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* Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea;
Department of Chemistry, University of Rochester, Rochester, NY 14642; and Departments of
Pathology and
Microbiology, School of Medicine, Division of Laboratory Medicine, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(2
8) 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.
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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides mimicking polysaccharide (PS)3 Ags can be readily produced and used various ways. The peptide mimics of PS Ags should be useful for studying differences in immune mechanisms used by PS and protein Ags. In addition, the peptide mimics should, unlike PS, elicit T cell help, immune memory, and strong Ab responses. Also, the peptides should be easier to manufacture and modify than PS Ags (7, 8). Thus, the mimotopes may be useful as vaccine components. However, despite reports of successful mimotope vaccines (4, 9, 10, 11), mimotopes often either are poorly immunogenic, elicit ineffective Abs, or induce B cell memory for ineffective Abs. Consequently, there is a need for further studies of peptide mimics of PS Ags before peptide mimic vaccines can reach their full potential.
Streptococcus pneumoniae is a well-known pathogen, causing several serious diseases in young children and the elderly (12). As a result, Ab responses to pneumococcal PS Ags have been extensively studied in the past. The peptide mimics can be easily compared with other pneumococcal Ags for their immunogenicity and for inducing protective Abs. Also, there is a need for more effective pneumococcal vaccines. The widely available pneumococcal vaccine containing capsular PS from 23 common serotypes (13) is not immunogenic in young children, and its effectiveness is reduced among the elderly (14). A new conjugate vaccine, although effective in young children (15), is not effective among the elderly (16) and is expensive to manufacture. We have studied the peptide mimics of pneumococcal capsular PS and have identified a peptide that can mimic pneumococcal PS as well as meningococcal PS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HmenB1 and HmenB3 are IgM mAb specific for Neisseria
meningitidis group B capsular PS (17). The 101.4.1 is
a mAb to N-acetyl-
-D-glucosamine from M.
Cunningham (University of Oklahoma, Oklahoma City, OK)
(18). Hyp6A1 is a mouse IgM mAb specific for pneumococcal
6A PS. Dob1 is a human IgG2 mAb binding to 6B PS (19).
Hyp6BM1, Hyp6BM7, Hyp6BM8, and Hyp6BM10 are mouse IgM mAb specific for
pneumococcal 6B PS (20). Dob1, Hyp6BM7, Hyp6BM8, and
Hyp6BM10 cross-react with 6A PS, an isopolymer of 6B PS. Hyp6BM1 does
not cross-react with 6A PS, and Hyp6A1 does not cross-react with 6B PS
(20). Hyp6BM1 and Hyp6BM8 were used for biopanning, and
the mAb were purified from the mouse ascites by
(NH4)2SO4
precipitation and by chromatography over a column of Sephacryl S-300HR
(Pharmacia Biotech, Uppsala, Sweden).
Production of phage clones expressing the mimotopes
Two phage libraries from New England Biolabs (Beverly, MA) were
used for our study. One contained linear peptides composed of 7 random
aa, and the other contained circular peptides of 9 aa; circularization
was achieved by a covalent bond between the cysteines at positions 1
and 9, and the 7 aa between the two cysteines are randomly chosen.
Biopanning the phage libraries was performed as described
(17). Briefly, 60-mm petri dishes (Nunc, Roskilde,
Denmark) were coated with mAb at a concentration of 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 (50 mM Tris-Cl (pH 7.5), 150 mM NaCl). For each
biopanning cycle, 
2 x 1011 PFU phage
were placed in the petri dish and incubated for 30 min. Following the
removal of unbound phage particles, bound phage particles were eluted
from the dish with 0.2 M glycine-HCl (pH 2.2), and the recovered phage
solution was neutralized with 1 M Tris-Cl (pH 9.1). The recovered phage
was expanded in number by growing with Escherichia coli for
a new cycle of biopanning. The biopanning was performed for three or
four cycles before individual phage clones were isolated.
