Jpn. J. Infect. Dis., 54, 167‑180, 2001

Review

QualityControl of Diphtheria Tetanus Acellular Pertussis
Combined (DTaP) Vaccines in Japan
Yoshinobu Horiuchi*, Motohide Takahashil , Toshifumi Kondal , Masaki Ochiai,
Akihiko Yamamoto, Michiyo Kataoka, Hiromi TbyoIZumi and Yoshichika Arakawal
Department ofSafeo) Research on Biologics andJDepartment of Bacterial and Blood Products, National Institute of lnfectious Diseases,

Gakuen 4‑7‑1, Musashimurayama‑shi, Too;0 208‑0011, Japan(Received March 30, 2001. Accepted October 1, 2001)
CONTENTS :412‑3. Effect of the toxdiding process on the
immunogeniclty and efrlCaCy Of adjuvant gel

1. 1mtroduction

2. A brief history of Japanese acellular pertussis

4‑2‑4. Establishing the clinical relevance ofvaccine development

3. A new trend in the quality control ofDTaP

toxoid potency tests

4‑2‑5. Other problems
4‑2‑6. Antitoxin titration

vaccines and its complications

3‑ 1. Testlng the consistency of products

3‑2. Accuracy of the results orclinical trials

4‑3. Tbxiclty

4. Quality control ofDTaP vaccines in Japan

4‑3‑1. Endotoxin

4‑3‑1‑1. The bacterial endotoxin test

4‑1. Potency of the pertussis component
4‑I‑1 ･ Intracerebral (ic) challengeヮode1

4‑3‑ 1 ‑2. Clinical relevance of the endotoxin test

4‑ 1 ‑2. Aerosol and intranasal (resplratOry)

4‑3‑ 1 ‑3. Necessary cautions for substitutlng atest method

challenge models

4‑3 ‑2. Lymphocytosis (or leukocytosis)‑promoting

4‑2. Potency tests of the diphtheria and tetanus

components of combined vaccines(LP) activity

4‑3‑3. Histamine sensitizing (HS) activity
4‑3‑4. Chinese hamster ovary (CHO) cell412‑1. Effect of whole cell pertussis vaccine on
the potency test of tetanus or diphtheria

toxoidclusterlng test

4‑2‑2. Effect ofadjuvant gel and animal model

5. Conclusionon the toxoid potency test

SUMMARY: Diphtheria tetanus.acellular pertussis combined (DTaP) vaccines have been successfullyused inJapan by controlling their potencleS and toxicities with animal models･ In accordance with the recent practical

introduction of DTaP vaccines of various formulations, a question has been raised in other nations as to theelrlCaCy Of a quality control system based on animal tests and standard preparations. The World Health Organiza‑tion issued its guidelines on the production and quality control ofacellular pe山ISSis vaccines in 1998 along withthe concept of quality control by ensurlng that production lots were consistent with clinical trial lots, rather thanby comparing them with standard preparations in traditional laboratory tests. However, because it is not feasible
to evaluate the combined use of vaccines from different manufacturers in a clinical study, the altemative trend of
quality control may glVe rise to a difficulty ln rationalizing the practical immunizations to use vaccines ofdifferent brands in a mixed consequence. A standardized national regulation system to ensure the equlValence ofapproved products would be essential for such an immunization practice. The success of the Japanese DTaP
vaccination suggests the possibility of an effective quality control of DTaP vaccines by meansof standardized
test systems･(Kendrick) test, whichinthe 1 950s had been proved to correlate

1. Introduction

well with clinical protection (1). The efficacy and safety of

the DTaP vaccines have been successfully controlled using

Diphtheria tetanus whole cell pertussis combined (DTwP)

vaccine was used effectively to prevent the diseases until it

animal models in Japan. The potency of the pertussis compo‑

was temporarily discontinued due to two cases of post‑vaccina‑

nent of the DTaP vaccines has been controlled by the modi丘edtion death in the mid‑ 1 970S. Subsequently, diphtheria tetanus

Kendrick test with the immunization period extended to 3

acellular pertussis combined (DTaP) vaccines were developed

weeks. The potency of diphtheria and tetanus toxoids in theand implemented in Japan in 198 I. In the course of the acellular

DTaP vaccines has been controlled by in vivo or in vitro

pertussis vaccine development, protective antlgenS Were iden‑

measurement of the antitoxin titers in the sera of immunizedtified by the Kendrick's mouse intracerebral (ic) potency

gulnea plgS Or mice. Animal tests have played crucial roles
also in ensunng the safety of the vaccines.

*Corresponding author: Tel: +8 11421561 1077 1 , Fax: +8 1‑42‑567‑

DTaP vaccines of variouspertussis antlgen compositions
and a4juvant gel contents have been developed and put into

0740, E‑mail: horiuchi@nih.gojp

167

clinical use in other nations during the past 1 0 years. Accord‑

achieve the goal. Kurokawa et al. (15,16,19) had developed

ingly, a question has been raised about the validity of thequantitative methods f♭r assaying the toxicity to mouse weight

pertussis icIPOtenCy test in predicting the clinical efflCaCy Of

gain (mouse body weight decreasing [BWD】 toxicity) andacellular vaccines. An altemative approach relying on theof acellular pertussis vaccine (2). ln this approach, the efrlCaCy

LP toxicity. BWD toxicity was assayed by measuring thetoxicityofa vaccine to mouse weight gain Pyer the 1 6‑h post‑
injection period to calculate the relative toxICltyof the vaccineof a routine product lot was suggested to be assured by ensur‑

referring to the dose‑response of a reference vaccine that haveresults ofclinical trials was suggested for the quality control

lng ltS COnSistency with the clinical trial lot. Attempts haveFn assigned unit value of the toxicity･ LP toxicitywas assayedln the same mice by measurlng the increase in peripheral

also been made to extend this new approach to the potency
tests of diphtheria and tetanus toxoids (73) in order to settle

leukocyte count on the third day of injection to calculate thethe problems concemlng the varied production processes andrelative toxIClty Ofthe vaccine referring to the dose‑response

adjuvant gel contents among the products from differentof the reference vaccine.

At the time of the above researches, the methods available

manu facturers.

According to the newly proposed concepts of standardiza‑

for assaying HS activlty Were those use the fatal sensitiza‑tion and quality control, the World Health Organization

tion of vaccinated mice to histamine challenge (histalnine‑(WHO) issued its guidelines f♭r the production and control ofsensitizing death [HSD]) (17,18). To achieve the goal of

the acellular pertussis component of lnOnOValent or combined

reducing the activlty tO One tenth of the average ofwhole cell

vaccines (WHO Guidelines) in 1998 (3). ln this paper, thevaccines, the assay method was required to have the sensitivity

concepts of the guidelines and their influence on the current

allowing quantitative discrimination of the DTaP vaccines thatvaccination system are discussed in comparison with Japanese

showed no fatal sensitization of mice to histamine challenge

concepts of quality control of DTaP vaccines･(20). Ishida et al. (21) developed a highly sensitive assay

method for HS activitybased on the change in rectal tempera‑ture of mice after histamine challenge on the fourth day of

2. A brief history of Japanese acellularpertussis

sensitization. The assay methods fわr BWD, LP and HS

vaccine development

activities played cmcial roles not only in the development or

Pertussis vaccination was temporarily suspended aRer two

the vaccine but also in quality control of the vaccine after its

cases of post‑vaccination death in 1974 and 1975 (4). As a result

clinical introduction (22)･ A lyophilized whole cell pertu苧Sisof the lowered vacclnation rate, by 1979 the annual number

vaccine was prepared for use as the reference preparatlOnof cases of whooping COugh had increased dramatically, to(Reference pertussis vチccine for toxicity tests, Lot 1) in the

13,000 cases (5). Shortly a洗er the cessation of vaccination a

toxicitytests･ This vacclne Was ass.1甲ed a BWD toxicityvalue

research team was set up to improve the vaccine. The researchof 1,380 BWDU/vialJts HS actlVlty Was determined to be

initially focYsed on isolating protective antigen(S) ofBondetella

60 HSU/vial by referrlng tO the Netherlands Reference 2,

pertussisuslng the Kendrick test. Two candidate antlgenS Werechosen as potential protective vaccine antlgenS. One was thewhose activitywas.stFted as 4･4 HSU/ml (23,24)I The identicalvalue as the HS actlVltywas assigned for the LP activityofthe

22S protective antlgen, Which was reported to show purelyreference vaccine ( 19)･ The initial limit values implemepted

protective activity (6,7) and the other was an antigen丘･action

for BWD, LP and HS toxicities of Japanese DTaP vacclneS
were 10 BWDU, 0.5 LPU and 0.8 HSU per milliliter, respec‑showing histamine s.ensitizing (HS), lymphocytosis (orleukocytosis)‑pr.0.motlng (LP), hemagglutinating (HA) andtively (19, 22).
The formulation of adjuvant gel was examined with regard

r!rotective PctivltleS (8‑1 I)･ ln the course of the investiga‑

tlOn, an antlgenfractionwith an appearance similar to that ofto the safety and potencies of the diphtheria, tetanus andthe 22S antlgen Was Shown to exert no protective activlty lnacellular pertussis components of the vaccine･ A rather low

the Kendrick test. Accordingly, the research was concentrated

concentration of the aluminum adjuvant gel was suggested

on establishing a method for isolating the fraction with HA,
LP and HS activities･ Although a fraction showlng Only HA
activitywas separated from the HA active fraction that also

to be adequate for the formulation of safe vaccines that confer

showed LP and HS activities in the early stage orthe study(1 2), attempts to develop a practical vaccine were made along

as 0.3 mgAl/ml (22).
The newly developed DTaP vaccine was put into publicwith the efforts to co‑purifyall the active substances. A soluble

use in 1981 a鮎r a clinical evaluation (25). The vaccine wassumcinet potencies or diphtheria and tetanus toxoids (68).
The upper limit of the adjuvant gel concentration was defined

fraction rich in the activities was obtained by extractlng

shown to be clinically safe (5,25). ln particular, it was shown

ammonium sulfate‑precIPltated culture supematant or whole

to be remarkably less pyrogenic (5). Public confidence in theculture ofB. pertussis Phase I Tohama strainwith concentrated

safetyofthe vaccine was quickly established, and the number

alkaline salt solution. The extract was found to lcontain aofwhooplng COugh cases quickly retumed to the level seen

considerable amount of endotoxin, which was removed by a

befわre the suspension or DTwP vaccination (5).

sucrose‑density.gradient centrifuge ( 1 3)I The fraction was lat?r
proved to consist Of mainly two different antlgenS, PertuSSIS

3. A new trend in the quality control ofDTaP vaccines

toxin (PT) and filamentous hemagglutinin (FHA) (14). The

and its complications

toxic activities of the fraction were detoxified by a formalde‑

hyde treatment (13)･ The DTaP vaccines w?re formulated

Followlng the recent clinical introduction of DTaP vaccines

uslng thus developed acellular pertussIS VaCClneS.

with various antlgen and adjuvant gel constituents in the

Limit values fわr the residual toxicities of the new vaccine

United States, as well as in Europeanand someAsian countries,

were another subject of the research. An initial goal was set

publications have been made by some researchers to proposeof reducing every measurable toxic activlty tO One tenth ofaltemative concepts of quality control of DTaP vaccines. The

the level of average whole cell vaccines. Consequently, highly

newly proposed concepts of quality control were based onsensitive and quantitative assay methods were required toensunng the consistency ofa production lot with clinical trial168

or standard preparations (2, 26)I In fact, the c.urrent icIPOtenCy

difficulties. Practically speaking, lt Will be all but impossible
to avoid uslng DTaP vaccines of different brands in a mixed

test might have a. validitylimitation in detectlng the protective

consequence for achieving thefull course of vaccination (30).

lots rather thanby companng the production lot with reference

potency of certaln antlgenS that have been claimed to confer

Although these new concepts seem to be taking hold in theprotection in humans or in mouse resplratOry infection models

quality control of DTaP vaccines, they will requlre Careful(27,28). The potency tests fわr diphtheria and tetanus toxoids

evaluations before full implementation. ln particular, certain

were also reported to have a complication due to varied produc‑

contradictions inherent in these concepts should be discussed

tion procedures and constituents including adjuvant (29).

in detail.

