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of June 17, 2017.
Antibodies to Keyhole Limpet Hemocyanin
Cross-React with an Epitope on the
Polysaccharide Capsule of Cryptococcus
neoformans and Other Carbohydrates:
Implications for Vaccine Development
Rena J. May, David O. Beenhouwer and Matthew D. Scharff
J Immunol 2003; 171:4905-4912; ;
doi: 10.4049/jimmunol.171.9.4905
http://www.jimmunol.org/content/171/9/4905
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Copyright © 2003 by The American Association of
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References
The Journal of Immunology
Antibodies to Keyhole Limpet Hemocyanin Cross-React with
an Epitope on the Polysaccharide Capsule of Cryptococcus
neoformans and Other Carbohydrates: Implications for
Vaccine Development1
Rena J. May,* David O. Beenhouwer,2*† and Matthew D. Scharff3*
E
ncapsulated organisms, such as Streptococcus pneumoniae, Haemophilus influenzae type B Neisseria meningitidis, group B streptococcus, and Cryptococcus neoformans, are responsible for many serious infections and for most
deaths from meningitis (1). Given their medical significance, there
is a great need for improved vaccine strategies against these organisms. The polysaccharide capsule of these organisms is the
primary virulence factor and, consequently, acapsular strains do
not cause significant disease. Purified capsular polysaccharide vaccines of S. pneumoniae and N. meningitidis can prevent disease
with homologous serotypes (reviewed in Ref. 2). However, purified polysaccharides are T cell-independent Ags that elicit an immune response lacking immunologic memory (3, 4). Furthermore,
capsular vaccines have not been effective in protecting those individuals most susceptible to infection, such as infants, people
over age 65, and those with impaired immunity (5).
Departments of *Cell Biology and †Medicine, Albert Einstein College of Medicine,
Bronx, NY 10461
Received for publication May 5, 2003. Accepted for publication August 18, 2003.
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
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This research was supported by grants from the National Institutes of Health: AI
01434 (to D.O.B.), T32 AG 00194 (to R.J.M.), and AI 43937 (to M.D.S.). M.D.S. is
further supported by the Harry Eagle Chair provided by the Women’s Division of the
Albert Einstein College of Medicine.
2
Current address: University of California, Department of Microbiology, Immunology and Molecular Genetics, 609 E. Charles Young Drive, MSB 1602G, Los Angeles,
CA 90025-1489.
3
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: [email protected]
Copyright © 2003 by The American Association of Immunologists, Inc.
Currently, glycoconjugate vaccines are the most effective means
of protecting individuals against infection by encapsulated organisms. Glycoconjugate vaccines are composed of capsular polysaccharide conjugated to an immunogenic protein carrier, such as tetanus toxoid (TT),4 or keyhole limpet hemocyanin (KLH) and
generate a T cell-dependent Ab response to the polysaccharide Ags
(4, 6, 7). Despite the success of the Haemophilus influenzae type
B glycoconjugate vaccine (8), there are still a number of potential
problems with the use of glycoconjugates. First, these vaccines
may not be sufficiently immunogenic in immunosuppressed and
elderly individuals (reviewed in Ref. 4). Second, glycoconjugates
are comprised of the entire capsular polysaccharide, which may
contain epitopes that elicit both protective and nonprotective Abs
(9 –11). In the case of C. neoformans, these epitopes have been
defined based on their reactivity with mAbs that do or do not
prolong the life of mice lethally infected with C. neoformans (12,
13). In addition, there is evidence that Abs directed against nonprotective epitopes of the polysaccharide capsule of Schistosoma
mansoni, N. meningitidis, and C. neoformans can block the efficacy of protective Abs and even enhance infection (11–15).
To address these issues, we and several others (4, 16 –21) have
searched for a method of generating a vaccine to encapsulated
organisms, such as C. neoformans, that is T cell dependent, highly
immunogenic, and will specifically direct the Ab response to a
protective epitope. mAbs against the glucuronoxylomannan
(GXM) component of the capsular polysaccharide can protect
4
Abbreviations used in this paper: TT, tetanus toxoid; BSA-g, BSA treated with
glutaraldehyde; GXM, glucuronoxylomannan; KLH, keyhole limpet hemocyanin;
KLH-g, KLH treated with glutaraldehyde; KLH-p, KLH treated with periodate; LAM,
lipoarabinomannan.
