Semi-Commercial-Scale Production of Japanese

Transcription

1 APPLuED MicROBioLOGY, June 1973, p Copyright i 1973 Americn Society for Microbiology Vol. 25, No. 6 Printed in U.SA. Semi-Commercil-Scle Production of Jpnese B Encephlitis Virus Vccines from Tissue Culture BALWANT SINGH, IK CHIN CHANG,I AND W. McD. HAMMON Deprtment of Epidemiology nd Microbiology. Grdute School of Public Helth, University of Pittsburgh, Pittsburgh, Pennsylvni Received for publiction 12 Jnury 1973 This study ws undertken to modify nd develop procedures for tissue culture-inctivted Jpnese B encephlitis (JBE) virus vccine production in lrge quntities. Vrious types of glss bottles were tried nd, considering mny dvntges, long cylindricl roller (CR) bottles were selected. Severl vribles were investigted including number nd volume of trypsinized cells to be seeded, volume of growth medium required for optimum cell growth, mount of clf serum, nd volume of hrvest medium for high-titer virus yield. A good confluent cell sheet in CR bottles ws obtined within week by incresing the clf serum from 4 to 10% nd when such tissue in CR bottle ws inoculted with 45,000 virl men tissue culture infective doses directly into the medium, the cytopthologicl effects (CPE) ppered on dy 5. High-titer virus yields were obtined when the hrvests were mde t 4+ CPE using medium 199 with 2% humn lbumin t ph 8.3 to 8.5. No pprecible gin in titer ws found from such hrvests by blending to relese intrcellulr virions. The production methods finlly dopted gve consistently good results, nd severl inctivted JBE virus vccine lots with minimum immunizing doses, rnging from to ml, were prepred using lrge number of CR bottles in simulted commercil-scle production system. Considering the dvntge of tissue culture s sfer nd possibly more potent source for vccine production, Rohityodhin nd Hmmon utilized primry hmster kidney cells (HKC) for obtining temperture-sensitive, ttenuted strin of Jpnese B encephlitis (JBE) virus from virulent isolte by seril pssge t reduced temperture nd plque selection (13, 14). Its biologicl properties nd stbility were tested; vrious chrcteristics nd specil mrkers were defined. Further evidence of modifiction of this virus strin ws derived from mosquito infection nd trnsmission tests nd its very low neurovirulence for lbortory nimls. This chnge in neurovirulence occurred with mintennce of high cell culture titers when incubted t permissive temperture, <37 C. It hs extremely low or bsent infectivity nd pthogenicity by the intrcerebrl (i.c.) route for monkeys, burros, "pthogen-free" wenling mice, nd hmsters, 'Present ddress: Michign Deprtment of Public Helth Lbortories, Lnsing, Mich nd usully hs n bsence of even infectivity by the subcutneous (s.c.) route for chimpnzees nd mn (1, 9, 13, 14). After thorough testing in severl species of nimls nd proper identifiction of this strin s JBE virus, it ws then inctivted with Formlin, for dditionl sfety, to produce n inctivted vccine for mn (3, 5, 16). Repeted doses of this vccine gve good ntibody responses of reltively long durtion when injected in different lbortory nimls, then in limited tril in mn, ll without locl or systemic rections (5, 6, 7, 9). The Investigtionl Drug Review Bord (AIDRB) of the U.S. Army reviewed nd pproved the production protocols. The Surgeon Generl of the Army uthorized these tests fter the protocols were forwrded to the Division of Biologics Stndrds, Ntionl Institutes of Helth nd recommended them for pprovl. The response to this vccine in nimls ppered to be essentilly the sme s tht of the live virus given in the sme mnner, pprently due to the extreme temperture sensitivity of the selected mutnt nd its inbility to 945

