.7o~rnat qf Biologi¢aI Standardi~,ati~n 5,979 7j 1-7

T h e p o t e n c y o f rabies vaccines as determined by t h e L,~ducti'on o f a n t i b o d y in ~ c e

Eugene I. Rosanoff'f

Eight Iota of D~c-k Embryo rabies vaccln~ and four XVi-38grown rabies ~ce.L-~e were assayed for potency by atttibody induction (AI) as well as by the convent~orta| N I H mouse test procedure. O u r resUlCs indicat~ that D u c k E m b r y o an(i,XVi-38 rabies ~accine A I

+pOteax~ values felt wJthk.x the maine ~ t L , xct Ixt,-agc as detected by the conventJo~a| N I H t~'t.

INTRODUCTION T h e v a | u ¢ o f t h e N I F I M o u s e P o t e n c y T e s t o f rabies vaccine (~V.H.O., 1973) is welt established. T h e test is usually a c o a r a t e a n d r e p r o d u c i b l e w h e n t h e s a m e lots of ~ c c i n e are assayed within t h e s a m e laboratory, Occasionally; ~,awever, variations in t h e p o t e n c y o f s a m p l e s f r o m t h e same:-]6t-have been n o t e d , w i t h p o t e n c y values differing f r o m l a b o r a t o r y to laboratory. A n o t h e r d r a w b a c k o f t h e N I H p o t e n c y test is t h e t i m e r e q u i r e d for testing, w h i c h is at least 28 days. Obviously, a m o r e r a p i d a n d e q u a l l y sensitive test for d e t e r m i n i n g rabies virus vaccine p o t e n c y is desirable. O n e m e t h o d u n d e r c o n s i d e r a t i o n in o u r laboratory, w h i c h r e q u i r e s c o n s i d e r a b I y less t i m e (14 days), i n v o l v ~ t h e i n d u c t i o n o f a n d b o d y in m i c e after ~nocu|at l o n o f i n a c t i v a t e d W i - 3 8 g r o w n rabies (PA~I) strain virus vaccine (XVRV). A n t i b o d y i n d u c t i o n p o t e n c i e s o b t a i n e d w i t h W R V as compareqd to D u c k E m b t T o G r o w n Vaccine (D]EV) are th~ s u b j e c t o f t h i s r e p o r t . * Received for publica~'o~ $ January 1978. ff R e s ~ r e h Virology M o ~ , Wyeth Laboramrie~ Inc., ]Ft.~tclnor,Pe~yIvat~ia I9088, U . S A . x

1

E. I. ROSANOFF

MATERIALS

AND

METHODS

Vacdrte centro& T h e Division of Biologic Standards Reference Rabies Vaccine L o t 182 .was obtained from due Burea,a of .Biologi~ (BuBio), FDA~ Rockville, P 4 a ~ l a n d , U.S.A., an d D u c k E m b r y o G ~ w n V a c ~ n e ( D E V ) was obtained by purchas from Eli Lilly & Co. Inc.

Antibody

( A I ) in ~ e

F e r u l e 12- t 6 g C h a d e s CDx mice divided into groups of I 6 w e ~ inoculated intraperitoneaI]y cat days 0 and 7 with 0-5 ~-nl~ a 1 : I0, I : 30, 1 : 9"0, I : 270 or I : ,8i0 d:Iution o f the ~ H 1~ Iot 182, XVi-38 rabies virus Vaccine (~,VR~,~)or Duck Emb~o Grown R Vaccine ( : B~o,0d obtained th e orbital sinus ~f i m mice on day 14 w ~ p to ser~Jm, inactivated at 56 ~C 30 mln - i a n d then stored at --60 *C # o r to use. S e r u m s a m p | ~ mA~ i m m u n i z e d with each dilution of v ~ n e were p o o l ~ for I n some experiments, of i m m u n i z e d mice were bled at i n t c ~ s up to 8 wee~#~ an d their s e ~ p r o c e ~ e d ~ d ~ c d b e d above. Sera w~re tested for an co'htent By th e ~ o u s e S e r u m Neutral Test ( after the m e t h o d by Atanasiu (1973), a n d / b y the F lu nt F o cu s Inhibition Test( ~ described by Smith, g a g e r & Baer (|973). T h e procedure :for the S t ~ dard N I H Potency T e s t was that emptoyed by Sel n (1973). Results were expressed as international units of antibody p e r millititer (i.u. ml-~). ,Antibody induction potency values were c,~lc~lated b y db¢idlng the total geometric mean amounts of antibody p r o d u c e d in all dilutions of the test vaccine by the total geometric mean-amounts of antibody p r o d u c e d in the ~ r r e s ~ n d i n g dilutions of the N I H reference vaccine extrapolated to ~ r o dilution. F o r these calculations, the m i n i m u m a m o u n t of antibody used was 0.2 Lu. rnl -~. CaIculations of ED~o and antigenic values (potencies) for the standard l q I H g,-otenc:ytest were perfoxmed after the methods of Spearma n (1908) and K a r h e r (I931). TABLX 1.

