Antibody Response to a Human Diploid Cell Rabies Vaccine

Transcription

1 APPLIE MIROBIOLOGY, Mr. 1974, P opyright 1974 Americn Society for Microbiology Vol. 27, No. 3 Printed in U.S.A. Antibody Response to Humn iploid ell Rbies Vccine V. J. ABASSO, M. B. OBKIN, R. E. ROBY, AN A. H. HAMMAR Microbiology Reserch, utter Lbortories, Incorported, Berkeley, liforni 9471 Received for publiction 28 ecember 1973 An experimentlly killed rbies virus vccine prepred in humn diploid cell strin (WI-38)-Wyeth rbies vccine (WRV)-ws used by vrious injection schedules in two seprte studies to define more closely in humn volunteer subjects n effective vccintion schedule for pre- or postexposure immuniztion, prticulrly for donors of rbies-hyperimmune plsm. To permit vlid comprisons between our results nd those of other workers, ntibody levels chieved were expressed in terms of interntionl units (LU) per milliliter of serum. Antibody response of previously nonvccinted persons were only modest fter single dose of WRV, never reching level higher thn.8 IU/ml over 56-dy testing period. Moreover, ntibody ws not detected t.16 IU/ml before the 14th dy, either fter single dose or fter two doses given 3 dys prt. The best response followed four doses of WRV given within.4 weeks. This schedule resulted in the highest rte of seroconversion to the t 6 IU/ml ntibody level required of potentil rbies-immune plsm donors. Giving the first vccine dose in luminum hydroxide diluent did not enhnce the ntibody response. There ws definite suggestion in the vrious injection schedules tht higher nd more sustined ntibody levels were reched when the intervl between the first nd second vccine doses ws longest. The greter immunogenicity of WRV s compred with duck embryo vccine ws best demonstrted by the fct tht single booster dose of duck embryo vccine to previously vccinted individuls resulted in only sevenfold ntibody rise during the following 56 dys, wheres booster dose of WRV elicited 69-fold rise. Al(OH)3 in the first dose of WRV hd no effect, but the enhncing effect of longer intervl between vccine doses ws noted once gin; 2 of 2 subjects who received three doses of WRV with 4 weeks between doses developed good levels of rbies ntibody, nd 19 exceeded 6 IU/ml. In previous, communictions, we reported the preprtion of rbies-immune globulin of humn origin (RIGH) nd the determintion of its optiml dosge in volunteer subjects ctively immunized with duck embryo rbies vccine (4, 8). Lter, we described the properties of RIGH nd pointed out mong other detils tht the immune plsm pool used for frctiontion must contin t lest 6 to 8 interntionl units (IU) of rbies ntibody to yield RIGH of cceptble potency (3). It goes without sying tht hyperimmuniztion is the only mens of inducing this level of rbies ntibody in prospective plsm donors. The choice of rbies vccines to be used in mn for hyperimmuniztion is limited. Vccines prepred from brin tissue of dult nimls re lible to cuse llergic postvccintion rections of the meningoencephlitis or polyneuritis type (1, 5). In view of this, vccine prepred from rbies-infected duck embryo tissues, nd inctivted with bet-propiolctone, ws introduced in 1956 (9). Grdully, this vccine cme to replce nervous tissue vccines in the U.S.A. nd is t present the only vilble licensed vccine for either pre- or postexposure immuniztion. Although this vccine proved to be gret del sfer thn those derived from nervous tissue, it presents number of disdvntges in the hyperimmuniztion of potentil rbies-immune plsm donors: (i) the gret number of injections needed; (ii) the undue occurrence of loclized rections; nd 553 (iii) the smll number of persons who develop the necessry level of rbies-neutrlizing ntibody, despite repeted booster injections. A bet-propiolctone-inctivted rbies vccine, prepred from virus grown in culture of humn diploid cells (WI-38), ws shown to hve higher ntigen index in mice thn duck embryo vccine nd to be immunogenic in monkeys (1, 12, 13). Another humn diploid cell vccine ws prepred by Wyeth Lbortories, Phildelphi, P., by inctivtion nd

