Clin. exp. Immunol. (1979) 35, 397-404.

Progressive rubella panencephalitis: immunovirological studies and results of isoprinosmie therapy J. S. WOLINSKY,*t P. C. DAU,*+ ELENA BUIMOVICI-KLEIN,§ J. MEDNICKt B. 0. BERG,* P. B. LANG§ & L. Z. COOPERS *Departments of Neurology, Pediatrics and Medicine, University of California, San Francisco, t Neurology Research Laboratory, Veterans Administration Hospital, San Francisco, t Cellular Immunology Laboratory, Children's Hospital, San Francisco, and § Department of Pediatrics, The Roosevelt Hospital, College of Physicians and Surgeons of Columbia University, New York, USA

Received 16 May 1978)

SUMMARY

Two patients with progressive rubella panencephalitis, one with and one without stigmata of congenital rubella, were treated for 9 months with isoprinosine and showed continued clinical deterioration. Immunovirological studies performed before, during and after treatment were unaffected by drug therapy. The virus was recovered on one occasion from the lymphocytes of one of these cases. Neither patient showed any major defects in cellular or humoral immunity. However, the lymphocytes of the patient with stigmata of congenital rubella failed to respond to rubella virus in vitro and had a heat stable, non-dialysable serum inhibitor of in vitro protein A stimulated proliferative responses. Both patients' serum interfered with the production of interferon by normal donor lymphocytes following stimulation with rubella and varicella virus antigen. Increasing serum titres of interferon which did not appear to be lymphoid or immune-specific in origin were found in these two cases. INTRODUCTION Progressive rubella panencephalitis (PRP) is a slowly progressive and fatal central nervous system (CNS) disorder (Wolinsky, 1978) that appears to be a late sequel of both congenital rubella (Townsend et aL., 1975; Weil et al., 1975a) and post-natal rubella infections (Lebon & Lyon, 1974; Wolinsky, Berg & Maitland, 1976). The disorder bears many features in common with subacute sclerosing panencephalitis (SSPE) (Wolinsky, 1978). Initially promising reports on the efficacy of isoprinosine in the treatment of SSPE (Mattson, 1975; Huttenlocker, 1975; Streletz & Cracco, 1977) prompted a clinical drug trial in our two surviving cases. This report summarizes the results of that trial and presents serial immunovirological data obtained on the patients and cellular immunological data obtained in healthy donors using serum from the two patients. MATERIALS AND METHODS Cases. The clinical summaries of the patients have been detailed in previous communications. In brief, the first child (case 1) was born in 1960 with stigmata of congenital rubella. Progressive neurological deterioration began at 12k years of age (Townsend et al., 1975). He was entered into the present study aged 15 years 10 months with microcephaly and growth retardation, severe mental retardation, spastic quadriparesis with inability to walk, optic atrophy and multifocal myoclonus. The second child (case 2) was born in 1953 without stigmata of congenital rubella and had a history of uncomplicated rubella infection at age 7 (Wolinsky et al., 1976). Neurological deterioration began at 19 years of age and has continued. At entry Correspondence: Dr J. S. Wolinsky, Johns Hopkins University, School of Medicine, 720 Rutland Avenue, Traylor Building 709, Baltimore, Maryland 21205, USA. 0099-9104/79/0030-0397$02.00 (C 1979 Blackwell Scientific Publications

