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Antibody response to tetanus toxoid inoculation in malarious and non-malarious Gambian children
364 TRANSACTIONS OF THE ROYAL SOCIETY OF T R O P I C A L .-~IEDICINE AND H Y G I E N E .
Vol. 56. No. 5. September, 1962.
ANTIBODY RESPONSE TO T E T A N U S TOXOID I N O C U L A T I O N IN MALARIOUS AND NON-MALARIOUS GAMBIAN C H I L D R E N BY
IAN A. McGREGOR Medical Research Council Laboratories, Gambia AN D
M.
BARR*
Wellcome Research Laboratories, Beckenham, England
In the Gambia, the African infant at an early age experiences heaxT and repeated malarial infections. Survival is conditional upon the development of an effective immunity against the disease, an event accompanied by a marked increase in serum ,(-globulin concentration and discernible in the third year of life (MCGREGOR et al., 1956; GILLES and MCGREGOR, 1959; MCGREGOR and GILLES, 1960). In the attainment of this immunity, the African child appears to develop defences which, primed and activated by frequent plasmodial challenges, may have, as part of their functional basis, tissues capable of prolific antibodv formation. COttENand McGR>;GoI~(in press) have demonstrated that healthy adult Gambians turn over -(-globulin at a daily rate that is eight times greater than that of Europeans. It is therefore conceivable that such primed defences, by responding to antigenic stimulation promptly and profusely, could be of considerable benefit to an individual encountering infection of a non-malarial nature. In the present investigation, malarious children in the third year of life, and exhibiting high serum T-globulin concentrations, were regarded as possessing primed immunity mechanisms. Children of identical age who had never experienced erythrocytic malaria and who, in consequence, exhibited relatively low levels of serum -(-globulin were considered to possess less well-developed immune defences. Both groups were immunized with tetanus toxoid, and antitoxin titres were subsequently assessed and contrasted. Tetanus toxoid was chosen as an antigen, since preliminary investigation of a sample of 50 adult Gambian sera revealed no evidence of detectable naturally occurring antitoxin. SUBJECTS AND METHODS All the children were resident in the Sukuta area of the Gambia, a region already described in detail (McGREGOR et al., 1956). They were observed in three groups, A, B and C. Group A (16 members) and Group B (14 members) had been kept malaria-free from birth by regular weekly chemoprophylaxis (chloroquine 150 rag. base for the former group, and * We gratefully acknowledge the assistance received from Dr. J. H. Humphrey, National Institute for Medical Research, Mill Hill, in planning this investigation; from Miss K. Edwards, M.R.C. I,aboratories, Gambia, in the field work and from Mr. K. Williams, M.R.C. Laboratories, Gambia, in technical and statistical matters.
IAN
A.
MCGRE(;OR
AND
M.
365
BARR
p y r i m e t h a m i n e 12.5 rag. for the latter). T h e 36 m e m b e r s of G r o u p C had received n o such regular c h e m o p r o p h y l a x i s , b u t had received a n t i m a l a r i a l t r e a t m e n t w h e n clinical illness i n d i c a t e d its need. M a l a r i a excepted, the m e m b e r s of the three g r o u p s appeared similar in all respects; they lived in the same e n v i r o n m e n t a n d c o n s u m e d almost identical diets. The investigation took place in the period October, 1958, to January, 1959. Each child received an inoculation of 0.5 mt. of tetanus toxoid followed by a second identical inoculation 6 weeks later. Ten to 14 days after the second inoculation, blood was taken from each child by venepuncture, allowed to clot and the serum obtained. The serum sample was divided into two parts; one was retained and subjected to filterpaper electrophoresis, while the other was forwarded by air in a refrigerated condition to the \Vellcome Research Laboratories, Beckenham, for determination of its tetanus antitoxin content. Electrophoresis of the serum proteins was effected in a horizontal tank containing a barbiturate buffer ( p H 8.6) to which a small quantity of fungicide (sodium salicylanilide) had been added. Serum 110-20 !xl.) was applied by coverslip to Whatman's No. 2 (Chromatography) filterpaper and a direct current (120 v.) passed through the paper for 20-22 hours. The paper was then dried in an air oven at 110-120";C. for 30 minutes, stained with asocarmine '13' (G.T. Gurr Ltd.), washed in distilled water and finally dried at 110°C. for 30 minutes. The stained electrophoretograms were expressed as reflectance density curves by scanning with a ret-lectance densitometer (Joyce, Loeble Ltd.) The yglobulin fraction was assessed as a percentage of total proteins and also of total globulins. The same hatch of tetanus toxoid was used for all inoculations. Antitoxin titration was performed in mice by the method described by (;LFNN'~ and STEVEXS (1938). The sera were tested at approximately twofuld differences and the results expressed in terms of International Units (1950). RI'SULTS
T a b l e I shows the results of electrophoretic separation of the s e r u m proteins. It will be n o t e d that the c h i l d r e n of G r o u p C possessed significantly higher m e a n s e r u m 7 - g l o b u l i n c o n c e n t r a t i o n s t h a n d i d c h i l d r e n of ( ; r o u p s A a n d B. F o r -.,-globulin expressed as a percentage of total s e r u m proteins the difference in m e a n values b e t w e e n G r o u p s A 'I'aBL8 I.