2 554 ABASSO ET AL. APPL. MIROBIOL. disggregtion of the virion with tri-(n)-butyl phosphte insted of bet-propiolctone (H. Tint, M. B. obkin, nd B. A. Rubin, submitted for publiction). In preliminry studies in mn, this vccine induced good booster response with single dose in those previously vccinted nd n cceptble ntibody response fter two doses in the mjority of those who were never vccinted before. Rections were miniml nd only locl (14). This pper summrizes the serologicl results of studies with Wyeth vccine, the object of which ws to define more closely n effective vccintion schedule for pre- or postexposure immuniztion, prticulrly for donors of rbieshyperimmune plsm. reful clinicl observtion reveled no dverse rections in these studies. A detiled description of the mild symptoms reported will be given elsewhere (J.. Loofbourow, V. J. bsso, T. Y. ooper, B. Smith, nd H. Hughes, submitted for publiction). MATERIALS AN METHOS A supply of rbies virus vccine (WRV), inctivted (Wyeth) (experimentl lot 372-A-11), ws obtined from Howrd Tint, Wyeth Lbortories, Phildelphi, P. The vccine consisted of n inctivted, concentrted, nd vcuum-dried extrct of humn diploid cell strin WI-38 which hd been infected with the PM strin of rbies virus. Inctivtion of the virus hd been crried out with.1% tri-(n)-butyl phosphte in the presence of.1% Tween 8. The vccine ws dispensed in 1-ml doses in disposble syringes (Tubex) nd ws reconstituted just before use with either sterile distilled wter or distilled wter contining.1% luminum hydroxide (Al(OH), diluent). At the time it ws used, it hd men potency rtio of 1 ginst the U.S. reference vccine 176, 178, or 179, obtined from the Bureu of Biologics (BOB), Food nd rug Administrtion, Rockville, Md. Rbies vccine, duck embryo origin (EV), ws obtined commercilly from lot well within the expirtion dte t the time of the study. linicl studies: study 1. The objective of the study ws to compre the rbies ntibody response of previously nonvccinted persons with vrious schedules of immuniztion, using one to four doses of WRV. The study ws conducted t the medicl fcility of the Arizon Stte Prison, Florence, Ariz. The study popultion consisted of informed nd consenting dults who were 21 to 55 yers old nd in good helth. The 5 subjects dmitted to the study were selected from mong those shown negtive for heptitis B ntigen nd with no history of sensitivity to ntibiotics or other llergies. Study 2. This study hd two objectives: (i) to obtin rpid mesure of the reltive ntigenicity of single doses of EV or WRV by compring secondry immune responses of previously vccinted volunteers; nd (ii) to study the immunogenicity of three doses of WRV t n intervl of 4 weeks between doses in previously nonvccinted subjects. This schedule is dptble to the vccintion of first-yer students seeking pre-exposure immuniztion. The study ws crried out t the owell Student Helth enter nd Hospitl of the University of liforni t vis with 4 informed nd consenting dult volunteers, 2 to 5 yers of ge, of either sex nd in good helth. Serum ntibody titrtion. The ntibody response of ech subject ws determined by mesuring the rbies-neutrlizing ntibody in ech serum specimen ccording to the procedure outlined by the BOB of the Food nd rug Administrtion for potency testing of ntirbies serum. This procedure ws crried out in Swiss Webster mice s previously described (4). The ntibody content of ech smple tested is reported in this pper s interntionl units of rbies ntibody per milliliter. omputtion of the interntionl units per milliliter is mde possible by the inclusion in ech test set of the U.S. stndrd ntirbies serum which is supplied by the BOB in dried form. When reconstituted for use, this serum hs designted ntibody content of 2 IU/ml. uring the course of the studies being reported, the men serum neutrliztion titer obtined by us with this serum ws 1:1, the vlue used in our computtions. To scertin the relibility of the mouse serum neutrliztion test results, rrngements were mde with the enter for isese ontrol (), Atlnt, G., to test in prllel with us 51 coded smples obtined from study 2. These smples represented three seril bleedings from 17 volunteers who received single booster doses of either EV or WRV. In terms of bsolute titers, those of were consistently twoto fivefold higher thn ours. These higher titers were lso reflected in the findings for the BOB stndrd serum, the men vlue of which ws 1:3 in their hnds. However, the differences between the two lbortories becme much smller when titers were converted to interntionl units per milliliter (Fig. 1). In fct, the two sets of results coincided remrkbly LL J.J _ E 2_ WYETH VAINE LiJ 1. - L).8 P _J i. -<UK EMBRYO.4 - VAINE 2 8 h RT -.2 /y TESTE AT UTTER ~~~~~~~~~~LABORATORIES Z.4- / TESTE AT ENTER < / * FOR ISEASE ONTROL Ut).2 W..1 <t o 1 2 FIG. 1. omprison of serum neutrliztion test results in two lbortories.

