1Departments of Biology and Chemistry, Knox College, Galesburg, IL, 2Department of Biochemistryand Molecular Biology, The George Washington University, Washington, D.C., 3Department ofMedicine/Internal Medicine, Wayne State University School of Medicine, Detroit, MI, USA
Effect of Peptide-Carrier Coupling on Peptide-SpecificImmune Responses
JANET E. KIRKLEyl, ALLAN L. GOLDSTEIN2, and PAUL H. NAYLOR3
Received April 10,2000 . Accepted in revised form November 17,2000
Abstract
Synthetic peptides are covalently linked to immunogenic carrier proteins to enhance the anti-peptideimmune response. To investigate whether the method of conjugation influences the immune response,we evaluated two distinctly different choices of linker for a peptide-carrier construct. HPG-30, a synthetic peptide derived from the p 17 gag protein of human immunodeficiency virus 1, was covalentlylinked to keyhole limpet hemocyanin by either glutaraldehyde or a maleimide ester. Glutaraldehydelinkage enhanced the anti-peptide antibody and native protein response compared to maleimide. Themaleimide-linked conjugate was more effective at inducing a peptide-specific cellular response. Thus,manipulation of the conjugation method can modify the magnitude and character of the immuneresponse to a synthetic peptide vaccine.
Introduction
The weakly immunogenic character of synthetic peptide immunogens is typically overcome by covalent coupling of the peptide to an immunogenic carrier protein. A varietyof methods for covalent attachment exists (reviewed in 1). These methods may employa functional group on a terminal residue or in internal residues as sites of attachment.The linking moiety may be homobifunctional, with the same reactive group at each end,or heterobifunctional, flanked by two different reactive groups. The space between thereactive groups may be long or short, with an aliphatic or aromatic character.
Selection of an appropriate coupling agent should incorporate several factors: conjugate stability and solubility, degree ofpeptide loading, effect on peptide or carrier immunogenicity and antigenicity, and possible undesired immunogenicity Qf the linker or themodified carrier. For example, less flexible linkers containing aromatic groups were moreimmunogenic in and of themselves than were aliphatic linkers (2). Glutaraldehyde coupling of a peptide to one carrier protein induced antibodies cross-reactive with glutaraldehyde-treated, heterologous carrier (3).
The coupling agent may have an effect on how many peptide moieties are attachedto each carrier molecule and the strength of the immune response to the coupled pep-
0171-2985/011203/04-601 $ 15.00/0
602 . J. E. KIRKLEY et al.
tide. RONCO et al. (4) found that coupling through succinimidyl 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (SMCC) resulted in fewer peptide moieties being attached to the carrier protein than coupling through glutaraldehyde, but the peptide-specific antibody response to the SMCC-linked conjugate was higher than the antibodyresponse to the glutaraldehyde-linked conjugate.
Because attachment to the carrier protein requires covalent modification of the peptide, the potential exists to alter its immunigenicity through chemical alteration of theresidues. SCHAAPER et al. (5) found that antibodies to the coupled peptide were directed towards residues at the end opposite to the one used for conjugation, a tendency thatcould have important ramifications for the immunodominant epitope bound by theantisera. Coupling of an intrinsically immunogenic peptide to a carrier can evendiminish the antibody response, as demonstrated by FRANCIS et al. (6).
Besides the size of the anti-peptide humoral response, two important considerations in evaluating an immune response raised by a synthetic peptide-carrier proteinimmunogen are native protein recognition and the induction of both B- and T-cellresponses. The current studies address these issues with a peptide-carrier constructconsisting of HGP-30, a human immunodeficiency virus 1 (HIV-l) P17-derived,30-amino acid peptide, conjugated to keyhole limpet hemocyanin (KLH). WhileHGP-30 has been shown to be weakly immunogenic by itself (7), a larger immuneresponse is seen following immunization with the peptide conjugated to KLH via glutaraldehyde (8, 9). The HGP-30-KLH conjugate was effective at generating a peptidedirected immune response in both mice and humans (10). Antibodies to the parentp 17 protein were also induced by the peptide-carrier construct (8), and the peptidealone (7).