Aaatibody mductlon poten~es of rgoics D E V preparations (geometric mean amounts)

Rabies antibody prod,aced Potency (Lu, m-1-z)

rs~St'~ Test*

NIH rcf.

RFFIT ref.

r~o.

I 2 3 4 5 6 7 8

37-9 46-0 65-2 62-7 86-3 I73-2 95-3 162-2

h~louse ~ntibody induction VRC¢.

MSN

RFFIT

Standard N I H test ure

I63-1 40"0 98~9 66~7 64-8 4S43 86~5 32-7

2~8 0~99 1-21 O-51 0-28 0~ 1-I5 0-52

2,0 0~70

0-50 0-61

:NIH Test

107-0 45"5 78~9 31-9 24-3 38-6 109-8 84-8

81-5 57"2 70-9 1 t 5-5 152-6 85-4 7t:0 45-7

r

T~t

1-39

0-58 0,42 0~53 1-20 0-71

0-36

0-57 ~33 &66 ~79

* Lot 7AP|SA used in Test ~o. I ; lot 8KX87A used in Tests 2-8. DEV = Duck E m h ~ o Grown Va~ine. 2

W

2 3 4 2

10701 10801 10901 | 1001

124,3 97.8 253"3 48'4

!0S5'9 927'6 592+7 250'3

Test va~.

96'7 t34,2 1~5"5 65.3

NIH Rd, vr~c.

"~

952'4 10164 35~'2 227.6

Test race,

........

REFIT

NIH Rd. vacc~

~

~

MSN . ~ ~ - ~

Potency (i.u. rolet)

9"85 (6~9 -!4'0) 7'56 (5-2~10.88) 2'62 (1"8~3'72) 3.48 (3.7.3-6"70)

Mome ~ti~ody induction ..................~ ................. -~- ~, MSN RFHT

.........................................

8'50 (3"9 -18-4) 9'~ (6'43-13'9) 2.55 (1+55-4,09) 5"1~ (4'02-6"$8)

~

~

6'24 (S'9 ~'6) 2'66 (2'0i~3'52) 2+26(1'27-4+00) 2 ' ~ (1'93-2"97)

Stand~d NIH test

Standards Reference Rabies Va~irie Lot I82, Bureau of B~ologtes, WRV = Wi.38 Gro~,n Rabie~Vaccine;() = Rang~of one sta;'~d~d deviation,

MSM ~ Mouse Serum Neutra~izatlon Test~ RFFIT -- R~bie~ F l u ~ c e n t Focus Inhibition Tes~ NIH Ref, ~ NIH Division of Bi01o#e

No. of t~

T~t vaccine (WRV) lot no,

Rabies mtlbody produced (i.u. ral-:t)

T^nLz 2. Antibody induction potencies of four WRV preparations (geometric me~ amoar,ts)

m

¢1

X 0,d 0

0 ,q

E. I. R O S A N O F F In: most e x p e r i m e n t , a teat consisted of simultaneous assays of one lot o f the Iq'I8 refere n d , one tot of V e R V and one lot d D E V ; the potency values o b t a i n e d ft~r ead~ vaccine ~ p e were grouped toge~.her, RESULTS T h e antlbody-inducing ability of and t h e N I H reference 14 post i m m u n ~ f i o n are showaa .in T a b l e L Eight s e p a ~ t e tests were p e r f o r m e d , seven on s a m # c s of t h e ~ m e D E V lot. Results of the M S N and R F F I T p~ocedures showed rcasonabM ag reemen t with ~respect to the amounts o f antibody detected in an y 'one test, Potency v a l u ~ Obtained b y antibody induction for aU eight D E V s~mples were comparable to those o ~ M n e d by t h e standard N I H mouse test. Analysis o~ these data by Student'z t - T e s t revealed that antibody induction wdues o b ~ n e d b y either M S N o r R F F I T did not differ significantly from those-of the standard. Ta bl e 2 s u m m ~ z ~ the 14 day antibodyoresponse of four .different ~VRVlots w h e n c o m p a r e d to the N I H reference lot 1822 Reasonably good correlation between the 1VISN and R_FFIT procedures: relative tO ~~antibody production and to potency v a l u ~ were eeldent. L o t 10801 ~aowed a slightly h i g h ~ potency ( ~ m p a r e d to the other three lots') by antibody induction ~ than b y t h e ~ r d ~';IIH procedure. LThe d a ~ in Table 3 show a.nti induction a n d ~ a n d a r d N I H ~ t e n c i ~ of all 8 D E V a n d . ~ l 12 ~VRV lots, O v e r ~ t geometric m ~ n potencies of W R V lots were five to sevenfold greater ~ a n t h c ~ of D E V lots ~ r d l e ~ o f the poten ~" p t o ~ d u r e u s e d ; I n addition, the geometric mean standard Y l H p o t e n ~ v a l u ~ for "~VRV lots 10701 t h r o u g h 11001 as previottsly d e t e r m i n e d (q.v. T ab le 2 ) s h o w r e ~ o n a h l e agreement ~-Hth those obtaL~ed by o u r ] a b o r a t o ~ ~ d by tla~ Bureau o£ Biologies for each a n d aH lots ( ~ a M e 4). TA s L E 3.