Mean D'-globulin values.
"¢-,~lobulin <% total sernm proteins ( ;rl,tip
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
v-~h>bulin ",, l~ltal ~lobulins
. . . . . . . . . . . . . . . . . . . . . . . . .
.
.~l can S. l). l{an•e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A
'
20.24
23.96
3.02
//
16 sui)jects
;.
.
.
~'lean . .
/
2.96
14 subjects .
.
.
.
.
.
.
~. 1). .
Range !
. . . . . . . . .
37.43
46.4
18.78
23.89 ge .
.
4.47
/
31 46
11
.
.
i
. . . . . . . . . . . . . . . . i. . . . . . . . . . i . . . . ." . . . . . . . . .
.
.
5 I . 18 . . . . . . . . .
i 4(~. 1S
I 3.<';7
28.84 .
.
.
.
.
.
.
C
,.
.
.
.
.
.
]
28.72
.
21 .
3(> subiccts .... ! denotes statistical significance. { between mean values.
.
.
.
.
.
35.60
/
45.74
]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . /
4.51
i' .
74
/ 40.3(>
34.88 53 . 70
5.99
/
/ 61.1 (>
.
366
A N T I B O D Y RESPONSE T O T E T A N U S T O X O I D I N G A M B I A N C H I L D R E N
and C is 4.49 times greater than the standard error of the difference, and between Groups B and C is 4.44 x S.E. For `(-globulin as a percentage of total globulins the difference in mean values between Groups A and C is 3.76 x S.E., and between Groups B and C is 4.58 x S.E. The difference in mean ,(-globulin values between Groups A and B is in no instance significant. The results of tetanus antitoxin titration are shown in Table II.
TABLE II.
The tetanus antitoxin titres of children 10-14 days after the second of two injections of tetanus toxoid. Number of children with tetanus antitoxin (Int. units/ml.) r ! ]>0.01 0.02 0.05 0.1 0.2 0.5 1 2 Group I Total <0.01 0.02 0.05 0.1 0.2 0.5 1 2 5 A
2
B
2
C
14
--
2
1
14
1
2
2
1
16
1
36
t I
Inspection revealed no appreciable difference between the results observed in Groups A and B and these were therefore combined for statistical analysis. Regarding titres of less than 0.01 unit per ml. as indicating non-responders, there were 4/30 such individuals in the combined group A + B, and 14/36 in Group C. These proportions are significantly different at the 2 per cent. level. In contrast, however, the distribution of titres among the responders (i.e., >0.01 unit per ml.) shows no difference between the malaria-free group (A + B) and the malarious Group C, the geometric mean titres being 0.19 unit per ml. for the former, and 0.18 unit per ml. for the latter.
DIscussioN It has been demonstrated that malaria is a major aetiological factor in the production of the hypergammaglobulinaemia so commonly observed in Gambian children (MCGREGOR et al., 1956; GILLES and MCGREGOR, 1959; McGREGOR and GILLES, 1960; COHEN and MCGREGOR, in press). The intriguing possibility exists, therefore, that children who survive the ravages of this disease may possess an efficient immunity mechanism which, primed by malaria, is of advantage in coping with other, non-malarial, infections. It was to investigate this possibility that the present investigation was made. The children of Groups A and B were known to have been malaria-free since birth. All members of Group C, however, had sustained malarial infections many times before the investigation began and, although their malarial state was not precisely known at the times of tetanus-toxoid inoculation, all were known to be parasitized at some time over the period of study.
