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Departments of
* Cell Biology and
Medicine, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. neoformans is an encapsulated yeast that causes a life-threatening meningoencephalitis in a significant number of AIDS patients in the U.S. and as many as 30% of HIV-infected people in Africa (16, 17, 18). The mortality rate of acute cryptococcal meningitis is 10–20% (19, 20), and those patients who survive must be maintained on life-long anti-fungal therapy. mAbs against the glucuronoxylomannan (GXM) component of the capsular polysaccharide can protect mice against infection with C. neoformans (21). However, infection with C. neoformans and immunization with GXM conjugated to tetanus toxoid (GXM-TT) induce both protective and nonprotective Abs (12, 22, 23). These different types of Abs bind to distinct epitopes on GXM (12, 24). This has led us to define protective and nonprotective epitopes on GXM based on their ability to bind mAbs that do or do not prolong the life of mice lethally infected with C. neoformans. Nonprotective Abs can block the efficacy of protective Abs, providing an explanation for why polyclonal immune serum from GXM-TT-immunized mice does not protect against infection (21, 22, 25). These results suggest that a vaccine that directs the immune response specifically against a protective epitope, thereby eliciting only protective Abs, would be more effective than a vaccine consisting of all the capsular epitopes.
Since oligosaccharides that can bind to anti-GXM Abs have not been
identified, we have previously sought to create an effective vaccine by
identifying a peptide mimic of a protective epitope of GXM. We have
screened phage peptide libraries with 2H1, one of our most protective
anti-GXM mAbs (26, 27). 2H1 is encoded by the
VH7183 and V
5.1 genes that dominate the murine
Ab response to GXM (28). In these earlier studies we
identified several peptides that were mimetics of GXM (Table I
) in that they competed for 2H1 binding
to GXM. However, these peptides were not mimotopes of GXM, since
immunization with three representative peptides conjugated to keyhole
limpet hemocyanin (KLH) did not induce significant anti-GXM titers
(27). Crystal structure analysis revealed that the
carboxyl-terminal portion of PA1 was not effectively engaging the H
chain portion of the 2H1 Ag binding pocket (29). We
postulated that if we could improve the fit of the peptide to the 2H1 H
chain complementarity-determining regions (CDRs), we would be able to
elicit a better 2H1-like response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The L200 library was created by inserting a modified version of the PA1 motif between the SfiI sites of the vector fUSE5, located at the amino-terminal part of the pIII coat protein giving the final amino acid sequence H2N-ADVA X6 TPXW[M/L][M/L] X6 AAG-g3p (30, 31). Briefly, 24 pmol of the oligonucleotide 5'-TATATAG GCC GAC GTG GCC [NNK]6 ACA CCT NNK TGG MTG MTG [NNK]6 NNG GCC GCT GGG GCCTTATTA (Genset, San Diego, CA) was amplified by PCR using Taq polymerase and two short biotinylated oligonucleotides, biotin-5'-TATATAGGCCGACGTGGCC and biotin-5'-TAATAAGGCCCCAGCGGCC (100 pmol each, in a final volume of 100 µl). After 10 cycles (94°C for 30 s/56°C for 60 s/72°C for 60 s), the resulting mixture was purified by filtration over a 10-kDa Minicon (Amicon, Beverly, MA), digested with SfiI, and incubated with streptavidin-coated beads to remove the cut ends. After phenol/chloroform purification and concentration over a 10-kDa Minicon, the resulting 67-base insert was ligated to 500 ng of SfiI-cut fUSE5 vector (26, 32) in a 4:1 ratio. A library of 186 million clones was generated after 10 electroporations into the bacterial strain MC1061, of which 6.5% (12 million) gave a positive signal with mAb 2H1 as detected by direct lift assay on bacterial colonies (for details on phage library amplification and lift assay see Ref. 26). Random sequencing of 10 clones of the original L200 library (data not shown) revealed correct insertion of the degenerated oligonucleotide and an over-representation of methionine residues vs leucine residues in the two degenerate positions, M/L.
