Antibody responses to non-immunogenic synthetic peptides induced by co-immunization with immunogenic peptides

8/6/2019 Antibody responses to non-immunogenic synthetic peptides induced by co-immunization with immunogenic peptides

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Immunology 1992 77 262-266

Antibody responses to non-immunogenic synthetic peptides induced byco-immunization with immunogenic peptidesC. D. PART IDO S, O . E . OBEID & M. W. STEW ARD Molecular Immunology Unit, Department of Clinical ScieLondon School of Hygiene an d Tropical Medicine, London

Accepted for publication II June 1992

SUMMARYChimeric pwptides comprising B- and T-helper cell epitopes from the proteins of infectious agentsrepresent immunogens with potential for use as new vaccines. However, it has become clear that theorientation ofth e epitopes. the presence of spacer residues and the number of copies ofthe epitopesinfluence the specificity, levels and affinity of the antibody produced following immunization withsuch constructs. Furthermore, the response to peptides is under genetic control leading to majorhistocompatibility complex (MHC)-linked non-responsiveness. In this study, we have investigatedthe potential of co-immunization of immunogenic peptides (to provide T-cell help) with non-immunogenic peptides (representing B-cell epitopes) to overcome the non-response to the latter. Forthis purpose, we have employed peptides representing T- and B-cell epitopes derived from thesequences ofthe fusion and haemagglutinin glycoproteins of measles virus. The results obtained showthat simple co-immunization of a B-cell epitope with a T-cell epitope results in the production ofantibody to the B-cell epitope without the requirement for covalent linkage ofthe two peptides. Thisapproach could thus be used to overcome the problem of poor imm unogenicity of peptides and willbe of f)otential value in the design of immunization strategies using synthetic immunogens.

INTRODUCTIONOUT current understanding of the induction of an antibodyresponse to a T-cell-dependent antigen suggests the requirementfor co-oi3eration and communication between B and Th cells.Early studies using haptenated molecules have shown that forthe induction of an immune response to the hapten it had to becovalently linked to a carrier mo lecule.' In addition, it has beenshown that immunogens are recognized not only by immuno-globulin receptors on B cells but also by T-cell receptors whichbind a processed fragment of the antigen in association withclass II major histocompatibility complex (M HC ) molecules onantigen-presenting cells.^ These results suggested that B- and T-cell epitopes need to be linked in order for Th cells to providecognate help for B-cell activation and antibody prod uctio n.' Onthe basis of these concepts, there have been numerous reportsshowing the successful construction of effective synthetic imm u-nogens by the combination of well-defined Th determ inants andB-cell sites representing neutralizing or potentially protectiveepitopes,** U nlike cognate help , T-cell-dependent B-cell acti-vation can also occur without the requirement for covalentlinkage of B- and Th-cell determinants (bystander help). Earlystudies showed that Th cells primed to a particular carrier.

support secondary antibody responses to a hapten linked tdifferent, non-cross-reactive anti gen .''" Jensen and K ap p" hshown that co-imm unization of non-imm unogenic pork inswith immunogenic sheep insulin (or foreign proteins), ovcomes non-responsiveness to the former, in mice of H-2'' and2^ haplotypes. In addition. Good et al.'^ have shown thapoor imm une response to a synthetic peptide from the malcircumsporozoite protein can be overcome by co-imm unizaof the peptide with keyhole limpet haemocyanin (KLH).On the basis of these findings we have investigated possibility of overcoming non-responsiveness to a synthpeptide by co-immunization ofthe peptide with an immunonic peptide. For this purpose, we used previously studpeptides derived from the fusion (F) and haemagglutinin surface glycoproteins of measles virus.The results obtained d emonstrate that simple co-immuntion of peptides representing B-cell epitopes with peptrepresenting Th determinants results in the productionantibody to the B-cell epitopes without the requirement forcovalent linkage of the B- and Th-cell epitopes. This approcan thus be used to overcome genetically controlled nresponsiveness to synthetic peptides.

MATERIALS AND METHODSCorrespondence; Professor M. W. Steward, Molecular Immunology Selection of peptides

U nit, Department of Clinical Sciences, London School of Hygiene and Stu die s on th e anti ge nic s tru ctu re of m easle s virus F an dTropical Medicine, London WCIE 7HT, U .K. glyc opro teins hav e show n several region s to represent po ten


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Antibody responses to non-immu nogenic synthetic peptides 26 3

ral protein PeptideImmunogenicity inBALB/c and SWR

240-252288-302258-277

49-72188-199-I --f-

Antigenic siteB

T/BT/BT/BB

and Th-ccll epitopes (Table I). Among these, peptide 240-252found to represent a B-cell epitope In the F protein and to beTO , CBA and C57BL/6 but not in BALB/c and

ferent H-2 type (ref. 14, C. D . Partido s, O. E. Obeid and M.results). The sequence 185-195 oftheB-celi epitojje which can

rom the H molecule has been shown to contain both B- andantigenic sites and was chosen for this study (C. D., O. E. O beid and M . W. Steward, unpublished results).

