Received 7 December 1998/Returned for modification 18 February
1999/Accepted 15 March 1999
 |
INTRODUCTION |
Streptococcus pneumoniae
has been classified into 90 different serotypes based on the structure
of its polysaccharide (PS) capsule (7). S. pneumoniae is a major causative agent for pneumonia, meningitis,
and sepsis among young children and older adults (4). Antibiotic treatment has become less effective since the prevalence of
antibiotic-resistant S. pneumoniae has become very high
(1). Thus, there is a great need for pneumococcal vaccines
effective among young children and older adults.
Antibodies to capsular PS provide protection against S. pneumoniae expressing the homologous or cross-reactive capsular
serotypes, and pneumococcal vaccines are designed to induce antibodies
to the capsular PS. The currently available vaccines contain the capsular PSs from 23 common serotypes of S. pneumoniae
(12). Because many PSs in the 23-valent vaccine are not
immunogenic in young children, PS-protein conjugate vaccines containing
several serotypes (e.g., serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F)
are being developed (16).
The development of new pneumococcal vaccines would be simplified
greatly if there was a simple serologic assay for vaccine-induced protective immunity. Previous studies suggested that the concentration of antibodies in the serum correlates with in vivo animal protection (8) and in vitro opsonic activity (14, 17), the
key step of protection in vivo. Yet, a recent epidemiological study
suggested that the antibody concentrations may not predict vaccine
efficacy against homologous serotypes (18). Also, the
correlation between antibody concentration and opsonic activity can be
low (r = 0.5) (10, 13). We have therefore
examined the antigen-binding properties of several sera with less
opsonic activity than expected on the basis of antibody concentration.
(Part of this material has been submitted as an abstract for the 199 meeting of the Society of Pediatric Research in New Orleans.)
| |
MATERIALS AND METHODS |
Human sera and bacteria.
Twenty-five healthy adults were
immunized once with a 23-valent pneumococcal vaccine (PNU-IMMUNE 23)
from Lederle Laboratories (Pearl River, N.Y.). Serum samples were
collected before and 1 month after vaccination and were stored frozen
at 
20°C until analysis. The following strains of S. pneumoniae were used: DS2214 (G. Carlone, Atlanta, Ga.), a
serotype 14 strain; WU2 (Janet Yother, Birmingham, Ala.), a serotype 3 strain; JY1119 (David Briles, Birmingham, Ala.) and JD908 (Janet
Yother), WU2 variants lacking PspA and the PS capsule, respectively;
Tre-108 (David Briles), a variant of D39 (serotype 2) lacking PspC
(3); and CSR-SCS2 (G. Schiffman, Brooklyn, N.Y.), a variant
lacking the capsule (15).
ELISA.
The amount of anti-capsular PS antibody was
determined by "sandwich-type" enzyme-linked immunosorbent assay
(ELISA). Briefly, the wells of Maxisorb plates (Nunc, Roskilde,
Denmark) were coated at 37°C with 10 µg of the capsular PS (6B
serotype)/ml overnight in phosphate-buffered saline, which was prepared
fresh with water from a Milli-Q UF water purification system
(Millipore, Bedford, Mass.) to minimize the background signal. All
pneumococcal capsular PSs were purchased from the American Type Culture
Collection (Rockville MD).
After being coated with the antigen, the plates were washed and blocked
with phosphate-buffered saline containing 1% bovine serum albumin
(Sigma Chemical Co., St. Louis, Mo.) and 0.05% Tween 20. A serum pool
(89-SF) (11) from C. Frasch of the Food and Drug
Administration (Bethesda, Md.) was used as the standard and was found
to contain 16.9 µg of immunoglobulin G (IgG) anti-6B as published
previously (11). All samples were absorbed with 10 µg of
C-PS (purchased from Statens Seruminstitut, Copenhagen, Denmark) per 20 µl of serum in a total volume of 1 ml of diluent for 30 min at room
temperature. The samples were then added to the wells, serially
diluted, and incubated for 2 h at room temperature. For the
inhibition ELISA, inhibitors were added to each well before the test
serum was added. The wells were washed and incubated with alkaline
phosphatase-conjugated goat antibody specific for human IgG (Sigma
Chemical). The amount of the enzyme immobilized to the well was
determined with para-nitrophenyl phosphate substrate (Sigma
Chemical) in diethanolamine buffer. The optical density at 405 nm was
read with a microplate reader (Cambridge Technology, Watertown, Mass.).
