Jpn. J. Infect. Dis., 53, 98‑106, 2000

Review

A Proposal for Safety Standards for Human Use of Cholera Toxin(or Escherichia coli Heat‑Labile Enterotoxin) Derivatives as an
Adjuvant of Nasal Inactivated Influenza Vaccine
Shin‑ichi Tamura* and Takeshi Kurata

Department of Pathology, National Institute ofInfectious Diseases,
Toyama I‑23‑I, Shinjuku‑ku, Too,0 I 62‑8640, Japan(Received June 15, 2000. Accepted July 28, 2000)

CONTENTS:
1. Introduction

2. Advantages of using CT ‑(or LT‑) adjuvant‑Combined nasal innuenza vaccine(1) Preclinical shdies(2) Clinical studies(3) Other advantages of CT (or LT) derivatives as an adjuvant

3･Assessment of CT (or LT) derivatives as nasal influenza vaccine adjuvants for human use(1) Abilityofvarious CT (or LT) mutants to enhance protective Ab responses(2)Assay for allergenicityof various CT (or LT) mutants(3) Assay system fわr toxicity of various CT (or Lr) mutants(4) Another assessment of toxicity of CT (or LT) mutants
4･ A proposal for safety standards for human use of CT (or LT) derivatives

StJMMARY: Cholera toxin (CT) and Escherichia coli heat‑1abile toxin (LT) are not only the causative agents ofdiarrhea but are also strong mucosal adjuvants which enhance immune responses to mucosally coadministeredbystander antlgenS･ One of the most promlSlng applications of these toxins would be as mucosal adjuvant of nasal

innuenza vaccine･ In comparison to current inactivated vaccines, the nasal vaccine provides superior cross‑protec‑
tion by inducing production or cross‑reacting anti‑Viral lgA antibodies in the respiratory tract even when thevaccine strain is different from the epidemic strain. On the use of the toxins as mucosal adjuvants in humans,

toxicityand allergenicity of the toxins are problems which impinge on safety. To resolve these problems, various
approaches have been attempted to produce less toxic and less allergenic CT (or LT) derivatives･ We now prop.ose
the following standards for humanuse of safer CT (or LT) derivatives as an adjuvant ofa nasalinnuenza vacclne.
Thus, CT (or LT) derivatives can be administered intranasally together with a current inactivated innuenza

vaccine, provided they meet the following criteria･ 1 ) A single dose of the derivatives, administered intranasallyby spraylng, Should be around 100 LLg/adult in a volume of less than 0.5 m1. 2) CT (or LT) derivatives should

retain the properties of the native CT (or LT), i･e･, the abilityto augment secretory IgA and serum IgG Ab
responses to viral surface glycoproteins, when administered intranasally togetherwith an inactivated influenza
vaccine･ 3) CT (or LT) derivatives should not induce IgE Ab responses to the vaccine, as well as to the CT (orLT) itself･ 4) The CT (or LT) should be nontoxic; the tPxicityofthe derivatives, as determined by the Yl 1 adrenal
cell as苧ay, Should not exceed 1/100 EC,o of the natlVe CT (or 1/1000 ECi of the native CT)･ 5) CT (or LT)

derivatlVeS Should not cause serious disease in gulnea PlgS When administered intranasally or intraperitoneally at
the dose used in humans (around 100 LLg).

subcutaneously･ These vaccines are shown to induce produc‑

1. Introduction

tion of high levels of serum antiviral IgG antibodies (Abs),

Innuenza is a highly contagious acute respiratory disease,which have a protective effect against homologous viralcaused by innuenza viruses that are implicated in both world‑

infection･ However, the vaccination is less effective against

wide pandemics and local outbreaks by altering their surface

heterologous viral infectionwithin the same subtype, whichhemagglutinin(HA) and neuramimidase either slightly orradically ( I )･ Due to the short incubation period, the immumity
raised within a few days afterinfection cannot prevent theonset of the respiratory symptoms. Therefore, an effective
immumitymust be induced in advance, by vaccination, in order

explains the ineffectiveness of the current influenza vaccine
when the vaccine strain is different from the epidemic strain.
On the other hand, natural innuenza viral infection has been

to prevent the disease. Current trivalent inactivated vaccines,

The cross‑protection induced by natural infection seems towhich contain A and Btypes of viruses, are administered

be largely due to the production of cross‑reacting IgAAbs toshown to be superior to current inactivated vaccines for
inducing crossIPrOteCtiOn against variant viral infections (2).

