs and m a n (unpublished
results). As seen in F/'g. 1, t he a m o u n t o f H b s A g required to obtain 5 0 % sere,conversion in m a n after one i nj e c t i on was approximately three times t h a t required in guinea-pigs an d 226
t M M U N O G E N I C I T Y OF HEVAC B 100 t i m e s t h a t r e q u i r e d in B a l b / C m i c e . I n v i e w o f these results, m a n a p p e a r e d to be m u c h less s e n s i t i v e t h a n g u i n e a - p i g s or m i c e . H o w e v e r , o n c e c o r r e c t e d for b o d y w e i g h t , t h e s e r e s u l t s l o o k t o t a l l y d i f f e r e n t a n d m a n a p p e a r s ro be 6 0 t i m e s a n d 3 0 t i m e s m o r e sensitive t h a n guinea-pigs a n d m i c e , r e s p e c t i v e l y . It is d i f f i c u l t to stress t h i s c o m p a r i s o n m u c h f u r t h e r in v i e w o f t h e d i f f e r e n t s l o p e s o f t h e d o s e - r e s p o n s e c u r v e s in t h e t w o a n i m a l s y s t e m s a n d in m a n . T h e l o w e r slope o f t h e d o s e - r e s p o n s e c u r v e in m a n c o u l d reflect g r e a t e r h e t e r o g e n e i t y o f t h e i m m u n e responsiveness of man compared with that of guinea-pigs or mice. The high slope of the d o s e - r e s p o n s e c u r v e in g u i n e a - p i g s is h o w e v e r o f g r e a t practical a d v a n t a g e as it gives m o r e accuracy to e x p e r i m e n t a l d e t e r m i n a t i o n o f I m D s s o .
Acknowledgements W e wish to t h a n k J. Flori and J. Arondel for skillful technical assistance. REFERENCES I. Barin F, Goudeau A, Coursaget, P, Maupas P. Large scale purification of Hepatitis B surface antigen (HBsAg). A n n Microbiol (Institut Pasteur) 1978: 129B, 87. 2. Mau1~,,s P, G o u d e a u A, Dubois F, Coursaget P, Barin F. Potency and efficacy of l i B vaccine applied to a h i g h risk population, a five year study. In: Maup~s P, Gue~ry P, eds. Hepatitis B vaccine. Inserum S y m p o s i u m No. 18. Elsevier/North Holland Biomedical Pres s, 1981. 3. Adamowicz P, Gerfaux G, Platel A, Muller L, Vacher B, Mazert M-C, Pruner P (eds 1981). Large scale productions of an Hepatitis B vaccine. In: Maupas P, Guesry P, eds. Hepatitis B vaccine. Inserum S y m p o s i u m No. 18. Elsevier/North Holland Biomedical Press, 1981. 4. Adamowicz P, Chabanier G, Hyafil F, Lucas G, Pruner P, Reculard P, Vinas R. Elimination o f serum i~roteins and potential virus contaminants d u r i n g Hepatitis B vaccine preparation. Vaccine, 1984; 2: 2 0 9 - 2 1 4 . 5. Crosnier J, J u n g e r s P, c o u r o u c e A-M, Laplanche A, Benharnou E, Degos F, Lacour B, Pruner P, Cerisier Y, Guesry P. ~Randomised placebo-controlled trial of Hepatitis B surface antigen vaccine in French haemodialysis units. Lancet i: 1981; 455--459. 6. Goudeau A, Dubois F, Barin F, Dubois M-C, Coursaget P. Hepatitis B vaccine: clinical trials in high risk settings in France. Devel Biol Stand 1983; 54: 2 6 7 - 2 8 4 . 7. Benhamou E, Courouce A-M, Jungers P, Laplanche A, Degos F, Brangier J, CrosnierJ. Hepatitis B vaccine: randomised trial o f i m m u n o g e n i c i t y in haemodialysis patients. Clin Nephrol 1984; 21: 143--147. 8. Coursagct P, C h i r o n J - P , Barin F, Goudeau A, Yvonnet B, Denis F, Lorrea P, N'Doye R, Diop-Mar I. Hepatitis B vacine: i m m u n i s a t i o n o f children and'newborns in a endemic area (Senegal). Devel 13/ol Stand 1983; 54: 2 4 5 - 2 5 7 . 9. Goudeau A, Lo K-J, Coursaget P, T o n g M-J, Yeh C-L, Tsai Y-T, Lee J - K , W u T-C, Yeh P-S-H, Lee S-D. Prevention o f Hepatitis B virus infection in children born to active and passive active i m m u n i z a t i o n . Devel Biol Stand 1983; 54: 3 9 9 - 4 0 4 . 10. W H O Requirements for Hepatitis B vaccine. W H O Tech Rep Set 1981; 658:731--156. 11. Finney D-J. Statistical M e t h o d in Biological Assay. 2nd edition. London: Charles Griffin, 1971. 12. Ge.rety R-J, Tabor E, Purcell R - H , Tyeryar F-J. Summary o f an international workshop on Hepatitis B vaccines. J Infect Dis 1979; 140: 6 4 2 - 6 4 8 .