For the purpose of comparing peptide-carrier linkers, we selected two very differentmeans of conjugation. One method was coupling via glutaraldehyde, which cross-linksbidirectionally through peptide and carrier lysyl residues. The other linker used was theunidirectional SMCC linker which is first attached to lysyl residues on the carrier protein and then reacted with an N-terminal cysteinyl residue on the peptide. We foundthat this difference in linkers profoundly affected the size and character of the resultingimmune response in mice.
Materials and Methods
Immunogens and antigens
HGP-30, HGP-18, and the maleimide-linked conjugate were provided by Viral Technologies(Washington, DC). Recombinant HIV-l IIIB p17 was a kind gift ofTransgene (Strasbourg, France).Monoclonal antibody specific for p17 was a kind gift of Prem Sarin. Aluminum hydroxide gel (alum)was purchased from Superfos (Denmark). QS-21 was a kind gift of Cambridge Biotech (now Aquila,Cambridge, MA, USA). HGP-30 was cross-linked to KLH (Calbiochem, LaJolla, CA, USA) (1: 1w/w) via glutaraldehyde (Kodak, Rochester, NY, USA). Cysteinated HGP-30 was cross-linked'tomaleimide-activated KLH (Pierce Chemical Co., Rockford, IL, USA) according to the manufactorer'sinstructions. Alum was mixed with immunogen to give a final 3: 1 alum: immunogen by mass. QS21 was mixed with immunogen according to instructions provided by the manufacturer. Antigens inthe serum antibody measurement and cell proliferation assays were unconjugated, uncysteinated peptide and underivatized carrier protein.
Effect of peptide linkage on immune responses . 603
Immunization
Female BALB/c mice were purchased from Harlan Sprague Dawley (Indianapolis, IN, USA) and started on study between 6 and 8 weeks ofage. For all studies, the mice were immunized at each time pointexcept the final one, with an injection volume of 200 JlI administered subcutaneously in the neckscruff The final booster was given intraperitoneally. For the time course study, mice were immunizedat 0, 4, and 8 weeks with 2.5 Jlg/mouse conjugate. The immunogen was administered mixed withalum in a 3: 1 alum: conjugate ratio by mass or mixed with QS-21 in a 1:2 QS-21 :conjugate ratio bymass. Mice received the conjugate in alum for all three immunizations, or they received the conjugatein alum for the first immunization and in QS-21 for the two subsequent boots. For the titration study,mice receiving the SMCC-coupled conjugate were immunized at 0 and 8 weeks with 2.5 Jlg/mouseconjugate in alum, and at 16 and 21 weeks with the same dose of conjugate in QS-21, while micereceiving the glutaraldehyde-coupled conjugate were immunized at 0, 8, and 16 weeks with 2.5 Jlgimouse conjugate in alum. Mice were bled through a tail vein every 2 weeks and sacrified 1 week afterthe final boost. All studies were performed using animal protocols approved by the InstitutionalAnimal Care and Use Committee.
Serum antibody measurement by enzyme-linked immunosorbent assay (ELISA)
96-well microtiter plates (Nunc, PGC Scientific, Gaithersburg, MD, USA) were coated overnight atroom temperature with antigen in phosphate buffered saline (PBS). Peptide antigens adsorbed to theplastic and were not used in conjugated form to coat the ELISA plates. Between each step, the plateswere washed in a Bio-Tek Instruments (Winooski Park, VT, USA) EL 403 microtiter plate washerwith PBS containing 0.050/0 v/v Tween 20. Plates were blocked for 2 hours at 37°C with 50/0 w/vdried, non-fat milk in PBS-Tween. Antiserum was assayed at a 11400 dilution in PBS unless otherwisenoted in the text or figure captions. Antiserum was incubated for 1 hour at 37°C. Peroxidase-conjugated goat anti-mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was diluted in PBS-Tween containing 1% bovine serum albumin and incubated 1 hour at 37°C. The TMBMicrowell Peroxidase Substrate System (Kirkegaard and Perry Laboratories) was prepared according tothe manufacturer's instructions and incubated at room temperature. Color development was stoppedwith the addition of phosphoric acid, and optical density was measured at 405 nm using a Dynatech(Chantilly, VA, USA) MR650 microtiter plate reader.