chemistry. In addition a chimeric peptide was produced infthe 288-302 peptide using a proline residue

esters by treat-

emoved and the peptide was cleaved in trifluoroacetic acidpurified by preparative high-perfor-id chrom atography (HPL C) and their purity assessedytical HPL C and amino acid analysis. A cysteine residuethe 240-252 and 288-P-240 peptides

responses to synthetic peptide 240-252 were measured6-8-week-old female mice.micrograms ofth e 240-252 peptide was injected as

with eithe r 100 /ig of 288-302 or 100 /ig 258-277emulsified in complete Freu nd's adjuvant (CFA ) (1:1).

Eleven weeks after priming, mice were boosted by the sameroute and w ith the same dose of peptides in incomplete Freun d'sadjuvant (I FA). Blood sam ples were collected every 2 weeks,centrifuged and sera were collected and stored frozen a t - 2 0 ' .For proliferative responses a group of four mice were immu-nized via the hind footpad with a mixture of 50 /ig ofthe 240-252peptide and 50 ng of the 258-277 peptide emulsified in CFA.Antibody assaysTitres of antibodies against the peptides were determined by anenzyme-linked imm unosorbent assay (EL ISA). Wells of micro-titre plates were coated overnight at 4" with 50 /il/well of 5 /ig/mlsolution ofthe relevant peptide in carbon ate-bicarb onate buffer0-1 M, pH 9-6. Plates were washed five times with tap water priorto and following blocking with 1% gelatin in phosphate-buffered saline (PBS) for 2 hr at 37 , dried and stored at 4'. Serawere titrated across the plate by serial twofold dilutions indiluent (PBS/0-25% gelatin/0-005% Tween 20) and incubatedfor 1 hr at 37 \ After washingfive imes with tap water 50 /il ofrabbit anti-mouse IgG-peroxidase conjugate (Nordic, Tilburg,The Netherlands) at a dilution of 1/1000 in the PBS/gelatin/Tween diluent was pipetted into each well and the platesincubated as before. After washing five times with tap water toremove unbound conjugate, bound enzyme was detected byadding 50 /(I/well of a chromogen solution (0-04% o-phenylene-diamine-1-0-004% hydrogen peroxide in 0 1 M citric acid/phosphate buffer pH 5-0). After 10 min, the colour reaction wasstopped with 25 //I/well of 2 M sulphuric acid and the absorba nceat 492 nm was measured on a Titertek Multiskan (FlowLabo ratories, Irvine, U.K .). Titres are expressed as logio of thereciprocal of the antibody dilution giving an OD greater than0-2 at 492 nm.Lymphocyte stimulation assayEight days after im munization, the draining lymph nodes wereremoved aseptically, pooled and the m ononuclear cells collectedby centrifugation. Viable, unfractionated lymph node cells(4 X 10') in 0-2 ml RPM I-1640 medium, supplemented with 1%autologous serum, 2 mM L-glutamine, 10 mM HEPES and 100/ig/ml antibiotics, were incubated in the presence of variousdoses of the peptides or medium alone in humidified 5% CO2atmosphere at 37 for 5 days. The cells were pulse-labelled with 1/iCi ['H]thymidine in 10 /il volumes/culture 16-18 hr beforeharvesting. Thymidine incorporation was assessed by liquidscintillation spectrometry and the results expressed as the meanc.p.m. from triplicate cultures SD.

R E S U L T SThe effect of co-immunization on the genetic control of antibodyresponses to the 240-252 peptidePeptide 240-252 is non-immunogenic in H-2^ and H-2^ micewhen administered free in Freund's adjuvant. However, immu-nization with a mixture of 240-252 and the immunogenic 258-277 peptide in CFA resulted in the induction of a primaryimmune response to bo th peptides in BALB/c and SWR/J mice(Fig. 1). A booster injection o fth e same mixture of peptides inIFA 11 weeks after priming resulted in a secondary antibodyresponse to both peptides. Thus the presence of B- and T-cell


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264 C. D. Partidos, O. E. Obeid & M. W. Steward

20 40 60 8 0 100 120

2 0 40 60 80 100Days post-immunization 120

Figure 1. Effect of co-imm unization on the immunogenicity o fth e 240-252 peptide in BALB/c (a) and SWR/J (b) mice. ELISA titres(mcanSD) in sera from sequential bleeds of groups of four miceinjected on Days 0 and 7 7 . Anti-240-252 titres in mice injected with 240-2 5 2 alone (): anti-240-252 titres in mice co-immunized with the 240-252and 258-277 peptides ( D ) ; anti-258-277 titres in mice co-immunized withthe 240-252 and 258-277 peptides ().