The amount of antibody in the sample was determined by comparing the
optical density of the sample to the curve that was constructed by a
linear interpolation of the data of the standard sample at multiple dilutions.
ELISA with a chaotropic agent.
ELISA was performed as
described above with the following modifications. The serum samples at
various dilutions were added to the ELISA wells for 2 h and
incubated at 37°C. The ELISA plates were washed before 0.1 ml of
buffer containing various concentrations of NaSCN was added. After 15 min of incubation, the plates were washed and alkaline
phosphatase-conjugated goat antibody against human IgG (Sigma Chemical)
was added.
Opsonophagocytic-killing assay.
The opsonophagocytic
activities of the samples were determined by the method of B. Gray
(5), with minor modifications. Briefly, 10 µl of bacterial
suspension (about 2,000 CFU) was incubated with 40 µl of
appropriately diluted antibody for 30 min at room temperature with
shaking. Pneumococci of serotype 6B (strain L82016) (2) were
obtained from D. Briles and were grown in Todd-Hewitt broth with 0.1%
yeast extract and kept frozen in aliquots in Hank's buffer with 15%
glycerol. After the 30-min incubation, this mixture was incubated with
40 µl of phagocytic cell suspension (about 1,000,000 HL-60 cells) and
10 µl of baby rabbit complement (Pelfreeze, Brown Deer, Wis.) for
1 h at 37°C with shaking. HL60 cells (a human promyelocytic cell
line from the American Type Culture Collection) were differentiated in
a medium containing 0.8% dimethyl formamide for 5 days (13,
14).
At the end of the 1-h incubation, 100 µl of normal saline was added
to each well and mixed. Ten microliters of the reaction mixture was
sampled and applied to a THY agar plate. The plates were incubated
overnight at 37°C in a candle jar, and the number of colonies of
surviving bacteria was determined. The opsonization titer of the serum
was determined as the dilution of the serum that results in half as
many viable bacteria as are seen with no antiserum.
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RESULTS |
IgG antibodies in some sera bind a novel epitope that is found in
capsular PS from multiple serotypes.
While comparing the
opsonophagocytic-killing capacity with the concentration of antibodies
to serotype 6B, we observed that some postvaccination sera have less
opsonic capacity than expected on the basis of the concentrations of
IgG anti-6B PS antibody (Fig. 1). To
investigate the epitopes recognized by the antibodies in such
"ineffective" antisera, we chose two serum samples (P3B and P4B
[Fig. 1]) and measured their anti-6B antibodies before and after
preabsorbing the sera with C-PS or other PS (Fig.
2). We found that preabsorption with the
conventional amount of C-PS (10 mg/liter) reduced the signal as
expected (Fig. 2) and that preabsorption with an additional (10-fold)
amount of C-PS did not reduce the signal (data not shown). Also,
additional preabsorption of the two sera (P3B and P4B) with serotype 14 pneumococcal PS (10 mg/liter) (Fig. 2) or with other poly-anionic PSs,
such as heparin (50 U/ml) or Hib-PS (10 mg/liter) (data not shown), did not reduce the signal. However, to our surprise, an additional preabsorption with 9V PS (10 mg/liter) reduced the IgG antibody binding
to 6B PS by about 70% for P4B and about 100% for P3B (Fig. 2).
In contrast, when a control serum (P23B) with an opsonic activity commensurate to its antibody concentration was preabsorbed with 9V, the
preabsorption did not reduce the amount of IgG antibody binding to 6B
PS. This finding suggested that the ineffective antisera may have IgG
antibodies binding a "novel epitope(s)," which is present in the
preparations of capsular PS of many serotypes.
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FIG. 1.
Opsonization titer (y axis) versus IgG
antibody concentration (x axis) for serotype 6B. P3B and P4B
serum samples are indicated with arrows. The correlation coefficient is
0.4. The regression line is as follows: log (opsonization titer) = 0.65 × log(IgG anti-6B) + 2. Abs, antibodies.
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FIG. 2.