Peptide library screening
2H1-binding phage were isolated by successive cycles of
selection and purification by incubation with 2H1 Fab. Fabs were made
using the ImmunoPure Fab preparation kit from Pierce (Rockford, IL).
For each round, between 5 x 105 and 1
x109 transducing units were incubated with
decreasing amounts of 2H1 Fab for 24 h at 37°C in a final volume
of 100 µl. Phage-2H1 complexes were captured by biotinylated
anti-mouse L chain (Southern Biotechnology Associates,
Birmingham, AL) bound to streptavidin-coated magnetic beads (Dynal
Biotech, Lake Success, NY), prepared as previously described
(26). For the initial screen, the capture time was 2 min,
and 50 µl of beads were used per incubation. Phage yields were
calculated for each capture; the incubation giving the lowest yield
>1% was chosen for the next round. A total of six rounds of selection
with decreasing amounts of mAb 2H1 were performed.
Peptide sequencing and preparation
Peptide sequences were inferred from DNA sequences of selected
phage as previously described (26). Peptides PA1, P206.1,
P206N, and P206C (Table II) and their
biotinylated counterparts were synthesized by the Proteomics Facility
at Albert Einstein College of Medicine. Peptide preparations were
analyzed by HPLC and mass spectroscopy. Control peptide P315 was
a gift from Dr. S. Nathenson (Albert Einstein College of Medicine).
P206.1-multiple antigenic peptide (P206.1-MAP) was also prepared in the
Proteomics Facility using solid phase methodology on an ABI 433A
peptide synthesizer (PE Applied Biosystems, Foster City, CA) in 0.1
mmol scale using F-moc chemistry. The F-moc 4 branch-MAP resin was
purchased from Advance ChemTech (Louisville, KY), and all other
reagents were purchased from PE Applied Biosystems. Peptide
resin was cleaved and deprotected by a mixture of trifluoroacetic
acid-thioanisole-1,2-ethanedithiol phenol-water for 2 h. Peptides
were precipitated by methyl-tert-butyl ether and washed.
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All mAb used in this study have been reported previously
(28). The reactivity of the peptides to several different
anti-GXM mAbs was analyzed by incubating 1 µg/ml mAb with
biotinylated peptide (1 µg/ml) bound to streptavidin-coated (1
µg/ml) ELISA plates. Binding of mAbs was detected with anti-mouse
L chain Abs conjugated to alkaline phosphatase. ELISA plates were
developed with 1 mg/ml p-nitrophenyl phosphate
(Sigma-Aldrich, St. Louis, MO) in 1 M diethanolamine and 0.25 mM
MgCl2, pH 9.8, and the absorbance was measured at
405 nm. Competition ELISAs were performed by first incubating
anti-GXM Abs or serum from immunized mice with different
concentrations of peptide for 2 h at 37°C. Samples were then
transferred to a GXM-coated plate, and binding was detected as
described above. Anti-GXM serum titer was determined by ELISA. Plates
were coated with 10 µg/ml of GXM prepared from C.
neoformans serotype A, strain NIH 371. The GXM was a gift from A.
Casadevall (Albert Einstein College of Medicine); it was prepared by
cetyltrimethylammonium bromide precipitation and did not contain
detectable protein contaminants. O-acetyl groups were
chemically removed from GXM prepared from C. neoformans
serotype D, American Type Culture Collection strain 24067 by treatment
with NH4OH (33). Serum samples were
first treated with 100 mM 2-ME to inactivate IgM. Sera were then
incubated in serial dilutions starting at 1/25 or 1/50. GXM binding was
then detected as described above with either a mixture of alkaline
phosphatase-conjugated anti-mouse IgG isotypes (Southern
Biotechnology Associates) or Abs to each individual isotype. P206.1
cross-reactivity with anti-GXM titers was determined by first
incubating sera with 0.01, 0.1, or 1 mg/ml P206.1 at 37°C for
1 h and then determining the anti-GXM titer as described
above. In all cases a dilution was considered positive when the OD
observed was 3 times greater than background. The presence of Abs
against the various peptides was similarly determined by screening
mouse sera on biotinylated peptide (10 µg/ml) bound to
streptavidin-coated (10 µg/ml) plates.
