Immunology 1991 73 249-254

Immunological evaluation of the multiple antigen peptide (MAP) systemusing the major immunogenic site of foot-and-mouth disease virus

M. J. FRANCIS,* G. Z. HASTINGS, F. BROWNt J. McDERMED,4 Y.-A. LU§ & J. P. TAM§Department of Virology R & D, Wellcome Biotechnology Ltd, Beckenham, Kent, U.K., JBurroughs Wellcome Co.,

Research Triangle Park, North Carolina and §The Rockefeller University, New York, New York, U.S.A.

Acceptedfor publication 11 March 1991

SUMMARY

The multiple antigenic peptide (MAP) system for presenting epitopes to the immune system has beenstudied with an immunogenic foot-and-mouth disease virus (FMDV) peptide comprising aminoacids 141-160 ofprotein VPl. Neutralizing antibody responses known to protect guinea-pigs againstchallenge infection were obtained with a single inoculation of 0-8-4 pg of peptide, presented as an

octamer or a tetramer, whereas 20 Mg of a dimer were required to evoke a similar level of antibody.A monomeric preparation did not elicit measurable levels of neutralizing antibody at doses up to 20pg. The octameric MAP was also immunogenic using an aluminium hydroxide adjuvant. Antibodieselicited by the octameric, tetrameric and dimeric constructs differed qualitatively in their reactionwith sequences within the 141-160 peptide. Those against the octamer reacted poorly with peptideswithin the 141-160 sequence, whereas those elicited by the tetramer and dimer reacted preferentiallywith the peptides covering the N-terminal region. The levels of neutralizing antibody obtained withthe octamer and tetramer compare favourably with those obtained when the FMDV peptide isattached to carrier proteins but are lower than. those obtained when it is presented as part of a

peptide-hepatitis B virus core particle. Nevertheless, the ability to elicit protective levels ofneutralizing antibody without the use of a carrier protein would be a distinct advantage in thedevelopment of synthetic peptide vaccines.

INTRODUCTION

There is increasing evidence that protective immune responsesagainst viral infections can be elicited by synthetic peptidescorresponding to critical amino acid sequences provided thatthey are presented appropriately to the immune system.' In thecase of the major immunogenic site of foot-and-mouth diseasevirus (FMDV) serotype 0, comprising residues 141-160 ofprotein VP1, protective levels of neutralizing antibody can beinduced in guinea-pigs following one inoculation of a peptideconjugated to keyhole limpet haemocyanin (KLH).2'3 Thispeptide was immunogenic in the absence of a carrier protein if itwas polymerized either with glutaraldehyde or air-oxidized afteradding a cysteine residue at each terminus.4 Subsequent workshowed that the unconjugated peptide was also immunogenicwhen delivered in liposomes5 or when allowed to form disul-

* Present address and correspondence: Dr M. J. Francis, Dept. ofVirology & Process Development, Pitman-Moore Ltd, BreakspearRoad South, Harefield, Uxbridge, Middlesex UB9 6LS, U.K.

t Present address: Dept. of Microbiology, University of Surrey,Guildford, Surrey GU2 5XH, U.K.

Abbreviations: FMDV, foot-and-mouth disease virus; MAP, mul-tiple antigen peptide; SN50, 50% serum neutralization end-point.

phide dimers via a non-natural cysteine residue added to thecarboxy-terminus.6,7 Furthermore, a tandem repeat of thispeptide with or without an added carboxy-terminal cysteine hasalso been shown to be as immunogenic and in some cases moreimmunogenic than the disulphide dimers (M. J. Francis et al.,unpublished data).8 This carrier-independent activity was due tothe presence of sites in the 20 amino acid sequence which elicitedneutralizing antibodies (B-cell epitopes) and sites capable ofproviding T-cell help for antibody production (Th-cell epi-topes).7

The marked immunogenicity of this peptide in the absenceof carrier proteins and the observation that polymeric deliveryappeared to enhance its activity led us to speculate that it wouldbe a valuable model for evaluating the multiple antigenicpeptide (MAP) system.9"'0 This system provides a method fordirect solid-phase synthesis of a peptide antigen on to abranching lysine backbone and has been used to produce severaloctameric constructs." In this paper we describe dose-responseexperiments with FMDV octameric MAP. We also show that atetrameric MAP is at least as immunogenic as the octamericMAP and that good antibody responses are obtained with twoinoculations of the MAP when aluminium hydroxide, which islicensed for use in man, is used as adjuvant. Finally we haveanalysed the MAP-induced antibody response serologically

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against various copy number peptides and nested sets ofpeptides within the 141-160 sequence.

