Antibody Responses in the Cerebrospinal Fluid of Cynomolgus

Transcription

1 INFECTION AND IMMUNITY, Feb. 1979, p /79/2-185/7$2./ Vol. 23, No. 2 Antibody Responses in the Cerebrospinl Fluid of Cynomolgus Monkeys After Intrcerebrl Inocultion with Prmyxoviruses KAZUYA YAMANOUCHI,* TAKESHI A. SATO, FUMIO KOBUNE, AND AKIRA SHISHIDO Deprtment ofmesles Virus, Ntionl Institute of Helth, Musshi-Murym, Tokyo 19-12, Jpn Received for publiction 3 October 1978 Cynomolgus monkeys with or without mesles ntibody were intrcerebrlly inoculted with mesles or cnine distemper viruses, nd ntibody responses in the cerebrospinl fluid (CSF) were investigted. In mesles ntibody-free monkeys, nturl infection with wild mesles virus or intrcerebrl inocultions with two ttenuted mesles vccines evoked primry ntibody responses to mesles virus in the ser but not in the CSF. In mesles-immune monkeys, intrcerebrl inocultion with the TYCSA strin of mesles virus produced significntly high titer of mesles ntibody in the CSF with miniml rise in the serum ntibody nd resulted in significnt decrese in serum/csf ntibody rtios. Intrcerebrl inocultion of neurotropic cnine distemper virus, the Onderstepoort strin, into mesles-immune monkeys cused production of both mesles nd distemper ntibodies in the CSF. Inocultion of mesles-immune monkeys intrvenously with mesles virus or intrcerebrlly with rubell virus, which hs no ntigenic reltion to mesles virus, filed to evoke mesles ntibody response in the CSF. These results indicted tht locl production of mesles ntibody in the CSF ws cused by stimulus within the centrl nervous system of mesles virus ntigen or cnine distemper virus ntigen tht prtilly cross-rected with mesles virus ntigen s secondry ntibody response. Virus infections in the centrl nervous system in chicken embryo cells. The infectivity titers of the (CNS) re generlly thought to cuse locl production of virus ntibody s indicted by the infective doses/ml, respectively. virus stocks were 16.3, 16.5, nd 158 5% tissue culture presence of ntibody t reltively high titers in (ii) Cnine distemper virus. The Onderstepoort the cerebrospinl fluid (CSF) compred with strin (21) of cnine distemper virus ws propgted tht in the in Vero serum (-6, 9, 11, 12). Demonstrtions of mesles ntibody t high titers in the (iii) Rubell virus. The TO-336 vccine strin (1) cells, nd the stock virus hd n infectivity titer of % tissue culture infective doses/ml. CSF of ptients with subcute sclerosing pnencephlitis re considered to be cused by kidney cells, ws obtined from Tked Chemicl of rubell virus, which ws grown in primry rbbit prolonged stimulus with mesles virus ntigen Industries, Ltd., nd its infectivity titer ws 18 5% within the CNS. In spite of the incresing interest in the level of virus ntibody in the CSF of Inocultion of monkeys. Cynomolgus monkeys tissue culture infective doses/ml. ptients with vrious neurologicl diseses, (Mcc fsciculris) were inoculted either intrcerebrlly or intrvenously. The intrcerebrl inocul- there hve been few experimentl studies on production of CSF ntibody in niml models tion ws conducted with.5 mil of ech stock virus into the bilterl thlmi. The intrvenous inocultion ws (1, 12, 21). mde with 1 ml of ech stock virus into the sphenous In the present study, we nlyzed mesles vein. ntibody response in the CSF of cynomolgus Smpling. Blood ws collected from the femorl monkeys, since monkeys were considered to be vein, nd the serum ws seprted. The CSF ws suitble experimentl niml due to their high obtined fronh the cerebellomedullry cistern nd centrifuged t 2, rpm for 1 min to confirm visully susceptibility to mesles virus nd to the ntomicl similrity of their CNS to tht of humns. Both smples were kept t C until tested. the bsence of contminting erythrocytes (RBC). Titrtion of virus ntibody. Hemgglutintion MATERIALS AND METHODS inhibition (HI) ntibody to mesles virus ws determined by our routine procedure (18); the ser were Virus. (i) Mesles virus. The TYCSA strin of treted with kolin nd RBC of Africn green monkeys mesles virus ws propgted in Vero cells, nd two to remove nonspecific inhibitors; thus, 1:8 dilution vccine strins, AIK-C (1) nd Schwrz, were grown served s the lowest dilution. Since the CSF is free of 185