Affinity determination
The kinetics of the interaction between 2H1 Fab and P206.1 were measured by optical sensor (Iasys; Fisons, Cambridge, U.K.). Biotinylated cuvettes (Fisons) were coated with streptavidin in PBS/0.05% Tween 20 until saturated. Biotinylated peptides (PA1, P206.1, P206C, or P206N) were then added at a concentration of 1 µM in PBS/0.05% Tween 20 and allowed to saturate the cuvette. Various concentrations of 2H1 Fab, ranging from 5–1000 nM, were added and allowed to bind. Cuvettes were regenerated using 10 mM HCl for 30 s. Curves were generated with Iasys software using two-phase association and dissociation protocols from which the equilibrium dissociation constant (Kd) was calculated.
Immunization
Peptides (1 mg) were conjugated to TT (3 mg; Connaught, Swiftwater, PA) in 0.25 ml of PBS, pH 7.5, with the slow addition of 120 µl of 5% glutaraldehyde. The conjugate was dialyzed at 4°C against sterile PBS for 3 days with daily changes. Six-week-old BALB/c mice (5–10 in each group) were immunized by i.p. injection with 100 µg of each peptide conjugate or 0.5 µg of GXM-TT (gift from J. Robbins, National Institutes of Health, Bethesda, MD) in emulsion with CFA on day 0 and with IFA on day 28. Mice were bled before the first immunization and on days 14 and 42 postimmunization.
Statistical analysis
Data were analyzed with StatView statistical software (SAS Institute, Cary, NC). ELISA data were compared using the Mann-Whitney U test for nonparametric data. A p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify peptides that would engage the 2H1 H chain CDRs more
effectively, we created an evolutionary sublibrary of PA1 using a
six-amino acid core peptide of the PA1 motif (TPXW[M/L][M/L]) that
was degenerate at residue 3 and had either methionine or leucine at the
fifth and sixth positions. Six random amino acids were added to either
end of the core motif (Fig. 1). This L200
library was subjected to six rounds of screening with progressively
decreasing amounts of 2H1 mAb. Random phage clones were sequenced from
each screen. Table III shows the amino
acid sequences from eight random phage clones (P206.1–6, P206.8, and
P206.11) isolated from the sixth and last phage screen. In this final
screen, the motifs represented by P206.1 and P206.6 were each
identified in three different clones. By ELISA, both motifs gave
stronger reactions than PA1, suggesting that they were binding 2H1 with
a higher affinity. The motif represented by P206.6 was present in the
second, fourth, and fifth rounds, while the motif represented by P206.1
appeared for the first time in this last screen. Since it required six
rounds of screening to focus the library on P206.1, this peptide was
synthesized for further analysis. The influence of amino acids on
either side of the core motif on the overall structure of the peptide
and its interaction with the Ab was difficult to predict. To examine
whether both amino- and carboxyl-terminal end randomization was
necessary for the higher binding to the 2H1 mAb and to study the roles
of these additional sequences, peptides were synthesized that retained
the core motif and had in addition either the amino-terminal (P206N,
Table II
) or carboxyl-terminal six amino acids (P206C, Table II) of
P206.1.
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ELISA data showed that 2H1 Fab bound to P206.1 
100-fold more
strongly than to PA1 (Fig. 2A). Competition ELISA (Fig. 2B) revealed that these new peptides inhibited the binding
of 2H1 Fab to GXM with an ID50 of 0.4 µM for
P206.1, 1.2 µM for P206N, and 12 µM for P206C compared with an
ID50 of 45 µM for PA1, which was very close to
the PA1 ID50 of 35 µM previously reported
(26). These peptides competed in a similar fashion with
the binding of 2H1 to P206.1 (Fig. 2C). In addition, all
three peptides blocked 2H1 binding to PA1 (data not shown). The control
peptide P315 had no effect on 2H1 Fab binding to GXM (Fig. 2B), P206.1 (Fig. 2C), or PA1 (data not shown) at
concentrations as high as 2.5 mM.