Amounts of IgG antibody binding to 6B-coated ELISA
plates in various serum samples following preabsorption with various
materials. The serum samples (P23B, P4B, and P3B) are identified on the
left. The preabsorbents, identified to the left of each bar, are none,
C-PS (10 mg/liter), capsular PS of serotype 14 (10 mg/liter), and
capsular PS of serotype 9V (10 mg/liter).
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To further investigate this novel epitope, we preabsorbed the three
postvaccination sera (P23B, P4B, and P3B) with C-PS and then performed
the ELISA for anti-6B antibody in the presence of varying
concentrations of different PSs in solution (Fig.
3). With the control serum sample (P23B),
the amount of IgG antibody binding to 6B PS-coated ELISA plates could
be almost completely inhibited at 0.5 mg of 6B PS/liter. No other PS
could reduce the P23B signal, even at 50 mg/liter. These observations
indicate that IgG anti-6B antibodies in P23B are 6B specific. However, when IgG anti-6B antibodies in P3B were analyzed, the optical density
could be reduced by pneumococcal capsular PSs of serotypes 2, 4, 9V,
19F (Fig. 3), and 3 (data not shown), as well as 6B PS itself (Fig. 3).
Pneumococcal capsular PS of serotype 14 could not reduce P3B signal
(Fig. 3). When P4B was analyzed, the capsular PS from all the serotypes
(except serotype 14) listed above inhibited more than half of the
binding. In all cases, the ELISA signal was not reduced even in the
presence of very high concentrations of C-PS. Thus, the novel epitope
is clearly distinct from C-PS, and the antibodies binding the novel
epitope account for almost 100% of IgG anti-6B for the P3B sample,
more than half for the P4B sample, and a negligible amount for the P23B
sample.
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FIG. 3.
Amounts of IgG antibodies in three serum samples which
bind to the ELISA plate coated with 6B PS in the presence (5 mg/liter)
of various inhibitors.
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The novel epitope is not on the capsular PS.
The capsular PS
used for our experiments is not chemically synthesized but is isolated
from the bacteria; thus, it may contain a contaminant(s) other than
C-PS. Our observations could be explained if the novel epitope is an
unidentified contaminant whose structure is conserved among different
serotypes of S. pneumoniae. To investigate this possibility,
we performed an ELISA with the three serum samples in the presence of
the lysates of various S. pneumoniae strains (Fig.
4). The binding of IgG antibodies in P3B
to 6B PS-coated plates could be inhibited with the lysates (0.03%
[vol/vol]) of various bacterial strains lacking either the capsular
PS, PspA (3), or PspC (3), although the
inhibition by SCR-SCS2, a noncapsular variant derived from S. pneumoniae serotype 2 (15) was not complete. For this
experiment, the bacterial lysate was prepared by repeatedly freezing
and thawing a bacterial suspension. Even though the purified capsular
PS of serotype 14 was not inhibitory (Fig. 3), the lysate of serotype
14 pneumococci could efficiently inhibit the binding of IgG antibodies
in P3B (Fig. 4A). Unlike S. pneumoniae, lysate of a
Streptococcus pyogenes strain was not significantly
inhibitory (data not shown). Consistent with the fact that only a part
of the antibodies in P4B bind the novel epitope, about 40% of antibody
remained bound even at the high concentration of bacterial lysates
(Fig. 4B). In contrast to P3B and P4B, no bacterial lysates could
inhibit the binding of IgG antibodies in P23B to 6B PS-coated ELISA
plates by more than 25% (Fig. 4C). Our findings taken together
strongly support the conclusion that the novel epitope(s) recognized by
IgG antibodies in P3B or P4B is not part of the capsular PS but an
unidentified bacterial antigen.
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FIG. 4.
Amounts of IgG antibodies in three serum samples (P3B,
P4B, and P23B) which bind to the ELISA plate coated with 6B PS in the
presence of various bacterial lysates. The bacterial strains used were
CSR-SCS2; WU2, a serotype 3 strain; JD908, a WU2 variant
expressing no capsular PS; JY1119, a WU2 variant lacking PspA; Tre108,
a variant lacking PspC; and DS2214, a serotype 14 strain.
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Antibodies can bind the novel epitope in the presence of a
chaotropic agent.