2 186 YAMANOUCHI ET AL. nonspecific inhibitors, it ws tested from the undiluted smple without pretretment. Neutrlizing ntibody to cnine distemper virus ws similrly determined in Vero cells by using the Onderstepoort strin s chllenge virus. Titrtion of nturl ntibody to GRBC. In preliminry studies on detection of nturl ntibodies in the ser of cynomolgus monkeys, presence of immunoglobulin G ntibody tht hemgglutintes goose RBC (GRBC) ws demonstrted in ll dult monkey ser exmined. This nturl ntibody ws not detected in the CSF of ny nimls except those contminted with RBC through improper puncture of the cistern by needle t the time of CSF smpling. Therefore, this hemgglutinting ntibody to GRBC ws used s reference ntibody to indicte mechnicl dmge to the blood-brin brrier. RESULTS Pttern of ntibodies to mesles virus nd GRBC in the ser nd CSF of meslesimmune monkeys. Fourteen monkeys nturlly infected with wild mesles virus during the qurntine were exmined for ntibodies to mesles virus nd GRBC in their ser nd CSF. After centrifugtion, the CSF were visully clssified into two groups, one without nd nother with blood contmintion. The results re shown in Tble 1. In group without blood contmintion, six CSF were completely negtive for mesles ntibody nd the other four contined ntibody t very low titers. Serum/CSF rtios of mesles ntibody were 256 to 512 or greter. Titers of nturl ntibody to GRBC in the serum vried from 16 to 128, wheres none of the CSF contined the ntibody. In group with blood contmintion, ll four monkeys hd ntibodies to both mesles virus nd GRBC in the serum nd CSF. Serum/CSF TABLE 1. rtios of both ntibodies closely correlted. Thus, the nturl ntibody to GRBC ws proven to be useful indictor for rtificil contmintion with serum ntibody in the CSF. CSF ntibody response fter nturl infection or fter inocultion with mesles vccines. Blood nd CSF smples were collected every other week during the qurntine period from group of monkeys which were newly imported from Southestern Asi. Since the previous serologicl exmintions indicted tht mesles HI ntibody usully ppered in the serum bout 2 weeks fter nturl infection (19), the time of nturl infection with wild mesles virus ws estimted retrospectively to be 2 weeks before the time of the ntibody rise in serum. None of the seven monkeys developed mesles ntibody in the CSF in spite of mrked rise of ntibody in the serum (Fig. 1). Two groups of mesles ntibody-free monkeys, consisting of four nimls ech, were intrcerebrlly inoculted with either AIK-C or Schwrz vccine, nd their ser nd CSF were exmined every other week for development of mesles HI ntibody. None of the nimls developed mesles HI ntibody in the CSF during n observtion period of 8 weeks fter inocultion (Fig. 1). These results indicted tht neither the nturl infection with wild mesles virus nor the intrcerebrl inocultion with live mesles vccines initited primry mesles ntibody response in the CSF, in contrst to mrked ntibody response in the serum. CSF ntibody response in mesles-immune monkeys fter intrcerebrl or intrvenous inocultion. Three groups of monkeys nturlly infected with wild mesles virus 2 to 3 months previously were intrcerebrlly or intr- Pttern of mesles ntibody nd nturl ntibody to GRBC in the ser nd CSF of monkeys nturlly infected with mesles virus Blood contrni- Mesles HI titer Nturl ntibody titer nin Monkey no. ntion Mon. Serum CSF Serum/CSF Serum CSF Serum/CSF INFECT. IMMUN <1 >512 6 <1 > , <1 > <1 > <1 > <1 >6 128 <1 > <1 >128 6 <1 > <1 > <1 > <1 > <1 > <1 > <1 > <1 >