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and Table IV). This data
confirmed that the amino-terminal amino acids are essential for the
high affinity of P206.1 for 2H1, whereas the carboxyl-terminal amino
acids are less important in this regard.
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We compared the binding of several closely related anti-GXM
mAbs (28) to the different peptides by ELISA. Compared
with PA1, P206.1 bound more strongly than PA1 to most of the mAbs
tested. The binding of the various peptides to some mAbs is shown in
Fig. 4A. P206.1 binds well to
all mAbs known to be protective against C. neoformans
infection in a passive immunization model (i.e., 2H1, 3E5, 18B7, and
12A1) (34, 35, 36) and does not bind to the nonprotective Abs
13F1 and 21D2 (22, 25). We tested peptide binding to
several other mAbs to GXM not shown in Fig. 4. 2D10 and 7G7 are shown,
as they were representative of peptide binding patterns seen with other
IgM and IgA mAbs to GXM; the difference in binding seen between these
mAbs and 2H1, 3E5, and 18B7 (all IgG1) may represent differences in
fine specificity, avidity, or both. P206N binds as well or better than
P206.1 to most mAbs, while P206C binds less well than both P206.1 and
PA1 to many of the mAbs. Neither P206N nor P206C binds to the
nonprotective mAbs. Competition ELISAs between the peptides and the
different mAbs were performed to confirm the binding data. A few of
these inhibition studies are shown in Fig. 4B. Although 3E5
binds to all the peptides, the competition data indicate that there is
a difference in binding among these peptides, with
ID50 values similar to those seen with 2H1 and
ranging from 1.0 µM for P206.1 to 2.2 µM for P206N and 10.1 µM
for P206C. While all these mAbs are encoded by
VH7183 and V5.1, the Abs assayed here differ
from each other by a number of somatic point mutations in their
CDRs, in particular within VH CDR2
(35), which has been shown to be critical for peptide
binding (37). These amino acid differences probably
contribute to the differences in binding characteristics of P206.1,
P206N, and P206C presented in Fig. 4A. These results
indicate that while the amino-terminal, but not the carboxyl-terminal,
extension promotes the broad specificity of P206.1, the amino acid
sequence of the Ab binding region also contributes to the fine
specificity for the peptides.
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We have previously reported that mice immunized with P601E or PA1
conjugated to KLH induced low (<1/200) or undetectable titers to GXM
(27). In our current studies mice were immunized with
P206.1 conjugated to several different carriers. Despite inducing
significant Ab titers to the peptide (Fig. 5A), neither P206.1 conjugated
to TT nor P206.1 expressed as a tetrameric MAP was able to induce
titers to GXM greater than those observed in mice immunized with TT
alone. This suggested that despite its high affinity for 2H1 and its
ability to block 2H1 binding to GXM, P206.1 is not a mimotope of GXM,
since it does not elicit an immunological response to the native
carbohydrate Ag.
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B). However, boosting with P206.1-TT induced
significant titers to GXM (1/50,000 to 1/280,000), which are very high
for an anti-carbohydrate response (GXM 2°, Fig. 5B).
None of these animals developed significant titers to GXM after the
primary immunization with GXM-TT (GXM 1°, Fig. 5B),
indicating that the GXM-specific Abs were activated in response to
P206.1 immunization. Furthermore, the anti-peptide titers to P206.1
observed following the single peptide immunization of the GXM-primed
mice were similar to the titers to GXM and higher than those seen in a
primary response to P206.1-TT (Fig. 5B). As expected from
this finding, free P206.1 inhibited the binding of the serum Abs to
GXM, while P315, an irrelevant peptide, did not (Fig. 5C).