Competition ELISA
The plate was coated with p 17 and blocked as described above for the measurement of serum anti'body. Mter blocking, the competing soluble antigens were added to the wells in 2-fold dilutions downfrom a starting concentration of 50 Jlg/ml in PBS. An equal volume of a p17-specific monoclonalantibody was then added to the wells to give a final dilution of 1/512,000. Mter a one-hour incubation at 37°C, the plate was washed and developed as described above for the measurement of serumantibody.
Measurement of antigen-specific cellular proliferation
After sacrifice, spleens were aseptically removed. Cells were separated from connective tissue, placedinto a single-cell suspension, and counted on a Coulter Counter (Coulter, Hialeah, FL, USA). Cellswere cultured with antigen in 96-well, tissue culture-treated plates (Costar Corp., Cambridge, MA,USA) containing RPMI 1640 supplemented with 100/0 CPSR-2 (Sigma Chemical Co., St. Louis,MO, USA), 10 U/ml recombinant interleukin-2 (a kind gift of Hoffman-LaRoche, Nutley, N], USA),50 IV/ml penicillin (Mediatech, Washington, DC), 50 Jlg/ml streptomycin (Mediatech), 2.5 Jlg/mlfungizone (lCN Biomedicals, Costa Mesa, CA, USA), and 2 mM I-glutamine (Mediatech). The covered plates were incubated for 6 days at 37°C and 50/0 CO2 • During the final 18 hours of incubation,the cells were pulsed with 1 JlCi/well 3H-thymidine (DuPont, NEN, Boston, MA, USA). Cells wereharvested onto filter paper and counted in scintillation cocktail. Stimulation index was calculated bydividing average counts per minute for triplicate wells containing antigen by average counts per min-
604 . J. E. KIRKLEY et al.
ute of triplicate wells containing no antigen (medium-alone background). The cell cultures responded vigorously to mitogenic stimulation (1 Jlgiml phytohemagglutinin, Wellcome Diagnostics,Dartford, UK) (data not shown).
Results
Comparison of glutaraldehyde and SMCC coniugation sites
The synthetic peptide HGP-30 was covalently attached to the carrier protein, KLH,with two different linkers. Glutaraldehyde is a homobifunctionallinker that cross-linksproteins and peptides through a Schiff base reaction with primary amines, mainly at theN terminal or the epsilon amine of lysyl residues (1). The conjugate made using glutaraldehyde to cross-link HGP-30 and KLH was designated HGP-30-KLHlys (K30lys).SMCC, a heterobifunctionallinker, cross-links primary amines on the carrier protein tocysteinyl residues on the peptide (1). A cysteinyl residue was added to the N terminal ifHGP-30 for use with the SMCC linker, and the resulting conjugate was designatedHGP-30-KLHcys (K30cys). Conjugation using glutaraldehyde results in a heterogeneous product because linkage between the peptide and carrier can occur at a variety ofresidues (Fig. IA). Also, peptide-peptide and carrier-carrier conjugates could form.SMCC, in contrast, gives a more homogeneous product because linkage can only occurbetween maleimide moieties on the carrier protein and the N-terminal cysteinyl residueincorporated in HGP-30 (Fig. IB).
Binding to a p17-specific monoclonal antibody as a criterion for immunoreactivityof the coniugates
The immunoreactivity of the conjugates was assessed in a competition assay using a p17specific monoclonal antibody and recombinant p17 protein. Soluble conjugate wasincubated in microtiter plates with the monoclonal antibody in the liquid phase and pI7adsorbed to the well surface. The K30lys conjugate competed more effectively againstthe immobilized p17 than did the K30cys conjugate. The K30lys conjugate inhibitedbinding of the monoclonal antibody to the immobilized recombinant antigen almostcompletely at 25 J..lg/ml and continued to inhibit binding until it had been diluted toless than 1 J..lg/ml (Fig. 2). In contrast, the K30cys conjugate substantially inhibitedmonoclonal antibody binding only at the highest concentration tested, and this effectdiluted out quickly. The carrier protein, KLH, did not inhibit binding of the monoclonal antibody to p17.