2 replicte t the restricting body temperture. Since n inctivted vccine prepred in cell culture by using n ttenuted strin of JBE virus would be sfer, both for those producing it nd the recipients, thn one of equl niml potency test similrly prepred in the mouse brin or chicken embryo with virulent strin, the former ws selected for production. The development of methods to prepre vccine in lrger quntities ws undertken so tht its efficcy might be tested in n dequte field tril nd so tht commercil production contrct might be redily nd quickly obtined should further lrge-scle use be needed t ny time. Lrge-scle production required severl modifictions from the pilot procedures described previously (2, 5). These minly concerned the cell culture growth, the virus inoculum, the size, shpe, nd movement of the continer, nd the method of virus hrvest, ll designed to obtin high-titered yield in lrge quntity with consistency nd economy of time nd mterils. This report describes the results of this developmentl reserch. MATERIALS AND METHODS Virus strin. The ttenuted JBE virus strin OCT-541, line 35 to 24 C, plque 4 to 5 described previously (1, 7, 14), hd two dditionl pssges in cell culture t 24 C. The seed virus suspension ws stored t -65 C nd contined men tissue culture infectivity doses (TCID50) per ml. Cell culture. The procedure for preprtion of primry HKC monolyers ws the sme s described previously (2, 16). The trypsinized pcked cells were mesured nd suspended first in 10 to 15 ml of outgrowth medium contining 10% inctivted clf serum nd 0.5% lctlbumin hydrolyste. Vigorous mixing under hood ws required to brek the clumps of cells. The cell suspension ws diluted further prior to seeding ccording to the type of bottles used nd the glss surfce re vilble for cell growth. For 32-oz (bout 945 ml), flt-surfce, prescriptiontype bottles, 1.0 ml of pcked trypsinized cells ws suspended in 300 ml of outgrowth medium. Ech bottle ws seeded with 50-ml smple, nd the medium ws chnged twice prior to virus inocultion (5). Proportiontely smller mounts, 30 nd 10 ml, were used for smller bottles, 16- nd 3-oz sizes (bout 473 nd 89 ml, respectively), nd lrger mounts, 400 ml, for lrge bottles (Pfizer bottles; 28 by 23 by 6 cm). Incubtion of these s well s other types of bottles described below, during cell growth, ws t 37 C. Other vessels used most were cyclindricl roller (CR) bottles 685 mm in length with 1,200-cm2 surfce re (Flow Lbortories Inc., Rockville, Md.). For CR bottles the trypsinized cells were dispersed t concentrtion of 1.5 ml of pcked trypsinized cells per 100 ml of outgrowth medium. Other concentrtions of trypsinized cells (1 nd 2 ml/100 ml of outgrowth SINGH, CHANG, AND HAMMON 946 APPL. MICROBIOL. medium) were lso tried but were less stisfctory. The diluted cells were mixed for 15 to 20 min on mgnetic stirrer, nd then 100 ml of suspended cells ws delivered into ech CR bottle with 25-ml utomtic syringe. The cells were distributed throughout the bottle by gentle shking nd rolling before plcing it on rotting drum (0.2 rpm). After 4 to 6 h, the bottles were removed from the drum nd gin gently shken to disperse cells more evenly nd to prevent the tendency for ggregtion of cells t certin res on the glss surfce. At 18 to 20 h fter seeding, n dditionl 100 ml of outgrowth medium ws dded to ech bottle. The first complete chnge of outgrowth medium ws mde 4 to 5 dys fter seeding, replcing it with 200 ml of the sme medium per bottle. A second medium chnge ws mde 2 to 3 dys lter with 200 ml of mintennce medium. A good cell sheet ws usully obtined 8 dys fter seeding. The outgrowth medium consisted of 10% clf serum; 1% vitmin stock (Microbiologicl Assocites, x 100 concentrtion); nd 0.02 g% NHCO, (0.5% of 4% stock solution), g% L-glutmine (1% of 2.92 g% stock solution), nd 86.5% of 0.5% solution of lctlbumin hydrolyste in Hnks blnced slt solution (HBSS) nd 1% ntibiotic mixture consisting of 20,000 U of Penicillin G, 1,000 U of polymyxin B sulfte, 4 mg of streptomycin, nd 2,500 U of mycosttin. The mintennce medium differed from outgrowth medium only in the mount of clf serum (4%) nd the ph (7.5). The exct composition of these medi hs been reported previously lso (10). Virus inoculum. Dilutions of the seed virus were prepred in HBSS contining 0.2% bovine lbumin. The mintennce medium ws removed from bottles, ech 32-oz (bout 945 ml) bottle ws inoculted with 0.5 ml of virus diluted to contin pproximtely 5,000 TCID5O, nd ech CR bottle ws inoculted with 4.5 ml of virus dilution contining 45,000 TCID50. The virus ws left to dsorb for 1.5 h t 30 C, s the bottles were rocked nd turned periodiclly. Ech 32-oz bottle then received 50 ml of mintennce medium nd ech CR bottle recieved 200 ml of the sme. The incubtion ws crried out t 30 C in the