Measles antibodies and histocompatibility types in multiple sclerosis

Journal of the Neurological Sciences, 1979, 43: 19-26 © Elsevier/North-Holland Biomedical Press

19

MEASLES ANTIBODIES A N D HISTOCOMPATIBILITY TYPES I N M U L T I P L E SCLEROSIS

MONA E. FEWSTER*, FRANCES R. AMES and M. C. BOTHA Department of ChemicalPathology, Universityof Stellenbosch, Tygerberg 7505, Neurology Department, Groote Schuur Hospital and Provincial Blood Grouping Laboratory, Observatory 7925, Cape Town (South Africa)

(Received 30 January, 1979) (Accepted 18 April, 1979)

SUMMARY Measles antibody titres and HLA antigens were determined in 71 White and 11 Coloured multiple sclerosis (MS) patients and 71 White and 11 Coloured age and sex-matched controls. Measles antibody titres were determined by the hemagglutination inhibition test and HLA antigens were determined serologically by a micro-lymphocytotoxicity test. Measles antibody titres were significantly higher in MS than in control cases and this was true for both female and male patients. No association was observed between the HLA antigens, especially HLA-A3 or HLA-B7 and measles antibody titres in the sera of MS patients or controls. INTRODUCTION Two factors which have been implicated in the vulnerability of subjects to MS have been tissue type and high titre of measles antibodies in the serum. The analysis of the relative risks of the major histocompatibility human lymphocyte antigens (HLA) in almost 2,000 multiple sclerosis (MS) patients compared with 12,000 controls in 14 different studies has shown overall significant increases of HLA-A1, A3, A10, B7 ,B8 and Bwl8 and significant decreases of HLA-A2, A28, B12, Bwl5, Bwl7 and Bw40 (Jersild 1978). In 1962, Adams and Imagawa reported higher titres of measles antibody in the sera of patients with MS than in control subjects. Since then, 29/38 studies have shown statistically higher measles antibody titres in sera of MS patients. Elevated titres to other viruses have been reported in MS but no other virus has been consistently implicated. In addition, a decrease has been shown in the * Supported by a grant from the Medical Research Council of South Africa. Correspondence to : Dr. Mona E. Fewster, Department of Neurology, U.C.L.A., Los Angeles, CA 90024, U.S.A.

20 effect of measles antigen on the cell-mediated response (Ciongoli et al. 1973; Utermohlen and Zabriske 1973). Some studies have shown a statistical relationship between higher measles antibody titres and certain HLA antigens in MS (Jersild et al. 1973; Cazzulo and Smeraldi, 1974; Paty et al. 1976; Fewster et ah 1977), but others have not confirmed this (Bertrams et al. 1973; Arnason et al. 1974, Whitaker et al. 1976; Ilonen et al. 1977; Stewart et al. 1977). The present study was undertaken to obtain additional information in an attempt to clarify the relationship and to determine whether the results of Fewster et al. (1977) could be confirmed in a geographic area with a low incidence of MS. METHODS

Subjects Seventy three unrelated White patients including 45 White cases born in South Africa and 28 immigrants, 26 of whom had settled in South Africa after the age of 15 yr and 12 Coloured patients with definite MS as determined on clinical grounds by accepted criteria (Schumacher et ah 1965) and in some cases by raised IgG in the CSF, donated blood for measles antibody determinations. Seventy three Whites, of whom 67 were born in South Africa, and 12 Coloured age- and sex-matched unrelated controls were hospital employees. The age- and sex-matched controls for the 45 White MS cases born in South Africa included 3 immigrants. Seventy one White MS patients including 43 White cases born in South Africa, 28 immigrants and 11 Coloured MS patients were HLA typed. All of the 71 White and 11 Coloured matched controls were born in South Africa. They were typed with the same antisera. Twenty three randomly selected normal Black (Bantu) hospital employees, previously HLA typed, donated blood for measles antibody determinations.

Hemagglutination Inhibition (HI) Test The blood was left at room temperature for 30 min to clot. The tubes were centrifuged at 200 x g for 10 min. The serum was removed and 1-ml aliquots were frozen and stored at --20 °C for analysis of approximately 25 samples simultaneously by the HI technique (U.S. Dept. of Health, Education and Welfare Standardisation, 1971 ; Dr. E. H. Lennette, State of California Department of Public Health, Viral and Rickettsial Disease Laboratory, Berkeley). Briefly, 0.1 ml of 1:8 dilution of coded serum was heat-inactivated (56 °C, 30 min) and natural agglutinins were absorbed with vervet monkey red cells (RBC). Serial 2-fold dilutions of serum were incubated with 4 HA units of measles antigen (Microbiological Associates) for 1 hr at room temperature, then with RBC for 1 hr at 37 °C. The measles HI titre is the highest serum dilution which completely inhibits hemagglutination of the indicator erythrocytes. The accuracy of the procedure was confirmed by showing reproducible titres with each serum sample analysed again at a different time.

21

Lymphocyte typing Ten ml of thrombolytic blood from each subject was delivered to the laboratory of Dr. M. C. Botha, normally within 1 hr and less than 3 hr of collection. The lymphocytes were isolated by the Ficoll-Hypaque method, frozen in liquid nitrogen and stored a t - - 196 °C. The lymphocytes were typed by the micro-lymphocytotoxicity test ( N A I A D 1966-1967). Typing sera were obtained from the N I H serum bank. All the samples were typed with identical reagents.

Statistical methods The classical g 2 test for homogeneity was used for tables larger than two-by-two tables. The g 2 test for association with Yates' correction factor for continuity was used for two-by-two tables. When the expected value was less than 5, Fisher's exact test was used. Geometric mean titres (GMT) were used to compare measles antibody titres because there is a constant ratio between successive dilutions in the H I test. The logarithms of reciprocals of the measles titres were used to determine whether the geometric mean titres for the MS and control samples differed significantly by the Student t-test. RESULTS Table 1 shows the numbers and ages in the various groups. The mean age of the MS patients was 43 yr. There were more females than males in all groups. When the measles antibody titres of 73 White MS patients are compared with those of 73 matched controls, the chi-square test for homogeneity shows a significant difference (Z 2 ---- l 1.347; 5 df; P < 0.05) and a significantly greater number of MS patients (38, 52.1 ~o than the controls (24, 32.9 ~o) have titres/> 1 : 64 (Z 2 Yates' ----4.738; 1 df; P < 0.05). This is also true for 42 White MS patients born in South Africa compared with 42 matched controls. A significantly greater number of MS patients (24, 57.1 ~ ) than the controls (13, 30.9~) have titres /> 1:64 (Z 2 Yates' ---- 4.8304; 1 dr; P < 0.05). There is no significant difference between titres /> 1 : 64 of the 12 Coloured MS cases and their controls, but the number of cases is too small to be meaningful. Table 2 shows that the geometric mean titre (GMT) of the 73 White MS patients (1:46.2) is highly significantly higher (P < 0.001) than the controls (1 : 27.0). When the measles antibody titres are analysed according to sex, the G M T of the 54 females is TABLE 1 SEX AND MEAN AGE FOR THE MULTIPLE SCLEROSIS PATIENTS Group

Total No.

Males

Females

Mean age (yr)

Range

Multiple sclerosis - - White South African born Immigrants Multiple sclerosis - - Coloured

74 46 28 12

19 11 8 2

55 35 20 l0

43 37 53 32

18-74 18-65 30-74 22-47

22 TABLE 2 SERUM TITRES OF MEASLES HEMAGGLUTINATION ANTIBODIES Group

Age, sex-matched, White Age, sex-matched, White female Age, sex-matched, White male Age, sex-matched, White South African born Age, sex-matched, White South African born Age, sex-matched, Coloured South African born

Geometric mean titres a MS

controls

46.2(73)~ 44.7(54) 39.8(19) 49.2(42) 49.2(42) 28.5(12)

27.0(73)c 30.0(54) 19.9(19) 25.8(42) 27.4(67) 22.6(12)

pb

<0.001 <0.05 ~0.02 <0.01 <0.0Ol n.s.

Reciprocal of serum dilutions. ~ Student's t-test. Number of subjects. significantly higher (P < 0.05) than the control females. The G M T of the 19 males is significantly higher (P < 0.02) than the control males. There is no significant difference between the G M T of the female and male patients. The measles antibody levels of 42 White South African born MS patients (1:42.2) are significantly higher than their matched controls (1:25.8). The level of significance increases with a larger number of controls, namely 67 instead of 42. There is no difference between the 12 Coloured cases and their controls. The G M T of 23 randomly selected healthy Black (Bantu) (1:25.9) with a range of age from 19 to 58 yr is not significantly different from that of the 67 healthy White South Africans (1:27.4). The G M T of the 28 immigrant MS cases was not determined, as South African born controls would not have been as meaningful as controls matched by place of birth, which were not available. Table 3 shows the tissue typing results. There are no significant differences in frequency between the 71 MS patients, including 43 MS patients born in South Africa and 28 immigrants and the 71 controls. As no HLA antigens showed altered frequencies in MS in this study, the measles antibody titres were compared with the 4 A and B locus antigens which had shown the greatest change in frequency in Jersild's review (1978), namely HLA-A2, HLA-A3, HLA-B7, HLA-B12 and also HLA-A3 or B7. Table 4 shows that there is no significant difference in G M T compared with the presence or absence of these antigens in 70 MS patients, including 42 White patients born in South Africa, or 70 White matched controls born in South Africa. There is, similarly, no association between the G M T of the 28 MS immigrants and these HLA antigens but the sample number is low. DISCUSSION The significantly higher measles HI antibody titres in the White MS patients compared with age and sex-matched controls, shown by the increase of titres /> 1 : 64 (P < 0.05) and the G M T (P < 0.01) confirms the overall results of 29/38 previous studies. As these differences are also significant for White South African born MS patients,

HLA-AI HLA-A2 HLA-A3 HLA-A9 HLA-A10 HLA-All HLA-A28 HLA-A~ol9 HLA-B5 HLA-B7 HLA-B8 HLA-BI2 HLA-B13 HLA-B14 HLA-Btol 5 HLA-BcoI6 HLA-BtoI7 HLA-Btol 8 HLA-Bo~21 HLA-Bco22 HLA-Bto27 HLA-Bto35 HLA-Bto37 HLA-Bta40 HLA-Bto41 HLA-Bta42

HLA antigen

19 28 20 15 3 7 10 18 4 20 10 14 4 5 10 5 4 5 2 5 9 7 2 7 2 1

52 43 51 56 68 64 61 53 67 51 61 57 67 66 61 66 67 66 69 66 62 64 69 64 69 70

26.8 39.4 28.2 21.1 4.1 9.9 16.4 25.4 5.6 31.0 14.1 19.7 5.6 7.0 4.1 7.0 5.6 7.0 2.8 7.0 2.7 9.9 2.8 9.9 2.8 1.4

26 29 18 14 l 9 7 18 4 30 12 14 5 2 7 6 6 9 4 2 5 7 1 4 5 0

45 42 53 57 70 62 64 53 67 41 60 57 66 68 64 65 65 62 67 69 66 64 70 67 66 71

--

+

%+

+

--

MS (N = 71)

Controls (N = 71)

36.6 40.8 25.4 19.7 1.4 12.7 9.9 25.4 5.6 42.2 16.9 19.7 7.0 2.8 9.9 8.5 8.5 12.7 5.6 2.8 7.0 9.9 1.4 5.6 7.0

%+

0.713 0

0.713

0.070 0.267 0 0 2.500 0.035 0 0 0.572 0.267 0.001

1.171 0 0.036 0

1.0 0.371 0.441 1.0

0.681 0.441

0.745

0.620

Xz P (Yates)

15 18 12 8 1 6 5 11 4 17 6 8 2 1 3 3 6 6 3 0 4 4 1 1 3 0

+

28 25 31 35 42 37 38 32 39 26 38 35 41 42 40 40 37 37 40 43 39 39 42 42 40 43

--

born in S. Aft.

MS (N = 43)

34.9 44.2 27.9 18.6 2.3 14.0 11.6 25.6 5.6 39.5 14.0 11.3 2.8 2.3 7.0 7.0 14.0 14.0 7.0 0 9.3 9.3

%+

0.531 0.044

0.008

0.501 0.004 0.003 0.007

1.0 0.255 0.364 0.377

0.194 0.407 0.363 1.0 0.127 0.327 0.364 0.155 0.764

0.150 0.230

Z2 P (Yates)

F R E Q U E N C Y O F H L A A N T I G E N S IN PATIENTS W I T H M U L T I P L E SCLEROSIS A N D I N C O N T R O L S

TABLE 3

11 10 6 6 0 3 2 7 0 13 6 6 3 1 4 3 0 3 1 2 1 3 0 3 2 0

+

17 18 22 22 28 25 26 21 28 15 22 22 25 27 24 25 28 25 27 26 27 25 28 25 26 28

--

immigrants

MS (N = 28)

39.3 35.7 21.4 21.4 0 16.3 9.7 25.0 0 46.4 21.4 21.4 10.7 3.6 14.3 10.7 0 10.7 2.3 9.7 2.3 10.7 0 10.7 9.7 0

%+

1.474

0.958 0.013 0.187 0.058

1.0 1.0 0.751 1.0

1.0 0.274

0.400 0.672 1.0 0.684 1.0 0.684

0.299

0.556 0.742 0.500

Z2 P (Yates)

TABLE 4 SERUM TITRES OF MEASLES HEMAGGLUTINATION INHIBITION ANTIBODIES AND HLA ANTIGENS IN PATIENTS WITH MULTIPLE SCLEROSIS AND CONTROLS Group

MS, white

Antigen

No.

Titres a . . . . . . . . . . . . < 8 16 32 64 128

;~256

Geometric m e a n titres a

HLA-A2

26

4

3

5

8

4

2

42.9

MS, born in

not H L A - A 2 HLA-A2

44 12

3 I

I1 1

8 2

11 6

6 2

5 0

44.5 48.0

So. Africa, white MS, i m m i g r a n t s

not HLA-A2 HLA-A2

30 8

2 2

7 0

5 3

7 3

4 0

5 0

49.5 29.3

Controls, born in

not H L A - A 2 HLA-A2

20 26

2 8

6 6

3 6

3 5

4 I

2 0

40.8 21.5

not H L A - A 2

44

8

8

11

1I

5

1

32.0

So. Africa, white MS, white MS, born in So. Africa, white MS, i m m i g r a n t s Controls, born in So. Africa, white MS, white MS, born in So. Africa, white MS, i m m i g r a n t s Controls, born in So. Africa, white MS, white MS, born in So. Africa, white MS, i m m i g r a n t s Controls, born in So. Africa, white MS, white