Co-lmmunogen Immunogeni.p. 240-252i.p. 288-P-240

i . p . 2 B 8 - 3 0 2 I. p . 2 4 0 - 2 5 2i.p. 2 8 8 - 3 0 2 S . C. 2 4 0 - 2 5 2

i.p. 2 4 0 - 2 5 2i.p. 2 8 8 - P - 2 4 0

i.p. 2 8 8 - 3 0 2 I .p . 2 4 0 - 2 5 2i.p. 2 8 8 - 3 0 2 S . C. 2 4 0 - 2 5 2

2 3 4 5g ^ anti-240-252 antibody trtre

Figure 2 . ELISA titres of antibody to the 240-252 peptide in BALB/and SWR /J (b) mice after co-immunization with the 288-302 peptidthe same or different sites. Results represent mean + S D of titreantibody from groups of four mice bled 2 weeks after the booimmunization.

240-252 peptide in non-responding mice. Thus, BALB/c mwere co-immunized with 240-252 and 258-277 peptides to this possibility. Table 2 shows that co-immunization did result in a detectable 240-252 specific proliferative response

TaUe 2 . Proliferative responses of lymph node cells from BALB/c micefollowing co-immunization with 258-277 peptide. Lymphocytes werestimulated i n vitro with varying concen trations of the 258-277 peptide,the 240-252 peptide and with peptide 284-302 as a control. Valuesrepresent the mean c.p.m. SD of ['HJTdR incorporation of triplicatecultures. Values in bold type represent significant stimulation

M g peptide/well 258-277 240-252 284-3022010

10 1Medium

31334220649,572 238733,647 522114,404 18 703215765

2664 7292 8 6 1 52939O3I1O53 6 2 1 12653215765

6262 2536439 3914332I1404083 5053215765

epitopes on the same molecule is no t a necessary requirement forthe induction of an antibody response to the 240-252 peptide.Tbe effect of co-iaununizatton of 24 0-252 w ith tiie 258-277 peptideon tlie proBferative responses of lymphocytes to IM^lSl peptidePreviously it has been shown that 240-252 peptide was able toinduce antibod y an d proliferative responses in mice of H - 2 ' , H-2^ and H-2'' haplotypes but not in strains with the H-2'' or H-2''haplotypes." Since co-immunization of 240-252 with the 258-277 peptide resulted in the induaion of anti-240-252 antibodyresponse (Fig. 1) the possibility exists that the 258-277 peptideenhanced the induction of T-cell proliferative response to the

Induction of antibody responses to the 240-252 peptide in nresponder BALB/c and SWR/J mice after co-immunization wthe immunogenic 288-302 peptideThe observa tion that non-respwnsiveness to the 240-252 pepcould be overcome by co-immunization with an immunogepeptide was further tested by using peptide 288-302 as a immunogen. This peptide behaves as a Th epitope in varimouse strains including BALB/c and SWR/J.'-''' Followingsame co-imm unization protocol as above, mice ofth e H-2'' H - 2 ' ' haplotype were shown to produce anti-240-252 antibresponses (Fig. 2). Furthermore, these responses were comable to the response induced by the chimeric construct 288240 in SWR/J mice but in BALB/c mice, responses wsignificantly lower. Both antigens must be injected in the ssite since intraperitoneal injection of the immunogenic 288-peptide followed by a subcutaneous immunization of 240-peptide did not result in an anti-240-252 response in either ostrains of mice tested (Fig. 2).

Induction of antibody responses to the 188-199 peptide afterimmunizadon with tiie immunogenic 49-72 peptideTo test further the validity ofthe observation that B - and T-epitopes do not need to be linked in order to develop a T-cdependent anti-peptide response in vivo, BALB/c and SWmice were immunized with a mixture containing the nimmunogenic B-cell epitope 188-199 and the imm unogenic7 2 peptides from the measles virus H g lycoprotein. As showFig. 3 both strains of mice developed a primary anti-188-


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Antibody responses to non-immuno genic synthetic peptides 26 5

20 40 60 80 IOO 120

20 40 60 80 IOO 120Days post-Immunization

Figure 3. Kinetics of the antibody response to the 188-199 peptide inBALB/c (a) and SWR/J (b) mice after immunization with the 188-199f)eptide alone (D) and after co-immunization with the 49-72 peptide (),Results represent mean + SD of ELISA titres of antibody in sequentialserum samples from groups of four mice.

