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,
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Departments of
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Microbiology and Immunology and
Medicine, Division of Infectious Diseases, Albert Einstein College of Medicine, Bronx, NY 10461;
Uniformed Services University, Bethesda, MD 20892; and
Biosynexus, Inc., Rockville, MD 20853
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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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GXM, like other capsular polysaccharides, is poorly immunogenic and induces a T-independent type 2 Ab response (13). Conversion of T-independent Ags to T-dependent immunogens by covalently linking polysaccharides to protein carriers has been successful for bacterial capsular polysaccharides. These conjugates can be highly effective vaccines as evidenced by the markedly enhanced response to polysaccharide-protein conjugate vaccines in children (14, 15, 16). In an effort to increase the immunogenicity of GXM, an investigational GXM-tetanus toxoid (GXM-TT) vaccine was produced (17) that was highly immunogenic (18) and protective against lethal cryptococcosis in mice (17). However, this vaccine was shown to elicit both protective and nonprotective Abs to GXM (9, 19). Although investigational studies with GXM-TT ceased by reasons unrelated to its efficacy (20), important work with this vaccine in mice established that protective and nonprotective Abs to GXM differed in their molecular structure, Id expression, and isotype (21, 22, 23). Since qualitative Ab characteristics are difficult to identify in polyclonal sera, the utility of GXM-based vaccines may be limited without the ability to design a polysaccharide-based vaccine with defined oligosaccharides that are known to induce only protective Abs. At present such epitopes are not available and there is no vaccine for C. neoformans. An alternative approach to vaccine design has been based on reports that peptides can mimic the conformation of polysaccharides (24, 25, 26, 27, 28) and involves the identification of peptide surrogates for unknown capsular polysaccharide Ags that can induce Ab responses to polysaccharide.
The mAb 2E9 is a human mAb to GXM (29) that prolongs the survival of mice with lethal cryptococcosis (11) and enhances the antifungal activity of human neutrophils in vitro (6). In view of its functional efficacy and shared Id expression with Abs to GXM found in human sera (11, 29), we used 2E9 to screen a peptide library and identified a peptide mimic of the GXM epitope it recognized. This 10-amino acid peptide, P13, inhibited the GXM binding of serum Abs from HIV-uninfected, but not HIV-infected individuals (25, 30), suggesting there was a qualitative (specificity) difference between Abs to GXM from susceptible compared with relatively resistant individuals. In this study, we determined whether P13 was a GXM mimotope and its efficacy as a peptide vaccine for C. neoformans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GXM was purified as described (31) from C. neoformans strain 24067 (serotype D; American Type Culture Collection, Manassas, VA).
2E9 and P13
The IgM/
human mAb 2E9 has been described previously
(29). The GXM peptide mimetic, P13 (GMDGTQLDRW), which was
selected from a random peptide phage display library by screening with
2E9 as described elsewhere (25), was synthesized for use
in the conjugates. To facilitate cross-liking, the P13 peptide was
synthesized with the sequence CAGA added to the amino terminus. This
peptide was prepared by Mike Flora (Biomedical Instrumentation Center,
Uniformed Services University, Bethesda, MD).
Preparation of P13-protein conjugates
To increase the immunogenicity of the peptide, conjugates consisting of P13 and various carriers were produced. The peptide was solubilized at 10 mg/ml in water containing 5% acetonitrile with gentle heating. After solubilization, the pH was raised to 7.0 by the addition of 70 liters of 0.75 M HEPES + 10 mM EDTA (pH 7.3). Reversed-phase HPLC indicated that the peptide was entirely oxidized. The peptide was reduced by the addition of 0.5 mol of tris(2-carboxyethyl)phospine (Pierce, Rockford, IL)/mol peptide. HPLC indicated 100% conversion of the peptide to the free thiol. P13 was conjugated to TT (a gift from SmithKline Beecham Biologicals, Rixensart, Belgium), BSA (Intergen, Purchase, NY), and high molecular weight amino-dextran (DEX) (32), each at 10 mg/ml in HEPES buffer at pH 8 were iodoacetylated (33) and combined with an excess of the reduced peptide. After an overnight reaction, the reaction was quenched by adding 2-ME to a concentration of 0.2 mM followed by gel filtration on an S200 HR column equilibrated with PBS to remove unconjugated peptide. The reduced peptide was also linked to BSA that was preadsorbed to the aluminum hydroxide adjuvant Alhydrogel (BSA2; Accurate Scientific, Westbury, NY), as described by Houen et al. (34). The total protein content of the conjugates was assayed using the bicinchoninic acid microassay (Pierce) using either BSA or TT as a standard. The TT, BSA, and BSA2 conjugates were used for vaccination and the DEX conjugate was used for ELISAs.