rottor. After 3 dys, the bottles were observed dily for cytopthologicl effects (CPE). Virus hrvest. About 5 dys fter virus inocultion, the first signs of CPE ppered in ech type of continer. The mintennce medium ws then removed, nd the monolyer ws wshed two times with HBSS by using bout 20 ml for ech 32-oz bottle, nd 50 ml for ech CR bottle. The bottles were replenished with the finl hrvest medium consisting of Prker medium 199 (stock concentrte X10, Microbiologicl Assocites) with 2% humn lbumin (HAL); the ph ws djusted to 8 to 8.5. Thirty milliliters of hrvest medium ws dded for ech 32-oz bottle nd 100 ml ws dded for ech CR bottle. The bottles were incubted gin t 30 C. After 10 to 16 h, the tissue monolyer showed CPE over more thn 75% of the re, nd virus ws hrvested fter shking to loosen the remining cells. For CR bottles, closed system ws devised for crrying out virus hrvest nd ll

3 VOL. 25, 1973 JBE VIRUS VACCINES 947 chnges of medium. The contents of ll bottles were pooled nd centrifuged t 100 x g for 30 min t 4 C, nd the superntnt fluid ws filtered through single membrne filter disk (0.22-Mm pore size; 142-mm dimeter; Millipore Corp.). Alterntively, centrifugtion ws performed t 4,080 x g for 30 min in Sorvll centrifuge nd ws not filtered fterwrds. Inctivtion. The filtered or clrified virus hrvest ws inctivted by using 10% Formlin t finl concentrtion of 1: 4,000 in wter bth t 37 C with mechnicl circultion nd precision temperture control. The required mount of 10% Formlin ws dded drop by drop s the flsk ws shken constntly for proper mixing (16). The inctivtion ws llowed to proceed for three times longer thn preceeding trils hd demonstrted ws the minimum period required to inctivte ll virl infectivity of lot of similr titer s mesured by test on 0.4 ml (four 0.1-ml tubes). The length of inctivtion vried with the titer of virus hrvest nd ws clculted ccordingly on the bsis of recorded experience (3). During inctivtion, smples were removed t vrious intervls, mpoules were prepred, nd the shell ws frozen nd kept t -65 C for titrtions of infectivity. Virus ssy. (A) Hemgglutintion titers. The test ws crried out ccording to modified method of Clrke nd Csls described previously (2). (B) Infectivity titrtions. Hlf-log virus infectivity titrtions were crried out in tubes of secondry HKC cells. HBSS contining 0.2% bovine lbumin ws used s diluent to prepre the seril virus dilutions. Four secondry HKC tubes were inoculted from ech of the series of hlf-log dilutions, with 0.1 ml of inoculum per tube, nd were incubted t 30 C. For tests during the ltter prt of inctivtion, when little or no virus ctivity ws expected, only undiluted mteril ws tested. The mintennce medium ws chnged fter bout 5 dys. The cultures were observed until CPE ws complete in ll positive tubes. The TCIDo ws clculted by the Reed Muench method s reported previously (2). Potency test. The minimum immunizing dose (MID) of the vccine ws determined by the mouse chllenge test s developed by Sbin et l. (15) nd modified only slightly by Drwish nd Hmmon (4). In brief, groups of young dult mice of stndrd ge nd weight re given two doses of vccine intrperitonelly (i.p.) t intervls of 3 dys in dilutions rnging from 1: 20 to 1: 1,280; subsequently, ll were chllenged by i.p., together with uninoculted controls, with highly virulent strin of JBE virus. The chllenge virus dose ws dequte to kill 90 to 100% of the control mice s reported previously (4, 17, 18). Chrcteriztion nd sfety tests. The method for identifiction of the live virus hs been reported in erlier publictions of this series (5, 9, 14). Sfety tests, including those to detect contminting viruses, were performed on ech vccine lot to be used for humn inocultion. These included tests prior to nd fter inctivtion. Requirements of the Division of Biologics Stndrds, Ntionl Institutes of Helth (12), were followed. As reported previously (5, 7, 8), virus hrvest both prior to nd fter inctivtion ws tested. These tests included inocultion of vrious cell cultures nd vriety of lbortory nimls s required for poliomyelitis nd mesles vccines. RESULTS Effect of concentrtion of clf serum in growth medium. The clf serum ws inctivted t 56 C for 30 min nd dded to the outgrowth medium in concentrtions