397

398

9. S. Wolinsky et al.

into this study aged 23, he had a severe dementia, dysarthria and spastic quadriparesis and could walk only with assistance. Appendicular ataxia, truncal titubation and mild optic disc pallor without visual loss also were present. Each patient received 100 mg/kg isoprinosine orally in divided daily dosages continuously for 9 months. Serological studies. Serum and cerebrospinal fluid (CSF) specimens were obtained prior to, and after 3 months, 6 months and at completion of drug therapy. Samples were divided into aliquots and stored at - 70'C prior to assay. Antibody assay were performed as described previously (Townsend et al., 1975). Antibodies of the IgM class were determined by a sucrose gradient technique (Vesikari et al., 1969). Oligoclonal antibody was sought by agarose gel electrophoresis of concentrated CSF (Johnson et al., 1977). Virological studies. Fresh samples of blood, CSF and urine were periodically assayed for rubella virus on BSC-1 monolayers using ECHO-1I virus interference challenge (Plotkin, 1969). Mononuclear cells, separated from heparinized whole blood on Ficoll-Hypaque gradients (Boyum, 1968), were cultured in suspension (Simons & Jack, 1968) and aliquots of supernatants were screened daily for the virus as described above. Alternatively, gradient purified mononuclear cells were co-cultivated with BSC-1 cells at a donor to indicator cell ratio of approximately 50:1 in Leighton tube cultures as a modification of a technique described by Cappel (1975). Supernatants of these cultures were passed at day 6 for free virus and the original cultures examined by immunofluorescent antibody technique (IFA) (Schmidt et al., 1966). The presence of virus on passage was confirmed by both interference of ECHO-1I virus challenge and direct IFA. Controls for these isolation studies included identically processed normal donor lymphocytes. Interferon assay. Serum samples obtained before, at 3 and 6 months of therapy, and after cessation of drug therapy were stored at - 70'C prior to assay. Interferon titres on these samples were expressed as the reciprocal of the highest dilution which protected 50% of the cells from the cytopathic effects induced by vesicular stomatitis virus in a human fibroblast cell line grown as a monolayer in microtitre plates as described previously (Buimovici-Klein, Weiss & Cooper, 1977). Cell-mediated immune assays. Delayed hypersensitivity responses at 24 and 48 hr to the intradermal injection of 0- 1 ml of candidin (1:100 dilution), coccidiodin (1:100), staphylococcal lysate (1:5), intermediate strength PPD, streptokinasestreptodornase, and mixed respiratory virus antigen were measured before and after 3 and 6 months, and after completion of drug therapy. Induration of 5 0 mm or more was considered to be a positive response. In vitro responsiveness of lymphocytes purified on Ficoll-Hypaque gradients were assessed by incubation with serial tenfold dilutions of concanavalin A (Sigma), pokeweed mitogen (Sigma), phytohaemagglutinin (Burroughs-Wellcome), and soluble protein A (Pharmacia). Cultures were set up in triplicate in microtitre plates (Falcon 3040) and contained 2x 105 lymphocytes in 0-2 ml of 85% minimum essential medium containing 2-0 mm glutamine, 50 units penicillin and 50 jug streptomycin per ml and 15% serum. After 3 days of incubation at 37°C in a humidified atmosphere of 5% CO2 and 95% humidified air and a 16 hr terminal labelling period with 0 05 pCi of 14C thymidine (sp. act. 49-2 mCi/mmol), the cells were harvested by collecting them on glass fibre filters using a Brandel Cell Harvester. The filters were dried, placed into plastic counting vials and incubated for 30 min at 37°C with 0 5 of Solulene (Pakard Instruments). 10 ml of a scintillation cocktail (Econoflour, New England Nuclear) were added and the radioactivity determined in a liquid scintillation counter. Supernatants from these cultures were stored frozen at - 70°C and assayed for the presence ofinterferon as described above and leucocyte migration inhibition factor (LMIF) by using the indirect assay modified after Claussen (1975), as previously described (Buimovici-Klein, Lang & Ziring, submitted for publication). Indicator polymorphonuclear leucocytes provided from a single healthy donor were incubated for 24 hr together with supernatants from either stimulated or unstimulated lymphocyte culture in wells prepared in petri dishes coated with agarose. The migration inhibition was expressed as a percentage reduction and was calculated as: /area of migration in stimulated supernatants - area of well\ X 100. Varea of migration in control supernatants - area of well Responses of nylon column purified lymphocytes to specific viral antigens were assayed in vitro as previously reported (Dau, Johnson & Spitler, 1976). Human embryonic lung fibroblasts infected with either the LEC strain of measles virus or mumps virus were used as stimulating antigens. Varicella virus complement fixation antigen was obtained from Microbiological Associates and used in a dose of 25 p1 per 1-0 ml culture. Rubella antigen was prepared from virus grown in BHK-21 cells, concentrated with ethylene glycol and partially purified by sucrose gradient separation as described previously (Buimovici-Klein et al., 1976). Results were expressed as a stimulation index obtained by dividing the counts per minute (ct/min) of radioactivity incorporated by lymphocyte cultures incubated with the specific virus by the ct/min incorporated by cultures incubated with control material not containing the specific virus. Supernatants from these cultures were stored frozen at - 70°C for assay of interferon and LMIF as described above. Blocking factor studies. Blocking factors were assayed in each patient's serum by setting up cultures in either 15% patient serum or 7.5% patient serum plus 7 5% pooled serum from six normal donors (NPS) and comparing the responses to those obtained from identical cultures set up in 15% NPS. Lymphoryte rosetting studies. The percentage of long and short incubation E-rosette-forming cells was measured according to Wybran et al. (1973). The percentage of EAC rosette-forming cells was determined according to Scheinberg & Cathcart

