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Primary and secondary immune responses of sheep to tetanus toxoid
J.
COMP.
1971. VOL.
PATH.
PRIMARY
249
81.
AND SECONDARY IMMUNE RESPONSES OF SHEEP TO TETANUS TOXOID
BY R. S. ROBERTS, S. G~~VEN U.N.D.P.IF.A.0.
AND E. E.
WORRALL
SheepDiseases Research Laboratories, Pcndik, Turk@
INTRODUCTION
Early researches on the rate of development of antibody in the circulation emphasised the priming effect of the first injection of antigen but they failed to explain the mechanism of the immune response (Holborow, 1967). The conclusions reached before the discovery of new methods of investigating the problem were that the pattern of the response varied from model to model (Wilson and Miles, 1964) and that in general, the secondary response was earlier, reached a higher peak and was more prolonged than the primary response (Sercarz and Byers, 1967). M ore recent researches suggest that the pattern of the two responses might have more definable characteristics. An opportunity occurred to investigate this problem by the immunisation of sheep against tetanus toxin and some definition of the characteristics of this response was possible. MATERIALS Sheep.
Two
groups
of yearling
AND METHODS pure-bred
Kivircik
males
from
the
Turkish
Government Farm at Inanli, Thrace. Housing and feeding. The animals were kept in groups, loose in a barn, with hay ad lib., and 250 g. of barley per day. Mice. The groups were obtained from a closed colony at the laboratories and weighed 18 to 22 g. Titration of antitoxin. Antibody was titrated in doubling dilutions, against a constant dose of toxin in pairs of mice. The test dose was in the region of L+‘/400 and contained about 15 minimum reacting doses. As a result of several hundred titrations against this test dose, the effects of adjacent doses of antiserum, near the neutralising point, could be distinguished. Intermediate values were interpolated according to a prescribed scale of toxin effect. The neutralising effects of the sera international units. Solutions were expressed as units, which were approximately of toxin were prepared daily from a concentrate in glycerol, and each batch was assayed for potency. Pre-vaccination sera from all sheep had some protective effect against toxin and this was recorded arbitrarily as O-005 unit and amounts of 0.01 of a unit and over were regarded as a response to the toxoid. The standard testing method was used, modified slightly to take a neutralising point with less toxin excess. Toxin and toxoid. The non-sporulating toxigenic Harvard strain of Cl. tetani from the State Serum Institute, Copenhagen, was grown in gauze-covered stainless steel containers for 6 days at 35OC. in a papain digest of lean meat adjusted to @45 l The work was supported by a U.N.D.P. Special Fund Project established through the Food and Agriculture Organisation of the United Nations.
250
IMMUNE
RESPONSES
OF
SHEEP
TO
TETANUS
TOXOID
per cent. N, with 0.5 per cent. NaCl and 1.0 per cent. reduced iron, and sterilised by autoclaving at 115OC. for 30 min. Test toxin was prepared by dialysing a filtrate of this culture against glycerol to reduce the volume. Toxoid was prepared by adding 0.3 per cent. of formalin to the filtrate and incubating at 37OC. for 28 days and purifying by dialysing under pressure to increase potency from 50 to 1500 Lf/ml. This concentrate was diluted in physiological saline solution to 10 Lf./ml. for immunisation of sheep. Immunisation. Two subcutaneous injections of 2 ml., i.e. 20 Lf, were given with an interval of 35 days. RESULTS
A group of 10 sheep was immunised with two injections of toxoid, and the antitoxin titre of the serum of each sheep was assayed at intervals of 2 or 3 days (generally 2 days) during the primary and secondary responses. The development of the primary response was later than expected. Samples taken on the 3rd, 5th and 7th days contained no antitoxin, and at the time of the last sample at 12 days, no decrease in titre had occurred; one sheep did not respond. The determination of the secondary response gave results similar to those of an almost identical experiment reported below and are, therefore, combined with it. In the second experiment a group of 10 sheep was immunised in a similar manner. Antitoxin was assayed (a) before injection, (b) 9, 11, 14, 16, 1% and 21 days after the first injection and (c) 3, 5, 7, 10, 12 and 14 days after the second injection. The results are shown in Fig. 1. The presence of only small quantities of antibody on the 9th day after the first injection supports the evidence of the first experiment that no antibody was formed in the first 7 days after the first injection and further suggests that the 9th day was the critical period when antitoxin production commenced. As one sheep died the secondary response in this experiment was observed on only 9 sheep. On the third day after injection the titres were almost the same as those of the last sample for the primary response. It is possible, therefore, that there had been a fall in titre to the primary response, and that some of the antibody was an early production in the secondary response. Between the 3rd and 5th days, antitoxin was produced in substantial quantities by 9/ 10 sheep in the first experiment and %/9 in the second; the remaining sheep in each experiment produced low levels of antibody between the 5th and 7th days. In both experiments, the primary and secondary responses occurred substantially within a 2-day interval between observations, but some sheep continued to produce antibody, high titres persisting for about 8 days. The decline did not deviate markedly from an even exponential rate and with little tendency to early rapid decline. There appeared to be no obvious difference between the two responses and no correlation in their magnitude; in general, the peak titre in the secondary response was only 2 or 3 times greater than that in the primary response. DISCUSSION
It is accepted that the loss of antibody from the blood is a complex phenomenon, regulated by a balancing between loss and new production, and it is widely held
R. S. ROBERTS
et al.
