Japan. J. Med. Sci. Biol., 42, 83-99, 1989.

COMPARISON OF IMMUNE RESPONSES TO DIPHTHERIA

AND TETANUS TOXOIDS OF VARIOUS MOUSE STRAINS

Shoichi KAMEYAMA, Fumiko NAGAOKA and Tyoku MATUHASI1

Department of Applied Immunology, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141, and 1Okinaka Memorial Institute for Medical Research, 2-2, 2-chome, Toranomon, Minato-ku, Tokyo 105

(Received August 14, 1989. Accepted October 30, 1989)

SUMMARY: Immune responses of 11 mouse strains with. known genetical characteristics and two outbred strains to diphtheria and to tetanus toxoids were compared. Both diphtheria and tetanus antitoxins were titrated by passive hemagglutination. From the pattern of the immune response, the mouse strains

tested may be classified into four groups. [1] Strains ddY (SPF) and ddY (cony) and those with haplotype H-2b, such as C57BL/6 and C57BL/10, were high responders to both toxoids. [2] Strains with H-2d, such as BALB/c, B10.D2 and DBA/2Cr, were intermediate responders to both toxoids. [3] Strains with H-2k, H-

2a or, H-2m, such as C3H/He, B10.BR, B10.BR/SgSn, B10.A/SgSnJ and B10.AKM/Ola, were high responders to diphtheria toxoid but low responders to tetanus toxoid. [4] The strain with H-2h4, B10.A (4R), was a poor responder to

both toxoids.

INTRODUCTION

Guinea pigs have commonly been used for the potency test of diphtheria and

tetanus toxoids, because of good correlation between the potency titrated in the

animal and the immune response of man to the toxoids (1-3). It is, however,

亀山昭一・長岡芙美子(国立予防衛生研究所体液 性免疫部)

松橋 直(沖 中記念成人病研究所 港区虎 ノ門2-2-2)

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rather difficult to carry out the test routinely in control laboratories because of the

high cost and short supply of the animals, especially in the developing countries

where the tests are very important to implement the Expanded Program on

Immunization effectively (4). It, therefore, is urgently necessary to develop a cost-

effective method for the potencty test of vaccines. Such situations lead us to use

mice for the tests (5).

The mouse method was adopted in the WHO Requirement for Tetanus Toxoid

(2). It was, however, sometimes pointed out that the relative potencies of tetanus

toxoid determined in mice were different significantly from those in guinea pigs

(6,7), and that the potencies in mice differed greatly depending on the mouse strain used (8,9). Further research is required by W. H. O. to solve these discrepancies

(2).

Nyerges (10) reported a mouse method for potency tests of diphtheria and

tetanus toxoids. We have developed independently a mouse method for titration of

diphtheria toxoid (11). The relative potencies determined by this method seemed

to agree fairely well with those determined by the conventional guinea-pig

method, provided that the tests are carried out within a laboratory with ddY strain

mice (11). Later, other reports appeared on the usefulness of mice for the potency

test of diphtheria toxoid (12-14). No report, however, has compared the immune

responses to diphtheria toxoid in various mouse strains. Therefore, it seemed

necessary to scrutinize the immune responses to various toxoid preparations of

various mouse strains before mice are adopted for the routine potency test.

MATERIALS AND METHODS

Mice: Female mice of 4 to 5 weeks old of 13 strains were used. Three strains,

B10.BR/SgSn, B10.A/SgSnJ and B10.AKM/O1a, were provided by the courtesy of

Professor Kazuo Moriwaki, National Institute of Genetics, Mishima-shi, Japan

and bred in this laboratory. The other strains were obtained from a farm, Japan

SLC, Inc., Mishima-shi, Japan.

Antigens: Batches A and B of commercial adsorbed diphtheria-purified

pertussis-tetanus combined vaccine (DPPTads) were used for immunization. The vaccines were prepared by a manufacturer in Japan and the compositions of the

preparations are shown in Table I.

Immunization: Ten mice were usually alloted to each group; less than 10 mice were used in some inbred strains. Each vaccine was diluted with 0.017 M

phosphate buffered saline containing 0.02 w/v % gelatin (pH 7.0) and 1 ml of each

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dilution was injected subcutaneously into each mouse. The immunized mice were bled from the heart under anesthesia on the stated days, and the control animals (unimmunized) were bled from the tail vein.

Titration of antitoxin: Diphtheria and tetanus antitoxins were titrated by

passive hemagglutination (PHA) with sheep red cells coated with highly purified diphtheria or tetanus toxoid as described elsewhere (15-17). Hemagglutination units were calculated relatively to the end points of Standard Diphtheria Antitoxin (National) or Reference Tetanus Antitoxin (National), and expressed in HAU/ml.

The antitoxin titers of some serum specimens were determined also by the in vivo toxin neutralization method to compare with the in vitro method. The rabbit intracutaneous method (18) and the mouse method were used for assaying diphtheria and tetanus antitoxins, respectively. The titers were expressed in International Units (IU/ml).

RESULTS

Relationship between Antitoxin Titer Determined by Passive

Hemagglutination and that by Toxin Neutralization

Diphtheria antitoxin titers in mice determined by PHA (HAU/ml) were

compared with those by toxin neutralizaiton (IU/ml) (Fig. 1). Figure 2 compares

those of tetanus antitoxin in mice. A good correlation was shown between PHA

and neutralization titers with both antitoxins in mice, as in guinea pigs (15,16).

Then, PHA was exclusively used for asaying antitoxins in mice in the present

studies.

Time Course of Antitoxin Production in Mice

Kinetics of production of diphtheria and tetanus antitoxins in mice were

investigated in five different mouse strains, ddY (SPF), ddY (conventional., cony

hereafter), C57BL/6, BALB/c and C3H/He. Each mouse of groups of 10 received

subcutaneously 1 ml of a dilution (1:40) of DPPTads B and bled at 1 week intervals.

The results are shown in Tables II and III and Figs. 3 and 4. Homogeneity of

variance was not denied at any point (p=0.05) (20).

As seen in Fig. 3, the immune responses of mice to diphtheria toxoid can be

classified in two patterns. In ddY (SPF, cony) and C57BL/6, antitoxin titers rose

quickly after immunization and then rather slowly for 5 weeks. On the other hand,

the responses of BALB/c and C3H/He were rather retarded during the early period

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Fig. 1. Correlation between toxin neutralization titer (IU) and hemag-

glutination titer (HAU) of diphtheria antitoxin. Sera from B10.BR/SgSn

(•œ) and ddY (SPF) (•›) were taken 4 weeks after primary injection. The

solid line shows the regression line between the two values. The

specimen with asterisk (*) could be omitted as an outlier by Grubb's

procedure (19).

and reached the level of the first group in 5 weeks. Likewise the response to

tetanus toxoid may be classified into three patterns, as shown in Fig. 4. In ddY

(SPF) and ddY (cony) strains, tetanus antitoxin was produced quickly at an early

stage and reached the maximum titer in 5 weeks of immunization. The immune

response of C3H/He to tetanus toxoid was poor, and the patterns of BALB/c and

C57BL/6 may be intermediate from results shown Fig. 5.

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Fig. 2. Relationship between toxin neutralization titer (IU) and

hemagglutination titer (HAU) of tetanus antitoxin. Sera of ddY (SPF)

were taken 4 weeks after primary (•›) and 2 weeks after secondary (•œ)

injections.

Dose-response Relationship in Mice to the Toxoids

Three mouse strains, ddY (SPF), BALB/c and C57BL/6, were used in this

experiment. DPPTads A was diluted serially at an equal logarithmic interval (-0.1

to -2.2). Each dilution was injected subcutaneously in a dose of 1 ml into at least 10

mice. Four weeks after injection, diphtheria and tetanus antitoxins were titrated

(Fig. 5). In the figure, geometric means of both antitoxin titers are plotted against

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Fig. 3. Immune responses of various mouse strains to diphtheria toxoid.

Geometric means of 10 animals were plotted.

•›•\ ddY (SPF) •œ------- ddY (cony) •¢•\-•\ C57BL/6

• •\ BALB/c •¡------- C3H/He

log doses of the vaccines. Homogeneity of variance in the antitoxin titers was not

denied between the doses of either toxoid at p=0.05. Titers of diphtheria and

tetanus antitoxins increased in proportion to the dose in each mouse strain. No

deviation from linearity of dose-response curves was significant in diphtheria or

tetanus antitoxin responses in any of the three strains, provided that a dose of -2.2

of diphtheria toxoid in ddY (SPF) and BALB/c and a dose of-2.2 of tetanus toxoid in

BALB/c are omitted from the calculation. The titers of antitoxins, however, varied

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Fig. 4. Immune responses of various mouse strains to tetanus toxoid.

Geometric means of 10 animals were plotted.

•›•\ ddY (SPF) •›------- ddY (cony) •¢•\-•\ C57BL/6

• •\ BALB/c •¡------- C3H/He

greatly depending on the strain. The responses to diphtheria toxoid were almost

similar in both ddY (SPF) and C57BL/6, and the response of BALB/c strain was

rather lower than the former two strains. The antitoxin responses to tetanus

toxoid differed from those to diphtheria toxoid. The antitetanus titers were high in

ddY (SPF), moderate in BALB/c and low in C57BL/6; the titers at middle dose (-

1.6) were 0.262, 0.048 and 0.01HAU/ml, respectively.

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Fig. 5. Dose-response curves for diphtheria and tetanus toxoids.

Geometric means of antitoxin titers of 10 animals were plotted to the

doses.

•›•\ ddY (SPF) •¢•\-•\C57BL/6 • •\BALB/c

Immune Responses of Thirteen Mouse Strains

to Diphtheria and Tetanus Toxoids

The immune response to diphtheria toxoid or tetanus toxoid was compared

among 13 mouse strains with known genetical characteristics. Each mouse of

groups of 10 animals of the respective strains was injected with 1 ml of a 40-fold dilution of DPPTads B, and antitoxin titers were determined in 4 weeks. A group

of ddY (SPF) was used as control of every experiment to compare among the

experiments carried out on different days. The results are shown in Table IV and

Fig. 6.

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Fig. 6. Comparison in diphtheria and tetanus antitoxin titers among

mouse strains.

Sera were taken 4 weeks after primary injection.

Geometric means of each mouse strain were plotted (from Table IV).

Bars

at each point are the 95% confidence intervals.

•› Diphtheria antitoxin •œ Tetanus antitoxin.

Homogeneity of variance among the strains was not denied (p=0.05) for

either antitoxin titer. Table IV and Fig. 6 show that the immune responses of mice

to diphtheria and tetanus toxoids differ greatly depending on the mouse strains

used. The strains may be classified into three (or four) groups from the pattern of

the response. The first group involves the strains showing high response to both

toxoids, such as ddY (SPF), ddY (cony), C57BL/6, C57BL/10, BALB/c, B10.D2 and

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Table V. Relationship between the primary immune responses to diphtheria or

tetanus toxoid and the genetic background of the mouse strain

For antitoxin titers refer to Table IV. *: Oblique bar indicates crossover position .

DBA/2Cr. The last three strains may be intermediate responders and belong to the

second group (Table V). The third group, consisting of B10.A/SgSnJ,

B10.AKM/O1a, B10.BR, B10.BR/SgSn and C3H/He, showed high response to

diphtheria toxoid but poor response to tetanus toxoid. The last (fourth) group is

low responder to both the toxoids, as B 10.A(4R).

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DISCUSSION

As described above, immune responses of mice to diphtheria and tetanus

toxoids differed remarkably depending on the mouse strains. In addition, the

patterns of the immune response were different from one toxoid to the other.

As stated above, the mouse strains may be classified into four groups from the

responses to the two toxoids. Although it is very difficult to discuss the

relationship between the immune response and the genetical back grounds of the

mouse as limited number of animals and of strains were used, it is of interest to

compare the immune responses of the starins with their H-2 haplotype as shown in

Table V, ddY strains and the strains with haplotype H-2b responded well to both

the toxoids, and those with H-2d showed the intermediate response to the toxoids.

The strains with H-2k, H-2a, or H-2m responded well to diphtheria toxoid, but

poorly to tetanus toxoid. The strain B10.A (4R) showing poor response to both antigens had haplotype H-2h4. Since B10.A/SgSnJ, kkkkk/dddd, provokes

relatively high response to diphtheria toxoid, the immune response gene to

diphtheria toxoid could be located between IK and IE subregions. On the other

hand, B 10.A (4R) with kk/bbbbbbb haplotype showed only a poor response to

diphtheria toxoid. It may be considered that the Ir gene to diphtheria toxoid is

located between IA and TB subregions and the gene would be injured during the

crossing over events in the B 10.A (4R) strain.

The antitoxin response to tetanus toxoid appeared to have some connection to

haplotype, too. H-2b strains gave as high response to tetanus toxoid as to

diphtheria toxoid. The very low antitoxin response of B10.A (4R) to tetanus toxoid

may suggest that the immune response gene is located within IA to TB subregions.

Although the haplotype of ddY strain has not been well analyzed, ddY strain could

be classified into a high responder. Further genetical analyses are necessary to

draw any definite conclusion on the immune responses of mice to the toxoids.

It may be possible that variation in the immunizability of mouse strains will

influence the results of potency tests of biological products. Ipsen (21) found a wide

variation in the immunizability of mouse strains to tetanus toxoid (adsorbed), but

the influence of such variation on the potency test was overcome by use of a

reference for the test. However, Wada et al. (8) stated that the potency of plain

tetanus toxoid relative to a reference varied significantly depending on the mouse

strain. Hardegree et al. (9) showed a similar result with adsorbed tetanus toxoid.

Murata et al. (6) showed a great difference between the potencies of tetanus toxoid

(plain) determined in guinea pigs and those in mice, and they showed also that

some contaminants in the toxoid preparation greatly affected the immune response

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of mice but not that of guinea pigs. It may be possible, however, that such a short

immunization period as two weeks influenced greatly the response of mice to the

toxoid. In this respect, van Ramshorst et al. (7) stated that with a single dose of

tetanus toxoid (adsorbed) the relative potencies were not different between the

mouse and guinea pig, but with combined vaccine (DPT) the potencies of tetanus

component determined in mice were about twice those determined in guinea pigs

with the immunization period of 4 weeks for both animals. Having taken such

variation in the potency by animal species, the WHO Expert Committee stated in

Requirement for Combined Vaccine (22) as follows:

•g The potency of tetanus component (of DPT combined vaccine) shall be less

than 40 IU per human single dose determined in guinea pigs, but 60 IU per dose, if

mice are used for the potency test.•h

The present authors recommend that selection of a suitable mouse strain is

necessary before adopting the mouse assay in place of the guinea pig assay, and

that the potencies obtained by the mouse assay should previously be checked by

comparing with those by the guinea pig assay.

The potencies of biological products are usually prescribed in acceptable

lowest levels, and the upper limits of the potencies are not taken into account in

the Requirement for many biological products. Since an increase in a single

human dose of diphtheria or tetanus toxoids by only twofold or so may sometimes

result in serious side reactions in vaccinees (23), it would be necessary to control

the upper limit and the acceptable lowest one as well of the potency to reduce side

reactions.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. R. Murata, Honorary member of National

Institute of Health, Tokyo, for his helpful advice and encouragement in performing

the present investigation. They are also grateful to Professor K. Moriwaki,

National Institute of Genetics, Mishima-shi and Dr. M. Nakagawa, Chief,

Laboratory of Experimental Animals 1, Department of Veterinary Science,

National Institute of Health, Tokyo, for the supply of mouse strains.

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REFERENCES

1. van Ramshorst, J. D., Cohen, H., Levine, L. and Edsall, G. (1973): J. Biol. Stand., 1, 215-220.

2. Wld. Hlth. Org. Expert Committee on Biological Standardization (1979): Tech. Rep. Ser., No. 638, p.90.

3. Wld. Hlth. Org. Expert Committee on Biological Standardization (1987): Tech. Rep. Ser., No. 760, 84-85.

4. Perkins, F. F. (1978): Develop. Biol. Standard., 41,291-294 (S. Karger, Basel, 1978).

5. Ministry of Health and Welfare (1989): Japanese Minimum Requirements for Biological Products, 55,105 (in Japanese).

6. Murata, R., Wada, E., Yamamoto, A. and Kubota, K. (1961): Japan. J. Med. Sci. Biol., 14, 121-129.

7. van Ramshorst, J. D., Sundaresan, T. K. and Outschoorn, A. S. (1972): Bull. Wld Hlth Org., 46, 263-276.

8. Wada, E., Yamamoto, A., Kubota, K. and Murata, R. (1961): Japan. J. Med. Sci. Biol., 14, 143-146.

9. Hardegree, M. C., Pittman, M, and Maloney, C. J. (1972): Appl. Microbiol., 24, 120-126.

10. Nyerges, G., Virag, E. and Lutter, J. (1983): J. Biol. Stand., 11,99-103.11. Kameyama, S., Muljati, Prijanto, Ito, A., Nagaoka, F, and Yamauchi, K.

(1983): Japan. J. Bacteriol., 38,466 (text in Japanese).12. Kreeftenberg, J. G., van der Gun, J. W., Marsman, F. R., Sekhuis, V. M.,

Bhandari, S. K. and Maheshwari, S. C. (1985): J. Biol. Stand., 13, 229-234.13. Hendriksen, C. F. M., van der Gun, J. W., Marsman, F. R. and Kreeftenberg,

J. G. (1987): J. Biol. Stand., 15,353-362.14. Maheshwari, S. C., Sharma, S. B., Ahuja, S. and Saxena, S. N. (1988): J. Biol.

Stand., 16, 139-146.15. Kameyama, S. and Kondo, S. (1975): Japan. J. Med. Sci. Biol., 28,127-138.16. Kameyama, S., Yamauchi, K., Yasuda, S. and Kondo, S. (1980): Japan. J.

Med. Sci. Biol., 33, 67-80.17. Kondo, S., Kameyama, S., Yasuda, S. and Nagaoka, F. (1977): Japan. J. Med.

Sci. Biol., 30,119-124.18. Jensen, C. (1933): Acta Pathol. Microbiol. Scand.,10, Suppl. 14,1-211.19. Grubbs, F. E. (1969): Technometrics, 11, 1-12.20. Bartlett, M. S. (1937): Proc. Royal Soc. London, A160, 268-282.21. Ipsen, J. (1954): J. Immunol., 72,243-247.22. Wld. Hlth. Org. Expert Committee on Biological Standardization (1979):

Tech. Rep. Ser., No. 638,103-104.23. Akama, K. and Yamamoto, A. (1971): Proc. Japan Med. J., 2465, 26-32 (text

in Japanese).

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