of 4 or 10%. Clf serum t the 10% level visibly enhnced the cell growth nd prticulrly improved the visibility of the cells by microscopy s compred with lower concentrtions. The cell sheet ws completely confluent in 8 dys. This effect ws more evident in the CR bottles. The virus yield from such tissue ws lso improved. In three different experiments, slightly higher infectivity nd HA titers were obtined when 10% clf serum ws used (Tble 1). Effect of size of virus inoculum nd volume of medium. A series of experiments ws performed with vrious sizes nd types of bottles, different input multiplicity doses, chnges in the cell growth medium, volumes of mintennce medium, nd time of chnge to 199 medium for virus hrvest. In generl, with lrger inocul of virus the CPE ppered erlier thn reported previously (2), nd with lower inocul the first ppernce of CPE ws 1 to 2 dys lter thn in the erlier study. The infectivity titers of virus hrvest obtined from different types of bottles under wht pproched optiml conditions for ech re shown in Tble 2. The highest titer ws obtined from CR bottles. This might be ttributed to the smll volume of hrvest medium used in reltion to the re of the cell sheet which ws mde possible with rottion. Time required for virus bsorption. Time required for virus bsorption before dding mintennce medium ws investigted with CR bottles. As shown in Tble 3, the results of experiment 1 nd 3 indicted tht virus could be dded directly to the medium without ny loss in finl titer. TABLE 1. Effect of clf serum concentrtion in outgrowth medium on finl virus yield Log,, TCID,,/ml Reciprocl of HA Clf of hrvest" titer per 0.4 ml serum (%) Expt 1 Expt 2 Expt 3 Expt 1 Expt Infectivity titrtions done in HKTC using four tubes per dilution nd hlf-log dilution fctor. The culture tubes were incubted t 30 C.

4 948 SINGH, CHANG, AND HAMMON APPL. MICROBIOL. Effect of cell scrping nd blending, nd ph on the virus hrvest. The necessity of scrping off the remining cells with rubber policemn to obtin unrelesed virus t the time of hrvest ws investigted in hrvest medium with ph of 8.4. Three different experiments showed tht by shking the bottle most of the cells could be dislodged from the glss. The ddition of cells broken nd removed by scrping did not significntly improve the titer of the hrvests (Tble 3). It ws lso found tht blending to rupture cells, s reported previously (2, 5), ws unnecessry under these conditions. Previous studies proved tht medium of ph 8 plced on the cells 6 to 24 h before hrvest gve higher virus titer yield thn medium of ph 7 (2). The effect of ph from 7.61 to 8.85 ws explored. As illustrted in Tble 4, the results indicte tht virus titers were highest when TABLE 2. TABLE 4. Effect of ph of 199 hrvest medium on virus yield in 32-oz bottles Expt no. ph Log,. TCID,./ml" ; Includes 2% bovine or humn lbumin depending on whether the lot ws expected to be used for mn. Titrted t 30 C in secondry HKTC using four tubes for ech hlf-log intervl dilution fctor. Virus yield from different types of bottles Surfce re Inoculum Vol of Vol of Virus yield" Type of for tissue per bottle mintennce hrvest (logutcid5/ bottle (cm2) (dilution) medium (ml) medlum 0.1 ml) 3 oz (c. 89 ml) ml OflO oz (c. 473 ml) ml of 10-' oz (c. 945 ml) ml of Pfizer ml of CRC ml of dilution contins 1,000 TCIDdO.1 ml. bfor infectivity titrtions four HKTC tubes were used per dilution with hlf-log dilution fctor; tubes were incubted t 30 C. C CR, Cylindricl roller. TABLE 3. Effect of virus bsorption nd scrping the remining cells from CR bottles on virus hrvest in 199 medium, ph 8.4 No. of Virus Tretment Virus yield" Expt. No. bottles bsorbed of cells (log10, TCID,00/0.l ml) pooled or not 1 3 Absorbed Scrpedc Absorbed Not scrpedd Not bsorbed Scrped Not bsorbed Not scrped Absorbed Scrped Absorbed Not scrped Absorbed Scrped Not bsorbed Not scrped 8.92 For virus bsorption, bottles were incubted t 30 C for 1.5 h nd rolled by hnd every 15 min prior to dding mintennce medium. For infectivity titrtions four HKTC tubes were used per dilution with hlf-log dilution fctor; tubes were incubted t 30 C. c Remining cells were scrped by using rubber policemn. d Virus hrvest ws mde fter shking the bottles to loosen the remining cells.