251
-04
3
a2 0 r04
0.2 EL4 0.2 0 D4 0.2 0
t
p
4
8
12 16 20 24 38 40 44 48 t Days = 0.005 u/ml. p=OXH u/ml. P = aO.01u/ml.
0
Fig. 1 Primary and secondary responses of sheep to tetanus toxoid. Toxoid was given on days 0 and 35(arrows).
that there is a suppressive mechanism operated by antibody and occurring early in the response. It would seem therefore that the measurement which is most acceptable as evidence of a corresponding cellular activity is the appearance of new antibody in the circulation. If the results of these experiments are interpreted in terms of evidence of new antibody, they disclose an interesting, possibly useful pattern : primed sheep differed from immunologically intact sheep in producing antibody between the 3rd and 7th days after injection of antigen. These results, and earlier unpublished work, have consistently disclosed a tendency for animals which eventually reach a high titre relative to others in a group to extend the producing period into a second and even a third observation interval. It is ti interesting speculation that this may be due to insufficiency of F
252
IMMUNE
RESPONSES
OF
SHEEP
TO
TETANUS
TOXOID
some ingredient, such as essential amino acid to enable the primed cells to discharge their function immediately on reaching maturity as antibody producers. It appears unwise to emphasise the relatively small difference seen here between the peak titres in the two responses. The doses of antigen were approximately those that would be used in the field for immunisation of any species of this weight and the first and second doses were equal. It is likely that a more imprcssive difference would follow other dosage rates. These results do however illustrate the difficulty of demonstrating this difference; if the primary response had been looked for in these two groups of sheep on any one day, from the 3rd to the 12th it would not have been found in all the sheep in either group, because the time of antibody production was unpredictable whereas in the secondary response it was found in all sheep at the conventionally expected times. When tetanus toxoid was injected without adjuvant into intact sheep the rate of the appearance of antitoxin in the circulation was invariably uniform in a large number of individuals and corresponded with knowledge which has been acquired recently by the use of more sophisticated techniques in the fields of cell biology and immunology. The immunological mechanism of these sheep was intact and they did not respond on the fifth or seventh days after the first injection of antigen i.e. they had no precursor cells. They did respond later, but the difference between the time of the response to the first injection and to the second was 4 to 8 days. This corresponds with the time claimed by Sercarz and Byers (1967) and by H amaoka, Kitagawa, Matsuoka and Yamamura (1969) for the appearance of memory; it suggests that this interval is occupied in processing of memory cells by macrophages in the radio-sensitive phase of the primary response (Pribnow and Silverman, 1967). A correlation between the magnitude of the primary and secondary responses would not be expected and variable times for the appearance of detectable antitoxin in the primary response would also be expected if precursor cells had to await this processing mechanism. In the secondary response, the interval of about 4 days from injection to antibody production accords with in vitro observation e.g. Sher and Harris (1968) and Marshall, Valentine and Laurence (1969), and the brief peak corresponds to the supposed 2 to 3 day life of an antibody cell as determined by tritium thymidine and fluorescent antibody methods (Holborrow, 1967). The rate of decline in titre immediately after the peak does not accord well with the evidence of an early suppressive mechanism in the immune response (Lang, Nase and Rajew&, 1969; Van, 1969). The evidence of classical immunology suggests different patterns of response to different antigens (Wilson and Miles, 1964), but the abundant literature of the past 3 years has failed to substantiate this concept. The observations of Halasa (1968) on the appearance of plaque-forming cells after the immunisation of rabbits with sheep erythrocytes, in trials in which the animals were bled at regular intervals, fall in line with the response of sheep to tetanus toxoid as reported in this paper. It must be emphasised, however, that our results pertain to one dose level and that different results might be obtained if either or both doses were varied.
R.
S. ROBERTS
et
d.
253
SUMMARY
After primary and secondary stimulation respectively with tetanus toxoid antitoxin appeared in the circulation of 19 sheep. The rate of antitoxin production, duration of the period of rapid production, persistence of a high titre, and the uniformity of the rate of its decline, were not significantly different after the primary and after the secondary responses. The primary response developed 9 to 15 days after injection : the secondary response occurred generally between the third and fifth days. Animals which have received a primary injection, can be differentiated from immunologically intact animals by ascertaining whether they produce antitoxin 7 days after one injection of toxoid. The difference in the magnitude of the two responses was less than would be anticipated from the generally accepted concepts of antibody production. The rate of the appearance of antitoxin in the circulation fits in with the observations of other workers using more sophisticated procedures. ACKNOWLEDGMENTS
The authors thank Mrs. Filiz Yazgan and Mr. Naci Guney for technical assistance. REFERENCES
Halasa, J. (1968). Nature, London, 217, 859. Ham;;kgaj T., Kitagawa, M., Matsuoka, Y., and Yamamura, Y. (1969). Immunology, Holborow,‘E. J. (1967). An ABC of M od ern Immunology, The Lancet Ltd.; London. Lang, W., Nase, S., and Rajewski, K. (1969). Nature, London, 223, 949. Marshall, W. H., Valentine, E. T., and Laurence, H. S. (1969). J. exp. Med., 130, 327. Pribnow, J. E., and Silverman, M. S. (1967). J. Immunol., 98, 225. Sercarz, E. E., and Byers, Vera, S. (1967). Ibid., 98, 836. Sher, Stephanie, E., and Harris, T. N. (1968). Ibid., 100, 699. Van, D. C. (1969). Ibid., 102, 451. Wilson, G. S., and Miles, A. A. (1964). Topley and Wilson’s Principles of Bacteriology and Immunity p. 1345 et seq., Arnold; London. [Receiued for publication, June 9th, 19701
COMP.
1971. VOL.
PATH.
PRIMARY
249
81.
AND SECONDARY IMMUNE RESPONSES OF SHEEP TO TETANUS TOXOID
BY R. S. ROBERTS, S. G~~VEN U.N.D.P.IF.A.0.
AND E. E.
WORRALL
SheepDiseases Research Laboratories, Pcndik, Turk@
INTRODUCTION
Early researches on the rate of development of antibody in the circulation emphasised the priming effect of the first injection of antigen but they failed to explain the mechanism of the immune response (Holborow, 1967). The conclusions reached before the discovery of new methods of investigating the problem were that the pattern of the response varied from model to model (Wilson and Miles, 1964) and that in general, the secondary response was earlier, reached a higher peak and was more prolonged than the primary response (Sercarz and Byers, 1967). M ore recent researches suggest that the pattern of the two responses might have more definable characteristics. An opportunity occurred to investigate this problem by the immunisation of sheep against tetanus toxin and some definition of the characteristics of this response was possible. MATERIALS Sheep.
Two
groups
of yearling
AND METHODS pure-bred
Kivircik
males
from
the
Turkish
Government Farm at Inanli, Thrace. Housing and feeding. The animals were kept in groups, loose in a barn, with hay ad lib., and 250 g. of barley per day. Mice. The groups were obtained from a closed colony at the laboratories and weighed 18 to 22 g. Titration of antitoxin. Antibody was titrated in doubling dilutions, against a constant dose of toxin in pairs of mice. The test dose was in the region of L+‘/400 and contained about 15 minimum reacting doses. As a result of several hundred titrations against this test dose, the effects of adjacent doses of antiserum, near the neutralising point, could be distinguished. Intermediate values were interpolated according to a prescribed scale of toxin effect. The neutralising effects of the sera international units. Solutions were expressed as units, which were approximately of toxin were prepared daily from a concentrate in glycerol, and each batch was assayed for potency. Pre-vaccination sera from all sheep had some protective effect against toxin and this was recorded arbitrarily as O-005 unit and amounts of 0.01 of a unit and over were regarded as a response to the toxoid. The standard testing method was used, modified slightly to take a neutralising point with less toxin excess. Toxin and toxoid. The non-sporulating toxigenic Harvard strain of Cl. tetani from the State Serum Institute, Copenhagen, was grown in gauze-covered stainless steel containers for 6 days at 35OC. in a papain digest of lean meat adjusted to @45 l The work was supported by a U.N.D.P. Special Fund Project established through the Food and Agriculture Organisation of the United Nations.
250
IMMUNE
RESPONSES
OF
SHEEP
TO
TETANUS
TOXOID
per cent. N, with 0.5 per cent. NaCl and 1.0 per cent. reduced iron, and sterilised by autoclaving at 115OC. for 30 min. Test toxin was prepared by dialysing a filtrate of this culture against glycerol to reduce the volume. Toxoid was prepared by adding 0.3 per cent. of formalin to the filtrate and incubating at 37OC. for 28 days and purifying by dialysing under pressure to increase potency from 50 to 1500 Lf/ml. This concentrate was diluted in physiological saline solution to 10 Lf./ml. for immunisation of sheep. Immunisation. Two subcutaneous injections of 2 ml., i.e. 20 Lf, were given with an interval of 35 days. RESULTS
A group of 10 sheep was immunised with two injections of toxoid, and the antitoxin titre of the serum of each sheep was assayed at intervals of 2 or 3 days (generally 2 days) during the primary and secondary responses. The development of the primary response was later than expected. Samples taken on the 3rd, 5th and 7th days contained no antitoxin, and at the time of the last sample at 12 days, no decrease in titre had occurred; one sheep did not respond. The determination of the secondary response gave results similar to those of an almost identical experiment reported below and are, therefore, combined with it. In the second experiment a group of 10 sheep was immunised in a similar manner. Antitoxin was assayed (a) before injection, (b) 9, 11, 14, 16, 1% and 21 days after the first injection and (c) 3, 5, 7, 10, 12 and 14 days after the second injection. The results are shown in Fig. 1. The presence of only small quantities of antibody on the 9th day after the first injection supports the evidence of the first experiment that no antibody was formed in the first 7 days after the first injection and further suggests that the 9th day was the critical period when antitoxin production commenced. As one sheep died the secondary response in this experiment was observed on only 9 sheep. On the third day after injection the titres were almost the same as those of the last sample for the primary response. It is possible, therefore, that there had been a fall in titre to the primary response, and that some of the antibody was an early production in the secondary response. Between the 3rd and 5th days, antitoxin was produced in substantial quantities by 9/ 10 sheep in the first experiment and %/9 in the second; the remaining sheep in each experiment produced low levels of antibody between the 5th and 7th days. In both experiments, the primary and secondary responses occurred substantially within a 2-day interval between observations, but some sheep continued to produce antibody, high titres persisting for about 8 days. The decline did not deviate markedly from an even exponential rate and with little tendency to early rapid decline. There appeared to be no obvious difference between the two responses and no correlation in their magnitude; in general, the peak titre in the secondary response was only 2 or 3 times greater than that in the primary response. DISCUSSION
It is accepted that the loss of antibody from the blood is a complex phenomenon, regulated by a balancing between loss and new production, and it is widely held
R. S. ROBERTS
et al.
251
-04
3
a2 0 r04
0.2 EL4 0.2 0 D4 0.2 0
t
p
4
8
12 16 20 24 38 40 44 48 t Days = 0.005 u/ml. p=OXH u/ml. P = aO.01u/ml.
0
Fig. 1 Primary and secondary responses of sheep to tetanus toxoid. Toxoid was given on days 0 and 35(arrows).
that there is a suppressive mechanism operated by antibody and occurring early in the response. It would seem therefore that the measurement which is most acceptable as evidence of a corresponding cellular activity is the appearance of new antibody in the circulation. If the results of these experiments are interpreted in terms of evidence of new antibody, they disclose an interesting, possibly useful pattern : primed sheep differed from immunologically intact sheep in producing antibody between the 3rd and 7th days after injection of antigen. These results, and earlier unpublished work, have consistently disclosed a tendency for animals which eventually reach a high titre relative to others in a group to extend the producing period into a second and even a third observation interval. It is ti interesting speculation that this may be due to insufficiency of F
252
IMMUNE
RESPONSES
OF
SHEEP
TO
TETANUS
TOXOID
some ingredient, such as essential amino acid to enable the primed cells to discharge their function immediately on reaching maturity as antibody producers. It appears unwise to emphasise the relatively small difference seen here between the peak titres in the two responses. The doses of antigen were approximately those that would be used in the field for immunisation of any species of this weight and the first and second doses were equal. It is likely that a more imprcssive difference would follow other dosage rates. These results do however illustrate the difficulty of demonstrating this difference; if the primary response had been looked for in these two groups of sheep on any one day, from the 3rd to the 12th it would not have been found in all the sheep in either group, because the time of antibody production was unpredictable whereas in the secondary response it was found in all sheep at the conventionally expected times. When tetanus toxoid was injected without adjuvant into intact sheep the rate of the appearance of antitoxin in the circulation was invariably uniform in a large number of individuals and corresponded with knowledge which has been acquired recently by the use of more sophisticated techniques in the fields of cell biology and immunology. The immunological mechanism of these sheep was intact and they did not respond on the fifth or seventh days after the first injection of antigen i.e. they had no precursor cells. They did respond later, but the difference between the time of the response to the first injection and to the second was 4 to 8 days. This corresponds with the time claimed by Sercarz and Byers (1967) and by H amaoka, Kitagawa, Matsuoka and Yamamura (1969) for the appearance of memory; it suggests that this interval is occupied in processing of memory cells by macrophages in the radio-sensitive phase of the primary response (Pribnow and Silverman, 1967). A correlation between the magnitude of the primary and secondary responses would not be expected and variable times for the appearance of detectable antitoxin in the primary response would also be expected if precursor cells had to await this processing mechanism. In the secondary response, the interval of about 4 days from injection to antibody production accords with in vitro observation e.g. Sher and Harris (1968) and Marshall, Valentine and Laurence (1969), and the brief peak corresponds to the supposed 2 to 3 day life of an antibody cell as determined by tritium thymidine and fluorescent antibody methods (Holborrow, 1967). The rate of decline in titre immediately after the peak does not accord well with the evidence of an early suppressive mechanism in the immune response (Lang, Nase and Rajew&, 1969; Van, 1969). The evidence of classical immunology suggests different patterns of response to different antigens (Wilson and Miles, 1964), but the abundant literature of the past 3 years has failed to substantiate this concept. The observations of Halasa (1968) on the appearance of plaque-forming cells after the immunisation of rabbits with sheep erythrocytes, in trials in which the animals were bled at regular intervals, fall in line with the response of sheep to tetanus toxoid as reported in this paper. It must be emphasised, however, that our results pertain to one dose level and that different results might be obtained if either or both doses were varied.
R.
S. ROBERTS
et
d.
253
SUMMARY
After primary and secondary stimulation respectively with tetanus toxoid antitoxin appeared in the circulation of 19 sheep. The rate of antitoxin production, duration of the period of rapid production, persistence of a high titre, and the uniformity of the rate of its decline, were not significantly different after the primary and after the secondary responses. The primary response developed 9 to 15 days after injection : the secondary response occurred generally between the third and fifth days. Animals which have received a primary injection, can be differentiated from immunologically intact animals by ascertaining whether they produce antitoxin 7 days after one injection of toxoid. The difference in the magnitude of the two responses was less than would be anticipated from the generally accepted concepts of antibody production. The rate of the appearance of antitoxin in the circulation fits in with the observations of other workers using more sophisticated procedures. ACKNOWLEDGMENTS
The authors thank Mrs. Filiz Yazgan and Mr. Naci Guney for technical assistance. REFERENCES
Halasa, J. (1968). Nature, London, 217, 859. Ham;;kgaj T., Kitagawa, M., Matsuoka, Y., and Yamamura, Y. (1969). Immunology, Holborow,‘E. J. (1967). An ABC of M od ern Immunology, The Lancet Ltd.; London. Lang, W., Nase, S., and Rajewski, K. (1969). Nature, London, 223, 949. Marshall, W. H., Valentine, E. T., and Laurence, H. S. (1969). J. exp. Med., 130, 327. Pribnow, J. E., and Silverman, M. S. (1967). J. Immunol., 98, 225. Sercarz, E. E., and Byers, Vera, S. (1967). Ibid., 98, 836. Sher, Stephanie, E., and Harris, T. N. (1968). Ibid., 100, 699. Van, D. C. (1969). Ibid., 102, 451. Wilson, G. S., and Miles, A. A. (1964). Topley and Wilson’s Principles of Bacteriology and Immunity p. 1345 et seq., Arnold; London. [Receiued for publication, June 9th, 19701