MATERIALS AND METHODS

Peptide synthesisThe synthesis ofa matrix core with peptide antigen attached wasaccomplished manually by a step-wise solid-phase procedure'2on t-butoxycarbonyl (t-Boc) Ala-OCH2-Pam resin in which0-05 mmol ofAla is present in 0-5 g of resin. The synthesis of thefirst and every subsequent level of the carrier core was achievedusing a 4 M excess of preformed symmetrical anhydride ofN-,NE-Boc-Lys(Boc) in dimethylformamide (HCONMe2, 12ml/g resin) followed by a second coupling viadicyclohexylcarbodiimide alone in CH2Cl2 to give, after depro-tection, a branched core matrix containing two, four or eightfunctional amino groups. The FMDV 01 Kaufbeuren VP1 141-160 sequence (VPNLRGDLQVLAQKVARTLP) was thenadded to the core matrix to produce octameric, tetrameric ordimeric MAP. A monomeric peptide was also synthesized bydirect attachment of the C-terminal proline to the resin. Theprotecting groups for the synthesis of the FMDV peptide wereBoc groups for the -amino termini and benzyl alcoholderivatives for most side-chain amino acids. For all residuesexcept arginine, asparagine, glutamine and glycine, a 1 hr firstcoupling monitored by a quantitative ninhydrin test was donewith the preformed symmetrical anhydride in CH2C12, and asecond coupling in HCONMe2. The coupling of Boc-Asn, Boc-Gln and Boc-Arg (Tos) was mediated by the preformed 1-hydroxybenzotriazole ester in HCONMe2. Boc-Gly was cou-pled with dicyclohexylcarbodiimide alone. The peptide chainswere capped on their -amino group by acetylation with 10%acetic anhydride and 10% diisopropylethylamine in HCONMe2at the completion ofthe MAP. The branched peptide oligolysinewas removed from the crosslinked polystyrene resin supportwith the low-high HF method to yield the crude MAP. Thecrude peptide and resin were then washed with cold ether/mercaptoethanol (99: 1, vol/vol, 30 ml) to remove p-thiocresoland p-cresol, and the peptide was extracted with 100 ml of 8 Murea/0-2 M dithiothreitol/0- 1 M Tris/HCl buffer, pH 8-0. Toremove all remaining aromatic by-products generated in thecleavage step, the peptide was dialysed in Spectrum Por 6tubing, 1000 MW cutoff, by equilibration for 24 hr with a de-aerated and N2-purged solution containing 8 M urea, 0-1 MNH4HCO3/(NH4)2CO3, pH 8-0, for 24 hr. The dialysis was thencontinued in 8 M and then in 2 M urea-all in 0-1 M NH4HCO3/(NH4)2CO3 buffer, pH 8-0, for 12 hr and then sequentially inH20 and 1 M HOAc to remove all urea. The MAP waslyophilized and purified batch-wise by high-performance gel-permeation chromatography. By this technique all peptideswere estimated to be > 60% pure. All purified materials werealso subjected to amino acid analysis and found to contain thepredicted amino acid composition.

Linear peptides covering the N- and C-termini of the 141-160 sequence, used in ELISA mapping studies, were synthesizedusing an adaptation of the Merrifield'2 technique described byHoughten.'3

AnimalsFemale Dunkin-Hartley guinea-pigs (Harlan Olac, U.K.),approximately 12 weeks old and weighing between 450 g and500 g, were used for all immunogenicity studies.