5 VOL. 25, 1973 JBE VIRUS VACCINES 949 ph from bout 8 to 8.5 ws used. A ph bove 8.5 did not pper to improve the finl virus hrvest. At ph of 8 to 8.5 most of the cells hd detched from the glss surfce t the time of hrvest. Apprently, this ph lso helped to relese virus prticles from the cells, since blending did not improve the titers. Effect of filtrtion. Different methods of removing cell debris from the hrvest fluid were tried. In previous studies, fter centrifugtion, Millipore filtrtion, using 0.45-m pore membrne in combintion with 0.22-m pore membrne, ws found most suitble. This observtion ws further investigted nd, s illustrted in Tble 5, 0.22-,um pore disk ws tried with nd without 0.45-;im disk in different experiments. In these selected sets, nd others, when the filter ws not primed, the loss of virus infectivy during filtrtion vried from 0.2 to 0.5 log/0.1 ml. However, these losses were reduced when filters were primed with bout 10 ml of Prker 199 medium contining 2% HAL. Amount of virus lost by discrding the centrifuged pellet of the finl hrvest. Previous tests indicted no pprecible difference in virus content between the superntnt of the centrifuged fluid hrvest (without cells scrped off) nd the homogenized superntnt of the totl hrvest (fluid hrvest plus cells scrped off). The virus in the sediment of the totl hrvest fter centrifugtion t 4,080 x g for 30 min ws now determined. The sediment ws treted by blending for 5 min to rupture ny intct cells, then diluted to the originl volume for titrtion. The infectious titer of the originl superntnt fluid ws 109 /ml nd ws for the resuspended sediment. The HA titers were 1:512 nd 1:32, respectively. It ws thus concluded tht over 99.9% of the totl infectivity ws present in the superntnt fluid nd tht n insignificnt proportion of the virus ws discrded in the sediment. Consistency of virus production in CR bottles. Suitbility of CR tube bottles for producing high titers of virus for inctivted vccine ws investigted. Virus hrvest ws obtined using the optiml methods described bove, i.e., 10% clf serum in outgrowth medium, virus inoculum contining 45,000 TCID5JCR bottle, hrvest medium with high ph (8.3 to 8.5), nd centrifugtion of virus hrvest t 4,080 x g, but no filtrtion. In repeted experiments, the virus yields were TCID5dO.1 ml or higher s shown in Tble 6. The inctivted vccine prepred from such virus hrvest gve MID vlues rnging from to 0.017, which re comprble to those reported previously when the vccine ws produced in smll lots using 3- or 16-oz bottles. These tests were performed by chllenge inocultion of mice in n ntigenic extinction type test (17). DISCUSSION For n inctivted vccine high-titer virus yield with minimum foreign protein is necessry. It is lso most desirble tht the method of vccine production be s simple s possible nd require minimum hndling nd processing. TABLE 5. Effect of Millipore filtrtion on infectivity titer of virus hrvest in 199 medium t ph 8.0 to 8.2 Expt Type of Before or fter Filters with or Porosity of no. hrvest" filtrtion without priming millipore Log60 TCID.dml filters 1 Fluid Before 9.60 Fluid After Unprimed 0.22 Am Am Fluid Before 8.72 Fluid After Unprimed 0.22Am Complete Before 8.83 Complete After Unprimed 0.22Mm Fluid Before 9.17 Fluid After Primed 0.22 Am Complete Before 9.17 Complete After Primed 0.22gm Fluid Before 9.25 Fluid After Unprimed 0.22Am Fluid Before 9.0 Fluid After Primed 0.22Mm 9.0 Complete hrvest ws mde by blending fluid nd cell hrvest. " A 10-ml smple of 199 medium contining 2% humn lbumin ws pssed through Millipore filters (to prime) before virus hrvest.

