Approaches to the treatment of central nervous system

autoimmune disease using speci®c neuroantigen

DAVID O WILLENBORG and MARIA A STAYKOVA

Neurosciences Research Unit, The Canberra Hospital, Woden, Australian Capital Territory, Australia

Summary The ultimate aim in the treatment of autoimmune disease is to restore self-tolerance to theautoantigen(s) in question. In lieu of this ideal result, the conversion of a destructive or pathogenic autoimmune

response into one of benign autoimmunity would also be highly desirable. In either case the use of the antigenicepitope, which is the target of the destructive immune response, would ideally be employed so as to givespeci®city to the protection without the need for long-term immunosuppression. This review describes a number

of di�erent approaches using various forms, doses, and routes of injection of speci®c neuroantigen to inhibit thedi�erent clinical varieties of autoimmune encephalomyelitis in a number of animal models; all done with the viewto translating the ®ndings into the clinic for the treatment of multiple sclerosis. We conclude that any treatment

strategy for multiple sclerosis (MS) must have a number of features: it must be clinically acceptable, speci®c, long-lasting, require only short-term treatment, able to shut o� ongoing disease, and have the potential to prevent ordeal with epitope spreading. Few of the approaches we describe ful®ll all of these criteria. We suggest that

investigations of new adjunctive agents to be used with a speci®c antigen be pursued, and that currently the use ofchimeric proteins or DNA vaccination with or without the new adjunctives may hold the most hope for the future.

Key words: autoimmune encephalomyelitis, multiple sclerosis, prevention, speci®c neuroantigen, suppression,therapy.

Introduction

Therapeutic intervention in autoimmune diseases shouldaim to restore self-tolerance to the autoantigen in question,or at the very least, convert autoimmune disease into non-

destructive autoimmunity. Ideally this would be done usingthe antigenic epitope(s) which is the target of the autoim-mune process such that the need for long-term immuno-

suppression is eliminated. Here we review the use of speci®cneuroantigen for the treatment of central nervous system(CNS) autoimmune disease. This review is not intended as acomprehensive review of the literature on the subject but is

an attempt to describe the varied approaches using speci®cantigen that have been explored by numerous investigatorsover a number of years. Some of the variables we have

considered are the dose and the form of the antigen used aswell as its route of delivery; the timing of antigen deliverywith respect to the natural history of the disease; that is,

does the treatment lead to prevention, suppression ortherapy of disease; the use of adjuncts; the type of diseaseinhibited; that is, acute, chronic or relapsing±remitting; and

the stability of the e�ect.In writing a paper such as this as part of an issue on

`Therapeutic Intervention in Multiple Sclerosis' the tacitassumption is that MS is an autoimmune disease and that

with the eventual identi®cation of the relevant autoanti-gen(s) it might be a simple matter of selecting one of the

approaches to be described here, for the successful treat-

ment of the disease. We would like to state at the outset ourreservations that we are not totally convinced that MS is, infact, primarily an autoimmune disease.1 It most certainly isin¯ammatory and demyelinating, but whether the in¯am-

mation is autoimmune in nature we feel is still an openquestion. Nonetheless, knowledge of how to manipulate theimmune system using speci®c antigen will have usages far

beyond the study of a single disease.

Early studies

Since its early description experimental autoimmune en-cephalomyelitis (EAE) was considered by many investiga-tors to be a reasonably good working model of human

demyelinating disease. It is not surprising, therefore, thatattempts to suppress disease went hand in hand with studiesdesigned to elucidate the pathogenic mechanism(s). As

much as 10 years before the description of what was themajor encephalitogen in CNS tissue (myelin basic protein;MBP)2,3 and even before the de®nitive description of EAE

as an autoimmune disease,4 investigators were using CNStissue itself to inhibit the development of disease. Interest-ingly, it was observed in these early studies that non-encephalitogenic fractions of CNS tissue were ine�ective

whereas encephalitogenic fractions were e�ective at inhibi-ting disease.5±7 This is, of course, explicable today in thelight of what is known about the need for determinants to

bind appropriately to restriction elements to induce animmune (or suppressive) response.

The general thesis in the early studies was that there was

a balance of power between the protective response and the

Correspondence: Dr David O Willenborg, Neurosciences Re-

search Unit, The Canberra Hospital, PO Box 11, Woden, ACT

2606, Australia. Email: <david.willenborg@anu.edu.au>

Received 10 November 1997; accepted 10 November 1997.

Immunology and Cell Biology (1998) 76, 91±103

inducing response produced by the same CNS material;the protective response was referred to as immunity and the

inductive response as hypersensitivity.8 Immunity wasthought to be mainly a function of antibody, and hyper-sensitivity one of the cellular responses linked with delayed-

type hypersensitivity (DTH). This concept was stronglysupported experimentally by the work of Paterson et al.whoshowed both the hypersensitivity component by transfer

with sensitized lymphocytes4 and the immune componentby the inhibition of EAE with transfer of serum.9

Another very useful concept developed in early workwas the distinction that was made with relation to timing of

inhibition. Prevention implied that the inhibiting regimenwas given prior to the challenge with an encephalitogenicinoculum; suppression implied that the treatment regimen

was given after the encephalitogenic inoculum but prior tothe onset of clinical symptoms; and therapy implied that thetreatment regimen was given after the onset of clinical signs

of EAE.10 These terms are still current and should, if theyare not, be used properly to indicate when treatment isgiven in studies of intervention strategies in EAE.

Neuroantigen with adjuvants in immunotherapy

A great many studies have been done using neuroantigen in

the form of either whole CNS tissue,10±12 fractions of CNStissue13±15 or puri®ed encephalitogenic proteins16±19 givenas an emulsion with incomplete Freund's adjuvant (IFA)

for the inhibition of EAE. Alvord et al.10 described the factthat CNS tissue given in water in oil emulsion to inhibitEAE in guinea-pigs was better than when it was given insaline; that prolonged treatment was better than short-term

treatment and that high doses of material were better thanlow doses. It was also shown that suppression was moder-ately better than prevention but that the combination

(treatment before and after sensitization) was most e�ec-tive. Discussion centred around the possibility that treat-ment produced antibodies which bound to antigen and

`neutralized' it both systemically and in the CNS. Mac-Pherson and colleagues13±15 reported that bovine spinal cordprotein (BSCP), which is immunochemically distinct frombovine myelin basic protein (MBP), could, when given with

IFA, be used to prevent, suppress and treat EAE in guinea-pigs. As there was no cross-reaction (at the antibody level)between BSCP and MBP it was postulated that BSCP and

MBP contain an identical amino acid sequence that isrecognized only by T cells.15

Swanborg17 employed puri®ed MBP for the inhibition of

EAE in guinea-pigs. Not only did the whole rat MBPmolecule prevent EAE when given in IFA prior to sensiti-zation (i.e. prevention) but so did the small rat MBP which

lacks a sequence of 40 amino acids which are present in thelarger molecule and which includes part of the major de-terminant which causes EAE in guinea-pigs.17 Other studiesshowed that purposely altering the single tryptophan resi-

due in MBP rendered the molecule non-encephalitogenicbut that this molecule could then be used to prevent EAEwhen given in IFA.16

Driscoll et al.19,20 used MBP in IFA to treat EAE instrain 13 guinea-pigs. Animals were given daily injections of

MBP/IFA s.c. beginning at the time after sensitization whenanimals began to show signi®cant weight loss. Most animals

treated in this way recovered from disease, whereas animalstreated with IFA alone did not. These experiments wereextended later to show that treatment of animals with MBP/

IFA after onset of actively induced disease rendered lymphnode cells from these animals unable to transfer disease tonaive recipients, suggesting that treatment `inactivated' the

sensitized cells.20 As well as acute EAE in guinea-pigs,chronic relapsing EAE (R-EAE) induced by immunizingjuvenile guinea-pigs could be suppressed by treatment withMBP/IFA. Success of suppression was dependent on time

after sensitization when treatment was begun.21

Interestingly, suppression with MBP/IFA has not beenuniformly successful. Hashim22 reported the failure of MBP

to prevent or suppress EAE in guinea-pigs when given inIFA. This was interpreted that the protective regions ofMBP were buried within the encephalitogenic determinants

and their protective function could only be realized aftersome sort of modi®cation. This report was part of an on-going debate at that time as to whether the protectivemoiety in MBP was, in fact, the encephalitogenic part of the

molecule or a non-encephalitogenic or modi®ed por-tion.13,16,18 The answer would seem to rest with the abilityof the molecule to bind to MHC class II with or without the

concomitant ability to bind to the T cell receptor (see sec-tion on peptide).

EAE has been studied in monkeys by several groups,

with the hopes of achieving disease conditions more closelyrelated to human MS23±27 especially with regards to treat-ment. Eylar and colleagues23±25 showed that monkeys

rapidly developed an acute and severe form of EAE andshowed clinical signs not unlike those of patients with MSundergoing acute exacerbations. This EAE proved fatal toall untreated monkeys whereas 70% of animals treated with

MBP/IFA after the onset of clinical signs suppressed dis-ease and the monkeys returned to normal. Further studiesshowed25 that recovered monkeys did not show spontane-

ous relapses but disease could be reinduced by further in-jections of MBP/CFA. This secondary disease was morerapid in onset but was again capable of being suppressed

with MBP/IFA.Of considerable interest is work in monkeys carried out

by Alvord et al.26 and Shaw et al.27 These investigatorsshowed that treatment of two strains of monkeys with high

doses of MBP in IFA failed to inhibit disease unless anadjunct was also used. The adjunct was, in fact, more ef-fective by itself than MBP given by itself.27 The adjunct

varied with the strain of monkey; in one strain it was anantibiotic (penicillin) and in the other a steroid. A closelook at the early studies of Eylar24,25 revealed that they too

used penicillin in the treatment regimen to prevent infec-tion. These results, of course, raise the question as to thepossible need for adjunctive treatments in any trials of

neuroantigen therapy of MS.

Resistance occurring naturally during the course of EAE

Although not studies on ways to inhibit EAE per se, work onthe natural history of the disease has revealed much about

92 DO Willenborg and MA Staykova

mechanisms which regulate disease. EAE in Lewis rats is anacute monophasic disease from which the animals recover.

They not only recover but fail to show spontaneous re-lapses. Furthermore, all animals develop a solid resistanceto active reinduction of disease.28 In an elegant series of

papers, Swanborg and his colleagues have described regu-latory/suppressor cells in recovered rats which are capableof transferring protection against active induction of dis-

ease.29±31 These cells were subsequently described as Tcells32 of the CD4+ phenotype which also inhibit the pro-duction of IFN-c but not IL-2 by EAE e�ector cells.33 Itwas further shown34,35 that this inhibition was due to the

secretion of TGF-b by the T regulatory cells. The value ofthis line of investigation has been that it has provided cluesas to how an animal spontaneously controls an autoim-

mune pathological response and converts it to a non-pathological state of autoimmunity.

Neuroantigen coupled to syngeneic cells

Neuroantigen administered when coupled to syngeneicsplenocytes has been shown to result in a dramatic inhibi-tion of clinical and/or histopathological signs of EAE in bothactive and passively induced disease.36±38 A variety of ne-

uroantigens have been used ranging from whole spinal cordhomogenate39 to MBP and PLP40,41 as well as peptidesderived from these molecules. In virtually all cases the an-

tigen has been coupled to syngeneic splenocytes using ethyl-carbodiimide (ECDI).

Intravenous or i.p. treatment of Lewis rats with guinea-

pig MBP (GP-MBP) or MBP68±88 conjugated splenocytes 1week before active immunization with the same neuroan-tigen in CFA, resulted in a signi®cant reduction in the in-

cidence and severity of both clinical and histopathologicalevidence of EAE.42,43 Following the same protocol, inhi-bition of the autoimmune disease of the peripheral nervoussystem, experimental autoimmune neuritis (EAN), could

also be achieved using splenocytes coupled to the peptide53±78 of the myelin P2 protein.44

That the e�erent limb of the response in EAE can also be

inhibited has been shown by Pope et al.45 EAE induced bytransfer of 4±5 ´ 107 GP-MBP-speci®c lymph node cellswas inhibited by injection of GP-MBP-conjugated spleno-

cytes 2 days after transfer of the encephalitogenic cells.Protection could also be provided by treatment as late as 1day prior to onset of disease. Protection was shown to be

dose dependent, dependent on route of injection of coupledcells (only i.v. was successful) and was exquisitely antigenspeci®c; that is, splenocytes coupled with guinea-pig or ratMBP (which are identical at the encephalitogenic 68±88

determinant) would both inhibit transfer with GP-MBP-speci®c T cells whereas cells conjugated with bovine orrabbit MBP (which di�er considerably from guinea-pig

MBP within the region of the encephalitogenic determi-nant) did not protect Lewis rats.