Anti-peptide assayA modification of the indirect ELISA technique described byVoller & Bidwell'4 was used to assay anti-peptide antibodyresponses. Briefly, microplates were coated overnight at roomtemperature with uncoupled synthetic peptide at a concentra-tion of 2 Mg/ml. The plates were washed and test serum samplesat a range of doubling dilutions from 1:10 were added. Afterincubation for 1 hr at 37°, plates were washed and anti-guinea-pig IgG-peroxidase conjugate was added. After a further hourat 370, the plates were washed and an enzyme substrate (0-04%o-phenylenediamine+0 004% hydrogen peroxide in phos-phate/citrate buffer) added. The resulting colour developmentwas stopped with 12 5% sulphuric acid after 3-5 min and theabsorbance at 492 nm measured in a Titertek Multiskan (FlowLaboratories, Irvine, Ayrshire, U.K.).

The A492 values obtained from doubling dilutions of post-inoculation samples were plotted against the logo reciprocalantiserum dilution and the antibody titres calculated by refer-ence to a negative standard (a 1:10 dilution of pre-inoculationserum). The results reported are the means of two tests, usingduplicate wells for each serum dilution in each test.

Neutralization assayThe neutralizing activity of serum samples against 100 TCIDsoof 0 Kaufbeuren virus was determined using a microneutrali-zation test in IBRS2 cells.'5 Each test was performed in duplicateand the results were recorded as the mean log,0 reciprocal of theserum dilution that gave confluent cell sheets in 50% of themicroplate wells (SNso).

RESULTS

Dose-response

Groups of four guinea-pigs were inoculated i.m. with octamericpeptide in doses ranging from 500 pg to 0-032 pg emulsified inIFA. Serum samples collected at 0, 14, 28 and 56 days wereanalysed for anti-peptide and virus neutralizing activity.

A peak anti-peptide titre of 3-7 log,0 was observed 28 daysfollowing inoculation ofa 20 pg dose (Fig. I a). A five- or 25-foldincrease in this dose produced similar, although reduced, titresof 3.5 logl0 and 3-2 logl0, respectively. A fivefold reduction to a4 pg dose resulted in a significantly lower 28-day titre of 2-9log,0. Further reduction to 0-8 pg delayed the anti-peptideresponse, although a titre of 2-5 logo was observed at 56 days.Doses as low as 0-16 or 0 0032 pg produced little or no primaryanti-peptide responses.

Neutralizing antibody response profiles were similar to theanti-peptide responses (Fig. lb). A peak titre of 2-3 log,0 SN50was observed at 28 days in the 20 pg dose group but increasingthe dosage to 100 or 500 pg produced no improvement. Indeed,as with the anti-peptide titres, lower peak titres of 1-8 or 1-7 log,0SN50, respectively, were observed. Reducing the dosage to 4 or0 8 pg also reduced the neutralizing antibody response to 1 9 or1-3 logl0 SNso, respectively, and no response could be detectedwith further reduction to 0 16 or 0-032 pg.

Effect of copy number

Having established the optimal dose for the octameric peptide,the effect of peptide copy number on immunogenicity was

250

Immunogenicity ofFMDV MAP

20jug

,l009g'500ULg

t0 8,HL

0 10 20 30 40 50 60

20 30 40Days post-inoculation

Figure 1. Immune response of guinea-pigs inoculated i.m. with octa-meric FMDV VP1 141-160 MAP in IFA at various doses ranging from500 pg to 0-032 pg. (a) Anti-peptide 141-160 Cys as measured by indirectELISA and (b) FMDV neutralizing antibody response.

studied. Nine groups of four guinea-pigs were inoculated i.m.with the tetrameric, dimeric or monomeric peptides each atthree different doses of 20, 4 or 0-8 pg and emulsified in IFA.Although in molar terms the monomer dose will contain twice asmany copies of the peptide as the dimer and four times as many

copies as the tetramer, all the preparations should containapproximately the same weight of the 141-160 sequence. Forcomparison a further group of guinea-pigs was inoculated with4 pg of the octamer. All animals were re-inoculated with thesame preparations at 84 days and serum samples collected at 7-28-day intervals were analysed for virus neutralizing activity.