Some antibodies against pneumococcal PSs have
very low avidity for 6B PS and do not opsonize well. Since these
low-avidity antibodies do not bind the antigen in the presence of a
chaotropic agent such as NaSCN, it is often added to the buffer used
for ELISA in order to measure only pneumococcal antibodies with high avidity (6). It is possible that the antibody to the novel epitope may have low avidity and may not bind to 6B PS in the modified
ELISA protocol employing NaSCN. When we directly examined whether NaSCN
prevents the binding of this antibody to immobilized 6B PS, to our
surprise, we repeatedly observed P3B to be more resistant to NaSCN than
P23B or 89-SF. The binding of P23B is reduced by more than 50% with
0.25 M NaSCN, whereas the binding of P3B is reduced by less than 20%,
even at 1 M NaSCN. Thus, the antibodies to the novel epitope can bind
the immobilized pneumococcal capsular PS even in the presence of a
chaotropic agent.
Many individuals have significant amounts of antibodies to the
novel epitope.
To determine the prevalence of the antibodies
binding the novel epitope, we studied 25 pre- and postvaccination serum
samples. This was done by performing the ELISA in the absence and
presence of 9V PS (10 mg/liter) in the reaction buffer with the immune sera that were already absorbed with C-PS. As can be seen in Fig. 5, preabsorption with 9V reduced the
apparent IgG anti-6B antibody concentrations by about twofold in 10 preimmune and in 4 postimmune serum samples. The apparent concentration
of anti-6B antibodies in the sample P3B (Fig. 5B) decreased almost
10-fold after absorption with 9V PS. Therefore, a recognizable fraction
of IgG "anti-6B antibodies" from adults actually bind the novel
epitope. In addition, the antibodies to the novel epitope affect the
determination of IgG anti-6B antibody levels more in preimmune sera
than in postimmune sera.
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FIG. 5.
IgG anti-6B antibody (Ab) concentrations with
(y axis) or without (x axis) preabsorption
with pneumococcal capsular PS of serotype 9V. (A) Preimmune sera; (B)
postimmune sera. The solid line indicates the identity, and the dotted
line indicates the 50% reduction in antibody concentrations following
preabsorption. absorp., absorption.
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Levels of antibodies binding the novel epitope can increase after
vaccination.
To determine if the antibodies to the novel epitope
increase in concentration following vaccination, we obtained preimmune sera from two individuals who had ineffective antibodies and four randomly chosen individuals who had effective antibodies. Anti-6B antibody concentrations were determined by ELISA with or without 9V PS in solution. Anti-6B antibody concentrations measured in the presence of 9V PS were considered to be 6B-specific antibodies (not
cross-reactive with 9V), and the difference in anti-6B antibody concentrations measured without or with 9V PS was considered to represent the antibodies recognizing the novel epitope (Table 1). Concentrations of antibodies to the
novel epitope can be negative if most of the anti-6B antibodies are
specific for 6B. The 6B-specific antibodies increased 1- to 10-fold
with vaccination, and in addition, we also found that the anti-6B
cross-reactive with 9V PS increased two- to threefold after
vaccination. Thus, the PS vaccine may stimulate the antibodies to the
novel epitope as well.
The novel epitope affects assays of anti-capsular PS for other
serotypes.
Pneumococcal conjugate vaccines will likely contain at
least seven serotypes (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F). To
examine if the antibody to the novel epitope may influence the
determination of the concentration of antibodies to capsular PS of
serotypes other than 6B, we performed the ELISA for anti-capsular antibodies for five serotypes (4, 14, 18C, 19F, and 23F) with serum
samples before and after absorption with 9V PS. For the ELISA assay of
serotype 9V, we absorbed the serum samples with 6B PS. As we have seen
with antibodies for serotype 6B, most data points were to the right of
the identity line, indicating that the concentration estimates became
smaller with absorption with an unrelated capsular PS (Fig.
6). For instance, for serotype 18C, 11 of
25 preimmune samples had more than half of the antibodies directed to
the novel epitope. Also, the absorption with unrelated PS reduced the
estimate of the antibody concentrations more for the preimmune samples
than for the postimmune samples. The exception to this general
observation is serotype 14, since the antibody concentration estimates
for serotype 14 did not change with additional absorption. It is likely
that the preparation of capsular PS of serotype 14 used for this study
does not contain the novel epitope but capsular PSs of all other
serotypes do.