These data further prove that the anti-P206.1 Abs were
cross-reactive with GXM. The anti-GXM response induced by P206.1-TT
was >90% IgG1 with small amounts of IgG2b, IgG2a, and IgG3,
consistent with a secondary immune response (data not shown). While
this was the first time we were able to induce a strong
anti-carbohydrate response using P206.1, and this response was very
good for a peptide mimotope for a carbohydrate Ag, the titers were
significantly lower than the anti-GXM titers from other experiments
in which animals were primed and boosted with the GXM-TT conjugate
(average of 1/1,000,000; data not shown). We conclude that after
priming with the glycoconjugate and boosting with the peptide
conjugated to TT, P206.1 behaved like a mimotope of GXM. P206.1 induces the production of Abs to a protective epitope
Protective mAbs 2H1, 3E5, 18B7, and 12A1 do not react with
de-O-acetylated GXM, while most nonprotective mAbs do
(38) (data not shown). About 5% of the Abs elicited by
priming with GXM-TT and boosting with P206.1-TT reacted with
de-O-acetylated GXM (Fig. 6).
This is compared with other experiments in which 20–60% of the GXM
Abs from animals primed and boosted with GXM-TT reacted with
de-O-acetylated GXM (Fig. 6). While we cannot be sure that
all the Abs that bind de-O-acetylated GXM are nonprotective,
it is clear that P206.1 directed the immune response away from
de-O-acetylated epitopes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5.1 (28, 39, 40). The restricted nature of this response is similar to that
reported for other polysaccharide Ags (41), suggesting
that only a few B cells expressing this particular combination of V
regions are responsible for the anti-GXM response. We hypothesized
that screening phage libraries with the anti-GXM mAb 2H1
(VH7183, V5.1) would identify a mimetic that
would elicit anti-GXM Abs using the same V regions. However, while
peptides PA1 and P601E, which were identified with 2H1, were mimetics
of GXM, they did not stimulate significant Ab titers to GXM
(27). The crystal structure of PA1 in the 2H1 Ag binding
pocket revealed that compared with other peptides that had been
cocrystallized with Abs, the carboxyl-terminal portion of PA1 does not
fully engage the 2H1 H chain CDRs (29). These structural
data were confirmed functionally by the finding that anti-peptide
Abs made in response to P601E immunization used a very similar
VL to 2H1, but used a different
VH (27). We therefore turned again to the power of screening large peptide libraries to find a more effective peptide mimotope. We designed an evolutionary phage peptide library using the PA1 core motif as a fixed sequence and randomized extensions of that motif at both the carboxyl- and amino-terminal ends. We screened for a peptide that had higher affinity for 2H1 and isolated P206.1, which has 80-fold higher affinity than PA1 for the selecting mAb. Like PA1, P206.1 inhibits the binding of 2H1 to GXM and binds to all mAbs known to be protective in a passive immunization model, but does not bind to nonprotective anti-GXM mAbs.
To better understand why P206.1 bound with a higher affinity than PA1 to the 2H1 Ag binding pocket, we created peptides similar to P206.1 that had either the carboxyl-terminal (P206C) or the amino-terminal (P206N) additions along with the core motif. In light of the data from the PA1/2H1 crystal structure and reports that H chain CDRs are the primary contributors to V region affinity (37), we expected that the carboxyl-terminal end of P206.1 would contribute to its higher affinity. However, our results indicate that the significant increase in affinity of P206.1 is mediated primarily by the amino-terminal amino acids, whereas the carboxyl-terminal amino acids are not important in this regard, since P206N had an affinity for 2H1 close to that of P206.1, while the affinity of P206C for 2H1 was similar to that of PA1. The additional amino-terminal amino acids are also primarily responsible for the ability of P206.1 to bind several different anti-GXM mAbs with a higher affinity than PA1. It is possible that the amino-terminal amino acids affect the way P206.1 folds into the 2H1 Ag binding site, thereby improving the fit of the peptide to the H chain CDRs. Alternatively, P206.1 may fold into the Ag binding site of 2H1 in a different conformation than PA1 or engage framework residues that border on the pocket. The only way to resolve these issues would be to analyze the crystal structure of P206.1 in 2H1. Despite its high affinity, P206.1 behaved like the lower affinity mimetic peptides and did not elicit a strong anti-GXM response in mice when conjugated to various carriers, even though it was able to stimulate a strong anti-peptide response. This result led us to a number of conclusions regarding the search for an effective peptide mimotope of GXM.
