The varicella zoster virus: A pilot trial of a potential therapeutic agent in multiple sclerosis

J Clin Epidemiol Vol. 50, No. 1, pp. 63-68, Cupyrighr 0 1997 Elsevier Science Inc.

0895-4356/97/$17.00 I’ll SO895-4356(96)00300-9

1997

ELSEVIER

The Varicella Zoster Virus: A Pilot Trial of a Potential Therapeutic Agent in Multiple Sclerosis R. T. Ross,’ Lindsay ‘SECTIONS ‘BIOSTATISTICAL

E. Nicolle,’

and Mary

Cheun$

OF NEUROLOGY, ‘INFECTIOUS DISEASES AND DEPARTMENT OF MEDICAL CONSULTING UNIT, COMMUNITY HEALTH SCIENCES, THE UNIVERSITY SCIENCES CENTRE, WINNIPEG, MANITOBA, CANADA R3A

MICROBIOLOGY, OF MANITOBA IR9

AND THE ANLI THE HEALTH

ABSTRACT.

There are a number of similarities in the geographic, latitudinal, and epidemiological features of multiple sclerosis (MS) and varicella (V). In the experimental model of MS, repeated high antigen doses (myelin basic protein) have deleted both the clinical and pathological manifestations of the disease. Therefore, it seemed appropriate to explore the effects of varicella zoster vaccine on patients with MS. Fifty patients with chronic progressive MS were vaccinated with attenuated varicella virus vaccine and followed for one year. Fourteen patients improved, four became worse, and twenty-nine were unchanged. All patients were seropositive for varicella before vaccination and all had a rise in varicella antibodies after the vaccinations. There were no major untoward results from the vaccine. Four patients developed mild chicken-pox after vaccination. This was a short-term pilot trial without control subjects. Thus, the apparently favourable clinical and magnetic imaging changes must be assessed with caution, as must the lack of adverse effects. Copyright 0 1997 Elsewier Science Inc. J CLIN EPIDEMIOL 50;1:63-68, 1997.

KEY WORDS.

Multiple

sclerosis,

varicella

vaccine,

varicella

zoster

virus,

magnetic

resonance

imaging

INTRODUCTION

METHODS

with slowly progressive multiple sclerosis (MS) developed varicella. Within weeks of the exanthem the MS signs and symptoms began to remit and the improvement continued. In 1962, another patient with the same disease contracted varicella and had a lengthy remission of his MS. Neither of these patients had deterioration of their MS in the next several years, both thought they had varicella as children. Reported as a Letter to the Editor [l], these observations elicited correspondence relating similar experiences. Recently, Merck Sharp &. Dohme developed and tested an attenuated live-virus varicella vaccine. Given this background, we tested the vaccine in subjects with multiple sclerosis to determine safety, immunogenicity, and the clinical course of MS after vaccination. Fifty subjects do not constitute an adequate cohort for vaccine safety testing. Currently these tests involve numbers of thousands of subjects. The present group was limited by the amount of vaccine provided. Two hundred and forty neurological assessments and 135 enhanced MRI examinations were performed. This provides reasonable assurance that no neurological damage occurred.

The protocol was approved by the University of Manitoba, Faculty Committee on the Use of Human Subjects in Research, the Research Committee of the Health Sciences Centre, Winnipeg, the American Food and Drug Administration, and the Health Protection Branch, Health and Welfare Canada, Ottawa. An open pilot study was designed to test the safety and possible efficacy of varicella zoster vaccine. Patients were eligible for enrollment if they had clinically definite MS [2,3]. All patients had evidence of deterioration in the 18 months before the trial. Patients were accepted with chronic progressive or relapsing progressive MS provided they had not had an acute relapse in the previous year. Chronic progressive MS was defined as steady deterioration in the patients’ ability to perform the activities of daily living due to increasing evidence of MS. Relapsing progressive MS was defined as multiple acute episodes of increased disability due to the disease with moderate or no recovery after each episode. The participants were between 18 and 60 years of age and they all understood and signed the Informed Consent Form. All patients were able to come to the outpatient department. No patients were paid for participating but travel, accommodation, and incidental expenses were reimbursed. It was emphasized to all patients that injecting a live virus into a person with a damaged or possibly damaged

In 1960

a male

patient

Address for correspondence: Professor R. T. Ross, Health Sciences Centre, GF535, 820 Sherhrook Street, Winnipeg, Manitoba, Canada R3A IR9. Accepted for publication 5 August 1996.