The results in Fig. 2 show that the primary response to theoctamer was similar to that seen in the previous dose-responseexperiment. This response was boosted slightly from 1-8 to 2-1logio SN5o following re-inoculation at 84 days. The primaryresponse to the 4 pg dose of tetrameric peptide was also similarto that obtained with the octamer. However, following re-

inoculation at 84 days a higher neutralizing antibody titre of> 2-8 log10 SN50 was obtained within 14 days. The 20 or 0-8 pgdose groups of the tetramer also displayed improved immuno-genicity over that observed with the octamer, giving primaryneutralizing antibody responses of >2-8 and 1-8 logl0 SN50,respectively, at 84 days. In contrast, the dimeric peptide inducedno primary or secondary neutralizing antibody response follow-ing an inoculation of 0-8 pg and only a secondary response (2-2

30 Octamer Teramer

0

.' 250-| |o 20pg peptide

o21-0 - / 1 * 08,.g peptide

0 135 -

_3

0 20 40 60 8Ot 0 20 40 60 8Qt

Days post-primary inoculation

Figure 2. FMDV neutralizing antibody response of guinea-pigs inocu-

lated i.m with octameric, tetrameric, dimenic or monomeric FMDV VPl

141-160 peptides in IFA at doses ranging from 20 6gto 0-8 pg.

-3-00

zco

D 2 00

.0

o 1-5C

._!0 1-0:3G)

z

IFA

A (OH)3

Re-inoculation

0 20 40 60 80Days post-primary inoculation

Figure 3. FMDV neutralizing antibody response of guinea-pigs inocu-lated i.m. with 20 pg ofoctameric FMDV VP1 141-160 MAP in IFA or

aluminium hydroxide [AI(OH)3] adjuvants.

log15 SN5o) was seen with the 4 pg dose. Furthermore, bothprimary (1-8 log1o SN50) and secondary (2-4 logl0 SN50) responses

to the 20 pg dose were lower than those obtained with theoctameric and tetrameric peptides using equivalent doses. Themonomeric peptide was non-immunogenic at all the dosesexamined.

Role of adjuvant

In previous experiments immunogenicity was examined usingpeptides emulsified in IFA. To examine the requirement of a

mineral oil-based adjuvant system, for the immunogenic activityofthe octameric MAP system, 20 pg of peptide were formulatedboth in IFA or with 0-4% aluminium hydroxide. Following i.m.inoculations of two groups of four guinea-pigs at 0 and 56 days,serum samples were analysed for neutralizing activity. Theresponse to the IFA emulsified peptide (Fig. 3) was similar tothat observed previously (2-6 log15 SN5o at 28 days). Incontrast, inoculation of the peptide with aluminium hydroxideadjuvant produced a significantly reduced and delayed primaryresponse, only reaching 0-8 log10 SN50 by 56 days. However,following re-inoculation, titres of > 2-0 log10 SN50 were

observed, although the response appeared to be declining after4 weeks.

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M. J. Francis et al.

4 5 Octamer

40 Tetramer

Dimer.23.5 -Monomer

Z. 251 X n + t+Re-inoculation

20

0 10 20 30 40 50 60 70 80 90Days post-primary inoculation

Figure 4. Analysis of anti-peptide antiserum from guinea-pigs inocu-lated with octameric FMDV VP1 141-160 MAP in an indirect ELISAagainst octameric, tetrameric, dimeric and monomeric preparations.

Serological analysis of anti-octamer antibody

The serological profile of octameric MAP antibodies in antiseracollected from immunized guinea-pigs was analysed in an

indirect ELISA against octameric, tetrameric, dimeric andmonomeric peptides. The results showed that octameric MAPantibodies preferentially recognized octameric peptide bound tomicroplates (Fig. 4). The level of activity against tetramericpeptide was 3-10-fold lower and against the dimeric andmonomeric preparations lower still. Interestingly, antibodies totetrameric and dimeric preparations showed a similar biastowards the octameric preparation in an ELISA, indicating thatincreased avidity of binding to the more complex MAP isincreasing the sensitivity of the assay (data not shown).