Chronic relapsing EAE (R-EAE) in SJL/J mice, which

presents with a severe initial disease followed by multiplerelapses and a progressive course, has also been successfullyinhibited using neuroantigen-coupled splenocytes.39±41,46±50

Splenocyte coupled with mouse spinal cord homogenate

(MSCH), MBP, PLP or immunodominant epitopes ofMBP or PLP have all been tested in this model. Thus, the

incidence of clinical and histological signs of active MSCHor PLP139±151 induced R-EAE, and accompanying neuro-antigen-speci®c DTH responses were dramatically reduced

after i.v. injection of MSCH, PLP or PLP peptide-conju-gated splenocytes 7±14 days before priming.38,41,51 MBPconjugated cells, however, were not e�ective in preventing

MSCH-induced R-EAE.47 Neuroantigen-coupled spleno-cytes were also e�ective in treating established disease. In-jecting coupled cells into SJL/J mice after the ®rst episodeof disease signi®cantly reduced the incidence and severity of

subsequent relapses.41,47

Using the model of R-EAE induced by MBP-speci®c Tcells it has been shown that MSCH-conjugated splenocytes

reduced the onset and the severity of relapses when ad-ministered after the ®rst paralytic episode but before theappearance of the ®rst relapse. In contrast, the i.v. injection

of MBP-splenocytes under similar conditions inhibited theinitial clinical relapse but not subsequent relapses, sug-gesting a possible change in the neuroantigen speci®city ofthe e�ector T cells (epitope spreading) in the later relaps-

es.40 Speci®city of the immunoregulation at the e�ectorstage of R-EAE was demonstrated in experiments whereMBP or MBP84±104 coupled cells, but not PLP-coupled

cells, suppressed R-EAE mediated by MBP-speci®c e�ectorT cells.47,49

In all cases of clinical prevention/suppression by antigen

coupled splenocytes, where it has been measured, there hasbeen a decrease in antigen-speci®c T cell proliferation andTh1-derived cytokine production from treated animals.

Prevention/suppression has also been shown to be dosedependent whereas the e�ectiveness of a given route of in-jection varies with the system used. In the Lewis rats,McKenna et al.42 (supra vidae) found that either i.v. or i.p.

injection would inhibit actively induced EAE whereas inadoptive transfer45 or R-EAE in the mouse39,49 only i.v.was e�ective. In all cases there does appear to be an ab-

solute requirement for ECDI in the coupling reaction be-tween neuroantigen and splenocytes for the induction oftolerance. Splenocytes incubated without ECDI bound the

same amount of MBP as did cells treated with ECDI;however, only those splenocytes treated with ECDI prior toincubation with antigen were capable of inducing tolerancein adoptive transfer recipients.45 Tolerance induction is,

however, independent of antigen processing by the sple-nocytes during the coupling reaction as treatment withantigen processing inhibitors (chloroquine) had no e�ect on

coupled splenocytes' ability to inhibit disease. The criticalrequirement for ECDI in the coupling process has led to theinterpretation that such a requirement is related to its abi-

lity to inhibit APC-derived costimulatory signals requiredfor T cell activation,52 thus leading to anergy in the Th1cells.53 Such a mechanism is certainly not the only one

operating in antigen/splenocyte tolerance induction asCD8+ spleen cells from mice tolerized by injection of PLP-coupled splenocytes can transfer resistance to EAE induc-tion in naive recipients, pointing to an active suppressor

e�ect.51

In a somewhat di�erent approach in rat EAE we54 havedescribed the development of a robust, long-lasting inhi-

Speci®c antigen as therapy for autoimmune disease 93

bition of EAE by injection of cell-bound neuroantigen.Lymphoid cells (spleen and lymph nodes) from rats re-

covered from actively induced EAE never (in our hands)directly transfer protection against EAE to naive recipients.Such cells, however, when incubated for 1 h with MBP (in

the absence of any coupling agent), washed and transferredi.v. at 3 ´ 108 per rat into naive recipients do protectagainst active challenge with MBP/CFA. Naive lymphoid

cells could substitute for cells from recovered rats but anessential ingredient in protection in both cases was theconcomitant transfer of anti-MBP antibody to recipientrats. Protection persisted for at least 2 months after the

treatment regimen, was antigen speci®c for the antigen withwhich the cells were incubated and protected against activebut not passive disease. We do not know the mechanism of

protection in this system but the need for antibody in therecipient might suggest some sort of opsonization of thecells leading to alterations in their tra�cking and distri-

bution such that they accumulate in lymphoid areas whereeither APC with `poor' costimulatory properties reside andpresent cell-bound antigen which leads to anergy or theyaccumulate in areas (e.g. the red pulp of the spleen) where

`suppressor' mechanisms are thought to be generated.Another variant on the theme of binding soluble antigen

to a carrier is the use of liposomes55±59 or MBP-galacto-

cerebroside complexes.60 We demonstrated that MBP-lip-osomes (3 : 7 phosphatidyl serine : phosphatidyl choline)injected s.c. were non-encephalitogenic in rats, did not in-

duce antibody or cellular reactivity to MBP nor did theyinhibit subsequent induction of active EAE. Such lipo-somes, however, when given i.v. just prior to onset of EAE,

suppressed disease. The same dose of soluble MBP had noe�ect.55 Treatment of rats earlier than day 10 had no e�ect.Others have been successful in inhibiting EAE in guinea-pigs by prior treatment with MBP-liposomes.56 The same

group also reported inhibition of EAE in Lewis rats. Inthese experiments liposomes were given 7 days prior tochallenge with encephalitogen whereas in our experiments

liposomes were given 4 weeks prior. The time di�erenceand/or a di�erence in the liposome composition may ac-count for the di�erent results. With respect to the latter

point, Stein et al.59 compared di�erent liposome prepara-tions and found that the best suppression of a R-EAEmodel was obtained when giving liposomes prepared fromwhole myelin. This may be similar to the results where

SCH-coupled splenocytes were most e�ective in inhibitingR-EAE.39

Treatment with soluble neuroantigen

Inhibition of EAE

Until rather recently, treatment with soluble neuroantigenshad not received the amount of study as that devoted todelivery of antigens in other forms. This may be partly due

to the belief that the short half-life of some antigens such asMBP in the circulation, due to the actions of proteolyticenzymes, would probably limit the success of such treat-ment. Nonetheless, successful treatment regimens have been

described. Swierkosz and Swanborg reported that treat-

ment of rats with very high doses of soluble MBP i.v. priorto induction of EAE would inhibit disease.61 This protec-

tion was short-lived and was not due to generation ofsuppressor cells.

Our laboratory has examined the use of soluble MBP as

a means of inhibiting EAE in Lewis rats. Taking into ac-count the short half-life of MBP in vivo, we chose to deliverit using mini-osmotic pumps which deliver a constant

amount over a 7 day period.62 This produced a solid, rap-idly developing and long-lasting resistance to the subse-quent induction of MBP-induced EAE. Inhibition was dosedependent (high doses required) and persisted for at least 5

months after the end of the short-term treatment; that is,when the rats were rested for 5 months prior to challengethey remained resistant to active challenge. Similar results

were obtained whether the MBP was infused s.c., i.v. or i.p.A large dose of MBP given as a single bolus was e�ective atinducing resistance but the e�ect was not as long-lived as

infusion of the neuroantigen (DO Willenborg and MAStaykova, unpubl. data, 1997). We examined the immunestatus of animals post-treatment with MBP but prior toencephalitogenic challenge and could not detect any T cell

priming against MBP based on antigen-speci®c prolifera-tion. That priming had taken place, however, was seen inantibody studies following encephalitogenic challenge

where non-treated animals showed no detectable antibodies7 days after encephalitogenic challenge but treated animalshave high titres at this time, indicating a memory response.

Studies on the mechanism of inhibition showed thatsuppressor cells were apparently not involved as we wereunable to transfer protection from donor animals receiving

MBP infusion to naive recipients, even with quite largenumbers of lymphoid cells (3 ´ 108), either ex vivo or an-tigen activated in vitro. The lack of active suppression wasalso supported by data where lymph node, spleen or thy-

mus cells from MBP-tolerized rats had no e�ect on thein vitro activation of MBP-speci®c T line cells. Studies oncytokine production by T cells following challenge of pro-

tected animals as well as studies on the isotype of anti-MBPantibodies produced suggested that pretreatment primedanimals such that subsequent challenge drove the immune

response down a non-pathogenic Th2 pathway rather thanan autoaggressive Th1 pathway; that is, deviated the re-sponse. Similar results to ours have been reported in Wistarrats receiving i.p. injections of MBP some 7±10 days prior

to encephalitogenic challenge. Protected animals showed ahigher incidence and level of IgG1 anti-MBP antibodiesthan untreated animals.63

It has been known for a number of years that the dose ofantigen can determine the type of immune responsemounted;64 that is, either humoral or cellular. The ability of

high-dose systemic MBP to deviate the response in ourmodel might be explained by the fact that the animals hadbeen primed for an antibody response following high-dose

infusion. The generation of a Th2 response is IL-4 depen-dent in both rats and mice.65,66 IL-4 is produced not only byCD4+ T cells but also by non-B, non-T cells of the mastcell-basophil lineage which have high-a�nity receptors for

IgE and IgG1. These antibody armed receptors, whencross-linked by multivalent antigen, trigger the mast cells orbasophils to release IL-4.67 In our system, priming by sol-

94 DO Willenborg and MA Staykova

uble MBP may arm the mast cells and basophils with anti-MBP antibody such that subsequent challenge with MBP-

CFA would result in cross-linking, IL-4 release and in amilieu of IL-4, the development of a Th2 response. Asimilar mechanism might be evoked in our results described

above using the transfer of cell-bound MBP along withimmune serum to induce resistance.54 The serum trans-ferred has subsequently been shown to contain high levels

of IgG1.Soluble antigen has been shown to lead to T cells en-

tering the lymph node paracortex, rather than the follicles,where they subsequently proliferate and disappear, leaving

a population of cells hyporesponsive to subsequent antigenchallenge.68,69 Alternatively, this enhancement of extrafol-licular activity by soluble antigen has been shown to lead to

an increased number of B cells which secrete antigen-spe-ci®c IgM and IgG1 antibodies70 Ð the isotype of antibodyfound in our studies.

The observation has been made that di�erent forms ofsoluble antigen can give similar results yet apparentlythrough di�erent mechanisms. Degermann et al.71 describedin an ovalbumin (OVA) T cell transgenic system that pre-

treatment of recipients of transgenic cells with soluble OVAor soluble OVA323±329 prior to immunization with OVA/CFA both inhibited the development of what would nor-

mally have been a Th1 response against OVA. Treatmentwith protein led to deviation to a Th2 pro®le in transgeniccells whereas treatment with peptide, although inhibitory of

Th1 responses, did not induce Th2 responsiveness. Whetherthis di�erence is related to di�erent types of APC prefer-entially presenting protein or peptide or simply a di�erence

in half-lives of the di�erent antigenic forms remains to bedetermined.

Two main APC costimulatory pathways (CD28/CTLA4± B71/B72 and CD40-gp39) have been identi®ed which are

crucial for T cell activation72,73 and a number of investi-gators have, therefore, explored the possibility of disruptingcostimulatory signals during antigen activation of T cells as

a means of inhibiting autoimmune disease in general, andEAE in particular.74±78 In one study, anti-B7-1 antibodyadministered to mice during disease induction reduced dis-

ease severity76 while in another, treatment with a CTLA-4fusion protein beginning prior to sensitization and contin-ued for 18 days totally inhibited the development of myelin-induced EAE in SJL/KJ mice.75 In yet another study,78

treatment of Lewis rats with anti-CTLA-4 Ig from 2 daysbefore to day 18 after immunization for EAE had a signi-®cant protective e�ect against the lethal e�ect of the dis-

ease. This protective e�ect could be reversed by giving therats daily i.p. administrations of human recombinant IL-2indicating a critical requirement for the costimulatory B7/

CD28 pathway in the development of this CD4+-mediatedautoimmune disease.

