KJU AD RESISTANCE TO VIRAL CHALLENGE IN THE DAYS IMMEDIATELY FOLLOWING VACCINATION. Jordi Casals-Ariet. Yale University.

Transcription

1 P-»-WÄ«*&lJJ«HlllH«JIU.UUr-l,U P5W»PH«PMPJP^B«B*W -» -- " «JPSLKU ' " ' "' - ' ' "- ' ' AD RESISTANCE TO VIRAL CHALLENGE IN THE DAYS IMMEDIATELY FOLLOWING VACCINATION Jord Casals-Aret Yale Unversty Prepared for: Army Medcal Research and Development Command May 974 DISTRIBUTED BY: KJU Natonal Techncal Informaton Servce U. S. DEPARTMENT OF COMMERCE 5235 Port Royal Road, Sprngfeld Va. 225

2 THIS DOCUMENT IS BEST QUALITY AVAILABLE. THE COPY FURNISHED TO DTIC CONTAINED A SIGNIFICANT NUMBER OF PAGES WHICH DO NOT REPRODUCE LEGIBLYo

3 j.^r^t-ww " >" +-.J~r~^Trr,,.- ~..,,jr,,t«^sh ajsprr.»?t--,-j?j-.v:^-..rmm^tp^, ,,-,-^ ^.-n - -.U-.JJ.jtl. - " -»,.-le ursod JlCUS.TV CLASS! FlCATlON Or" ".'HIS PAGU f«7cn /> F.nlrtrd) REPORT DCCU^EKTATOM PAGE '.. SE.PC...T NUMSCr 2. COVT ACCESSION NO I READ INSTRUCTIONS BEFORE COMPLETING rorm NUM3EH 4. TlTLK,"-n.J 'udtloj Resstance to vral challenge n the days mmedately followng vaccnaton Annual Progress Report COVERED 6. PERFORMING ORC. REPORT NUMBER 7. AUTnOr*J Jord Cnsals-Aret P RrOHM!NC GHGAM I ATlQN NAME AND ADDRESS ^ Unvjrs-y 60 College Street Connectcut C2.-.r?»u-I.SG OFFICE NAME AND ADDRESS Deparor.-:u of the Army ITS Arssy y.c±zc\ Research and Development Command Sas.-._ --.jrcr.. DC zuj-+ f*. MONITORING AGENCY KÄME A AOORESSflf dlttamu horn Contntlnt Olle») 8. CONTRACT OR GRANT NUMBER/«; DADA7-72-C-209 «0. PROGRAM ELEMENT. PfOjE-T. TASK AREA a WORK UNIT NUMBERS 2. REPOR DATE May 974. NUMBER «f PAGES *33 IS. SECURITY CLASS. (»I thlm tmport) 5a. DECLASSl FlCATlON/DOWNGRADING SCHEDULE I '.6. DSTS.ä.jTON STATEMENT (a.' thlm Report) Approved for publc release; dstrbuton unlmted. > IT. DISTRIBUTION STATEMENT fo/ «ft««««tract «tw«ja s«* 20, */ mtttmt mm R*p*n) -: I!3. SüP>-t.tM<.STARY NOTES t!5. KEv t - -z_ %?. 'c.-r. nu* <w» r***r#» «/l»»i «** **.«"*» *»v*!4****f* h? b*?t rtyfr^frf^ antbodes blockng of antbodes nonspecfc resstance vrema.f.nutf «-. r«v«.* ««**«/ n«c»*««/y «no* dentty by Mock number) en J. /naned wen I7D yellow fever vrus, ntracerebrally challenged from.-~ lecer wth the French vscerotropc stran of yellow fever, were lly protected; when the challenge was wth a neurotropc stran, ^röwecten was observed. The former stran was slow to nvade and :'.-. wth an average survval tme 2 to 2% tmes longer than that of '.. Challenge wth a small dose of VEE vrus of mce vaccnated.ccne revealed a moderate but sgnfcant protecton 24 hours, but ft 7 ".. J EDITION OF > NOW tft IS OBSOLETE "-NATONÄ! TECHNICAL INFORMATION SERVICE SECURITY CLASSIFICATION OF THIS PACE (TRrtn DM /»«* Spfn«! -nt^---^. ---

4 fr**,.-^-^jf^^ytt ^,^ SECURITY CLASSIFICATION O* TNIS PAStjfAtj Palm BufrwQ /^C not 7 days, after vaccnaton; tha la another example of non-specfc early resstance after vaccnaton. Cytoxan gven repeatedly at the tme of vaccnaton and challenge to nhbt early non-specfc resstance after vaccnaton. It would seem that wth an adequate model t can be shown that ths early resstance s ndependent of the Imnune system. Resstance followng vaccnaton of gunea pgs wth EEE vrus s apparent n anmals challenged 3 or more days later; wth *n earler challenge resstance seems to exst but the small numbers of.mmals permtted no statstcally sgnfcant estmates. Ttraton of antbodes followng challenge of gunea pgs wth arbovruaes has been used to determne development of early and late resstance followng vaccnaton. The vaccne, when effectve, blocks the producton of antbodes; observatons have been made wth EEE and JE vruses. MCUMTT CkASMnCATMN OP THIS PASCfllM«

5 H -2- SUMMARY Ilce vaccnated wch 7D yellow fever vrus, ntracerebrally challenged from to 4 days later wth the French vscerotropc stran of yellow fever, were substantally protected; when the challenge was wth a neurotropc stran, Asb, no protecton was observed. The former stran was slow to nvade and cause death wth an average.survval cne 2 to 2 /2 tmes longer than that of Asb stran. Challenge wth a small dose of VEE vrus of mce vaccnated wth JE vaccne revealed a moderate but sgnfcant protecton 24 hours, but :ot 7 Jays, after vaccnaton; ths s another example of non-specfc early resstance after vaccnaton. Cytoxan gven repeatedly at the tme of vaccnaton and challenge faled Co nhbt early non-specfc resstance after vaccnaton. It would seem that wth an adequate model t can be shown that ths early resstance s ndependent of the mmune system. Resstance followng vaccnaton of gunea pgs wth EEE vrus s appr.rent n anmals challenged 3 or more da>s later; wth an earler challenge resstance seems to exst but the small numbers of anmals permtted no statstcally sgnfcant estmates. Ttraton of antbodes followng challenge of gunea pgs wth arbovruses has been used to determne development of early and late resstance followng vaccnaton. The vaccne, when effectve, blocks tne producton of antbodes; observatons have been made wth EEE and JE vruses. FOREWORD In conductng the research descrbed n ths report, the nvest- «ntor adhered to the "Gude for Laboratory Anmal Facltes and Care", as promulgated by the Commtcee on the -Gude for Laboratory Anmal Resources, Natonal Academy of Scences-Natonal Research Councl.