Potency of ~ E V and WRV as determined by v~fibody i n d u ~ o n and N I H mouse ~ t e n c y test procedures (i.u. ~A -I) D E V (g'teSts)

WI~IV (12 tests)

0-71 (0"30-I-64) 0.83 (0.48-1-40)

5-12 (2-55-10.3I) 4.57 ( 2 ~ 8.49)

0-57 (0"38~-84)

2.84 (I-71- 4.69)

I . Antibody induction:

MSN RFFIT 2. N I H potency test:

V ~ u ~ are ~ r e g s e d as overall g e o ~ ¢ S dcvlatioa (in ~ m ~ , s ~ ) . T A B L ~ 4.

Geome~ic mm

m~

lpo~:enei~ wi*h a ~rig~ of ~ e

s ~ n d a r d N I H potency V ~ u ~ of W R V l o t s (i,u. m l -~) .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

VVRV lot no.

t No. of tests

Geome~e me~

Range of one S.D.

10701 10801 1~01 1t ~ I

5 6 7 6

7-I8 2:35 2"25 2-72

3-66-t4-12 1"47-- 3"74 1"43-- 3"54 1-93- 3=86

3"~

1"6~

To~a

24

.

"--

5"7g

No. of tests

Go0me~i¢

:

"---:: ..........

m~

Range o f one S.D.

2

6'41

4-77-8-61

2 4 3

3-65 3"20 I'80 '3"22

3;'58-3"72 2"3~'46 t"41--2"51

11

* ~ B i o Test ~ t a , k ~ y suppli~ by D r Edward Fitzge~d, ~ a u N I H , Bethesda, MD, I j . S ~ .

4

:7::

BuBio t ~ *

Veyeth t e ~

I ;9~5'27

of Biology,

2"27 0.33

9,9 1.0

56

1,0

WRV 11001 DBV 8g_X87A

10,8 0.60

9'68 0'82

7.08 0"80

WRV 10801 DEC 9PWgSA

Vacdn¢ 1o~.no. 3'8g 2;83 0"22

0.64

9.01 4'03 0"88

0.64

~.20 0,5$

0.36

9'[ |

* Geometricm~r,.~. For leg~,ds s~:~T,~e 2,

3'9l 0'74

O,S¢

7'75

6"12 0,98

0'53

3'62

6"3 0'68

2'84 0 '92

1"t8 0'23

6"88 0"55

1'5 0'64

2'5 0.51

......................... ~=.................................. ,,_..._ ........................................... ~ NIH Day 2I Day ~ Day 42 Day $6 ~tand~rd D~y 14 ..........:';.,....................... ................, - ~ test MSN RFFIT MSN RFFIT MSN RFFIT MSN RFFIT ~'ISN RFFIT pr:~ed~re

M o u s e antibody induction

Potency' (i,u, ml-~)