DNA sequencing of phage peptide
To determine the DNA sequence of the mimotope peptide, phage clones were expanded by growing them in 1 ml E. coli cultures for 4–5 h at 37°C. The bacteria present in the culture were removed by a brief centrifugation (10,000 x g for 30 s). A total of 500 µl bacteria-free culture supernatant was then mixed with 200 µl 20% PEG/2.5 M NaCl solution, and the mixture was centrifuged (10,000 x g) for 10 min to precipitate the phage as a pellet. The pellet was isolated and resuspended with 100 µl Tris buffer with iodide (10 mM Tris-Cl (pH 8), 1 mM EDTA, 4 M NaI) and 250 µl absolute ethanol. The phage DNA was then washed with 70% ethanol, dried, and resuspended in 30 µl TE buffer (50 mM Tris and 10 mM EDTA, pH8). A total of 5 µl DNA suspension was subjected to dideoxy termination reaction using DNA sequencing kit (PerkinElmer, Norwalk, CT). The sequencing primer are position -96 from New England Biolabs, and AmpliTaq DNA polymerase FS. The sequence of the mimotope was obtained by running the above reaction products through an automated DNA sequencer from PE Applied Biosystems (Foster City, CA).
Bacteriophage-binding assays
A sandwich type ELISA was performed, as described below. Hyp6BM1 or 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 washing, and serially diluted phage clones (ranging from 6 x 1010 to 3.7 x 106 PFU/well) were added to the wells and incubated for 1.5 h at room temperature. After the unbound phage particles were washed, peroxidase-labeled anti-phage mAb (Pharmacia Biotech) was added. Bound peroxidase was quantitated with tetramethyl benzidine (Kirkegaard & Perry Laboratories, 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 x 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
influenzae type b, and capsular PS of S. pneumoniae
serotypes 1, 3, 7F, and 23F. E. coli K1 PS is
(2, 3, 4, 5, 6, 7, 8)-linked polysialic acid and identical in structure to N.
meningitidis group B capsular PS (21, 22).
Synthesis of peptides
The peptides used for this study were NLpeptide6 (HSACTTPGPWFC), NLpeptide10 (YHSNIKFNPPG), NLpeptide9 (HSACTGPGSWFCG), NLpeptide11 (CHSHYHKFG), and NLpeptide12 (YSACTTPGPWFC). NLpeptide6, NLpeptide9, and NLpeptide12 were circularized at two cysteine sites. NLpeptide10 and NLpeptide12 were also referred to as PepM1L3 and PepM8C1, respectively. The core sequence of the peptides was identified with an underline and derived from the peptide inserts of PhaB3C1 (17), PhaM1L3, PhaM1L9, or PhaM8C1. The amino acid Y or C at the N-terminal was added for the purpose of radiolabeling or conjugation with carrier protein. The remaining amino acids in either sides of the core peptide were derived from the sequence of the phage protein pIII flanking the peptide inserts. These peptides were synthesized by Biosynthesis (Lewisville, TX) or Biomolecules Midwest (Waterloo, IL).
NMR spectroscopy
Solutions (2.5 mM) of NLpeptide6 and NLpeptide9 were prepared in PBS. The 1-H TOCSY spectra of both peptides were obtained on a 500-MHz Varian INOVA nuclear magnetic resonance (NMR) spectrometer (Varian, Palo Alto, CA) using a standard pulse sequence (23, 24). Data were processed and analyzed using FELIX 97 software (Accelrys, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biopanning of the phage library displaying the linear peptides
with Hyp6BM1-coated petri dishes readily enriched for the Ab-specific
phage clones and 17 phage clones were chosen randomly after three or
four cycles of biopanning. Biopanning the same phage library with
Hyp6BM8-coated petri dish did not enrich for the Ab-binding clones.
When a second phage library expressing the circular peptide was
biopanned, 6 clones binding to the plates were selected at random. In
addition to these 23 clones selected by panning, a phage clone was
chosen from each library (PhaM8-CNTL and PhaM1-CNTL) without any
selection to use as negative controls. PhaM1-CNTL expresses the linear
peptide and PhaM8-CNTL, a circular peptide. Binding patterns of select
phage clones were shown in Fig. 1
.
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. Fig. 2A shows the data for
PhaM1L3; Fig. 2B shows the data for PhaM8C1. The inhibition
of binding was almost complete in the presence of 0.5 mg/L 6B PS, but
it was not demonstrable even in the presence of 100-fold more unrelated
PS. Interestingly, PhaM8C1, but not PhaM1L3, was partially inhibited
with 6A PS, which is an isopolymer of 6B PS. These findings indicate
that all 23 biopanned clones bound to the Ab in an Ag-specific
manner.
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Determination of the sequence of the peptide inserts in the selected phage clones
Although the 23 phage clones were isolated from separate phage
plaques, it is expected that many clones are duplicate clones sharing
the clonal origin and expressing the identical peptide sequence. To
identify the duplicate clones, the nucleotide sequence of the inserted
DNA of all the cloned phages was determined and the DNA sequences were
translated into amino acid sequences (Table I). As expected, many clones were found
to have the identical sequences. Among the phage clones obtained with
Hyp6BM1, five clones (29%) expressed the sequence SHHKFSP, and PhaM1L1
clone was chosen as the representative of the five clones for
additional studies. Two more linear sequences, represented by PhaM1L2
and PhaM1L3 clones, were also found within multiple clones. All six
phage clones obtained with Hyp6BM8 expressed the identical sequence
CTTPGPWFC, and PhaM8C1 clone was chosen as the representative
clone.
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). This HS in the viral protein
may participate in the formation of the epitope of PhaM1L9 because it
is selected by PhaM1L8 (HSHYHKF) as a part of the peptide insert.
Phenylalanine was in all phage clones. Proline is very common, and two
proline residues are found in several mimotopes, including PhaM8C1. Peptide inserts are responsible for the binding of phage clones
Although the heptameric peptide inserts are most likely
responsible for the binding of the phage particles to the pneumococcal
Abs, it is possible that other parts of the phage protein may also be
critical to the binding. To directly investigate the role of the
peptide inserts in binding, three peptides were chemically synthesized
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 PS Ags
(data not shown) perhaps because the PS bound to the Ab too strongly
for the peptides to displace it from the Ab. However, all three
peptides (NLpeptide10, NLpeptide11, NLpeptide12) that were examined
inhibited the phage clones from binding to the mAb used for their
biopanning in a dose-dependent manner (Fig. 3). These data, along with the fact that
6B PS can inhibit the binding of the phage clones, suggest that the
peptide inserts are directly involved in the binding.
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The finding that the peptide insert of PhaM8C1 is identical to a meningococcal mimotope identified with HmenB3, a mAb against N. meningitidis group B PS, was unexpected. One explanation for the above finding is that HmenB3 and Hyp6BM8 may have the identical or very similar V region structures. To examine the possibility, we determined the DNA sequence of the V regions of HmenB3 and Hyp6BM8, and their sequences were deposited in GenBank. The accession numbers are: AF486641 for VH of Hyp6BM8, AF486642 for VL of Hyp6BM8, AF486643 for VH of HmenB3, AF486644 for VL of HmenB3. Hyp6BM8 VH region is 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 and a V2 family gene for HmenB3. In addition,
there are differences 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 epitopes
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. coli K1 PS, but not with an excess
amount of 6B PS (Fig. 4A). E. coli K1 PS is
chemically identical to N. meningitidis group B PS
(25). Conversely, PhaM8C1 clone bound to Hyp6BM8, and its
binding was efficiently inhibited with 6B PS, but not even with a large
amount of E. coli K1 capsular PS (Fig. 4B). This
observation showed that PhaM8C1 clone does bind to pneumococcal as well
as meningococcal Abs in an Ag-specific manner.