6 -3- TABLE OF CONTENTS SUMMARY 2 FOREWORD 2 TABLE OF CONTENTS 3 BODY OF BEPORT 4 I. Statement of problem... 4 II. Background 4 III. Approach 5 IV. Materal«and method» 5 Anmala 5 Vruses... 6 Vaccnes... «6 Vaccnaton 6 Challenge 6 Admnstraton of Cytoxan 7 Antbodes 7 Interpretaton of challenge results... 7 V. Results 7 Effect of Cytoxan on early-phase resstance.. 7 Early resstance aganst yellow fever and.. VEE vruses n alee 9 Early resstance aganst EEE vrus t.... gunea pgs, followng vaccnaton... 0 Development of early resstance aganst JE.. vrus In gunea pgs, sfter vaccnaton.. 2 Development of complement-fxng antbodes.. n gunea pgs followng noculaton of * several group B arbovruaea 4 VI. Dscusson 4 VII. Conclusons 5 BIBLIOGRAPHY 6 TABLES 7 DISTRIBUTION LIST 32

7 -4- BODY. OF REPORT I. Statement of problem Vaccnaton aganst vrus nfectons of man s an accepted preventve procedure when the vrus n queston s unversally prevalent n tme?nd space. Arbovrus nfectons are, n general, geographcally restrcted; n addton, they are numerous and may present antgenc varetes. Under the crcumstances, even avalable vaccnes or other* that may be developed n the future ere to be recoaaended only when travellng nto an endemc zone or n the face of outbreaks n heretofore free areas. In such stuatons, for a ratonal use of the vaccne t would be helpful to have nformaton concernng the tme needed, n days or hours, for substantal effectve mmunty to develop followng vaccnaton. II. Background Over 350 arbovrus serotypes are currently recognzed of whch only a snal number, 20 to 25, are known to cause dsease of man to an extent or severty whch warrant efforts to develop and apply preventve measures, among whch s vaccnaton. Among these vruses, n addton to yellow fever for whch an effectve vaccne s n use are: the equne encephalts vruses -EEE, WEE, and Venezuelan (VEE); St. Lous (SLE), Russan tck-borne (RSSE), Japanese (JE), a vaccne for whch s wdely used n Japan, Kyasanur Forest dsease (KFD) and the four dengue serotypes, ether as classcal dengue or n the forms of uengue henorrhagc fever and dengue shock svndrome. It s concevable that nfecton by Crmean hemorrhagc fever-congo (CHF-C) vrus a.ay, n the future, be added to the lst n vew of ts wde area of dstrbuton and the severty of the dsease that t can cause n certan countres. Sauattcoua oz sequental nfecton of an ndvdual by antgencally closely related vruses, or ther antgens, may be harmful, as has Ueeu suggested to explan the dengue shock syndrome (,2). Recent expermental work n ths laboratory (3) shows that wth Lagos bat vrus there s a delay n the evoluton of llness n mmunosuppresseo mce compared wth that n mmuno-competent controls; and that the admnstraton of hypermmune serum to the nfected jounosuppressed anmals results n an acceleraton of ther llness. It would seem therefore that unless a vaccne gves effectve substantal protecton when admnstered almost smultaneously wth the actual nfecton, the use of a vaccne may be nadvsable. It has been shown n ths laboratory (see frst year's annual progress report, and 4) that mce vaccnated once wth formaln-

8 J -5- nactlvated suspensons of souse bran tssue Infected wth EEE, VEE, JE, and Banz vruses were protected aganst a challenge gven less than 24 hours after vaccnaton. The protecton had two phases, the frst lastng from 24 to 48 hours after vaccnaton whch was nonspecfc,.e., appeared between antgenlcally unrelated vruses; ths was followed by a specfc phase demonstrable only aganst the homologous vrus challenge (possbly also aganst related vruses; ths was not nveatgated). There was evdence that the early phase was assocated wth an nterferon-llke actvty whch was manfested by the serum of vaccnated mce«an mmunosuppressant, Cytoxan (cyclophosphamlde), was effectve, as antcpated, n suppressng resstance to a challenge n the course of the specfc phase; ts effect on the early, nonspecfc phase had been unpredctable.. Approach The prncpal goal of the work descrbed n ths report has been to determne whether development of early resstance followng vaccnaton could be observed n a speces other than the mouse; the f.uaea pg was chosen. The susceptblty of gunea pga to arbovruses mportant aa cause of dsease of man yet relatvely safe to work wth n the laboratory s, f anythng, lower Chan that of mce; t would be necessary to work wth large numbers of anmals whch s an Impossblty mposed by cost and the avalable housng. It was decded, afcer prelmnary tests, to try to develop crtera other than death or survval as a measure of resstance and susceptblty; vremla and, partcularly, antbody development have been used. In contnung our studes wth mce as expermental anmals, two challenge vruses were added wth whch lttle or no nformaton s avalable: VEE and yellow fever. All the laboratory personnel have been vaccnated aganst these vruses and are known to be mmune, Fnally, addtonal studes have been conducted on the effect of Cytoxan on the early phase of resstance followng vaccnaton. IV. Materals and Methods Anmals. Mce derved from the harlea Rver CD(R)- stran were used, random bred n a barrer colony mantaned n thla laboratory. In experments n whch VEE was the challenge vrus were used mce from 80 to 90 days old; for test usng other challenge vruses the mce were between 30 and 40 days old. Mce from to 4 days old were employed for preparaton of vaccnes, ttrston of neutralsng (N) antbodes and preparaton of vrus stocks sad complement-fxng (CF) antgens.

