Antibody Responses in the Cerebrospinal Fluid of Cynomolgus

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1 INFECTION AND IMMUNITY, Feb. 1979, p /79/2-185/7$2./ Vol. 23, No. 2 Antibody Responses in the Cerebrospinal Fluid of Cynomolgus Monkeys After Intracerebral Inoculation with Paramyxoviruses KAZUYA YAMANOUCHI,* TAKESHI A. SATO, FUMIO KOBUNE, AND AKIRA SHISHIDO Department ofmeasles Virus, National Institute of Health, Musashi-Murayama, Tokyo 19-12, Japan Received for publication 3 October 1978 Cynomolgus monkeys with or without measles antibody were intracerebrally inoculated with measles or canine distemper viruses, and antibody responses in the cerebrospinal fluid (CSF) were investigated. In measles antibody-free monkeys, natural infection with wild measles virus or intracerebral inoculations with two attenuated measles vaccines evoked primary antibody responses to measles virus in the sera but not in the CSF. In measles-immune monkeys, intracerebral inoculation with the TYCSA strain of measles virus produced a significantly high titer of measles antibody in the CSF with a minimal rise in the serum antibody and resulted in a significant decrease in serum/csf antibody ratios. Intracerebral inoculation of a neurotropic canine distemper virus, the Onderstepoort strain, into measles-immune monkeys caused production of both measles and distemper antibodies in the CSF. Inoculation of measles-immune monkeys intravenously with measles virus or intracerebrally with rubella virus, which has no antigenic relation to measles virus, failed to evoke a measles antibody response in the CSF. These results indicated that local production of measles antibody in the CSF was caused by a stimulus within the central nervous system of measles virus antigen or canine distemper virus antigen that partially cross-reacted with measles virus antigen as a secondary antibody response. Virus infections in the central nervous system in chicken embryo cells. The infectivity titers of the (CNS) are generally thought to cause local production of virus antibody as indicated by the infective doses/ml, respectively. virus stocks were 16.3, 16.5, and 158 5% tissue culture presence of antibody at relatively high titers in (ii) Canine distemper virus. The Onderstepoort the cerebrospinal fluid (CSF) compared with strain (21) of canine distemper virus was propagated that in the in Vero serum (-6, 9, 11, 12). Demonstrations of measles antibody at high titers in the (iii) Rubella virus. The TO-336 vaccine strain (1) cells, and the stock virus had an infectivity titer of % tissue culture infective doses/ml. CSF of patients with subacute sclerosing panencephalitis are considered to be caused by a kidney cells, was obtained from Takeda Chemical of rubella virus, which was grown in primary rabbit prolonged stimulus with measles virus antigen Industries, Ltd., and its infectivity titer was 18 5% within the CNS. In spite of the increasing interest in the level of virus antibody in the CSF of Inoculation of monkeys. Cynomolgus monkeys tissue culture infective doses/ml. patients with various neurological diseases, (Macaca fascicularis) were inoculated either intracerebrally or intravenously. The intracerebral inocula- there have been few experimental studies on production of CSF antibody in animal models tion was conducted with.5 mil of each stock virus into the bilateral thalami. The intravenous inoculation was (1, 12, 21). made with 1 ml of each stock virus into the saphenous In the present study, we analyzed measles vein. antibody response in the CSF of cynomolgus Sampling. Blood was collected from the femoral monkeys, since monkeys were considered to be vein, and the serum was separated. The CSF was a suitable experimental animal due to their high obtained fronh the cerebellomedullary cistern and centrifuged at 2, rpm for 1 min to confirm visually susceptibility to measles virus and to the anatomical similarity of their CNS to that of humans. Both samples were kept at C until tested. the absence of contaminating erythrocytes (RBC). Titration of virus antibody. Hemagglutination MATERIALS AND METHODS inhibition (HI) antibody to measles virus was determined by our routine procedure (18); the sera were Virus. (i) Measles virus. The TYCSA strain of treated with kaolin and RBC of African green monkeys measles virus was propagated in Vero cells, and two to remove nonspecific inhibitors; thus, a 1:8 dilution vaccine strains, AIK-C (1) and Schwarz, were grown served as the lowest dilution. Since the CSF is free of 185