Experimental groups and immunizations
Groups of five female BALB/c or CBA/n mice (6–8 wk; The Jackson Laboratory, Bar Harbor, ME) were injected s.c. with the peptide conjugates, Alhydrogel, or PBS at the base of the tail in three different experiments. In experiment 1, there were four groups of BALB/c mice that received 100 µg of Alhydrogel, 100 µg of Alhydrogel and 50 µg of P13-TT, or 100 µg of Alhydrogel with 50 µg of P13-BSA or P13-BSA2, respectively. In experiment 2, there were four groups of BALB/c mice that received 100 µl of PBS, 100 µg of Alhydrogel, 100 µg of Alhydrogel and 10 µg of P13-TT, or 100 µg of Alhydrogel and 10 µg of P13-BSA2, respectively. In experiment 3, there were three groups of CBA/n mice that received 100 µg of Alhydrogel, 100 µg of Alhydrogel and 10 µg of P13-TT, or 100 µg of Alhydrogel and 10 µg of P13-BSA2, respectively. In experiment 1, mice were bled and vaccinated again s.c. every 14 days for 4 wk and then again 6 wk later, 10 wk after the initial vaccination on day 70. In experiments 2 and 3, the mice were bled and vaccinated again s.c. every 14 days for 6 wk. Sera were obtained on days 7, 21, 35, 63, and 77. In all, the mice received a total of four injections.
ELISAs
The titer of Abs to P13 and GXM in sera from the vaccinated mice
was determined before and 2 wk after each vaccination. The titer of Abs
to GXM was determined by ELISA as described previously (11, 35). ELISAs were performed in 96-well polystyrene plates
(Corning Glass Works, Corning, NY) coated with 10 µg/ml GXM 24067.
Wells were incubated with serial dilutions of the serum samples diluted
1:50 with 1x PBS/0.1% Tween 20 for 1 h at 37°C, washed, and
incubated with alkaline phosphatase-labeled goat anti-mouse IgM and
IgG (Fisher Biotech, Orangeburg, NY). The bound Abs were detected by
incubating the plates with p-nitrophenyl phosphate substrate
buffer (Sigma, St. Louis, MO) at pH 9.8. The negative control consisted
of wells without serum. The positive control was the mouse mAb 2H1
(IgG/
) (provided by A. Casadevall, Albert Einstein College of
Medicine). Absorbances were measured at 405 nm using an MRX ELISA
reader (Dynex Technologies, Chantilly, VA). For each sample, the
average absorbance of paired duplicates at each dilution was determined
after subtracting two times the background (the absorbance of wells
without serum). Titers were defined as the greatest 3-fold dilution
which produced an absorbance >0.1. The concentrations of selected sera
relative to the mouse mAb 18B7 (IgG1/) (36) was also
determined as follows: GXM-coated plates were incubated with dilutions
of sera and 18B7 starting at a concentration of 10 µg/ml, the plates
were washed and incubated with alkaline phosphatase-conjugated goat
anti-mouse IgG1, washed, and developed as above. The concentration
of IgG to GXM was extrapolated from a standard curve generated with
18B7 based on the OD of sera at a given dilution. The mAb 18B7 was
chosen as a reference Ab since it is now in use in a phase I clinical
trial in human cryptococcosis.