64

blood-brain-barrier might be hazardous. Premenopausal women were accepted if they had a negative pregnancy test and would use contraception for the following two years. Patients were excluded if they had taken azathioprine, corticosteroids, or any other immune suppressing agent 30 days before entry into the trial. Patients were excluded if they had ever received cyclophosphamide or mediastinal radiation. All patients had an Expanded Disability Status Scale (EDSS) [4] of 2.0 or greater at entry and were free of any other major disease. They all agreed to take no new medication apart from analgesics (excluding acetylsalacylic acid) during the trial. Investigations at entry included a complete blood count, urinalysis, biochemical profile, chest x-ray, electrocardiogram, and quantitative humoral varicella antibody testing by the enzyme linked immunosorbent assay (ELISA). Using the EnzygnostB kit, Anti-VZV/IgG (Hoechst-Bering), antibodies were measured at entry and at second, fourth, and final neurological examinations. Magnetic resonance imaging (MRI) of the head with enhancement was performed at entry, six months, and 12 months after the first examination. The MRI examinations were performed on a 2.0 Tesla (T) Siemens Helicon imager operating at 1.5 T. All patients had a sagittal localizer spin echo sequence (TR 200, TE 15, 1 excitation) and an axial T2 weighted sequence oriented to the long axis of the corpus callosum of 5 mm. thickness with a 0.2 to 0.3 interslice gap and scanning parameters of 2500/22, 90/l (repetition time/echo time/excitations). Tl weighted axial images of 5-mm thickness and an interslice gap of 0.3 and parameters 600/15/2 were performed before and after the infusion of Gadopentetate Dimeglumine (Magnevist, Berlex Canada Inc.) at 0.1 millimoles per kilogram. The radiologists were uninformed as to the diagnosis or clinical activity of the disease and had no contact with the neurologist during the trial. MRI findings were not incorporated into the patients’ records until the trial was completed. The number of lesions greater than 5.0 mm in diameter consistent with MS and the number of enhancing lesions were assessed by three independent neuroradiologists. One was internal and two external to the hospital performing the MRIs. Consistent interobserver differences were recorded but each was systematically different. The number of lesions and the number of enhancing lesions are reported as the mean of the three observers. All 240 neurological assessments were done by the same person (RTR) without reference to prior EDSS scores or concurrent MRI findings.

Vaccine Merck Sharp & Dohme Research Laboratories supplied 250 X 0.7 ml vials of OKA/Merck Varicella Vaccine, Lot 1164/ c-T684, plaque-forming units (PFU) unknown, and 250 X 0.7 ml vials of sterile diluent, being distilled water without

R. T. Ross et al.

preservatives. The vaccine was stored at -20°C and the diluent refrigerated. The vaccine was reconstituted by adding a11 of a vial of diluent to a vial of lyophilized vaccine and agitating the mixture thoroughly. Then 0.5 ml of the mixture was injected subcutaneously within five minutes of reconstitution. After nine patients had been given this dose (six patients had two vaccinations and three had one only) the dose was doubled, i.e., two vials were reconstituted for each injection and 0.5 ml from each (total dose 1 .O ml) was used for each vaccination. A trained and experienced nurse gave all vaccinations. The first vaccine was given after the initial laboratory, clinical, and MRI examinations were completed. The second vaccination was given six weeks later. All antibody determinations were done by the Cadham Provincial Laboratory on all sera together at the end of the trial. At each vaccination, patients were given a vaccination report card and a clinical thermometer. The report card listed all known post-vaccinal symptoms. Patients were asked to take their temperature at the same time every day, record it, note any symptoms for 30 days after vaccination, and return the card. Statistical

Methods

Repeated Measures analysis of variance were used to compare repeated assessments over time. Spearman-Rank correlation coefficients evaluated associations between different variables. Exact permutation tests were used to evaluate sparse contingency tables for differences in proportions within categories. Significance levels were set at alpha = 0.05 2-tail.