3 VOL. 27, 1974 HUMAN IPLOI ELL RABIES VAINE 555 well when so expressed, considering the low degree of precision of the mouse test. RESULTS Study 1. The 5 volunteers ccepted in the study were rndomly ssigned to one of the ten groups detiled in Tble 1. In ll cses except group X, WRV ws reconstituted in sterile distilled wter. The subjects in group X received the first injection of vccine (dy ) reconstituted in Al(OH)3 diluent, nd they received the lter injections of vccine reconstituted in distilled wter. All injections were mde intrmusculrly in the deltoid re in 1-ml volumes. On the dys specified in Tble 1, 1 ml of whole blood ws withdrwn from ech prticipnt, nd the ser recovered from the specimens were frozen nd shipped to the lbortory for lter ntibody level determintion. All smples from the sme individul were tested t one time. Prevccintion ser were tested in dilution rnge strting t 1:2 (permitting detection of.4 IU/ml), wheres lter specimens were tested in dilution rnges strting t 1:8, permitting estimtions of ntibody levels s low s.16 IU/ml. At different times erly in the study, four individuls withdrew from it becuse of vrious resons, but none becuse of n undue rection to the vccine. One ech of the four individuls hd been ssigned to group II, III, V, or VIII (Tble 1). The ntibody responses of groups I to X re presented in Tble 2 nd in Fig. 2 to 4. onsidering the lck of precision of the mouse Group No. test, firly consistent results were obtined for the individul subjects of ech group. onsequently, lthough the smll size of the group precluded rriving t definitive conclusions concerning n optiml schedule of immuniztion, number of vlid observtions cn be mde. As expected from their history, ll prticipnts in the study were negtive for rbies ntibody before vccintion. In evluting the serologicl response to preexposure rbies vccintion, ny mesurble ntibody level is considered to hve conferred on the individul n effective degree of resistnce to nturl infection (2). In this respect, ll but 2 of the 46 prticipnts in study 1 responded to vccintion with WRV, regrdless of the number of vccine doses or the schedule of injection. One of the two who filed to respond t the.16 IU/ml level hd received one dose of vccine (group I, Fig. 2) nd the other three vccine doses (group VII, Fig. 3). From the stndpoint of hyperimmuniztion of plsm donors, however, it ws cler tht the ntibody response ws insufficient, either fter 'single dose of WRV (group I, Fig. 2) or fter two doses given 3 dys prt (groups II nd VI, Fig. 2 nd 3). It ws lso cler tht single dose of vccine resulted in only modest ntibody response over 56-dy testing period (group I, Fig. 2). The groups receiving two doses of vccine (II, III, nd IV, Fig. 2) responded to essentilly the sme level of rbies ntibody. However, there ws suggestion tht, even though the rise of ntibody ws slower, it reched higher level nd ws more sustined when the intervl TABLE 1. WRV study 1: immuniztion nd bleeding schedules of 1 groups of volunteer subjects No. in group No. of vccine doses y of vccine injection nd/or bleeding(s)b I 5 1 S-V s S s s S S II 4 2 S-V S-V S S S S III 4 2 S-V S-V S S IV 5 2 S-V S S S S S-V S V 4 3 S-V S-V S-V S S VI 5 3 S-V S-V S S S-V S VII 5 3 S-V S-V S-V S VIII 4 3 S-V S S-V S-X IX 5 4 S-V S-V S-V S-V S X 5 4 S-V S S S-V S-V S-V S At the initition of the study, ech group consisted of five individuls. 'S-V, Blood smpling followed shortly by vccine dministrtion; S, blood smpling only. c In ll cses except group X, ech dose consisted of 1 ml of vccine reconstituted in sterile distilled wter. All injections were given intrmusculrly in the deltoid re. Subjects in group X received on dy 1 ml of vccine reconstituted in A1(OH). diluent, lso intrmusculrly. Lter doses to members of this group were reconstituted in sterile distilled wter.