3 VOL. 23, <8 < _~~~~~ AIK-C vccine Schwrz vccine so _* *- - Se _ < _ ~ / ~ ~~~ * _ /. - / go &8 Be ANTIBODY RESPONSES IN CSF <8 <1 / Nturl infection Weeks pi FIG. 1. Antibody response to mesles virus fter nturl infection with wild mesles virus or fter intrcerebrl inocultion with ttenuted mesles vccines, strins AIK-C nd Schwrz. Symbols: HI ntibody in the serum (); HI ntibody in the CSF (). venously inoculted with the TYCSA strin of mesles virus. The time course of the secondry ntibody response fter intrcerebrl inocultion in the first group is demonstrted in Fig. 2. Four monkeys lcked mesles ntibody in their CSF, nd two hd the ntibody t titer of 1 before inocultion with the TYCSA strin. All the monkeys except one whose CSF could not be exmined becuse of contmintion with blood developed mesles HI ntibody in the CSF 1 week postinfection (p.i.). The mesles ntibody titer in the CSF reched the highest titer 1 to 2 weeks p.i. in most of the monkeys. In contrst, serum ntibody response exhibited pttern different from tht of CSF ntibody; most of the serum ntibody remined unchnged 1 week p.i., with slight increse 2 weeks p.i. Thus, serum/csf ntibody rtios generlly tended to decrese in most of the monkeys; men rtio of before inocultion decresed to 1, 1, 126, nd 72 t 1, 2, 3, nd weeks p.i., respectively. Frctiontion of the CSF smples on sucrose density grdient disclosed HI ntibody ctivity only in 7S frctions. Durtion of CSF ntibody response ws exmined in the serum nd CSF smples collected from four mesles-immune monkeys in the second group up to 1 weeks fter intrcerebrl inocultion. CSF ntibody persisted for t lest 1 weeks (Fig. 3). Intrvenous inocultion ws mde into the third group, consisting of four monkeys, but none of them developed ntibody in the CSF when exmined t 2 nd weeks p.i. These results indicted tht ntigenic stimulus within the CNS is required for the production of mesles ntibody in the CSF. CSF ntibody response fter inocultion with cnine distemper virus. Four monkeys nturlly infected with wild mesles virus 2 to 3 months previously were intrcerebrlly inoculted with the Onderstepoort strin of cnine distemper virus. A typicl pttern of primry ntibody response to cnine distemper virus ws demonstrted in the serum (Fig. ). Moreover, reltively high distemper ntibody ws observed in the CSF. Therefore, repliction of cnine distemper virus within the CNS of mesles-immune monkeys ws suggested. No significnt chnge in the titers of mesles HI ntibody in the serum ws noticed, wheres rise of mesles ntibody in the CSF ws noticed in ll four monkeys. Consequently, serum/csf rtios of mesles ntibody mrkedly decresed from over 256 to men rtios of 16 to 6 fter inocultion with cnine distemper virus. Thus, it ws suggested tht the repliction of heterologous cnine distemper virus provided ntigenic stimulus to ntimesles virus memory B cells within the CNS. CSF ntibody response fter inocultion with rubell virus. To eliminte the possibility tht mechnicl dmge to the CNS by intrcerebrl inocultion might be responsible for n pprent development of mesles ntibody in the CSF, s demonstrted in the preceding experiments, rubell virus, which is not serologiclly relted to either mesles or cnine distem-

4 188 YAMANOUCHI ET AL. INFECT. IMMUN. No.1163 No.116 No * U- -- At ---O-~~ * --. A_~~~ %._ b- 1. i:. _: No.1166 U- No 1167 *U- N." No.1168 * _/.-- u U.. ".l" M I Weeks Pi FIG. 2. Antibody response to mesles virus in mesles-immune monkeys fter intrcerebrl inocultion with the TYCSA strin of mesles virus. Symbols: HI ntibody in the serum (); HI ntibody in the CSF (); serum/csf rtio of HI ntibody (). No No Downloded from _. 256 _ - I..5. c I o No '. _- } o o~~~ 6 _ *~~~~~~~~~- U-*- _-_ -9_ No. 1562, o o on April 5, 219 by guest Weeks pi FIG. 3. Antibody response to mesles virus in mesles-immune monkeys fter intrcerebrl inocultion with the TYCSA strin of mesles virus. Symbols: Sme s in Fig. 2.