9 -O- Cunea pgs were purchased fron conaercal dealers or from the laboratory anmal supply faclty mantaned at Yale Medcal School The gunea pgs were albno, Hartley stran, ether males (average weght 5ÜÜ gr.) or females ( gr.). Vruses, the followng vruses and strans have been employed: EEE, Alabama stran , 6th to 0th mouse passage; VEE TC 80, attenuated stran developed by WBAIR by tssue culture passage to use as vaccne, was employed at mouse passage one or two; VEE, Tr-donkey 945 stran, mouse passage 0-2; JE, Nakayama stran, mouse passage unknown but probably between 00 and 200; Banz, 336 stran, mouse passage 6-9; yellow fever, 7D stran, vaccne suppled by Merrell-»\atonal Laboratores, mouse passage or 2; and yellow fever, French vscerotropc stran, a stran whch has been mantaned by monkey-toaonkey njecton of nfected monkey serum wth never before and ntracereoral passage n mce; for the current testa, a mouse passage or 2 has been used. Vaccnes. Yellow fever 7D vaccne was used fully actve, all other vaccnes were nactvated; 0.5 percent formaln was added to a 0 percent suspenson of nfected mouse bran tssue n physologcal salne. The suspenson was held at 4*C for a mnmum of 7 days before use, when safety testng ndcated that no vrus was present. The vaccne was used for perods up to 2 months and was always kept at 4*C. In our prevous experence, the vaccnes were stored at -60*C. Ths procedure has been dscontnued as t has resulted n, at least, one falure to mmunze anmals; t was observed that on thawng some of the vaccnes kept for several months there appeared heavy flucculaton or clumpng whch may have resulted n denaturaton of the antgen. The tters of the vrus n the vaccnes (IC LD 5 Q/ml) pror to nactvaton, determned by ntracerebral (c) ttraton n newborn nce were: EEE, 0 8 -lq-$; VEE (TC80), 0»; JE, 0 8 ; and yellow fever (7Ü), VacaactUou. Hce were vaccnated by the ntrapertoneal (p) route of noculaton: 0.2 ml of vaccne was gven, ether undluted or at dluton :0 n physologcal salne. Gunea pgs were gven the vaccne by the ntramuscular (m) route; 0.5 ml of vaccne was njected nto each hnd leg for a total of ml of vaccne, undluted or at dluton :0; the amount of vaccne was dvded and gven n two stes to onmse leakage outsde the muscle tssue. When n an experment there were groups of anmals of dfferent tme ntervals between vaccnaton and challenge, the vaccne was gven on dfferent days and all groups, ncludng controls, were challenged on the same day. Challenge. Mce were challenged ether by the subcutaneous (sc) or c routes of noculaton. For the ac challenge ale«were noculated

10 tm/mmtt n M>-W-!t!llP»Jm««IWII ' ' ~~" ~- ~ ~ - ~ - -..,,. '"' "'" "-,- < Ü.2 or Ü.3 ml of a vrus suspenson n the ngunal regon; ether anesthesa was used to prevent strugglng and to expedte the procedure; the c challenge conssted of 0.03 ml of a suspenson, also under deep anesthesa. Gunea pgs were challenged by the p route, by njectng jal of the gven vrus suspenson; ths route was used n preference to the sc route a more natural route because exploratory tests wth EEE vrus showed that the former route resulted n a hgher mortalty over a range of dlutons, from 0" 2 to 0" 6. Vrus suspensons employed n a challenge were, as a rule, ttrated c n newborn mce. Admnstraton of Cytoxan. Ths drug (Mead and Johnson) was resuspended, as prescrbed, n sterle water at the tme when used; the resultng soluton contaned 20 mg of cyclophosphamde n ml. The drug was gven once or repeatedly, as planned, by the p route n an amount of 50 mg/kg body weght each tme; ths amount was contaned n a volume of ether 0.2 ml or 0.25 ml, dependng on the age, sex, or weght of the anmals and equaled 4 or 5 mg of Cytoxan to a mouse for each njecton. Antbodes. Complement-fxng (CF) antbodes were determned by the method routnely used n ths laboratory (5), consstng essentally on a sem-mcrotest wth a total reagent volume of 0.5 ml n each well, or 6 drops, wth ncubaton of the frst phase for 8 hours at 4*C. The sera were nactvated at o0*c for 20 mnutes and used n Increasng twofold dlutons begnnng at :4 or :8. One or two dlutons of antgen were used, contanng 4 and 6 unts of antgen, respectvely; as the tters gven by the sera aganst the two doses of antgen were very smlar, eventually only one dose was used, 6 unts. A control antgen,.e., an antgen prepared wth an unrelated vrus was used n all tests. Neutralzng (N) antbodes were determned by the c route of noculaton n newborn mce; undluted serum and vrus n ncreasng ten-fold dlutons wer* ncubated for 2 hr at 37"C pror to noculaton. Interpretaton of challenge results. In experments n whch death or survval was the bass for evaluaton of the effectveness of a vaccne or treatment, the ch square test n 2 X 2 tables wth Yates' correcton for small numbers was used. V. Results Effect of Cytoxan on aarly-p.ase resstance. On the assumpton that nconclusve results obtaned earler may have been occasoned by an excessve challenge dose, the dosage of vrus n the current experments