0022-1767/03/$02.00
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Cryptococcus neoformans causes a life-threatening meningoencephalitis in AIDS patients. Mice immunized with a glycoconjugate
vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to
tetanus toxoid produce Abs that can be either protective or nonprotective. Because nonprotective Abs block the efficacy of
protective Abs, an effective vaccine must focus the Ab response on a protective epitope. Mice immunized with peptide mimetics
of GXM conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde developed Abs to GXM. However, control peptides
P315 and P24 conjugated to KLH also elicited Abs to GXM. GXM-binding Abs from mice immunized with P315-KLH were
inhibited by KLH treated with glutaraldehyde (KLH-g), but not by P315. Furthermore, KLH-g inhibited binding of GXM by
serum of mice immunized with GXM-TT, indicating that glutaraldehyde treatment of KLH reveals an epitope(s) that cross-reacts
with GXM. Vaccination with KLH-g or unmodified KLH elicited Abs to GXM, but did not confer protection against C. neoformans, suggesting the cross-reactive epitope on KLH was not protective. This was supported by the finding that 4H3, a nonprotective mAb, cross-reacted strongly with KLH-g. Sera from mice immunized with either native KLH or KLH-g cross-reacted with
several other carbohydrate Ags, many of which have been conjugated to KLH for vaccine development. This study illustrates how
mAbs can be used to determine the efficacy of potential vaccines, in addition to describing the complexity of using KLH and
glutaraldehyde in the development of vaccines to carbohydrate Ags. The Journal of Immunology, 2003, 171: 4905– 4912.
4906
KLH CROSS-REACTIVITY WITH CRYPTOCOCCAL POLYSACCHARIDE CAPSULE
Materials and Methods
Peptides
The identification of peptides PA1 (LQYTPSWMLV), P206 (FGGETFT
PDWMMEVAIDNE), P206N (FGGETFTPDWMMEV), and P206C (AF
TPDWMMEVAIDNE) have been described (19 –21, 23). Peptides PA1,
P206, P206N, and P206C and their biotinylated counterparts were synthesized by the Laboratory for Macromolecular Analysis at Albert Einstein
College of Medicine (AECOM). Peptide preparations were analyzed by
HPLC and mass spectroscopy. Control peptide P315 (CKVMVHDPH
SLA), the carboxyl-terminal domain of murine MHC class I H2Kk (32),
was a gift of S. Nathenson (AECOM), and control peptide P24A
(ANDYTTEYASASVRGRFIVS), a peptide mimetic of phosphorylcholine
(17), was a gift from B. Diamond (AECOM).
Immunizations
Peptides (1 mg) were conjugated to KLH (3 mg; Pierce, Rockford, IL) in
0.25 ml PBS, pH 7.5, with the slow addition of 120 ␮l 5% glutaraldehyde
(Sigma-Aldrich, St. Louis, MO), as described (33). Conjugates were then
dialyzed at 4°C against sterile PBS for 3 days with daily changes. KLH and
BSA used for in vitro studies and control immunizations were treated with
glutaraldehyde in the same manner (termed KLH-g or BSA-g). Six-weekold BALB/c mice (National Cancer Institute, Bethesda, MD) were maintained in a pathogen-free barrier facility and immunized via i.p. injection
with 100 ␮g of each peptide-KLH conjugate or 0.5 ␮g of GXM-TT (gift
of J. Robbins, National Institutes of Health, Bethesda, MD) in emulsion
with CFA (Difco, Detroit, MI) on day 0 and with IFA (Difco) on day 28.
Mice were bled before the first immunization and on days 14 and 42
postimmunization. Protective efficacy of immunizations was assessed by
infecting mice with 1 ⫻ 106 CFU C. neoformans strain 24067 i.v. on day
56, and survival was monitored.
Carbohydrates
C. neoformans strain 24067 (serotype D) was provided by A. Casadevall
(AECOM). Total cryptococcal polysaccharide was prepared by ethanol
precipitation from late log phase cultures, and polysaccharide concentration was determined by the phenol-sulfuric acid method (34). GXM was
purified from whole polysaccharide by cetyltrimethylammonium bromide
precipitation, and did not contain detectable protein contaminants (35).
Mycobacterium tuberculosis-derived lipoarabinomannan (LAM) was a gift
from A. Freedman (AECOM). All other carbohydrate reagents were ob-
tained commercially: Lewis Y tetrasaccharide (Calbiochem, San Diego,
CA), LPS from Escherichia coli serotype 055:B5 (Sigma-Aldrich), S.
pneumoniae vaccine containing capsular polysaccharide of 23 different serotypes (Pnu-immune 23; Lederle, Pearl River, NY), and GD3 disialodiammonium salt extracted from bovine buttermilk (Calbiochem)
Periodate treatment
KLH (Pierce) was treated with 0.2 M sodium periodate in 0.01 M sodium
acetate buffer (pH 4.5) overnight at 4°C (36). The reaction was stopped by
the addition of an equal volume of 0.05 M sodium borohydride for 30 min
at room temperature. Periodate-treated KLH (KLH-p) was then dialyzed
against sterile PBS for 48 h with daily changes.