4 556 t _N c E L. t E -o Qc cc :H 3 /A x._~ 3 I. IU _ co r_._ I (A cc I>1 -c. cn IE (1).I I) -r _o E ABASSO ET AL. l 6 A i l! A -]N -4 ( -4 cq O co 1- ' -4 n -4 ov L O - o ov -I o ) O - O lk 1 cs cs t- c'i c O - (I1Mq Al V V V V V V V V V V - - I I--I I--I 66 -! I;--! -! ! I--I v v v VV v v vvv vvivivv vvvv vv v v Vv -VV VV vv VV r t]rt "It LO 'ot Ln LO Lo NO 2 2 l -4 14~ 2 I O c~~z 4- st, s M z g Nl > oc > m i v oq 6 ;~6 ~~~ o ccq cq cq 4 cq o M o()l cq U:cl l _ -c ii c I q. 6 I., 6 4 l ci I cy Ql l -L ocia -O 6q~ 6 6 6I - _ -O.-O -l l-o -lo (> > 1 6 l V ) 1. 1 Ov Vl 1. 1 OV O V > 1. 1 OV ) 166 l116.1 V Vl V Vl 1 1 V Vl IV 11l 1 l V L YI' Y.J 1J "I " l lo 'll I'll "It " lo o "It 1 APPL. MIROBIOL. - S. _ O._ X cnc -c 3.- o o vq) Vo cc - 13 cz Z t4) >._4- cc4-~, cc o, r. crs- - Zc O z 2= O ~ ~b 3~ c c bc bc bc bc b c b c b c ~~~~~~ ~ 13 ci cis Z Z occ (A L S 4 : I., P P-4 > > x x > >

5 VOL. 27, LJ.3 n) ' A.1 4. \-.4 Z.4 1(1.2 HUMAN IPLOI ELL RABIES VAINE,q/ _~I ON 4yS o AZ) - -&--GROUP II *,/ <;<jp*5 VAINE ON AYS -3 I AYS AFTER INITIAL VAINE OSE FIG. 2. Antibody response to one or two doses of Wyeth rbies vccine (study 1). ') 4 2 cr 8 O 2 I<.4 GROUP 21TI VAINE ON AYS , TO 8 84 AYS AFTER INITIAL VAINE OSE FIG. 3. Antibody response to three doses of Wyeth rbies vccine (study 2). J 1. k cr 2. V).8 m z < c cr to.ol,oine ON ~~~~~~~~ 1~~~~~~~~N W _ ISTER SES.. Z ^ ~~~~~~YS23 As S AYS AFTER INITIAL VAINE OSE - - FIG. 4. Antibody response to four doses of Wyeth rbies vccine compred with response to 16 doses of duck embryo vccine (study 2). between the two vccine doses ws longest (group IV, Fig. 2). This suggestion cme lso from mong the groups receiving three doses of vccine (Fig. 3), where group VIII hd the TABLE 3. WRV study 2: ntibody response fter one booster dose (1) of Wyeth vccine Subject Antibody (MU/mW)8 fter booster on dv: (1) A <.4 < B <.4 < < <2.56 <2.56 <.4 < E < < 1.28 F < G.6 1. < 2.56 < H I J Geometric menc All subjects received 1 ml of vccine reconstituted in distilled wter intrmusculrly immeditely fter blood ws drwn for -dy serum specimen. Subjects were selected on the bsis of hving received rbies vccine 6 or more months previously. Interntionl units of rbies ntibody per milliliter of serum bsed on U.S. Stndrd Antirbies Serum lot no. 4. c lculted by considering <.4 or <.8 s zero nd including them in the computtion; other < vlues were omitted from the computtion. TABLE 4. Subject WRV study 2: ntibody response fter one booster dose (1) of EV Antibody (IU/ml)b fter booster on dy: (l) K <.4 < L < <.8 <.8 M <.4 <.6 <.8 <.8 <.8 N < <.8 < NTc.8 P < <1.28 Q < 1.28 < 1.28 R S.8 < T Geometric mend All subjects received 1 ml of vccine subcutneously immeditely fter blood ws drwn for -dy serum specimens. Subjects were selected on the bsis of hving received rbies vccine 6 or more months previously. 'See Tble 3. c NT, Not tested. d See Tble 3. longest intervl between doses one nd two nd reched the highest ntibody levels. This ws observed in study 2 lso, in the groups receiving