6 950 SINGH, CHANG, AND HAMMON APPL. MICROBIOL. TABLE 6. Infectivity titers of different lots of virus hrvest from cylindricl roller bottles nd MID fter inctivtion Dte of virus No. of Trypsinized Virus hrvestb MIJY hrvest bottles cells per bottle Log,, TCID,JO.1 m1' inctivted (ml) (h) (l 8/19/68d /18/68d < /1/68d /23/68d /18/ /20/ /13/ /7/ /14/ /15/ /3/ /20/ /23/ Four HKTC tubes were used per dilution with hlf-log dilution. 'Inctivted using 1: 4000 Formlin t 37 C. c MID determined by mouse potency test. Two equl doses of vccine on dy 0 nd 3, nd chllenge on dy 7. dvirus hrvest prepred by scrping the remining cells from glss surfce. The experiments reported in this vccine development project were designed to chieve these ims. A systemtic study of growth experiments, prticulrly in lrge bottles using Pfizertype nd CR bottles with 640 nd 1,200 cm2 surfce res, respectively, ws undertken in order to dpt vccine production from smll to semi-commercil scle. A consistently high-titered virus yield from Pfizer bottles could not be obtined. We believe the difficulties were (i) the lck of n dequtely flt bottom, resulting in the need to employ lrge volume of medium to cover ll tissue dequtely, nd (ii) high rtio of ir volume to tissue surfce. In both respects, the long CR bottles offered n dvntge. The CR bottles provided lrge surfce-to-medium volume rtio (1,200 cm2 per 100 ml of medium) nd low rtio of ir volume to tissue surfce. The rottion of bottles insured contct of minimum mount of medium with ll cells. The tremendous tissue surfce in CR bottles necessitted chnge in volume of outgrowth medium nd in the concentrtion of clf serum needed for high-qulity contiguous cell growth. The exct function of clf serum for HKC growth ws not studied but, s reported in Results, incresing the clf serum in the outgrowth medium beyond 4% produced cells which gve n incresed yield of virus fter inocultion. Mny workers hve ttempted to explin the function nd the role of serum for the growth of mmmlin cells. It hs been suggested tht serum cts s reservoir for essentil nutrients nd tht serum proteins protect cells ginst n unfvorble environment. It hs lso been reported tht serum is required to inctivte the residul trypsin remining from enzymtic digestion of monkey kidney cells nd the proteolytic enzymes subsequently synthesized by the cells (19). Experience with HKC culture showed tht the monolyers were dversely ffected when serum-free medium ws used from the time of inocultion, resulting in lower virus yield, nd tht 2% HAL could not substitute for serum during the period of erly virus growth (2). Consequently, virus ws inoculted in the presence of clf serum, but t the first sign of CPE the serum-contining medium ws removed nd substituted with serum-free Prker 199 medium contining 2% HAL to be hrvested fter 10 to 16 h. It ws shown erlier tht the JBE virus is relesed grdully over considerble period of time from cell sheet nd tht the stbility of this relesed virus is enhnced by n lkline ph (2). It ws shown tht medium with ph of 8, plced on the cells 6 to 24 h before hrvest, yielded higher titers of virus thn t low ph. This ws exmined further to see if the optimum ph ws being used. It ws found tht pek yield occurred between ph 8.3 nd 8.5. This rnge is now stndrd. Generl prctice in vccine mnufcture is, t the time of hrvest, to scrpe ny remining cells from the glss surfce nd then rupture them by some mens to obtin unrelesed virus prticles. However,

7 VOL. 25, 1973 JBE VIRUS VACCINES 951 these steps require more hndling time nd risk contmintion. The high ph (8.3 to 8.5) detched most of the cells nd probbly ruptured most cells. In ny cse, no significnt increse in infectious titer ws obtined by further mechnicl scrping or rupturing. Since we found tht the immunogenicity of n inctivted virl vccine ws directly proportionte to the infectious virus titer of the hrvest prior to inctivtion, it ws of prime importnce tht whtever method ws used for the removl of cell debris nd virl ggregtes should not decrese the titer (17). In our studies, virus hrvest ws originlly filtered but, despite