Adiuvant dependence of coniugate in inducing an antibody response
HGP-30-specific antibodies in the serum of animals immunized with the glutaraldehyde-linked conjugate, K30lys, were detectable after two subcutaneous injections (Fig.3A). In contrast, mice immunized with K30cys in alum did not mount a detectable antibody response against the peptide until after the third immunization, which was administered intraperitoneally. By Student's t-test, the HGP-30-directed antibody responseinduced by K30lys was significantly higher than the response induced by K30cys at4 weeks (p =0.0014), 6 weeks (p =0.0039), and 8 weeks (p =0.0045) after the initial
Effect of peptide linkage on immune responses . 605
A. HGP-30-KLHlys
rSVHQRIElrTrALD~EEEQN~S~~rN N N N N NNNII II II II II II II II(j <J (j <J (j (j<J n::I: ::I:::I:::I: ::I: ::I:::I:::I:n n n n n nnn:I: :I::I::I: :I: :I::I::I:n n n n n nnn:I: :I::I::I: :I: :I::I::I:n n n n n nnn:I: :I::I::I: :I: :I::I::I:n n n n n nnn:I: :I:::I:::I: ::I: ::I:::I:::CII II II II II 1111 IIN N N N N NNN\ 1__1 I I_LU
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Fig. 1. Two different methods of coupling HGP-30 to the carrier protein are shown. The peptide wascoupled by glutaraldehyde (IA) or by SMCC (IB). Glutaraldehyde potentially can cross-link usingany lysyl residues and the N-terminal amino group in the peptide, while the SMCC is restricted to anN-terminal cysteinyl residue.
immunization. The difference in the antibody response induced by the two conjugatesfollowing the initial immunization was adjuvant dependent. Changing the adjuvant toQS-21 for the booster immunization resulted in an HGP-30-specific antibody responseinduced by K30cys that was not significantly different from the peptide-specific antibody response to K30lys (Fig. 3B), except at 4 weeks (p =0.01). Previous studies withQS-21 and K30lys found this adjuvant to be stronger than alum (11).
Native antigen recognition by antibodies raised by the two conjugates
The HGP-30-specific antisera were analyzed for reactivity with the native protein, pI?,and with HGP-I8, a peptide incorporating the central 18 residues of HGP-30. HGP-
606 . J. E. KIRKLEY et al.
Ec
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---+- K30cys -+- K30lys -+- KLH
Fig. 2. K30lys inhibited binding of a p17-specific monoclonal antibody more effectively than K30cysdid. A P17-specific monoclonal antibody was incubated for one hour in a well containing p 17 absorbed to the solid phase and a soluble competitor. The competitors tested were K30cys (triangles),K30lys (circles), and KLH (crosses). The competitor concentration started at 25 llg/ml and was diluted down in 2-fold increments as indicated on the X axis. Results are shown as optical density at405 nm.
Table 1. Cross-reactivity of antisera raised by each conjugate a.
Weeks post initial immunizationConjugate Boosteradjuvant
6 8 9 6 8 9
No. positive foranti-p 17 antibodyb
No. positive foranti-HGP-18 antibodyb
K30cys alum 0 0 1 0 0 1K30lys alum 2 3 5 3 3 5
K30cys QS-21 1 1 1 1 2 2K30lys QS-21 4 4 5 4 4 5
a Serum analyzed by ELISA at a 1/400 dilution.bOut of 5 mice per group.
18 contains the epitope ALDKIEE, which was recognized by a neutralizing monoclonalantibody generated through immunization of BALB/c mice with purified viral protein(12). Using antisera from rabbits immunized with KLH-HGP-30Iys, this 7-residuesequence was determined to be the immunidominant B cell epitope in HGP-30 (9).