2 186 YAMANOUCHI ET AL. nonspecific inhibitors, it was tested from the undiluted sample without pretreatment. Neutralizing antibody to canine distemper virus was similarly determined in Vero cells by using the Onderstepoort strain as a challenge virus. Titration of natural antibody to GRBC. In preliminary studies on detection of natural antibodies in the sera of cynomolgus monkeys, presence of immunoglobulin G antibody that hemagglutinates goose RBC (GRBC) was demonstrated in all adult monkey sera examined. This natural antibody was not detected in the CSF of any animals except those contaminated with RBC through improper puncture of the cistern by needle at the time of CSF sampling. Therefore, this hemagglutinating antibody to GRBC was used as a reference antibody to indicate mechanical damage to the blood-brain barrier. RESULTS Pattern of antibodies to measles virus and GRBC in the sera and CSF of measlesimmune monkeys. Fourteen monkeys naturally infected with wild measles virus during the quarantine were examined for antibodies to measles virus and GRBC in their sera and CSF. After centrifugation, the CSF were visually classified into two groups, one without and another with blood contamination. The results are shown in Table 1. In a group without blood contamination, six CSF were completely negative for measles antibody and the other four contained antibody at very low titers. Serum/CSF ratios of measles antibody were 256 to 512 or greater. Titers of natural antibody to GRBC in the serum varied from 16 to 128, whereas none of the CSF contained the antibody. In a group with blood contamination, all four monkeys had antibodies to both measles virus and GRBC in the serum and CSF. Serum/CSF TABLE 1. ratios of both antibodies closely correlated. Thus, the natural antibody to GRBC was proven to be a useful indicator for artificial contamination with serum antibody in the CSF. CSF antibody response after natural infection or after inoculation with measles vaccines. Blood and CSF samples were collected every other week during the quarantine period from a group of monkeys which were newly imported from Southeastern Asia. Since the previous serological examinations indicated that measles HI antibody usually appeared in the serum about 2 weeks after natural infection (19), the time of natural infection with wild measles virus was estimated retrospectively to be 2 weeks before the time of the antibody rise in serum. None of the seven monkeys developed measles antibody in the CSF in spite of a marked rise of antibody in the serum (Fig. 1). Two groups of measles antibody-free monkeys, consisting of four animals each, were intracerebrally inoculated with either AIK-C or Schwarz vaccine, and their sera and CSF were examined every other week for development of measles HI antibody. None of the animals developed measles HI antibody in the CSF during an observation period of 8 weeks after inoculation (Fig. 1). These results indicated that neither the natural infection with wild measles virus nor the intracerebral inoculation with live measles vaccines initiated a primary measles antibody response in the CSF, in contrast to a marked antibody response in the serum. CSF antibody response in measles-immune monkeys after intracerebral or intravenous inoculation. Three groups of monkeys naturally infected with wild measles virus 2 to 3 months previously were intracerebrally or intra- Pattern of measles antibody and natural antibody to GRBC in the sera and CSF of monkeys naturally infected with measles virus Blood contarni- Measles HI titer Natural antibody titer nain Monkey no. nation 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 < <8 <1 _~~~~~ AIK-C vaccine Schwarz vaccine so _* *- - Se _ < _ ~ / ~ ~~~ * _ /. - / go &8 Be / Natural infection ANTIBODY RESPONSES IN CSF Weeks pi FIG. 1. Antibody response to measles virus after natural infection with wild measles virus or after intracerebral inoculation with attenuated measles vaccines, strains AIK-C and Schwarz. Symbols: HI antibody in the serum (); HI antibody in the CSF (). venously inoculated with the TYCSA strain of measles virus. The time course of the secondary antibody response after intracerebral inoculation in the first group is demonstrated in Fig. 2. Four monkeys lacked measles antibody in their CSF, and two had the antibody at a titer of 1 before inoculation with the TYCSA strain. All the monkeys except one whose CSF could not be examined because of contamination with blood developed measles HI antibody in the CSF 1 week postinfection (p.i.). The measles antibody titer in the CSF reached the highest titer 1 to 2 weeks p.i. in most of the monkeys. In contrast, serum antibody response exhibited a pattern different from that of CSF antibody; most of the serum antibody remained unchanged 1 week p.i., with a slight increase 2 weeks p.i. Thus, serum/csf antibody ratios generally tended to decrease in most of the monkeys; a mean ratio of before inoculation decreased to 1, 1, 126, and 72 at 1, 2, 3, and weeks p.i., respectively. Fractionation of the CSF samples on a sucrose density gradient disclosed HI antibody activity only in 7S fractions. Duration of CSF antibody response was examined in the serum and CSF samples collected from four measles-immune monkeys in the second group up to 1 weeks after intracerebral inoculation. CSF antibody persisted for at least 1 weeks (Fig. 3). Intravenous inoculation was made into the third group, consisting of four monkeys, but none of them developed antibody in the CSF when examined at 2 and weeks p.i. These results indicated that antigenic stimulus within the CNS is required for the production of measles antibody in the CSF. CSF antibody response after inoculation with canine distemper virus. Four monkeys naturally infected with wild measles virus 2 to 3 months previously were intracerebrally inoculated with the Onderstepoort strain of canine distemper virus. A typical pattern of primary antibody response to canine distemper virus was demonstrated in the serum (Fig. ). Moreover, relatively high distemper antibody was observed in the CSF. Therefore, replication of canine distemper virus within the CNS of measles-immune monkeys was suggested. No significant change in the titers of measles HI antibody in the serum was noticed, whereas a rise of measles antibody in the CSF was noticed in all four monkeys. Consequently, serum/csf ratios of measles antibody markedly decreased from over 256 to mean ratios of 16 to 6 after inoculation with canine distemper virus. Thus, it was suggested that the replication of heterologous canine distemper virus provided antigenic stimulus to antimeasles virus memory B cells within the CNS. CSF antibody response after inoculation with rubella virus. To eliminate the possibility that mechanical damage to the CNS by intracerebral inoculation might be responsible for an apparent development of measles antibody in the CSF, as demonstrated in the preceding experiments, rubella virus, which is not serologically related to either measles or canine distem-

4 188 YAMANOUCHI ET AL. INFECT. IMMUN. No.1163 No.116 No a 16. * U- -- a At ---O-~~ * --. A_~~~ %._ a b- 1. i:. _: No.1166 U- a No 1167 *U- N." No.1168 * _/.-- u U.. ".l" M I Weeks Pi FIG. 2. Antibody response to measles virus in measles-immune monkeys after intracerebral inoculation with the TYCSA strain of measles virus. Symbols: HI antibody in the serum (); HI antibody in the CSF (); serum/csf ratio of HI antibody (). No No _. 256 _ - o 6 _ *~~~~~~~~~- U-*- _-_ -9_ a a - a I..5. No No c '. _-a 6 I. } o o~~~, o o Weeks pi FIG. 3. Antibody response to measles virus in measles-immune monkeys after intracerebral inoculation with the TYCSA strain of measles virus. Symbols: Same as in Fig. 2.