There is abundant evidence that peptides can serve as mimics and even
mimotopes for carbohydrate Ags (42, 43). As reported by
Scott et al. (44, 45), there appears to be a difference
between a structural mimic and an immunologically functional mimic. We
were not searching for a peptide that would mimic the structure of GXM,
but, rather, for a peptide that would best mirror the Ag binding pocket
of 2H1 and thus serve as a functional mimotope. Because of its high
affinity, it is possible that P206.1 can achieve a conformation that is
a true mirror image of the 2H1 binding pocket. However, this would have
to be a rare conformation, since P206.1 alone elicits high
anti-peptide titers, but negligible Ab to GXM. Since peptides are
likely to bind Ab through a different set of V region interactions than
does the native carbohydrate Ag (29, 46), we did not
expect these peptides to be structural mimics of GXM. This is true of
the anti-GXM family of Abs, since a single amino acid change in the
VH CDRs is sufficient to alter the Ab specificity
for the peptides used here, whereas such differences are not sufficient
to alter the Ab specificity to GXM (37). This has been
further illustrated by the binding of our peptides to a panel of
anti-GXM mAbs (Fig. 4). These Abs have all undergone somatic
mutation, and the different amino acid point mutations in their CDRs
(35) probably contribute to the differences in binding
characteristics to peptides P206.1, P206N, and P206C. Since these Abs
retain binding to GXM, GXM and the peptides are probably reacting with
a different set of amino acid residues in the 2H1 binding site. This
dissociation between Ab binding to a peptide vs a carbohydrate has
serious implications in the search for peptide mimotopes of
carbohydrate Ags; when isolating a higher affinity peptide, one selects
for interactions with the Ab paratope that may be very different from
the carbohydrate-Ab interactions. This is, in fact, what we observed
with P206.1.
Since the response to GXM is highly restricted, and all of the
anti-GXM mAbs we have identified have somatic mutations
(28), we hypothesized that P206.1 was not eliciting an
anti-GXM response because at any given time there were not
sufficient B cells expressing a surface Ab that cross-reacted with
P206.1 and GXM. In retrospect, this is not surprising, since 2H1 has
undergone somatic mutation and affinity maturation (28),
and these amino acid differences are likely to form a paratope that
differs significantly from that formed by the canonical anti-GXM
germline VH7183 and V5.1 genes expressed on
the surface of naive circulating B cells. A peptide that binds to
somatically mutated V regions could be restricted in its ability to
recognize unmutated anti-GXM Abs. This suggests that the best way
to identify an immunologically functional peptide mimotope of GXM that
would stimulate naive B cells making 2H1-like Abs to GXM is to screen
peptide libraries with an mAb expressing the germline
VH7183 and V5.1 genes. This idea is supported
by the success of several groups in identifying mimotopes to
carbohydrate Ags selected by screening libraries with low affinity IgM
or IgA mAbs (47, 48, 49).
Although P206.1 was not able to elicit an immune response to GXM from
naive mice, we wanted to determine whether this peptide could be used
as a means of selectively stimulating B cells producing protective Abs
from a diverse anti-GXM B cell repertoire. We therefore immunized
mice with a low dose of GXM-TT to expand the pool of memory B cells
making both protective and nonprotective anti-GXM Abs. After
boosting these mice with P206.1, either alone or attached to various
carriers, we found that P206.1-TT stimulated significant anti-GXM
titers with the characteristics of a secondary immune response.