NLpeptide6 can assume multiple well-populated conformations
The inhibition studies described above suggest that distinct
structures are binding the different mAb reactive with PhaM8C1. One way
this could happen would be if PhaM8C1 can express two or more
conformations, with each expressing a different epitope. To examine
this possibility, we examined two circular peptides, NLpeptide6 and
NLpeptide9, with two-dimensional (2-D) NMR 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 tryptophan found in both peptides. TOCSY
cross-peaks between the ring HN (chemical shift range, 10–10.3 ppm)
and Hd (chemical shift range, 7.1–7.3 ppm) are shown in Fig. 5. For NLpeptide9, three Trp ring HN-Hd
cross-peaks are observed with the intensity ratio of 4:1:0.25 (Fig. 5, upper panel). One cross-peak is significantly more intense
than the others, indicating that one conformation is favored for
NLpeptide9. In contrast, NLpeptide6 shows three strong Trp HN-Hd
cross-peaks (Fig. 5, lower panel) with the intensity ratio
of 2:1:1, indicating the existence of three well-populated forms. TOCSY
peak patterns for other residues in both peptides also indicate the
presence of multiple conformations present in similar relative amounts.
The existence of multiple conformers was shown not to be a result of
peptide aggregation; the relative intensities of resonances assigned to
different conformers were found to be invariant over a range of peptide
concentrations (0.15–3.4 mM) for both NLpeptide6 and NLpeptide9.
Rather, multiple conformers may arise from proline
cis-trans isomerization: the proline resonances
show the largest chemical shift differences among conformers, and the
analysis of 2-D ROESY data detects both NMR patterns of cis-
and trans-forms of proline (L. Zhong and K. L. Bren,
unpublished observations). Taken together, we conclude that
cis-trans-isomerization of proline residues gives
rise to more well-populated conformers for NLpeptide6, which has two
proline residues, than for NLpeptide9, which has only one proline.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Thus, peptide inserts expressed by our phage
clones are the mimotopes for 6B PS. Although both groups of phage clones bind to the Abs in an Ag-specific manner, the two groups are clearly different in their binding characteristics. PhaM8C1 clone binds to Hyp6BM8 as well as other anti-6B Abs, whereas the linear PhaM1L3 (the linear mimotopes) binds only to Hyp6BM1, the mAb used for the selection. Perhaps the linear mimotope can assume multiple conformations (NMR data not shown), and only a very small fraction of the linear mimotope can bind to the Ab. Consequently, the binding can be demonstrated only with the Ab used for the biopanning. In contrast, PhaM8C1 is a circular peptide that can assume only a limited number of conformations and may behave like a hapten expressing the 6B PS epitope. Because Hyp6BM8 binds to both 6A and 6B PS, PhaM8C1 may express the epitope shared by both 6A and 6B PS. This appeared to be the case because both 6A and 6B PS could also inhibit the binding of the PhaM8C1 to Hyp6BM8.
When the sequences of the individual mimotopes of Hyp6BM1 were determined, areas of consensus were readily recognizable. KF was expressed on 82.4% of the linear mimotopes. In some cases, K is replaced with R, another positively charged amino acid. KF often appears in association with NX, suggesting NXKF is a broader consensus sequence for the linear peptide mimotopes. These consensus sequences were not previously known, and the consensus sequences already associated with PS mimotopes, such as WXY, were not observed. When single amino acids are examined, proline is found to be common: all but three mimotopes have proline, and five mimotopes have two prolines. In addition, our sequence contained a large number of aromatic amino acids. F could be found in all phage clones. PhaM8C1 clone has two, and PhaM1L2 and PhaM1L7 clones have three aromatic amino acids. Aromatic amino acids have been noted for various PS mimotopes of group C meningococcal PS (26), group B streptococcal type III capsular PS (27), and other PS (26, 28). Taken together, these results indicate that our mimotope sequences are distinct from other PS mimotopes.