In the studies just described, blocking costimulatory

signals was done during or after the challenge with neuro-antigen in CFA. We adopted a di�erent approach in whicha second signalling molecule, LFA-1,79 was blocked duringthe treatment with soluble neuroantigen and prior to

challenge with an encephalitogenic inoculum.80 Low dosesof MBP were infused into Lewis rats over a 1 week periodduring which time three i.p. injections of anti-LFA-1 anti-

body were given. Following such treatment, animals wereresistant to induction of EAE with MBP/CFA and this

resistance persisted for a minimum of 1 month after ces-sation of treatment. Studies on the mechanism indicatedthat, in a similar vein to infusion of high doses of MBP

alone, treatment primed animals such that upon subsequentencephalitogenic challenge the immune response to self-antigen went down a non-destructive (Th2) rather than a

destructive (Th1) pathway. Much lower doses of speci®cantigen were required when combined with anti-LFA-1treatment.

The lack of a good model of R-EAE in the rat has thus

far prevented us determining if the use of short-term pulsetreatment with anti-LFA-1 antibody in conjunction withsoluble antigen is e�ective in deviating an already existing

immune response. The description by Miller et al.77 thatgiving anti-B7-1 antibody alone to mice after disease onsetblocked epitope spreading and subsequent relapses, sug-

gests such an approach might be feasible.We can only speculate on how anti-LFA-1 treatment

apparently deviated the response from a Th1 to a Th2.Previous studies have shown, however, that anti-LFA-1

antibody can activate B cells to both proliferate and up-regulate MHC class II antigen.81 The injection of anti-LFA-1 antibody at the same time as MBP could lead to

activation of B cells with subsequent presentation of MBPthrough the B cells. Such presentation has been shown tofavour Th2 di�erentiation.71,82,83

Suppression and/or therapy of EAE with soluble antigen

Suppression or therapy of EAE with soluble neuroantigenhas also had limited success in a number of studies. Levine

et al. reported successful treatment of EAE in rats using i.v.injection of MBP beginning on the day of ®rst clinicalsigns.84 The most successful results were obtained whenrepeated doses were given at 2 day intervals. Levine and

Sowinski85 then revisited studies on therapy of EAE withsoluble MBP following unsuccessful studies by others onthe use of MBP in the therapy of MS.6,87 They showed that

the most successful treatment of EAE with soluble MBPwas when it was given i.v. This is in contrast to the humanstudies where MBP was given i.m. or s.c. It has also been

pointed out26 that the dose of MBP used in human MStrials was in the order of 1000 times less (on a per kg basis)than the dose used in experimental studies in animals.

Therefore, the di�erence in route of injection, the dose ofantigen and perhaps the lack of adjunctive material27 mayhave been the limiting factor in the failure of therapy in MS.

Another experimental approach for suppression of dis-

ease has been the administration of soluble neuroantigendirectly into the target organ, the CNS. A single dose of12.5 mg MBP, injected into the cerebrospinal ¯uid via the

lateral ventricles shortly before or on the day of EAE onset,was e�ective and decreased the clinical and histologicalsymptoms of MBP-induced EAE in Lewis rats.88 Suppres-

sion of passive EAE in rats can be achieved if MBP isinfused as reported above80 and e�ector cells are trans-ferred within 1 week after treatment. If cell transfer wasdone more than 1 week after treatment there was little, if

any, protection.

Speci®c antigen as therapy for autoimmune disease 95

Critch®eld et al.89 and Racke et al.90 used a passivetransfer system in mice in which naive recipients develop

severe disease after 7±10 days. High doses of MBP wereinjected i.v., three times, twice daily at three di�erent timepoints after the transfer of in vitro MBP-activated en-

cephalitogenic T cells; that is: (i) immediately after the celltransfer; (ii) at the beginning of the acute EAE; (iii) afterrecovery from the acute EAE; and (iv) at the beginning of

the ®rst EAE relapse. In situations (i) and (ii) when ad-ministration of MBP was early in the course of the diseasethere was a dramatic reduction in pathological changeslater in the course of disease, while in (iii) and (iv) there was

a suppression in the severity of R-EAE. Parallel in vitrostudies were done on cells from mice transgenic for a TCRthat recognized MBPAc1-11. The in vitro studies indicated

that exposing activated T cells to high antigen doses led toIL-2 production and rapid cell cycling leading to cell deaththrough apoptosis. Extrapolating from the in vitro data it

was suggested that in vivo, high antigen dose leads to de-letion of reactive cells. Certainly activation-induced celldeath (AICD � T cell elimination by apoptosis) may havebeen the mechanism considering the fact that in order to get

passive EAE the cells must be activated prior to transfer.Challenging with high-dose antigen then in vivo would re-ligate the T cell receptor, up-regulate CD95 (Fas/APO-1),

which interacts with its ligand and leads to cell death.91

We would suggest another possibility for the ability, notonly of soluble neuroantigen, but other forms of antigen to

prevent passive transfer or shut o� an ongoing EAE re-sponse. The concept of an alternative target structure forantibody or immune cells was ®rst suggested by Ferraro5,6

and later experimentally tested by Simon and Nowoczek.92

The concept is simply that the presence of large amounts ofantigen in a particular area may divert immune cells awayfrom the target; that is, the CNS, and prevent disease by

preventing the requisite number of cells from entering theCNS. In our passive transfer experiments into animals inwhich MBP was infused at high dose it may well be that the

cells tra�cked to the area of the pump where antigen mayhave still been present at the time of injection. The fact thatthere was no protection when cells were transferred more

than 1 week after cessation of treatment would support thisconclusion. In the work of Racke et al.90 the high doseinjections of MBP were given beginning at the time oftransfer of encephalitogenic cells. No disease occurred and

they described a decrease in e�ector cells in the spleen oftreated mice. The conclusion was deletion of e�ector cells.The cells may, however, simply have been diverted to other

lymphoid tissue (which was not examined) where they in-teracted with antigen, some perhaps dying via apoptosiswhile others revert to memory cells.

A recent novel approach to suppression and therapy ofEAE is that taken by Elliott et al.93 With the knowledgethat there is a marked heterogeneity of MS patients' T cell

proliferative responses to the two major proteins of themyelin sheath, MBP and PLP, with respect to epitoperecognition, and the existence of progressive T cell reper-toire diversi®cation of such recognition (epitope spreading)

they chose to use two neuroantigens in the form of a chi-meric fusion protein of MBP and PLP, which containmultiple epitopes. This fusion protein (MP4) was en-

cephalitogenic in SJL/J mice when injected along with CFAand produced a disease with a clinical severity quite similar

to that with PLP139±151. When MP4 was injected i.v. twiceper day on days 5, 7, and 9 after immunization withPLP139±151 or MP4/CFA, the subsequent clinical paralysis

was completely prevented. The e�ect appeared to be long-lasting and after cessation of the therapy there was no ev-idence of disease for over 100 days. In addition, a rechal-

lenge 6 weeks after recovery of the control group fromPLP139±151 EAE resulted in a second severe episode of EAEwith an accelerated onset, while all mice in the originalMP4-treated group were resistant to reinduction of disease.

MP4 could also suppress passively transferred EAE.Twice per day i.v. administration of MP4 on days 5, 7, and9, after cell transfer completely prevented the development

of passive acute and relapsing EAE induced by either MBPor PLP-speci®c T cells. Moreover, MP4 ameliorated EAEinitiated by a cotransfer of both MBP- and PLP-activated T

cells where i.v. treatment with only MBP or PLP was inef-fective. Furthermore, MP4 could also alter the course of R-EAE; that is, treat ongoing disease. Treatment given ondays 24, 26, and 28 when the mice were in remission from

the initial episode, prevented subsequent relapses over thenext 3 months while control animals showed several re-lapses. Further studies showed MBP- and PLP-speci®c T

cell proliferative responses were signi®cantly inhibited inMP4-immunized mice treated with soluble MP4 and thatthe chimeric MP4 protein was functionally more stable

in vivo than either MBP or PLP peptides, supporting thesuggestion that the therapeutic potency of tolerogenic pep-tides might correlate with the duration with which func-

tional peptide±MHC complexes can be detected in vivo afterpeptide injection.94 It was also demonstrated that systemicMP4 administration can be e�ective even in Fas-de®cientmice; that is, the eventual mechanism of protection was not

by Fas receptor-mediated activation-induced cell death.95

The resistance of MP4-treated mice to reinduction ofdisease some months after the initial immunization raised

the possibility that i.v. injection of the protein may be ca-pable of mediating clonal deletion of developing MBP- andPLP-speci®c T cell precursors during their maturation in

the thymus. Studies in mice have shown that systemic an-tigen injection can result in depletion of thymocytes of bothimmature and mature phenotypes.96 However, in the MP4system, studies of immune reactivity, other than disease

occurrence, following rechallenge were not done and,therefore, deviation of the response from a Th1 to a Th2 hasnot been ruled out.

Irrespective of the operative mechanism, these resultssuggest that the use of a soluble form of chimeric proteincan induce tolerance to multiple epitopes on MBP and PLP

and may be more e�ective than the use of individual pep-tides in Ag-speci®c therapy where determinant spreadingmight occur.

Neuroantigen peptide-based immunotherapy

The central role of the products of the MHC genes in de-termining immune reactivity has, of course, now been widelyaccepted.97 This, plus the association of certain alleles of

the MHC complex with certain autoimmune diseases, has

96 DO Willenborg and MA Staykova

suggested that the products of these alleles may be recog-nized by T lymphocytes and, therefore, be central to the

disease process.98 With our increasing knowledge of therequirements for peptide binding to the MHC,99±101 anumber of investigators have proposed the blocking of the

binding site of MHC by antagonist peptides, particularlypeptides of high a�nity, as a means of intervention in au-toimmune disease.98,102±115

These intervention strategies have employed ®ve similaryet distinct approaches based on the type and form ofpeptide used: (i) non-encephalitogenic peptides which are,however, part of the encephalitogenic protein;107 (ii) foreign

non-self peptides from proteins which are unrelated to theencephalitogenic protein;109,113,114 (iii) synthetic peptides ofimmunodominant determinants;115 (iv) altered encepha-

litogenic peptides Ð popularly known as altered peptideligands (APL);105,110,111,116 and (v) encephalitogenic pep-tide±MHC complexes.104,106,108

Sakai et al. described two synthetic peptides of MBP,N1-20 and AcN9-20 which competed with the en-cephalitogenic peptide, Ac1-11, in in vitro T cell responsesin the SJL/J (I-Au) mouse.107 These peptides are mutant

constructs of the encephalitogenic molecule which do notoccur in nature; however, they also had the ability tocompete with the self-antigen in vivo resulting in both de-

creased in vitro proliferative responses of lymphoid cells toAc1-11 and protection against clinical signs of EAE. Thesein vivo results were obtained by delivering the inhibitory

peptide and the encephalitogenic peptide emulsi®ed to-gether in CFA. This strongly suggests that the mechanismof protection is competition between peptides for MHC.

Foreign non-self proteins114 or peptides109,113 can alsoin¯uence the interaction between T cells and encepha-litogenic molecules presented by MHC. Gautam and Glynnhave shown that MBP-reactive T cells capable of transfer-

ring EAE to naive Lewis rats can be inhibited from doing soif OVA, preprocessed by macrophages, is incubated alongwith MBP and dendritic cells during the pulsing stage of

antigen presentation.114 Further studies showed that a de-®ned peptide of OVA323±339, which is structurally unrelatedto the encephalitogen (in this case MBPAc1-11 in the SJL/

J ´ PLJ F1 mouse), and is non-immunogenic as well, iscapable of binding to the MHC I-Au.113 When this peptidewas administered as co-immunogen with Ac1-11 to (PLJ ´SJL/J) F1 mice it inhibited EAE. Lamont et al. screened a

number of non-self peptides for inhibition of I-As-restrictedantigen presentation to a T cell hybridoma recognizing animmunodominant epitope in the I-As haplotype.109 From

the panel of peptides one was chosen which had a higha�nity for I-As. When this was co-administered withPLP139±151 in CFA it was capable of inhibiting EAE in the

SJL/J mouse. The peptide was also able to inhibit inducedEAE when it was given (in CFA) 1 day prior to PLP139±151.Longer times between competing peptides were not tried.

Unresponsiveness was not long-lived in that mice protectedby pretreatment 24 h prior to primary challenge were notprotected when a second challenge with PLP139±151 wasgiven 30 days later. These data also suggest that the

mechanism of protection is MHC blockade.The use of synthetic peptides corresponding to the

immunodominant region of native proteins has been used

to induce unresponsiveness to both themselves and to thenative protein and has been successfully employed in the

therapy of EAE. Thus, MBP peptide Ac1-11 can be ad-ministered to mice in IFA either several weeks previously or10 days after induction of EAE with whole MBP and be

shown to inhibit disease.115 The proposed mechanism ofprotection was suggested to be anergy in proliferative, an-tigen-speci®c T cells.