Furthermore, this anti-GXM response consisted primarily of Abs
that, like other protective anti-GXM Abs, did not bind
de-O-acetylated GXM, suggesting that the peptide boost had
specifically focused the Ab response on a protective epitope. While we
have not yet made mAbs from the mice that were primed with GXM-TT and
boosted with P206-TT, we have made a few mAbs from mice that were
primed with GXM-TT and boosted with P206.1 conjugated to KLH. Two of
these mAbs cross-reacted with GXM and P206.1, and both expressed
VH7183 and V5.1 (our unpublished data). Even
though a different carrier was used to produce these mAbs, this
suggests that the serum response described in this paper would also be
encoded by the same V region genes that are used by 2H1, like most of
the other Abs made in response to GXM-TT (28, 39).
The results presented here indicate that evolutionary phage peptide sublibraries can be powerful tools for isolating high affinity peptides. This technique may prove useful in identifying other high affinity peptide-Ab interactions for use as biochemical, diagnostic, and therapeutic reagents. The evolutionary sublibrary technique we have employed may also be effective in isolating high affinity peptides involved in other protein-protein interactions (reviewed in Ref. 50). While we did identify a high affinity peptide that discriminates between protective and nonprotective Abs, immunization with this peptide did not lead to significantly higher anti-GXM titers than immunization with the lower affinity parent peptide. However, this high affinity peptide was effective as a secondary immunogen in selectively stimulating those B cells producing Abs to a protective epitope from among a broad pool of B cells primed by a glycoconjugate vaccine known to contain both protective and nonprotective epitopes. Furthermore, our results suggest that if we primed with a peptide that reacted with germline-encoded Abs, a secondary Ab response could then be specifically directed to a protective epitope by immunization with higher affinity peptides obtained from an evolutionary peptide sublibrary such as P206.1. These results have implications for the design of peptide mimotope vaccines to carbohydrate Ags.
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Acknowledgments |
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Footnotes |
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2 D.O.B. and R.J.M. contributed equally to this work.
3 Current address: Department of Microbiology, Immunology, and Molecular Genetics, University of California, 609 East Charles Young Drive, MSB 1602G, Los Angeles, CA 90025-1489.
4 Current address: Sidney Kimmel Cancer Institute, 10835 Altman Row, San Diego CA 92121.
5 Address correspondence and reprint requests to Dr. Matthew D. Scharff, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: scharff{at}aecom.yu.edu
6 Abbreviations used in this paper: TT, tetanus toxoid; CDR, complementarity-determining region; GXM, glucuronoxylomannan; KLH, keyhole limpet hemocyanin; MAP, multiple antigenic peptide.
Received for publication July 10, 2002. Accepted for publication October 17, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 M. N. Dharmasena, D. A. Jewell, and R. K. Taylor Development of Peptide Mimics of a Protective Epitope of Vibrio cholerae Ogawa O-antigen and Investigation of the Structural Basis of Peptide Mimicry J. Biol. Chem., November 16, 2007; 282(46): 33805 - 33816. [Abstract] [Full Text] [PDF]
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 D. O. Beenhouwer, E. M. Yoo, C.-W. Lai, M. A. Rocha, and S. L. Morrison Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection Infect. Immun., March 1, 2007; 75(3): 1424 - 1435. [Abstract] [Full Text] [PDF]