An unexpected finding was that PhaM8C1 is identical in sequence to a
meningococcal mimotope, PhaB3C4 (17). PhaB3C4 phage clone
was obtained in our prior studies with a mAb against NMGB capsular PS,
(2, 3, 4, 5, 6, 7, 8)-linked polysialic acid (17). PhaM8C1 clone was
found to bind to both mAb in an Ag-specific manner, indicating that the
sequence identity was not based on trivial technical accidents (e.g.,
contamination in the phage clone). Also, this observation is not based
on the similarity in the two PS molecules or two mAbs. No serological
relationship has been noted between the two PS, even though these two
PS have been extensively studied serologically for their pathogenic
importance. The two mAbs were formed with totally different V region
gene families, and their complementarity-determining regions were found
to be quite distinct when the DNA sequences of the
VL and VH regions of the
two Abs were determined.
The explanation is most likely based on the peptide mimotope itself. For instance, PhaB3C4 may express multiple epitopes at all times. The circular mimotope is relatively small and would behave like a hapten expressing one epitope. Nevertheless, it may be possible that the top side of the circular mimotope may bind one Ab, but the bottom side of the mimotope may bind another. Alternatively, the mimotope may express multiple conformations over time by rapidly switching from one conformation to another. Although the circular mimotope forms a small molecular ring with little conformational freedom, PhaM8C1 mimotope has two proline residues that can slowly (in 10–100 s) switch between cis and trans conformations. This is consistent with the fact that proline is often important in the overall conformation of a protein molecule (29). PhaM8C1 is shown to have multiple conformations in our NMR studies, although one conformation is preferred over the other. We propose that one conformation seen in the NMR pattern mimics pneumococcal PS, and the other mimics meningococcal PS. While we believe that the two major conformations are involved in the reactions, it is possible the third conformation may be stabilized by the Ab, becomes the dominant conformation in the presence of Ab, and participates in the binding. Further studies are necessary to examine these possibilities.
We believe that peptides with proline residues may not be so desirable as mimotope vaccines because proline can assume multiple semistable conformations. This belief is reinforced by the observation that a short peptide containing several proline residues (PPPGMRPP) can elicit autoantibodies binding Sm and nRNP (30, 31). Immediately following the immunization with the peptide, the animals initially produce Abs binding the peptide. In the later phase of immunization, the animals produce Abs to multiple epitopes found in Sm and nRNP molecules. If the animals have the proper genetic background, the immunized animals may develop systemic lupus erythematosus disease. Although it is unclear how this Ag-spreading phenomenon occurs, the existence of distinct, semistable conformations associated with the presence of multiple proline residues in the peptide may contribute to this phenomenon.
Over the last several years, peptide mimotopes have been studied extensively for their usefulness as a vaccine. While there are successful examples reported in the literature, there are also studies showing limitations in this approach. For instance, mimotope vaccines may produce Abs binding the native Ag, but without protective function. These peptides could lead to the induction of inappropriate memory B cells. Our study now suggests that the mimotope vaccines should be tested for the induction of unintended Abs. Although peptide mimotopes are a promising new approach for vaccines, their immunological properties should be carefully investigated before their use as vaccines.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Moon H. Nahm, Department of Pathology, School of Medicine, Division of Laboratory Medicine, University of Alabama, 845 19th Street South, BBRB 614, Birmingham, AL 35294. E-mail address: nahm{at}uab.edu
3 Abbreviations used in this paper: PS, polysaccharide; 2-D, two-dimensional; NMR, nuclear magnetic resonance.
Received for publication January 14, 2002. Accepted for publication March 27, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-D-glucosamine in reaction with Abs and lectins, and induces in vivo anti-carbohydrate Ab response. J. Immunol. 153:5593.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), PhaM1L9 (•), or PhaM1-CNTL
(
) phage clones were placed in Hyp6BM1-coated wells. PhaM8C1 (
)
or PhaM8-CNTL (
) phage clones were placed in Hyp6BM8-coated wells.
PhaM1-CNTL and PhaM8-CNTL do not bind to either Ab and were used as
negative controls. The amount of phage particles bound to the wells was
determined with HRP-conjugated anti-phage Ab, as described in
Materials and Methods.
) in
solution. PhaM8C1 phage was added to Hyp6BM8 (A)- and
HmenB3 (B)-coated microwells.
H (y-axis) of the single
tryptophan present in the two peptides. Contour lines indicate the
magnitude of the cross-peaks.