The titer of Abs to P13 was also determined by ELISA. Polystyrene ELISA plates (Corning Glass Works) were coated overnight with 10 µg/ml P13-DEX at room temperature. The plates were washed five times with 1x PBS/0.1% Tween 20 and incubated with 3-fold dilutions of the serum samples beginning with a dilution of 1:50 in 1x PBS/0.1% Tween 20 for 1 h at 37°C. The bound Abs were detected as described above.
IgG subclass determinations
The IgG subclass of Abs to GXM was determined by ELISA. Polystyrene microtiter plates (Corning Glass Works) coated with 10 µg/ml GXM 24067 were incubated with dilutions of the serum samples for 1 h at 37°C, washed, and incubated with alkaline phosphatase-labeled goat anti-mouse IgG1, IgG2a, and IgG2b combined and IgG3 (Fisher Biotech). The bound Abs were detected by incubating the plates with p-nitrophenyl phosphate substrate buffer (Sigma) at pH 9.8, and the plates were read as above.
Inhibition of serum Abs with GXM 24067 and P13
These studies were performed by inhibition ELISA as described elsewhere (25). Sera from the three mice in each group from experiment 1 that had the highest titer of Abs to each of the conjugates on day 35 were used. The sera were incubated in duplicate at a final dilution of 1:500 with 500 µg/ml GXM 24067 or 0.25 mM P13-DEX or 500 µg/ml Streptococcus pneumoniae serotype 14 polysaccharide (American Type Culture Collection) overnight at 4°C. The solutions of sera and inhibitors was then incubated with plates coated with GXM (0.5 µg/ml) or P13-DEX (10 µg/ml) for 1 h at 37°C. The plates were washed and the bound IgG was detected by ELISA as described above. The serum dilution used was selected because it represented 50% saturation of GXM binding by ELISA under the conditions described.
C. neoformans infection and passive immunization
C. neoformans serotype D (strain 24067) was used for protection experiments. This strain was chosen because it has been used extensively to study the protective efficacy of Abs to C. neoformans, including 2E9 (11) and a mouse mAb that is currently in phase I clinical trial in humans (36). A colony from a culture of C. neoformans was grown in Sabouraud dextrose medium (Difco, Detroit, MI) for 18 h at 37°C. Yeast cells were washed five times with sterile PBS and counted using a hemocytometer. In experiment 1, each group of vaccinated mice received 5 x 106 C. neoformans i.v. in 100 µl of PBS on day 70 after the first vaccination in experiment 1. In experiments 2 and 3, each group of mice received 5 x 106 C. neoformans i.v. on day 66 after the first vaccination. The passive immunization experiment was performed as follows: the yeast, grown, washed, and diluted as described, were suspended in pooled sera from each of the vaccinated groups at a final serum dilution of 1:20. A solution containing 5 x 106 C. neoformans was administered to groups of five mice i.v. The control for this experiment was serum from mice that were vaccinated with Alhydrogel alone. All mice were monitored daily for survival.
Serum GXM determinations
Serum samples were obtained from the mice once a week after infection with C. neoformans and analyzed for the presence of GXM by a capture ELISA as described previously (11, 35). Briefly, 5-µl aliquots treated with 200 µg/ml proteinase K (International Biotechnology, New Haven, CT) were incubated with plates coated with the murine mAb to GXM, 2H1. The plates were washed and GXM from strain 24067 was incubated with the plates. The plates were washed and incubated with the human IgM mAb 2E9, alkaline phosphatase-labeled goat anti-human IgM (Fisher Biotech) and developed with p-nitrophenyl phosphate substrate (Sigma). Absorbances were determined as described above. GXM levels were determined by comparing the absorbance of the samples to those obtained with known concentrations of GXM 24067. Sera from experiment 1 from days 7 and 14 after infection were also studied for the presence of Abs to GXM by ELISA as described.