(1974).

Progressive rubella panencephalitis

399

RESULTS Both patients showed continued clinical deterioration despite 9 months of daily therapy with isoprinosine. Case 1 developed retinal changes compatible with the retinitis of congenital rubella, became increasingly less responsive to environmental stimuli, developed more prominent myoclonus and has subsequently required institutionalization for chronic care. Case 2 showed increasing optic disc pallor in association with deterioration of visual acuity and has become essentially mute and wheelchair bound. No untoward effects of drug therapy apart from an anticipated mild elevation of serum uric acid levels were observed. Drug therapy did not appear to influence any of the immunological variables studied. Rubella virus was isolated from gradient purified mononuclear cells of case 1 by co-cultivation techniques 1 week after the cessation of drug therapy. All other attempts at viral isolation were negative. F (b) 'F (a)

I

0 ._

E

4'

-

(c)

(d) /

t_ 'I

go

0

c 2 -P

Jag FIG. 1. In vitro lymphocyte proliferation response to standard mitogens. (a) Phytohaemagglutinin, (b) Con A, (c) pokeweed mitogen, and (d) protein A. (a) Response of normal donor lymphocytes in 150%) ), and case NPS± 1 s.d. of the mean value. Response of lymphocytes in 15% NPS for case 1(I 2 (a - - -A) with vertical bars representing± s.d.

7. S. Wolinsky et a!.

400

Serum and CSF haemagglutination inhibition (HAI) antibody activity to rubella virus has remained stable in both cases since initial diagnosis and did not change during treatment (case 1: serum 256, CSF 16; case 2: serum 128, CSF 16). Serum HAI antibody of the IgM class was detected over the previous 2 years at dilutions up to 1:4 in both patients and was present in trace amounts at initiation of the drug trial, but was not seen on subsequent testing. Rubella specific IgM was never seen in CSF. Oligoclonal IgG banding patterns in CSF samples from both patients were qualitatively unchanged throughout the study. Interferon was present in the pre-treatment sera of both patients (4 and 6 units) and remained stable or increased over the observation period (16 and 6 units, respectively). Both patients showed positive delayed hypersensitivity reactions to at least one of the skin test antigens used. Each patient had normal numbers of T lymphocytes (70-8 1% and 64-70%; normal = 63 + 7-8%) and active T lymphocytes (20-38% and 23-34%; normal = 29+7.2%), with increased proportions of EAC rosette-forming cells (29-40% and 18-28%; normal = 17-2+4.8%), but few or no null cells (normal = 17+ 10-2%). The proportion of cells containing Fc receptors on their surfaces was shown to be increased (23% and 26%) upon direct IFA testing with a polyvalent antiserum against IgG, IgA and IgM, further suggesting increased numbers of circulating B lymphocytes. Lymphocyte proliferative responses in vitro were normal or near normal for most mitogens when tested in NPS. However, case 1 had consistently depressed responses to protein A at concentrations up to 100 yug/ml. These responses were similar over the entire observation period and are illustrated by a typical set of data from the sixth month of drug therapy (Fig. 1). Similar mitogen stimulated responses were found when the tests were carried out in the presence of either fresh autologous serum, heat-inactivated autologous serum, or a 1:1 mixture of NPS and autologous serum, except that the responses of cells from case 1 to protein A in the presence ofautologous serum were even more depressed. The responses to protein A (Fig. 2) indicated that a heat stable, non-dialysable inhibitor of the proliferative responses of normal donor lymphocytes to protein A stimulation was present in the serum of case 1 (Fig. 2), but not