6 558 ABASSO ET AL. APPL. MIROBIOL. the sme immuniztion schedule s group VIII. The best response in study 1 ws tht of group IX (four injections of WRV within 4-week period) with ntibody levels of 3 to 2 IU/ml in the 56-dy bleedings, nd with two of the five subjects responding with 6 or more IU/ml tht we require of immune plsm donors. Group X, which received n identicl schedule of four injections except for the first dose in Al(OH)3 diluent, responded mesurbly less well. This difference could suggest tht Al(OH)3 cted more s inhibitor in this cse thn s enhncer of the immune response, but s will be seen in study 2, this ws most probbly chnce occurrence due to the smll number of prticipnts in ech group. Finlly, to fford direct comprison of the response which followed four doses of WRV (group IX) to tht elicited by 16 doses of EV (14 dily doses plus booster doses on dys 1 nd 2 fter the lst dily dose), our findings in eight volunteers from previous study (4) were plotted in Fig. 4 fter conversion to interntionl units per milliliter. It is seen tht even though ntibody levels rose to higher level with 14 EV doses during the first 3 weeks, this level ws lter surpssed by group IX fter dministrtion of the third nd fourth doses of WRV. The highest geometric men ntibody level chieved with EV ws bout 3 IU/ml, wheres tht ttined in group IX ws 6.4 IU/ml. Study 2. This study consisted of the following two prts. (i) Booster effects of WRV nd EV. In the first prt of the study, 2 persons who hd received rbies vccine erlier, but not during the previous 6 months, were ssigned rndomly to two groups of 1 ech. Those in one group received one booster dose of WRV reconstituted in distilled wter, wheres those in the other group were given one dose of EV. On the dys specified in Tbles 3 nd 4, 1 ml of blood ws withdrwn from ech subject, nd the serum ws processed nd tested s described for study 1. The ntibody responses re presented in Tbles 3 nd 4 nd Fig. 5. The rndomness of the distribution of subjects between the two groups is supported by the ntibody sttus of the prticipnts before they received the booster dose of vccine. In ech group, only 4 of the 1 hd mesurble serum ntibody levels (>.4 IU/ml). The geometric men ntibody level ws.5 IU/ml in the WRV group nd.7 IU/ml in the EV group. The highest ntibody responses occurred t 7 to 28 dys for both groups, depending upon the individul subject. Among the ten subjects receiving EV, five chieved n ntibody level of 1 IU/ml or higher in one or more post-booster smples, but only one of the five reched level of 6 IU/ml or higher, nd this in only one of the four postbooster ser tested. In contrst, nine of the ten subjects who were given WRV ttined ntibody levels of 1 IU/ml or higher in one or more post-booster smples, nd five of the nine reched levels of 6 IU/ml or higher in one or more of the post-booster ser. The greter immunogenic effect of WRV cn best be summrized by the geometric men ntibody levels chieved by the two groups of volunteers:.9 to 3.4 IU/ml on the 7th through 56th dys fter WRV s ginst.32 to.48 IU/ml fter EV during the sme period. (ii) Responses of previously nonvccinted subjects to three doses of WRV. In the second portion of this study, previously nonvccinted subjects were rndomly ssigned to one of two groups. Ech member of one group received three doses of WRV reconstituted in distilled wter t 28-dy intervls (Tble 5). Ech member of the other group ws given three doses of WRV; the first ws reconstituted in Al(OH)3 diluent, nd the lst two were reconstituted in distilled wter (Tble 6). On the dys specified in the tbles, blood ws drwn nd the serum ws processed s described bove. The ntibody responses of the subjects re shown in Tbles 5 nd 6 nd Fig. 6. All prticipnts were free of mesurble rbies ntibody t the strt of the study. The ntibody responses were very similr in the two groups. After one dose of vccine in distilled wter, 7 of 11 subjects developed more thn 1 IU/ml of l <E L LU. J) OkJ Z.4 V),2 LUj O r "'YET'l V4II,4L, _ (.t.-mtr.o _ VAINE m < <EO.I I I *. I s 2 3 4, SO 6 7 BOOSTER AYS AFTER INJETION OSE FIG. 5. Antibody response to booster dose of Wyeth or duck embryo vccine in prevccinted subjects (study 2).

7 VOL. 27, 1974 HUMAN IPLOI ELL RABIES VAINE 559 TABLE 5. WRVstudy 2: ntibody response ofpreviously unvccinted volunteers fter three doses (1) of Wyeth vccine Subject Antibody (IU/ml)b fter initil vccine dose on dy: (1) (1) 56 (1) <.4 <.16 < < < > < < < < < < < < Geometric menc All vccine doses were reconstituted in distilled wter nd given intrmusculrly in 1-ml mounts. 'See Tble 3. c See Tble 3. TABLE 6. WRVstudy 2: ntibody response ofpreviously unvccinted volunteers fter three doses (1) of Wyeth vccines Subject Antibody (IU/ml)" fter initil vccine dose on dy: (1) 14 28(1) 56(1) <.4 < <.4.16 < < < < < < < < Geometric menc The first vccine dose ws reconstituted in Al(OH), diluent. The second nd third doses were reconstituted in distilled wter. All injections were given intrmusculrly in 1-ml mounts. I See Tble 3. c See Tble 3. serum, nd of those receiving the first dose in Al(OH) 3 diluent, 4 of 9 showed this response. After two doses of vccine, 1 of 11 subjects who received vccine in distilled wter nd ll 9 who received the first dose of vccine in Al(OH)3 diluent exceeded the 1 IU/ml level. Subsequent bleedings showed tht ll prticipnts hd responded to levels higher thn 1 IU/mi. The highest level (25 IU/ml) ws chieved by two of them. The geometric men vlues in the tbles depict well the similrity of responses to the two schedules of vccintion, ruling out ny beneficil contribution by Al(OH),. As in Fig. 4, the responses which followed 16 doses of EV in previous study (4) were lso plotted in Fig. 6 for the purpose of comprison. As ws noted erlier, the response to EV ws higher thn tht elicited by one dose of WRV during the first 4 weeks, but it ws overtken rpidly fter the second nd third doses of WRV. Highest geometric men ntibody levels chieved were 3 IU/ml of serum for EV nd 9.56 IU/ml for WRV. ISUSSION The precision of results of serum ntibody titrtions in mice is generlly considered to be low. In terms of bsolute titers, the differences obtined on the sme smples in our lbortory nd t re cse in point. It is difficult,

8 56 LI Q II -i L- ;f Hi, HI I-4., (,.. 2 _4~~~~~~~- _ /-OTH- WYETF VAINE GROUPS REEivE A NE OSES ON AYS -O * AQUEOUS VAINE AY A1(o 2) VAINE AY I* Ev GROUP REEIVE AILY OSES BETWEEN AYS AN 1-, AN BOOSTER OSES ON A, S 23 AN 33 I A4 S 6 7c 84 9 znc AFTER JITI/-AL VANE OSE FIG. 6. Antibody response to three doses of Wyeth rbies vccine in previously nonvccinted subjects (study 2). ABASSO ET AL. therefore, if not impossible, to'mke ccurte comprisons from one test to nother in the sme lbortory or from one lbortory to nother. The inccurcy of the comprison becomes even greter when different lbortories use different test systems to determine the levels of rbies ntibody. For exmple, Wiktor et l. (14) ttempted to compre the immune response they obtined to one booster dose of WRV in group of previously vccinted subjects with tht reported by others (3) for group of previously nonvccinted individuls fter complete