11 E^JHK r " " a^^^bg^j^^^^^^ -8- was adjusted to yeld between 3 and 0 LD^Q. Test. Three hundred and twenty (320) alee were dvded n four groups, as shown n Table.. Groups and 3* each consstng of 64 mce were gven JE vaccne n the mornng, 0.2 ml p, whle groups 2 and 4 each consstng of 96 nce, were left unvaccnated. Sx hours later, groups «wd 2 were gven 5 ag Cytoxan p, but not groups 3 and 4. Twenty-four hours after vaccnaton all nce were challenged wth EEE vrus, 0.2 ml sc, n the dlutons and wth the result shown n Table. As the table ndcates the admnstraton of Cytoxan dd not affect n the least the non-specfc resstance nduced by the vaccne aganst a challenge gven 24 hours later. Wth the amount of Cytoxan gven, furthermore, there was no dscernable change n the susceptblty of the unvaccnated nce to EEE vrus. Test 2. On the assumpton that f Cytoxan had a depressng effect on early-phase non-specfc resstance t nght be more easly uncovered by ncreasng the dosage, the experment outlned n Table 2 was carred out. Mce recevng Cytoxan were gven 2 njectons of the drug, one each on days and 3; mce recevng no Cytoxan were njected, nstead, physologcal salne on those days. EEE vaccne was gven to the correspondng groups, and 3 on day, after the njectcr. of Cytoxan or salne. On day 2,.e., 24 hours after vaccnaton, all nce were challenged sc wth Banz vrus at dluton 0~8, whch was antcpated to contan between and 5 IP LD30. w *h the result shown n Table 2. The results n Table 2 confrm once more our prevous observatons of the development of a non-specfc resstance nduced by EEE vaccne aganst Banz vrus; even, though the dfference between vaccnated and controls, groups 3 and 4, s moderate consderng the amount of vrus n ehe challenge, 3 LD50, the ch square value s sgnfcant, 5.7. The admnstraton of Cytoxan dd not abolsh the resstance nduced by the vaccne; Cytoxan ncreased somewhat the susceptblty of the control, unvaccnated mce, so that the noculated challenge contaned now LDJQ, and aganst ths challenge, the Cytoxan treated, vaccnated mce nad a cortalty 2 of 32 whch dffered from that seen n the controls, U of 32 by a dfference whch has a ch square value of 6.. Test 3. In a test descrbed n last year's report, repeated njectons of Cytoxan over a 6-day perod gave nconclusve results on the early phase resstance. In a current test, a large amount of the drug was njected to two groups of nce (see Table 3), no. and 2; tr.e drug n amounts of 4 mg per mouse average body weght, 25 gr was gven on days, 3, 4, 5 and 8, whle two other groups, no. 3 and 4, receved no Inoculum. Ths amount of Cytoxan was toxc resultng n sgns of llness followed by death n 9 of 64 nce; the Cytoxan assocated deaths occurred or days 8 and 9. Vaccnated nce, groups and 3, receved JE vaccne on day 3, and ell nce were challenged wth JE vrus on day 4, or 24 hours after vaccnaton; the result of the challenge s shown n Table 3.

12 -9- Cytoxaa-noculated nce whch ded as a result of Che actoo of.at drug have been elmnated fron the fgures gven n the table. There was no dmnshed early resstance n the mce gven Cytoxan; n fact snce the susceptblty of the mce to the vrus ncreased through the acton of the drug, the early resstance Induced by vaccnaton was f anythng ncreased. Ths s n sharp contrast wth the known effect of Cytoxan on the mmune response followng vaccnaton wth a challenge wth the homologous vrus gven 7 days later, as was descrbed n last year's report. Early resstance aganst yellow fever and VEE vruses n mce. Yellow fever and VEE are nfectons of consderable mpr ance n relaton to ths project. Unfortunately t has not been po»..ble to develop wth yellow fever gven to mce a model smlar to that uued wth other arbovruses; yellow fever vrus fals to kll mce after perpheral noculaton of even the largest amounts, 0 or 0 7 IC LD50. Attempts have been made to detect, shortly after vaccnaton, ndcatons of resstance aganst an c challenge, even though ths was consdered to be an entrely artfcal model. The results of two experments n whch the Asb stran was used for the challenge are gven n Table 4. The mce had been vaccnated once wth lve 7D vrus, approxmately 0 6 LD^Q, gven p and challenged by c route on the ndcated days. The LD50 of the vrus n the control mce was ID" 8 «2 n test no. and Q-?«8 n test no. 2. As the table shows, not the slghtest protecton was observed aganst Q2.2 or 03.2 LD^Q 0 f vrus, ether or 7 days after vaccnaton. Wth a smaller dose of challenge vrus, test no. 2, the mce vaccnated 7 days before challenge revealed a sgnfcant degree of protecton aganst 6 LD50 (dluton U~ 7 ); the ch square between vaccnated and controls (24/29, 3d/4U) was.; there may also have been some slght protecton In the next hgher dluton, 0~ 8, wth a ch square value of 4.8. No protecton whatsoever was observed when mce were challenged 24 bouts followng vaccnaton. Snce tests wth Asb stran as challenge showed lttle promse, tests were next done wth the French vscerotropc stran; snce t had never before been propagated n mce the assumpton was made that t may be less nvasve for ths host than the Asb neurotropc stran and offer, therefore, a better chance of survval to vaccnated mce. The dfference n actvty n mce of the two strans followng c noculaton s shown n Table 5, n whch have been summarsed the results obtaned n several experments. It can easly be calculated from the data n the table that the LD50 of the Asb stran was 0~ 8 *0/ 0.3 ml; that of the French vscerotropc stran 0" «2/0.3 ml. It s of nterest to note that the average survval tme (AST) of the mce that succombed was at all dosage levels twce as long wth the French vlscerotropc stran than wth the Asb. The result of a challenge test wth the French vlscerotropc stran s gven n Table 6. Groups of mce were vaccnated once wth lve 7-0 vrus at 4 consecutve days before challenge and another unvacdnated group served as control. From the outcome n the controls c can be calculated that the D 5 Q was 0"" 6 «2 ; all vaccnated mce,