Statistical analysis
Within group comparisons, e.g., pre- vs postvaccination and postvaccination titers at different times, were analyzed with the Wilcoxon matched pairs test. Between group comparisons were analyzed with the Mann-Whitney U test. The survival of the different groups of mice in the protection experiments was compared with the Kaplan-Meier log rank survival test. A p value of < 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vaccination of BALB/c mice with each peptide conjugate resulted in
an increase in the titer of Abs to P13 and GXM as compared with
preimmune Ab levels and the Alhydrogel control group in all three
experiments. The data shown are from experiments 1 and 2 (Fig. 1
). Similar results were obtained in
experiment 3 (data not shown). For experiment 1, on days 7 and 21, the
P13-TT, P13-BSA, and P13-BSA2 groups had significantly higher IgM to
P13 than the Alhydrogel group (p < 0.02) (Fig. 1A). For all groups, the IgM titer to P13 at days 7 and 21
was greater than at day 35 (p < 0.05) and
greater on day 7 than on day 21 or 35 (p <
0.05). Between group comparisons revealed that the P13-TT group had a
greater IgM titer to P13 on day 7 than the day 21 and day 35 titer of
the P13-BSA and P13-BSA2 groups (p < 0.05).
The P13-BSA group had a greater IgM titer to P13 on day 7 than the day
21 and 35 titer of the P13-TT and P13-BSA2 groups
(p < 0.05).
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B). On day 21, the P13-TT, P13-BSA, and P13-BSA2 groups had
a significantly higher IgG titer to P13 in sera than the Alhydrogel
group (p < 0.03). On day 21, the P13-TT,
P13-BSA, and P13-BSA2 groups had a significantly higher IgG titer to
P13 than on day 7 (p < 0.05). On day 21, the
P13-BSA had a significantly lower titer than on day 77
(p < 0.05) and the P13-BSA2 group had a
significantly higher titer than on days 35 and 77
(p < 0.03). Between group comparisons revealed
that on day 21 the P13-BSA2 and P13-BSA groups had a significantly
higher IgG titer to P13 than the P13-TT group
(p < 0.04). The P13-BSA group had a
significantly higher IgG to P13 than the P13-TT and P13-BSA2 on days 7,
21, and 35 (p < 0.05). On day 77, the P13-TT
group had a lower IgG titer to P13 than the P13-BSA group, but a higher
titer than the P13-BSA2 group. Abs to GXM in conjugate-vaccinated mice
The mice vaccinated with P13-TT, P13-BSA, and P13-BSA2 had IgM
titers to GXM that were two to four times higher than their
prevaccination levels (p < 0.05) and the titer
of the Alhydrogel group (p < 0.04) (Fig. 1C). On day 7, the P13-BSA2 group had a significantly higher
IgM titer to GXM than on day 35 (p = 0.04).
There was no significant difference in IgM titer to GXM for the P13-TT
and P13-BSA groups on days 7, 21, and 35. Between groups, there was a
significantly higher IgM titer to GXM found on day 7 in the P13-BSA2
when compared with day 35 P13-TT and the P13-BSA titer
(p < 0.05). Other between group comparisons
revealed no significant differences.
The IgG titers to GXM of the vaccinated groups were increased in the
conjugate-vaccinated groups as compared with their prevaccination titer
and the Alhydrogel group, depending on the time of observation (Fig. 1D). Mice that were vaccinated with P13-TT and P13-BSA had a
similar IgG titer to GXM on days 7, 21, and 35. Mice that received
P13-BSA2 had a higher titer of IgG to GXM on day 35 compared with days
7, 21, 63, and 77 (p < 0.04). On day 77, the
P13-BSA group had a significantly higher IgG titer to GXM than on days
7, 21, 35, and 63 (p < 0.01) and a
significantly higher titer than all of the other groups, except the
P13-BSA2 group on day 35 (p < 0.01). The
P13-BSA2 group had a significantly higher IgG titer to GXM on day 21
than the P13-TT group on days 35, 63, and 77 (p
< 0.03).