5 VOL. 23, 1979 ANTIBODY RESPONSES IN CSF 189 No No Ia a 12[ 251 6H Is all Air A._ l1 * - No. 131 a82 No m 9 -- A~~~~ Weeks pi FIG.. Antibody response to measles and canine distemper viruses in measles-immune monkeys after intracerebral inoculation with the Onderstepoort strain of canine distemper virus. Symbols: Measles-HI antibody in the serum (a); measles-hi antibody in the CSF (); distemper-neutralizing antibody in the serum (A); distemper-neutralizing antibody in the CSF (A); serum/csf ratio of measles-hi antibody (-). per virus, was intracerebrally inoculated into nine measles-immune monkeys with mean measles HI titers of 8, and the serum and CSF were collected 3 weeks p.i. All nine monkeys developed rubella HI antibody, with a mean titer of 1 in the serum, but showed antibody production at a minimal level, ranging from <1 to 2, in the CSF. The measles HI antibody titer remained unchanged in the serum and was completely absent in the CSF. DISCUSSION Primary and secondary antibody responses in the CSF were investigated in monkeys by intracerebral inoculation with measles or canine distemper viruses. A primary antibody response was not evoked in the CSF after natural infection with wild measles virus or by inoculation with two measles vaccines, whereas the Onderstepoort strain of canine distemper virus induced a marked rise in distemper antibody in the CSF. These measles vaccine strains had been proven not to induce significant histological lesions in the CNS of monkeys when examined by intracerebral inoculation (unpublished data). In contrast, the Onderstepoort strain was shown to replicate to high titers and to induce disseminated histological lesions in the CNS of monkeys (21). In subacute sclerosing panencephalitis and acute measles encephalitis in which growth of measles virus was suspected, relatively high measles antibody in the CSF compared to that in the serum has been reported (5, 11, 15). Therefore, lack of CSF antibody response after intracerebral inoculation with the measles vaccine strains can be taken as indirect proof of the lack of virus replication within the CNS. A significant rise in measles antibody in the CSF compared with that in the serum was noticed when measles-immune monkeys were intracerebrally inoculated with measles virus. These monkeys were naturally infected (possibly through the respiratory route) 2 to 3 months before the inoculation. Therefore, it may be speculated that measles virus antigen stimulated measles virus-specific B memory cells as a secondary antibody response. Since the procedure of intracerebral inoculation results in the introduction of virus into the blood at the same time (7), antigenic stimulus should have been given also to the lymphoid system outside the CNS. Nevertheless, serum antibody was not significantly increased in general, in contrast to a marked rise in CSF antibody. Preexisting mea-

6 19 YAMANOUCHI ET AL. sles antibody at high titers in the serum before virus inoculation might have masked a subsequent antibody rise in the serum as a secondary response. Decrease in serum/csf antibody ratios can be caused by local production of antibody within the CNS, by mechanical damage of the bloodbrain barrier, or by increased permeability of the blood-brain barrier mediated through inflammation. The intracerebral inoculation of measles-immune monkeys with rubella virus failed to result in a rise of measles antibody in the CSF. Therefore, mechanical damage by intracerebral inoculation seems to be unlikely. The absence of natural immunoglobulin G antibody to GRBC in the CSF in contrast to high antibody titers in the serum may eliminate a possibility of serum antibody leaking into the CSF. Moreover, a secondary antibody response in the CSF was not evoked by intravenous inoculation. These results strongly suggest that measles and canine distemper viruses introduced into the CNS provided an antigenic stimulus leading to in situ secondary antibody response. Development of distemper antibody to high titers in the serum and CSF in measles-immune monkeys after intracerebral inoculation indicated that canine distemper virus replicated in the CNS of these monkeys. In a separate experiment (data not shown), the presence in the CNS of histological lesions similar to