IAN A. MCGREGOR AND M, BARR
367
The children in Group C, despite their significantly higher concentration of serum 2,-globulin, showed a poorer antibody response to inoculation with tetanus toxoid than did children in Groups A and B. The high incidence of non-responders in the malarious group, coupled with the similarity of the distribution of antitoxin titres in both the protected and unprotected groups, suggests that freedom from malaria increases the responder rate, but not the magnitude of antibody response. The latter was practically identical in both groups. It has long been known that "crowding-out" of antibody formation may take place if an antigen is injected into an animal when active production of another antibody is occurring (BFNJAMINand WITZINGER,1912; GLENNY, HOPKINS and WADDIN(;TON,1925). It appears possible, therefore, that the comparatively large number of poor- and non-responders in Group C were actively producing antibodies to some natural infection, probably malaria, at the time of immunization with tetanus toxoid. The results, therefi)re, do not support a hypothesis that malaria confers an ultimate benefit on survivors by priming immunity mechanisms. The children in Group C, however, were only in the third year of life and were still actively coping with moderately dense parasitaemia. Perhaps a different result might have emerged had older more immune individuals with less dense parasitaemia been studied. As large-scale immunizing campaigns against diseases such as tuberculosis, whooping cough and measles, are now being advocated in tropical Africa and since plasmodial infection is both frequent and heavy in the age-groups in which immunization is desirable, the possibility that malaria may interfere with antibody response to specific vaccines should be borne in mind. Similarly, in the field trials of new and experimental vaccines in malarious regions, prudence would appear to require the inclusion of a group of children previously freed from malaria bv antimalarial drugs. REFERENCES E. & W1TZINGER, V. (1912). Z. Kinderheilk, 3, 73. & MCGREGOR,I.A. £),mposium on Immunity to Protozoal Diseases, I.ondon, 1961. (In press). GtLI.V.s, H. M. & McGREGOR, I. A. (1959). Ann. trop. Med. Parasit., 53, 492. GLEXNY, A. "F., HOPKINS, B. E. & WADDI×GTON,H. (1925). J. Path. Bact., 28, 305. .... & STEVENS, M. F. (1938). J. R. Army reed. Cps., 70, 308. MCGRt..'(;OR, I~ A. & GILLES,H. M. (1960). Ann. trop. Mled. Parasit., 54, 275. , - - - - , WALTERS,J. H., DAXlES,A. 11. & PEARSON,F.A. (1956). Brit. reed. J., 2, 686. t:{ENJAMIN, COHEN, S.
Vol. 56. No. 5. September, 1962.
ANTIBODY RESPONSE TO T E T A N U S TOXOID I N O C U L A T I O N IN MALARIOUS AND NON-MALARIOUS GAMBIAN C H I L D R E N BY
IAN A. McGREGOR Medical Research Council Laboratories, Gambia AN D
M.
BARR*
Wellcome Research Laboratories, Beckenham, England
In the Gambia, the African infant at an early age experiences heaxT and repeated malarial infections. Survival is conditional upon the development of an effective immunity against the disease, an event accompanied by a marked increase in serum ,(-globulin concentration and discernible in the third year of life (MCGREGOR et al., 1956; GILLES and MCGREGOR, 1959; MCGREGOR and GILLES, 1960). In the attainment of this immunity, the African child appears to develop defences which, primed and activated by frequent plasmodial challenges, may have, as part of their functional basis, tissues capable of prolific antibodv formation. COttENand McGR>;GoI~(in press) have demonstrated that healthy adult Gambians turn over -(-globulin at a daily rate that is eight times greater than that of Europeans. It is therefore conceivable that such primed defences, by responding to antigenic stimulation promptly and profusely, could be of considerable benefit to an individual encountering infection of a non-malarial nature. In the present investigation, malarious children in the third year of life, and exhibiting high serum T-globulin concentrations, were regarded as possessing primed immunity mechanisms. Children of identical age who had never experienced erythrocytic malaria and who, in consequence, exhibited relatively low levels of serum -(-globulin were considered to possess less well-developed immune defences. Both groups were immunized with tetanus toxoid, and antitoxin titres were subsequently assessed and contrasted. Tetanus toxoid was chosen as an antigen, since preliminary investigation of a sample of 50 adult Gambian sera revealed no evidence of detectable naturally occurring antitoxin. SUBJECTS AND METHODS All the children were resident in the Sukuta area of the Gambia, a region already described in detail (McGREGOR et al., 1956). They were observed in three groups, A, B and C. Group A (16 members) and Group B (14 members) had been kept malaria-free from birth by regular weekly chemoprophylaxis (chloroquine 150 rag. base for the former group, and * We gratefully acknowledge the assistance received from Dr. J. H. Humphrey, National Institute for Medical Research, Mill Hill, in planning this investigation; from Miss K. Edwards, M.R.C. I,aboratories, Gambia, in the field work and from Mr. K. Williams, M.R.C. Laboratories, Gambia, in technical and statistical matters.