Fig. 1E shows the titers of IgG to P13, and Fig. 1F shows the titers of IgG to GXM for control, P13-TT, and
P13-BSA2-vaccinated mice obtained in experiment 2. The titers of both
the P13-TT and P13-BSA2 mice were greatest on day 35 compared with the
same and the other conjugate on days 7, 21, 49, and 63,
p < 0.05 Wilcoxon ranked sum test and Mann-Whitney
U test, respectively. Overall, the titer of the
P13-TT-vaccinated mice was greatest on day 77 (14 days after
infection), p < 0.05 Mann-Whitney U test.
There was an increase in IgG to GXM on days 7 and 14 after C.
neoformans infection on day 56 (days 63 and 70 from the first
vaccination) (Fig. 1F). No increase in IgG to P13 or IgM to
either P13 or GXM was noted after infection (data not shown). The
concentrations of serum IgG to GXM in sera with titers of 1:1250 and
1:900 on day 35 after the first vaccination were 150 and 100 µg/ml,
respectively, relative to standard reference curves generated reference
mAb 18B7 (data not shown). It should be noted that these are not actual
IgG concentrations, but rather the serum IgG concentrations calculated
relative to a high-affinity mAb to GXM that is in use in a clinical
trial.
In summary, the data from both experiments indicate that all three conjugates were immunogenic, including the BSA2 conjugate that was produced using a solid-phase approach. The subclass determinations revealed that mice that received each conjugate produced predominantly IgG1 to GXM, with little to no detectable IgG2a and 2b or IgG3 (data not shown).
Serum titers of Abs to GXM of C. neoformans-infected mice
C. neoformans-infected mice in the Alhydrogel group had
Ab titers to GXM of 
1:50 (the lowest limit of detection with the
ELISA used). The infected mice in the TT, BSA, and BSA2 groups had
detectable IgG, but not IgM to GXM in their sera on day 7 after
infection (Fig. 1F). The titers after infection cannot be
compared with the titers before infection, because of the presence of
GXM in the serum after infection (see Fig. 3). Immune complexes of GXM
and Ab to GXM may interfere with the detection of Abs to GXM by ELISA.
Thus, Ab levels may be underestimated. The Abs to GXM found on days 7
and 14 were IgG1 and IgG2a, with the titer of IgG2a more than IgG1;
there was little to no detectable IgM to GXM at either time (data not
shown).
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The serum samples were incubated with P13-DEX or GXM 24067 to
characterize the specificity of their IgG to P13 and GXM. Serum
inhibition of GXM and P13 binding varied depending on which conjugate
the mice received (Fig. 2). In experiment
1, P13-DEX inhibited 94% of IgG to P13 and 65% of GXM Abs,
respectively, in sera from mice that were vaccinated with P13-TT (Fig. 3A), and GXM inhibited 78 and
67% of IgG to P13 and IgG to GXM in sera from these mice, respectively
(Fig. 2A). P13-DEX inhibited 84% of IgG to and 48% of GXM
Abs in sera from mice that were vaccinated with P13-BSA (Fig. 3B), and GXM inhibited 56 and 40% of IgG to P13 and IgG to
GXM in sera from these mice, respectively (Fig. 2B). P13-DEX
inhibited 87% of IgG to P13 and 52% of IgG to GXM, respectively, in
sera from mice that were vaccinated with P13-BSA-2 (Fig. 2C), and GXM inhibited 46 and 37% of IgG to P13 and IgG to
GXM in sera from these mice, respectively (Fig. 2C). The
40–70% inhibition of binding of the Abs to P13 by GXM and of the Abs
to GXM by P13, depending on the conjugate, is consistent with the
conclusion that these Abs reacted with both P13 and GXM. In experiment
2, inhibition of serum Ab binding to GXM by soluble GXM was studied at
each time after vaccination and after infection (Fig. 2D).
The percent inhibition for the P13-TT and P13-BSA2 conjugates was
similar. There was no inhibition of binding to either P13 DEX or GXM by
serotype 14 pneumococcal capsular polysaccharide (data not shown).