gret vriety of methods used, there ws usully substntil loss in infectivity. It ws found tht centrifugtion for 30 min t 4,080 x g ws sufficient to ccomplish the sme purpose without ny detectble loss in virus titer. Becuse centrifugtion ws permitted to substitute for filtrtion in the mnufcture of inctivted mesles virus vccine in the United Sttes, this chnge ws considered cceptble, prticulrly since the JBE virus (OCT-541) used is very highly ttenuted, temperturesensitive strin shown to be essentilly nonpthogenic for mn nd vrious lbortory nimls (1, 8, 13, 14). We hve, therefore, dopted centrifugtion s substitute for filtrtion. This is one of the resons we fvor selection of virulent strins for inctivted vccines. The cceptble MID for licensing requirements of the U.S. Public Helth Service of the previously licensed vccine is 0.02 ml or less. The inctivted JBE virus vccine prepred for using these modifictions in production method gve product rnging from to 0.017, well within this limit. ACKNOWLEDGMENTS This investigtion ws crried out under the sponsorship of the Commission on Virl Infections, Armed Forces Epidemiologicl Bord, nd ws supported by the U. S. Army Medicl Reserch nd Development Commnd, Deprtment of Army, under Contrct No. DA MD-2042, nd the Public Helth Service reserch grnt AI from the Ntionl Institute of Allergy nd Infectious Diseses. We thnk R. D. Bhn nd Mry White for their technicl ssistnce. LITERATURE CITED 1. Berge, T. O., nd D. A. Stevens, Ctlogue of viruses, rickettsie, chlmydie, (4th ed.) p. 25. Americn Type Culture Collection. Rockville, Md. 2. Drwish, M. A., nd W. McD. Hmmon Studies on culture inctivted vccine for mn. (1) Obtining mximum titers of virus using n ttenuted strin of OCT-541. J. Immunol. 96: Drwish, M. A., nd W. McD. Hmmon Studies on culture inctivted vccine for mn. (2) Chrcteristics of inctivtion of n ttenuted strin of OCT-541. J. Immunol. 96: Drwish, M. A., nd W. McD. Hmmon, Studies on culture inctivted vccine for mn. (3) Potency of n ttenuted strin of OCT-541. J. Immunol. 96: Drwish, M. A., nd W. McD. Hmmon Studies of culture inctivted vccine for mn. (4) Preprtion nd chrcteriztion of vccine lot for humn tril. Amer. J. Trop. Med. Hyg. 16: Drwish, M. A., W. McD. Hmmon, nd G. E. Sther Studies on Jpnese B encephlitis virus vccines from tissue culture. IX. Humn responses to hmster kidney tissue culture inctivted vccine. Amer. J. Trop. Med. Hyg. 16: Hmmon, W. McD., M. A. Drwish, G. E. Sther, nd B. Singh Response in dults to n inctivted cell culture vccine employing highly ttenuted strin, OCT P1.4-5, p Immuniztion for Jpnese encephlitis. Igku Shoin, Ltd., Tokyo. 8. Hmmon, W. McD., S. Rohityodhin, nd J. S. Rhim Studies on Jpnese B encephlitis virus vccines from tissue culture. IV. Preprtion nd chrcteriztion of pool of ttenuted OCT-541 line for humn vccine tril. J. Immunol. 91: Hmmon, W. McD., S. Rohityodhin, nd J. S. Rhim Studies on Jpnese B encephlitis virus vccines from tissue culture. IV. Preprtion nd chrcteriztion of pool of ttenuted OCT-541 line for humn vccine tril. J. Immunol. 91: Ngi, K., nd W. McD. Hmmon Plque studies with certin group B rboviruses. I. Jpnese B encephlitis virus strins on hmster kidney nd chick embryo tissue cultures. Proc. Soc. Exp. Biol. Med. 117: Ntionl Institutes of Helth Minimum requirements: Jpnese encephlitis vccine, chick embryo type, dried, p August Public Helth Service Publiction no Revision, by Division of Biologics Stndrds of the Ntionl Institutes of Helth on Biologicl Products. 13. Rohityodhin, S., nd W. McD. Hmmon Studies on culture. II. Development of n ttenuted strin of virus. J. Immunol. 89: Rohityodhin, S., nd W. McD. Hmmon Studies on Jpnese B encephlitis virus vccine from tissue culture. m. Further selection nd testing of ttenuted virus lines from OCT-541. J. Immunol. 89: Sbin, A. B., C. E. Duffy, J. Wrren, R. Wrd, J. L. Peck, Jr., nd I. Ruchmn, The St. Louis nd Jpnese B types of epidemic nd encephlitis. Development of noninfective vccines: report of bsic dt, J. Amer. Med. Ass. 112: Singh, B., M. A. Drwish, nd W. McD. 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