227
Immunogenicity of hepatitis B vaccine in g u i n e a - p i g s , m i c e a n d m a n * Marie-Christine Mazert, t Liliane Rabier, t Marc Girardt and Alain Goudeau~
T h e i m m u n o g e n i c i t y o f the h e p a t i t i s B vaccine Hevac B Pasteur was assayed in g u i n e a - p i g s . T h e r e p r o d u c i b i l i t y o f the test was d e t e r m i n e d by c o m p a r i n g the ImDso oft-~o samples of vaccine in 12 and e i g h t g r o u p s o f 4 0 animals each. T h e variation coefficient between g r o u p s was a p p r o x i m a t e l y 3 0 % , s i m i l a r to t h a t found for other vaccines assayed in biological tests in vivo. The s e n s i t i v i t y o f the test was d e t e r m i n e d by c o m p a r i n g the dose response curves in mice, g u i n e a - p i g s and h u m a n s . T h e results s h o w e d t h a t one I m D s o in m a n (alter one injection) was a p p r o x i m a t e l y equivalent to 3 ImDso in g u i n e a pigs and to 100 l m D s 0 in Balb/C mice. S t a b i l i t y o f Hevac B at 37°C was assayed by d e t e r m i n i n g in guinea=pigs the ImDs0 of a series o f samples o f vaccine t h a t h a d been i n c u b a t e d at 37°C for varying a m o u n t s o f time. T h e i m m u n o g e n i c i t y o f t h e vaccine was f o u n d to decay w i t h a half-life o f t e n to 15 days at 37°C.
INTRODUCTION Following t he first e x p e r i m e n t a l H e p a t i t i s B vaccines c o n t a i n i n g purified H B s A g , 1.2 the Hevac B Pasteur Vaccine ( I n s t i t u t Pasteur P r o d u c t i o n , France) was the first vaccine available on the m a r k e t , c o n t a i n i n g a 9 5 - 9 9 % pure preparation o f H B s A g , treated u l t i m a t e l y w i t h formalin. 3.4 T h e i m m u n o g e n i c i t y o f H e v a c B has been tested in m u l t i p l e clinical trials in France, 5-7 Africa s a nd A s i a ? Usually, from 90 to 9 5 % o f the vaccinees developed a long-tasting a n t i - H B s antibodies response after three injections of 5 gLg H B s A g each, given at zero, one a nd t w o m o n t h s , respectively, a n d followed by a booster injection at 12 m o n t h s . H o w e v e r , the need to c o m p a r e the i m m u n o g e n i c i t y of successive vaccine * Received for p u b l i c a t i o n 28 A u g u s t 1984. t I n s t i t u t Pasteur P r o d u c t i o n , 3, I3
~ ) 1985 The International Associatioa of Biological gtandardization
221
/~f.-C. M A Z E R T ET AL. b a t c h p r e p a r a t i o n a n d to assess t h e b a t c h to b a t c h c o n s i s t e n c y o f t h e p r o d u c t i o n process, as well as t h e necessity to f o l l o w t h e effect o f a g e i n g o n t h e p o t e n c y o f t h e vaccine a n d ro be able to c o n t r o l t h e v a l i d i t y o f a g e d b a t c h e s o f vaccine, call for a s u i t a b l e l a b o r a t o r y test. F u r t h e r m o r e , t h e fact t