Antibodies from mice immunized with the K30lys conjugate three times in alum,which had produced a better anti-HGP-30 response than the H30cys conjugate, alsocross-reacted strongly with both p17 and HGP-18 (Table 1). Antisera from mice immu-
Effect of peptide linkage on immune responses . 607
A.Ec
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-.- K30cys -.- K30lys -+- ControlALUM ALUM ALUM
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Fig. 3. Linkage through the N-terminal resulted in a lower anti-peptide antibody response. Mice wereimmunized three times with 2.5 pg/mouse K30cys (triangles) or K30lys (circles) at 0, 4, and 8 weeks asindicated by the arrows (SQ= subcutaneous injection, IP = intraperitoneal injection). After the initialimmunization with alum, mice received the conjugate mixed either with alum (3A) or QS-21 (3B) at the4- and 8-week boosts. Serum antibody response to HGP-30 was measured by ELISA. Results shown arethe average of 5 mice in a group and are expressed as optical density. Control mice (crosses) received anamount of adjuvant equal to that given to the immunized mice. Serum was analyzed at a 1/400 dilution.
nized with K30cys three times in alum did not recognize either pI7 or HGP-I8 verystrongly, consistent with their poor titer against HGP-30. However, even when the morepotent QS-21 adjuvant was used, similar levels of antibody recognition of either p17 orHGP-I8 did not develop (Table 1). Antibodies from mice immunized with K30lys werealways more cross-reactive with either the parent protein or the immunodominant epi-
608 . J. E. KIRKLEY et al.
A.EcII) 1.400~ 1.20
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_ HGP-30 ~ rp17 HGP-18antibody antibody antibody
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_ HGP-30 ~ rp17 o HGP-18antibody antibody antibody
Fig. 4. At a titer where the anti-peptide response was equal between the two conjugates, the crossreactivity of the antisera was still not similar. The 9-week bleed of the mice whose antibody responsesare shown in Figure 3 and Table 1 was analyzed by ELISA at a 1/40 dilution for the ability to bindHGP-30 (diagonal), pI? (cross-hatch), or HGP-18 (stipple). Mice were immunized with either K30cysor K301ys, as shown on the X axis, with either alum (4A) or QS-21 (4B) as the boosing adjuvant.Results shown are the average of 5 mice per group and are expressed as optical density. Statistical analysis was by Student's t-test. The star indicates a significant difference (p < 0.05).
tope than antibodies from mice immunized with K30cys. A response was consideredpositive if the optical density (00) was at least 0.2. For comparison, the highest background ODs for adjuvant-immunized control mice over these three timepoints were asfollows: for rp17, alum control 0.011 ± 0.022, QS-21 control 0.001 ± 0.002; for HGP18, alum control 0.013 ± 0.010, QS-21 control 0.022 ± 0.017.
Effect of peptide linkage on immune responses . 609
Fig. 5. A stronger adjuvant resulted in a higher anti-peptide titer with the K30cys conjugate but nota higher anti-pI? titer. The antibody response against HGP-30 (5A) and pI? (5B) was titered andanalyzed by ELISA. Mice were immunized with either K30cys (triangles) or K30lys (circles). The micereceiving K30cys were immunized twice with alum, then twice with QS-21. The mice receivingK30lys were immunized three times with alum. The reciprocal, in 100s, of the serum serial dilutionis shown on the X axis. Results shown are the average of 5 mice per group and are expressed as optical density.
The antibody response was re-assessed at a ten-fold higher serum concentration todetermine if any antibodies weakly reactive with pI? or HGP-I8 could be found in thesera from the K30cys-immunized animals. At a 1/40 serum dilution, the HGP-30 reactivity was similar in mice immunized with either conjugate (Fig. 4A alum, Fig. 4B, QS21). With both adjuvant regimens, the recognition of pI? and HGP-I8 by K30cys-
610 . J. E. KIRKLEY et al.
induced antibody was improved at the higher serum concentration, but was still lessthan that of the K30lys-induced response. We interpret this as indicating that p 17- andHGP-18-binding antibodies were present in low amounts that were undetectable at the1/400 dilution. The difference in antibody response to p17 or HGP-18 using the twodifferent conjugates was still significantly different (p < 0.05) by Student's t-test. Use ofQS-21 in the second and third immunizations with K30cys appeared to induce a morestrongly cross-reactive antisera to both p17 and HGP-18 than use of alum, but the highstandard deviation made the difference not statistically significant at this serum concen-tration. .
A more intensive immunization regimen was employed with K30cys in which animals received K30cys twice in alum and twice in QS-21. K30lys-immunized animals, bycontrast, received immunogen 3 times in alum. When sera from each group of micewere titrated, the average anti-HGP-30 titer of the mice immunized with K30cys inalum and then in QS-21 was approximately 1: 100,000, while that of the mice thatreceived K30lys in alum only was a much weaker 1: 3,200 (Fig. SA). However, the titerof the anti-pl7 response for either group of mice was approximately 1: 1,600 (Fig. 5B).
Enhancement of peptide-specific proliferative responses through N-terminal conjugation
Splenocytes from the immunized mice were cultured with unconjugated HGP-30 peptide,and the proliferative response to the free peptide was measured. The best proliferativeresponse occurred in splenoeytes from animals that were immunized with the K30cys conjugate (Fig. 6). Three out of 5 mice immunized with the maleimide conjugate in eitheralum or QS-21 had a stimulation index (SI) of2 or greater in response to 0.5 mg/ml pep-
6
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0K30cys K30cys K30lys K30lys
alum QS-21 alum QS-21+ A 6- B 0 C v 0 0 E
Fig. 6. Linkage of the peptide through its N terminal resulted in a cellular response to the peptide.Mice were immunized at 0, 4, and 8 weeks with K30cys or K30lys as described for Figure 1. Conjugate and boosting adjuvant are shown on the X axis. Proliferation of splenocytes was measured 1 weekafter the final immunization. Wells contained unconjugated, uncysteinated HGP-30 at a 500 }lg/mlfinal concentration. Results shown are the stimulation indices of individual mice A through E, as indicated in the figure legend. A stimulation index of 2 or higher, as marked by the horizontal line, wasconsidered a positive result.
Effect of peptide linkage on immune responses . 611
tide. The stimulation indices of responding mice immunized with K30cys in QS-21 werenot as high as those seen with cells from mice immunized with the conjugate in alum. Incontrast, none of the animals immunized with the K30lys conjugate in alum or QS-21showed a proliferative response. By Wilcoxon rank sum test, the difference between theK30cys-immunized and K30Iys-immunized animals who received alum as the adjuvantwas statistically significant (p = 0.05). By the same test, there was no statistically significantdifference between the animals immunized with either K30cys or K30lys in QS-21.
Splenoeytes from most of the immunized animals proliferated when cultured withKLH, which served as a positive control for the induction of a proliferative response (datanot shown). Splenoeytes from adjuvant-immunized control mice did not proliferate inresponse to HGP-30 or KLH (data not shown).
Discussion
The role of the carrier protein in the generation of an immune response to a conjugatedmoiety can range from inducing necessary T-cell help to serving as a increaser of massand/or repetition, depending on the immunogenic character of the conjugated material(reviewed in 13). For a short, weakly immunigenic synthetic peptide, such as HGP-30, thecarrier protein may serve multiple functions, such as enhancing uptake by antigen-presenting cells and stimulating a eytokine-rich environment by activating carrier-specific helperT cells, that combine to improve the immune response to the peptide.
The results of our comparison of two different peptide-carrier linkers agree with previous reports that the method selected for conjugation of the peptide to the carrier can haveprofound effect on the immune response. In terms of the peptide-specific antibodyresponse, our observation that glutaraldehyde linkage through lysyl residues resulted in amore immunogenic conjugate than SMCC linkage through the N-terminal residue doesnot agree with the results of a study by RONCO et al. (4) who found the opposite effectwhen conjugating a peptide representing the V3 loop from HIV-l BRU gp120. Thislack of agreement suggests that linker selection may depend on the peptide and· thuswarrant analysis on a case-by-case basis.