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 F. Perosa, E. Favoino, M. A. Caragnano, and F. Dammacco Generation of biologically active linear and cyclic peptides has revealed a unique fine specificity of rituximab and its possible cross-reactivity with acid sphingomyelinase-like phosphodiesterase 3b precursor Blood, February 1, 2006; 107(3): 1070 - 1077. [Abstract] [Full Text] [PDF]
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M.-J. Clement, A. Fortune, A. Phalipon, V. Marcel-Peyre, C. Simenel, A. Imberty, M. Delepierre, and L. A. Mulard Toward a Better Understanding of the Basis of the Molecular Mimicry of Polysaccharide Antigens by Peptides: THE EXAMPLE OF SHIGELLA FLEXNERI 5A J. Biol. Chem., January 27, 2006; 281(4): 2317 - 2332. [Abstract] [Full Text] [PDF]
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L. Yuan, I. Kurek, J. English, and R. Keenan Laboratory-Directed Protein Evolution Microbiol. Mol. Biol. Rev., September 1, 2005; 69(3): 373 - 392. [Abstract] [Full Text] [PDF]
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M. Torres, R. May, M. D. Scharff, and A. Casadevall Variable-Region-Identical Antibodies Differing in Isotype Demonstrate Differences in Fine Specificity and Idiotype J. Immunol., February 15, 2005; 174(4): 2132 - 2142. [Abstract] [Full Text] [PDF]
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U. K. Buchwald, A. Lees, M. Steinitz, and L.-a. Pirofski A Peptide Mimotope of Type 8 Pneumococcal Capsular Polysaccharide Induces a Protective Immune Response in Mice Infect. Immun., January 1, 2005; 73(1): 325 - 333. [Abstract] [Full Text] [PDF]
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P. Torregrossa, L. Buhl, M. Bancila, P. Durbec, C. Schafer, M. Schachner, and G. Rougon Selection of Poly-{alpha} 2,8-Sialic Acid Mimotopes from a Random Phage Peptide Library and Analysis of Their Bioactivity J. Biol. Chem., July 16, 2004; 279(29): 30707 - 30714. [Abstract] [Full Text] [PDF]
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 G. Zhang, P. H. H. Lopez, C. Y. Li, N. R. Mehta, J. W. Griffin, R. L. Schnaar, and K. A. Sheikh Anti-ganglioside antibody-mediated neuronal cytotoxicity and its protection by intravenous immunoglobulin: implications for immune neuropathies Brain, May 1, 2004; 127(5): 1085 - 1100. [Abstract] [Full Text] [PDF]
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R. W. Maitta, K. Datta, A. Lees, S. S. Belouski, and L.-a. Pirofski Immunogenicity and Efficacy of Cryptococcus neoformans Capsular Polysaccharide Glucuronoxylomannan Peptide Mimotope-Protein Conjugates in Human Immunoglobulin Transgenic Mice Infect. Immun., January 1, 2004; 72(1): 196 - 208. [Abstract] [Full Text] [PDF]
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 N. K. Vyas, M. N. Vyas, M. C. Chervenak, D. R. Bundle, B. M. Pinto, and F. A. Quiocho Structural basis of peptide-carbohydrate mimicry in an antibody-combining site PNAS, December 9, 2003; 100(25): 15023 - 15028. [Abstract] [Full Text] [PDF]
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S.-S. Tang, W.-S. Tan, S. Devi, L.-F. Wang, T. Pang, and K.-L. Thong Mimotopes of the Vi Antigen of Salmonella enterica Serovar Typhi Identified from Phage Display Peptide Library Clin. Vaccine Immunol., November 1, 2003; 10(6): 1078 - 1084. [Abstract] [Full Text] [PDF]
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R. J. May, D. O. Beenhouwer, and M. D. Scharff Antibodies to Keyhole Limpet Hemocyanin Cross-React with an Epitope on the Polysaccharide Capsule of Cryptococcus neoformans and Other Carbohydrates: Implications for Vaccine Development J. Immunol., November 1, 2003; 171(9): 4905 - 4912. [Abstract] [Full Text] [PDF]
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M. A. Johnson, M. Jaseja, W. Zou, H. J. Jennings, V. Copie, B. M. Pinto, and S. H. Pincus NMR Studies of Carbohydrates and Carbohydrate-mimetic Peptides Recognized by an Anti-Group B Streptococcus Antibody J. Biol. Chem., June 27, 2003; 278(27): 24740 - 24752. [Abstract] [Full Text] [PDF]
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