but with a milder nature than those observed in the case of measles antibody-free monkeys (21) was also confirmed. It is interesting that a marked rise in both measles and distemper antibodies was observed in the CSF of measles-immune monkeys after intracerebral inoculation with canine distemper virus. Between measles and canine distemper viruses, common antigenicity is demonstrated mainly in the nucleocapsids, whereas only partial correlation is present in the virus envelope (3, 13, 2). Thus, canine distemper virus can produce only a minimal level of measles neutralizing antibody and fails in the production of measles HI antibody in both dogs and monkeys (19, 2). Accordingly, an apparent rise in measles HI antibody in the CSF of measles-immune monkeys after intracerebral inoculation with canine distemper virus can be shown to be caused by the anamnestic response in which canine distemper virus antigen stimulated measles virus-specific B memory cells, as reported in mice immunized sequentially with related influenza virus hemagglutinin (16). It is possible that the intrathalamic inoculation used in the present study eventually resulted in introduction of virus inoculum into the third ventricle underlying the thalamus. The INFECT. IMMUN. cells lying on the ventricular surface of the hypothalamic ependyma of the tegu lizard were demonstrated to have macrophage-like phagocytic activity (2). Actively phagocytizing cells were also demonstrated on the ependymal surface after mumps virus infection (17). Therefore, it may be concluded that the virus particles were immobilized by the ventricular phagocytes and antibody response was subsequently initiated. A preliminary experiment (unpublished data) in which intracisternal inoculation with measles virus resulted in development of a significantly higher antibody response in the CSF than that by intrathalamic inoculation may indicate efficient uptake of virus particles by the ventricular phagocytes. ACKNOWLEDGMENTIS This study was supported by a research grant from the Intractable Diseases Division, Public Health Bureau, Ministry of Health and Welfare, and by a Grant-in-Aid for Intractable Disease Research from the Ministry of Education, Science and Culture, Japan. LITERATURE CITED 1. Albrecht, P., T. Burnstein, M. J. Klutch, J. Hicks, and F. A. Ennis Subacute sclerosing panencephalitis: experimental infection in primates. 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7 VOL. 23, 1979 ANTIBODY RESPONSES IN CSF 191 Coppola Local antibody response to experimental poliovirus infection in the central nervous system of rhesus monkeys. Infect. Immun. 8: Orvell, C., and E. Norrby Further studies on the immunologic relationships among measles, distemper, and rinderpest viruses. J. Immunol. 113: Shishido, A., and M. Ohtawara Development of attenuated rubella virus vaccines in Japan. Jpn. J. Med. Sci. Biol. 29: Skoldenberg, B., A. Carlstr6m, M. Forsgren, and E. Norrby Transient appearance of oligoclonal immunoglobulins and measles virus antibodies in the cerebrospinal fluid in a case of acute measles encephalitis. Clin. Exp. Immunol. 23: Virelizier, J.-L, A. C. Allison, and G. C. Schild Antibody responses to antigenic determinants of influenza virus hemagglutinin. H. Original antigenic sin: a bone-marrow-derived lymphocyte memory phenomenon modulated by thymus-derived lymphocytes. J. Exp. Med. 1: Wolinsky, J., J. Baringer, G. Margolis, and L Kilham Ultrastructure of mumps virus replication in newborn hamster central nervous system. Lab. Invest. 31: Yamanouchi, K., A. Fukuda, F. Kobune, M. Hayami, and A. Shishido Serologic response in monkeys inoculated with rinderpest and measles viruses. Am. J. Vet. Res. 3: Yamanouchi, K., A. Fukuda, F. Kobune, M. Hikita, and A. Shishido Serologic survey with the sera of monkeys in regard to their natural infection with measles virus. Jpn. J. Med. Sci. Biol. 22: Yamanouchi, K., F. Kobune, A. Fukuda, M. Hayami, and A. Shishido Comparative immunofluorescent studies on measles, canine distemper, and rinderpest viruses. Arch. Gesamte Virusforsch. 29: Yamanouchi, K., Y. Yoshikawa, T. A. Sato, S. Katow, F. Kobune, K. Kobune, N. Uchida, and A. Shishido Encephalomyelitis induced by canine distemper virus in non-human primates. Jpn. J. Med. Sci. Biol. 3: