A novel concept in mucosal adjuvanticity: The CTA1-DD

adjuvant is a B cell-targeted fusion protein that incorporates

the enzymatically active cholera toxin A1 subunit

LENA AÊ GREN,1 BJOÈ RN LOÈ WENADLER2 and NILS LYCKE1

1Department of Medical Microbiology and Immunology, University of GoÈteborg, GoÈteborg and 2Astra HaÈssle AB,Department of Molecular Biology, MoÈndal, Sweden

Summary A promising novel concept in mucosal adjuvant research is demonstrated here. The adjuvant and

toxic e�ects of the cholera toxin (CT) have been successfully separated in a gene fusion protein, CTA1-DD. Thisprotein consists of the ADP-ribosylating A1 subunit of CT linked to a synthetic analogue of protein A. TheCTA1-DD protein was found to exert comparable adjuvant activity to that of CT after systemic as well as

mucosal immunizations with soluble protein antigens, such as KLH or ovalbumin (OVA). However, contrary toCT it was completely non-toxic. The CTA1-DD approach to the construction of a potential vaccine adjuvant isunique and highly promising. Conceptually, the CTA1-DD fusion protein demonstrates that: (i) contrary to CT

the CTA1-DD is a highly targeted adjuvant, directed to B cells and possibly other antigen-presenting cells; (ii) it ispossible to introduce ADP-ribosyltransferase activity into cells via an alternative pathway to the GM1 receptorpathway used by CTB; (iii) the adjuvant e�ect of CTA1-DD, and possibly also of CT, depend on the enzymaticactivity; and (iv) one possible mechanism, shared by CT, that may explain the adjuvant e�ect of CTA1-DD is its

ability to induce expression of the costimulatory molecule CD86 on B cells.

Key words: ADP-ribosylation, cholera toxin, costimulation, mucosal, targeted adjuvants.

Introduction

Many diseases are caused by micro-organisms that infect(or gain access to) the body via the mucosal membranes.

Globally each year, millions of people su�er and die fromdiarrhoeal-, respiratory or sexually transmitted diseases.1

Prevention against these diseases is much in need. The most

e�ective means to stimulate immune protection at mucosalmembranes is by local vaccination.2 However, the devel-opment of mucosal vaccines has been hampered by the lackof e�ective immunomodulators/adjuvants. The present re-

view will describe some of the recently reported promisingattempts to exploit the bacterial enterotoxins, cholera toxin(CT) and the heat-labile toxin (LT) from Escherichia coli, as

mucosal adjuvants.3,4 Two di�erent strategies will be dis-cussed that demonstrate that it is possible to separate tox-icity from adjuvanticity in these molecules.

With the growing understanding of surface receptorstructures, signal transduction and functions of secondmessenger systems in eukaryotic cells, a new realm of reg-ulatory mechanisms has attracted the interest of many sci-

entists. Intervening with the intracellular controllingsystems appears to be an attractive and highly e�caciousway to alter immune reactivity or subdue unwarranted

reactions to environmental factors or microbial infections.In clinical medicine it has become increasingly important to

control in¯ammatory reactions in autoimmunity andallergy, to enhance anti-tumour immunity as well as toincrease e�ciency in vaccine take.5 Therefore, exploring thepossibilities of immunomodulation, through membrane

receptors, signal transduction or second messengers, mayopen up new therapies and strategies to prevent or treatdiseases in which the immune system is part of the patho-

genic mechanism. In particular, this strategy o�ers thepossibility to design second-generation vaccines with pre-dicted e�ects on critical immune events. As will be

described in the present review, targeted actions of CT orLT on antigen-presenting cells (APC) may selectivelyup-regulate B7.2 (CD86) expression and costimulation,thereby dramatically augmenting T cell priming and vac-

cine e�cacy. By being highly selective, such targeted vac-cines would require less antigen and fewer doses ascompared to current vaccines. They would also be more

economical, safer and easier to deliver.

Mucosal vaccines

Exploitation of the mucosal immune system o�ers severaladvantages from a vaccine point of view. Mucosal vaccinesmay achieve both systemic and local mucosal immune

protection against infectious micro-organisms. There is agrowing interest for intranasal, genital tract, rectal and oralvaccines and the possibility to use such vaccines to protect

against infectious diseases a�ecting not only mucosal sur-faces, but also against diseases like HIV, chlamydia, tetanusetc. Except for the oral polio vaccine, few mucosal vaccines

Correspondence: N Lycke, Department of Medical Microbiol-

ogy and Immunology, University of GoÈ teborg, S-413 46 GoÈ teborg,

Sweden. Email: <nils.lycke@microbio.gu.se>

Received 18 March 1998; accepted 23 March 1998.

Immunology and Cell Biology (1998) 76, 280±287

that use live vectors are used clinically today. Although, ingeneral, live vectors have been found to be more e�cient

delivery systems for oral antigen than vaccines based onnon-living vectors, the clinical use of live vaccines is inmany cases still unclear. For example, concern has been

raised as to their safety, stability and cost. This hasprompted research with the aim of identifying mucosalimmunomodulators/adjuvants that could ®nd general use

in non-replicating mucosal vaccines. However, most solubleprotein antigens are normally poor immunogens at mucosalsurfaces, and when administered perorally they fail tostimulate signi®cant immune responses. On the contrary,

feeding conventional protein antigens will induce oral tol-erance, which is characterized by systemic unresponsivenessto a second challenge with the antigen.6 Thus, unless an

adjuvant is incorporated in the mucosal vaccine, systemictolerance rather than local IgA immunity may be induced.

Should oral tolerance be a concern in mucosal vaccine

development?

With few exceptions, most soluble protein antigens givenorally induce tolerance. It is considered that induction of

tolerance has developed as a means to protect against un-toward reactions to food antigens or to the commensal¯ora.7 Despite many years of research focused on ®ndingwhat distinguishes the induction of oral tolerance from that

of mucosal IgA immunity, relatively little is known. Al-though it is well established that adjuvants are required forstimulation of mucosal IgA responses (whereas oral toler-

ance is readily induced simply by feeding antigen), a basicunderstanding of the regulatory pathways involved is stilllacking. One concern is that a mucosal vaccine that stim-

ulates IgA immunity and concomitantly promotes systemictolerance, may put people at risk to develop uncontrolledinfections if pathogens breach the mucosal barrier. An

unwanted consequence of this scenario would be that hostresistance against pathogenic infections may be reducedrather than increased following oral immunization. Such ane�ect would preclude the use of oral immunization against

HIV, chlamydia and many other infections that evade themucosal barriers. However, most mucosal adjuvants de-scribed so far abrogate oral tolerance while promoting se-

rum IgG and gut mucosal IgA immunity; for example,poly(D,L-lactide-co-glycolide) (PLG) microparticles, im-mune-stimulating complexes (ISCOMs) or the enterotox-

ins, CT and LT.3,8,9 This has raised the question as towhether it is, in fact, necessary to break induction of oraltolerance to achieve mucosal IgA immunity. Current in-formation appears to suggest that this is the case and that

the two phenomena, oral tolerance and mucosal IgA im-munity, are reciprocally regulated and mutually exclu-sive.6,10 Given that this assumption is correct, it will be of

critical importance to evaluate oral vaccine adjuvants withregard to a dichotomy between induction of oral toleranceand local IgA immunity.

Cholera toxin is a powerful mucosal adjuvant

Cholera toxin is perhaps the most potent mucosal adjuvant

we know of today and it has lately attracted much atten-

tion.3,4 The toxin not only greatly enhances mucosal andsystemic cellular and humoral immunity, but it also pre-

vents the induction of oral tolerance when co-administeredorally with unrelated antigens.11 Cholera toxin is composedof ®ve enzymatically inactive, non-toxic B subunits (CTB)

held together in a pentamer surrounding a single A subunitthat contains a linker to the pentamer via the A2 fragment(CTA2) and the toxic, enzymatically active, A1 fragment

(CTA1) of the molecule. Thus, CT forms an AB5-he-terohexamer for which the crystal structure recently waspublished.12±14 The toxic CTA1 has strong ADP-ribosyltransferase activity and is thought to act on several G

proteins. The activity is strongest on the Gsa protein whichis ADP-ribosylated at Arg201.15 This results in activationof adenylate cyclase and the subsequent increase in intra-

cellular cAMP.14 The CTB binds to the ganglioside GM1receptor, present on most mammalian cells includinglymphocytes and gut epithelial cells and, thereafter, CTA is

translocated into the cell membrane/cytosol of the cellwhere the CTA1 and CTA2 are dissociated. The profusediarrhoeal response in cholera is thought to result fromCTA1-induced increased cAMP levels in the intestinal

epithelium. An important issue is, therefore, whether theimmunomodulating property of CT may be separated fromthe toxic property.

Cholera toxin is both structurally and functionallyclosely related to LT.14,15 In fact, LT has been found tohave comparable or even better adjuvant and immunogenic

properties after systemic as well as after mucosal adminis-tration compared to CT.16,17 This is not surprising sinceX-ray crystallography of CT and LT has revealed that these

proteins are highly homologous and demonstrate verysimilar chemical structures.15,18 Although the B subunits ofCT and LT have been found to di�er in their amino acidsequence by � 20%19 and LTB can bind, not only GM1-

ganglioside, but other gangliosides and galactoproteins, wemost often view these toxins as functionally similar. Forexample, disrupting the binding of CTB or LTB to the

GM1 receptor by introducing a mutation in position G33E/D will equally reduce immunogenicity of both molecules.20

However, other reports on mutations in the same position

in the A1 subunit S63K, have demonstrated stronger ad-juvant and immunogenic capability of LTK63 compared toCTK63, which may indicate that CT and LT are func-tionally di�erent.17

Understanding immune regulation at mucosal surfaces

A prerequisite for any attempt to construct immunomod-ulators is to understand the fundamental basis of immuneregulation. Although much has been learnt about antigen

processing and presentation and induction of T and B cells,there is still much research to be done before we can designvaccines or treatments of diseases based on predictedmechanisms of action. Two areas of particular importance

for immunomodulation are cell±cell interactions and cyto-kine regulation as we envisage them in T±B cell coopera-tion, APC costimulation and CD4+ Th1 or Th2

di�erentiation. Mouse strains with targeted gene disrup-tions have provided new powerful tools in immunology.Our group has used several di�erent strains of mice with

CTA1-DD adjuvant 281

de®ciencies in T cell subsets, cell±cell interactions or speci®ccytokines, such as IL-4, to evaluate regulatory pathways in

mucosal immunity.21 These investigations have providedstrong support for the notion that induction of mucosalIgA immune responses and oral tolerance are strictly reg-

ulated. Three main ®ndings are worth mentioning: induc-tion of mucosal IgA responses following oral immunizationis dependent on IL-4 and Th2 cells;22,23 CD8+ T cells ap-

pear to exert local suppression of immune responses;10,24

and costimulation through CD86 may be particularly im-portant for stimulation of mucosal immunity.25 We willreturn to how these regulatory mechanisms can be exploited

to achieve strong IgA immunity using our newly developedconcept for a mucosal adjuvant.

The use of CT adjuvant in these di�erent models pro-

vided evidence to suggest that an important e�ect of apowerful mucosal adjuvant may be to augment the functionof the APC and, in particular, the B lymphocyte.26 There-

fore, we constructed a fusion protein that speci®callytargeted the enzymatically active CTA1 to the B cell antigenreceptor (BCR), hoping to achieve a potent immunomod-ulating compound that would mimic the adjuvant e�ects of

CT, but which would be safe and completely non-toxic.

A novel concept in mucosal adjuvanticity

Given that the assumption was correct that the APC is themain target for CT and responsible for the adjuvant e�ects

in vivo, we constructed a fusion protein that targeted CTA1to the APC. The rationale for this strategy was two-fold:®rst, we had performed experiments in 1992 with LT and anon-ADP-ribosylating mutant of LT, LTE112K, which

clearly demonstrated that the enzymatic activity was ab-solutely required for an adjuvant e�ect following oral ad-ministration.27 In a di�erent system, Liang et al. had

produced similar evidence suggesting that the adjuvantactivity of CT was lost if the enzymatic activity was dis-rupted by glutaraldehyde treatment of the holotoxin.28

Second, we were convinced that the toxic activity of CTwould preclude clinical use of CT as a vaccine adjuvant,because as little as 5lg of CT had caused enterotoxic re-actions in human volunteers.29 Therefore, we reasoned that

a logical solution to the problem of separating enterotox-icity from adjuvanticity was to target the CTA1 subunitaway from most cells by excluding the promiscuous CTB in

the construct. The theory was that if we could target CTA1

to the APC, a non-toxic but highly adjuvant active mole-cule could be constructed. Another rationale for this ap-

proach was that we wanted to test whether we couldspeci®cally target B cells and induce activation of costim-ulatory molecules in these cells that would turn the B cells

into e�ective APC for priming of naive CD4+ T cells.

The CTA1-DD adjuvant

The gene encoding the ADP-ribosyltransferase-activeCTA1 subunit was fused together with the gene for a carrierpeptide that targets to the APC, or more speci®cally the

BCR (Fig. 1). To achieve the targeting strategy we selectedas carrier moiety a synthetic analogue of Staphylococcusaureus protein A, the D fragment, which was known to

avidly bind to both the Fc and Fab fragments of immu-noglobulin, thereby allowing binding to all isotypes.30,31

After some initial failures, the construct came to contain adimer of D, CTA1-DD, to obtain a highly enzymatically

active and su�ciently stable molecule with relatively goodwater solubility.25 On a molar level, the CTA1-DD fusionprotein had 50% of the ADP-ribosylating ability of the

intact holotoxin (Fig. 2). Thus, the CTA1-DD constructproved that it is possible to fuse enzymatically active bac-terial enterotoxins to novel carrier molecules and still retain

good enzymatic function. This in itself was an importantachievement because it enables further exploitation of theconcept of targeted immunomodulation using enzymati-

cally active subunit molecules.As aforementioned, the rationale for choosing the DD

element in the construct was to speci®cally target not onlyimmunoglobulin but the BCR so that we would induce

activation of the B cell, turning it into an e�cient APC forpriming of CD4+ T cells. Although DC are consideredprimary APC for stimulation of naive CD4+ T cells, also B

cells when activated have been ascribed potent APC func-tions.32±34 While DC constitutively express costimulatorysurface molecules such as CD80 or CD86 which, according

to the two-signal model for T cell activation are requiredfor IL-2 production and proliferation, also B cells can beinduced to do so. Our earlier studies had demonstrated thatCT, but not CTB, e�ectively induced the expression of

CD80 and CD86 on resting B cells.25 A similar e�ect of CTon CD86 expression in macrophages was recently reportedby Cong et al., suggesting that also other APC than B cells

exhibit enhanced CD86 expression after exposure to CT.35

Figure 1 Schematic drawing of the pCTA1-DD construct. The pCTA1-DD construct contains the CTA1 gene (aa 1±194) cloned at

HindIII-BamHI and two D fragments from the staphylococcal protein A gene under the control of the trp promotor. The abbre-

viations used, other than restriction enzyme sites are: Ptrp, trp promoter.

282 L AÊgren et al.

Targeting of the CTA1-DD adjuvant to B cells

By targeting resting IgD+ or IgM+ B cells with CTA1-DD,we hoped to mimic the e�ect of CT on these cells. Theexperimental analysis revealed that CTA1-DD, bound to

membrane IgD+ or IgM+ cells both in vitro and in vivo,clearly documenting that CTA1-DD, indeed, targeted Bcells. In fact, CTA1-DD could be retrieved on splenic B

cells 2±4 h after injection. Furthermore, radioactively la-belled CTA1-DD was found to recirculate for several hoursin serum and accumulate in the spleen rather than in the

liver or kidney after injection, implying that CTA1-DD didnot form immune complexes with soluble Ig in serum toany greater extent (L AÊ gren & N Lycke unpubl. data,1998). To our satisfaction, we found that splenic IgM+ B

cells that were treated with CTA1-DD in vitro were alsoinduced to express high levels of membrane CD86.25 Thus,CTA1-DD appeared to promote very similar e�ects as CT

on B cell activation and expression of costimulatory mol-ecules. Whether CTA1-DD also interacted with other APC,either directly or if complexed with free soluble immuno-

globulin via Fc receptors could not be observed, but moreconclusive experiments are underway.