Altering encephalitogenic peptides by a single aminoacid residue can have profound e�ects on their immuno-logical properties. The MBP peptide Ac1-11 is en-cephalitogenic in (PLJ ´ SJL/J)F1 mice. Substituting an

alanine for residue 3 (Ac1-11[3A]) or residue 6 (Ac1-11[6A])produces peptides which are able to bind to I-Au as well asthe native peptide Ac1-11, are immunogenic but are non-

stimulatory for Ac1-11-speci®c T cell clones,103 suggestingthat the inability to stimulate is due to residues 3 and 6 ofAc1-11 determining TCR interactions rather than I-Au in-

teractions. Substituting alanine for residue 4, however,(Ac1-11[4A]) produces a peptide which binds I-Au 10 to100-fold better than the native peptide, stimulates Ac1-11-speci®c T cell clones in an enhanced fashion, but is non-

immunogenic and non-encephalitogenic.103,105 De®ningresidue 3 as a T cell recognition determinant and residue 4as the I-Au binding determinant made it possible to design a

peptide (Ac1-11[3A,4A]) that binds strongly to I-Au butdoes not stimulate the encephalitogenic T cells.103 Inter-estingly, this peptide does not inhibit EAE induced with the

native Ac1-11 whereas Ac1-11[4A] does.105 Ac1-11[4A] canbe administered some weeks prior to immunization or justat the onset of disease and will inhibit the development of

clinical signs of EAE. The fact that Ac1-11[3A,4A] andAc1-11[4A] are structurally similar and bind to I-Au withequal a�nity suggests that inhibition of EAE by Ac1-11[4A] is not due simply to competitive binding to I-Au.

That the altered peptide ligand (APL) approach is notsomehow unique to MBP Ac1-11-induced EAE in the(PLJ ´ SJL/J)F1 mouse was shown by the ability of altered

MBP peptide 87±99 to alter EAE in the Lewis rat.110 Thepeptide was altered at seven distinct sites by substituting al-anine for the native amino acid; four sites were those which

interact with the MHC and three with the TCR. Some ofthese peptides competed very e�ectively with native p87±99for binding to MHC and could also antagonize the responseof T cells to native peptide. Only one, however, (p87±99

[91K>A])was able to prevent and reverseEAEeven thoughthis peptide was less e�ective in the above two parameters.This indicated that the extent of MHC blockade or TCR

competition does not predict success in treating disease.One further example indicating the apparent universal

nature of APL therapy is the demonstration that proteo-

lipid protein-induced EAE in the SJL/J mouse can also beinhibited by treatment with the PLP139±151 altered at vari-ous sites.116 Thus, alterations at both residues 144 and 147,

the two principal TCR contact residues, generated a peptidewhich, interestingly, inhibited not only PLP139±151-inducedEAE when animals were pretreated with the peptide, butalso inhibited EAE induced with the PLP178±189 and the

completely unrelated encephalitogen MOG92±106. Thispeptide apparently is able to induce regulatory cells capableof producing bystander suppression.

Speci®c antigen as therapy for autoimmune disease 97

In the majority of peptide therapies thus far described,the peptides were given in either IFA or CFA in order to

induce the protective e�ect. The use of such adjuvantswould seem to preclude such an approach in the treatmentof human autoimmune diseases. Some studies110,111 have

shown, however, that soluble peptides can be e�ective in thetreatment of EAE.

Soluble MBP87±99 or altered MBP87±99[91K>A] could

inhibit EAE induced by transfer of activated MBP88±99-speci®c T cells when given on day 2 or days 2 and 4 aftertransfer.110 Importantly, MBP87±99[91K>A] could alsoreverse disease when given after the onset of clinical signs.

More recently111 another altered peptide MBP87±99[97R>A] has been shown to also be able to inhibit EAE whengiven in soluble form. Comparing MBP87±99[97R>A] with

the activity of MBP87±99[91K>A] it appeared that treat-ment with the latter induced cytokine shifts from Th1 toTh2 in the target T cells while the former caused deletion of

MBP87±99 responsive cells.A ®nal approach using peptides therapeutically is to

employ the peptide complexed to the respective MHCmolecule.104,106,108 Complexing either MBP91±103 or

PLP139±151 with soluble I-As protected against EAE in theSJL/J mouse induced by the respective peptide-speci®c Tcell lines or active peptide immunization.106 Similar ap-

proaches have been reported for the therapy of experi-mental myasthenia gravis.104,108 Currently, a clinical trial isbeing done in which detergent-solubilized MHC from MS

patients with the DR2 haplotype are loaded with theMBP84±102 (thought to be the dominant MBP peptide inman) and injected in soluble form back into the patient (EG

Spack, pers. comm. 1997). The complexes appear to have ahalf-life of hours rather than minutes for peptide alone,have good biodistribution and are well tolerated by thepatients. In an open trial of 11 patients, six developed

peptide-speci®c responses and some may have shown adecrease in MBP-reactive T cells. A larger controlled trial isunderway but results are not yet available.

In summary, peptide therapy can be used to inhibit andeven reverse the clinical course of EAE. Peptides can beadministered as non-encephalitogenic peptides from the

encephalitogenic molecule, as totally unrelated foreignpeptides, as encephalitogenic peptides in native form, as al-tered peptide ligands or as native encephalitogenic peptide/MHC. Administration of peptides can be done with CFA or

IFA or, in some cases, in soluble form. The mechanismsinvolved in inhibition may be competition for the MHCbinding site, deletion of speci®c antigen-reactive T cells,

deviation of the immune response from a pathogenic Th1 toa non-pathogenic Th2 response, or bystander suppression.

The limitations of some of these approaches with respect

to application to human autoimmune diseases are apparent.Methods which appear to act by MHC blockade109,113,114

would require repeated and continual delivery of peptides.