IAN
A.
MCGRE(;OR
AND
M.
365
BARR
p y r i m e t h a m i n e 12.5 rag. for the latter). T h e 36 m e m b e r s of G r o u p C had received n o such regular c h e m o p r o p h y l a x i s , b u t had received a n t i m a l a r i a l t r e a t m e n t w h e n clinical illness i n d i c a t e d its need. M a l a r i a excepted, the m e m b e r s of the three g r o u p s appeared similar in all respects; they lived in the same e n v i r o n m e n t a n d c o n s u m e d almost identical diets. The investigation took place in the period October, 1958, to January, 1959. Each child received an inoculation of 0.5 mt. of tetanus toxoid followed by a second identical inoculation 6 weeks later. Ten to 14 days after the second inoculation, blood was taken from each child by venepuncture, allowed to clot and the serum obtained. The serum sample was divided into two parts; one was retained and subjected to filterpaper electrophoresis, while the other was forwarded by air in a refrigerated condition to the \Vellcome Research Laboratories, Beckenham, for determination of its tetanus antitoxin content. Electrophoresis of the serum proteins was effected in a horizontal tank containing a barbiturate buffer ( p H 8.6) to which a small quantity of fungicide (sodium salicylanilide) had been added. Serum 110-20 !xl.) was applied by coverslip to Whatman's No. 2 (Chromatography) filterpaper and a direct current (120 v.) passed through the paper for 20-22 hours. The paper was then dried in an air oven at 110-120";C. for 30 minutes, stained with asocarmine '13' (G.T. Gurr Ltd.), washed in distilled water and finally dried at 110°C. for 30 minutes. The stained electrophoretograms were expressed as reflectance density curves by scanning with a ret-lectance densitometer (Joyce, Loeble Ltd.) The yglobulin fraction was assessed as a percentage of total proteins and also of total globulins. The same hatch of tetanus toxoid was used for all inoculations. Antitoxin titration was performed in mice by the method described by (;LFNN'~ and STEVEXS (1938). The sera were tested at approximately twofuld differences and the results expressed in terms of International Units (1950). RI'SULTS
T a b l e I shows the results of electrophoretic separation of the s e r u m proteins. It will be n o t e d that the c h i l d r e n of G r o u p C possessed significantly higher m e a n s e r u m 7 - g l o b u l i n c o n c e n t r a t i o n s t h a n d i d c h i l d r e n of ( ; r o u p s A a n d B. F o r -.,-globulin expressed as a percentage of total s e r u m proteins the difference in m e a n values b e t w e e n G r o u p s A 'I'aBL8 I.
Mean D'-globulin values.
"¢-,~lobulin <% total sernm proteins ( ;rl,tip
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
v-~h>bulin ",, l~ltal ~lobulins
. . . . . . . . . . . . . . . . . . . . . . . . .
.
.~l can S. l). l{an•e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A
'
20.24
23.96
3.02
//
16 sui)jects
;.
.
.
~'lean . .
/
2.96
14 subjects .
.
.
.
.
.
.
~. 1). .
Range !
. . . . . . . . .
37.43
46.4
18.78
23.89 ge .
.
4.47
/
31 46
11
.
.
i
. . . . . . . . . . . . . . . . i. . . . . . . . . . i . . . . ." . . . . . . . . .
.
.
5 I . 18 . . . . . . . . .
i 4(~. 1S
I 3.<';7
28.84 .
.
.
.
.
.
.
C
,.
.
.
.
.
.
]
28.72
.
21 .
3(> subiccts .... ! denotes statistical significance. { between mean values.
.
.
.
.
.
35.60
/
45.74
]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . /
4.51
i' .
74
/ 40.3(>
34.88 53 . 70
5.99
/
/ 61.1 (>
.
366
A N T I B O D Y RESPONSE T O T E T A N U S T O X O I D I N G A M B I A N C H I L D R E N
and C is 4.49 times greater than the standard error of the difference, and between Groups B and C is 4.44 x S.E. For `(-globulin as a percentage of total globulins the difference in mean values between Groups A and C is 3.76 x S.E., and between Groups B and C is 4.58 x S.E. The difference in mean ,(-globulin values between Groups A and B is in no instance significant. The results of tetanus antitoxin titration are shown in Table II.