RESULTS Enrollment began in March 1992 with the final enrollment in October 1992. The last patient completed the study in December 1993; the average period of observation was 14 months. The cohort demographics are in Table 1. Fifty subjects entered the study and 45 completed it. The five who were lost failed to appear for subsequent clinical assessments (n = 2), could not tolerate the MRI examinations (n = I), or quit the trial of their own volition (n = 2). There were 10 males. Vaccination Forty-seven patients were vaccinated twice. Two of these people did not complete the trial. Three were vaccinated once and withdrew. The five who left the study were followed for one year and reported no untoward effects related to the vaccine. Sixty-one vaccinations were uneventful. Thirty-two vaccinations were followed by pain, tenderness, and/or swell-

Varicella

Zoster

TABLE

1.

Virus/Multiple

65

Sclerosis

Details of the cohort n

Age

(years)

20-29 30-39 40-49 50-59 Disease

17 21 11 duration

(years)

o-9 10-19 20-29 >30 EDSS

17 21 8 4

at entry

2-3.5 4-6 6.5-7 7.5-8 8.5 Ahbreviatiun:

12 12 8 17 EDSS

= Expanded

disability

status scale

ing, and/or redness for two to three days at the injection site after the first or second or both vaccinations. Two patients had elevated temperatures of l-15°C with the local symptoms. Generalized muscle aching and stiffness for four to six days followed two vaccinations. Photophobia, headache, stiff neck, and coryza-like symptoms followed four vaccinations. Four patients developed minor episodes of probable varicella three to six days after vaccination. Two of these patients were not seen, but they described itchy patches of vesicles on their limbs and trunk, probably less than 20 vesicles in all, and no constitutional symptoms. The other two patients were seen. One had 25 vesicles on limbs and abdomen and felt generally unwell. The vesicles were clinically consistent with varicella. The culture was negative. The fourth patient had about 20-25 grouped vesicles on a red base on the back, buttocks, shoulder, and thigh. The patient’s temperature was normal, the lesions were not itchy, and there were no constitutional signs or symptoms. A culture of the vesicle fluid was negative. Of these four patients, one changed from an EDSS rating of 2.5 to 1.5; one changed from 3.0 to 1.5; one from 6.0 to 2.0; and one remained at 6.5. Varicella

Antibodies

Varicella antibodies were measured by the ELISA method on all patients at entry, three, six, and 12 months after the first vaccination. The EnzygnostB Anti-VZV IgG kit (Hoechst-Bering) was used. There were no relationships between the antibody level at entry and the patient’s age, duration of disease, or level of disability. The mean levels of antibodies are shown in Table 2. All patients were seropositive on entry and all had a significant rise in antibodies

following vaccination. The rise persisted for 7 to 12 months and was inversely proportional to the antibody level at entry. The patients who received the smaller dose of vaccine had no significant difference in their antibody responses compared with those receiving the larger dose.

Clinical

Changes

This study was a safety trial without control subjects. Observer errors and placebo effects also make the interpretation of any clinical changes very difficult. They are reported here for completeness and shown in Fig. 1. Fourteen patients (30%) improved, 4 became worse, and 29 were unchanged. This may be of little significance. Three patients changed 0.5 grade as measured by the EDSS and should be ignored. The clinically improved patients do not differ from the others by age (p = 0.97), disease duration (IJ = 0.91), gender (p = 0.63), difference in varicella antibodies at entry (p = 0.19), or changes in varicella antibody level at 6 months (p = 0.15) or 12 months (1, = 0.22). However, 62% of those with entry EDSS < 5.5 improved while only 13% of those with EDSS > 5.5 improved (p < 0.001). Some improvement was transient and more evident in the earlier examinations than at the end of the trial. Analysis of EDSS ratings showed the greatest changes from entry at 6 and 9 months (p < 0.0001) compared to the 12 month evaluation (p < 0.0011). The number of improved patients was compared with the placebo group of the Canadian Cooperative Study [5] as historical controls. This latter study also examined patients with chronic progressive MS and showed 2% of the placebo group improved after 12 months. The difference is significant (p < 0.0002).