3‑1･ Testing the consistency of products

In 1998, WHO issued the guidelines f♭r the acellular

lt has been considered to have a reliable method f♭r directly

pertussis vaccine (3) based on the fわllowing rationale.

1 ) Each DTaP product produced by a different manufTacturer

testlng the consistency of the antlgen composition of final

should be considered unlque due to the differences inbulks･ Tわward this end, intensive studies were made during

production processes, antlgenS and adjuvant‑gel constitu‑

the development of the acellular vaccine in Japan. However,ents.

an applicable method for testing the final bulk could not beestablished due to the aggregation of antlgensby the detoxi‑2) Their quality, therefわre, camot be evaluated in a labora‑

tory test uslng a universal standard preparation.

fication process. It was concluded that the test for antlgen

consistency could be correctly applied only to materials before

3) Accordingly, their quality should be evaluated by ensuring
their consistency with a clinical trial lot whose e爪cacy

detoxification. It was assumed necessary, therefore, to consider

and safety have been clinically evaluated.

the possibility or imposlng an in‑process control test uslng

The above concepts have led to remarkable changes in thereference antlgenS and standardized methods, in splte Of the

traditional methods of vaccination and vaccine qualitycontrol,

possibility of practical inconsistency between the materials

as summarized in Table I. We note that products from different

before detoxification and those in final bulk. Another possi‑
bilityWould be to test antibody production by vaccine antigens
in approprlate animals. However, this would not be accurate

manufacturers should also be considered unique because no

chance ofclinical evaluation would be probable on the effectof use of vaccines of different brands in a mixed consequence

enough fわr provlng the exact consistency of products.

for primary and booster doses.lmplementatiOn of such concepts

Another concem regarding the test fわr consistency is thebrings an important change to the traditional vaccination

stabilization of a clinical trial lot as the stan血rd preparation.

practice to allow uslng a combination of different vaccine

lt is stated in the WHO Guidelines that it is necessary tobrands for pnmary and booster immuni2:ation series. The new

ensure consistency between a routine product lot and clinicalconcepts requlre that DTaP vaccines of the same brand betrial lot in order to assur.e efficacy (2,3)･ A difficultywould

used for all doses in the vaccination series (30)･ Howe†er,∬ise, however, in prepanng stable vials, if by lyophilization.

full implementation of the new system may cause practlCal

It would not allow ones to use the preparations ofexactly the

Table I , Comparison between the current Japanese quality control system and the WHO Guidelines

Current Japanese system New trends inthe WHO Guidelines
A quality control system using laboratory

A quality control system based on ensumg

models established or evaluated according toconsistency withclinical triallots whose
safetyand efficacy has been assured

clinical rclcvance.

clinically.(2,3)

Because results of a clinical trial would also

Should adhere strictly to tbc production

have limited accuracy, lt lS nCCCSSaIY tO

process used for the manufacture of theRole orcomplement them with results or post‑

vaccine lots used to prove cfricacy andclinical trials andmarketing suⅣcillance and or the tests i一l

safctyin clinical trials. Laboratory tests

laboratory tests

laboratory model systems.

should show cqulValcnce bctwcen vaccine
lots and those lots known to bc clinicallyeffcctive and safe.(3)Vaccine brandand quality

Under the assumption that appropnatc test

Bccausc ofthc differcnce in antlgen

systems can bc cffCctivc in predicting thecomposition (and lack of the chance to

cfrlCaCy Or Safety of a product, aH vaccines

cvaluate the cffcct of using vaccines ofpasslng aPPrOPnatCIy lmPIcmcntcd laboratory

different brands in amixcd sequence in atests are considcrcd to have similar lcvcls ofclinical trial) vaccines from different

safctyand efficacy.

manufacturers should be assumed diffcrcr)t
and unjquc products.(3)AH vaccines passlng aPPrOPnately

Bccausc data are insufncicnt rcgardingthC

implcmcntcd laboratory tests can bc usedsafety, immunogenicityand efficacy of using

Vaccine brandcvcn in amixed scqucncc in the vaccination

vaccincs舟om different manufacturers in aand useSCrl CS.

mixed sequence, the same brand of vaccine
is recommcndcd to be used for all doses inthe vaccination series.(30)

Modifications of
production process
or fbrmulatioll

Any modifications of production process

Any change in manufacture or formulationmust bc rationalized clinically. Howcvcr, it isshould bc carefully cvaluatcd clinically or, atalso acceptable to make the modification

least, by dcmonstratlng CqulValencc ofaccording to the results of laboratory tests

approprlately defined criteria of

whose clinical rclcvance have been proved

characteristics withthat of clinicaHotsknOwn

appmpn atcl y.

to bc clinically safe and effective.(3)169

same characteristics both for a reference and for a clinical
trial due to the difrlCulty in proving the influence of lyophiliza‑tion as negligible.

3‑2. Accuracy of the results of cJinical trials
The reliability orthe results ora clinical trial is orprimaryめ0

importance for the system prescribed in the Guidelines. In

20000

め
a

general, however, the results of a clinical trial have limited
validity ln accurately predicting the outcomes or a massO

immunization due to the usually limited sample sizes or ethical‑;:.?.'==7

DisOontin uati

I 0000of vaccinatio

restrictions in the inclusion ofa placebo. Furthermore, estimatesof DTaP

vacci nationof vaccine efrlCaCy would differ depending on the diagnostic
criteria applied. The results would also be affected by the｡‑■｡.一...∫/ ヽ･･･t････.........

variation in clinical diagnoses, even when made and recorded inaccordance with the WHO's case definition (3 1 ,32). Further‑

1960

1965

1970

1975

1980

1985

1990

1995

2000

Year

more, an estimate of vaccine efficacy, and therefore of thedifference in infTection rate between the vaccinated and placebo‑

Fig, I. Annual whooplng COughcases in Japan. Pcrtussis vaccination

administered cases, would not be constant even in a slngle

was suspended in I 975 due to two cases of post‑vaccination death.

study･ Instead, it would vary depending on the selected obser‑

Wbooplng COugh cases quickly incrcascd to over I 3,000 per year by
1979, then dcclincd due to the rcsumcd immunization with DTaP

vation period after vacc.ination (33)･ EfflCaCy estimates on

vaccinc. The annual incidcncc has dcclincd steadily since 1979,

the same product can slgnificantly vary in different trials

indicatlngthc efrlCaCy Ofthc acellular pcrtussis vaccine,(34,35)･ Therefわre, we need to be cautious not to rely too

much on the absolute figures of the estimated vaccine efGcacy

reproducibility (modified Kendrick test). This modification

Pr safety･ This fact emphasizes the importance of post‑market‑

was later proved to glVe an improved immune response tolng SuⅣeillance.

In addition, it has become increasingly difficult to carry

PT constituFntS Of the vaccine in mice (39)･

out clinical trials. Although animal testlng lS O洗en criticized

Thusdeveloped Japanese acellular pertussis vaccines wereas a means ofevalua.ting vaccine quality (26), it is necessary

found to compnse mainly FHA and PT. Although the vaccinesfrom different manufacturers had varylng contents Of FHA,
PT and other trace antige?S, such as agglutinogens (14), allto develop useful animal model systems not only for quality
control, but also to improve or develop new vaccines (2).

were found to have equlValent potency ln the modified

Kendrick test (40). With regard to clinical efficacy, the two

4･ Quality control ofDTaP vaccines in Japan

Japanese acellular vaccines having the most divergent antlgen

One of the fundamental policies for DTaP vaccination incompositions, namely TJtype and B‑type vaccines, were both

Japan has been to apply a unified vaccination program inwhich all vaccines are controlled to have the consistent qual‑

proved to be clinically effective (33,36,38,41‑43). Japaneseacellular vaccines of T‑type, B‑type and intermediate antigen

ityirrespective of the manufacturer, as explained in Table 1.

compositions approved by the modified Kendrick test showedThis quality control has been realized uslng laboratory tests

Pomparable clinical etrlCaCy (44)･ As shownin Fig･ 1 , pertussis

with reference or standard preparations. Accordingly, Japa‑
nese DTaP vaccines from six different manufacturers have

reduced the cases of whooping cough in Japan, further

been used in an interchangeable manner in the course ofsuggesting the validityofthe modi丘ed Kendrick test. However,prlmary and booster immunizations. The system has been
accepted as the realistic practice in the routine vaccination.

doubts have been raised in regard to the ability or the ic

Immunization resumed with the acellular vaccines effectively

protection test to predict the clinical protective potency of

As no chance would be generally expectable to evaluate
the effect of using vaccines of different brands in combination,

antlgenS Other than PT. As possible altematives, lntranaSal

the Japanese system needs to base upon the assumptlOn that

No agreement has been reached so far on the immunization

the vaccines from all the six manufacturers are equlValent in

血ses, schedule or challenge doses丘)r an adequate stan血rdiza‑

their efficacy and safety once they passed the approprlately

tion of the resplratOry infection models.

and aerosol infection models have been examined (27,28).

implemented quality control tests. Effective model systems

4‑I‑I. Ic challenge modelare of primary Importance for quahty control and successful

The orlglnal Kendrick test was approved based on theroutine vaccinations･ Japanese quality control or DTaP

results of a field trial of the British Medical Research Coun‑cil (MRC field bial) (1) and has been effectivelyused to control

vaccines has largely depended on animal models and has been

4‑1･ Potency of the pertussis component

whole cell vaccines. However, the immune response of miceto acellular vaccines has been suggested to be different from

Development of the Japanese acellular pertussis vaccinethat of mice to whole cell vaccines. Whole cell pertussis

successful thus far (5,36‑38).

began with identifying a pertussis bacterial fraction showing

vaccines generate both cell‑mediated and humoral immune

protective activlty･ This was accomplished by means or a

responses, while acellular vaccines induce mainly humoral

Kendrick test in which protective activlty Was measured as
the degree to which a glVen fraction protected immunizedimmune response (27,45,46)･ T‑cell dependent humoral
lmmune response has been suggested to play a cmcial role inmice against an ic Injection ofvirulent B. pertussis at 2 weeks

the protection against ic challenge (27,45). ln particular, thepost‑immunization. The test had been used throughout the