In preliminary experiments, we have also compared the

direct functional e�ects of CTA1-DD on B cells with thoseobserved with CT, assuming that shared e�ects may pointto the mechanism responsible for adjuvanticity. Strikingly,we have found that both CTA1-DD and CT, but not CTB,

support B cell proliferation in the presence of ionomycin,which represents an inductive pathway distinct from theBCR or that used by LPS36 (N Lycke, unpubl. obs., 1998).

This pathway was reported to be cAMP independent, whichmay indicate that CTA1-DD and CT have access to a

pathway that does not involve Gsa, the classical G proteinfor CT. Whether this pathway is critical for adjuvanticity ispremature to say, but preliminary studies demonstrate thatCTA1-DD may not stimulate increases in intracellular

cAMP (L AÊ gren & N Lycke unpubl. obs., 1998). Further-more, CT as well as CTA1-DD do not negatively a�ectinteractions with B cells through the CD40 pathway. By

contrast, CT strongly reduces B cell proliferation afterstimulation via the LPS receptor or the BCR. Interestingly,we observed that CTA1-DD and CT in the context of CD40

stimulation had an enhanced anti-apoptotic e�ect on Bcells, which may explain why CT as well as CTA1-DD werefound to promote germinal centre reactions in the B cell

follicles of the spleen and lymph nodes following immuni-zation (L AÊ gren & N Lycke unpubl. obs., 1998).

CTA1-DD is a powerful non-toxic vaccine adjuvant

The goal was to construct a compound that functionallywould mimic the adjuvant e�ect of CT without being toxic

to the host. With the CTA1-DD fusion protein we weresuccessful in achieving these goals. First, we evaluated theCTA1-DD concept using a panel of established tests fortoxicity, frequently used with CT or LT. The CTA1-DD

fusion protein exhibited no toxic e�ects in any of the assaysystems. It was clearly not enterotoxic, not even in highdoses, since it failed to cause intestinal ¯uid secretion in the

loop test (Fig. 2). Furthermore, it did not elicit footpadoedema, as did CT (Fig. 3). Also, it failed to induce cAMPas evaluated in the thymocyte GM1 receptor-mediated

toxicity assay (Fig. 2). Thus, we had successfully con-structed a completely non-toxic fusion protein that carriedthe full enzymatic activity of the CTA1 subunit.

Figure 2 Analysis of toxicity and enzymatic activity. Toxicity was analysed using the intestinal loop test and the result was

expressed as mg/cm of ¯uid accumulation. The loops were challenged with PBS, 2.5lg of cholera toxin (CT) or 25.0 lg of CTA1-DD

per loop and the ¯uid accumulation was assessed 4 h later. Enzymatic activity (i.e. ADP-ribosyltransferase activity) was determined

by the NAD-agmatin assay.25 The cAMP induction assay was performed as described47 and cAMP increases were determined by a

radio-immunoassay (Amersham). Values are given as mean�SDpmol/107 cells. These values are representative of two experiments.

CTA1-DD adjuvant 283

The adjuvant e�ect of CTA1-DD is comparable to that

of CT

The augmenting e�ect of CTA1-DD on immune responseswas comparable in both magnitude and quality to that ofCT.25 We found that the CTA1-DD adjuvant, together

with conventional antigens such as KLH or OVA, en-hanced speci®c antibody responses and T cell priming e�-cacy to a comparable degree to that of intact CT. The

adjuvant e�ect was impressive, both following mucosal andsystemic immunizations, and the e�ect was also similar tothat of CT with regard to qualitative e�ects on serumisotype distribution and priming of CD4+ T cells. As il-

lustrated in Fig. 4, the enhancing e�ect of CTA1-DD onanti-KLH antibody responses following i.p. immunizationswas comparable to that of CT. The CTA1-DD fusion

protein, therefore, demonstrates that an e�ective mucosaland systemic adjuvant, which in contrast to CT is com-pletely non-toxic, can be constructed by fusions with the

CTA1-encoding gene. The targeted CTA1-DD represents anovel concept in adjuvant design and it convincingly illus-trates that toxicity of CT and LT can be separated from

their adjuvant function.

Is enzymatic activity required for adjuvanticity?

Despite several years of research and many reports ondi�erent immunomodulating properties it is still unclearwhich entity of CT is critical for adjuvanticity. Most in-

vestigators would agree that the adjuvant e�ect requires theintact holotoxin rather than the non-toxic B subunit, whichhas been found to possess only poor or no adjuvant ability.3

Whether the enzymatic activity of CTA1 is needed is cur-

rently an issue of intense investigations. Two di�erentapproaches have been attempted to advance our under-standing in this ®eld. The ®rst approach uses site-directed

mutagenesis of the CTA1 subunit to partly or completelydisrupt the enzymatic activity of the holotoxin.37±41 This

results in a holotoxin, AB5-heterohexamer, which retains

the CTB-binding element. Several of these mutants haverecently been found to exert adjuvant function althoughthey lack ADP-ribosylating activity. As outlined in pre-

vious sections we chose a di�erent approach by expressingthe intact enzymatic activity of CTA1 as a gene fusionprotein, CTA1-DD, devoid of the CTB-binding ele-ment.25,31 The CTA1-DD molecule therefore represents an

adjuvant that exclusively carries the CTA1, whereas allother constructs are AB5-heterohexamers. We can then usethe CTA1-DD adjuvant to ask to what extent the ADP-

ribosylating activity of the CTA1 subunit is required foradjuvanticity.3

To directly address this question we introduced muta-

tions in the CTA1 to disrupt the enzymatic activity (L.AÊ gren, unpubl. data, 1997). We found that following sys-temic immunizations with the mutant CTA1E112K-DDadmixed with KLH or OVA, poor systemic responses and

no adjuvant e�ect were observed (Fig. 4). On purpose, weselected the E112K mutation because that was the one usedby Yamamoto et al., as well as the fact that we had pre-

viously used the LTE112K mutation, which failed to showadjuvanticity following oral immunization.27

Several groups have contributed new and provocative

®ndings to the ®eld by using site-directed mutagenesis toconstruct CT or LT molecules with no-to-variable degreeof enzymatic activity in the A1 subunit. After our initial

report in 1992 on the lack of an oral mucosal adjuvante�ect of LTK112,29 many other single amino acid muta-tions of the A1 subunit of CT or LT have been reported. A

Figure 3 A mouse footpad oedema test is illustrated. Footpad

swelling in the mouse foot oedema assay was measured in mm

and expressed as mean�SD after 24 h of immunization. The

mouse to the right demonstrates extensive swelling of the foot

after s.c. injection of 1lg of cholera toxin (CT), while the mouse

on the left was given 10lg of CTA1-DD. The left foot on each

mouse served as a negative control and was injected with PBS. Figure 4 CTA1-DD, a novel targeted adjuvant that requires

the ADP-ribosyltransferase activity. Mice were given two i.p.

doses with 100 lg of KLH plus adjuvant and 7 days after the

last dose, serum was collected and analysed for KLH-speci®c

total Ig-antibody log10 titres. The response to antigen alone,

KLH, was compared to that with KLH plus 1 lg of cholera

toxin (CT), 20lg of CTA1-DD or 20lg of the enzymatically

inactive mutant, CTA1E112K-DD, added as adjuvants. This

experiment is one of three giving similar results.

284 L AÊgren et al.

list of some of the most important include mutations atArg-7, Asp-9, His-44, Gln-49, Thr-50, Ser-61, Ser-63, His-

70, Glu-79, Val-97, Glu-112, Pro-106, Ser 114, Arg l46 andArg-192.17,37±39,42±44 These mutants have been found tohave no or diminished or even altered ADP-ribosyltrans-

ferase activity/pattern and the ability to enhance eithermucosal or systemic immune responses has varied greatly.The results from these studies are, therefore, not conclusive.

Whereas some investigators report no relationship betweenadjuvanticity and the ability to ADP-ribosylate, othersdocument a close link between these two properties. Theadjuvant activity of most mutants has been assessed after

intranasal immunization and only a few have been tested byoral administration.40 This has to be taken into account inthe evaluation as well as the fact that even CTB has been

ascribed signi®cant adjuvant properties after intranasalimmunization.45,46 At present it appears that both the CTand LT mutants that completely lack ADP-ribosyltrans-

ferase activity host adjuvant properties and that these mu-tants are signi®cantly stronger adjuvants than the CTB orLTB molecules. However, while Douce et al. argue thatthese mutants are not as e�ective as the holotoxins given

intranasally, Yamamoto et al. demonstrated that the CTmutants, CTLE 112K and CT61F, given s.c. or orally wereequally potent adjuvants compared to CT.17,41 Our results

with the CTA1E112K-DD fusion protein directly prove thatthe ADP-ribosyltransferase activity is required for adju-vanticity of CTA1-DD and points to the idea that the intact

holotoxin may have two di�erent mechanisms to provideadjuvanticity: one restricted to the AB5 complex and an-other dependent on the ADP-ribosyltransferase activity.

However, one may speculate that, because the CTB andLTB have been found to be less e�ective adjuvants ascompared to the enzymatically dead mutants or the nativetoxins, it would indicate that the holotoxin AB5 structure

somehow is central to adjuvanticity. If, as argued by thedata from Yamamoto et al., the ADP-ribosyltransferaseactivity is not required,40,41 then what is so critical about

the AB5 structure? One aspect may be stability and resis-tance to proteolysis of mutant holotoxins which has beenfound to correlate with adjuvant activity.44 This would

ascribe a stabilizing, structural function to CTA1, ratherthan an active component of the AB5 complex. In this way,the interaction of the CTB with the cell membrane wouldstill be the main mechanism for adjuvanticity, unless CTA1

is found to structurally associate with molecules that arecritical for immune regulation, such as membrane signalingreceptors. Future studies will hopefully disclose the reason

for the discrepant results.

Conclusion

The novel CTA1-DD adjuvant enhanced speci®c antibodyresponses and T cell priming after systemic as well as mu-cosal immunizations. The adjuvant e�ect of CTA1-DD was

quantitatively and qualitatively similar to that of CT. Themost striking e�ect of this B cell-targeted adjuvant was itsability to augment costimulation including up-regulation of

surface expression of the CD86 molecules on naive IgM+ Bcells. Furthermore, the adjuvant e�ect was dependent on

the enzymatic activity of CTA1 because a mutated, enzy-matically inactive, CTA1E112K-DD construct failed to

exert adjuvanticity. These results provide the basis forfurther exploitation of our innovative concept and thepossibility to circumvent the toxic e�ects of CT simply by

taking away the CTB moiety. Also, this strategy of allowingfor targeting of the immunomodulating activity to de®nedcell populations, will help determine the mechanism for the

adjuvant e�ect of CT.Our studies with the CTA1-DD adjuvant indicate that:

(i) it is possible to introduce ADP-ribosyltransferase ac-tivity into cells via an alternative pathway to the GM1 re-

ceptor pathway used by CTB; (ii) the adjuvant e�ect of CTappears to depend on the CTA1 molecule rather than thewhole toxin; and (iii) the enterotoxicity and the adjuvant

function may, indeed, be separable entities. Preliminaryresults with a mutated non-Ig-binding D fragment hasrevealed that also the binding to Ig is critically required for

an adjuvant e�ect of CTA1-DD (L AÊ gren & N Lycke un-publ. data, 1998).

Acknowledgements

We are grateful to Lena Ekman and Karin SchoÈ n for skilledtechnical assistance. The study was supported by: the WHO

GPV-Transdisease-Vaccinology Programme, the SwedishMedical Research Council, the NIH through grant no. 1R01 AI 40701±01, the Swedish Cancer Foundation, the

Martin Bergvalls and the Nanna Svartz Foundations.

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CTA1-DD adjuvant 287