ELISAs
Sera from immunized animals were treated with 0.1 M 2-ME at 37°C for
1 h to dissociate pentameric IgM. GXM ELISAs were performed by coating plates with 10 ␮g/ml of GXM; KLH ELISAs were prepared by coating
plates with 10 ␮g/ml of KLH (KLH-g or KLH-p described above). To
analyze binding to various carbohydrates, ELISA plates were coated with
either 10 ␮g/ml GD3, 10 ␮g/ml Lewis Y, 10 ␮g/ml LPS, 10 ␮g/ml PC, or
3 ␮g/ml LAM. BSA-coated wells served as controls on each microtiter
plate. For all ELISAs, sera were incubated in serial dilutions starting at
1/25 or 1/50. GXM and KLH binding were detected with a mixture of
alkaline phosphatase-conjugated anti-mouse IgG isotypes (Southern Biotechnology, Birmingham, AL), or Abs to each individual isotype. Microtiter plates were developed with 1 mg/ml p-nitrophenyl phosphate (SigmaAldrich) in 1 M diethanolamine and 0.25 mM MgCl2, pH 9.8, and the
absorbance was measured at 405 nm. Ab reactivity to peptides was assayed
on biotinylated peptide (1 ␮g/ml) bound to streptavidin-coated (1 ␮g/ml)
ELISA plates. Binding of GXM mAbs (12, 37–39) (0.5 ␮g/well) to KLHg-coated plates was detected using a mixture of alkaline phosphatase-conjugated anti-mouse IgG isotypes, and developed as described above. Competition ELISAs were performed by first incubating serum dilutions with
different concentrations of peptide, BSA-g, KLH-g, or KLH-p, fully solubilized in PBS, for 2 h at 37°C. Concentrations of KLH and KLH-g greater
than 5 mg/ml come out of solution, thus limiting the competition assay.
Samples were then transferred to an ELISA plate, and GXM binding was
detected as described above. In all cases, a dilution was considered positive
when the OD observed (minus the BSA-reactive background) was 3 times
greater than background. The 4H3 competition ELISA was performed in a
similar manner: Serum dilutions were incubated on a GXM-coated microtiter plate at 37°C for 1 h. mAb 4H3 (1 ␮g/ml) was then added to each well,
and the plates were incubated at 37°C for an additional hour. Binding of
4H3 to GXM was detected using alkaline phosphatase-conjugated antimouse ␭ and developed as described above.
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. Survival data were subjected to Kaplan-Meier analysis,
and statistical significance was determined by the log rank (Mantel-Cox)
test. A p value of ⬍0.05 was considered statistically significant. Error bars
represent an SE of 1.
Results
KLH-g elicits Abs to GXM
We have previously screened phage peptide display libraries and
identified peptide mimetics of GXM that bound with high affinity
to protective, but not to nonprotective, mAbs to GXM (20, 21, 23).
Two of these peptides, PA1 and P206 (20), were conjugated to
KLH with glutaraldehyde and injected into mice. As a negative
control, mice were immunized with KLH conjugated to an irrelevant peptide, P315, which does not bind any mAb to GXM (20,
23). As illustrated in Fig. 1A, immunization with P315-KLH resulted in IgG titers against GXM comparable to those in mice
immunized with P206-KLH, the highest affinity peptide mimetic
of GXM yet identified (20), and greater than those elicited by PA1,
a lower affinity peptide mimetic of GXM (21). In contrast, the
anti-peptide response to P315 was significantly lower than that of
animals receiving other peptide-KLH conjugates (Fig. 1B).
To rule out the possibility that P315 was playing a unique role
in eliciting Abs to GXM, a second experiment was done using
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mice against infection with C. neoformans (22). Although glycoconjugates using oligosaccharides that reacted with protective
mAbs might serve this purpose, such oligosaccharides have not
been identified. Peptide mimetics of polysaccharides offer an attractive alternative because they: 1) induce a T cell-dependent response, 2) are small and present few epitopes, 3) can be identified
using high throughput methods, and 4) are easy and cost effective
to produce (reviewed in Ref. 21). Therefore, we sought to create an
effective vaccine by identifying a peptide mimic of a protective
epitope of GXM (23). Phage display peptide libraries were
screened with 2H1, a protective anti-GXM mAb, and a number of
peptides were isolated (19 –21, 23).
To study their efficacy as vaccines, peptide mimetics were conjugated to KLH, and mice were then immunized with these conjugates. Carrier proteins such as TT, KLH, OVA, and BSA are
commonly conjugated to small Ags, thereby providing the hapten
with a strong T cell epitope (24). The hemocyanin of the keyhole
limpet marine mollusk (KLH) has a remarkable immunostimulatory effect because it can activate both humoral and cellular immunity (25). Due to these properties, many vaccine studies have
used KLH as the immunogenic carrier. The haptens used together
with KLH have varied, from capsular polysaccharides (26) to peptide Ags (19, 27), anti-idiotypic Abs (28, 29), as well as a number
of tumor-associated carbohydrates (30, 31). We report in this study
that peptide mimetics of GXM did not elicit a strong response to
GXM when they were conjugated to KLH. Rather, KLH contained
polysaccharide that elicited an anti-GXM response to nonprotective epitopes. These results have significant ramifications for vaccine development.
The Journal of Immunology
4907
P24A, a peptide mimetic of phosphorylcholine (17), as an alternative negative control. Additional mice were immunized with two
other high affinity peptide mimetics of GXM, P206N, and P206C
(20) conjugated to KLH, and with native KLH as an additional
control. The two control Ags, P24A-KLH and native KLH, elicited
IgG titers to GXM that were equal, and in some cases better than
the Ab response elicited by the high affinity peptide mimetics (Fig.
1C). The animals immunized with the different peptides all
mounted a significant Ab response to the peptide Ag (Fig. 1D).
Competition studies revealed that soluble peptides inhibited binding of sera to homologous peptide-coated ELISA plates; soluble
peptides did not inhibit binding of sera to GXM (data not shown
and see below).
Because KLH alone could elicit an Ab response to GXM, the
ability of KLH to inhibit the binding of Abs to GXM in the serum
of a P315-KLH-immunized mouse was analyzed (Fig. 2A). Native
KLH weakly inhibited the binding of serum Abs to GXM. However, KLH-g effectively inhibited binding of serum Abs to GXM.
The difference between native and glutaraldehyde-treated KLH
implies that glutaraldehyde treatment changes the structure of
KLH, creating or revealing cross-reactive epitopes to GXM. Although it is difficult to make quantitative comparisons between
molecules that may have different epitope densities, KLH-g inhibited the binding of serum Abs to GXM more efficiently than GXM
itself (Fig. 2A). The average m.w. of KLH is twice that of GXM,
but even if this difference was corrected for, the inhibition would
be comparable. KLH-g did not bind to GXM-coated plates (data
not shown), ruling out the possibility that KLH-g was merely act-
ing as a lectin. P315 alone did not inhibit the binding of serum Abs
to GXM (data not shown). These data suggested that KLH, the
carrier used in these immunizations, was eliciting the anti-GXM
response. This was confirmed when mice immunized with KLH-g,
in the absence of any peptide, mounted an Ab response to GXM
that was even greater than animals immunized with the various
peptide conjugates or KLH alone (compare Figs. 1C and 2B).
GXM and KLH share a carbohydrate epitope
Animals immunized with the various peptide conjugates mounted
an anti-KLH-g IgG response that was 10-fold lower than the antipeptide response (data not shown). To further establish that the
anti-GXM Abs elicited by the peptide-KLH conjugates were stimulated by KLH, competition studies were conducted with sera from
mice immunized with the various conjugates. KLH-g inhibited an
average of 60% of GXM binding in the serum of mice immunized
with the different peptide mimetics conjugated to KLH (Fig. 3).
KLH-g also inhibited an average of 70% of the serum GXM-binding IgG from mice immunized with GXM-TT (Fig. 3), even
though these animals had never been exposed to KLH.
To examine whether the glycosylated regions of KLH were responsible for the cross-reactivity between KLH and GXM, KLH
was treated with periodate (KLH-p), which cleaves the vicinal hydroxyl groups of carbohydrates on the surface of KLH (36).
KLH-p also inhibited the binding of sera to GXM, but was less
efficient than KLH-g (Fig. 3). BSA-g does not inhibit the binding
of GXM-reactive Abs to the same extent as KLH-g (Fig. 3). These
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FIGURE 1. Average serum IgG titers 14 days after the primary and secondary immunizations. Mice (n ⫽ 10 per group) were immunized i.p. with the
various peptides conjugated to KLH in CFA (1o), followed by IFA (2o). Serum from each mouse was assayed for IgG Abs on an ELISA plate coated with
either GXM or the peptide Ag received during immunization. A, Anti-GXM titer. ⴱ, Statistical significance was noted between 2o P315 and 2o PA1 (p ⫽ 0.003).
B, Anti-peptide titer. ⴱ, Statistical significance of p ⬍⬍ 0.05 was noted between 2o P315 and 2o PA1 and P206. Anti-GXM (C) and anti-peptide (D) titers from
a second experiment using a different control peptide (P24A) and amino-terminal (P206N) or carboxyl-terminal (P206C) peptides of P206 (20).
4908
KLH CROSS-REACTIVITY WITH CRYPTOCOCCAL POLYSACCHARIDE CAPSULE
FIGURE 2. A, KLH-g inhibits P315KLH serum binding to GXM. Serum (1/200 dilution) from a mouse immunized with P315KLH (Fig. 1, A
and B) was incubated at 37oC for 2 h with KLH, GXM, or KLH-g and then
transferred to ELISA plates coated with GXM. B, KLH-g elicits high serum titers to GXM (ⴱ, p ⬍⬍ 0.05). Average serum IgG anti-GXM titer
taken 14 days after the secondary immunization. Mice (n ⫽ 10 per group)
were immunized i.p. with P206-KLH conjugate or KLH-g in CFA (1o),
followed by IFA (2o). Serum from each mouse was assayed on an ELISA
plate coated with GXM.
results further support the conclusion that KLH surface carbohydrates contribute to the cross-reactivity between KLH and GXM,
but also suggest that glutaraldehyde can modify proteins to produce an epitope(s) that cross-reacts with GXM.
KLH cross-reacts with a nonimmunodominant, nonprotective
epitope of GXM
If KLH-g shares an epitope(s) with GXM, then immunization with
KLH-g should boost the anti-GXM titer of mice previously immunized with GXM. However, when mice were immunized with
GXM-TT and then boosted with either GXM-TT or KLH-g, the
GXM titer was not significantly enhanced in those animals that
received KLH-g, compared with mice that were primed and
boosted with GXM-TT (Fig. 4). These data imply that although
Abs to KLH-g can cross-react with GXM, the cross-reactive
epitope on KLH-g is not recognized by a sufficient number of
memory B cells elicited by GXM-TT to boost the anti-GXM
response.
Due to the lack of structural data with regard to GXM and KLH
epitopes, the easiest way to determine which epitope of GXM
cross-reacted with KLH-g would be to screen the sera of immunized mice on a panel of KLH- and GXM-derived oligosaccharides. However, such oligosaccharides do not exist, so we chose to
identify the cross-reactive epitope by studying the binding of a
panel of mAbs to GXM to KLH-g (Fig. 5). mAbs 2H1, 18B7,
2D10, and 12A1 did not bind to KLH-g or KLH-p. These mAbs
recognize the same, or a closely related, protective epitope on
GXM, and are members of the group II anti-cryptococcal mAbs
that express VH7183 and V␬5.1 (12, 39). These Abs prolong the
life of lethally infected animals (11, 38, 40, 41). In addition,
KLH-g did not bind to either 13F1 and 21D2, two nonprotective
group II mAbs (11, 12). However, mAb 4H3 bound strongly to
KLH-g, and very weakly to KLH-p, providing evidence that a
GXM cross-reactive epitope on KLH that binds to 4H3 is a polysaccharide. The 4H3 is encoded by VH441 and V␭1 and does not
compete with group II Abs for binding to GXM (42). This Ab
recognizes a different epitope on the surface of GXM than the
dominant 2H1 epitope of GXM and does not protect mice against
C. neoformans infection (38, 42). mAb 3E5, another group II
member, reacted weakly with KLH-g and more strongly with KLH-p,
suggesting that it was recognizing still another epitope on KLH. Sera
from mice immunized with either native KLH or KLH-g could inhibit
the binding of mAb 4H3 to GXM (Fig. 6). These findings suggest that
at least some of the epitopes on KLH that cross-react with GXM can
elicit nonprotective Abs. This was confirmed when mice immunized
with KLH-g were not protected against lethal C. neoformans infection
(Fig. 7), despite the appreciable titers of Ab to GXM stimulated in
these animals (Fig. 2B).