DTaP vaccine containlng genetically detoxified PT has beencourse ofdeveloplng the vaccine as the only laboratory method

shown to be highly protective in the ic potency test and wasfor identifying candidate antlgenS for an effective vaccine. In

reported to confer a strong cell‑mediated immunity both in

the process, the interval between iThmunization and challenge

mice and humans (52).

was extended from the onglna1 2 weeks to 3 weeks to improve

Both PT immunization and passive anti‑PT antibody are170known to confer sufficient protection agalnSt ic challenge

intranasal challenge model from the ic challenge model would(47,48). PT has also been reported to show a synergistic effectbe that antlgenS Such as FHA, pertactin or flmbriae could bewhen combined wi血such other antlgenS aS FHA or 69K outer

protective without the aid ofPT (59164)･ However, CPution

membrane protein (69k OMP), both or which show marginal

should be taken when interpretlng the results of the resplratOry

protection when given alone (49,50). The role ofPT is, there‑

challenge model, since this model was reported to show nofore, suggested to be crucial for the protection in the Kendrickcorrelation with the clinical efficacy of whole cell vaccines

test. ln addition, Pillemer 's Stromata Protective Antigen (SPA),

and also to have fわund boiled whole cell vaccines protective

which is assumed to comprlSe mainly bacterial components(65). ln addition, ethical restrictions will prevent quantita‑
tive determination of the clinical relevance of this model forwith HA activlty and was, consequently, shown to have HS
activity, has been proved to be highly protective bothinKendrick

predicting the efficacy of vaccines. Further study will be
required to evaluate and standardize the respiratory infection

test and clinically based on MRC trial (5 1).

model for any of its routine utilizations.

Regarding the i‑unization of mice,也e optlmum immuni‑

zation period for the maximum anti‑PT production by PT

4‑2. Potency tests of the diphtheria and tetanus compo‑

toxoid was reported to be 28 days (39). The immunization

nents of combined vaccines

Vaccination with diphtheria and tetanus toxoids has played aperiod for the modified Kendrick test, therefore, is assumed to

key role in the effective prophylaxis agalnSt these diseases. The

be more appropnate f♭r optlmal anti‑PT production companng

than the orlglnal Kendrick test. A definite correlation wasseen between the logarithmically transfbmed potency values

potencies of the toxoids have been controlled by measurlng

neutralizing antibody production in gulnea plgS Or mice. The

at 2 weeks and 3 weeks post‑immunizations on 13 batches ofmajor concems raised thus far on the reliability of the potency

DTaP and three batches ofDTwP vaccines (unpublished). The

tests have related to 1) immunizing animal species or strains

resulting equation fわr the relationship between the log‑potency

and the clinical relevance of their lmmuPe responses; 2) thevalues or immunizations of 3 weeks (Y) and 2 weeks (X)influence of the adjuvant effect of the alumlnumSalt or pertussis

was Y ‑ 0.864X + 0.259 with a correlation coeWICient ofO.67 1

component; and 3) methods for titration of the serum.antitoxin･(P ‑ 0.0044).lmmunization of3 Weeks was suggested to giveThe results of the assay to measure antitoxin inductlOn mightslightly higher potency values than that of 2 weeks. However,be affected by the species or strains of immunizing animals
and by an adjuvant effect of the vaccine constituents. It would

this dilrerence did not reach the level of statistical significance.

The results of the modified Kendrick test obtained by thealso be important to use a method fb∫ semm antitoxin titra‑

Japanese National Control Laboratory were highly consistenttion that can co汀eCtly detect the toxin一meutralizing antibody

with those of manufacturers (unpublished).

(66).

There is currently a trend to simplifypotency tests.S? as toA method f♭r sequential sampling lnSpeCtion has been

to avoid approvlng a Vaccine with a potency ofbelow 4.0

confimlthe potency ofa product only to be above the mlnlmallyrequired level. Regarding the safety of the vaccination,

units/ml. All Japanese acellular vaccines from six manufac‑

however, it is also necessary to establish that the potency lS

turers, including T‑type and B‑type (14), were shown to havenot so high as to cause?V?r‑immunizations (67,68)･ Accurate

similar potencies by the modi丘ed Ken血ick test (40) and havequantifying of potency lS Important in this regard･

applied丘)r intelpretlng the results of the test since 1 98 1 so as

been proved clinically effective (33,38,4 1‑44,53,54).

Guinea pigs have been used fわr the immunization because

The ability of the ic potency test to quantitatively predict

their immune response to the toxoid antlgenS appears tO be

the clinical protective potency of acellular pertussis vaccines

similar to that ofhumans (69). Many works have been pub‑has not beenfully established. It is necessary for establishinglished regarding mouse models for testlng the potency ofa quantitative clinical relevance as fb∫ whole cell vaccine totoxoid vacc.ines (70･71,73)･ Care must be taken in selecting

carry out a clinical trial similar to the MRC trial uslng Vaccines

the叩prOpnate mouse Strains, since the immune response towith widely varied levels of protective potency, lnCluding

toxoid antigens may differ from that of guinea pi.gs (72)･
4‑2‑I. Effect of whole cell pertussis vacclne On thethose with very low potency. For ethical reasons, however, it
is not possible to conduct such clinical trials. The ic challenge

potency test of tetanus or diphtheria toxoid

model is assumed worthwhile to be further explored into its

The adjuvant effect of constituents in combined vaccines

mechanisms of protection for its valuable facilitation as thewould affect the potency of toxoids. The pertussis component

in DTwP vaccines shows a slgnificant adjuvant effect on the

only available potency test method.

4‑1‑2･ Aerosol and intranasaI (respiratory) challenge

toxoid jmmunizations both in animals and humans (68). ln

the case of tetanus toxoid in DTwP vaccines, Murata et al.

modelsln models of resplratOry infection, the protective efrlCaCy(74) proved the e氏cacy of the potency test in predicting clinicalof a vaccine is evaluated only with respect to the bacterial

immunogenicity by showlng a Clear co汀elation between theclearance from lungs, rather than in terms ofprotectionfrom

amount of immunized intemational units (IU) of tetanus

mortality, since mice generally survive the challenge eventoxoid and the neutralizing antibody titers in the sera of infants

after receivlng much higher doses of pertussis bacteria than(Fig･ 2)･ They ㌣sed the potency values of the tetanus toxoid

in the ic challenge model. One particular advantage of the
resplratOry infection model over the ic challenge model is thatin DTwP vacclneS measured agalnSt a plain preparation of
National Standard Tetanus Toxoid based upon the report on

the susceptibilityof mice is not limited to particular challenge

parallelism of the doseィesponse regressions of the tetanus

strains ofB. pertussis. As is the case in the ic challenge model,

toxoids in plain and DTwP vaccines (75, 76)･ Kondo et al･

it was reported that anti‑PT or PT alone was able to confer(67) proposed a simple method fわr estimating the potency orprotection in the aerosol or intranasal challenge model (55,56).

tetanus toxoid in DTwP vaccines uslng the results measured

Celトmediated immunlty has been reported to play a cmcial

agalnSt the plain standard in accordance with the parallelism

role also in bacterial clearance from the lungs of infected miceof the dose‑response regressions. The standardization of tetanus(46,57). Nonspecific cellular immunity has also been noted
to play a role (58). A possible difference of the aerosol or

toxoid in DTwP vaccines could proceedusing the data depicted

in Fig. 2 and the method ofKondo et al. (67), irlyophilized171

not only the immunogeniclty Of the toxoid antlgen but also

the efrlCaCy Ofthe aluminum adjuvant on the toxoid (72,80)･
SIUeJu! JOt!1uXOlpue OM

This is why it has been di氏cult to consider the immunochemi‑

cally assayed antlgen COntentS, Such as the value of limes

flocculation (LF), as representing immunogenicity. The
difficulty of consistently controlling toxoiding processes intum makes it difficult to estimate the potency of丘nal products

uslng the potency values of bulk materials measured before

blending with adjuvant gel because of the influence of■linconsistent characteristics of the resulting toxoid moleculeson the efficacy ofadjuvant (81). Although it may be possible
to directly evaluate the structural validityof the toxin neutraliz‑
ipg epitope (82), the results will not be sulrlCient for quantita‑
10

tively predicting the adjuvant effect on the toxoid immuniza‑

100

tion.

Total immunizing dose of tetanus toxoid (lU)

4‑2‑4. Establishing the clinical relevance of toxoid
Fig, 2. Relationship bctwecnthe amount ofimmunizcd tetanus toxoid

potency tests

in lU and the scrum antitoxin in infants. The clinical rclcvancc ofthc

The cu汀ent potency tests evaluate the overall immun0‑

potcncy test results was proved for tetanus toxoid in DTwP vaccinc･

geniclty Of toxoid vaccines and would be useful to predictclinical efficacy if a correlation between the potency test

(ref.74)

results and clinical immunogeniclty COuld be demonstrated,combined toxoids were available. Although no such data haveas.in the case of tetanus toxoid in DTwP vaccines (74)･ 1n
splte Of a possible difference in adjuvant effects between

been reported for diphtheria toxoid, it would also seem worth‑aluminum salt and whole cell pertussis vaccine (79), there

reference preparations of DTwP and plain diphtheria tetanus

while to explore the standardization or diphtheria toxoid inseems to be a possibility of correlating the potency estimates

DTwP vaccines in this regard.
4‑2‑2. Effect ofadjuvant gel and animal model on theand clinical immunogenlClty Of adsorbed toxoid vaccines.
Because a clear co汀elation between the potency of tetanus

toxoid and the clinical immunogenicityofDTwP vaccine wastoxoid potency test

Controlling for the effects of the adjuvant gel would

proved ever uslng the tetanus toxoids prepared by various

requlre an additional standardization strategy. The adjuvant

procedures Including those suggested to affect differently theeffect may differ in different animal species or strains (72,77)･
Simultaneous dilutions of aluminum a4juvant and toxoid
antlgenS would affect the slope of the dose‑response regressions

characteristics of toxoid molecule (74). In addition, thein the potency assays and, consequently, would influence the(68)･ Further investigation will be nPcessary, howeyer, fordifference in adjuvant contents was shown to have little effecton the immunogenicity both in guinea pigs (79) and humans

relative potency values (78)･ Factors possibly influencing theidentifying approprlate animal strains and immunlZation

slope need to be carefully validated so as to minimlZe theprocedures ‑ including that fわr making graded doses ‑ So as

deviation of the potency test results from those reflectlng theto maximally renect the clinical immunization procedure･ A

outcomes of clinical immunization. Although aluminum
adjuvant also has a slgnificant effect on the clinical immuni‑
zations, differences in its content, so far as within a practicalrange, no slgnificant difference was detected in the clinicalpotency test involving Immunization with serially varied
volumes of undiluted adsorbed vaccine would seem to be

immunogeniclty Or adsorbed diphtheria and tetanus toxoidsof the possible difference between the effects of dilutions ofprobably due to a wide variation in the sensitivity ofhumans

antlgen and adjuvant on the immunization. It would also be(68)･ AluminuP salt showed a remarkable adjuvant effect onimportant to evaluate boostlng ln a potency test, Since boostlng

the immunlZatlOn Of tetanus toxoid in mice and gulnea plgSis used clinically and has been shown to result in antitoxin(66,79). Guinea pigs were shown to respond to an almost

levels in gulnea plgS that are highly similar to those in humans

similar extent to adsorbed tetanus toxoids having markedly

and that differ considerablyfrom those obtained with a slngle

different contents of aluminum adjuvant gel (79). ln adose (66,79).

worth investlgatlng aS a possible altemative to the cumnt
immunization uslng Serial dilutions of the vaccine because

4‑2‑5. Other problems

comparison of the adjuvant effects of aluminum hydroxide

The immunogenicityof toxoid might be affected by otherantigens･ such as Haem.ophilus injluenzae.type b (Hib)and calcium phosphate on tetanus toxoid, gulnea plgS Showeda much more human‑like response to aluminum hydroxide

and calciumphosphate than did mice (66). On thi other hand,polysaccharide. Conjugation Or Simultaneous ImmunizationofHib polysaccharide and tetanustoxoid was reported to show

the response of gulnea Pigs tO both these adjuvants on diph‑theria toxoid was more similar to that of mice than to that ofprofound effects on the immummogeniclty Of both antlgenS in
mice without affectlng the immunogenicities of diphtheria

humans. However, the difference Bomthe response ofhumans

and pertussis components (83,84)･ However, n? such immuno‑

became less evident in gulnea PlgS after a booster immuniza‑

potentiation †as noted in infants (85) or gulnea Pigs (86)･

tion (66)･ When introducing mワuse models, it is of primary

lmpOrtanCe tO Select the appropnate test conditions and mouse

Again, selectlng apprOprlate Strains would be very lmpOrtantstrains having a responsiveness to adsorbed toxoid vaccines

for the potency test in mice. Meaningful potency estimationfor Hib‑conjugated tetanus toxoid in DTwP‑Hib vaccines insimilar to that ofgulnea plgS.

4‑2‑3･ Effect of the toxoiding process on the immun0‑

mice seems to be possible only on血e vaccine befわre or wi血out

genicity and efrlCaCy Of adjuvant gel
The process of toxoiding affects the molecular characteris‑

conjugation. However, the dose‑response curve of tetanus
toxoid in DTwP vaccine containlng the conjugate of Hib‑

tics of the resulting toxoids and has been reported to influence

Specific polysaccharide or oligosaccharide did not seem to172

即 50 仰 幻 20 1 0

deviate slgnificantlyfrom parallelism with that of the control

DTwP vaccine in mice of the CBA strain irrespective of theincreased extent of antitoxin production (83)･ If this is true,

estlmation of the potency of tetanus toxoid in DTwP‑Hib
conjugate vaccines might be possible by the method ofKondo
et all (67) using a reference preparation ofDTwP vaPcine andthe data orpotency measurements on DTwP vacclneS Withand without the Hib‑conjugate.
412‑6. Antitoxin titration

ゝ3uanb巴山

With regard to etrlCaCy, it is important to evaluate the ability
oftoxoids to induce toxin一meutralizing antibody. ln vivo toxin
0.5

1.0

challenge methods have been loslng Support for reasons of

1.5

2.0

Log‑BWDU/mI

animal welfare. ln vitro assay methods have been developed

for measurlng the serum antitoxin titer. Passive hemagglutina‑

tion (87), enzyme linked immunosorbent assay (EuSA) (88),Fig. 3. Mouse body weight dccrcasing (BWD) toxicity ofJapancsc
DTwP and DTaP vaccines. The average BWD toxicityofthc DTaP

toxin binding inhibition (ToBI) (89) and passive particle

vaccines was rcduccd to approximately I /1 0 0fthat ofDTwP vaccines.

agglutination (90,9 1 ) assa.ys are the methods based on antigen‑antibody binding. The mlCrO Vero cell culture method (92) isan in vitro toxin neutralizing assay method. Because it is(95). This had been initially achieved by the BWD toxicity

essential that the potency test be capable or evaluatlng thetest (21 )･Asreptesented in Fig･ 3, the avera.ge BWD toxicity

production or toxin‑neutralizing antibodies, every ln Vitro
assay method based on binding with the toxin molecule needs

that ofwhole cell vaccines. The BWD toxiclty test is capable

to be vahdated carefully before utilization. Generally, suchof detectlng the toxiclty Of endotoxin to mouse weight galn

validation has been made by provlng a correlation with an induring the 1 6 h after injection but is possibly affected by othervivo toxin‑neutralizing assay. The immunizlng efGcacy of
toxoid would be affected by various uncontrollable factors in
the toxoiding process to glVerise to final products ofvarylng

constituents, such as aluminum adjuvant gel and active PT

toxicitytest with some other test method, particularly for DTaP

potency (80)･ The mi.cro Vero cell culture method is highly

vaccines (1 06).

of acellular vacclneS Was reduced to approxlmately one‑tenth(106). We should therefわre consider replacing the BWD

sensitive and approprlate in this regard. The TbBl assay may

4‑3̲1̲1. The bacterial endotoxin test: The endotoxin

also become a promlSlng binding assay in this context, throughcontent of Japanese DTaP vaccines can also be measured by
the endotoxin test method (22) usiTg Limulus amaebocyte

the use of a monoclonal antibody recognlZlng the epltOpe Of
toxic action f♭r sensitizing micro‑wells of plates. ELISA and

Lyzate (LAL) reagents whose reactlVity toト3‑β‑D‑glucan

passive particle agglutination assays may also becomehas been removed or suppressed (96‑100) to avoid aヮypromlSlng direct binding assays by using SPeCific peptides

possible confusion due to reaction with the non‑pyrogenlC

exhibiting the responsible structure for the toxic action of
toxin molecules.

substance. However, a special precaution has been shown tobe necessary when applying the endotoxin test to DTaP

4‑3. Toxicity

vaccines. In an enhancement/suppression experiment to detect

The toxicityofDTwP vaccines has been controlled as overall
toxicity by means of the mouse body weight gain (MWG)

endotoxin added to DTaP vaccines, some commercial DTaP
vaccine batches slgnificantly suppressed the endotoxin test,

test, in which the toxiclty Ofa vaccine is evaluated by theas shown in Table 2 (the amount ofendotoxin detected is

toxiclty tO Weight galn Ofmice over the first 7 days post‑

represented as a percent of the added endotoxin). ln spite ofvaccination. Even though the MWG test has a long history ofsafetycontrol for the pertussis vaccine, it has been discredited
by reports that PT toxicityaccelerates the weight gain of mice,
thereby improving the results for toxic vaccines (1061108).

Table 2. Interfering cffcct ofDTaP on LAL*
activity of endotoxin

It is therefわre considered important to evaluate each of the

DTaP Lot

toxicities separately (1 6, 1 9) for meaningful toxicity tests.
Toxicitytestsfor vaccines generally need to be implemented% dctcctcd

AaCDEFGHI∫KLM

1 1 一l 1 1 1 1 4 ‑ ‑ 2 2 2 ′LU つJ tJ つJ一1 2

0 5 8 6 0 6 L 5 5 8 L 5 8 7 0ノ 0ノ 7 ′b 4 ′b つJ /b 7 2 0 4

for all measurable toxic activities unless proven to have noclinical relevance. It is difrlCult to prove the climical relevance
*

* *

*

*

*

or a biological activity, particularly without its quantitative

assay results (94). Kurokawa et al. analyzed bacterial factorsof pertussis vaccine influencing the MWG test and reported
that endotoxin, heat‑labile dermonecrotic toxin (HLT) and PT

were the major moieties affecting the test at 1, 2 and from 3days a洗er injection, respectively (16,I 9,2 1 ). They developed
quantitative assay methods f♭r these activities and the methods

were later implemented for the quality control of DTaP
vaccines in the Minimum RequlrementS Of Biological Products

oHapan (Minimum Requirements) in 1981 (19,22).
LO EU/ml ofcndotoxin spiked to the DTaP

4‑3‑1. Endotoxim

vaccine lots was dctcctcd by the cndotoxin test

One of the characteristics of Japanese acellular pertussis

to cxprcss as pcrccnt ofthc spiked cndotoxin.

vaccine is that it results in a slgnificantly reduced febrile

辛 LAL: Limulus amacbocytc Lyzatc

reaction rate, as low as l% for a febrile reaction over 37.5oC** Significantly suppressed173

Vaccine and a clinical adverse effectwithout quantitative assay
results (94)･ Qualitative observations in F clinical study may

25

tend to be biased depending on血e obseⅣlng perSO‑el° quan‑
>

O
⊂
0

20

titative data such as temperature in a febrile reaction can be
more reliable and are useful for analyzlng the relationship

旨15with the quantitative laboratory assay results of biologlCal

4)
L

activities. We believe that the quantitative quality control ofLL

10final products would be meaningful in this regard.

4‑3‑I‑3. Necessary cautions for substituting a test5

method: Introduction of a new test method, particularly of a
toxiclty test, requlreS Careful validation. ln the case of the

0
‑2.0

‑1.5

‑1.0

‑0.5

0.0

0.5

1.0

1.5

2.0

2.5

endotoxin test, at very least a limit value fわr approval or

Log‑EU/ml

rejection should be implemented so as not to approve a productthat should have been rejected in the pyrogen test because

Fig. 4. Endotoxin content orJapancsc DTaP vaccines. The cndotoxin

contcnt showed a divcrsc distribution. The DTaP vaccines ofthc carly

safety of such products has not been proved clinically. The

1 980s contaimcd considcrablc amounts or cndotoxin, but thcsc wcrc

results of the different assays will inevitably vary. Thus the

reduccd to a lcvcl orO.1 EU/ml in more rcccnt batchcs.

new limit should be defined so as not to falsely approve apyrogenic product even when its endotoxin test result showsa maximal variation. From the polnt Of view of statistics, thethe strong suppression of endotoxin test, endotoxin added toextent of variation or accuracy or an assay result would

vaccine batches I), ∫ and M was fわund to have retained its

depend on the test design, lnCluding the number ordoses andbiologlCal activlty aS measured by the pyrogen test, thetheir intervals. Use of a smaller number or doses or intervals

lethality of D‑galactosamine‑treated mice and the method ofwill produce less accurate results. Consequently, the limit for

TNF‑ a induction in a mouse monocytlC Cell line, RAW264.7

rejection or acceptance should not be defined according tocells (109)･ Caution is thus suggested when applying thethe ordinal significance‑of‑difference bases to avoid a betterchance ofapproval due to a less accurate measurement. hstead,endotoxin test to DTaP vaccines.
Japanese DTaP vaccines show a diverse distribution ofendotoxin content, as shown in Fig. 4. A rather high level ofendotoxin was detected in the DTaP vaccine batches of thethe limit should be defined according to the allowable varia‑
tion of results under a defined accuracy, therefore a defined

design, of a test.

early 1980S, but in more recent batches the level declined

In addition, special care should be taken when substitutlng

remarkably to below 0. 1 EU/ml.

an in vitro test f♭r an in vivo test. An in vitro test generally

4‑311‑2. clinical relevance of the endotoxin test: Thecannot detect a possible in vivo synerglStic e飴ct of vaccine

quantitatively estimated endotoxin contents of DTaP vaccinesconstituents on the target toxic activity･ It mTy require a

could be utilized for analyzlng the relationship with febrile

preliminary quantitative evaluatlOn Of the synerglSm tO define

reaction rate ofvaccinees, as shown in Fig. 5. Here, the probit

the limit value not only f♭r regulating the amount or toxic

transformed rate of vaccinees showlng a febrile reaction ofsubstance but also to regulate the extent of the resulting toxic

37.50C or more is shown vertically and the endotoxin content

effect. The prescribed limits of endotoxin contamination inin vaccine batches is shown horizontally. The data summarizedinterferon Injections, human albumin and purified plasmain Fig･ 5 could be utilized for estimatlng a VaCCine's febrileprotein fraction lnJ eCtionsinthe Minimum RequlrementS Werereaction rate, provided its endotoxin content was available.

defined by taking the synerglStic effect and variation of theClinical studies have become increaslngly difficult to conduct.

test results into consideration so as not to allow the approval

And even in cases where clinical data is available, it is notof possibly pyrogenic products (22)･ In the case of endotoxin

possible to identify a correlation between a toxic activlty Of

in DTaP vacclneS, nO SynergyStic effect has been detected sofar. Therefore, it would seem reasonable to set the limit forendotoxin so as not to allow approval of a batch that has notbeen emplnCally shown to be safe ‑ i.e., one that may contain

excess endotoxin than previous batches.

4‑3‑2. LP activity
Leukocytosis or lymphocytosis is a characteristic symptomof whooping cough patients and PT isknown as the responsible
agent. Leukocytosis can be induced in mice by whole cell

pertussis vaccine or PT, and a method for quantitative measure‑ment of the actiyitywas described by Kurokaw.a pt al･ (101)･
The assay was lmPlemented for testing the toxIClty Of DTaP

vaccine in Minimum RequlrementS in 1 98 1. The activlty Can
be assayed quantitatively ln mice by comparlng peripheralleukocytosis induced by a vaccine batch with that induced

10by the serial dilutions of the reference vaccine on the third

EU/ml

day of injection. Figure 6 represents accumulated results of

the assay on commercial DTaP and DTwP vaccines. The LP

Fig. 5. Relationship of cndotoxin content and the rate of febrilercsponsc in vaccinecs. A significant comlatiom was dcmonstratcd

activity of the acellular vaccines was also shown to have been

bctwccn cndotoxin content in DTaP vaccine batches and the probit‑transformed rate of febrile rcsponsc of 37.50C or more in the

reduced to approximately one‑tenth that of whole cell vaccines.

The LP activities ormost of the DTaP vaccines of the early
1980s were over 0.25 LPU/ml, but these values declined to avaccinccs･ 1¶c equation could be utilized f♭r cstimatlng the rate orthc

fcbrile rcsponsc by a vaccine batch with known endotoxin content.

17454320 0 0 0⊂=コAceIIular

E国Whole ceJl
40 30 20

辛 :Selq軌吋rjmit of herectal temperature method

ゝ3uanbeJj

ゝ3UOnba丘
HSD如

1

‑1.0

‑0.5

0.0

0.5

Log‑LPU/ml
Fig. 6. Lymphocytosis (or lcukocytosis)‑promoting (LP) activity or‑1.5

Japancsc DTwP and DTaP vaccines. The LP activlty Ofthc DTaP
vaccincs was reduced to 1/10 that orDTwP vaccines.

‑1.0

‑0.5

0.0

0.5

1.0

Log‑HSU/ml
Fig. 7. Histamine sensitizing (HS) activityofJapancse DTaP and DTwP
vaccincs as mcasurcd on the fourth day of scnsitization. The scnsi‑

level below or equal to the sensitivlty limit. Accordingly, lt

tivitics orassay methods fわr HS activlty are Shown fわr purpose orcomparison. The rcctaltcmperaturc method could adcquatcly cvaluatc

would be regarded possible currently to simplify the test

the HS activityofJapancsc DTaP vaccinesthat showed no histamine

adequately, Instead of carrylng Out a quantitative measurement,

to prove only that the activlty Of a batch of DTaP vaccine

scnsitizing death of mice. The HS activlty OfDTaP vaccines wasshown to bc reduced to less than I/10 the average leveI for DTwP

does not significantly exceed the sensitivity limit under a valid

VaCClneS.

test condition. Fur仙emore, it would be assumed possible todiscontinue the test if LP activity was proved to be more labile

Rectar terTIPOraturO method HSD method

to the treatment of detoxification than HS activity under any
possible variation of the detoxifying process.

4‑3‑3. HS activity
As we attempted to develop acellular vaccines with average
toxicities of less than one tenth of the average of whole cell

vaccines, we faced a serious difrlCulty. Particularly for HS
toxicity, the traditional assay method us中g HSD was found

not adequate due to its insufficient sensltlVity and accuracy

even in mice of the HSFS/N line of the N:NIH(SW) strain,
which are suggested to be the most sensitive to HS activlty(102)･ A highly sensitive and accurate assay method for HS
activity in which rectal temperature change lS measured a洗er

1
1

histamine challenge on the fourthday of vaccine Injection was

2

3

4

2

3

4

5

5

Experirnent

Expe ri me nt

developed by lshida et al. in 1979 (2 I). The rectal temperature

method was sensi.tive enough for quantifying even the activity

Fig. 8. Comparison of the accuracy of the rectal temperature method tothat of the histamine‑sensitizing dcadl (HSD) method. A DTaP vaccineof vaccines causlng nO HSD. Figure 7 shows the frequency
distribution of HS activlty OfDTaP and DTwP vaccines. The
sensitivity limit (HSDIO) and the median sensitizing dosebatch showing a considerable level of HS activitywas assayed fivetimcs. The estimated HS activityand its 95% confidence interval
obtained in each measurement arc indicated by the closed box andattached vertical bar, respectively. Both methods were proven to be(HSD50) forthe method ofHSD and the sensitivity limit for

highly reproducible. The rectal temperature method was shown to

the rectal temperature method are also shown in the figure

be more accurate thanthe HSD method and was suggested to requlrC

together with the current acceptance/rejection limit for DTaPa smaller numb(汀Oranimals.

vaccines. The regulation limit fわr HS activlty lmplemented
in 1981 was 0.8 HSU/ml, and was revised to the cu汀ent 0.4

HSU/ml in 1991. The average HS activlty OfDTaP vaccines

In the mid‑1980S, it was discovered that the chemicallywas shown to be less than one tenth of the average ofwhole

detoxified PT in DTaP vaccines may r?vert to toxicity during

cell vaccines.

long storage, and the Minimum RequlrementS Were revised

A commercial batch or DTaP vaccine with considerable
HS activlty Was assayed repeatedly five times by the rectal

accordingly in 1991 to implement the relevant toxicity test,
namely, the HS test f♭r the incubated preparation in which atemperature method and by the method of sensitizlng death.

vaccine is tested after incubating at 37oC for 4 weeks.lmple‑

Although both methods were shown to be highly reproducible,
the rectal temperature method was found to have a slgnificantly

mentation of the new regulation had a considerable effect on
the residual HS activity Of DTaP vaccines. The level of thebetter accuracy than the sensitizlng death method, as seen in

residual activlty declined remarkably after 1990 Without

Fig･ 8･ This finding emphasizes the advantage of the measure一

slgnificant change in potency, as measured by the modified

meれt on continuous variables, such as rectal temperature,

Kendrick test. Furthermore, no slgnificant change in the clinicalrather than discrete variables, such as survival rate, which gives

efficacy ofDTaP vaccines was noted, as seen in Fig. 1. Thismight suggest that the modified Kendrick test could beused

only one data uslng Several animals. Consequently, the rectal
temperature method was expected to glVe more accurate

to evaluate the effects of a modification of the production

results uslng a Smaller number or animals.

process, if any, on the potency of acellular pertussis vaccines.

175

Table 3. Comparison oftoxicitytests for detecting residual
pcrtussis toxin activity

4.5

CHO celrclustcrization HS activity
4.4
Formalin'
trcatmemt

4.3

fわr

13.7

4･2

a:
4.1

4.0

dose

clustcrizlngRelative

Relative

activity

activity

pg/ml

1.000

0.1%

58

mg/ml

0.000236

0.164

0.3%

58

mg/ml

0.000236

0.073

0.8%

530 mg/m1=ヒ8Minimun0.000026

1.000

0.064

*:Treated at 37oC fわr 24 b** : CHO cell: Chincsc hamster ovary cell
3.9

3.8

0.01

0.1

1

HSU/mland according efficacy limitations into consideration.

10

1

HSU/ml

5. Conclusion

Fig. 9. Correlation bctwccn the occurrence rate of crythema and thehistaminc seTsitizing (HS) activities of4 day‑ and 1 1 day一号cnsitiza‑

Acellular pertussis vaccines or various fわrmulations aretions. HS actlVity was shown to be a cause of the local rcact10m at thepnmary dose of immunization. The rate of occurrcncc oferythcma

currently available. A clinically relevant method or testlng

ofovcr 101mm diameter was well corrclatcd to both HS activities
measurcd on the fourth and eleventhdays of sensitization.

brands can be used interchangeably in the course of primary

their potency lS required in order that vaccines of different

and booster irrmunizations. A clinical study similar to the MRC
trial on vaccines with widely different potencies will be needed

Iwasa et al･ (103) described a nPvel HS activity ofa DTaP

to establish the quantitative clinical relevance of potency‑test‑

vaccine showing a Peak sensitizatlOn in mice after more thanlng methods such as the Kendrick test. Cumntly, however,

10 days of injection (late‑appearipg HS lLHS] activity)I
Commercial DTaP vaccines were Incubated at 37oC for 4

such clinical trials are not possible. Because the Kendrick
test is the only method with proven clinical relevance for testing

weeks and their HS activitywas measured on the fourth andwhole cell vaccines, it will be of particular importance to

1 1 th days of sensitization and compared with that of vaccines

analyze the mechanism of the Kendrick test to open the way

kept at 4oC. The incubation was shown to markedly increase

for its application to acellular pertussis vaccines. The role of

the LHS activity, While no effect could be detected for theHS activlty measured on the fourth day of sensitization.
The clinical relevance or HS activlty Was also analyzed
using the accumulated data of the activityofcommercial DTaP

PT ill both the Kendrick testand clinical protection and the factthat the current intra‑muscular irrmunization is not appropnate

for theinduction oflocal irrmunityor secretory immunogulObulin
would suggest the efrlCaCy Ofthe Kendrick test.

vaccines. Both HS activities measured on the fourth and I lth

Eradication of tetanusis desperately hoped for and diphtheria

days of sensitization showed a slgni丘cant co汀elation with thewould once again become epidemic if effective immuniza‑rate of erythema of over 10 mm in diameter among pnmary‑tion was lost (67,93). In addition, because safetyis a concem

dose vaccinees, as shown in Fig. 9. A better co汀elation was

due to the potential fわr over‑immunization, the potency ofseen with L出S activity than with the activity of ordinal fbur‑

toxoid vaccines needs to be precisely controlled･ And although

day sensitization. Although the mechanism of the delayed
HS by DTaP vaccines has not yet been clarified, these findingssuch control presents many difrlCulties, there also seem to be

viable solutions, as discussed in this paper.

might suggest that fわur‑day sensitization is not adequate f♭r

Toxicity tests for ya?cines need to be implemented for every

detecting.residual HS activityof the DTaP vaccines contain‑

detectable toxic actlVlty, With the exceptlOn Of those shown

lng Chemically detoxified PT.

to have no clinical relevance. The efrlCaCy Of every toxIClty

4‑3‑4･ Chinese hamster ovary (CHO) cell clustering

test also needs to be carefully validated through a consider‑ation of its possible mechanisms. It is of particular importancetest

Hewlett et al. (104) described the clustering ofCHO cells
by PT in 1 983. Attempts have been made to apply the method

before introducing an in vitro test method. In addition, before

for evaluatlng the residual PT toxIClty ln aCellular pertussis

replacing a traditional test with a new testing method, it isto ensure a definite correlation with the relevant in vivo test

vaccines (52,105). The assay method was prescribed in the

crucial to define the limit for acceptance/rejection so as not

WHO Guidelines in 1998 as a sensitive technique f♭r evaluat‑

to approve a product that should have been rejected in thelng the detoxification of PT. However, the assay seemed

traditional test. Quantitative test results are essential for

inadequate for evaluatlng the safety of acellular pertussis

establishing clinical relevance. However, tests could be simpli一

vaccines containlng Chemically detoxi丘ed PT. The CHO‑cell凸ed approprlately to prove only not slgnificantly exceedingclusterlng aCtivlty Of PT fわund decrease to 1/4,000 0r lessthe least detectable level ira toxicity of products was reduced

even by mild treatment with 0.1% orfbmalin at 37oC f♭r 24

to below the detectable limit.

h, but to retain a considerable level of its in vivo HS activlty(Table 3)･ Therefore, in spite of its excellent sensitivity, the

REFERENCES

CHO cell assay was deemed inadequate for evaluatlng thedetoxification of in vivo toxIClty Of chemically detoxified PT

1. British Medical Research Council (1956): Vaccinatiopin acellular pertussis vaccines. However, this method might
beuseful for ensurlng the purity of the culture ofB. pertussis

tion in children and results of laboratory tests. Br. Med.

producing genetically detoxified PT. It is important for thevalidation of a test method to take the possible mechanism

2. KreeRenberg, ∫. G. (1 999): Standardization of acellularagalnSt Whooplng COugh: Co汀elation between protec‑

J., 2, 454‑462.

176

pertussis vaccine. Jpn. J. Med. Sci. Bio1., 37, 41181.
20･ Horiuchi, Y･ and lida, T･ (1988): Determination of thepertussis vaccines. BiologlCals, 27, 1 15‑1 17.

3l Arcinlega, J. L., Corbel, M., Delleplane, N., Dobbelaer,R., Griffiths, E., Heron, I., Ivanoff, B., KreeRenberg,
H., Mastrantonio, P., Meade, B., Milstein, I., Robertson,
S., Robinson, A., Sato, H., Sato, Y., Schwanig, M. andeplnePhrine‑refractory hypoglycaemic actlVlty Of wholecell pertussis vaccine in mice. J. Biol. Stand., 16, 239‑
248.

Tiru, M. (1998): WHO guidelines fわr the production

21. Ishida, S., Kurokawa, M., Asakawa, S. and lwasa, S.

and control of the acellular pertussis component of(1979): A sensitive. assay method for the histamine‑monovalent or combined vaccines. BiologlCals, 26, 1 95‑

sensltlZlng factor uslng Change in rectal temperature of204.

mice after histamine challenge as a response. J. Biol.

4･ Kanai, K･ (1980): Japan's experience in pertussis epide‑

Stand., 7, 2ト29.

miology and vaccination in the past thirtyyears. Jpn. J.

221 Ministry ofHealth and Welfare, Japanese Govemment(1 986): The Minimum Requirements or Biological Pro‑

Med. S°i. 丘iol., 33, 107‑143.

5･Kimura, M･and Kuno‑Sakai, H. (1987): Experiences withducts of Japan 1986. Ministry of Health and We脆re,acellular pertussis vaccine in Japan and epidemiology

Tokyo.

ofpertussis. Tokai J. Exp. Clin. Med., 12, 2631273.

23･ Cohen, H. H. (1963): Development ofpemlSSis vaccine

6. Sato, Y and Nagase, K. (1965): Studies on protective

production and control in the National lnst血te ofPublicantlgen Of Bordetella pertussis: isolation of protective

Health in the Netherlands during the years 1950‑ 1 962.

Antonie van Leeuwenhock, 29, 183‑201.

antlgen by sucrose density gradient centrifugation and

24. Pittman, M. (1975): Determination of the histaminesome properties of the antlgen. Jpn. ∫. Med. S°i. Bio1.,

18, 173‑174.

sensitizlng unitage ofpertussis vaccine. J. Biol. Stand.,

7･ Sato, Y･ and Nagase, K｣. (1967): Isolation of protective

3, 185‑191.

25. Sato, Y.,Kimura, M. and Fukumi, H. (1984): Develop‑

antlgen from Boydetella pertussis. Biochem. Biophys.

Res. Commun., 27, 195‑201.

ment of a pertussis component vaccine in Japan. Lancet,

8･ Keogh, E･ V･ and North, E･ A. (1948)･･ The haem*glutinin

1, 122‑126.

of Haemophilus pertussis. I. Haemagglutlnin as a

26･ Arciniega, J･ L･(2000):What's in a name? The mea.ning

protective antigen in experimental murine pertussis. Aust.

of the ten "potency", as applied to immunologlCals

J. Exptl. Biol. Med. Sci., 26, 315‑322.

for humanuse, and its implications for altematives andvalidation. p. 979‑986. In Balls, M., van Zeller, A. M.
and Hadler, M. E. (eds.), Progress in the Reduction,

9. Niwa, M., Hiramatsu, T., Kawasaki, N. and Kuwajlma, Y.
( 1 965): On the relationship between histamine sensitiz‑lng factor and mouse protective antigen. Jpn. J. Med.

Refinement and Replacement ofAnimal Experimenta‑
tion. EIsevier Science B. V.

Sci. Bio1., 18, 175.
10. Pieroni, R. E., Broderick, E. J.and Levine, L. (1965): The

27. Mills, K. H., Ryan, M., Ryan, E. and Mahon, B. P.

soluble protective antlgen and the histamine‑sensitiz‑(1 998)‥ A mufine model in which protection correlates

lng factor ofBoydetella pertussis. J. 1mmuno1., 95, 643‑

with pertussIS Vaccine efficacy ln Children reveals

650.

complementary roles fわr humoral and celトmediatedimmunityin protection against Boydetella pertussis.

1 1l Sato, Y･, Arai, H･ and Suzuki, K･ (1974): Leukocytos享sI

Infect. Immun., 66, 594‑602.

promoting factor ofBordetella pertussis IIl. Its identitywith protective antlgen. Infect. Immun., 9, 801‑810.

28. Robinson, A., Ashwor也, L A., BaskeⅣille, A. and Irons,

12･ Nakase, Y., Doi, M.and Kasuga, T. (1975): Hemaggluti‑

L. I. (1985): Protection against intranasal infection of

natlng Substance ofBoydetella pertussis. Symp. Toxins,

mice with Boydetella pertussis. Dev. Biol. Stand., 6 I ,165‑172.

28, 57‑59.

29･ Lymg, J･ and Heron, 1･ (1991): Quantitative estimation of

13. Chazono, M., Yoshida, Ⅰ., Konobe, T. and Fukai, K.

diphtheria and tetanus toxoids. 5. ComparatlVe assays(1988): The purification and characterization of anacellular pertussis vaccine. J. Biol. Stand., 16, 83‑89.

in mice and in gulnea‑plgS Of two diphtheria toxoid

14･ Kamiya･ H･ and Nii, R･ (1988): Overv.iew of currently
available Japanese acellular pertussIS Vaccines andpreparations. Biologicals, 4, 327‑334.

30. The Advisory Committee on Immunization Practices

future problems. Tokai J. Exp. Clin. Med., 13 Suppl,(2000) : Use of diphtheria toxoid‑tetanus toxoid‑acellular

45‑49.

pertussis vaccine as a five‑dose series. Supplemental

recommendations of the Advisory Committee on lmmu‑
nization Practices (ACIP). Morb. Mortal. Wkly Rep.,

15. 1wasa, S., Ishida, S., Asakawa, S. and Kurokawa, M.
( 1 968): Lymphocytosis‑promoting factor produced by
BoTldetella pertussis. Partial purification and character‑

49(RR‑13), 1‑8.
31･ Miller, E･ (1999): Overview of recent clinical trials of

ization･ Jpn. ∫. Med. S°i. 丘iol., 21, 363‑368.

16. Ishida, S. (1968): Characterization orthe body weightacellular pertussis vacclneS. BiologlCals, 27, 79‑86.

decreaslng tOXicities in mice by the lymphocytosis‑

32. Sclmitt, H. J., Schuind, A., Knuf, M., Zepp, F., Beutel,

promotlng factor and the heat‑labile toxin ofB. pertzLgSis

K., Wirsing Yon Konig, C. H., Neiss, A., Bock, H.し.,and endotoxin. Jpn. ∫. Med. S°i. 丘iol., 21, 115‑135.

Bogaerts, H. and Clemens, R. (1996): Acellular pertussis

17･ Preston, N･ W･ (1959): Factors influencing the assay of

vaccines: The rationale for an efficacy trial in Germany.

the histamine‑sensltlZlng factor ofHaemophilus pertusIJ. Infect. Dis., 175(Suppl. 3), S287‑290.
33. Heinlnger, U., Cherry, J. D., Stehr, K., Schmitt‑Grohe,sis. J. Pathol. Bacterio1., 78, 217‑224.

18. Ishida, S., Kurokawa, M. andAsakawa, S. (1976): A

S., Uberall, M., Laussucq, S., Eckhardt, T., Meyer, M.

new biologlCal assay method f♭r histamine‑sensitizlng

factor uslng Survival lime as a response. Jpn. J. Med.

and Gombein, J. (1 998): Comparative efficacy of theLederle/Takeda acellular pertussis component DTP

Sci. 丘iol., 29, 139‑150.

(DTaP) vaccine and Lederle whole‑cell component DTP

19. Kurokawa, M. (1984): Toxicity and toxicity testing ofvaccine in Geman children after household exposure.

177

ratory infection model. Infect. lmmun., 61, 399‑410.

Pertussis ⅥlCCine Study Group. Pediatrics, 102, 546‑

47. 1ida, T., Horiuchi, Y. and lshii, K. (1989): Passive protec‑

553.

34･ Schmitt, H･ J･, Wirslng VOn Konig, C. H., Neiss, A.,

tiOn of mice by two antlgenic preparations ofBordetella

Bogaets, H., 占ock, H.し., Schulte‑Wisseman, H., Gahr,

pertussis compnslng mainly pertussis toxin or filamen‑
tous hemagglutinin as measured by two intracerebral

M., Schult, 良., Folkens, J. U., Rauh, W. and Clemens,

R･ (1996): EtrlCaCy Ofacellular pertussis vaccine in earlymouse protection tests. Yokohama Med. Bull., 40, 37‑

childhood after household exposue. JAMA, 275, 37‑49.

48. Manghi, M. A., Delleplane, N. 1., Eriksson, P. V, Di

41.

35. Greco, D., Salmaso, S., Mastrantonio, P., Giuliano, M.,
Tozzi, A. E., Anemona, A., Ciofi degli Atti, M. L,
Giammanco, A., Panel, P., Blackwelder, W. C., Klein,
D. L and Wassilak, S. G. (1996): A controlled trial or

Paola, G･ and De Dios, F･ H･ (1990)‥ Antibody resppnヲe

to pertussis toxin and filamentous haemagglutinln ln

NIH mice immunized with the international standard
for pertussis vaccine. BiologlCals, 18, 165‑172.

two acellular vaccines and one whole‑cell vaccine

49. Robinson, A. and Hawkins, D. C. (1983): StnユCture andagalnSt pertuSSis. Progetto Pertosse Working Group. N.

biologlCal properties of solubilized envelope proteinsof BoTdetella pertussis. Infect. lmmun., 39, 5901598.

Engl. ∫. Med., 334, 341‑348.

36･ Mortimer, E･ A･ Jr･ (1988): Preliminary report on hous9‑

50･ Robinson, A･ and Irons, L l･ (1983): Synergi*c effecthold contact studies of the preventlOn Of pertussis lnof Boydetella pertussis lymphocytosisIPrOmOtlng factor

Japanese children by Takeda diphtheria and tetanus

on protective activities of isolated Bordetella antlgen

toxoids and acellular pertussis vaccine combined. Tbkai

in mice. Infect. Immun., 40, 523‑528.

J. Exp. Clin. Med., 13 Supp1., 29‑34.
37. Isomura, S., Suzuki, S. and Sato, Y (1985): Clinical

5 1. Pillemer, L. (1954): Protective antigen ofHaemophilus

efncacy of the Japanese acellular pertussis vaccine a洗er

52･ Redhead, K･ (1996): Developments in pertus苧is vaccines

intrafamiliar exposure to pertussis patients. Dev. Biol.

leading to reduction and replacement of in VIVO teStlng.

pertussis. Lancet, I, 1257‑1260.

38. 且ato, T., Kaku, H. andArimoto, Y (1988): Protection

Dev. Biol. Stand., 86, 263‑270.
53. Ad Hoe Group for the study ofpertussis vaccines (1988):

agalnSt PertuSSis by Takeda's acellular pertussis vaccine :Placebo‑controlled trial of two acellular pertussis

household contact studies in Kawasaki City, Japan.

vaccines in Sweden‑protective efficacy and adverse

Tbkai J･ Exp. Clin. Med., 13 Suppl, 35‑38.

events. Ad Hoe Group for the Study ofPertussis Vaccines.

Stand., 61, 531‑537.

39･ Tiru, M･, Jaatmaa, E･, Gillenips, P･andAskelof, P･ (1985):[published emtum appears in Lancet, 1(8596), 1238,

Optimization of immunizatlOn SChedule to standardize1988] Lancet, 1(8592), 955‑960.

antibody response in mice to pertussis vaccines. Dev.
Biol. Stand., 61, 469‑475.
40. Sato, Y and Sato, H. (1988): Further characterization

54･ Trollfors, B" Taranger, J., Lagergard, T., Lind, L.,ofJapanese acellular pertussis vaccine prepared in 1 988

or a pertussis‑toxoid vaccine. N. Engl. ∫. Med., 333,

Sundh, V, Zackrisson, G., Lowe, C. U., Blackwelder,
W and Robbins, ∫. 良. (1995): A placebo‑controlled trial

by 6 Japanese manufacturers. Tokai J. Exp. Clin. Med.,1045‑1050.

13 Suppl, 79‑88.

55. Robbins, ∫. a., Pittman, M., Trollfbrs, 良., Lagergard,

41. 01in, P. (1991): The Swedish efficacy trial ofacellular

T. A., Taranger, J. and Sclmeerson, R. (1993): Primum

pertussis vaccines‑update and time for renection. Dev.
Biol. Stand., 73, 33‑36.

non nocere: a pharmacologlCally inert pertussis toxoid
alone should be the next pertussis vaccine. Pediatr.

lnfect. Dis. J., 12, 795‑807.

42. Che汀y, ∫. D., Mortimer, E. A., Hackell, ∫. G. and Scott,56. Shahin, R. D., Witvliet, M. H.andManclark, C. R. (1990):

J. Ⅴ (1991): Clinical trials in the United States andJapan with the Lederle‑Takeda and Takeda acellular

Mechanism of pertussis toxin B oligomer一mediatedpertussis‑diphtheria‑tetFnus (APDT) vaccines･ Theprotection agalnSt Bordetella pertussis resplratOry

Multicenter APDT Vacclne Study Groups. Dev. Biol.

infection. Infect. Immun., 58, 4063‑4068.

57. Redhead, K., Watkins, J., Bamard A. and Mills, K. H.

Stand., 73, 51‑58.

43･ Heinlnger, U･, Stehr, K., Christenson, P. and Cherry, J.
D･ ( 1 999): Evidence of efficacy of the Lederle/Takeda

pertussis respiratory infection in mice is dependent onacellular pertussis component diphtheria and tetanus

induction of cell‑mediated immunlty. Infect.lmmun.,(1993): Effective immunization Tgainst Bordetella61, 3190‑3198.

toxoids and pertussis vaccine but not the Lederle whole‑

58. Torre, D., Speranza, F., Pugliese,A., Perversi, L., Marone,
P. and Ferrario, G. (1996): Effects of recombinant

cell component diphtheria and tetanus toxoids andpertussis vaccine agalnSt Bordetella parapertussis
infection. Chn. Infect. Dis., 28, 6021604.
44l lsomura, S･ (1988): Clinical studies on elrlCaCy and safTetypuri.ne (rm) interleukin‑ 12 and rm interferon‑gammaln mlCe infected with Bofldetellapertussis. J. Infect. Dis.,
174, 786‑789.

oracellular pertussis vaccine. Tbkai ∫. Exp. Clin. Med.,

13 Suppl, 39‑43.

59. Shahin, 良. D., Brennan, M. ∫., Li, Z. M., Meade, 良. D.

45. Wiertz, E. ∫., Walvoort, H. C., Van Loveren, H., Van

and Manclark, C. 良. (1990): Characterization of the

Straaten‑Van De Kappelle, 1., Van Der Gun, J. W. andprotective capaclty and immunogeniclty Or the 69‑kD

Kreeftenberg, J. G. (1990): Acellular and whole cellouter membrane protein ofBordetella pertussis. J. Exp.

pertussis vaccines protect against the lethal effects ofMed., 171, 63‑73.
60. Roberts, M., Cropley, I., Chatfield, S. and Dougan, G.

intracerebral challenge by two different TICell dependent

humoral routes. BiologlCals, 18, 173‑180.

( I 993): Protection of mice against respiratory Bordetella

pertussis infection by intranasal immunization with P. 69

46. Mills, K. H., Bamard, A., Watkins, ∫. and Redhead, 良.

and FHA. Vaccine, 1 1, 866‑872.

( 1 993)‥ Cell‑mediatedl immunityto Bordetella pertussif:

61･ Jones, D･ H., McBride, B. W., Jeffery, H., Ohagan, D.

role ofThl cells in bacterial clearance in a murine respl‑

178

T., Robinson, A. and Farrar, G. H. (1995): Protection ofJapanese).

mice from Boydetella pertussis resplratOry infection

76. Kubota, 良., Wada, E., Yamamoto, A. and Murata, 良.

uslng microencapsulated pertussis fimbriae. Vaccine,(1962): Studies on the combined vac(空e (pertussis‑

diphtheria‑tetanus). ll. 0n the antigenlCity of tetanus

13, 675‑681.

62. Kimura, A., Mountzouros, K. T, Relman, D. A., Falkow,

toxoid. Jpn. ∫. Bacterio1., 17, 184‑188 (in Japanese)i

S･ and Cowell, J･ L･ (1990): Bordetella pertus.sis

77･ Lyng, J･ and Heron, 1･(1991)‥ Quantitative estimation offilamentous hemagglutinin: evaluation as a protectlVe

diphtheria and tetanus toxoids. 5. ComparatlVe assays

antlgen and colonization factor in a mouse resplratOryin mice and in gulnea‑PlgS Of two diphtheria toxoid

infection model. Infect. lmmun., 58, 7‑16.

preparations. BiologlCals, 19, 327‑334･

63. Mooi, 已 良., Jansen, W H., Br皿lngS, H., Gielen, H･, van

78. Lyng, J. and Heron, 1.(1992): Quantitative estimationder Heide, H. G., Walvoort, H. C.and Guinee, P. A. (1992):

of diphtheria and tetanus toxoids. 6. Use of different

Construction and analysis Of Bordetella pertussis

antibody titration methods fわr evaluation of immun0‑

mutants defective in the production offimbriae. Microb.

geniclty ln animals during potency assay of diphtheria

Pathog., 12, 127‑135.

toxoid. BiologlCals, 20, 101‑108.

64. van den Berg, B. M., Beekhuizen, H., Mooi, F･ R･and van

79. Yamamoto, A., Rondo, S., Kameyama, S. and Murata,

Furth, R. ( 1999): Role of antibodies against Bordetella

R. (1 978): Study on adjuvants for human prophylactics.

pertussis virulence factors in adherence ofBoydetella

1. Comparison of efficiencies of different adjuvants at

pertussisand BoTldetella parapertussis to human bronchialvarious stages of immunization with tetanus and

65l Standfast, A･ F･ B. (1958): The comparison between field

80. Knight, P. A. (1986): Are potency tests f♭r tetanus

epithelial cells. Infect.lmmun., 67, 1050‑1055･

diphtheria toxoids. Jpn. ∫. Med. S°i. 丘iol., 3 1, 263‑276･

trials and mouse protection tests agalnSt intranasal andvaccines really necessary? Dev. Biol. Stand･, 64, 39‑

intracerebral challenges with Boydetella pertussis.lmmu‑

45.

81. Holt, L., Bames, J. M., Bousfield, G., Spiller, V. andnology, 2, 135‑143.

66. Aggerbeck, H. and Heron, I. (1996): Potency tests of

Croarke, F･ (1959): A com.pariso.n of the antigenic

diphtheria, tetanus and combined vaccines suggestion

characteristics or mixed antlgenS ln the child and thefor a simplified potency assay. Dev. Biol. Stand･, 86,

gulnea‑plgS. Bull. WHO, 20, 1121‑1132.
82. Knight, P. A. (1996): Opportunities to reduce the use of

297‑301.

animals in the potency and rtoxiclty teStlng Of toxoid

67. Rondo, S., Kameyama, S. and Murata, 氏. (1974):

vaccines. Dev. Biol. Stand., 86, 185‑197.

Standardization and control of toxoid components in

83. Redhead, K., Sesardic, D., Yost, S. E., Attwell, A. M.,

the combined vaccine (DPT). Jpn. ∫. Med. S°i. 丘iol.,27, 315‑319.

Watkins, J., Hoy, C. S., Plumb, ∫. E. and Corbel, M･ J･

68. Someya, S., Mizuhara, H., Murata, R., Kondo, S.,( 1 994): Combination ofDTP and llaemopgihLH'njluenzae
type b conjugate vaccines can affect laboratory evalua‑

Kameyama, S., Kurokawa, M., Yamamoto, M., Kaneko,

Y., Murata, T., Kokubu, Y., Asano, S., Hori, M.,
Matsumura, T., Murata, F. and Horayama, M. (1978):tion or potency and immunogenicity. Biologicals., 22,

Studies on the effectiveness and untoward reaction of

84. Redhead, K., Sesardic, D., Yost, S. E., Attwell, A. M.,

combined diphtheria and tetanus toxoid. Bull. Inst. Publ･

Watkins, J., Hoy, C. S., Plumb, J. E. and Corbel, M. J.

Health, 27, 136‑145 (in Japanese).
69･ Ipsen, J･ (1953): Bio‑assay of four te.tanus.toxoids

conjugate vacclneS With diphtheria‑tetanus‑pertussis339‑345.

( 1 994): Interact.ion ofHaemopgilus injluenzae type b
vaccine in control tests. Vaccine, 12, 1460‑1466.

(aluminium precipitated) in mice, gulnea‑plgS and

85. Granoff, D. M., Anderson, E. L., Osterholm, M. T.,

humans. ∫. Immunol0., 70, 426‑434.

70. Nyerges, G., Virag, E. and Lutter, J･ (1983): The potency
testlng Of diphtheria and tetanus toxoids as detemined

Holmes, S. ∫., McHugh, ∫. E., Belshe, 良. B., Medley, F･and Murphy, T･ V･ (1992): Difference in the immuヮ0‑

genicity of three Haemophilus injluenzaetype b conJu‑

by the induction of antibody in mice. ∫. 丘iol. Stand., 1 1,

99‑103.

gate vaccines in infants. J. Ped., 121, 1871194･

86･ Gupta, R･ K･,Anderson, R･, Cecchini, Rost B･, Griffin,

71. Kreeftenberg, J. G., van der Gun, J., Marsman, F･ R･,
Sekhuis, V M., Bhandari, S. K. and Maheshwari, S. C.

P. Jr., Benscoter, K., Xu, J., Montanez‑0riZ, L and Siber,
G. R. (1996): Development of a guinea‑pig model for(1985): An investigation ofa mouse model to e9mate
the potency of the diphtheria component in vacclneS･ J･

potency/immunogenicityevaluation of diphtheria, tetanusacellular pertussis (DTaP) and Haemophilus injluenzae

Biol. Stand., 13, 229‑234.

72. Lyng, J., Heron, I. and Ljungqvist, L.(1990): Quantita‑

type b polysaccharide conjugate vaccines･ Dev･ Biol･Stand., 86, 283‑296.

tive estimation of diphtheria and tetanus toxoids. 3.

87. Kameyama, S. and Kondo, S. (1975): Titration of tetanus

Comparative assays in mice and in gulnea‑pigs Of two
tetanus toxoid preparations. BiologlCals, 1 8, 3‑9･

antitoxin by passive hemagglutination. I. Titration of

73. KreeRenberg, J. G. (1986): Report of an informal meeting

gulnea‑plg antitoxins at various periods of immuniza‑

about alternative methods for the potency control oftion. Jpn. J. Med. S°i. 丘iol., 28, 127‑138･

88. Stifner‑Rosenberg, G. and Fey, H･ (1977): Measurement

the diphtheria and tetanus components in vaccines. Dev･

Biol. Stand., 65, 261‑266.

or human tetanus antitoxin uslng aS enZyme‑linked

immunosorbent assay. Schweiz. Med. Wochenschr., 6,

74. Murata, R., Someya, S. and Kaneko, J･ (1970): Standard‑

1101‑1104.

ization of tetanus toxoid combined with pertussis

vaccine. Jpn. J. Med. Sci･ Biol･, 23, 211‑216･

89. Hendriksen, C., van der Gun, ∫., Nagel, ∫. and

75. Kameyama, S.,Akama, K., Ito,A. and Murata, R･ (1962):

Kreeftenberg, J. (1988): The toxin binding inhibition

Studies on the combined vaccine. I. 0n the antlgenlClty

test as a reliable in vitro altemative to the toxin neutraliza‑

ofdiphtheria toxoid. Jpn. ∫. Bacterio1., 17, 149‑153 (intion test in mice for the estimation of tetanus antitoxin

179amebocyte lysate test blocking beta‑glucan一mediatedin human sera. ∫. Biol. Stand., 16, 287‑297.

90. Miyamura, K., Sadahiro, S., Konda, T., Takahashi, M.,
Fujino, R., Nishimura, Y., Miyakoshi, H., Horiuchi, K.,
Furuya, Y, Kubota, T., Watanabe, H., lnouye, S. andpathway by carboxymethylated curdlan and its applica‑tion. Jpn. ∫. Bacterio1., 45, 903‑91 1 (in Japanese).

loo. Oishi, H., Fusamoto, M., Hatayama, Y., Tsuchiya, M.,

the gelatin‑particle‑agglutinatlOn test fわr titration of

TAkaoka, A. and Sakata, Y. ( 1 988):Anautomated analysis
systeP Of Limulus amebocyte lysate (LAL)‑endotoxin

antibodies agalnSt diphtheria, pertussis and tetanus

reactlOn kinetics uslng turbidimetric kinetic assay.

toxins. Jpn. J. Med. Sci. Bio1., 48, 49‑59.
91 ･ de Saint Martin, J･, Eyquem, A., Turpin, A･ and Bizzini,Chem. Pham. Bull. (Tbkyo), 36, 3012‑3019.
101. lwasa, S., Ishida, S., Asakawa, S. and Kurokawa, M.

Yamazaki, S･ (1995): Developヮent and usefulness of

B.(1975): Titration of antibodies to tetanus toxoid by( 1 963): LymphocytosisIPrOmOting factor produced by

agglutination of purified tetanus toxoid sensitized latex

Bordetellapertussis. Jpn. J. Med. Sci. Bio1., 21, 363‑
368.

particles. Vox. Sang., 28, 238‑242.

92. Miyamura, K., Tajiri, E., lto, A., Murata, R. and Kondo,
R･ (1974): Micro. cell culture method for determination

102. Manclark, C. 良., Hansen, C. T., Treadwell, P. E. and

Pittman, M. (1975): Selective breeding to establish aof diphtheria toxln and antitoxin titers uslng Vero cells.

standard mouse fわr pertussis vaccine bioassay. ll.

11･ Comparison with仇e rabbit skin method and practical

8ioresponses of mice susceptible and resistant toapplication for sero‑epidemiologlCal studies. J. Biol.

sensitization by pertussis vaccine HSF. ∫. Biol. Stand.,Stand., 2, 203‑209.

3, 353‑363.

93. Dittmann, S.,Wharton, M., Vitek, C., Cio仕i, M., Galazka,

103. 1wasa, S., Ishida, S.,Asakawa, S. andAkama, 良.(1985):

A･, Guichard, S., Hardy, I., Kartoglu, U., Koyama, S.,

A curious histamine‑sensitizlng aCtivlty Shown by the

Kreysler, J., Martin, B., Mercer, D., Ronne, T., Roure,
C., Steinglass, R., Strebel, P., Sutter, R. and Trostle, M.

newly‑developed Japanese acellular pertussis vaccine.

(2000): Successful control of epidemic diphtheria in the104･ Hewlett, E･し., Sauer, K. T., Myers, G.A., Cowell, ∫. L

Dev. 丘iol. Stand., 61, 453‑460.

states of the Fo‑er Union of Soviet Socialist Republics:
lessons learned. J. Infect. Dis., 181 Suppl 1, S10‑22.

and Guemnt, 良. (1983): Induction ofa novel molph0‑

10glCal response in Chinese hamster ovary cells by

94. Baraff, L J., Manclark, C. R., Cherry, J. D., Christenson,
P･ and Marcy, S･ M･ (1989): Analysis of adverse rea9‑

pertussis toxin. Infect.lmmun., 40, I 198‑1203.

tions to diphtheria and tetanus toxoids and pertussIS

105･ Gupta, R. K., Saxena, S. N., Sharma, S. B. andAhuja,
S･ (1991): Production ofa safe, potent and immu?0‑

vaccine by vaccine lot, endotoxin content, pertussis

genic partially purified acellular pertussis vaccine uslng

vaccine potency and percentage of mouse weight galn.

simple indigenous techniques. Dev. Biol. Stand., 73,

Pediatr. Infect. Dis. J" 8, 5021507.

205‑222.

95. Kimura, M. and Kuno‑Sakai, H. (1990): Developments

106. Horiuchi, Y., Takahashi, M., Asada, S. and lshida, S.

in pertussis immunisation in Japan. Lancet, 336, 30‑(1994)= Incr?ased levels of active pertussis toxin may
aid a pertussIS Vaccine to pass the mouse body weight

32.

96･ Kawai, T. (1985): A new chromogenic endotoxin‑

galn test. BiologlCals, 22, 243‑248.

107. Gupta, R. K., Saxena, S. N., Sharma, S. B. andAbuja,
S･ (1988): The effects of purified pertussis components

specific assay uslng reCOmbined limulus coagulation

enzymes and its clinical applications. Clin. Chin. Acta.,
149, 55‑ 65.

and lipopolysaccharide on the result of the mouse weight

97. Obayashi, T., Tamura, H., Tanaka, S., Ohki, M.,
Takahashi, S., Arai, M., Masuda, M., Tanaka, S. andgaln test. ∫. Biol. Stand., 16, 32ト331.

108. Redhead, K. and Seagroatt, V. (1986): The effect oflwanaga, S･ ( 1 993): Limulus test fわr detecting bacterial

purified components of Bordetella pertussis in theendotoxins. Methods Enzymo1., 223, 358‑364.

weight galn test for the toxicity teStlng Of pertussis

98･ Tanaka, S. and lwanaga, S.(1993): Limulus test for

vaccines. ∫. Biol. Stand., 14, 57‑65.

detectlng bacterial endotoxins. Methods Enzymo1., 223,

109. Ochiai, M., Yamamoto, A., Kataoka, M., TbyoIZumi, H.
and Horiuchi, Y. (200 1 ): Interfering effect of diphtheria‑
tetanus‑acellular. pertussis (DTaP) Vaccines to the3581364.

99. Tsuchiya, M., Takaoka, A., Tokioka, N. and Matsuura, S.

bacterial endotoxln test. Biologicals, 29 (in press).

( 1 990): Development of an endotoxin‑specific Limulus

180