TAegg 5. Aa~tibodyreduction poteneie~ of WRV and DEV as a function of tinge

Z

O

>

O

Z

O

E. I. R O S A N O F F T w o e x p e r i m e n ~ were pe ~ d ne the duration of antigenic response in rru\~-e ~/ter in ~'a of the N I H ~ he, or lots 10801 ~ d 11@~1. The resul~ d that the ratio o f t h e test vaccine ~ y to.°referen:ee antibody rent,tined tel ~ , n ~ n t at each o f the ~ m p l i n g p e r i o d , Le. days 14, 2 I , 28, 42 gad 56 5). T h u s , tire earliest llng ~ r i o d (14 would a p p ~ to be ~. DISCUSSION In this paper we have r ~ the nt o f antibody produced in mice inoculated e i d e r with or ~ t h W R V to the amount of antibody p r o d u c e d ~ mice inoculated with t h e N I H Reference Rabies V a e d n e . %Ve a good eorr~dation the two laboratory assay re8 ( M S N and R F F I T ) u ~ d for the detection o f rabies antibody. I n addition, our findings from 10 separate assays of D E V d n g three different Iots and from !4 ~ a y s of W R Y ring four different vaccL.e lots, indicated t_hat the ratios o f t ~ t vaccine ~ d b o d y / r e vaccine antibody produced in 14 .days yielded potency values comparable to t h o ~ o b t ~ e d by the standard 28 day N I H poteficy test p r o ~ d u r e . T h e ~ m e advantage gMned by use of the antibody induction test can be e ~ l o i t e d only by the P ~ F I T a ~ a y p r o ~ d u r e . Our experience w i t h t h e antibody induction test has shown us that the same m i ~ bled ~ day 14 may also be used for a confirming standard N I H p o t e n ~ test if t h e animaLs are bled ~ r e f u l l y . T h e reserve of sera obtained at 14 days also a l b w s for repeat A I tests as needed and as a back-up to t h e standard N I H t ~ t . T h e fact that one of the %VP,V lots (10801) had a slightly higher potency- b y the antib o d y . ~ d u c t i o n test than b y the standard N I H p r o c e d u ~ is not distnarbing since %VRV lots made in our laboratory, which have shown a range in poten~¢ from 1.0 t o 7-2 (by the standard N I H mouse test procedure), have also not ~ d d e d sign~c~andy different antibody levels in h u m a n -¢oIunteers (PloLkin, %¥iktor, K o p Rosm~off & T i n t 1976). Similar o b s e r ~ d o n s in other human e l i n i M trifls have been reported in a %V.H.O. consultation puMiea~on (t975), and in pfi lated with ceil culture of duck embryo o r "~Vi-38 ~vely b y Lavender (t973) and Sikes, . , "~riktor & (1971), ~. ne appli o f the antibeMy i test tb~n t h e standard N I H mouse t ~ t for the measurement o f eies o f rabies es witt a reality as data ac~tmulate to rabiIity of these ~ ' o U~.

REFERENCES

A t a n ~ u , P. (1973)~ assay and p o t e n ~ test of abies se-_~am~ d . In: Techniques in 3rd. ed. % ~ l d H-ealth Organization rY~o Series No. 2AI,pp. 3 1 ~ 3 1 8 . K a t h y , G. (193i). B e i t r a g ~ r yen B e h a n d u ~ n g p h a ~ claer Relhenvcr=~uc.he. Archiv..far Experimenttlle und logic 162, 480-483. Lavender, J. F. (I973). I m m u n e r~pons6 K,a primates vaccinated ~ t h dudg errd~ryo e ~ culture vacc~ine. Applied togy, 2S, 327-331. Plotkin, S. A., %Viktor, T. J,, K o p r o ~ k i , H.',' Rosanoff, E. L & Tint, H. (1976). lm_mu_nization s c h e d u l ~ for the ~ew human diploid cell vacc~e against rabies. Americar~ ffournaI of E.~z'de~fology 1 ~ , 75-80. Sdigmann, E. B. (1973). ~ e N ! H t ~ t for p o t e n t . I n : ~boratory Ttchrdques in ~.aba'es, 3rd ed. V~ror]d Health Orgarfizafion Monograph S e 6 ~ NO. 23, pp. 279-286. W.I-LO. 6

POTENCY OF ~ABIES VACCINES R. K., Clearf, ~#~rF., Koprowskl, H., ~'{ktoe~ T . J. ~ Kap]an, ~'|. D. (t97f). Effective protcc~on of r n o n k e ~ against death from street virus by p ~ t exposure ~drnlnistratlon of fis~u¢ cu]roee, rabies vaccine. Bulletin of the 0 tion 4~, 1I-1. Smith, J. S., ~rager, P. A. & Baee, G. 1V[.(1973). A rapid tissue culture test ?or determining rabi¢~ ncutralizLng antibody. I n : Laboratory Technique, in Rabies, 3tO ed. Vv'orld Health Org~izatiort Monograph Series No. 23, pp. 3 5 ~ 3 5 7 , XV.H.O. Spearrnmq, C. (1908). T h e .method of 'Right and Wrong C~es" (constant stimuli) with Gauss' f o ~ u l a e , British Journal of Psychology 2, 227--242. V~.H.O. (1973). "Vaccine s a f e ~ mrld potency tes~. I n : ~ o r a t o r y Ted, m'qnes in Rabies~ 3rd ed; ~,Vofld Health Organization Monograph Series No. 23, pp. 269-286.. WV.H.O. "vV.H/O. Consu|tafion (1975). ]RelaOrtof consultation on tables vaccine prepared on human ~#oid ~n ¢~r~. "vv~LaridH e a t h Organization Document HP]75.3. VV.H.O.

7