Peptide mapping ofMAP antibodies

Guinea-pig antisera against octameric, tetrameric and dimericpeptides were tested on four separate occasions for their activityagainst N-terminal and C-terminal sets of peptides within the141-160 sequence. Indirect ELISA titres against a 141-160 Cyspeptide were within twofold of each other, ranging from 3-5 to3-8 loglo. These served as 100% reference points for all othermeasurements taken.

The antibodies against the octamer showed a slight C-terminal bias (19%); however, reactivity against all of thepeptides within the 141-160 region was low (Table 1). Bycontrast the tetramer antibodies preferentially recognized theN-terminal peptides with 58% of activity against 141-149 Cys.The dimer antibodies also recognized N-terminal peptidespreferentially (13-64%). However, a significant response was

seen against the C-terminal peptide 150-160 (36%). The totalactivity observed against the non-overlapping 141-149 and 150-160 peptides of 100% suggests that antibodies elicited by thedimer preparation are more reactive with shorter linear peptideswithin the 141-160 region than antibodies against the more

complex tetramer and octamer preparations.

DISCUSSION

Previous results with peptides from the major immunogenic siteof foot-and-mouth disease virus have shown that glutaralde-

Table 1. Peptide mapping of guinea-pig MAP antibodiesagainst C- and N-terminal sets of peptides within the

141-160 sequence of FMDV VP1 serotype 01

Anti-peptide antiseraPeptide

on ELISA plate Octamer Tetramer Dimer

141-160 Cys 100* 100 100141-151 Cys 4 25 16141-149 Cys 11 58 64141-147 Cys 4 21 13150-160Cys 19 10 36152-160 Cys 15 6 21154-160 Cys 3 4 8

* Relative percentage ELISA activity taking 141-160Cys titre as 100%.

hyde and Cys-Cys polymerization,4 liposome presentation,5disulphide dimerisation6'7 and synthesis of tandem repeats(M. J. Francis et al., unpublished data)8 appear to enhanceimmunogenicity. In this study we have shown that presentationof multiple copies on a polylysine backbone using the MAPsystem9"10 can further enhance the immune response. Prelimin-ary experiments in guinea-pigs using less well characterizedoctameric MAP preparations revealed that a similar correlationbetween neutralizing antibody and protection to that reportedpreviously by Francis et al.'6 exists in this species. However,experiments will need to be carried out in target species in order toconfirm the protective effect. This is particularly important inview of the poor correlation between anti-peptide neutralizingantibodies and protection in cattle reported previously.6 Thus, aslittle as 0 8 jg ofoctamericMAP can elicit a primary anti-peptideresponse and protective levels of neutralizing antibodies (M. J.Francis et al., unpublished data)'6 and only 20 Mg are required tomake this response optimal. Furthermore, it is interesting toobserve that a lysine dimeric peptide is significantly moreimmunogenic than a monomer and that a tetramer is five- to 10-fold better than the dimer. Indeed, the FMDV tetramericMAP isas good if not better than the octameric MAP in eliciting a virusneutralizing antibody response. Thus, further complexity ofpresentation beyond a tetramer, for FMDV peptide deliveryusing this system, would appear to be unnecessary.

Although it appears that the octamer preparation is prefer-entially recognized in an indirect ELISA by octameric MAPelicited antibodies, as has been observed previously by Tam &Zavala,'7 there is no clear explanation from our antigenicmapping studies for the different responses to the octameric andtetrameric MAP or why both preparations are significantlymore immunogenic than a dimer. However, the antibodieselicited by the octamer, tetramer and dimer preparations differsignificantly in their specificity for shorter linear peptidestretches within the 141-160 sequence. There appears to be areduced recognition of N-terminal residues, thought to beinvolved in virus neutralization,'8-" by octamer antibodies,suggesting that the increased complexity of this peptide whencompared to the tetramer may be detrimental to its biologicalactivity. It also appears that the C-terminal residues in thetetramer and octamer preparations are poorly recognized by the

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Immunogenicity ofFMDV MAP 253

immune system, when compared to the dimer. However, ingeneral the observed results are consistent with other MAPmodel immunogens. Residues located at the N-terminal anddistal from the core matrix are usually most antigenic due totheir flexibility and accessibility. Residues located at the C-terminal and proximal to the core matrix are less antigenic. Suchis the case for the tetramer. Indeed it appears that the flexibilityof the dimer results in the induction of anti-peptide antibodiesthat can be easily mapped using short linear stretches of aminoacids, while an increase in copy number results in a moreorganized and complex molecule and thus antibodies that donot react well with short linear peptides bond to an ELISA plate.