5 VOL. 23, 1979 ANTIBODY RESPONSES IN CSF 189 No No [ 251 6H - I- Is ll Air A._ l1 * - m 9 No No A~~~~ Weeks pi FIG.. Antibody response to mesles nd cnine distemper viruses in mesles-immune monkeys fter intrcerebrl inocultion with the Onderstepoort strin of cnine distemper virus. Symbols: Mesles-HI ntibody in the serum (); mesles-hi ntibody in the CSF (); distemper-neutrlizing ntibody in the serum (A); distemper-neutrlizing ntibody in the CSF (A); serum/csf rtio of mesles-hi ntibody (-). per virus, ws intrcerebrlly inoculted into nine mesles-immune monkeys with men mesles HI titers of 8, nd the serum nd CSF were collected 3 weeks p.i. All nine monkeys developed rubell HI ntibody, with men titer of 1 in the serum, but showed ntibody production t miniml level, rnging from <1 to 2, in the CSF. The mesles HI ntibody titer remined unchnged in the serum nd ws completely bsent in the CSF. DISCUSSION Primry nd secondry ntibody responses in the CSF were investigted in monkeys by intrcerebrl inocultion with mesles or cnine distemper viruses. A primry ntibody response ws not evoked in the CSF fter nturl infection with wild mesles virus or by inocultion with two mesles vccines, wheres the Onderstepoort strin of cnine distemper virus induced mrked rise in distemper ntibody in the CSF. These mesles vccine strins hd been proven not to induce significnt histologicl lesions in the CNS of monkeys when exmined by intrcerebrl inocultion (unpublished dt). In contrst, the Onderstepoort strin ws shown to replicte to high titers nd to induce disseminted histologicl lesions in the CNS of monkeys (21). In subcute sclerosing pnencephlitis nd cute mesles encephlitis in which growth of mesles virus ws suspected, reltively high mesles ntibody in the CSF compred to tht in the serum hs been reported (5, 11, 15). Therefore, lck of CSF ntibody response fter intrcerebrl inocultion with the mesles vccine strins cn be tken s indirect proof of the lck of virus repliction within the CNS. A significnt rise in mesles ntibody in the CSF compred with tht in the serum ws noticed when mesles-immune monkeys were intrcerebrlly inoculted with mesles virus. These monkeys were nturlly infected (possibly through the respirtory route) 2 to 3 months before the inocultion. Therefore, it my be speculted tht mesles virus ntigen stimulted mesles virus-specific B memory cells s secondry ntibody response. Since the procedure of intrcerebrl inocultion results in the introduction of virus into the blood t the sme time (7), ntigenic stimulus should hve been given lso to the lymphoid system outside the CNS. Nevertheless, serum ntibody ws not significntly incresed in generl, in contrst to mrked rise in CSF ntibody. Preexisting me-..

6 19 YAMANOUCHI ET AL. sles ntibody t high titers in the serum before virus inocultion might hve msked subsequent ntibody rise in the serum s secondry response. Decrese in serum/csf ntibody rtios cn be cused by locl production of ntibody within the CNS, by mechnicl dmge of the bloodbrin brrier, or by incresed permebility of the blood-brin brrier medited through inflmmtion. The intrcerebrl inocultion of mesles-immune monkeys with rubell virus filed to result in rise of mesles ntibody in the CSF. Therefore, mechnicl dmge by intrcerebrl inocultion seems to be unlikely. The bsence of nturl immunoglobulin G ntibody to GRBC in the CSF in contrst to high ntibody titers in the serum my eliminte possibility of serum ntibody leking into the CSF. Moreover, secondry ntibody response in the CSF ws not evoked by intrvenous inocultion. These results strongly suggest tht mesles nd cnine distemper viruses introduced into the CNS provided n ntigenic stimulus leding to in situ secondry ntibody response. Development of distemper ntibody to high titers in the serum nd CSF in mesles-immune monkeys fter intrcerebrl inocultion indicted tht cnine distemper virus replicted in the CNS of these monkeys. In seprte experiment (dt not shown), the presence in the CNS of histologicl lesions similr to but with milder nture thn those observed in the cse of mesles ntibody-free monkeys (21) ws lso confirmed. It is interesting tht mrked rise in both mesles nd distemper ntibodies ws observed in the CSF of mesles-immune monkeys fter intrcerebrl inocultion with cnine distemper virus. Between mesles nd cnine distemper viruses, common ntigenicity is demonstrted minly in the nucleocpsids, wheres only prtil correltion is present in the virus envelope (3, 13, 2). Thus, cnine distemper virus cn produce only miniml level of mesles neutrlizing ntibody nd fils in the production of mesles HI ntibody in both dogs nd monkeys (19, 2). Accordingly, n pprent rise in mesles HI ntibody in the CSF of mesles-immune monkeys fter intrcerebrl inocultion with cnine distemper virus cn be shown to be cused by the nmnestic response in which cnine distemper virus ntigen stimulted mesles virus-specific B memory cells, s reported in mice immunized sequentilly with relted influenz virus hemgglutinin (16). It is possible tht the intrthlmic inocultion used in the present study eventully resulted in introduction of virus inoculum into the third ventricle underlying the thlmus. The INFECT. IMMUN. cells lying on the ventriculr surfce of the hypothlmic ependym of the tegu lizrd were demonstrted to hve mcrophge-like phgocytic ctivity (2). Actively phgocytizing cells were lso demonstrted on the ependyml surfce fter mumps virus infection (17). Therefore, it my be concluded tht the virus prticles were immobilized by the ventriculr phgocytes nd ntibody response ws subsequently initited. A preliminry experiment (unpublished dt) in which intrcisternl inocultion with mesles virus resulted in development of significntly higher ntibody response in the CSF thn tht by intrthlmic inocultion my indicte efficient uptke of virus prticles by the ventriculr phgocytes. ACKNOWLEDGMENTIS This study ws supported by reserch grnt from the Intrctble Diseses Division, Public Helth Bureu, Ministry of Helth nd Welfre, nd by Grnt-in-Aid for Intrctble Disese Reserch from the Ministry of Eduction, Science nd Culture, Jpn. LITERATURE CITED 1. Albrecht, P., T. Burnstein, M. J. Klutch, J. Hicks, nd F. A. Ennis Subcute sclerosing pnencephlitis: experimentl infection in primtes. Science 195: Bleier, R., R. Albrecht, nd J. A. F. Cruce Suprependyml cells of hypothlmic third ventricle: identifiction s resident phgocytes of the brin. Science 189: Breese, S. S., Jr., nd C. J. de Boer Ferritintgged ntibody cross-rections mong rinderpest, cnine distemper, nd mesles viruses. J. Gen. Virol. 2: Cppel, R., L. Thiry, nd G. Clinet Virl ntibodies in the CSF fter cute CNS infections. Arch. Neurol. 32: Connolly, J. H., M. Hire, nd D. S. M. Hdden Mesles immunoglobulins in subcute sclerosing pnencephlitis. Br. Med. J. 1: Deibel, R., nd G. D. Schryver Virl ntibody in the cerebrospinl fluid of ptients with cute centrl nervous system infections. J. Clin. Microbiol. 3: Fenner, F., B. R. McAusln, C. A. Mims, J. Smbrook, nd D.. White The biology of niml viruses, 2nd ed., p Acdemic Press Inc., New York. 8. Kiessling, W. R., W. W. Hll, L L. Yung, nd V. ter Meulen Mesles-virus specific immunoglobulin- M response in subcute sclerosing pnencephlitis. Lncet i: McCllum, F. O., L J. Chinn, nd J. V. T. Gostling Antibodies to herpes-simplex virus in the cerebrospinl fluid of ptients with herpetic encephlitis. J. Med. Microbiol. 7: Mkino, S., K. Sski, N. Nkmur, et l Studies on the modifiction of the live AIK mesles vccine. II. Development nd evlution of the live AIK-C mesles vccine. Kitsto Arch. Exp. Med. 7: Norrby, E., H. Link, J. E. Olsson, M. Pnelius, A. Slmi, nd B. Vndvik Comprison of ntibodies ginst different viruses in cerebrospinl fluid nd serum smples from ptients with multiple sclerosis. Infect. Immun. 1: Ogr, P. L, S. S. Ogr, S. Al-Nkeeb, nd P. R.

7 VOL. 23, 1979 ANTIBODY RESPONSES IN CSF 191 Coppol Locl ntibody response to experimentl poliovirus infection in the centrl nervous system of rhesus monkeys. Infect. Immun. 8: Orvell, C., nd E. Norrby Further studies on the immunologic reltionships mong mesles, distemper, nd rinderpest viruses. J. Immunol. 113: Shishido, A., nd M. Ohtwr Development of ttenuted rubell virus vccines in Jpn. Jpn. J. Med. Sci. Biol. 29: Skoldenberg, B., A. Crlstr6m, M. Forsgren, nd E. Norrby Trnsient ppernce of oligoclonl immunoglobulins nd mesles virus ntibodies in the cerebrospinl fluid in cse of cute mesles encephlitis. Clin. Exp. Immunol. 23: Virelizier, J.-L, A. C. Allison, nd G. C. Schild Antibody responses to ntigenic determinnts of influenz virus hemgglutinin. H. Originl ntigenic sin: bone-mrrow-derived lymphocyte memory phenomenon modulted by thymus-derived lymphocytes. J. Exp. Med. 1: Wolinsky, J., J. Bringer, G. Mrgolis, nd L Kilhm Ultrstructure of mumps virus repliction in newborn hmster centrl nervous system. Lb. Invest. 31: Ymnouchi, K., A. Fukud, F. Kobune, M. Hymi, nd A. Shishido Serologic response in monkeys inoculted with rinderpest nd mesles viruses. Am. J. Vet. Res. 3: Ymnouchi, K., A. Fukud, F. Kobune, M. Hikit, nd A. Shishido Serologic survey with the ser of monkeys in regrd to their nturl infection with mesles virus. Jpn. J. Med. Sci. Biol. 22: Ymnouchi, K., F. Kobune, A. Fukud, M. Hymi, nd A. Shishido Comprtive immunofluorescent studies on mesles, cnine distemper, nd rinderpest viruses. Arch. Gesmte Virusforsch. 29: Ymnouchi, K., Y. Yoshikw, T. A. Sto, S. Ktow, F. Kobune, K. Kobune, N. Uchid, nd A. Shishido Encephlomyelitis induced by cnine distemper virus in non-humn primtes. Jpn. J. Med. Sci. Biol. 3: Downloded from on April 5, 219 by guest