Although different amounts of conjugate were administered, the
differences in the degree of inhibition in experiments 1 and 2 for the
BSA2 conjugate are not understood.
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Serum GXM concentrations were analyzed in C.
neoformans-infected mice. The GXM concentrations of mice in
experiment 2 are shown in Fig. 3. The concentration of GXM increased
from 5 to 33 µg/ml in the PBS and 4 to 33 µg/ml in the Alhydrogel
groups from day 7 after infection until death (Fig. 3). For the P13-TT
group, the serum GXM concentration increased at a slower rate. On day 7
after infection, the mice had 3 µg/ml GXM in their serum which
increased to 8 and 10 µg/ml on days 14 and 21, respectively; 20 and
20 µg/ml on days 28 and 35, respectively; and 35 and 32 µg/ml on
days 42 and 49, respectively (Fig. 3). For P13-BSA2, the concentration
of GXM in the serum remained relatively the same on days 7, 14, and 21
after infection and increased to 25 µg/ml on day 28; to 30 on day 35;
to 34 on day 42, and to 43 on day 49, and was 33 and 33 µg/ml,
respectively, on days 56 and 63 after infection. All mice succumbed
within 1 wk of serum GXM measurements 33 µg/ml or greater.
Survival of C. neoformans-infected mice
Administration of each peptide conjugate was associated with
prolonged survival of C. neoformans-infected mice in all
three experiments. In experiment 1, the mean survival was 28 days for
the Alhydrogel group, 52 days for the P13-BSA group, 70 days for the
P13-BSA2 group, and 72 days for the P13-TT group (Fig. 4A). In experiment 2, the mean
survival was 22 days for the PBS group, 24 days for the Alhydrogel
group, 44 days for the P13-TT group, and 48 days for the P13-BSA2 group
(Fig. 4B). In experiment 3, the mean survival was 24 days
for the Alhydrogel group, 48 days for the P13-TT group, and 54 days for
the P13-BSA2 group (Fig. 4C). Survival was significantly
greater for each of the conjugate-vaccinated groups as compared with
the Alhydrogel group (p < 0.001, Kaplan-Meier
log rank survival test). There was no difference in survival between
each of the vaccinated groups in any experiment
(p > 0.05, Kaplan-Meier log rank survival
test). The results of the passive protection experiment revealed a
statistically significant prolongation of survival for the mice that
received pooled sera from mice that had been vaccinated with P13-TT and
P13-BSA2 as compared with mice that received sera from mice that had
been vaccinated with Alhydrogel (p < 0.03,
Kaplan-Meier log rank survival test). The mean survival was 10.5 days
for the Alhydrogel group and 12.5 days for the P13-TT and P13-BSA-2
groups (Fig. 4D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The differences in the kinetics of the conjugate-induced Ab response to P13 and GXM suggest that the Ab response to the peptide and polysaccharide may be regulated differently, although this was not directly investigated in the study. A convincing secondary response to GXM was only observed after the second vaccination for the BSA2 conjugate in both experiments, after the fourth vaccination for the BSA conjugate in experiment 1, and after the second vaccination for the TT conjugate in experiment 2. The induction of an earlier secondary response by BSA2 is notable in view of the fact that it was produced using a solid-phase approach that has been proposed to enhance the immunogenicity of peptide Ags (34). The explanation for why this novel method did not result in a markedly enhanced response to either P13 or GXM is unknown. Multiple vaccinations with the TT conjugate failed to induce a secondary response to GXM, and the secondary response to the BSA2 conjugate was earlier, of lesser magnitude, and short lived. Thus, the P13 conjugate-elicited response to GXM resembled the so-called incomplete T-dependent responses characterized by a lack of secondary boosting that have been noted by others for polysaccharide-protein conjugates in mice (44) and humans (45).