13 ' '^ftvjflf^h^.t ^^^l^^^^^^^^^ßj^ß. ^äälzzm*** -0- ncludng those vaccnated 24 hours before challenge, were substantally protected aganst. LD 50 (dluton Q~*) and LD 50 (dluton 0-5). Vaccnaton and challenge wth VEE vrus. Groups of mce were vaccnated once wth ether VEE (TC80) or JE (Nakayama) formaln nactvated vaccnes on days and 7 before challenge wth VEE (Tr donkey) vrus. The LD50 of a stock of the latter after sc noculaton n 80- to 90-dayold mce had been prevously determned as beng 0*9.6. x n the present test, dluton of vrus 0*' was used as challenge; the result s shown n Table 7. The challenge dluton contaned 2.5 SC JUD50; all mce vaccnated wth VEE (TCöü) vrus were soldly protected and 7 days after vaccnaton. It s also shown n the table that the JE vaccne had a dstnct protectve effect when gven 24 hours before challenge (8/32 vaccnated compared wth 26/32 controls, ded, ch square - 8.0) but not when admnstered 7 days before challenge. Early resstance aganst EEE vrus n gunea pgs, followng vaccnaton. Intrapertoaeal noculaton of gunea pgs wth EEE vrus results n fatal nfecton n only a fracton of the anmals; n lmted observatons, t appeared as though the lethalty was the same over a range of vrus dlutons, from 0" 2 to 0". Although a smlar stuaton occurs n adult mce noculated subcutaneously, t s possble by usng relatvely large numbers of them to evaluate the sgnfcance of results on a statstcal bass; the use of such large numbers s not realstc wth gunea pgs. Ualy vrema ttratons as an ndcaton of nfecton wth EEE could, probably, be used; t was not, however, a practcal procedure wth our gunea pgs experments. Attempts have been made to determne «nether development of antbodes n survvors followng challenge of vaccnated aad control anmals could be utlzed as a crteron of resstance or susceptblty to the challenge. Gunea pgs were challenge from to 7 days after a sngle vaccnaton, consstng of 2 m njectons, 0.5 ml each, gven at the same tme. The p challenge, ml of dluton 0~5 0 f nfected mouse bran, contaned on the average J.0->*' CLU 50» when ttrated n newborn mce. In all except one test, homologous EEE vaccne-was used; n a test, heterologous JE vaccne was employed. Gunea pgs that ded followng challenge generally dd so between days 5 and 8 wth lttle dfference n AST amongst the varous groups. The results of a number of experments are presented n Tables a, 9, and 0. Tne overall mortalty of unvaccnated controls was 68.5 percent (Table lc>; n separate experments t was (Table 8) from 53 to 80 percent. Admnstraton of JE vaccne 24 hours before challenge dd not reduce the mortalty apprecable, the dfference between 6.5 and 68.5 percent beng nsgnfcant; even f the comparson s made (Table ) between the mortaltes n JE vaccnated gunea pgs and n the controls of the test, number 4, n whch the JE vaccnated anmals wave Included, the dfference from 6.5 to 80 percent, although apprecable, «not sgnfcant wth

14 -l- the numbers of anmals n the groups. Gunea pgs vaccnated 7 days before challenge were soldly mmune. Vaccnaton 3 or 4 days before challenge (Table 3) resulted n sgnfcant protecton, even wth the small numbers of anmals used. Vaccnaton or 2 days before challenge whle gvng nu statstcally sgnfcant protecton n the separate ndvdual tests had a defnte postve trend; the mortalty of the combned - and 2-day old pre-challenge vaccnated groups was 3.6 percents whle that of the total control group was 68.5 percent. A number of gunea pgs that survved challenge were bled between 8 and 24 days later.; also bled at the same tme were vaccnated but unchallenged anmals. The sera were tested for CP and N antbodes wth the result shown n Table 9. The results of the CF tests are partcularly Interestng. Followng challenge of unvacclnated anmals, 5 survvors tested had generally developed antbodes at hgh tters, :64 and hgher; n contrast, vaccnated unchallenged gunea pgs had ether no detectable antbodes or only at low tter, :8. Vaccnated gunea pgs challenged 7 days after vaccnaton responded In a manner smlar to that of the vaccnated unchallenged anmals: they ether had no detectable antbodes, 2 or 5, or, wth only one excepton, the tters were low. Gunea pgs vaccnated wth EEE vaccne and challenged from to 4 days later fell n an ntermedate area; few were negatve, a number developed elevated tters, but the majorty presented only moderate tters. Interestngly, gunea pgs gven the heterologous vaccne, JE, 24 hours before challenge also fell n the ntermedate area. The results of the N test, n terms of neutralsaton ndces of undluted sera, are less sharply defned than those of the CF test when comparng the unvacclnated controls, vaccnated and challenged 7 days later and unchallenged vaccnated anmals; the trend, however, parallels the results of the CF test. Concevably, the use of serum dlutons n the K test would be a more accurate pcture. The results of all the challenge, CF and N tests have been assembled and summarzed In Table 0. Owng to the fact that some of the smlar categores conssted of small number», they have been combned s the table. Furthermore, and n order to underscore certan trends, the results of the CF test have been dstrbuted u three classes: negatve or less than :8 tter; ntermedate tters, :8, :6, :32; and hgh tters, :64, :28 and hgher. The results of the N tests have been dvded n two groups: Nl values of 3.5 or lower In one, 3.6 or hgher n the other. The observatons on development of resstance to a challenge wth EEE vrus n gunea pgs shortly alter vaccnaton can be summarzed as follows. Based on survval, statstcally sgnfcant protecton was apparent by the 3rd day after vaccnaton wth the homologous vaccne; and, whle only on the borderlne of statstcal sgnfcance, there was an ndcaton that protecton may appear or 2 days after vaccnaton. Mom-

15 " * " """" *&%& -2- spectflc protecton was not observed when JE vaccne was gven 24 hours before challenge. The dstrbuton of CF antbody tters n survvng gunea tgs presented a pattern whch corresponded closely to that observed n terns of survval to the challenge: unvaccnated controls, wth the hghest norcalty, had cost CF tters n the hgh-tter class, 9 or 5; soldly protected gunea pgs vaccnated 7 days before challenge, wth zero mortalty, had taost CF tters n the negatve class, 2 of 5: In ths cwv resembled the vaccnated unchallenged anmals whch had 5 of 7 n ths category. Gunea pgs vaccnated from } lo 4 days before challenge, whch had shown followng challenge mortaltes of 2.5 and 3.ö percent, ntermedate between controls 68.5 percent and fully protected anmals G percent had also an ntermedate poston n the CF test results; the majorty, 2 of 22, were n the :8 to :32 group. Fnally, the gunea pgs vaccnated wth JE vaccne whch showed no protecton (or at most the merest ndcaton) had tters also n the ntermedate group. Tne results of the l tests showed that the HI could be placed n tvo sets; one wth the hgher values, ncluded the sera from challenged uvaccnated controls, anmals vaccnated wth E vaccne to 4 days before challenge and anmals vaccnated wth JE vaccne 24 hours before challenge; the second set wth the lower values was made up of the sera from the fully protected gunea pgs vaccnated 7 days before challenge and those from vaccnated unchallenged anmals. Davelopaent of early resstance aganst JE vrus n gunea pgs, : vaccnaton. Perpheral noculaton of aval üble strans of JE vrus to gunea pgs dd not, as antcpated, cause aeath or vsble sgns of llness. In attempts to develop a vaccnaton-challenge aodel n ths speces, prelmnary studes were made to establsh certan practcal parameters followng noculaton of the vrus by p route; ttraton of vrema and development of CF and N antbodes. After noculaton of a large dose of vrus equvalent to 0 newborn r-ouso IC LD-,0, Table, there followed a short perod of vrema wth we ngüest tters 24 hours after noculaton. It was not clear whether the vrus recovered n the blood represented persstng vrus or actual multplcaton of the noculum. No.attempt has been made thus far to utlze vrema or ts suppresson to estmate resstance to a challenge; the use of ths procedure n a group of 30 or 40 gunea pgs for several consecutve days s not easy, but t may be attempted later. Investgatons on the development of CF and K antbodes gave the results shown n Table 2. As can be seen n the table, noculaton of JE vrus over a dosage from 0 (dluton 0" 4 ) to 0 2 (dluton 0" 8 ) newborn mouse IC LD 50 tc gunea pgs resulted n a systematc, orderly development of CF antbodes; wsh the sera secured 2 days after noculaton ''dlutons o"-* to 0* 8 ), a sharp, clear ttraton of nfactvty of the noculum was obtaned, whch n the present example gave an IP ID50 (ntrapertor.eal nfectve dose 50) of 0~ 7 ' 2. Assumng that challenge of vac-?