Another interesting feature of the MAP peptides is theirability to elicit significant levels of neutralizing antibodiesfollowing two inoculations with aluminium hydroxide, the onlyadjuvant currently licensed for use in man. FMDV peptideshave previously been shown to be poorly immunogenic wheninoculated with this adjuvant7 unless they were coupled to aprotein carrier.4'5 Therefore, in this respect the octameric MAPpreparation is behaving like a carrier-linked peptide, possiblydue to its improved adsorption to the Al(OH)3 when comparedto that of the 141-160 Cys (M. J. Francis, unpublishedobservation).

Since the MAP system was designed to replace moreconventional methods of peptide presentation using proteincarriers,9 it is interesting to compare the relative immunogeni-city of a 4 yg dose ofMAP with other conjugated forms of thesame FMDV peptide. Previous reports have shown that 100 pgor more of KLH-linked peptide were required for the inductionof significant levels of neutralizing antibody.4'2' This wasimproved approximately 10-fold by presentation of multiplecopies of peptide as a fusion protein with fl-galactosidase.22Using this system only 10 pg ofFMDV peptide component wererequired to elicit 2-0 logl0 SN50. The most immunogenicpreparation described so far is another fusion protein, that ofpeptide fused to the N-terminus of hepatitis B core antigen(HBcAg).23 This 23,500 MW polypeptide spontaneously as-sembles into 27 nm particles with approximately 180 copies ofthe FMDV peptide repeated over the surface. Such particles arehighly immunogenic with a peptide dose as little as 0 2 pgeliciting protective levels of neutralizing antibody. This markedimmunogenicity is principally due to their polymeric nature24and the presence of a number of well defined helper T-cellepitopes in HBcAg.25 Therefore, although the MAP system is animprovement on KLH coupled peptide or fl-galactosidasefusion proteins it is nevertheless 20-fold less immunogenic thana delivery system based on HBcAg fusion particles. It seemslikely that the helper T-cell epitope(s) for guinea-pigs containedwithin the 141-160 sequence7 are insufficient to allow completerestoration of its inherent immunogenic activity within theFMDV particle and that the incorporation of further Th-cellsequences within the MAP is likely to improve activity.26 In thecase ofFMDV, Th-cell sequences appropriate for target specieswould also need to be included in a fully synthetic product toovercome a restricted response to the 141-160 sequence.7" Inthis respect it has been shown that MAP may be designed withadded Th-cell epitopes that enhance the immune response to apoorly immunogenic B-cell epitope.27

In conclusion we have shown that the MAP approach topeptide presentation significantly improved the immunogenicityof an uncoupled peptide from FMDV. However, it appears that

a tetrameric structure is sufficient for an optimal response. SuchMAP are highly immunogenic when inoculated with mineral oiladjuvant and also display marked immunogenicity following adouble inoculation regimen with aluminium hydroxide. Theyclearly offer the opportunity to design totally synthetic pro-ducts, avoiding the requirement for carrier coupling, and couldform the basis of future peptide vaccines.

ACKNOWLEDGMENTS

The authors would like to thank Andrew Syred for peptide synthesis andLorraine Joyce for typing the manuscript. Funding was provided by USPH Al 28701 to James Tam.

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9. TAM J.P. (1988) Synthetic peptide vaccine design: Synthesis andproperties ofa high-density multiple antigenic peptide system. Proc.natl. Acad. Sci. U.S.A. 85, 5409.

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13. HOUGHTEN R.A. (1985) General method for the rapid solid phasesynthesis of large numbers of peptides: specificity of antigen-antibody interactions at the level of individual amino acids. Proc.nat!. Acad. Sci. U.S.A. 82, 5131.

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