3 0

22

0

0'1

1.0

10

100

01

1.0 Protein A

10

(jag)

100

0.1

1.0

10

100

FIG. 2. In vitro lymphocyte response to protein A stimulation. (a) and (b): (n) Represents the mean+ 1 s.d. for normal donor lymphocytes in 15% NPS, in (c) it is the mean response+ s.d. for normal donors in 7-5% ( )) NPS+7-5% dialysed normal control serum. (a) The response of case 1 lymphocytes in 15% NPS ( 0). (b) The response of normal donor lymphocytes in and in 7-5% NPS+7-5% homologous serum (a 7.5% NPS-L7-5% fresh case 1 serum (D - - - L) and in 7-5% NPS+7-5% case 1 sera heat-inactivated for 30 i). (c) The response of normal donor lymphocytes in the presence of 7-5% NPS Imin at 560C (a *). All vertical 7-5% case 1 sera dialysed against four changes of phosphate buffered saline at 40C (* bars signify± s.d.

401

Progressive rubella panencephalitis

in the serum of case 2. Similar results were seen before, during and after drug therapy. In some experiments the inhibitory effect was partially overcome at high concentration of protein A (10-100 pg per culture). Lymphocytes from case 1 consistently failed to show an in vitro proliferative response to rubella virus antigen while those from case 2 showed responses similar to those of controls (Fig. 3). There was no change in this pattern of response when cells were either washed six times in serum-free media, or preincubated for 1 hr in medium containing 10% NPS before stimulation with rubella virus antigen. Neither patient's serum inhibited the positive proliferative response of immune donor lymphocytes to rubella virus (Fig. 3). Lymphocytes from case 1 did show significant responses to measles, mumps and varicella virus antigens; case 2 also responded to varicella antigen. Supernatants of lymphocyte cultures from both cases did not contain either interferon or LMIF after challenge with rubella antigen (Table 1). Interferon and LMIF were present in lymphocyte cultures from two immune donors when stimulated by rubella and varicella virus antigens in the presence of 6

4

0

-E 2

-

0~~~~ 0~~~~~

A

C

B

FIG. 3. In vitro lymphocyte responses to partially purified rubella virus antigens. Vertical bars are the mean responses+ s.d. for lymphocytes from healthy donors with serological evidence of prior rubella virus infection. (o) Responses of lymphocytes from case 1; (A) the responses of case 2. Condition A is the response in the presence of 15% NPS, B is the response in 7-5% NPSA-7-5% case 2 serum, and C the response in 7-5% NPS4-

7-5%

case

1

serum.

TABLE 1. Interferon (IF) and leucocyte migration inhibition factor (LMIF) production after rubella virus (RV) and varicella virus (HVZ) stimulation in the presence of pooled normal donor sera (NPS) and patient's sera

Origin of lymphocytes

Antigen

Case 1 Case 2 Donor I Donor 2 Case I Case 2 Donor 1 Donor 2 *

NPS+1I

RV RV RV RV HVZ HVZ HVZ HVZ =

NPS

NPS+1*

NPS+2t

IF§ LMIFT

IF LMIF

IF LMIF