MRI

Findings

Forty-four patients completed five clinical assessments and three enhanced MRI examinations. One patient with clinically definite MS had three normal MRI scans, leaving the results of 132 scans on 44 patients for analysis (Fig. 2, A and B). The entry examination provided a baseline and confirmed the diagnosis [6,7]. The MRI scans were assessed as to number of lesions and number of enhancing lesions at entry and after 6 and 12 months of the study. There was no communication between clinical and MRI assessors at anytime during the study. The three groups of patients (clinically unchanged, improved, or worse) had a statistically significant, different number of lesions at entry and this was maintained throughout the study (Fig. 2 A). The clinically worse and unchanged groups had a significant increase in number of lesions over the duration of the study, while the improved group did not (p < 0.01). The improved and unchanged groups had significantly fewer enhancing lesions than the

R. T. Ross et al.

66

TABLE sclerosis

2. Mean

level

(and

standard

deviation)

of varicella

antibodies

(elisa)“

in 47 patients

Post 3 Monthsh

Entry 1.284

1.662

-c (0.551)

2

Bladder

chronic

(0.477)’

group at entry and at all times (p < 0.016)

level

at entry;

multiple

vaccination

1.598

I = 0.70,

?

14 Months

(0.478)’

1.440

-c (0.481)’

p = 0.0001.

(Fig.

Function

Bladder function improved more than any other abnormality and the improvement persisted. Twelve patients had no bladder complaints at entry, 16 had no improvement in their bladder complaints, and 3 became worse. Seventeen patients had major improvement in bladder function and nine of these became symptom-free in this system. These patients were able to abandon self-catheterization, protective clothing, and restrictive fluid intake, etc. There was no obvious correlation between the improvement in bladder function and the improvement in any other system or systems. The use of anticholinergic and other medications was closely monitored. No patient added or deleted medications during the trial.

INITIAL

GMONS

1

12MONS

4 ,

i 3t- ...I1 t

DISCUSSION

I

r

The two original MS patients with adult varicella are a clinical observation and there is little more to say about them. Both had varicella consistent with Heberden’s description

INITIAL,

Expanded Disability SWUS kore Change 12 Months After Em-f

progressive

6 Months

“The antibody levels are expressed as optical density absorbent values. hSecond vaccination given at this time. ‘P = 0.001 from entry level. The rise correlated inversely with the

worsening 2 B).

with

GMONS

12MONS

FIGURE 2. (A) Mean number of MRI lesions consistent with MS as determined by three independent radiologists. The scans were performed before and 6 and 12 months after vaccination with attenuated varicella vaccine and are further classified by the patient’s clinical state, i.e., improved (black bars), same (shaded bars), or worse (open bars). (B) The mean number of gadolinium enhancing MRI lesions. The details are the same as in panel A.

-2.0 -1.5 -1.0 -0.5 0.0 +0.5 +1.0 f1.5 +2.0 f2.5 +3.0

0 0 2.0-3.0 (N=5)

3.5 IN=71

4.0-5.5 (N=4

6.0 (N=8)

6.5 (N=3)

7.0-7.5 (N=15)

8.0-8.5 (N=5)

Expanded Disability status score Grading at Entry and Number cd Subjects by Grade

FIGURE 1. Changes in EDSS of 47 patients with multiple sclerosis 12 months after vaccination with attenuated vat-icella virus. (Decreases in status score represent improvement; increases represent worsening.) From [4].