series of EV injections. They contrsted the 1:2, geometric men ntibody titer reched in 35 dys by the WRV group, s mesured by the plque reduction technique in grose-suspended bby hmster kidney cells (BHK 21/13S), to the 1:145 geometric men ntibody titer chieved by the EV group 4 dys fter initition of the vccintion series, but mesured by serum neutrliztion test in mice. The conditions in these two studies were too different for vlid conclusion to be derived from this comprison. omprison of results mong lbortories cn be mde more meningful by inclusion of defined reference ntibody preprtion in ech lbortory nd in ech test, regrdless of the test system. This cn even be more meningful if bsolute titers re henceforth converted to defined ntibody unit vlue, s is vilble for the Stndrd Antirbies Serum vilble from the Public Helth Service or from the World Helth Orgniztion. By this procedure, we were ble to demonstrte remrkbly good greement between our results nd those obtined t, despite the differences in bsolute ntibody titers. This consistency is lso borne out by the results shown in the tbles nd APPL. MIROBIOL. figures of this report obtined over severl months of testing. For the ske of more vlid interprettion of results mong rbies workers throughout the world, we urge tht rbies ntibody levels be henceforth expressed by ll in terms of interntionl units per milliliter of serum. Turning to the reltive effects of WRV nd EV, the results of our study demonstrte unequivoclly the greter immunogenicity of the former. This ws best illustrted by the secondry immune response of those previously given rbies vccine. Strting with essentilly the sme pre-booster ntibody level, the WRV group experienced 69-fold ntibody rise on the 28th dy fter the booster, wheres the highest ntibody rise ws only sevenfold in the EV group on the 14th dy. A prompt ntibody induction is mndtory in individuls exposed to rbies. When vccintion lone is considered, this my pose specil if not insurmountble problems. Stvitsky (11) found in rbbits tht the induction period for serum ntibody rnges between 3 nd 7 dys, depending on the nture of the ntigen, the site of ntigen injection, nd the presence of djuvnt which could enhnce the prolifertion of reticulum cells. Studies s systemtic s this hve not been crried out with rbies vccine in mn, but number of workers hve reported tht fter dily doses of nervous tissue or duck embryo vccine, the erliest ntibody detection occurred 6 to 8 dys fter the initil dose, nd this only in smll percentge of recipients (2, 4, 6, 7). Whether more potent rbies vccines or different injection schedules could mterilly shorten the induction time remins to be determined. However, detectble level of circulting ntibody cn be fforded within 24 h fter injection of n pproprite quntity of rbies immune globulin of humn origin (4). Where rpidity of ctive ntibody induction is not criticl, s in pre-exposure immuniztion, or when hyperimmuniztion of prospective donors of rbies immune plsm is crried out, it seems tht schedule of two or three doses of vccine hving the potency of the WRV used in our studies cn be very effective, lthough three doses my be required for hyperimmuniztion. In study 2, three doses of WRV induced circulting ntibody in ll 2 recipients, regrdless of whether the first vccine dose ws reconstituted in wter or in Al(OH)3 diluent (Tbles 5 nd 6). In fct, in 19 of the 2 recipients, the ntibody reched the level we require of potentil rbies immune plsm donors, i.e., 76 IU/ml of serum. Thus, three vccine doses spced 4