response which was greatly enhanced after a secondary immuni-zation,DISCUSSION

The results reported in this study demonstrate that covalentlinkage of B and Th epitopes is not a necessary requirement forthe generation of a T-cell-dependent antibody response in vivoto non-immunogenic synthetic peptides representing B-cellantigenic sites. That a Th-cell epitope such as 288-302 or 258-277 is needed to overcome non-responsiveness to the B-cellepitope 240-252, is shown by the failure of H-2'' and H-2'' mice todevelop an antibody response unless a Th-cell epitope isprovided, following either covalent linkage or co-immunization.The results presented here confirm early observations by Go odet al.'^ that co-immunization of a non-immunogenic peptidefrom thecircumspwrozoite protein of malaria with KLH (a largemolecule providing T-helper epitopes), overrides the geneticallycontrolled unresponsiveness to the peptide. However, thissystem suffers from the following limitations: (1) the unsuitabi-lity of KLH for human use; (2) the prolein carrier may induceepitopic suppression of the antibody response,'' and (3) thisstrategy will not prime the host for a T-cell memory response tothe pathogen. Thus, our findings extend these early observationsby overcoming the requirement of a carrier by the use of asynthetic peptide representing a Th-cell epitope from thepathogen concerned. These results are consistent with thosefrom a recent preliminary study in which the induction of anti-hapten antibodies did not require covalent linkage between thehapten and the peptide behaving as Th-cell determinant,"

A possible explanation for these findings is that the peptidescross-link via free sulphydryl (SH) groups on each peptide.However, since the T-cell epitope peptides 288-302 and 258-277do not have free SH groups this explanation is not a ppropria te.The inability of the 240-252 peptide to induce antibodyresponses in BALB/c and SWR/J mice may be due to defects inprocessing and presentation o fthe peptide by B cells or failure tomake functional complexes with MH C m olecules. The formerpossibility is unlikely since anti-240-252 antibody responseshave been induced in both strains of mice after immunizationwith the chimeric construct 288-P-240, The 240-252 peptide isunable to induce proliferative responses in the above strains"which suggests that it cannot form functional complexes withMH C molecules. The observation that the Th-cell determ inants(288-302 or 258-277) can provide help when they are co-immunized with the p)eptide 240-252 at the same site suggeststhat the local environment where the immunogens are adminis-tered is crucial for the 'comm unication' between B and Th cells.On the basis of our current understanding for the generation ofhelp for a B-cell response it appears that during co-immuniza-tion, clones of B cells will recognize the immunogenic peptides(e,g, 258-277) and subsequently process and present them inconjunction with class II molecules to Th cells. Subsequent T-cell activation will provide the necessary help to the 258-277 B-cell clones for antibody production through the classicalpathway of cognate help which requires the interaction ofthe T-cell receptor with the M HC-peptide com plex on 258-277 specificB cells. In the subsequent effector phase of help, the interactionof activated Th cells and B cells is not MH C restricted andsoluble factors such as interleukin-2 (IL-2), IL-4, IL-5, inter-feron-y (IFN-y) will provide the necessary signals not only to the258-277 specific B cells but also to B-cell clones which havebound the 240-252 peptide via their surface immunoglobulin.This would thus result in anti-240-252 antibody production(bystander help). The concept of bystander help as a possiblemechanism for overcoming non-responsiveness is further sup-ported by the finding that co-immunization of the 258-277peptide with the 240-252 peptide does not result in a detectableproliferative response to the 240-252 peptide. In addition, sinceaqueous mixtures of the peptides were co-emulsified in CFA,individual droplets may well have co-entrapped both peptideswhich may well have facilitated their uptake by the same B cells,M HC restriction is a major concern for the prospects ofth edevelopment of a synthetic peptide vaccine because peptidesmay only be recognized by a limited number of individuals in thegenetically heterogeneous human population. Recently, anumber of peptides with the ability to bind to multiple MHCalleles have been described b oth in m ouse and hum an s tudie s,"" These findings have led to the construction of chimericsynthetic vaccines by pairing well-defined Th dete rminants w ithB-cell antigenic sites by polymerization,^^ covalent linkage' orco-linear sy nthe sis .'" However, it was subsequently found thatchemical linkage has the disadvantage of the uncontrollednatu re ofth e reaction and the risk of affecting the antigenicity ofthe peptides," Moreover, orientation of the epitopes has beenshown to have a profound effect on antibody specificity andantibody affinity," ^* suggesting that the stoichiometry and theamino acid composition of the epitopes determines the waychimeric peptides are processed and presented. The findingdescribed in this report that genetically controlled unresponsive-ness to synthetic peptides can be overcome by co-imm unization


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266 C . D. Partidos, O. E. Obeid & M. W. Stewardo f peptides representing B- and T-cell epitopes rather than bycovalent linkage ofthe epitopes, provides a new approach to theuse of synthetic peptides as potential vaccines.

ACKNOWLEDGMENTSThis work was supported by Proteus Molecular Design Limited and by agrant from Action Research. This is paper 591 from the Department ofClinical Sciences.

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Antibody responses to non-immunogenic synthetic peptides induced by co-immunization with immunogenic peptides