the Lviral HA molecules in the respiratory tract (3). The cross‑*Corresponding author: Tel: +81‑3‑5285‑1 1 1 1 , Fax: +81‑3‑5285‑

reactivity is derived from the polymeric nature of SIIgA

1 1 89, E‑mail: stamura@nih.gojp(secretory IgA), which generates a greater reactivityfor the98innuenza virus than serum IgG. This fact suggests that theshown in Fig. 1 , intranasal vaccination with A/Guizhou‑Xdevelopment of an immunization procedure to stimulate

vaccine 4 weeks prior to a homologous viral challenge‑whichmucosalIgA production would improvethe protective efficacy

was administered in a small volume in an attempt to induceof cu汀ent inactivated vaccines. In this regard, intranasal

upper respiratory infecdon‑provided almost complete protec‑

TYLerefore, We attempted to administer a vaccine together withtion against infection. In our experimental system, virus titersof lessthan1 00･5 EID5. Were regarded as complete protection.
In parallel with the protection, production of bothHA‑reactive
IgA and IgG Abs were induced in the upper respiratory tract･cholera toxin (CT) as a mucosal adjuvant (5).

Each of two vaccines derived from a varlant Within the sameimmunization with a vaccine has been advocated as a meansof inducing IgA and systemic IgG against influenza virus (4).
However, a vaccine alone carLnOt easily generate S‑IgA Abs.

CT, which is produced by Vibrio cholerae, is the causative

subtype, the A/Fukuoka (H3N2) and A/Sichuan (H3N2)

agent of cholera, while Escherichia coli heat‑Iabile enterotoxin

vaccines, also provided almost complete cross‑protection,(I:T), whoseamino acid sequence shows 80% homology tothat

accompanied by the producdon ofa low level of cross‑reactlngof CT, is one of the causative.agents oftraveler's diarrhea (6‑IgA to A/Guizhou‑X HA molecules in the upper resplratOry

8). At the same time, both toxlnS a陀StrOng muCOSal adjuvants

tract. A different subtype of viral vaccine, A/PR8 (HINl),which enhance immune responses to mucosalIy co‑administeredbystander antigens (9‑12). CT, as well as LT, consists ofa

gave slight crossIPrOteCtion, whereas a differenttype of viral
vaccine, B/Ibaraki, failed to glVe Protection. Subcutaneousbinding reg10n Offive B subunits, into which the active toxic

vaccination with AノGuizhou‑X vaccine 4 weeks prlOr tO acomponent, the A subunit, is inserted. CT (or LT) binds to ahomologousviral challenge provided almost complete protec‑
tion, accompanied by the production of anti‑HA IgG Abs inthe upper respiratory tract. However, the A/Fukuoka and A/

specific receptor, i.e., GMl ganglioside, via the B subunits
on the cell membrane. This enables translocation of the A
subunit across the membrane into the cell, where it activates

Sichuan vaccines, as well as the PR8 and B/Ibaraki vaccines,

adenylate cyclase. The activation of adenylate cyclase results

failed to provide cross‑protection and did not induce antibody

in an increase in the level of intracellular cyclic adenosine

pro duction.

monophosphate, which induces diarrhea and other biologicalWe drew the followlng conclusions from these results.

activities ( 1 3); the intracellular mechanisms by which CT (orIntranasal immunization wi仇the cu汀ent inactivated vaccineLT) augments immune responses remain to be clarified.

together with CTB*(or LTB*) provides effective cross‑Since 1972, the mucosaI adjuvants, CT and LT have beenprotection against variants within a subtypeof influenza A

widely investigated (9‑14). We have also been engaged inviruses or within type B virusesinthe upper respiratory tract.

the development of an adjuvant‑combined nasal influenza

The cross‑protection in the upper resplratOry tract lS Provided

vaccine, which has become one of the most successful appli‑

mainly by cross‑reacting anti‑HA IgAAbs, whereas the cross‑cations orthe toxin in a human mucosal vaccine (5,15‑17).

protection in the lower resplratOry tract is provided by the

Although both toxins function as mucosal adjuvants, the use

IgG Abs (34). We further examined the vaccination conditionsof toxins as adjuvants ofmucosal vaccines for humans is not

to apply the adjuvant‑combined vaccine to humans. In terms

feasible owing to their toxicity. In addition, these toxins haveof providing cross‑protection agalnSt lethal infection, slngle
immunization has been showh to be less effective than two

another potential drawback of possibly inducing lgE Ab
responses to bystanderantigens, as well as to the toxins them‑doses or vaccine, i.e., pnmary Immunization with a low doseselves (18,19). Thus, two problems which impinge on theor vaccine and CTB* and second immunization with anothersafety of these toxins, toxicity and allergenlClty, Should be

vaccine alone (35･ 36)･ In addition･ two doses oftrivalent

examined carefully when these toxins are used as adjuvants

vacclneS conferred cross‑protection agalnSt Various strains

ofmucosalvaccines for humanS. One approach being exploredof viruses. Based on these studies, we theorized that applica‑

to resolve the problem is to use the nontoxic B subunits (CTB

tion of an a4juvant‑combined vaccine to humans could beor LTB) alone (20), or B subunits coupled chemically or by

performed effectivelyand safely via two successive intranasal

gene fusion to v?cPines (21‑24)･ Another approach is to use
B subunits contalnlng a trace amount Ofthe toxin (0. 1 ‑0.5%)

with a CTB* (or LTB*)‑combined trivalent vaccine (prepared(CTB* or LTB*), because the B subunit and a trace amountfrom HINl , H3N2 and B‑type viral strains, recently circulat‑

immunizations at 4‑week intervals, the first immunizationof the holotoxin act synergistically as an adjuvant, inducing

ing in humans) mixed with CTB* (or LTB*) and the second
Immunization with the trivalent vaccine (35,36).

mucosal immune responses to the vaccine (25‑27). A third
app.roach is to use a chemically modified (28) orヮutated(2) clinical studies

We conducted the first field trial to evaluate the efrlCaCy Of

toxln Without toxicity but retaining its adjuvant actlOn (29‑33). At present, there are no safetystandards on the use ofCT

the LTB*‑Combined nasal innuenza vaccine dming the winter(or LT) derivatives as a mucosal adjuvant for humans,

or 1993‑1994 (16). A cu汀ent inactivated trivalent vaccine

although there is much data onthese derivatives. The objectives

composed ofA/Yamagata (H I N 1 ), A/Kitakyushu (H3N2) andof this review are to propose safetystandards for human use

B侶angkok innuenza virus strains, was administered intrana‑of CT (or LT) derivatives as an adjuvant of nasal innuenza

sally into volunteers by spraylng With or without recombinant
LTB*. Vaccination was performed twice, 4 Weeks apart. Titersof salivary S‑IgA and serum hemagglutination‑inhibiting (HI)Abs were measured before and 8 Weeks after the prlmary

vaccine and to present the experimentalsystem and data on
which this proposal is based.

vaccination. The volunteers were divided into two groups:

2. Advantages of using CT‑ (or LT‑) adjuvant‑combined

73 volunteers were admimistered the LTB *‑combined vaccine,nasal im爪uenZa Vaccine

and 49 volunteers were administered the vaccine alone. For(1) Preclinical studies

the IgAAb responヲC, a 1 ･41fold or greater increasでin umits of

We compared the protective efficacy of nasal immuniza‑

specific IgAper mlCrOgram Oftotal IgA after vacclnation wastion with the CTB'(or LTB')‑ combined vacci.ne with thatregarded as a positive response. This was because only lessof subcutaneous immunization in BALB/c mlCe (34).As

than lO% of the vaccinees, who received a subcutaneous99

I mm unization

vaccine with CTB'

RouteCha]tenge

NGLJizhou‑X HA‑reactive Ab Virus liters (ElDs｡;10n)vdth virLJS.

(EuSA:ng/mouse) in nasal wash in nasal wash

NGLJizhou‑X(4 weeks)

0

IO

20

30

40

50

0

1

2

3

4

5

I.川.川.川.川.川〟Gu izhou ‑X(H3N2)〟Fukuoka (H3N2)
A/Sichuan〟PR8+ + 十 十(H3N2)(HI Nl)a/I baTakJ'■ oq o o"M w o

十十十+

A/Guizhou ‑X (H3N2)〟Fukuoka (H3N2)〟Sichuan〟PR8(H3N2)(HI Nl)

十 十

L JgA

口19G

a/I barakj

Fig･ 1 ･ Protection agalnSt influenza A virus infection and production of cross‑reactive anti‑HA lgAand lgG Abs in the upper

resplratOry tract in mice immunized intranasally or subcutaneouslywith different adjuvant‑combined vaccines･ Themicewere immunized intranasal1y or subcutaneouslywith0.5 LLg Of a vaccine combinedwith 1 LLg Of CTB* (CTB containing
about 0. 1% ofCT) 4 weeks previously. The immunized mice were infected intranasally witha small volume (I LEI x 2) ofa mouse‑adapted A/Guizhou‑X virus (H3N2) S†spension. Three days later, their nasal virus titers were assayed･ Eachcolumn represents the mean± SD ofnasal virus tlterS (EID5｡) ofall mice in each group ofsix･ An asterisk (+) represents astatistically signi丘Cant difference舟om a non‑immunized control at P < 0･05･

Injection of the same trivalent vaccine, showed a 1.4‑fold

In addition, because we did not compare the e爪cacy of仙e

increase in their salivary IgA titer. For the HI Ab response, a4‑fold or greater increase in HI Ab titer after vaccination was

nasal vaccinewith that of subcutaneous Injection of theregarded as a positive response. The results show that in the

not assess the superiorlty Of the nasal vaccine to the current

group administered the nasal LTB'‑combined vaccine, 37

subcutaneous vaccine. Further field trials are required tocurrent inactivated vaccine in the second field trial, We could(50.7%) and 36 (49.3%) of73 vaccinees showed positive IgA

assess the effectiveness of the nasal LTB*‑combined innuenza

and HI Ab responses to one or more of仇e vaccine strains,

VaCClne.

respectively. On the other hand, inthe group glVenthe vaccine(3) Other advantages of CT (or lX) derivatives as analone, 16 (32.8%) and 15 (30.6%) of49 vaccinees showed

adj uyantCT and LT act similarly as mucosal adjuvants of the nasal

positive IgA and HI Ab responses. There was a statisticallyslgnificant difference between these two groups･ Thus, theinnuenza vaccine, although more LT is required for positive

nasal LTB*‑combined vaccine could induce both nasal lgAand serum lgG Ab responses at levels higher than the nasal

mucosal adjuvant activities than CT (26, 27)･ Both toxins

vaccine alone. Whether the increase in the salivary lgAnasal vaccine. (i) In mice, the abilityofCTB* (or LTB*) toAb titer was effective in preventlng influenza could not be

augment mucosal immune responses to the vaccine isdetermined inthe present study because there was no epidemic

generated only when the derivatives were administeredappear to have the following advantages as adjuvants of thesimultaneouslywith the vaccine via the same route (37). (ii)of innuenza during the testperiod.
In the winter of 1994‑1995, we conducted a second field

The mechanisms by which the CTB*‑Combined vaccine

trial to evaluate the efrlCaCy Of the nasa) LTB*‑combined

augments mucosal Ab responses inmice can be explained byinnuenza vaccine (Fig. 2) (17). The conditions of vaccina‑

the stimulation of irLnate immunityby CT (or LT) derivatives

tion in the second field trial were the same as those in the(38). (iii) CTB*(or LTB*) (around 1 LLg) does not cause any

first, except that B/Mie/ 1 /93 was used instead of B侶angkok/

damage to the nasal mucosa ofmice･ In addition, 0･ I LLg Of

1 63/90. Contraction ofinnuenza was monitored up to 8 Weeks

CTB* does not cause any local brain血mage, i.e., so‑called

after the second vaccination. Outbreaks of H3N2‑subtype
virus and B‑type virus occurred over the trial period. The

encephalopathy, even when inj ected intracerebrally into mice

results showed that 3 of 18 vaecinees (16･7%) who received

to mice as an adjuvant for nasal vaccination, without inhibi‑

the nasal LTB *‑Combined vacclne COntraCted innuenza, whiletion of preexisting immumity to the CTB (or LTB) (41,42)･

6 of 14 nonvaccinees (42.7%) contracted innuenza. There
was no statistical)y slgnificant difference between the two(V) The adjuvant effect of CTB* is not genetically restricted
by MHC genes in Tlice (43); this finding would favor thegroups because the number of subjects was small. Thus, We

combined applicat10n Of current vaccines with CTB* to

can only speculate on the effectiveness of the nasal LTB*‑

genetically heterogeneous humans･ (vi) CT induces almost
no IgE Ab responses to bystander antigens, as well as to CT(39,40). (iv) CTB* (or LTB*) could be repeatedly administeredcombined influenza vaccine (estimated effectiveness, 6 I %)･

100

Vaccln宅地on
1) tdvaLent vacchw (140 〃g) ± LTB'(100 JJg): rLTB containing l% rLT〟YamagataB2m9; HI NI

NKW15的3; H3N2
B/…ie/1 β32)細Ⅶ1aSaJ spray. 125 pl x 23) Firstvaccination S∝ond vacdnation

.I

4w

I

4W

.

4W

.

Contraction of influenza: mo帽than 4‑fbkJ rise in serum HI titerand presence of dinical symptoms

Group

NumberofAgedis廿ibu60n

Morbidity

volunteers (fema一e) (means ± SD)

T,oB･e̲C.mbined vaccine 1.3g‡

of州uenza

37:揺.1.･; 36制篭:写芸壬'EfFectiveness = (42.9 ‑ 16.7/42.9) x 100 = 61.0 (%)
Fig. 2. Preliminary rleld trial using nasal IJB+‑combined innuenza vaccine (1994‑1995). A trivalent inactivated vaccine,composed ofA/Yamagata/32/89 (Il lN I), A/Kitakyushu/I 59/93 (H3N2) and B/Mie innuenza virus strains, as well as theadjuvant, r∝ombinant LTB supplemented with1 % recombinant LT (LTB+), were administered intranasally into 1 8 volunteers(mean age, 37.7 ± 1 1.3). The control group was composed of 14 non‑vaccinated volunteers (mean age, 44. 1 ±1 1.3). Vaccina‑tion was perf♭med twice 4 weeks apart.

itself, when used as an adjuvant for nasal influenza vaccine

after infection, nasal wash, )ung wash and serum specimens

in mice, as described in the fわllowing section (44).

were collected to measure either virus titers based on 50%
egg‑infecting dose (EID50) or anti‑HA IgA and IgG Abs using
ELISA. The dose of adjuvant or vaccine (0.1 LLg per 20‑g

3. Assessment of CT (or LT) derivatives as nasal

weight of mouse) and the volume of the vaccine solution (1influenza vaccine adjuvants for human use

Lil per each nostril) used in this experiment correspond to

The human field trials showed that the adjuvant‑combined

100 jig per 20 kg body weightand 1 ml per nosbilinhumanS,

vaccine induced production of a slgnificantly high level ofrespectlVely. The method used in mice was similar to thatanti‑HA secretory IgA and systemic lgG, comp.ared with thevaccine alone, suggesting that this is effective ln Preventing

LTB contalnlng 0.5‑1% LT, together with 140 LLg Of trivalent

innuenza (16, 1 7). Although no serious adverse effects asso‑
ciated withthe vaccine were observed inthe prelimlnary humantwo‑dose regimen (twice at 4‑week intervals) (1 6, 1 7). Thus,

used in preliminary human廿ials, i.e., 100 LLg of recombinant

innuenza vaccine per 250 LEI, was sprayed intranasally in a

the data obtainedinthis screenlng System uSlng BALB/c miceshould provide clues for estimating the efrlCaCy Of adjuvant‑combined vaccines for humans. Figure 3 showsthat relatively
high anti‑HA IgA and IgG Ab responses wereinduced in micetrials, further trials on the reduction of the toxicity andallergenlClty Or these toxins will be required to avoid any

untoward effects of adjuvant‑combined vaccines. We prepared
four CT mutants, namely, CT7K (Arg to Lys substitution atamino acid position 7from the N‑terminus of the A l丘agment

immunized with也e CT 1 1 2K‑combined vaccine, al仇ough仇eof the A subunit) (45), CT61F (Arg to Phe substitution at

Ab responses were approximately one‑third lower than thoseamino acid position 61from the N‑terminus) (3 I), CTl 12K

with the native CT. Table I shows that the anti‑HA IgA and(Glu to Lys substitution at amino acid position 1 12 from theIgG Ab levels, induced in mice immunized with the CTl I 2K‑

N‑teminus) (32) and 1 1 8E (Glu to Gly subst血tion at amino

combined vaccine, were suWICient to provide almost completeacid position 1 12 from the N‑terminus), as useful adjuvant
models since all of these were less‑toxic than LTB*. Then we

protection agalnSt the viruSinfection. In addition, the Ab titers

examined experimental systems for assesslng the effecdvenessCTl 1 2K.‑combined vaccine roughly comSpOnded to thoseand the degree of protection in mice immunized with theand safetyof the toxin derivatives as adjuvants for nasal

inmi.ce immunizedwith the LTB‑ 1 % LT (LTB*)‑combined

innuenza vaccines (44).

vacclne (Fig. 3 and Table 1). These results suggest thatCTl 12K, as well as LTB‑1% LT, could be used effectively as(1) Ability of various CT (or IX) mutants to enhancesafer adjuvants for nasal innuenza vaccines in humans.

protective Ab responses
BALB/C mice were used to assess the efficacy ofmucosal
adjuvant activides ofCT mutantsinmice, becausethey showedintermediate levels of response to either CT derivatives or
influerLZa HA molecules (43 ,46). Mice were immunizedintra‑
nasally (1 lil into each nostril) with PR8 vaccine (0.1 LLg)

To assess the efrlCaCy Of mucosal adjuvant activities of CT
mutants, the ability of the mutants to induce delayed‑typehypersensitivity (DTH) reaction is also available. Mice are
irrmumized intranasally (1 lil into each nosbil) with influerL2a

togetherwith CT (or LT) derivatives (0. I LLg) and boosted in

vaccine (0. 1 LLg) together with CT (or IJ) derivatives (0.1
JLg). Seven days aRer the vaccination,thepeak DTH response,

the same manner 4 Weeks later. Two weeks after the second

which is elicited by injecting the vaccine into the footpad ofvaccination,the immunized mice were challengedwitheither

the immunized mouse, is induced (47). Twenty‑four hours

a small volume or a lethal dose of PR8 virus. Three days

after the elicitation, footpad swelling is measured asanindex

101

AntLPFt8 HA lgA bl nasal wash tpghTtQ AntLPR8 HA lgQ bl SOrum (pgmO

AdjLNant 0. 1 pg
(bin vacclno 0. 1 ll由

0

O.2

0.4

0.0

0,8

0

60

1

00

1

50

200

native CT (Slgrna)

recombinant CT
rocombinant CTB
CT7K

CT61 FCT1 12KCT1 18E
controJ (‑)

recombinant LTD‑1% LJ

Fig･ 3･ Secondary anti‑PR8 HA IgA and IgG Ab responses in mice immunized twice intranasallywith0･l LLg Ofvarious CT
derivatives and O･l LLg of PR8 viral vaccine, at 4‑Week interval･ Nasal wash and serum samples were collected from theimmunized mice 2 weeks after the second immunization. Each column represents the mean Ab liter ± SD of fivemice ineach group measured by ELISA･Anasterisk (') represents a statistically signiBcant difference from a control group immunizedwithvacclne alone at P < 0.05.

Table I ･ Protection against PR8virus infection in mice immunized intranasally with CT
mutant‑combined innuenza vaccine

Nasal immunizationa Virus infection Nasalvirus titerb Lungvirus titerb
vaccine(0･l+十十+++++

LLg)

CT

(0.1

LLg)

PR8

native CT (Sigma)(EID,.:++++++++

recombinant CT

Ion)(EID5.:

Ion)

<0.5+

<0.5*

<0.58

<0.58

CT7K

0.5±0.2書

N.D.

CT61F

<0.5*

N.D.

CTl12K<0.5*

0.7±0.3*

CTl18E

0.6±0.3書

0.9±0.9書

3.0±0.3

6.5±0.7

recombinant LTB‑ 1 % LT

<0.5*

<0.5*

a Mice were immunized by intranaSal admimistration of a CT mutant (0. I LLg) togetherwith influenza PR8 virus
vaccine (0･ 1 JLg), twice at 4‑Week interval･ Two weeks a触r the second immunizadon, themice were challenged

by intranasal administration of either a small or a large volume of PR8 virus suspension･ Three days aRer thechallenge, nasalor lung washes &om the immunized mice were assayed for virus titer.
b values represent mean± SD of each group of丘ve mi去e.

* Statistically significant difference from control mice immunized with vaccine alone at P < 0.05.
N.D∴not done.

ofDTH reaction･ This assay can detect the abilityof CT (orLT) derivatives to enhance Im.mune responses Within 8 days･

wAiqJTyya?cnctiioe'払. Footp23.A SWelli器̀× u16?.? mm'8.

Figure 4 shows that in mlCe Immunized with the CTl 12K‑
combined vaccine, a relatively high DTH response, which isslightly lower than that in mice immunized with the nativemCT (Sigma)

CT‑combined vaccine, is induced. The DTH response detectedby this assay seems to be mediated by CD4'T helper cells
that are capable of producing IFN‑T (47).

rCT E112K

Strictly speaking,further studies in humanswill be needed
to conrlrm that the dose/body weight for adjuvant activityis(‑)

sufficient･ In one previous study, 1 00 LLg of recombinant CTB,
which is estimated to correspond to 0. I LLg ofCTB in BALB/

Fig･ 4･ DAH reaction in mice immunized intranasally with both 0.I LLg

c mice, was used as a strong mucosal immunogen in humansofCT derivatives and O･l LLg OfPR8virus vaccine. Six days after(20), although less than1 LLg of CTB had no adjuvant activity

immunization, DAH reaction was elicited by injectlng PR8 virus

in BALB/c mice (26). This finding suggests that humans are

vaccine intothefootpad. Twenty‑four hours a洗er elicitation,the加tpad

thickness (swelling) was measured as an index ofDTH reaction. Eachmore sensitive to CT derivatives than BALB/c mice. Ifthis isbar represents the mean footpad swelling (± SE) offive mice.

the case, the eWICaCy Of CT derivatives as mucosal adjuvants,
as detected in this experiment uslng BALB/c mice, will bemuch higher in humans･ ln any case,the experimental system

the case of antlgenS Other than the current innuenza vaccine

used for Tssessing the ability of CT (or LT) derivatives toremains to be examined.

enhance Immune reSPOnSeSwill be useful to select ideal(2)Assay for allergenicity of various CT (or LT) mutants

candidates for effective adjuvants for nasal influenza vaccines.

A possible drawback of CT as a mucosal adjuvant is its

However, whether this system is useful for assessing the abilityof CT (or IJ) derivatives to enhance immune responses inpotential to induce IgE Ab responses to bystander antigens,

as well as to CT itself(18, 19). Under the experimental condi‑

102dons described above, sera can be obtained for assesslng the

CAMP‑stimulation test (55) and theAl)P‑ribosyltransferase

ability ofCT (or LT) derivatives to induce IgE Ab responses
using ELISA or passive cutaneousanaphylaxis (PCA) (48,49).
Table 2 shows bothtotal IgE and anti‑CTB IgE Ab responses

activityassay (56). Ofthese, the Y‑1 adrenal cell assay is theassess the toxicityof the CT mutants. Brieny, mouse Y‑I

simplest and most sensitive (53). Thus, We used this assay toin mice immunized intranasally with 0. I LLg of a CT mutant

adrenal tumor cells were cultured in 96‑Well nat bottom plates

and vaccine in a two‑dose regimen (44). One microgram of

at a concenFation of 104 cellsper well, to which the CT (ornative CT‑, recombinant CT‑ or CT7K‑combined vaccineLT) derivatlVeS Were added. A触r ovemight incubation, theinduced only a low level of total IgE Ab responses. Whengiven

cells were examined by light microscopy to determine thein doses of 1 LLg or 0. I LLg, neither the native CT‑combined

minimum concentration of the toxin required for greater thanvaccine northe other CT derivative‑combined vaccine induced
IgE Ab responses to CTB. On the other hand, frequent intra‑

to initiate cell rounding (ECi). Table 3 shows the data for

50% cell rounding PC5.)and the toxin concentration required

peritoneal injections of 2 LJg of native CT (positive control)

toxicity of也e CT mutants on VI cells. All fわur CT mutants

could induce both total IgE and anti‑CTB IgE Ab responses

examined were less toxic than LTB*.

(4) Another assessment of toxicity of CT (or LT) mutants(PCA titer lessthan25). These findings suggest that 0. 1 LLg Of
native CT produces only a low level oftotalIgE Ab responses
even after several administrations. Moreover, Yamamoto et

To ensure the safetyof CT (or LT) derivatives for humanuse, we must consider another toxicityassessment factor. CT

al. have already shownthat the abilityofnative CT (0.5 LLg)

(or IX) derivatives should not cause serioTS disea守e in guinea

to induce IgE Ab responses is slgnificantly higher than that

plgS When administered intranasally or lntraPerltOneally at
the dose used in humans (around loo LLg) (57).

ofCT61F (5 LLg) in C57BL/6 mice (31). Thus, when the CT
mutants, which produce IgE Abs at a lower level than thenative CT,are used at a low dose (0.I LLg),they caTmOt induce
IgE Ab responses even after several vaccinations.

4. A proposal for safety standards for human use ofCT (or LT) derivatives(3)Assay system fTor toxici吋ofvarious CT (or IlT) mutants
There are several assay systems f♭r assessing血e toxicity

From the results described above, it was shown that theof CT (or LT) derivatives (50‑56): the vascular permeability

effectiveness and safetyof CT (or LT) derivatives as a nasal

test (50), the rabbit ileal loop test (5 1 ,52), the Y‑1 adrenal cell

influenza vaccine adjuvant for humanuse can be assessed byassay (53), the Chinese hamster ovary cell assay (54), thethe following assays. (i) The ability ofCT (or LT) derivatives

Table 2. IgE responses induced by CT mutants

Total IgE (ng/ml)a Anti‑CTB IgE (PCA:2n)
CT derivatives

1

JLg

0.1

LLg

1

LLg

0.1

FLg

native CT (Sigma)

178±85<160<1recombimant CT

259±74< 1 60く く く く く 1 1 l 一l 1

CT7K

236̲+12

<1 60

<1

CT61F

<160

<160

<1

CTl12K

<160

<160

く1

CTl18E

<160

<160

<1

vaccine alone

<1 60

<1 60

<1

positive

controlb

53

6±23

5

<1

<1

4

8 Mice were immunized by intranasaladministration ofa CT mutant (I Fig or 0. 1 LLg)
together with inactivated innuenza vaccine (I JLg or 0.I JLg), twice at a 4‑weekinterval. Two weeks a洗er the second immunization, sera丘omthe immunized mice

were assayed for total IgE and anti‑CTB IgE Ab responses.
b positive control sera were obtained 2 weeks aRerthe last immunization舟om mice

immunized by intraperitoneal injection of native CT (2 LLg, Sigma) 3 times at a 2‑
week interval.

Table 3. ToxicityofCT mutants on Y‑I cells
EC5.a(pg/we ll )ECibbg/wet 1 )native CT (Sigma)

26

(1)￠

0.2

recombinant CT

240

(1/9)

5

(1)C

(1/25)

recombinant CTB< I 000000

CT7K (Are.Lys)

11100(1/427)1200

CT6 1 F (Are‑Phe)

19300(1/742)3700

CTl 12K (Glu.Lys)

19300(1/742)5100(I/25500)

CT1 18E (GlyーGlu)

11100(1/427)3700(1/18500)

recombinant LTB‑1% LT

2 140(1 /82)46(> I /38462)< 1000000(>1/5000000)(1/6000)(1/18500)(1/230)

A Concentration of CT (or Lr) derivatives inducing rounding of 50% ofcells.
b concentration of CT (or LT) derivatives initiating rounding ofY‑ 1 cells.

c The values in parenthesis representthe ratio of the toxicity ofCT (or LT) derivatives to thetoxicity of native CT (Sigma).

103

protection against virus infection can be assayed in BALB/C
mice immunized intrapasally with O･ 1 LLg ofbothCT (or LT)

thanLTB*, as described in the safety standaJ'ds ofCT (or LT)
derivatives. (iv) Vaccination is carried out before the onset of
epidemic of influenza in two successive intranasal administra‑

derivatives and vacclne in a two‑dose reglmen at 4‑weektions at 4‑week intervals, pnmary Immunization with theto enhance immune responses against vaccine and to provide

intervals, or in BALB/C mice which received the first intra‑

adjuvant‑Combined vaccine and a secondary immunization

nasal血nIumi血ion with O･ 1 LLg Ofboth CT (or IJT) derivatives

with the vaccine alone. This two‑dose reglmen would be usedand vacclne, and the second intranasal immunization withfor naivepeople who have not yet contracted innuenza. The

O･ 1 LLg of the vaccine alone at 4‑week inteTals (unpublishedsingle‑dose reglmen uSlng the vaccine alone would bedata). (ii) The ability ofCT (or LT) derivatlVeS tO induce IgE

recommended for those who have already contracted influ‑

Ab responses against CT (or LT) derivatives?r vaccine

enza.

(allergenicity) Canalso be assayed in BALB/C mice Immunized

Further detailed field trials are required to confirm theintranasally with 0.1 LLg Ofboth CT (or LT) derivatives andeffectiveness of the adjuvant‑Combined inactivated innuenza

vaccine in a two‑dose regimen at 4‑week intervals. (iii) The

vaccineinpreventing orinattenuating illnesswithout negative

adrenal cells･ (iv) The toxicityof CT (or LT) derivatlVeS.Can

effects. The adjuvant‑combined nasal influenza inactivatedvaccine will be useful in preventing innuenza and its compli‑

also be assayed based ontheir ability to caヮse disease ln gulnea

cations not only in chil血en and adults, but also in the elderly

pigs When administered intranasally or lntraPeritoneally at

at highrisk (those with conditions such as diabetes, chronic

toxicity of CT (or LT) derivatives can be assayed u守ing Yl 1

obstructive pulmonary disease or congestive heart failure)

the dose used in humans (around 100 LLg).

who cannot receive an attenuated innuenza virus vaccine. nle"SafetyStandards for Human Use ofCT (or LT) Derivatives

From the assessment of the effectiveness and safetyof CT(or LT) derivatives as.a nasal influehza vaccine adjuvant, we

asanAdjuvant of Nasal Inactivated Influenza Vaccine" Will be
useful in the realization of such an effective nasal innuenza

canpropose the followmg …SafetyStzmdwds for HumanUse ofCT (or LT) Derivatives as an Adjuvant ofNasaI InactivatedInfluenza Vaccine"･ CT (or LT) derivadv.es canbe administeredvaccine in the near future.

intranasally together with a current lnaCtivated influenza

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106