9 VOL. 27, 1974 HUMAN IPLOI ELL RABIES VAINE 561 weeks prt ppered to result in higher ntibody levels thn when three or four doses were given within shorter intervls (Tble 2). As regrds the effect of Al(OH)3, the conditions of its use in our studies my not hve been optiml. AKNOWLEGMENTS We thnk Howrd Tint, Wyeth Lbortories, Phildelphi, P., for supplying the humn diploid cell rbies vccine used in this study; J.. Loofbourow nd T. Y. ooper, Brbr Smith, nd Helen Hughes, University of liforni t vis, for their most vluble coopertion in one prt of this study; W. J. lemns nd R. L. Hyde, Florence, Ariz., for their unstinting coopertion in the other prt of this study; George M. Ber, enter for isese ontrol, Atlnt, G., for testing some of the serum smples for rbies neutrlizing ntibody; Sndr Miller nd Betty hin for cpble technicl ssistnce; the stff members nd students of the University of liforni t vis; the inmtes of Arizon Stte Prison, without whose voluntry prticiption these studies would not hve been possible; nd George A. Hottle nd Lynde I. Petter for vluble support in the preprtion of this mnuscript. LITERATURE ITE 1. Abdusslm, M., nd K. Bogel The problem of ntirbies vccintiion, p Pn Americn Helth Orgniztion Scientific Publiction no Wshington,.. 2. Atnsiu, P., M. Bhmnyr, M. Bltzrd, J. P. Fox, K. Hbel, M. M. Kpln, R. E. Kissling, A. Komrov, H. Koprowski, P. Lpine, F. Perez Gllrdo, nd M. Scheffer Rbies neutrlizing ntibody response to different schedules of serum nd vccine inocultions in non-exposed persons. Bull. W.H.O. 14: bsso, V. J Properties of rbies immune globulin of humn origin. J. Biol. Stndrd. 2: bsso, V. J., J.. Loofbourow, R. E. Roby, nd W. Anuskiewicz Rbies immune globulin of humn origin: preprtion nd dosge determintion in nonexposed volunteer subjects. Bull. W.H.O. 45: Fenje, P The problem of ntirbies vccintion, p In Americn Helth Orgniztion Scientific Publiction no Wshington,.. 6. Httwick, M. A., R. H. Rubin, S. Music, R. K. Sikes, J. S. Smith, nd M. B. Gregg Postexposure rbies prophylxis with humn rbies immune globulin. J. Amer. Med. Ass. 227: Hosty, T. S., R. E. Kissling, M. Scheffer, G. A. Wllce, nd E. H. ibble Humn ntirbies gmm globulin. Bull. W.H.O. 2: Loofbourow, J.., V. J. bsso, R. E. Roby, nd W. Anuskiewicz Rbies immune globulin (humn)-clinicl trils nd dose determintion. J. Amer. Med. Ass. 217: Peck, F. G., Jr., H. N. Powell, nd. G. ulbertson uck embryo rbies vccine. J. Amer. Med. Ass. 162: Sikes, R. K., W. F. lery, H. Koprowski, T. J. Wiktor, nd M. M. Kpln Effective protection of monkeys ginst deth from Street virus by post-exposure dministrtion of tissue-culture rbies vccine. Bull. W.H.O. 45: Stvitsky, A. B In vitro studies of the ntibody response, p In W. H. Tliferro nd J. H. Humphrey (ed.), Acdemic Press Inc., New York. Advnces in immunology, vol Wiktor, T. J., nd H. Koprowski, Successful immuniztion of primtes with rbies vccine prepred in humn diploid cell strin WI-38. Proc. Soc. Exp. Biol. Med. 118: Wiktor, T. J., F. Sokol, E. Kuwert, nd H. Koprowski Immunogenicity of concentrted nd purified rbies vccine of tissue-culture origin. Proc. Soc. Exp. Biol. Med. 131: Wiktor, T. J., S. A. Plotkin, nd. W. Grell Humn cell culture rbies vccine-ntibody response in mn. J. Amer. Med. Ass. 224: