Peptide Mimotopes of Pneumococcal Capsular Polysaccharide of 6B Serotype: A Peptide Mimotope Can Bind to Two Unrelated This information is current as of October 1, 2021. Jeon-Soo Shin, Jigui Yu, Jisheng Lin, Linghao Zhong, Kara L. Bren and Moon H. Nahm J Immunol 2002; 168:6273-6278; ; doi: 10.4049/jimmunol.168.12.6273 http://www.jimmunol.org/content/168/12/6273 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Peptide Mimotopes of Pneumococcal Capsular Polysaccharide of 6B Serotype: A Peptide Mimotope Can Bind to Two Unrelated Antibodies1

Jeon-Soo Shin,* Jigui Yu,‡ Jisheng Lin,‡ Linghao Zhong,† Kara L. Bren,† and Moon H. Nahm2‡§

Two groups of bacteriophage clones displaying the antigenic properties of serotype 6B pneumococcal capsular polysaccharide (PS) were obtained from different phage libraries expressing random heptameric peptides. One group, biopanned with a mouse mAb (Hyp6BM1), is comprised of 17 phage clones expressing 10 unique sequences of linear peptides. The other group, selected with another mAb (Hyp6BM8), contained six clones, all of which expressed the identical circular peptide. Phage clones expressing the linear peptides (e.g., PhaM1L3) bound only to Hyp6BM1, but not other 6B PS-specific mAb, and their binding could be inhibited Downloaded from with pneumococcal capsular type 6B PS only. In contrast, a phage clone expressing the circular peptide (PhaM8C1) cross-reacted with several other 6B PS-specific mAbs, and their binding could be inhibited with pneumococcal capsular PS of 6A and 6B serotypes. Two short peptides, PepM1L3 and PepM8C1, reflecting the peptide inserts of the corresponding phage clones, could inhibit the binding of the two clones to their respective mAb. Interestingly, the peptide insert in PhaM8C1 was identical to that in PhaB3C4, a previously reported mimotope of ␣(238) polysialic acid, Neisseria meningitidis group B PS. Indeed, PhaM8C1 bound to HmenB3 (a meningococcal Ab), and their association could be inhibited with ␣(2–8) polysialic acid, but not with 6B PS. http://www.jimmunol.org/ Conversely, ␣(2–8) polysialic acid could not inhibit the binding of PhaM8C1 to Hyp6BM8. The two-dimensional nuclear magnetic resonance studies indicate that PepM8C1 peptide can assume several conformations in solution. The ability of this peptide to assume multiple conformations might account for its ability to mimic more than one Ag type. The Journal of Immunology, 2002, 168: 6273–6278.

s different peptides can assume diverse conformations, a should be useful for studying differences in immune mechanisms large peptide library expressing random sequences used by PS and protein Ags. In addition, the peptide mimics should contain peptides that can conformationally should, unlike PS, elicit help, immune memory, and strong

A by guest on October 1, 2021 mimic virtually any ligand or Ag. This idea has been shown to Ab responses. Also, the peptides should be easier to manufacture have practical application, with the recent demonstration that pep- and modify than PS Ags (7, 8). Thus, the mimotopes may be useful tide mimics of antigenic specificities can be efficiently identified as components. However, despite reports of successful using large peptide libraries. For instance, random peptides can be mimotope (4, 9Ð11), mimotopes often either are poorly displayed on a coat protein of a bacteriophage, and biopanning can immunogenic, elicit ineffective Abs, or induce memory for be used to isolate phage clones expressing peptides of a desired ineffective Abs. Consequently, there is a need for further studies of binding specificity (1Ð3). This approach has been successfully peptide mimics of PS Ags before peptide mimic vaccines can used to identify various antigenic mimics (mimotopes). The mi- reach their full potential. motopes have been used to examine Ab fine specificity (4), to Streptococcus pneumoniae is a well-known pathogen, causing examine the requirement for B cell stimulation by Ags (5), and to several serious diseases in young children and the elderly (12). As elicit desirable Abs (6). a result, Ab responses to pneumococcal PS Ags have been exten- Peptides mimicking polysaccharide (PS)3 Ags can be readily sively studied in the past. The peptide mimics can be easily com- produced and used various ways. The peptide mimics of PS Ags pared with other pneumococcal Ags for their immunogenicity and for inducing protective Abs. Also, there is a need for more effec- tive pneumococcal vaccines. The widely available pneumococcal *Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea; vaccine containing capsular PS from 23 common serotypes (13) is †Department of Chemistry, University of Rochester, Rochester, NY 14642; and De- not immunogenic in young children, and its effectiveness is re- partments of ‡Pathology and ¤Microbiology, School of Medicine, Division of Labo- ratory Medicine, University of Alabama, Birmingham, AL 35294 duced among the elderly (14). A new conjugate vaccine, although Received for publication January 14, 2002. Accepted for publication March 27, 2002. effective in young children (15), is not effective among the elderly (16) and is expensive to manufacture. We have studied the peptide The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance mimics of pneumococcal capsular PS and have identified a peptide with 18 U.S.C. Section 1734 solely to indicate this fact. that can mimic pneumococcal PS as well as meningococcal PS. 1 This work is supported by National Institutes of Health Grant AI-31473 (to M.H.N.) and a Howard Hughes Biomedical Research Support Program grant (to K.L.B.). Materials and Methods 2 Address correspondence and reprint requests to Dr. Moon H. Nahm, Department of Antibodies Pathology, School of Medicine, Division of Laboratory Medicine, University of Al- abama, 845 19th Street South, BBRB 614, Birmingham, AL 35294. E-mail address: HmenB1 and HmenB3 are IgM mAb specific for Neisseria meningitidis [email protected] group B capsular PS (17). The 101.4.1 is a mAb to N-acetyl-␤-D-glu- 3 Abbreviations used in this paper: PS, polysaccharide; 2-D, two-dimensional; NMR, cosamine from M. Cunningham (University of Oklahoma, Oklahoma City, nuclear magnetic resonance. OK) (18). Hyp6A1 is a mouse IgM mAb specific for pneumococcal 6A PS.

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 6274 A PEPTIDE MIMOTOPE BINDING TO TWO DIFFERENT Abs

Dob1 is a human IgG2 mAb binding to 6B PS (19). Hyp6BM1, Hyp6BM7, was identified with an underline and derived from the peptide inserts of Hyp6BM8, and Hyp6BM10 are mouse IgM mAb specific for pneumococ- PhaB3C1 (17), PhaM1L3, PhaM1L9, or PhaM8C1. The amino acid Y or C cal 6B PS (20). Dob1, Hyp6BM7, Hyp6BM8, and Hyp6BM10 cross-react at the N-terminal was added for the purpose of radiolabeling or conjugation with 6A PS, an isopolymer of 6B PS. Hyp6BM1 does not cross-react with with carrier protein. The remaining amino acids in either sides of the core 6A PS, and Hyp6A1 does not cross-react with 6B PS (20). Hyp6BM1 and peptide were derived from the sequence of the phage protein pIII flanking Hyp6BM8 were used for biopanning, and the mAb were purified from the the peptide inserts. These peptides were synthesized by Biosynthesis mouse ascites by (NH4)2SO4 precipitation and by chromatography over a (Lewisville, TX) or Biomolecules Midwest (Waterloo, IL). column of Sephacryl S-300HR (Pharmacia Biotech, Uppsala, Sweden). NMR spectroscopy Production of phage clones expressing the mimotopes Solutions (2.5 mM) of NLpeptide6 and NLpeptide9 were prepared in PBS. Two phage libraries from New England Biolabs (Beverly, MA) were used The 1-H TOCSY spectra of both peptides were obtained on a 500-MHz for our study. One contained linear peptides composed of 7 random aa, and Varian INOVA nuclear magnetic resonance (NMR) spectrometer (Varian, the other contained circular peptides of 9 aa; circularization was achieved Palo Alto, CA) using a standard pulse sequence (23, 24). Data were pro- by a covalent bond between the cysteines at positions 1 and 9, and the 7 aa cessed and analyzed using FELIX 97 software (Accelrys, San Diego, CA). between the two cysteines are randomly chosen. Biopanning the phage libraries was performed as described (17). Briefly, 60-mm petri dishes Results (Nunc, Roskilde, Denmark) were coated with mAb at a concentration of Selection of phage clones and analysis of their binding 100 mg/L in 0.1 M NaHCO3 (pH 8.6). The mAb-coated petri dishes were blocked with 0.5% BSA in 0.1 M NaHCO3 and washed with 0.1% TBST properties (50 mM Tris-Cl (pH 7.5), 150 mM NaCl). For each biopanning cycle, ϳ2 ϫ 1011 PFU phage were placed in the petri dish and incubated for 30 Biopanning of the phage library displaying the linear peptides with min. Following the removal of unbound phage particles, bound phage par- Hyp6BM1-coated petri dishes readily enriched for the Ab-specific ticles were eluted from the dish with 0.2 M glycine-HCl (pH 2.2), and the phage clones and 17 phage clones were chosen randomly after Downloaded from recovered phage solution was neutralized with 1 M Tris-Cl (pH 9.1). The three or four cycles of biopanning. Biopanning the same phage recovered phage was expanded in number by growing with Escherichia library with Hyp6BM8-coated petri dish did not enrich for the coli for a new cycle of biopanning. The biopanning was performed for three or four cycles before individual phage clones were isolated. Ab-binding clones. When a second phage library expressing the circular peptide was biopanned, 6 clones binding to the plates were DNA sequencing of phage peptide selected at random. In addition to these 23 clones selected by pan-

To determine the DNA sequence of the mimotope peptide, phage clones ning, a phage clone was chosen from each library (PhaM8-CNTL http://www.jimmunol.org/ were expanded by growing them in 1 ml E. coli cultures for 4Ð5hat37¡C. and PhaM1-CNTL) without any selection to use as negative con- The bacteria present in the culture were removed by a brief centrifugation trols. PhaM1-CNTL expresses the linear peptide and PhaM8- ϫ ␮ (10,000 g for 30 s). A total of 500 l bacteria-free culture supernatant CNTL, a circular peptide. Binding patterns of select phage clones was then mixed with 200 ␮l 20% PEG/2.5 M NaCl solution, and the mix- ture was centrifuged (10,000 ϫ g) for 10 min to precipitate the phage as a were shown in Fig. 1. pellet. The pellet was isolated and resuspended with 100 ␮l Tris buffer with All 23 biopanned clones bound to the selection Abs in a dose- iodide (10 mM Tris-Cl (pH 8), 1 mM EDTA, 4 M NaI) and 250 ␮l absolute dependent manner, and they did not bind to the Abs in the presence ethanol. The phage DNA was then washed with 70% ethanol, dried, and of 6B PS in solution (data not shown). The data for two biopanned resuspended in 30 ␮l TE buffer (50 mM Tris and 10 mM EDTA, pH8). A total of 5 ␮l DNA suspension was subjected to dideoxy termination reac- clones (PhaM1L3 and PhaM8C1) are shown in Fig. 2. Fig. 2A shows tion using DNA sequencing kit (PerkinElmer, Norwalk, CT). The sequenc- the data for PhaM1L3; Fig. 2B shows the data for PhaM8C1. The by guest on October 1, 2021 ing primer are position Ϫ96 from New England Biolabs, and AmpliTaq inhibition of binding was almost complete in the presence of 0.5 mg/L DNA polymerase FS. The sequence of the mimotope was obtained by 6B PS, but it was not demonstrable even in the presence of 100-fold running the above reaction products through an automated DNA sequencer more unrelated PS. Interestingly, PhaM8C1, but not PhaM1L3, was from PE Applied Biosystems (Foster City, CA). partially inhibited with 6A PS, which is an isopolymer of 6B PS. Bacteriophage-binding assays These findings indicate that all 23 biopanned clones bound to the Ab A sandwich type ELISA was performed, as described below. Hyp6BM1 or in an Ag-specific manner. Hyp6BM8 mAb was absorbed on 96-well microtiter plates (Nunc) at a concentration of 10 mg/L in carbonate-bicarbonate buffer (pH 9.6). The plates coated with Ab were blocked with 2% skim milk in PBS after wash- ing, and serially diluted phage clones (ranging from 6 ϫ 1010 to 3.7 ϫ 106 PFU/well) were added to the wells and incubated for 1.5 h at room tem- perature. After the unbound phage particles were washed, peroxidase-la- beled anti-phage mAb (Pharmacia Biotech) was added. Bound peroxidase was quantitated with tetramethyl benzidine (Kirkegaard & Perry Labora- tories, Gaithersburg, MD) substrate. The sandwich type assay was modified in some cases, as below. To evaluate cross-reactive binding of the phage clones to other mAbs, the plates were coated with HmenB3, Hyp6A1, Hyp6BM7, Hyp6BM10, or 101.4.1. To determine the specificity of the binding of the phage particles to mAb, phage particles (1 ϫ 109 PFU/well) were added to the wells in the presence of varying concentrations of inhibitors. Used as inhibitors were synthetic peptides or PS. The PS used as inhibitors include E. coli K1 PS, pneumococcal cell wall polysaccharide, capsular PS of Hemophilus influ- enzae type b, and capsular PS of S. pneumoniae serotypes 1, 3, 7F, and 23F. E. coli K1 PS is ␣(2Ð8)-linked polysialic acid and identical in struc- ture to N. meningitidis group B capsular PS (21, 22). FIGURE 1. The amount of bacteriophage bound to the ELISA wells Synthesis of peptides (y-axis) vs the number of phage particles in each well (x-axis). PhaM1L3 (f), PhaM1L9 (F), or PhaM1-CNTL (Ⅺ) phage clones were placed in The peptides used for this study were NLpeptide6 (HSACTTPGPWFC), Œ ‚ NLpeptide10 (YHSNIKFNPPG), NLpeptide9 (HSACTGPGSWFCG), Hyp6BM1-coated wells. PhaM8C1 ( ) or PhaM8-CNTL ( ) phage clones NLpeptide11 (CHSHYHKFG), and NLpeptide12 (YSACTTPGPWFC). were placed in Hyp6BM8-coated wells. PhaM1-CNTL and PhaM8-CNTL NLpeptide6, NLpeptide9, and NLpeptide12 were circularized at two cys- do not bind to either Ab and were used as negative controls. The amount teine sites. NLpeptide10 and NLpeptide12 were also referred to as of phage particles bound to the wells was determined with HRP-conjugated PepM1L3 and PepM8C1, respectively. The core sequence of the peptides anti-phage Ab, as described in Materials and Methods. The Journal of Immunology 6275

Table I. Mimotope peptide sequences of Hyp6BM1 and Hyp6BM8

mAb Phagea Peptide Sequenceb No. of Clonesc

Hyp6BM1 PhaM1L1 hsSHHKFSPggg 5/17 PhaM1L2 hsWNPKFHWggg 3/17 PhaM1L3 hsNIKFNPPggg 2/17 PhaM1L4 hsNPKFSPPggg 1/17 PhaM1L5 hsNWKFAPPggg 1/17 PhaM1L6 hsNWRFHDTggg 1/17 PhaM1L7 hsHFHYFSPggg 1/17 PhaM1L8 hsHSHYHKFggg 1/17 PhaM1L9 hsHYHKFggg 1/17 PhaM1L10 hsSMDPRFYggg 1/17 Hyp6BM8 PhaM8C1 hsacTTPGPWFcggg 6/6

a The names of phage clones with linear (L) and circular (C) random peptides. b Sequences of clones from heptapeptide insert phage library after third biopan- ning by Hyp6BM1 and Hyp6BM8 are shown. Flanking sequences are shown in small letters and inserted sequences are shown in capital letters. c Number of identical peptide sequences vs number of isolated clones. Downloaded from

clones. All six phage clones obtained with Hyp6BM8 expressed the identical sequence CTTPGPWFC, and PhaM8C1 clone was chosen as the representative clone. A totally unexpected observation was that the PhaM8C1 insert sequence is identical to the insert sequence found for the phage clone PhaB3C4, which was obtained previously with a mAb http://www.jimmunol.org/ against NMGB PS (17). Consistent with the sequence data, the PhaM8C1 clone was observed to bind to Abs to meningococcal FIGURE 2. The amount of phage bound to the microwells in the pres- group B PS as well as Abs to pneumococcal 6B PS (see Fig. 4). ence of 50 mg/L (filled bars) or 0.5 mg/L (hatched bars) pneumococcal capsular PS of different serotypes. The serotypes were indicated for each In addition to the identical sequences of some of the clones, group at the x-axis. Pneumococcal cell wall polysaccharide and H. influ- there are also shared amino acid sequence motifs among different enzae type b PS were used as irrelevant PS controls. A, Microwells were clones. NXKF motif was found in seven nonidentical phage clones coated with Hyp6BM1 and had 1010 PFU PhaM1L3 phage particles. B, (41%). Dimeric motif KF was very common and was found in 14 Microwells were coated with Hyp6BM8 and contained 109 PFU PhaM8C1. clones. In two additional clones, K, of the KF motif, was replaced by guest on October 1, 2021 with R, another cationic amino acid. HS motif is also present in the viral protein III adjoining the amino end of the peptide insert (Ta- The binding specificity of clones PhaM1L3 and PhaM8C1 was ble I). This HS in the viral protein may participate in the formation examined in greater detail by testing their binding to other pneu- of the of PhaM1L9 because it is selected by PhaM1L8 mococcal Abs. PhaM1L3 demonstrably bound only to Hyp6BM1, (HSHYHKF) as a part of the peptide insert. Phenylalanine was in the mAb used for the selection of PhaM1L3, and did not bind all phage clones. Proline is very common, and two proline residues several other mAbs binding 6B PS. Thus, PhaM1L3 appears to be are found in several mimotopes, including PhaM8C1. specific for a unique (idiotypic) determinant of the Ab used to select the clone. In contrast, PhaM8C1 bound to various anti-6B Peptide inserts are responsible for the binding of phage clones Abs. Although PhaM8C1 was selected with Hyp6BM8, it actually Although the heptameric peptide inserts are most likely responsi- bound better to the anti-6B mouse mAb Hyp6BM7 and Hyp6BM1 ble for the binding of the phage particles to the pneumococcal Abs, than to Hyp6BM8. It did not bind to Dob1 (data not shown), which it is possible that other parts of the phage protein may also be is a human mAb to 6B PS (19). PhaM8C1 also failed to bind to an critical to the binding. To directly investigate the role of the pep- unrelated Ab, 101.4.1. Thus, PhaM8C1 is broadly reactive with tide inserts in binding, three peptides were chemically synthesized several mouse anti-6B Abs, but its binding is selective. and were examined for their ability to inhibit the binding of the Abs to their Ags. Peptides did not inhibit the binding of Abs to 6B Determination of the sequence of the peptide inserts in the PS Ags (data not shown) perhaps because the PS bound to the Ab selected phage clones too strongly for the peptides to displace it from the Ab. However, Although the 23 phage clones were isolated from separate phage all three peptides (NLpeptide10, NLpeptide11, NLpeptide12) that plaques, it is expected that many clones are duplicate clones shar- were examined inhibited the phage clones from binding to the ing the clonal origin and expressing the identical peptide sequence. mAb used for their biopanning in a dose-dependent manner (Fig. To identify the duplicate clones, the nucleotide sequence of the 3). These data, along with the fact that 6B PS can inhibit the inserted DNA of all the cloned phages was determined and the binding of the phage clones, suggest that the peptide inserts are DNA sequences were translated into amino acid sequences (Table directly involved in the binding. I). As expected, many clones were found to have the identical sequences. Among the phage clones obtained with Hyp6BM1, five V region structures of Hyp6BM8 and HmenB3 are distinct clones (29%) expressed the sequence SHHKFSP, and PhaM1L1 The finding that the peptide insert of PhaM8C1 is identical to a clone was chosen as the representative of the five clones for ad- meningococcal mimotope identified with HmenB3, a mAb against ditional studies. Two more linear sequences, represented by N. meningitidis group B PS, was unexpected. One explanation for PhaM1L2 and PhaM1L3 clones, were also found within multiple the above finding is that HmenB3 and Hyp6BM8 may have the 6276 A PEPTIDE MIMOTOPE BINDING TO TWO DIFFERENT Abs

derived from a J606 family VH gene and JH1 gene, whereas HmenB3 VH region is formed with a J558 VH gene and JH4 gene. ␬ The VL region is formed with a V 1 family gene for Hyp6BM8 andaV␬2 family gene for HmenB3. In addition, there are differ-

ences in DH regions and somatic mutations between the two Abs. Thus, the V regions of the two mAb display large differences and show little similarity.

PhaM8C1 expresses both meningococcal and pneumococcal We next examined the binding of PhaM8C1 to HmenB3 and Hyp6BM8. PhaM8C1 did bind to HmenB3-coated plates readily (Fig. 4), but it did not bind to another meningococcal Ab, HmenB1 (data not shown). Its binding to HmenB3 was inhibitable with E. FIGURE 3. The amount of phage bound to Ab-coated microwells in the coli K1 PS, but not with an excess amount of 6B PS (Fig. 4A). E. presence of varying concentrations of synthetic peptides in solution. The coli K1 PS is chemically identical to N. meningitidis group B PS synthetic peptides, identified for each experimental group at the x-axis, (25). Conversely, PhaM8C1 clone bound to Hyp6BM8, and its were present at 1 mM (open bars), 0.1 mM (gray bars), or 0 mM (filled binding was efficiently inhibited with 6B PS, but not even with a bars). PhaM1L3, PhaM1L9, or PhaM8C1 phage clones were used respec-

large amount of E. coli K1 capsular PS (Fig. 4B). This observation Downloaded from tively for the experimental group, using PepM1L3 (NLpeptide10), NLpep- showed that PhaM8C1 clone does bind to pneumococcal as well as tide11, or PepM8C1 (NLpeptide12) peptides. PepM1L3 and NLpeptide11 meningococcal Abs in an Ag-specific manner. peptides were added to Hyp6BM1-coated microwells, and PepM8C1 pep- tides were added to Hyp6BM8-coated wells. NLpeptide6 can assume multiple well-populated conformations The inhibition studies described above suggest that distinct struc- identical or very similar V region structures. To examine the pos- tures are binding the different mAb reactive with PhaM8C1. One http://www.jimmunol.org/ sibility, we determined the DNA sequence of the V regions of way this could happen would be if PhaM8C1 can express two or HmenB3 and Hyp6BM8, and their sequences were deposited in more conformations, with each expressing a different epitope. To

GenBank. The accession numbers are: AF486641 for VH of examine this possibility, we examined two circular peptides, Hyp6BM8, AF486642 for VL of Hyp6BM8, AF486643 for VH of NLpeptide6 and NLpeptide9, with two-dimensional (2-D) NMR HmenB3, AF486644 for VL of HmenB3. Hyp6BM8 VH region is spectroscopy. NLpeptide6 is based on PhaM8C1 insert, and NLpeptide9 is a control peptide that is also circular. To simplify the identification of the major conformations of the two peptides, we examined the proton NMR TOCSY patterns of the single tryp-

tophan found in both peptides. TOCSY cross-peaks between the by guest on October 1, 2021 ring HN (chemical shift range, ϳ10Ð10.3 ppm) and Hd (chemical

FIGURE 4. The amount of phage bound to the microwells in the pres- ence of varying concentrations of 6B PS (F) and E. coli K1 PS (E)in FIGURE 5. Proton TOCSY 2-D NMR cross-peaks between the ring HN solution. PhaM8C1 phage was added to Hyp6BM8 (A)- and HmenB3 (B)- (x-axis) and ␦H(y-axis) of the single tryptophan present in the two pep- coated microwells. tides. Contour lines indicate the magnitude of the cross-peaks. The Journal of Immunology 6277 shift range, ϳ7.1Ð7.3 ppm) are shown in Fig. 5. For NLpeptide9, Aromatic amino acids have been noted for various PS mimotopes three Trp ring HN-Hd cross-peaks are observed with the intensity of group C meningococcal PS (26), group B streptococcal type III ratio of 4:1:0.25 (Fig. 5, upper panel). One cross-peak is signifi- capsular PS (27), and other PS (26, 28). Taken together, these cantly more intense than the others, indicating that one conforma- results indicate that our mimotope sequences are distinct from tion is favored for NLpeptide9. In contrast, NLpeptide6 shows other PS mimotopes. three strong Trp HN-Hd cross-peaks (Fig. 5, lower panel) with the An unexpected finding was that PhaM8C1 is identical in se- intensity ratio of 2:1:1, indicating the existence of three well-pop- quence to a meningococcal mimotope, PhaB3C4 (17). PhaB3C4 ulated forms. TOCSY peak patterns for other residues in both pep- phage clone was obtained in our prior studies with a mAb against tides also indicate the presence of multiple conformations present NMGB capsular PS, ␣(2Ð8)-linked polysialic acid (17). PhaM8C1 in similar relative amounts. The existence of multiple conformers clone was found to bind to both mAb in an Ag-specific manner, was shown not to be a result of peptide aggregation; the relative indicating that the sequence identity was not based on trivial tech- intensities of resonances assigned to different conformers were nical accidents (e.g., contamination in the phage clone). Also, this found to be invariant over a range of peptide concentrations (0.15Ð observation is not based on the similarity in the two PS molecules 3.4 mM) for both NLpeptide6 and NLpeptide9. Rather, multiple or two mAbs. No serological relationship has been noted between conformers may arise from proline cis-trans isomerization: the the two PS, even though these two PS have been extensively stud- proline resonances show the largest chemical shift differences ied serologically for their pathogenic importance. The two mAbs among conformers, and the analysis of 2-D ROESY data detects were formed with totally different V region gene families, and their both NMR patterns of cis- and trans-forms of proline (L. Zhong complementarity-determining regions were found to be quite dis- and K. L. Bren, unpublished observations). Taken together, we tinct when the DNA sequences of the VL and VH regions of the Downloaded from conclude that cis-trans-isomerization of proline residues gives rise two Abs were determined. to more well-populated conformers for NLpeptide6, which has two The explanation is most likely based on the peptide mimotope proline residues, than for NLpeptide9, which has only one proline. itself. For instance, PhaB3C4 may express multiple epitopes at all times. The circular mimotope is relatively small and would behave Discussion like a expressing one epitope. Nevertheless, it may be pos- By biopanning phage libraries with two IgM mAbs against S.

sible that the top side of the circular mimotope may bind one Ab, http://www.jimmunol.org/ pneumoniae type 6B PS (20), we have produced two groups of but the bottom side of the mimotope may bind another. Alterna- phage clones binding to anti-6B Abs. Their binding is specific tively, the mimotope may express multiple conformations over because the phage clones examined did not bind to irrelevant IgM time by rapidly switching from one conformation to another. Al- Abs and their binding was inhibited by pneumococcal capsular PS though the circular mimotope forms a small molecular ring with of 6B serotype, but not by pneumococcal PS of unrelated sero- little conformational freedom, PhaM8C1 mimotope has two pro- types. Furthermore, the peptide insert was directly involved in the line residues that can slowly (in 10Ð100 s) switch between cis and binding because synthetic peptide can inhibit the binding of the trans conformations. This is consistent with the fact that proline is phage clones to the two mAbs (Fig. 3). Thus, peptide inserts ex- often important in the overall conformation of a protein molecule

pressed by our phage clones are the mimotopes for 6B PS. by guest on October 1, 2021 (29). PhaM8C1 is shown to have multiple conformations in our Although both groups of phage clones bind to the Abs in an NMR studies, although one conformation is preferred over the Ag-specific manner, the two groups are clearly different in their other. We propose that one conformation seen in the NMR pattern binding characteristics. PhaM8C1 clone binds to Hyp6BM8 as well as other anti-6B Abs, whereas the linear PhaM1L3 (the linear mimics pneumococcal PS, and the other mimics meningococcal mimotopes) binds only to Hyp6BM1, the mAb used for the selec- PS. While we believe that the two major conformations are in- tion. Perhaps the linear mimotope can assume multiple conforma- volved in the reactions, it is possible the third conformation may be tions (NMR data not shown), and only a very small fraction of the stabilized by the Ab, becomes the dominant conformation in the linear mimotope can bind to the Ab. Consequently, the binding can presence of Ab, and participates in the binding. Further studies are be demonstrated only with the Ab used for the biopanning. In necessary to examine these possibilities. contrast, PhaM8C1 is a circular peptide that can assume only a We believe that peptides with proline residues may not be so limited number of conformations and may behave like a hapten desirable as mimotope vaccines because proline can assume mul- expressing the 6B PS epitope. Because Hyp6BM8 binds to both tiple semistable conformations. This belief is reinforced by the 6A and 6B PS, PhaM8C1 may express the epitope shared by both observation that a short peptide containing several proline residues 6A and 6B PS. This appeared to be the case because both 6A and (PPPGMRPP) can elicit binding Sm and nRNP (30, 6B PS could also inhibit the binding of the PhaM8C1 to 31). Immediately following the immunization with the peptide, the Hyp6BM8. animals initially produce Abs binding the peptide. In the later When the sequences of the individual mimotopes of Hyp6BM1 phase of immunization, the animals produce Abs to multiple were determined, areas of consensus were readily recognizable. epitopes found in Sm and nRNP molecules. If the animals have the KF was expressed on 82.4% of the linear mimotopes. In some proper genetic background, the immunized animals may develop cases, K is replaced with R, another positively charged amino acid. systemic lupus erythematosus disease. Although it is unclear how KF often appears in association with NX, suggesting NXKF is a this Ag-spreading phenomenon occurs, the existence of distinct, broader consensus sequence for the linear peptide mimotopes. semistable conformations associated with the presence of multiple These consensus sequences were not previously known, and the proline residues in the peptide may contribute to this phenomenon. consensus sequences already associated with PS mimotopes, such Over the last several years, peptide mimotopes have been stud- as WXY, were not observed. When single amino acids are exam- ied extensively for their usefulness as a vaccine. While there are ined, proline is found to be common: all but three mimotopes have successful examples reported in the literature, there are also studies proline, and five mimotopes have two prolines. In addition, our showing limitations in this approach. For instance, mimotope vac- sequence contained a large number of aromatic amino acids. F cines may produce Abs binding the native Ag, but without pro- could be found in all phage clones. PhaM8C1 clone has two, and tective function. These peptides could lead to the induction of in- PhaM1L2 and PhaM1L7 clones have three aromatic amino acids. appropriate memory B cells. Our study now suggests that the 6278 A PEPTIDE MIMOTOPE BINDING TO TWO DIFFERENT Abs mimotope vaccines should be tested for the induction of unin- 14. Shapiro, E. D., A. T. Berg, R. Austrian, D. Schroeder, V. Parcells, A. Margolis, tended Abs. Although peptide mimotopes are a promising new R. K. Adair, and J. D. Clemens. 1991. The protective efficacy of polyvalent pneumococcal polysaccharide vaccine. N. Engl. J. Med. 325:1453. approach for vaccines, their immunological properties should be 15. Shinefield, H. R., and S. Black. 2000. Efficacy of pneumococcal conjugate vac- carefully investigated before their use as vaccines. cines in large scale field trials. Pediatr. Infect. Dis. J. 19:394. 16. Shelly, M. 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