KLH shares epitopes with many carbohydrate Ags
The existence of a shared epitope between GXM and KLH alerted
us to the possibility that the glycosylated regions of KLH may
cross-react with other carbohydrate Ags. The serum from mice
immunized with KLH-g was screened against a number of carbohydrates, some of which have been conjugated to KLH and used
successfully as vaccines (Fig. 8) (7, 30, 31, 43). None of the preimmunization sera reacted with Lewis Y tetrasaccharide, LAM,
GXM, GD3, LPS, or Pnu-immune 23 (data not shown). In addition
to binding to GXM, the sera from the secondary immunization
with KLH-g bound each of these carbohydrates (Fig. 8). To determine whether this finding was limited to epitopes of KLH-g, we
immunized mice with unmodified KLH and their sera reacted with
all the carbohydrate Ags, albeit with lower Ab titer (Fig. 8). This
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FIGURE 3. Inhibition studies of sera from mice immunized with the
various peptide conjugates. Serum (1/200 dilution) from mice immunized
with the different peptide KLH conjugates (n ⫽ 10 mice/group) was incubated at 37oC for 2 h with 1.25 mg/ml of KLH-g, KLH-p, or BSA-g and
then transferred to ELISA plates coated with GXM. The anti-GXM titer
that remained after inhibition is presented as a percentage of anti-GXM
titer before inhibition. ⴱ, Statistical significance of p ⬍⬍ 0.05 was noted
between the KLH-g-treated sera and the KLH-p-treated sera (315, p ⫽
0.0002; PA1, p ⫽ 0.0002; P206, p ⫽ 0.0004; GXMTT, p ⫽ 0.0143). ⴱⴱ,
Statistical significance of p ⬍ 0.05 was noted between the KLH-g-treated
sera and the BSA-g-treated sera (PA1, p ⫽ 0.0015; P206, p ⫽ 0.0001;
GXMTT, p ⫽ 0.0339).
The Journal of Immunology
result implies that regardless of whether KLH is treated with glutaraldehyde or not, there are epitopes on the surface of KLH that
cross-react with a variety of different carbohydrate Ags.
Discussion
In an attempt to create effective peptide vaccines that will prevent
infections, we have used the GXM capsular polysaccharide of C.
neoformans as our model system. mAbs were generated that identified protective and nonprotective epitopes of GXM. One such Ab
that reacted with a protective epitope was used to screen phage
peptide libraries for peptide mimetics. Mice immunized with the
GXM mimetic peptides conjugated to TT (20) did not generate
significant anti-GXM Ab responses. However, when mice were
first primed with the GXMTT glycoconjugate to generate memory
B cells to GXM and then boosted with the P206-TT conjugate,
high titers of IgG Ab to protective epitope(s) of GXM were obtained (20). We have attempted to extend those studies by developing strategies that would allow us to immunize naive mice with
the peptide mimotope conjugates. Because it is now difficult to
obtain carriers such as TT or diptheria toxin, a more readily available carrier such as KLH was tested. KLH is a large (⬎2000 kDa),
highly glycosylated protein frequently used as a carrier protein in
animals and humans because of its potent immunogenicity. Several
groups developing carbohydrate vaccines have reported success
when using KLH as the carrier protein (7, 30, 31, 43).
In the studies described in this work, peptide-KLH conjugates
were prepared with a glutaraldehyde conjugation method commonly used with candidate vaccines (17, 29, 44). Glutaraldehyde
is a bifunctional coupling reagent that links two compounds primarily through their amino groups. The peptides we used did not
contain internal lysines, and therefore, the main reactive group
should be at the amino terminus. We used P315 and P24A as
control peptides, and were surprised to find that naive mice immunized with these irrelevant peptides conjugated to KLH
mounted an Ab response to GXM. This response was composed
primarily of the IgG1 and IgG2a isotype (data not shown), and
these Abs were inhibited from binding to GXM by KLH-g. The
results from this and other inhibition studies led us to conclude that
neoepitopes generated by treating KLH with glutaraldehyde are
cross-reactive with GXM. Further studies revealed that native
KLH also had epitopes that cross-reacted with GXM.
FIGURE 5. Nonprotective mAb 4H3 binds to KLH-g. Binding of various anti-GXM mAbs (39) to KLH-g- or KLH-p-coated ELISA plates.
Each mAb was assayed in triplicate, and background binding to BSA was
subtracted to get the final OD. Ab isotype is indicated below, and known
nonprotective Abs are underlined.
It has been known for some time that KLH, which is itself a
highly glycosylated protein, can cross-react with a number of carbohydrate Ags. Although the nature of the oligosaccharides on the
surface of KLH has not been completely elucidated, those identified to date have been shown to contain unique oligosaccharide
patterns, resulting in novel structures (45, 46). These novel structures, together with the high levels of cross-reactivity with a variety of carbohydrate Ags, may contribute to the immunogenicity
of KLH (47). In 1974, Olsson et al. (48) discovered that patients
immunized with KLH had a reduction in recurrence of bladder
carcinomas. The cross-reactive epitope was identified (49), and
clinical trials with Immucothel, a KLH subunit, are reported (50).
In addition, a fucose epitope on the surface of S. mansoni has been
found to be cross-reactive with KLH (46, 51). Immobilized KLH
or anti-KLH Abs have been used to diagnose acute vs chronic
infections of S. mansoni (52).
Because these studies indicate that the cross-reactive nature of
KLH is not limited to GXM, sera from mice immunized with
KLH-g and KLH were screened against a variety of different carbohydrate Ags, a number of which have been conjugated to KLH
(7, 30, 31), and are either being studied as candidate vaccines or
are in clinical trials (43). The average titers to the various carbohydrate Ags were greater in the sera of mice immunized with
KLH-g compared with untreated KLH, possibly because crosslinking the KLH with glutaraldehyde and modifying the primary
amines may either reveal more cross-reactive epitopes or render
KLH more immunogenic. It is unlikely that the glutaraldehyde
structure alone is responsible for the cross-reactive nature of
KLH-g, and carbohydrates on the surface of KLH must cross-react
with GXM because BSA-g, an irrelevant protein, cannot inhibit the
binding of GXM-specific Abs as effectively as KLH-g (Fig. 3), and
mice immunized with native KLH induce anti-GXM Abs (Fig. 1C)
that are inhibited by mAb 4H3 (Fig. 6). It is possible that glutaraldehyde treatment of other heavily glycosylated carrier proteins
may result in the induction of cross-reactive Abs to
carbohydrate Ags.
To determine whether the cross-reactivity observed with GXM
was also due to a carbohydrate epitope on native KLH, KLH was
subjected to periodate oxidation, which has been shown to disrupt the
polysaccharide structure on the surface of KLH (36). If carbohydrates
are subjected to one round of oxidation and reduction, this procedure
will destroy the immunogenicity of the carbohydrate, while not
disturbing the amino acids or linear stretches of internal sugar residues
(53). This method has been used in other systems to determine
whether carbohydrate epitopes bind to mAbs (53).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 4. Serum studies from mice primed with GXM-TT and
boosted with KLH-g. Average serum IgG GXM titers 14 days after the
primary and secondary immunization. Mice (n ⫽ 10 per group) were immunized i.p. with 0.5 ␮g of GXM-TT in CFA, followed 3 wk later with
either 75 ␮g KLH-g or 0.5 ␮g GXM-TT in IFA. Serum from each mouse
was assayed on an ELISA plate coated with GXM.
4909
4910
KLH CROSS-REACTIVITY WITH CRYPTOCOCCAL POLYSACCHARIDE CAPSULE
Treating KLH with periodate reduced its ability to inhibit serum
binding to GXM by ⬃50%, indicating that some of the crossreactive epitope(s) must be carbohydrate in nature. KLH-p does
not inhibit all of the serum reactivity to GXM, and there are a
number of explanations for this finding. First, it is possible that a
protein epitope of KLH also contributes to the cross-reactivity with
GXM, because periodate treatment does not disturb protein
epitopes (53, 54). Second, while most of the reactivity of mAbs to
carbohydrate is lost following periodate treatment (53, 54), polyclonal sera retain some binding to the carbohydrate Ag even after
oxidation (55, 56). Third, it is possible that, as is seen with 3E5
IgG1 (Fig. 5), and as documented in other cases (57, 58), periodate
oxidation may unmask other carbohydrate residues, thus exposing
epitopes that can bind to Abs in polyclonal sera.
Because oligosaccharides of KLH and GXM were unavailable,
and simple carbohydrate molecules representing the known carbohydrate motifs of KLH did not inhibit the binding of KLH-specific
Abs to GXM (data not shown), we used a panel of anti-GXM
mAbs to further characterize the cross-reactive epitope between
KLH and GXM. Of all the anti-GXM mAbs screened against
KLH-g and KLH-p, mAb 4H3 bound most strongly to KLH-g and
did not bind to KLH-p. In addition, the polyclonal sera from mice
immunized with either KLH-g or KLH inhibited the binding of
4H3 to GXM, indicating that the 4H3 epitope is at least one of the
epitopes that is cross-reactive between KLH and GXM. mAb 4H3
is encoded by different V regions than the dominant group II Ab
response to GXM and recognizes a different epitope than these Abs
(12, 39). mAb 4H3 has been shown to be nonprotective (42), and
the presence of a 4H3-reactive epitope on KLH is consistent with
the finding that mice immunized with KLH-g were not protected
against the infection with C. neoformans. The Abs made in response to GXM-TT are highly restricted to VH7183 and V␬5.1
(39). Immunization with KLH induced a 4H3-like response to
GXM, thus shifting the GXM Ab specificity away from the highly
restricted response characterized by VH7183- and V␬5.1-encoded
Abs. The KLH cross-reactive epitope, which is defined by mAb
4H3, will not cross-react with the VH7183- and V␬5.1-encoded
Abs made in response to GXM-TT, providing an explanation for
why animals primed with GXM-TT and boosted with KLH-g
mount a limited anti-GXM response. Although a majority of the
GXM-reactive Abs in these animals had undergone class switching
and were of the IgG1 isotype (data not shown), it is possible that
FIGURE 7. Immunization with KLH or KLH-g does not protect mice
from cryptococcal infection. BALB/c mice (n ⫽ 5 per group) were immunized i.p. with PBS, KLH, or KLH-g in CFA, followed by IFA. Mice were
infected i.v. on day 56 with 1 ⫻ 106 CFU of C. neoformans strain 24067.
Survival was monitored daily.
the limited response to GXM was due to the lack of KLH-specific
T cells, because class switching can occur even in the absence of
T cell help (reviewed in Ref. 59). Despite the potent immunogenicity of KLH, and the possibility that by the time these mice were
bled (day 14 after immunization), T cells specific for KLH had
been activated, sufficient amounts of T cell memory may not have
been induced, providing an additional explanation to the weak secondary response mounted in these immunized animals.
Taken together, these findings lead us to conclude that caution
needs to be taken when creating vaccines for carbohydrate Ags
using KLH as the carrier. This is not the first study showing that
one needs to be careful when dealing with the cross-reactive nature
of KLH. Recently, there have been reports cautioning against the
use of KLH in the diagnosis of S. mansoni because Abs to KLH
have been detected that are not specific to this parasite and crossreact with other worms, leading to false positives (60). In addition,
KLH cross-reacts with nonprotective S. mansoni epitopes, and not
the protective ones (10). Just as GXM contains protective and nonprotective epitopes, similar findings have been reported with other
carbohydrate Ags (14, 15). There is a possibility that other, yet
unknown, nonprotective epitopes that cross-react with KLH exist
on carbohydrates of other organisms, possibly resulting in deleterious effects.
There is also the possibility that the carbohydrate residues on
KLH could act as a polyclonal activator, as has been shown with
other carbohydrate epitopes (61). Whether or not KLH activates B
cells by binding to Toll-like receptors or by some other method,
the Abs to KLH generated in such a response would derive from
a diverse group of variable genes and limit the ability to generate
a focused response to the Ag of choice (reviewed in Ref. 62). Due
to its strong immunogenicity and highly cross-reactive nature,
KLH also has the potential of causing carrier-induced suppression.
The immune system’s continual exposure to ubiquitous carbohydrate Ags may result in a population of memory B cells that are
cross-reactive to KLH. The existence of KLH-specific B cells may
result in an inhibitory effect on the production of Abs to a new Ag
that has been coupled to KLH (reviewed in Ref. 63). Whether this
suppression is a result of clonal dominance (64), T cells (65), or
macrophages (66), a pool of circulating KLH-reactive B cells
could result in a weakened Ab response to any Ag that has been
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FIGURE 6. Serum from mice immunized with KLH or KLH-g can inhibit 4H3 binding. Serial dilutions of sera from mice immunized with KLH
or KLH-g were incubated at 37oC for 1 h on a GXM-coated ELISA plate.
A total of 1 ␮g/ml of 4H3 was then added to each well, and the 4H3 that
bound to GXM was assayed with a mouse anti-␭ alkaline phosphataseconjugated Ab. GXM-reactive serum that bound to mouse anti-␭ alkaline
phosphatase-conjugated Ab in the absence of 4H3 was subtracted.
The Journal of Immunology
4911
9.
10.
11.
12.
13.
14.
FIGURE 8. Immunization with KLH or KLH-g induces cross-reactive
Abs to different carbohydrates. Sera from mice (n ⫽ 5 per group) immunized with either KLH-g or KLH assayed on ELISA plates coated with the
indicated carbohydrates. There were no detectable titers to any of the carbohydrates in the preimmune sera (data not shown). Background binding to
BSA was subtracted to get the final IgG titer.
15.
16.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Acknowledgments
We thank Jieru Zhang and Susan Buhl for expert technical support. We
also thank Drs. Betty Diamond, Arturo Casadevall, and Liise-anne Pirofski
for helpful comments and critical review of this manuscript.
27.
28.
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