h a t t h e r e c o m m e n d e d d o s e o f I q B s A g r e q u i r e d for v a c c i n a t i o n o f h u m a n s a g a i n s t h e p a t i t i s B varies f r o m a p p r o x i m a t e l y o n e to sixfold f r o m o n e m a n u f a c t u r e r to a n o t h e r , s u g g e s t s t h a t t h e s a m e a m o u n t o f a n t i g e n c o u l d e x h i b i t q u i t e d i f f e r e n t levels o f i m m u n o g e n i c i r y , p e r h a p s as a c o n s e q u e n c e o f tLe d i f f e r e n t e x t e n t o f d e n a t u r a t i o n o f t h e a n t i g e n in t h e d i f f e r e n t c o m m e r c i a l p r e p a r a t i o n s o f vaccine. T h i s in t u r n , w o u l d i m p l y t h a t t h e a m o u n t o f a n t i g e n in a g i v e n vaccine p r e p a r a t i o n c a n n o t be t a k e n as an a c c u r a t e m e a s u r e o f its efficacy. F o r t h e s e reasons, it was felt to be o f i n t e r e s t to set u p a n a n i m a l test to evaluate t h e i m m u n o g e n i c i t y o f t h e H e v a c lff vaccine. I n t h e p r e s e n t s t u d y we i n v e s t i g a t e d t h e r e p r o d u c i b i l i t y a n d s e n s i t i v i t y o f a p o t e n c y t e s t in g u i n e a - p i g s a n d t h e d o s e response c u r v e in m a n , m i c e a n d g u i n e a - p i g s . T h e g u i n e a - p i g s y s t e m w~s t h e n used to s t u d y t h e s t a b i l i t y o f t h e v a c c i n e d u r i n g accelerated a g e i n g at 370(2. MATERIALS
AND
METHODS
Immunogenicity tests in the animal Commercial samples o f t h e H e v a c B P a s t e u r vaccine c o n t a i n e d 5 / z g o f H B s A g a d s o r b e d o n t o 1 m g o f A I ( O H ) 3 in a v o l u m e o f 1 m l . P o t e n c y tests w e r e carried o u t a c c o r d i n g to t h e W H O r e q u i r e m e n t s for h e p a t i t i s B vaccine, lo L a b o r a t o r y a n i m a l s u s e d were 300-350 g, male guinea-pigs (HartIey strain, randomized, ten guinea-pigs per g r o u p ) . G u i n e a - p i g s w e r e i n j e c t e d by t h e s u b - c u t a n e o u s r o u t e w i t h u n d i l u t e d a n d serial d i l u t i o n s o f t h e v a c c i n e in 0 " 9 % N a C l . A n i m a l s w e r e b l e d after 28 days a n d their sera w e r e a n a l y s e d i n d i v i d u a l l y for a n t i - H B s A g a n t i b o d i e s . A n t i b o d y t i t r e s w e r e d e t e r m i n e d by r a d i o i m m u n o a s s a y ( R I A ) u s i n g t h e A u s a b k i t ( A b b o t t ) . M e a n t i t r e s w e r e e x p r e s s e d as g e o m e t r i c m e a n s o f t h e titres o f t h e a n i m a l s in each g r o u p . I m m u n o g e n i c doses 5 0 ( I m D s o ) w e r e t a k e n as t h e a m o u n t o f H B s A g r e q u i r e d for o b t a i n i n g a p o s i t i v e a n t i b o d y response in 5 0 % o f t h e a n i m a l s i n j e c t e d , as c a l c u l a t e d by l o g i t t r a n s f o r m a t i o n . 11 A p o s i t i v e r e s p o n s e was d e f i n e d as a titre o f at least 50 R I A units. Immunogenicity results wen compared with those obtained with one batch of H e v a c B v a c c i n e u s e d t h r o u g h o u t as a reference s t a n d a r d a n d t h e R e l a t i v e P o t e n c y ( R P ) was c a l c u l a t e d . Experimentalsamples w e r e p r e p a r e d c o n t a i n i n g e i t h e r 5, 1- 25 or 0 - 3 1 2 / ~ g o f H B s A g , all a d s o r b e d o n t o 1 m g o f A l ( O H ) s in a v o l u m e c~f I m l . G u i n e a - p i g s were i n j e c t e d by t h e s u b c u t a n e o u s r o u t e w i t h 1 m l o f each ~accine. M i c e w e r e i n j e c t e d by t h e i n t r a p e r i t o n e a l r o u t e , u s i n g serial d i l u t i o n s o f v a c c i n e in 1 m g / m l o f A I ( O H ) 3 . T h e mice used were six-week-old females (Balb/C strain, randomized, 20 mice per group). I m m u n o g e n i c i t y r e s u l t s w e r e c o m p a r e d w i t h t h o s e o b t a i n e d in m a n . A p o s i t i v e r e s p o n s e was d e f i n e d as a t i t r e o f at least 50 R I A u n i t s . Immunogenicity in man T h r e e g r o u p s o f 8 0 y o u n g a d u l t s w e r e i n j e c t e d by t h e s u b c u t a n e o u s r o u t e w i t h 1 m l o f v a c c i n e c o n t a i n i n g e i t h e r 5, 1-25 o r 0 - 3 1 2 / z g o f H B s A g . I n j e c t i o n s w e r e at days O, 30 a n d 6 0 . A n t i b o d y t i t r e s w e r e d e t e r m i n e d at days 3 0 , 6 0 a n d 9 0 by ILIA, u s i n g t h e A u s a b k i t ( A b b o t 0 . T i t r e s w e r e e x p r e s s e d i n I n t e r n a t i o n a l U n i t s (IU) by comparison with the International Reference Preparation of Hepatitis B Immunog l o b u l i n s . A p o s i t i v e a n t i b o d y r e s p o n s e was d e f i n e d as a t i t r e o f at least 3 m l U / m L 222
I M M U N O G E N I C I T Y OF H E V A C B RESULTS
Reproducibility of poteucy test in guinea-pigs T h e I m D s o o f t w o b a t c h e s o f H e v a c B v a c c i n e w a s d e t e r m i n e d b y 12 a n d e i g h t s u c c e s s i v e assays, u s i n g 4 0 g u i n e a p i g s p e r assay ( T a b l e 1). T h e I m D s o w a s c o m p u t e d for e a c h a s s a y as t h e a m o u n t o f H b s A g ( / z g ) s u f f i c i e n t t o i n d u c e s e r o c o n v e r s i o n in 5 0 % o f t h e i n j e c t e d a n i m a l s . S t a n d a r d d e v i a t i o n s o f m e a n I m D s 0 w e r e 0 " 9 0 a n d 0 - 7 7 /~g f o r batch A and batch B, respectively. Coefficients of variation between individual assays w e r e c o m p u t e d . T h e f i g u r e s o b t a i n e : l w e r e 3 0 % for b a t c h A a n d 3 5 % for b a t c h B . These figures are quite comparable to those obtained in the laboratory when performing r o u t i n e p o t e n c y t e s t o n o t h e r v a c c i n e s s u c h as r a b i e s or t e t a n u s v a c c i n e s . T h e R P o f b a t c h B , in t e r m s o f b a t c h A t a k e n as a r e f e r e n c e , w a s 1 - 2 5 ( a v e r a g e o f e i g h t assays) w i t h an a v e r a g e S D o f 0 " 3 2 . T h e c o n f i d e n c e l i m i t s ( P = 0 " 9 5 ) o f t h e R P a r e i n d i c a t e d in T a b l e 1. T h e c o e f f i c i e n t o f v a r i a t i o n b e t w e e n t h e i n d i v i d u a l d e t e r m i n a t i o n s o f t h e R P o f batch B was 26%. This observation implies that ImDsso should always be determined in a c o m p a r a t i v e f a s h i o n . Batch to batch consistency and the RPs of successive vaccine batches were determ i n e d o n s a m p l e s f r o m 12 s u c c e s s i v e p r o d u c t i o n b a t c h e s ( T a b l e 2). T h e a v e r a g e R P o f t h e 12 b a t c h e s w a s 1-3 in r e l a t i o n t o a r e f e r e n c e ~-accine p r e p a r a t i o n , t h e S D w a s 0 - 3 6 and the variation coefficient 27%. Analysis of the data revealed that most of the v a r i a t i o n s o b s e r v e d w e r e d u e to s t a t i s t i c a l f l u c t u a t i o n in t h e a s s a y s ( p e r h a p s i n r e l a t i o n to the animals) and not to lack of batch-to-batch consistency. A great part of the v a r i a t i o n s in t h e I m D s o v a l u e s w a s n o t r e f l e c t e d in v a r i a t i o n o f t h e R P - T h i s f u r t h e r strengthens the conclusion that ImDsso should always be determined comparatively. TABLE I. R e p r o d u c i b i l i t y o f p o t e n c y test in guinea-pigs: the ImD~0 o f two batches o f H e v a c B vaccine (A and B) was assayed as described in Materials and M e t h o d s using ten g u i n e a - p i g s p e r dilution I m D s o (/zg) Assay no. 1
Batch A 4-2
Relative p o t e n c y o f b a t c h B ~*
Batch B --
2 3 4 5 6 7 8 9 10 11 12
3"5 2"5 4-3 1-8 3-8 3" I 3-5 2-4 1-6 2-2 2-9
-a . . 2-5 3-8 2-4 2-2 1-9 1-3 1-6 1-7
Average
3"0
2- 2
N .
. 0-7 1-0 1-3 1-6 1-2 1-2 1-4 1-7
(0-4-1-3) (0-5-2.0) (0-8-2-0) (0-9-3-1) (0-5--3"7) (0-5--3-9) (0-7--3-(3) (1-0-3-0) l- 25
*~Figures in parentheses indicate the confidence limits (P -----0-95) o f the RP data. 223
I~i.-C. MAZERT ET AL,. TABLE 2. Batch-to-batch consistency of potenty test: the ImDso of 12 successive vaccine batches was measured and compared to that of a reference vaccine as described in the legend o f Table 1
Batch no.
ImDso (/~g)
2 3 4 5 6 7 8 9 10 11 12 13
1-4 1"7 3"5 2"9 3"5 3"6 1"9 2-2 3-8 2-6 1-6 3-3
Average
2"7
Relative porency*'~ 1-8 (1-2--2-5) ND~: 1-2 (0-7-2.2) 1-2 (0-6-2"5) 1-25 ( 0 - 9 - t - 8 ) 1-2 ( 0 - 7 - 1 . 8 ) 1-0 (0-6-1-7) 1-25 ( 1 - 0 - t - 5 ) 1-0 (0-6-1-7) 1-2 (0-7-2-0) 2-2 (1-3--4-5) 1-2 (0-7-2-0) 1- 3
"* The SD of average relative potency was 0-36. ~" Figures in parentheses indicate the confidence limits (P -----0-95) of the RP data. :[: 1MD -----Not detezmined.
Comparisou of dose-response effects iu guinea-pigs, mice a n d m~o T h r e e e x p e r i m e n t a l v a c c i n e p r e p a r a t i o n s c o n t a i n i n g 0 " 3 1 2 , 1-25 a n d 5 / z g H B s A g were injected into three groups of ten guinea-pigs, and into three groups of 80 a n t i - H B s A g - n e g a t i v e y o u n g m e n . T h e i n j e c t i o n s w e r e r e p e a t e d t w i c e at m o n t h l y i n t e r v a / s i n t h e m e n . P a r t o f t h e v a c c i n e was d i l u t e d w i t h a s u s p e n s i o n o f I m g / m l o f Al(OH)3 and samples containing 0-109, 0-078 and 0-312/a.g HBsAg, were injected into three groups of 20 mice each. A n t i - l - I B s A g a n t i b o d i e s w e r e m e a s u r e d 2 8 d a y s l a t e r in all t h e a n i m a l s , a n d o n e m o n t h after e a c h o f t h e successive i n j e c t i o n s in t h e m e n . R e s u l t s w e r e e x p r e s s e d as p e r c e n t a g e s o f s e r o c o n v e r s i o n (Fig. 1) o r a s a v e r a g e a n t i - H B s A g a n t i b o d y t i t r e s (Fig. 2). I n t h e case o f t h e m i c e , s o m e a n t i b o d y t i t r e s w e r e h i g h e r t h a n t h e c u t - o f f for s e r o c o n v e r s i o n b u t c o u l d n o t b e d e t e r m i n e d a c c u r a t e l y in v i e w o f i n s u f f i c i e n t s e r u m r e c o v e r y . T h e real level o f a n t i - H B s A g a n t i b o d i e s in m i c e m u s t , t h e r e f o r e , be h i g h e r t h a n i n d i c a t e d . A c u t - o f f f o r s e r o c o n v e r s i o n o f 3 m l U was u s e d i n m a n , as c o m p a r e d to 5 0 R I A u n i t s ( a p p r o x i m a t e l y 8 m I U ) ' i n g u i n e a - p i g s a n d m i c e , as is u s u a l in clinical s t u d i e s . 6-8 T h e r e s u l t s in F i g . 1 s h o w t h a t t h e i m m u n o g e n i c i t y o f H B s A g i n g u i n e a - p i g s was a p p r o x i m a t e l y t h r e e t i m e s h i g h e r t h a n in m a n (after o n e i n j e c t i o n ) a n d 100 t i m e s l o w e r t h a n i n m i c e . T h i s is i n c o n t r a d i c t i o n w i t h t h e r e p o r t t h a t g u i n e a - p i g s r e s p o n d e d r e a d i l y t o l o w d o s e s o f H B s A g a n d c o u l d n o t b e u s e d f o r . p o t e n c y tests. 12
Stability of H B s A g immuuogenicity Samples~ o f o n e v a c c i n e b a t c h w e r e s t o r e d at 3 7 ° C f o r s e v e n , 14 a n d 2 8 d a y s , after w h i c h t h e y w e r e assayed for r e l a t i v e p o t e n c y in g u i n e a p i g s by c o m p a r i s o n w i t h s a m p l e s
224
IMMUNOGENICITY
OF "HEVAC B
991 i 90
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Fi 8 . 1. P e r c e n t a g e s e r o c o n v e r s i o n in g u i n e a - p i g s a n d m i c e after one i n j e c t i o n o f w c c i n e a n d in m a n after one, t w o o r t h r e e i n j e c t i o n s . × , M i c e , o n e i n j e c t i o n ; A , g u i n e a - p i g s , o n e i n j e c t i o n ; e , m a n , one injection; o , m a n , ~wo i n j e c t i o n s ; • m a n , t h r e e i n j e c t i o n s .
o f t h e s a m e b a t c h o f v a c c i n e s t o r e d at 2 - 8 ° C . T h e results s h o w e d t h a t t h e immunogenicity of the samples decreased regularly with time. The slope of the curve relating l o g o f R P t o t i m e (in days) at 3 7 ° C was - - 0 - 0 2 , c o r r e s p o n d i n g to a half-life o f a b o u t 15 d a y s (Fig. 3). T h e e x p e r i m e n t was r e p e a t e d w i t h t w o o t h e r b a t c h e s o f vaccine: t h e slopes o f t h e curves o f i m m u n o g e n i c i t y d e c a y at 3 7 ° C w e r e - - 0 - 0 3 a n d - - 0 - 0 2 , c o r r e s p o n d i n g to half-lives o f t e n a n d 15 days, r e s p e c t i v e l y (results n o t s h o w n ) . T h e a v e r a g e R P o f t h e three b a t c h e s o f v a c c i n e w e r e 0 - 5 + 0- 12, 0 - 3 4- 0 - 2 a n d 0 - 2 + 0 - 1 6 after seven, 14 t0oo
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F i g . 2. M e a n a n t i - H B s A g a n t i b o d y t i t r e s in g u i n e a - p i g s a n d m i c e after one i n j e c t i o n o f vaccine a n d in m a n a f t e r o n e , t w o a n d t h r e e i n j e c t i o n s . X , M i c e , o n e i n j e c t i o n ; Ak, g u i n e a - p i g s , one inject/on; e , m a n , o n e i n j e c t i o n ; o , m a n , t w o i n j e c t i o n s ; A , m a n , t h r e e i n j e c t i o n s . M e a n a n t i - H B s A g t i t r e s w e r e expressed as m / U / m l for m a n o r r a d i o i m m u n o a s s a y u n i t s / m l for g u i n e a - p i g s a n d m i c e .