The difference in the cross-reactivity of the antiserum raised by each conjugate withthe native p 17 protein was not the result of a difference in anti-peptide titer. A K30cysinduced antiserum of similar HGP-30-specific titer as the K30lys-induced antiserumstill did not bind the parent protein as well, and the antiserum raised by K30cys that hada similar level of p17-reactivity as the antiserum generated by K30lys had an HGP-30titer that was considerably greater. This result indicates a distinct immunogenic difference between the two conjugates that may have arisen from different conformationalappearances of the peptide. The difference in epitope recognition may reflect a difference in clonal engagement of B cells, with the K301ys, but not the K30cys, being recognized by B cells whose antibodies will bind the parent protein and immunodominantepitope. Further support for this explanation comes from the results using adjuvants ofdifferent potency. While the use of the stronger QS-21 adjuvant was able to increase theanti-HGP-30 response generated by K30cys to the magnitude of the response inducedby K301ys, the cross-reactivity of the antisera with either the native protein or theimmunodominant epitope remained significantly lower when the immunogen was theSMCC-linked conjugate.
612 . J. E. KIRKLEY et al.
Conjugation through glutaraldehyde seems likely to result in a more constrained conformation because attachment to the carrier protein can be through several internal residues, thus anchoring the peptide in more places and restricting its freedom of movement. In contrast, the SMCC linkage being through the N-terminal residue only wouldallow the peptide more freedom of movement. Studies of protein antigenicity have indicated that recognition by antibody positively correlates with mobility of the antigenicregion (14, 15). Synthetic peptides coupled through the C terminal raised cross-reactiveantisera against the more mobile segments of the native protein better than peptidesdirected at less mobile segments (14). The region ofp17 represented by HGP-30 (aa 85115), therefore, could be a conformationally constrained one, thus reducing the crossreactivity of antiserum raised against the N-terminal coupled peptide. Conjugation ofHGP-30 through internal residues may result in an epitope that physically resembles thecorresponding segment in the folded p17 protein.
One intriguing result of these studies was the ability of the K30cys conjugate to primefor a peptide-specific proliferative response in vitro. HGP-30 by itself: when injected witha sufficiently strong adjuvant, has been shown to be immunogenic in BALB/c mice (7).It therefore must contain a T cell epitope for that haplotype in addition to the B-cellepitope(s). The region of p17 contained within HGP-30 has been predicted to contain aT cell epitope (16) on the basis of amphipathic score (17). HGP-30 contains an epitoperecognized by lymphocytes from HIV-l-infected individuals in proliferation studies (18,19) and cytotoxicity studies (20). A B-cell epitope within the same region was also predicted (16), and HGP-30 is recognized by antisera from infected individuals (21).
Interestingly, there is a four-residue motif contained within HGP-30 that is the samepattern as a common sequence in T cell epitopes identified by ROTHBARD and TAYLOR(22). This pattern of two hydrophobic residues in a row flanked on either side by ahydrophilic residue is seen at amino acids 14-17 (EALD) in the non-cysteinated HGP30. On either side of this motif: there is a lysyl residue. Coupling through these lysylresidues may be why glutaraldehyde conjugation diminishes recognition of HGP-30 asa T cell epitope, simply through physical interference with peptide binding to the majorhistocompatibility complex II protein (MHC II). In the K30cys conjugate, these lysylresidues are not covalently modified, and no physical hindrance thus exists to preventassociation of the peptide with the MHC II molecule.
Although identification of binding motifs in MHC II ligands is not as straightforwardas their characterization in MHC I-binding peptides due to greater variability in peptidelength (23), analysis ofpeptides able to bind human or mouse MHC II has identified certain chemical features (reviewed in 23, 24), and sequence motifs have been used successfully to predict MHC II-binding peptides (25). Analysis ofIAd-presented peptides generated from endogenous protein revealed a common 6-residue core (26) containing apreviously identified canonical binding sequence (25, 26). Since both IAd and lEd binding motifs contain basic residues (28), covalent modification oflysyl residues in HGP-30by glutaraldehyde could interfere with its association with MHC II. The identification ofa few residues critical for binding, such as has been done for IEk (29), helps characterizethe residue preferences of pockets in the peptide-binding groove (reviewed in 24). Thestrong preference of two of three lEd pockets for basic residues (24) further highlights thepotential disruption of MHC II binding by covalent modification of lysyl residues.