16 -3- cruc-j annals resulted n suppresson of antbody development n a.smlarly systematc fashon, ths model could be proftably used for the study of the problem at hand. The X test, wth undluted serum and vrus dlutons, gave an answer smlar to that of the CF test n a much shorter tme; all annals that n tme became postve by CF test at 2 days, were already postve by K test at 0 days. The N test may, however, have dsadvantages: the speed and ntensty of the response may oblterate subtle quanttatve dfferences, unless the serum-dluton method s used; and the labor and means needed far outweght those requred for the CF test. Vaccnaton and challenge. Havng establshed that antbodes developed n a systematc fashon followng noculaton of JE vrus and as t appeared that a dluton 0~ of mouse bran vrus was effectve but not excessve nfectve dose, lo** 2 IP ID50» experments were next carred out to see how vaccnaton would alter the response to a challenge measured by CF antbody development. In the frst of 2 experments, Table 3, undluted vaccne was gven to a group of gunea pgs 7 days (#3) and to another 4 days (#4) before challenge; another group, #2, were vaccnated along wth group #3 and not challenged; and another group, #, were held as unvaccnated controls. All gunea pgs were bled, only once, 5 days after challenge, ncludng group #2 whch was not challenged. The result of the CF test wth the sera s shown n Table 3. The unvaccnated controls, group #, had 2 anmals wth no detectable antbodes, presumed to have escaped nfecton; 6 of the remanng 7 anmals had cters of :64 or :28. The vaccnated, unchallenged gunea pgs, group #2, responded to vaccnaton wth tters usually n the mddle range, J :32, wth one showng a tter of :64* Groups #3 and #4, vaccnated and challenged 7 and 4 days later respectvely, responded almost exactly lke group #2, gven vaccne and no challenge. It appears, therefore, that challenge of unvaccnated gunea pgs resulted n a few nstances n no response; n most cases nfecton occurred and resulted n CF tters of :64 and hgher; whereas vaccnated and challenged anmals responded as though they had been vaccnated only,.e., they were protected. The second experment, Table 4, was tmlar to the frst wth the excepton that the vaccne was used at dluton :0 and the gunea pgs were bled 24 and 24 days after challenge. It s clearly apparent that CF antbody tters of the several groups dffered but lttle 4 days after challenge; on the later bleedng, however, the tters of the unvaccnated controls had ncreased; those of all the vaccnated anmals whether challenged or not had,f anythng, decreased wth the result that n group, 5 of 8 gunea pgs had tters of :64 or hgher, whle n groups 2, 3, and 4 all anmals had tters :6 and lower. The protectve effect of the vaccne n groups 3 and 4 s evdent; t seems, furthermore, that whle antbodes sttrbutable to vaccnaton had reached ther peak on day 4 (relatve to the challenge) the antbodes due to nfecton were not fully developed at that tme.

17 _ ^ tpjbmh» > 5,...,.,.,.., _.. pssp -u- Dcv.lopment of complement-fxng antbodes la gunea pg«followng noculaton of several group B arbovlruses. Exploratory efforts to employ development of CF antbodes n gunea pgs as a sgn of nfecton followng noculaton of group B vruses other than JE, have net wth vared. success; the results of these attempts are sramarzed n Table 5. None of the vruses used caused death or apparent llness n any of the anmals. Bans! vrus was partcularly effectve; even after njecton of as lttle as 0**' newborn mouse IC LD50 all gunea pgs had hgh antbody tters 2-22 days after Inoculaton; evdently ths vrus n gunea pgs gves an excellent model ot nvestgate the present problem. The other vruses used were not very successful n nducng CF antbodes. Only 4 aamals of 6 njected wth yellow fever vrus had antbodes 2 or 22 days later and only one had a reasonably hgh tter, :64. Inoculaton of Powassan vrus to 7 gunea pgs resulted n only one postve serum, :32, tucea weeks later. The low tters, :8 or :6, observed n 3 anmals gven the largest of the two doses of dengue 2 vrus (dluton 0~ 2 ) may have been a response to the mass noculated rather than the result of vrus multplcaton; further studes are needed to settle ths queston, ncludng tests on sera taken later than 5 days after noculaton. VI. Dscusson In observatons prevously reported (Annual Report, Hay 973) resstance to a challenge wth VEE vrus was detected n mce vaccnated to 4 days earler wth the homologous vrus, but not wth a heterologous one; the challenge dose was 00 ID$Q and the vaccne Colorado tck fever vrus. In the current experments an early resstance aganst a VEE vrus challenge gven 24 hours after vaccnaton wth JE vrus vaccne was noted; whle the vrus n the vaccne may have nfluenced the outcome, t appears more logcal to assume that the smaller amount of vrus n the challenge, 2.5 LD50» *? have been th * prncpal reason for the early non-specfc protecton. Yellow fever vrus s of great mportance n connecton wth the present project; no observatons had been attempted n the past due to nablty to challenge mce effectvely by perpheral route. In spte of ther artfcalty, studes were made on the development of resstance to an c challenge wth the vrus, whch have revealed an nterestng fact: effectve protecton was observed 24 hours and later after vaccnaton when the challenge was a stran, French vscerotropc, whch had not been propagated n mce before, whle no protecton to speak of was observed when the challenge was a thoroughly mouse adapted stran, Asb neurotropc. The determnng factor may well, have been that the vscerotropc stran was a slow actng vrus In the mouse, whle the Asb stran was quck actng; concevably, the vscerotroplc stran beng less rapdly nvasve allowed for Immunty due to the vaccne to overtake and block th» lethal actvty of the vrus.

18 -5- The effect of Cytoxan, gven repeatedly at the tae of vaccnaton a:\d challenge,.' usng moderate to low doses c challenge vrus, seems to have been characterzed wth the model used: It has faled to nhbt te early resstance to a challenge after vaccnaton. Snce, n fact, Cyco:;un ncreased the mortalty of perpherally nfected mce, the detecton of early resstance was made easer. The current observatons strengthened the pror concluson that the early phase of protecton followng vaccnaton s not, or s not entrely, explcable on an mmune mechansm. Investgaton of early resstance n gunea pgs s made dffcult by ehe low, or lack of, susceptblllty n terms of lethalty to same of the vruses that we had planned to study. The number of anmals needed wth a vrus whch klls only a proporton of controls, such as EEE, s much too large to use ths speces as a practcal method to nvestgate small degrees of protecton followng vaccnaton. Other parameters, for example, ttraton of vlreaa followng challenge of gunea pgs wch JE vrus, offered other possble ways of study. However, a smpler method appears to be the ttraton of antbodes n survvng challenged anmals; studes wth the CF test show a marked dfference n the degree of response n varously treated groups of anmals: vaccnated only, challenged only, and vaccnated and challenged at several days later. Development of antbodes, or resstance to a second challenge, n expermental anmals after a non-fatal nfecton s a method often used to detect nfecton. In the experments reported the procedure has been appled and the results have been nterpreted to ndcate blockng of napparent nfecton n anmals whch, judgng by the response In unvaccnated controls should have developed antbodes. The method requres a well balanced combnaton of amount of vaccne, dose of challenge, and tae after challenge when the survvors are bled; otherwse the dfferences between the responses of the several groups may be less apparent. VII. Conclusons. Early resstance,.e. resstance 24 hours after vaccnaton, aganst a small dose challenge wth VEE vrus was observed n mce vacc.-u.ted wth VEE TC80 vrus. A sgnfcant resstance aganst the same challenge was noted n mce vaccnated 24 hours earler wth JE vaccne, but not when ths vaccne was gven 7 days before challenge. 2. Vaccnaton of mce wth lve 7D stran of yellow fever resulted n early resstance, to 4 days later, aganst an ntracerebral challenge wth yellow fever, French vscerotropc stran, but not aganst the Asb neurotropc strat;. 3. Admnstraton of Cytoxan to mce mmunzed wth arbovruses dd not suppress or dmnsh early resstance to a homologous or heterologous challenge gven 24 hours later.

19 ' ~"'. I Substantal resstance to a challenge wth EEE vrus vas observed n gunea pgs vaccnated wth homologous vaccne 3 or more days before challenge; a snal degree of protecton seemed apparent n anmals vaccnated or 2 days before challenge but ts statstcal sgnfcance was borderlne. 5. Gunea pgs that survved a challenge wth EEE or JE vruses developed hgh ttered CF antbodes; anmals that were vaccnated but not challenged developed low ttered CF antbodes. Vaccnated and challenged gunea pgs developed antbodes wth ttets smlar to those present n vaccnated only anmals. 6. Ttraton of antbodes n vaccnated gunea pgs followng a non-lethal challenge can be used as a method for detectng the effcacy of the vaccne. BIBLIOGRAPHY. U&lstead, S.B. Observatons related to pathogeness of dengue henorrhagc fever. IV. Hypothess and dscusson. Yale J. Bol. Med., 42: , Hammon, W. McO. Observatons on dengue fever bengn protector and kller: A Dr. Jekyl and Mr. Hyde. Am. J. Trop. Med. Hyg., 8: 59-65, Tgnor, G.H., R.E. Shope, R.K. Gershon, and B.H. Uaksman. Ismunopathologc aspects of nfecton wth Lagos bat vrus of the rabes serogroup. J. Immunol., 2: , Casals, J., S.M. Buckley, and D.W. Barry. Resstance to arbovrus challenge n mce mmedately after vaccnaton. Appled Mcrobol., 2a: , Casals, J. Immunologcal technques for anmal vruses. In "Methods n Vrology", K. Maramorosch and H. Koprowsk, eds. Vol. Ill: 3-98, Academc Press, New York, 967.

20 -7- Table Effect of Cytoxan on early phase resstance followng vaccnaton of Mce: vaccne Jl vrus, challenge EEE vrus Group No. Beglaen Vaccne Result of challenge, day after vaccnaton EEE vrus dluton 0-5 0" 0-7 Cytoxan JE 6/64 2 Cytoxan none 53/64 4/6 2/6 3 No Cytoxan JE 4 No Cytoxan none /64 47/64 9/6 2/6 Mce dead/mce challenged

21 ".. '. ' ^m^^m^w^m^m^^mm ^)mj^ß^^j^!*^ f^ßfm wlk» n o c (S K A «-* S» a n g o a & g s» M K ^!? * s o n» 0 Ö a %?? y* 0 < * 3 C I S r» f s 0 8

22 -9-. Table 3 Effect of large dosag«of Cytoxan oo early phase resstaaee followng vaccnaton of nce: vaccne and challenge J5 vrus Group Ko. leglaen vaccne Result of challenge, day after vaccnaton, JE vrus dlutons 0-6 o-7 Q-8 ch square 0-6 Cytoxan, 5 njectons JE ~~ 4/28 5".5 2 Cytoxan, 5 njectons Hone, 27/27 8/6 7/6 3 Ko Cytoxan JE 0/3* 2/6 4/ Ho Cytoxan None 2/32 Mce dead/mce challenged c 3.