TABLE II.
The tetanus antitoxin titres of children 10-14 days after the second of two injections of tetanus toxoid. Number of children with tetanus antitoxin (Int. units/ml.) r ! ]>0.01 0.02 0.05 0.1 0.2 0.5 1 2 Group I Total <0.01 0.02 0.05 0.1 0.2 0.5 1 2 5 A
2
B
2
C
14
--
2
1
14
1
2
2
1
16
1
36
t I
Inspection revealed no appreciable difference between the results observed in Groups A and B and these were therefore combined for statistical analysis. Regarding titres of less than 0.01 unit per ml. as indicating non-responders, there were 4/30 such individuals in the combined group A + B, and 14/36 in Group C. These proportions are significantly different at the 2 per cent. level. In contrast, however, the distribution of titres among the responders (i.e., >0.01 unit per ml.) shows no difference between the malaria-free group (A + B) and the malarious Group C, the geometric mean titres being 0.19 unit per ml. for the former, and 0.18 unit per ml. for the latter.
DIscussioN It has been demonstrated that malaria is a major aetiological factor in the production of the hypergammaglobulinaemia so commonly observed in Gambian children (MCGREGOR et al., 1956; GILLES and MCGREGOR, 1959; McGREGOR and GILLES, 1960; COHEN and MCGREGOR, in press). The intriguing possibility exists, therefore, that children who survive the ravages of this disease may possess an efficient immunity mechanism which, primed by malaria, is of advantage in coping with other, non-malarial, infections. It was to investigate this possibility that the present investigation was made. The children of Groups A and B were known to have been malaria-free since birth. All members of Group C, however, had sustained malarial infections many times before the investigation began and, although their malarial state was not precisely known at the times of tetanus-toxoid inoculation, all were known to be parasitized at some time over the period of study.
IAN A. MCGREGOR AND M, BARR
367
The children in Group C, despite their significantly higher concentration of serum 2,-globulin, showed a poorer antibody response to inoculation with tetanus toxoid than did children in Groups A and B. The high incidence of non-responders in the malarious group, coupled with the similarity of the distribution of antitoxin titres in both the protected and unprotected groups, suggests that freedom from malaria increases the responder rate, but not the magnitude of antibody response. The latter was practically identical in both groups. It has long been known that "crowding-out" of antibody formation may take place if an antigen is injected into an animal when active production of another antibody is occurring (BFNJAMINand WITZINGER,1912; GLENNY, HOPKINS and WADDIN(;TON,1925). It appears possible, therefore, that the comparatively large number of poor- and non-responders in Group C were actively producing antibodies to some natural infection, probably malaria, at the time of immunization with tetanus toxoid. The results, therefi)re, do not support a hypothesis that malaria confers an ultimate benefit on survivors by priming immunity mechanisms. The children in Group C, however, were only in the third year of life and were still actively coping with moderately dense parasitaemia. Perhaps a different result might have emerged had older more immune individuals with less dense parasitaemia been studied. As large-scale immunizing campaigns against diseases such as tuberculosis, whooping cough and measles, are now being advocated in tropical Africa and since plasmodial infection is both frequent and heavy in the age-groups in which immunization is desirable, the possibility that malaria may interfere with antibody response to specific vaccines should be borne in mind. Similarly, in the field trials of new and experimental vaccines in malarious regions, prudence would appear to require the inclusion of a group of children previously freed from malaria bv antimalarial drugs. REFERENCES E. & W1TZINGER, V. (1912). Z. Kinderheilk, 3, 73. & MCGREGOR,I.A. £),mposium on Immunity to Protozoal Diseases, I.ondon, 1961. (In press). GtLI.V.s, H. M. & McGREGOR, I. A. (1959). Ann. trop. Med. Parasit., 53, 492. GLEXNY, A. "F., HOPKINS, B. E. & WADDI×GTON,H. (1925). J. Path. Bact., 28, 305. .... & STEVENS, M. F. (1938). J. R. Army reed. Cps., 70, 308. MCGRt..'(;OR, I~ A. & GILLES,H. M. (1960). Ann. trop. Mled. Parasit., 54, 275. , - - - - , WALTERS,J. H., DAXlES,A. 11. & PEARSON,F.A. (1956). Brit. reed. J., 2, 686. t:{ENJAMIN, COHEN, S.