[8] and both had clinically definite MS by the standards of the day. Both thought they had chicken-pox as children. No one was injured by the vaccine. There was better than 95% compliance by the patients in completing their postvaccination report cards. Many of the patients had active, enhancing, MRI lesions at the vaccination time. It is known that new enhancing MRI lesions are short-lived (230

Varicella

Zoster

Virus/Multiple

Sclerosis

days?) [9], and correlate with fresh intense inflammatory activity, perivascular cuffing, edema, and parenchymal mononuclear cell infiltration at the margins of the lesions [lo]. The ELISA antibody assay reveals optical density absorbent values and is not a quantitative measure of antibody change. There were no differences in antibody levels at entry, responsiveness, or persistence between the improved and unimproved patients. It has been reported by others that this vaccine will produce substantial antibody increases in seropositive adults as well as boosting cellular immunity

illI. The use of varicella zoster live-virus vaccine may have slowed the rate of deterioration and improved the clinical outcome in this study beyond what might be expected by chance. The MRI findings paralleled the clinical results at the end of the trial. Because new, active MRI lesions are seen so often in clinically quiescent MS [12,13,14] it is possible that a diminishing number of enhancing lesions represents diminishing activity of the disease. The urinary bladder improvement was not restricted to the 14 clinically improved patients. Five of the 14 had no bladder symptoms, three experienced no change in major urgency and incontinence, and six had major urgency and incontinence that was completely relieved at the end of the study. No studies of bladder dynamics were made at entry. Therefore, there is no information or speculation as to the mechanism behind the improvement in this symptom. It is a generally accepted view that some exogenous agent acquired in childhood probably leads to MS years later [ 151. The measles and canine distemper viruses have been prime contenders but studies have been unable to confirm an association. Some epidemiological and geographic factors common to MS and varicella suggest varicella zoster virus may be a candidate. Both diseases have relatively few years of maximum age-specific incidence (varicella 5-9 years [ 161, MS 3 l-33 years [ 171). Both are common in the same places and both are rare in other places [ 181 although there are many exceptions, i.e., Japan has a high varicella seropositivity [19] and a low prevalence of MS (20). The effects of migration and the age-at-migration “protective” effect on the subsequent chances of developing MS strongly supports the possibility that some childhood infectioushisease has an etiologicai role in MS [21,22]. A study of a Hutterite population in Western Canada supported the correlation. In spite of pure Austrian racial origin and life-long residence in a high risk MS area, their prevalence of MS is significantly less than a control group, as is their prevalence of varicella and herpes zoster [23]. The inverse correlation, namely, low varicella prevalence/serpositivity with high MS prevalence is apparently not known. There are other links between varicella roster virus and MS. There is controversial evidence of an increased incidence of herpes zoster in MS [24,25,26]. Ten of the 50 patients in the current study had a history of herpes zoster, and

67

one had two attacks. In five patients, the disease occurred between ages 10 and 20, in two in their mid-20s, and in the others it was at 35, 49, and 50 years of age. If the varicella zoster virus has an antigenic role in the evolution of MS there is an explanation why a subsequent antigen dose might ameliorate the pathological mechanism of MS. Felton demonstrated “immunologic paralysis” (IP) in normal adult mice by administering large doses of pneumoccocal polysaccharide [27], as did Sercarz and Coons [28] and Mitchison [29] using bovine serum albumin. They showed that the mechanism was inhibition of antibody formation and not neutralization while the responsiveness to other antigens persisted, confirming the highly specific nature of the paralysis (Mitchison [29]). More recently, Critchfield et al. have applied similar mechanisms in experimental allergic encephalomyelitis (EAE) [30]. They studied the paradoxical, high dose, T-cell suppression in two in vitro models as well as in two susceptible mouse strains. They found that T-cell death was a consequence of interleukin2-stimulated cell cycling and T-cell receptor re-engagement at high antigen doses. Autoreactive T-cells were deleted and the clinical and pathological signs of EAE in mice were diminished after repeated dosing with myelin basic protein. If some aspect of the varicella zoster virus genome has antigenic properties in initiating the MS process, it is possible that a subsequent episode of clinical varicella or high dose varicella antigen might produce T-cell deletion and ameliorate the disease. This might also worsen the disease. With the evidence that live varicella virus vaccination may have produced clinical and MRI improvement in some of the patients in this trial, further investigation with a blinded, placebo-controlled study seems warranted. Patients in the two groups should have matched EDSS ratings with no ratings greater than 4.0 at entry. The tempo of the MS can be more accurately followed by monthly MRI studies for three months prior to and during the trial. With the development of varicella zoster virus subunit vaccines it may be possible to induce a higher and more persistent state of immune paralysis than is possible with attenuated live varicella zoster virus vaccine. This work was supported by The Murphy Foundation, The Health Sciences Centre Research Foundation, The Manitoba Medical Service Foundation, and Merck Frosst Canada Inc. It is a pleasure to thank Gail La&y for coordinating the inuestigation, administrating the funds, and the innumerable day-to-day tasks of the trial over a two and one-half year period, plus many hours in library searches and manuscript preparation. Ruth Loewen has done exemplary work as the Study Nurse. She has given all vaccinations and been a&able to all the patients for initial responses to multiple enquiries and encouraged patients to complete and return their post-vaccination records. The authors wish to thank Doctors A. I. Gomori, M. R. Young, C. N. Bourque, M. D. Fast, F. S. Dominique, A. Auty, N. Pillay, A. Nath, M. J. Newman, D. E. Hobson, I. B. Rusen, R. C. Bright for their referrals of p attents f or I, 03s~‘bl e inclusion in the trial.