Another explanation for the T cell recognition of the peptide in the N-terminal conjugate construct may have to do with antigen processing and presentation. TAKAHASHI
Effect of peptide linkage on immune responses . 613
et al. (30) identified thiol proteases as being necessary for the presentation of spermwhale myoglobin and found that one enzyme in particular from this category, cathepsin B, could produce ex vivo fragments of protein recognizable by myoglobin-specifichelper T cell clones. More recent evidence has strongly associated this particular cathepsin with antigen processing (31, 32, 33), Cathepsin B's substrate specific is believed tobe two basic residues in a row (34). Conjugation through the lysyl residues in HGP-30could prevent appropriate processing by this enzyme and thus inhibit presentation as aT cell epitope and promote recognition as a B cell epitope. In this case, T cell help wouldoccur via cytokines generated by the response to the KLH carrier.
The decreased size of the peptide-specific antibody response to the SMCC-linked peptide conjugate could be due to a lower peptide: carrier ratio. PEETERS et ale (2) found that,among soluble peptide-carrier conjugates, antibody titer had a positive correlation withpeptide loading density. (We observed no solubility difficulties with either conjugate.)Because the size of the KLH carrier prohibits effective quantitation of the peptide: carrierratio by the usual method of amino acid analysis (2, 3), we used an alternative method toaddress this issue. Analysis of each conjugate's antigenicity by competition ELISA showedthat soluble K30cys conjugate was a less effective competitor for binding to a p17-specific monoclonal antibody than was the K30lys conjugate on an equal-mass basis. This method would be unable to distinguish between a difference in peptide: carrier ratio or a difference in antigenic appearance as the potential cause of this observed effect. However, theK30lys could be diluted 8-fold more than the K30cys before reaching an equivalentdecrease in inhibitory activity. Amino acid analysis of the two conjugates found less thana 2-fold difference in the amount of peptide loaded, with the glutaraldehyde-linked conjugate having more attached peptide (data not shown). Because the magnitude of the difference in antigenicity between the two conjugates exceeds the magnitude of difference inattached peptide, it is likely that linkage of the HGP-30 peptide to the carrier through itsinternallysyl residues affected its ability to bind p17-specific monoclonal antibody. Glutaraldehyde attachment may have resulted in the peptide assuming a confomation more likethat found in the native protein and thus more recognizable by the p 17-directed monoclonal antibody. After two immunizations with alum, the glutaraldehyde-linked conjugateraised a peptide-specific antibody titer 7-fold higher than the SMCC-linked conjugate, anincrease greater than would be expected if peptide loading were the sole cause of differences in conjugate activity. Additionally, with the appropriate immunization protocol, theK30cys was able to induce an anti-HGP-30 titer equivalent to or even stronger than thatinduced by the K30lys conjugate. Yet this high-titer antisera raised against the end-linkedpeptide did not bind either the native protein or the immunodominant epitope verystrongly, further supporting the explanation that peptide in the K30cys conjugate had adifferent physical appearance, resulting in antibody that recognized p17 poorly.
When a peptide has only haptenic character, then measurement of peptide-specificantibody and quantitation ofpeptide-directed antiserum recognition ofnative protein areprobably sufficient for the analysis of different peptide-carrier conjugation techniquesand the identification of the most effective one. When the peptide additionally has thecapacity to stimulate a T cell response, then, as seen with these studies investigatingHGP-30-KLH constructs, additional information is needed before the best linker can beselected. These studies provide evidence that the method of covalent attachment of thepeptide to the carrier protein affects important characteristics of the resulting immuneresponse, such as native protein recognition and anti-peptide proliferative response.
614 . J. E. KIRKLEY et al.
Acknowledgements
These studies were supported in part by Viral Technologies, Inc., and by a fellowship from the Institutefor Advanced Studies in Immunology and Aging (JEK).
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Dr. JANET E. KIRKLEY, Knox College, 2 E. South St., Galesburg, IL 61401, USA, Phone: (309) 3417308, Fax: (309) 341-7718, e-mail: [email protected]