225
bI.-C. MAZERT
liT A L .
\ t/2
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10 20 Length o f storage at 37¢'C (days)
~50
Fig. 3. Decay ofimmunogenicity at 37°C. A batch o f H e v a c B vaccine was i n c u b a t e d at 37°C a n d the I m D s 0 was d e t e r m i n e d i n guinea-pigs at each o f the i n d i c a t e d ' t i m e s a n d c o m p a r e d to that of the same vaccine k e p t at zi°C.
and 28 days at 37°(=, respectively. F u r t h e r data is presently being collected in order to obtain better statistical precision. W e have also followed the t i m e course of i m m u n o g e n i c i t y decay o f the vaccine at 20°C. Pr e l i m i na r y results indicate that the Hevac 13 vaccine is very stable d u r i n g prolonged storage at this t e m p e r a t u r e (non-observable loss o f potency after four m o n t h s storage).
DISCUSSION Comparisons o f close-response effects of Hevac B in guinea-pigs, mice and m e n show that gui ne a - pi gs are a suitable m o d e l to evaluate t h e i m m u n o g e n i c i t y for hepatitis B vaccine. T h u s , the close o f H e v a c B required for 50% seroconversion in guinea-pigs, at 28 days after injection, was approximately one-third o f that required in man. T h e same dose, w h e n injected three times, at m o n t h l y intervais, in m a n , achieved m o r e than 9 5 % seroconversion. R e p r o d u c i b i l i t y o f the pot e nc y test in g u in ea-p ig s was fairly good, as judged from the fact t h a t the coefficient o f variation b etween e i g h t successive determinations of the relative potency o f t w o vaccine batches was 269~, using ten guinea-pigs per d i l u t i o n of each vaccine. T h e relative pot e n cy o f 12 successive product!on batches o f vaccine showed a variation coefficient of 2 7 % , indicative o f good batch to batch consistency. T h e p o t e n c y test in guinea-pigs was used to measure the stability o f the vaccine under conditions of accelerated a g e i n g at 37°(=. I m m u n o g e n i c i t y o f the vaccine was found to decay exponentially w i t h a half-life o f t e n to 15 days at 37°(=. T h e vaccine appears to be very stable at 20°C, as j u d g e d from p r e l i m i n a r y results obtained in guinea-pigs. Such a h i g h stability is c o n f i r m e d by the fact that vaccine samples-that were kept for up to four years at 4°(: are still fully i m m u n o g e n i c in guinea-pigs and m a n (unpublished
results). As seen in F/'g. 1, t he a m o u n t o f H b s A g required to obtain 5 0 % sere,conversion in m a n after one i nj e c t i on was approximately three times t h a t required in guinea-pigs an d 226
t M M U N O G E N I C I T Y OF HEVAC B 100 t i m e s t h a t r e q u i r e d in B a l b / C m i c e . I n v i e w o f these results, m a n a p p e a r e d to be m u c h less s e n s i t i v e t h a n g u i n e a - p i g s or m i c e . H o w e v e r , o n c e c o r r e c t e d for b o d y w e i g h t , t h e s e r e s u l t s l o o k t o t a l l y d i f f e r e n t a n d m a n a p p e a r s ro be 6 0 t i m e s a n d 3 0 t i m e s m o r e sensitive t h a n guinea-pigs a n d m i c e , r e s p e c t i v e l y . It is d i f f i c u l t to stress t h i s c o m p a r i s o n m u c h f u r t h e r in v i e w o f t h e d i f f e r e n t s l o p e s o f t h e d o s e - r e s p o n s e c u r v e s in t h e t w o a n i m a l s y s t e m s a n d in m a n . T h e l o w e r slope o f t h e d o s e - r e s p o n s e c u r v e in m a n c o u l d reflect g r e a t e r h e t e r o g e n e i t y o f t h e i m m u n e responsiveness of man compared with that of guinea-pigs or mice. The high slope of the d o s e - r e s p o n s e c u r v e in g u i n e a - p i g s is h o w e v e r o f g r e a t practical a d v a n t a g e as it gives m o r e accuracy to e x p e r i m e n t a l d e t e r m i n a t i o n o f I m D s s o .