R. T. Ross et al.

68

Doctors Blake McClarty, Scott Sutherland have thors greatly appreciate

Gregory McGinn,

independently their efforts.

assessed

William Gordon and MRZ findings. The au-

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16. Preblud SR, D’Angelo LJ. Chickenpox in the United States 1972-1977. From the Center for Disease Control. J Inf Dis 1979; 140(2): 257-259. 17. Acheson ED. The Epidemiology of Multiple Sclerosis. In: Multiple Sclerosis: A Reappraisal. McAlpine D,: Lumsden CE, Acheson ED, Eds. Edinburgh and London: E. S. Livingston Ltd., 1965: 16. 18. Ross RT, Cheang M. Geographic similarities between varicella and multiple sclerosis. An hypothesis on the environmental factor of multiple sclerosis. J Clin Epidemiol 1995; 48(6): 731-737. 19. Takahashi M. Herpes: Varicella-zoster virus. In: Laboratory Diagnosis of Infectious Diseases: Principles and Practice, Vol. II-Viral, Rickettsial and Chlamydial Diseases. Lennette EG, Halonen P, Murphy FA, Eds. New York: Springer Verlag; 1988: 261-283. 20. Kuroiwa Y, Igata A, Itahara K, Shinzaburo K, Tsubaki T, Toyokura Y, Shibasaka H. Nationwide survey of multiple sclerosis in Japan. Neurology 1975; 25: 845-851. 21. Elian M, Dean G. Multiple sclerosis among the United Kingdom-born children of immigrants from the West Indies. J Neural Neurosurg Psychiat 1987; 50: 327-332. 22. Elian M, Nightingale S, Dean G. Multiple sclerosis among United Kingdom-born children of immigrants from the Indian subcontinent, Africa, and the West Indies. J Neural Neurosurg Psychiat 1990; 53: 906-911. LE, Cheang M. Varicella zoster virus and 23. Ross RT, Nicolle multiple sclerosis in a Hutterite population. J Clin Epidemiole ogy 1995; 48(11): 1319-1324. JAR, Peters TJ. Herpes zoster and multiple sclerosis. 24. Lenman Brit Med J 1969; 2: 218-220. 25. Ragozzino MW, Kurland LT. Epidemiologic investigation of the association between herpes zoster and multiple sclerosis. Neurology 1983; 33: 648-649. 26. Martyn CN. The epidemiology of multiple sclerosis. In: McAlpine’s Multiple Sclerosis. Matthews WB, Compston A, Allen IV, Martyn CN, Eds. Edinburgh: Churchill Livingstone; 1991: 35. DD. The significance of antigen in animal tissues. J 27. Felton Immunol 1941; 61: 107-117. 28. Sercarz E. Coons AH. Snecific inhibition of antibodv formation during immunological paralysis and unresponsiveness. Nature (Land) 1959; 184: 1080-1082. 29. Mitchison NA. Induction of immunological paralysis in two zones of dosage. Proc Royal Sot (Land) 1964; 161: 275-292. 30. Critchfield JM, Racke MK, Zuniga-Pflucker JC, Cannella B, Raine CS, Goverman J, Lenardo MJ. T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis. Science 1994; 263: 1139-1142. I