MS, born in So. Africa, white

MS, i m m i g r a n t s

Controls, b o r n in So. Africa, white

HLA-A3

19

1

4

4

8

2

0

39.8

not H L A - A 3 HLA-A3

51 12

6 1

10 1

9 2

11 0

8 2

7 0

45.6 48.0

not H L A - A 3 HLA-A3 not H L A - A 3 HLA-A3

30 7 21 20

2 0 4 6

7 3 3 4

5 2 4 6

7 2 4 2

4 0 4 I

5 0 2 1

49.5 29.0 40.3 23.4

not H L A - A 3

50

l0

10

11

14

5

0

29.4

HLA-B7 not H L A - B 7 HLA-B7

30 40 16

2 5 I

7 7 2

5 8 3

9 10 5

4 6 3

3 4 2

51.6 42.8 56.2

not HLA-B7 HLA-B7 not H L A - B 7 HLA-B7

26 14 14 20

2 1 3 5

6 5 I 4

4 2 4 4

8 4 2 5

3 1 3 I

3 1 I 1

45.2 35.3 39.0 27.9

not H L A - B 7

50

I1

10

13

I1

5

0

27.5

HLA-B12

13

3

2

2

3

1

2

37.6

not H L A - B 1 2 HLA-B12

57 7

4 2

12 I

11 0

16 2

9 0

5 2

45.5 43.0

not H L A - B I 2 HLA-B12 not HLA-BI2 HLA-BI 2

35 6 22 12

I I 3 2

7 1 5 5

7 2 4 2

II 1 5 3

6 1 3 0

3 0 2 0

44.2 32.0 38.7 22.6

not H L A - B 1 2

58

14

9

15

13

6

1

28.7

30

3

7

5

10

2

2

42.5

40

4

8

7

9

7

5

41.7

20

2

3

3

7

3

2

44.2

22

I

5

4

6

3

3

49.7

10

1

4

2

3

0

0

26.0

HLA-A3 or B7 not H L A - A 3 or B7 HLA-A3 or B7 not HLA-A3 or B7 HLA-A3 or B7 not H L A - A 3 or B7 HLA-A3 or B7

18

3

3

3

3

4

2

43.5

24

7

5

5

5

1

1

23.7

not HLA-A3 orB7

46

9

9

12

1l

5

0

29.2

Reciprocal of s e r u m dilutions.

25 the differences have been shown for the first time, as far as we know, in a country with a low risk of MS (Dean 1967). Although both the female and male MS patients had a higher measles antibody G M T than their matched controls, there was no relationship between measles antibody GMT and sex in the MS group as shown also by Ammitzboll and Clausen (1972) and Fewster et al. (1977). Higher measles antibody levels in female patients, however, have also been shown (Henson et al. 1970; Brody et al. 1972; Panelius et al. 1973; Paty et al. 1976). Detailed case histories of 7 Coloured South African MS patients out of a population of 2,434,000 Coloured, were first reported by Ames and Louw (1977). In the present study, although the number of cases (12) is too small for a valid statistical comparison of GMT, it is of interest that the mean age (32) and GMT (1:22.6) of the Coloured South African control group is similar to the mean age (37) and GMT (1:25.8) of the 42 White South African matched controls. Similarly, the GMT of 23 randomly selected Blacks (Bantu) (1:25.9) does not differ from that of the White South African control group. Their mean age was 40 yr (range 19-58 yr). The GMT of 111 American MS patients (1:43.5) and the GMT of 76 controls (1:29.8) analysed in Los Angeles by the same procedure and the same brand of reagents (Fewster et al. 1977) is surprisingly similar to the G M T values of 49.2 and 27.4 in the respective South African born cases. The relationship between histocompatibility and control of the immune response has received increasing attention but the relationship between the elevation of measles antibodies in MS and HLA antigens provides conflicting results. Arnason et al. (1974) suggested that the increase in measles antibody levels in MS may reflect the increased frequency of HLA-A3 among MS patients rather than a direct association of measles virus with MS. However, the present report is the 6th study which has failed to show any relationship between measles antibody levels and an increased frequency of HLAA3 or HLA-B7 in MS or control cases. As this study is from a low risk area for MS, it tends to reduce the likelihood of an overall association of measles antibody levels with these antigens. On the other hand, it is possible that HLA-Dw2, which has the strongest known association with MS (Jersild 1978) will prove to have a more consistent degree of association. The studies by Myers et al. (1976), Stewart et al. (1976) and Ilonen et al. (1977), however, which show no association between elevated measles antibody titres and HLA-Dw2, do not substantiate this. ACKNOWLEDGEMENTS The authors acknowledge the technical assistance of Carolyn Winchester, Derek Taljaard and Elizabeth Campbell. The Institute for Biostatistics of the South African Medical Research Council provided statistical advice. Standard serum with high measles HI antibody titre and some reagents were provided by Dr. G. W. Ellison, U.C.L.A., Los Angeles, CA. REFERENCES Adams, J. and D. T. Imagawa (1962) Measles antibodies in multiple sclerosis, Proc. Soe. exp. Biol. (N. Y.), 8 : 562-566.

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