Acknowledgements W e wish to t h a n k J. Flori and J. Arondel for skillful technical assistance. REFERENCES I. Barin F, Goudeau A, Coursaget, P, Maupas P. Large scale purification of Hepatitis B surface antigen (HBsAg). A n n Microbiol (Institut Pasteur) 1978: 129B, 87. 2. Mau1~,,s P, G o u d e a u A, Dubois F, Coursaget P, Barin F. Potency and efficacy of l i B vaccine applied to a h i g h risk population, a five year study. In: Maup~s P, Gue~ry P, eds. Hepatitis B vaccine. Inserum S y m p o s i u m No. 18. Elsevier/North Holland Biomedical Pres s, 1981. 3. Adamowicz P, Gerfaux G, Platel A, Muller L, Vacher B, Mazert M-C, Pruner P (eds 1981). Large scale productions of an Hepatitis B vaccine. In: Maupas P, Guesry P, eds. Hepatitis B vaccine. Inserum S y m p o s i u m No. 18. Elsevier/North Holland Biomedical Press, 1981. 4. Adamowicz P, Chabanier G, Hyafil F, Lucas G, Pruner P, Reculard P, Vinas R. Elimination o f serum i~roteins and potential virus contaminants d u r i n g Hepatitis B vaccine preparation. Vaccine, 1984; 2: 2 0 9 - 2 1 4 . 5. Crosnier J, J u n g e r s P, c o u r o u c e A-M, Laplanche A, Benharnou E, Degos F, Lacour B, Pruner P, Cerisier Y, Guesry P. ~Randomised placebo-controlled trial of Hepatitis B surface antigen vaccine in French haemodialysis units. Lancet i: 1981; 455--459. 6. Goudeau A, Dubois F, Barin F, Dubois M-C, Coursaget P. Hepatitis B vaccine: clinical trials in high risk settings in France. Devel Biol Stand 1983; 54: 2 6 7 - 2 8 4 . 7. Benhamou E, Courouce A-M, Jungers P, Laplanche A, Degos F, Brangier J, CrosnierJ. Hepatitis B vaccine: randomised trial o f i m m u n o g e n i c i t y in haemodialysis patients. Clin Nephrol 1984; 21: 143--147. 8. Coursagct P, C h i r o n J - P , Barin F, Goudeau A, Yvonnet B, Denis F, Lorrea P, N'Doye R, Diop-Mar I. Hepatitis B vacine: i m m u n i s a t i o n o f children and'newborns in a endemic area (Senegal). Devel 13/ol Stand 1983; 54: 2 4 5 - 2 5 7 . 9. Goudeau A, Lo K-J, Coursaget P, T o n g M-J, Yeh C-L, Tsai Y-T, Lee J - K , W u T-C, Yeh P-S-H, Lee S-D. Prevention o f Hepatitis B virus infection in children born to active and passive active i m m u n i z a t i o n . Devel Biol Stand 1983; 54: 3 9 9 - 4 0 4 . 10. W H O Requirements for Hepatitis B vaccine. W H O Tech Rep Set 1981; 658:731--156. 11. Finney D-J. Statistical M e t h o d in Biological Assay. 2nd edition. London: Charles Griffin, 1971. 12. Ge.rety R-J, Tabor E, Purcell R - H , Tyeryar F-J. Summary o f an international workshop on Hepatitis B vaccines. J Infect Dis 1979; 140: 6 4 2 - 6 4 8 .
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