The P13-induced Ab response to GXM was characterized by rapid kinetics and a lack of boosting, findings that were also reported for a T-dependent response to polysaccharide on a bacterial surface (46). This suggests the possibility that, although it is elicited by a peptide, the P13-induced response to GXM may be regulated like a response to the native polysaccharide. Notwithstanding, the conjugates appeared to prime for an amnestic response based on the observation that the IgG titer to GXM increased after C. neoformans infection (upon exposure to either soluble GXM or the capsular polysaccharide of encapsulated C. neoformans), without a concomitant increase in IgG to P13 or IgM to GXM. A peptide-mimotope-induced amnestic response to group A Neisseria meningitidis has also been reported (27). The following evidence suggests that the P13 conjugates elicited a T-dependent response: 1) the P13 conjugate elicited a memory response, 2) IgG1 was the predominant IgG subclass to GXM, and 2) an Ab response to GXM was induced in CBA/n mice, a strain that is unable to mount Ab responses to T-independent Ags (44, 47). However, more work is needed to determine the structural correlates and mechanisms that regulate mimotope-induced T-dependent Ab responses to polysaccharide.
The specificity studies with the conjugate-induced sera indicated that at least three populations of Abs were elicited by vaccination: Abs to P13; Abs to GXM; and Abs reactive with both P13 and GXM. Interestingly, the TT was the least antigenic conjugate based on the quantitative response. However, the greatest inhibition of GXM binding of conjugate-induced Abs to GXM was among those elicited by P13-TT, suggesting that Ab quantity and the qualitative characteristic of Ab specificity were not correlated. The lower titer of TT-induced IgG to GXM, particularly after the fourth vaccination, may have been the result of carrier-induced suppression, which can decrease the production of Ag-specific Abs (48). This possibility notwithstanding, the protective efficacy of the TT conjugate was similar to the others, again suggesting a dissociation between quantitative and qualitative Ab features with respect to priming for and/or the generation of Abs with the qualities found among protective Abs to GXM (10, 19). Interestingly, inhibition studies with sera from experiment 2 taken at different times after vaccination revealed a remarkably similar degree of inhibition of serum Ab binding to GXM, suggesting there may not have been an affinity (or specificity) shift in which cross-reactive Abs became Abs to GXM. Although this concept is speculative and requires further investigation, it raises the possibility that Abs to P13 and GXM may be structurally unique and derived from different Ab subsets. The latter is supported by studies showing a discontinuity between the polysaccharide epitopes recognized by peptide epitopes and Abs to the same polysaccharide determinant (38).
In both experiments 1 and 2, despite the administration of different amounts of different conjugates, the response to GXM after multiple vaccinations was greater than the response to P13. At present, the reason for this finding as well as the characteristics of a peptide mimetic that make it a mimotope are unknown. Regarding the latter, a GXM mimetic reported by another group that was selected by a mouse mAb did not elicit an anti-GXM response, although it induced Abs that shared molecular characteristics with the selecting mAb (37). It has also been reported that peptide mimics of a measles virus glycoprotein and group B N. meningitidis did not induce an antimeasles (49) or antimeningococcal response (42), respectively. However, peptide mimics of serotype A meningococcal capsular polysaccharide (27), the Shigella flexneri serotype 5a LPS (41), LewisY carbohydrate Ags (50, 51), group B streptococcal capsular polysaccharide (28), a herpes simplex virus glycoprotein (52), and a respiratory syncytical virus-associated glycoprotein (53) have been found to elicit anticarbohydrate responses. Notably, several peptide mimics that were subsequently found to be mimotopes reacted with immune or hyperimmune serum (often from another species than the selecting mAb) to the relevant capsular polysaccharide (27, 28, 54), and we had previously shown in two different studies that P13 inhibited the GXM binding of serum Abs from normal, but not HIV-infected, individuals (11, 30).