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FIG. 6.
Concentrations of IgG antibody (Ab) to capsular PS with
(y axis) or without (x axis) additional
preabsorption with an irrelevant pneumococcal capsular PS for preimmune
sera or for postimmune sera. The serotype specificity of the antibodies
is indicated at the top of each panel. The solid line indicates the
identity, and the dotted line indicates the 50% reduction in antibody
concentrations following preabsorption.
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| |
DISCUSSION |
In this study we demonstrated that the IgG antibodies to a novel
epitope constitute the majority of IgG antibodies to pneumococcal capsule in many prevaccination sera as well as in occasional
postvaccination sera. The antibodies to the novel epitope affect
estimation of the antibodies not just to 6B PS but to PSs of many other
serotypes that are included in the conjugate vaccines, except for
serotype 14. The significant effect of the novel epitope was
independently observed by the two laboratories collaborating in this
project, using different sets of reagents. The presence of the novel
epitope was also independently reported by Coughlin et al. in nonimmune sera after our manuscript was prepared for publication (3a). Furthermore, the antibodies to the novel epitope are nonopsonic and may
not be protective. Therefore, the presence of antibodies to the novel
epitope must be appreciated in evaluating new pneumococcal vaccines.
In several situations, the impact of antibodies to the novel epitope on
estimating the concentration of antibodies to the capsular PS may be
magnified. The impact could be large in the studies of some populations
(e.g., young children) who are poorly responsive to PS. The impact
could be magnified when the capsular PS-specific antibody response is
weak, such as with serotype 6B. It should be more significant in
estimating the antibody levels in preimmune sera than in postimmune
sera, which contain generally higher levels of antibodies to capsular
PS. Also, the impact could be significant in postimmune sera if the
pneumococcal vaccines used were contaminated with the novel epitope
more than usual, since the novel epitope appears to be immunogenic.
The exact nature of the novel epitope is unclear. However, it is not
part of the capsular PS but is likely a contaminant found in the
commercially available vaccine-grade preparations of capsular PS. The
novel epitope does not appear to be C-PS, a well known and common
contaminant in most pneumococcal capsular PS (9). Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed
several contaminant molecules that varied among different capsular PS
preparations, but the magnitude of antibodies to the novel epitope did
not correlate with the presence of a specific contaminant visualized in
the gel (data not shown). The novel epitope is not expressed on
pneumococcal proteins like PspA or PspC, and the novel epitope was
also resistant to trypsin or papain (data not shown). Interestingly,
the novel epitope appears to be absent in the preparations of capsular
PS of serotype 14. Type 14 capsular PS is biochemically quite different
from the PSs of other serotypes, and its purification method is
different from that of other capsular PSs.
In order to improve our ability to measure vaccine-induced protective
immunity, ELISA assays for pneumococcal antibodies have been
extensively standardized and are being further modified not to measure
low-avidity (and nonopsonic) antibodies. For instance, antibodies to
C-PS are routinely neutralized by preabsorption, and a chaotropic agent
is often used to measure only the high-avidity antibodies by ELISA
(6). The antibodies to the novel epitope not only interfere
with the standardized ELISA assays but also bind to the ELISA plates
even in the presence of the chaotropic agent. One way of improving the
specificity of the standardized ELISA is to preabsorb the serum samples
with an unrelated capsular PS in addition to C-PS. While this
modification should be evaluated further, the increasing complexity of
ELISAs used for estimating the concentrations of protective antibodies
further emphasizes the need for standardizing the ELISA and for
supplementing the ELISA results with an independent measure of
protective immunity (e.g., opsonophagocytosis assay results).
This work was supported by funds from the National Institutes of
Health (AI-31473 and AI-85334) and a grant from the World Health
Organization (VRD-V23/181/72). M.H.N. is partially supported by NIAID
contract N01 AI 45248.
We thank P. Anderson, J. B. Robbins, P. Allen, D. Briles,
and J. Treanor for their critical readings. We also thank J. Yother for her generous gift of the bacterial strains and J. Olander and C. Fristschle for their continued interest in this project.
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Abstract
Introduction