This problem is not insurmountable and, in fact, Copoly-mer 1, which may exert its limited bene®cial e�ect by actingas a competitor for MHC binding, is currently in the clinicfor the therapy of MS and is, in fact, given on a daily basis

(see article by Bashir and Whitaker, this issue).117 A ther-apy which would inhibit reactivity to multiple neuroanti-gens following treatment with a single epitope would be a

most desirable one. Unfortunately, such a therapy as re-cently reported required the use of CFA.116

Miscellaneous approaches

A unique approach related to the use of speci®c neuroan-

tigen is that of DNA-based immunization. With DNA-based therapy, immunization is achieved not by the injectionof antigen but by the injection of bacterial plasmid DNA

encoding the gene(s) for those antigens. The genes are en-coded under a mammalian promoter/enhancer that enablesthe gene(s) to be expressed in mammalian cells.118 Several

methods have been developed for delivery of DNA vaccinessuch as i.m. injection, liposomes and particle-mediated genetransfer (`gene gun') into the skin. There is evidence that the

various delivery systems can produce di�erent immune re-sponses to the same gene products.119,120

Most DNA vaccination has been directed at inducingprotective immunity to infectious agents; however, we have

applied this approach to the inhibition of autoimmunedisease.121 Using `gene gun' delivery of the MBP genecloned into the expression vector pJW4303 to immunize

rats we have shown that rats convert to seropositivity toMBP, the antibody produced is of the IgG1 isotype, and therats are subsequently resistant to challenge with MBP-

CFA. The mechanism of protection would appear to be thedeviation of the immune response from a normally de-structive Th1 to a non-destructive Th2 type. We have now

to determine if such treatment can be e�ective in a chronicrelapsing type of disease.

Exceptions to therapeutic bene®t of administering

speci®c antigen

Not surprisingly treatment with speci®c antigen has not

always been successful in autoimmune disease as pointedout above.22 Not only has lack of success been reported butalso adverse e�ects. Oral delivery of antigen has been

shown to lead to tolerance in a number of systems;122

however, Blanas et al. have reported that such adminis-tration of autoantigen in mice was found to induce a cy-totoxic T lymphocyte response that could, in point of fact,

lead to the onset of autoimmune diabetes.123

Another example is in a primate model of EAE inducedwith myelin oligodendrocyte glycoprotein (MOG).124

Marmosets which received soluble recombinant MOG i.p.from day 7 to day 18 after immunization with MOG/CFAwere protected against acute disease but after cessation of

treatment developed a rapidly progressive lethal demy-elinating disease. The MOG-speci®c T cell proliferationresponses and the lymphocyte cytokine pro®le showed atemporal (during the treatment) shift from a Th1-like to a

Th2-like pattern. The four- to eight-fold increase in serumanti-MOG antibodies contributed to the development ofmultiple lesions in the cerebral hemispheres and spinal cord

(anti-MOG antibodies unlike anti-MBP and anti-PLP an-tibodies have demyelinating activity125). Thus, the induc-tion of a MOG-speci®c Th2 response may exacerbate

autoimmunity by enhancing production of pathogenic au-toantibodies.

98 DO Willenborg and MA Staykova

Conclusions

Themechanisms involved in the prevention, suppression and

therapy of EAE by administering speci®c neuroantigen(s)are varied and may be attributed to clonal deletion, anergy,ignorance, immune deviation, a combination of these or

other, as yet, unknown mechanisms. Which mechanism isoperative is probably determined by which APC deals withthe antigen and in which cytokine environment, both of

which, in turn, are dependent on the dose, the route, the useof adjunctive, the form, and the timing of that antigen.

Confronted with this formidable array of variables inanimal studies, even assuming knowledge of the responsible

neuroantigen, it is extremely di�cult to attempt to design atherapeutic strategy for the human disease MS. Any strat-egy would ®rst and foremost have to be clinically accept-

able. It should then have a number of ideal features, such asextreme speci®city; a long-lasting e�ect; a short-termtreatment regimen; ability to shut o� ongoing disease and/

or at least prevent relapses; and the capability of controllingpotential epitope spreading.

Some of the studies described in this paper come close toful®lling these criteria while others lack one or more re-

quirements. Our studies, for example, with soluble antigeninfusion or second signal blockade would be clinically ac-ceptable, are long lasting and require only a short-term

treatment. Unfortunately, it has not yet been shown thatsuch treatment would be e�ective in altering disease in apresensitized individual. Intraventricular injections of an-

tigen would be clinically unacceptable as would the use ofIFA, calling into question many of the various peptideapproaches. Antigen-coupled splenocytes are clinically ac-

ceptable, require one injection, shut o� ongoing disease and,in some cases, prevent relapses. The stability of the e�ect isnot known. The chimeric protein approach93 would seem tocurrently ®ll most of the criteria, exhibiting even a long-

lived e�ect and the apparent control of epitope spreading.The fact that some of the peptide studies116 showed es-

sentially all the requirements except for the use of IFA

suggests that perhaps a search for clinically acceptable ad-juvants which produce the same e�ect when used with thepeptides should be pursued. We, in fact, have looked ex-

perimentally at two adjuvants, gamma-inulin and a variantcalled algammulin126,127 which have been used clinicallyand whose composition is thought to be adjustable suchthat a Th1 or Th2 response to a given antigen can be se-

lectively achieved. In pilot experiments, injecting MBP-gamma-inulin s.c. produced no clinical or histological signsof EAE in rats and established a state of resistance to

subsequent challenge with MBP in CFA. Work remains,however, to establish if the other requirements of an idealstrategy are ful®lled.

Returning ®nally to the chimeric protein93 and the DNAvaccination121 approaches, we suggest that in the long termthese will be the most e�ective. Stringing a number of

proteins together or producing a plasmid with a number ofgenes for those proteins and inducing some form of toler-ance to a number of epitopes would deal with the problemof epitope spreading. Such molecules would also be useful

in treating what may, in fact, be quite separate forms ofMS. Recent studies by Berger et al.128 have looked at the

topographic distribution of in¯ammatory lesions in EAEinduced by T cell lines with di�erent antigen speci®cities Ð

MBP, MOG, MAG, S-100 protein and GFAP Ð andclearly shown that lesion distribution and composition weredetermined by antigen speci®city. This suggests that, for

example, spinal MS may have MBP as a target antigenwhile cases with prominent eye involvement may haveS-100 as the target, and again involvement of periventric-

ular or cerebellar white matter may result from anti-MAGor -MOG activity.

Acknowledgements

This work was supported by the National Health andMedical Research Council of Australia (DOW), National

Multiple Sclerosis Society of Australia (DOW & MAS) andthe Canberra Hospital Private Practice Trust Fund (DOW& MAS).

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