At present, the structural and immunologic aspects of antigenic mimicry that translate into immunologic mimicry and the functional correlates of each are unknown. However, available data suggest that commonly used parameters such as high-affinity peptide reactivity with the mAbs used for selecting peptide mimetics and/or a high titer antipeptide response are insufficient and perhaps not predictive of whether a peptide mimic is a carbohydrate mimotope. We believe that findings from this study suggest two possible criteria that may define a polysaccharide mimotope: 1) a peptide mimetic that is a mimotope may induce the generation of a greater antipolysaccharide than antipeptide response, and 2) there may be an association between the ability of a mimetic to function as a mimotope and cross-reactivity with immune serum. In light of the many polysaccharide mimetics that have been found not to be mimotopes, the poor immunogenicity of native capsular polysaccharides and the protective efficacy of the P13 conjugates in this study, we propose that an investigation of the feasibility of these criteria in vaccine design be undertaken.
In this study, all vaccinations were performed with adjuvant, and the protein carriers did not influence the efficacy of the conjugates. Although the mechanism of protection is unknown, the data suggest that it was likely to be Ab mediated. Prolongation of survival was slightly longer when a 5-fold greater amount of conjugate was administered, but this was only associated with a modestly higher titer of IgG to GXM. A minimum Ab concentration and affinity was needed for mimotope-induced protection against viral pathogens (52, 54). We found that the concentration of IgG to GXM with binding characteristics similar to a high-affinity mouse mAb that is in use in a clinical trial in humans was comparable to that which conferred passive protection against C. neoformans in mice (8). Irrespective of Ab quantity, qualitative characteristics such as Ab isotype, Id and specificity also determine Ab efficacy against C. neoformans (9, 10, 21, 36, 55). For example, IgG2a to GXM mediated superior opsonization and reduction in fungal burden in one mouse model of C. neoformans (7, 56), and IgG1 to GXM was highly effective in another model (9) and is the isotype of the mouse mAb used in a clinical trial (36). We found both IgG2a and IgG1 to GXM with little to no IgM after infection of conjugate-vaccinated mice, suggesting that protection may have been mediated by IgG. In addition, conjugate-vaccinated mice died soon after their serum levels of GXM increased to that of the controls at the time they died. In light of evidence that Abs to GXM reduce GXM antigenemia in C. neoformans-infected mice (55, 57, 58), the reduction in fungal burden in the P13 conjugate-vaccinated mice may be consistent with Ab-mediated protection. Finally, the fact that a statistically significant increase in survival was observed when immune serum was administered to naive infected mice suggests that conjugate-mediated protection was Ab mediated.
The data presented in this study support the conclusion that P13 is a polysaccharide mimotope that elicited a protective response against C. neoformans in mice, making it the only available vaccine for this fungal pathogen at the present time. Since P13 was reactive with human serum Abs to GXM, but the variable gene usage of human Abs to GXM is highly restricted (11, 30) and serum reactivity with P13 has discriminated between susceptible and relatively resistant patient populations, P13 may represent a conformational mimic of a GXM epitope that can stimulate a protective Ab response. Thus, the P13 conjugates represent rationally designed candidate vaccines for C. neoformans, although the GXM epitope that P13 mimics is unknown. Despite significant gaps in our knowledge regarding the molecular and functional characteristics of polysaccharide mimotopes, this study encourages further investigation to ascertain the mechanism(s) by which certain peptide mimics of polysaccharide Ags are mimotopes that can stimulate protective immunity.
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2 Address correspondence and reprint requests to Dr. Liise-anne Pirofski, Division of Infectious Diseases, Room 402 Forchheimer, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
3 Abbreviations used in this paper: GXM, glucuronoxylomannan; TT, tetanus toxoid; DEX, dextran.
Received for publication August 7, 2000. Accepted for publication October 19, 2000.
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, p < 0.05; Mann-Whitney
U test. *, p < 0.05; Wilcoxon
matched pairs test (see text for comparisons). For each group,
n = 5 mice. In experiment 1, the mice were injected
s.c. on day 1 and then again on days 14, 28, 42, and 70 before
infection on day 77; in experiment 2, the mice were injected s.c. on
day 1 and then again on days 14, 28, and 42 before infection on day
56.