23 J -20- Table 4 Vaccnaton of mce wth lve yellow fever (7D) vrus and ntracerebral challenge wth yellow fever (Asb) vrus Test no. Vaccnaton, days before challenge 7 Challenge, dluton Mce dead/mce challenged

24 I -2- Xable 5 Tters and average survval tmes (AST) followng ntracerebral noculaton of yellow fever, stran Asb and French vscerotropc, to nce 5 to 7 weeks old Stran Asb ; Mortalty AST Vrus dj Lluton o- 2 o' 3 IO" 4 I 0~ 5 0" 6 o-' t 28/28 3/3 54/ I 0-8 o" 9 ( 27/56(2/ French vse. Mortalty AST 2/ /2 9*7 W / / / /28 Mce dead/mce noculated AST, n days

25 -22- Table 6 Vaccnaton of nce Wth lve yellow fever (7D) vrus and ntracerebral challenge wth yellow fever (French vscerotropc) Vrus Vaccnaton, days before challenge 0-5 Challenge, dluton /32 0/32 3 2/3 0/32 2 5/28 /32 3/32 /30 No vaccne 28/32 2/32 2/6 0/3 Mce dead / mce challenged.

26 rr -23- Table 7 Early resstance followng vaccnaton of alee aganst VEE Vrus subcutaneous challenge Vaccnaton Challenge, dluton Vaccne Days before challenge ID" 9 o- 0-0 VEE 7 0/32 0/32 - JE 7 24/30 8/32 No vaccne 26/32 /5 0/6 Mce dead / nce challenged.

27 -24- Table 8 Challenge of gunea pgs wth EEE vrus soon after vaccnaton wth ether EEE or JE vruses l Test Vaccnaton N'o.! Vaccne Days before ; challenge ''. 2! EEE 2 None! EEE 7 None Number of Anmals Result of challenge Dead Survved Z Mortalty Sgnfcant results, ch square 9.06 * 4 EEE 3 None « JE None

28 -25- Table 9 Antbody development aganst EEE vrus n survvng gunea pgs followng vaccnaton and challenge wth EEE vrus Vaccne Days between vaccne and challenge Number Tested Complement-fxaton Test No. wth ndcated tter Number Tested < Neutralzaton Test 2.9 or < Number wth NI or > None controls EEE no challenge EEE 7! EEE 3 or 4! EEE or 2 j JE X j Complement-fxaton tters expressed as recprocal of tter; 28, ndcates :28 or hgher. NI, Neutralzaton Index gven by undluted serum.

29 f I = -26- Tacle 0 Summary of result«wth gunea pgs vaccnated and challenged wth EEE vrus: Protecton end Antbodes Vaccne Challenge, days after vaccne Number of anmals Challenge <7 Teet N Test Deed sur- X lter Nl vved mortalty <8 8,6 64, or 32 or > or < 3.6 or > None Controls EEE None EEE EEE EEE 3 or 4 or \ JE CF Tost: Recprocal of ttere. Nl, Neutralxeton Index of undluted

30 g'-y-^ggdbthtml^j-^tf^""^ -27- Table Vrema n gunea pgs followng ntraperltoneal uoculacon of Japanese encephalts vrus Gunea pg Days tested after noculaton an. cter* ,4.0 none ± none *Tlter: log Q ;+, aouse ded of 8 noculated undluted blood; none, no nce ded of 8 Inoculated. The noculua» ml dluton 0"*, contaned approxmately 0* IC U>5o by tltraton n newborn mce.

31 -28- Table 2 development of antbodes n gunea pgs followng lntrsperltoneal noculaton of Japanese encephalts vrus Juaber of Anaals Dluton! Day after nocu-atedjnoculaton (ICLD^Q)! when bled 0-4(0 6 ) t 4 0 CF test N test T * 5 (0 5 )j ; ;o-*(o 4 ) ! I j j { 0"" 7 (0 3 )j * j 0 l < {2.8+! t 3.8+ <.0,3.8+ 4! 0** (lo 2 )! <.0 <.0 <.o CF test: recprocal of sern tlter; 0, no fxaton at dluton :8. JJ test: neutralsaton ndex, ntracerebral test n newborn nce.

32 -29- Table 3 Development of complement-fxng antbodes n gunea pgs followng vaccnaton and challenge wth Japanese encephalts vrus Vaccne Challenge, Number CF test, tter 0. day8 after o of vaccne o u Anmals < None Yes» controls Yes None Yes ; Yes S!» CF tter: recprocal of tters. Vaccne: ml, undluted.

33 -30- Table 4 Development of coaplenent-fxng antbodes n gunea pgs followng vaccnaton and challenge wth Japanese encephalts vrus 3 O u o Challenge, Nuabcr Day bled CF test, tter { Vaccne days after of after vaccne anaals challenge < {256! None! Yes I Yes None 8 (4) 4 3 (24) 6 2 t 3 Yes 4 I CF test: recprocal of tters. Vaccne: ol, dluted :0. 24! Yes " ! t (! (4), (24): not challenged gunea pgs bled on the those that had been challenged. days as

34 »JBJPJIPPUFWWIB m^'\% -- M '*' " gbgbg^aegpg^pggg^ BgWff -3- Table 5 Development of complement-fxng antbodes n gunea pgs followng ntraperltoneal njecton of group B srbovruses Vrus Dluton of vrus noculated (mouse IC LD50) Number «ra» anmals... Day bled after Inoculaton and CF tters 0 or 5 2 or 22 Banz 0-6(0 4» 7 ) 4 28, 28, 256, (0 3.7) 4 64, 28, 28, 28 lo-^uo 2 ' 7 ) 4 L , 256, 256 Dengue 2 (NGO) 0-2 (0 6 ' 7 ) 3 3 6, 6, 8 0, 0. 0 Powassan lo^do 6-7 ) 7 0, 0, , 0 Yellow fever (French vscerotropc) lo^do 5 ) 0" 5 (0 4 ) , 0, , 0, , 0, 0 32, 0, 0 8, *, 0, 0 0, 0, 64, 0 lo^do 3 ) 4 0, 0, 0, 0 8, 0, 0, 0 CF tters: recprocal of tter; 0, negatve at dluton :4,