INFECTION AND IMMUNITY, Jan. 2011, p. 314–320
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 1
Older Adults Have a Low Capacity To Opsonize Pneumococci Due to
Low IgM Antibody Response to Pneumococcal Vaccinations?†
Saeyoung Park and Moon H. Nahm*
University of Alabama at Birmingham, Birmingham, Alabama 35294
Received 16 July 2010/Returned for modification 22 September 2010/Accepted 19 October 2010
Since the 23-valent pneumococcal polysaccharide vaccine (PPV23) is less effective for older adults than for
young adults, it is important to investigate the immunologic basis for the reduced efficacy of PPV23 among
older adults. We determined the effectiveness of PPV23 among young (n ? 55) and older (n ? 44) adults by
measuring the serum IgG, IgM, and IgA concentrations and opsonic capacities against serotypes 14, 18C, and
23F. While young and older adults showed no difference in levels of IgG antibodies against pneumococcal
polysaccharide (PPS), older adults had lower IgA and IgM antibody levels than young adults for all three
serotypes. In both age groups, anti-PPS IgA or IgM antibody levels were much lower than anti-PPS IgG antibody
levels. Young adults showed higher opsonic capacities than older adults for serotypes 14 and 23F. In order to
anti-PPS IgA or IgM antibodies were removed from immune sera by affinity chromatography. The difference in
opsonic capacity between young and older adults disappeared for serotypes 14 and 23F (but not for serotype 18C)
when IgM antibody was removed. However, there was no significant difference between the two age groups when IgA
antibody was removed. In conclusion, even though anti-PPS IgG antibody levels are high compared with anti-PPS
IgM antibody levels, the low levels of anti-PPS IgM antibody alone can explain the functional difference observed
between young and older adults immunized with PPV23 with regard to some pneumococcal serotypes.
Streptococcus pneumoniae (pneumococcus) is a significant
cause of morbidity and mortality due to bacterial meningitis and
sepsis, particularly in young children and elderly adults (12). Also,
it is receiving increased attention as the pathogen responsible for
a large number of deaths among influenza-infected persons dur-
ing influenza epidemics (2, 18, 22, 26, 45). To control pneumo-
coccal infections, a 23-valent pneumococcal polysaccharide vac-
cine (PPV23) and three conjugate vaccines are currently available
for immunizing old adults and children, respectively (1, 8, 43).
Although conjugate vaccines have been shown to be highly
effective among children (11, 31), the protective efficacy of
PPV23 is less clear among adults, and its efficacy decreases
dramatically with increased age (14, 34, 36, 37).
To investigate the immunologic basis for its reduced efficacy
for the aged, the immunogenicity of PPV23 in old adults has
been studied extensively (32, 33, 37). Previous immunogenicity
studies of immune sera have focused on measuring both op-
sonic capacity and levels of IgG antibodies against pneumo-
coccal polysaccharide (PPS), since anti-PPS antibodies provide
protection primarily by opsonizing pneumococci, and anti-PPS
IgG antibodies account for the majority of anti-PPS antibodies
in the immune sera (5, 24, 29, 44). Several studies by different
investigators and with different analytical approaches have
shown that old adults elicit anti-PPS IgG antibody levels equiv-
alent to those of young adults but that old adults’ sera are less
opsonic than young adults’ sera (32, 34, 37).
The findings of the immunogenicity studies have often been
interpreted as showing a reduction in antibody affinity or avid-
ity due to aging, since animal studies have shown that aging is
associated with changes in antibody V-gene expression and
fewer somatic mutations (4, 20, 41). However, the role of
anti-PPS IgM or IgA antibodies, which are made in smaller
amounts than IgG antibodies, has not been investigated pre-
viously. It is important to clarify their role, since IgA antibodies
have been shown to inhibit the opsonic capacity of IgG anti-
bodies (16, 17, 35), and IgM antibody molecules are more
efficient than IgG antibodies in opsonizing pneumococci (28,
42). Therefore, we have investigated the roles of anti-PPS IgM
and IgA antibodies produced in response to PPV23 in both
young and old adults.
MATERIALS AND METHODS
Serum samples. Two groups of anonymous serum samples were obtained. One
group was obtained from L. Jackson (Seattle, WA) and contained sera from 45
old adults, 70 through 79 years of age (mean age ? standard deviation [SD],
74.9 ? 2.4 years), who were immunized with PPV23 (Pneumovax; Merck & Co.,
Inc., Whitehouse Station, NJ) 4 weeks prior to phlebotomy (15). The exclusion
criteria for this study population were nursing home residence, immunocompro-
mise, chronic anticoagulation or a known bleeding disorder, asplenia, active
cancer, liver or renal failure, known hypersensitivity to any pneumococcal vac-
cine component, and receipt of a diphtheria toxoid-containing vaccine in the
previous 6 months. All elderly adults received one dose of PPV23 at least 5 years
prior to enrollment (15). The other group was from M. Blake (Bethesda, MD)
and contained sera from 55 young college students who were bled 4 weeks after
immunization with PPV23. These students had not received PPV23 previously.
ELISA for anti-PS antibodies. The amount of anti-PPS IgA, IgG, or IgM
antibody was determined by a “sandwich”-type third-generation pneumococcal
antibody enzyme-linked immunosorbent assay (ELISA) (48). Briefly, the wells of
microtiter plates were coated at 37°C with capsular polysaccharide (PS; usually
1 to 10 ?g/ml) (ATCC, Rockville, MD) for 5 h in phosphate-buffered saline
(PBS) with 0.02% NaN3. Next, the plates were washed with Tris-buffered saline
(10 mM Tris with 150 mM NaCl and 2 mM KCl at pH 7.4) containing 0.1% Brij
35 (Sigma-Aldrich, St. Louis, MO). All serum samples were preabsorbed with 5
* Corresponding author. Mailing address: University of Alabama at
Birmingham, Department of Pathology, BBRB 614, 1530 Third Ave.
South, Birmingham, AL 35294-2170. Phone: (205) 934-0163. Fax:
(205) 975-2149. E-mail: email@example.com.
† Supplemental data for this article may be found at http://iai.asm
?Published ahead of print on 1 November 2010.
?g of cell wall polysaccharide (C-PS) (Statens Serum Institut, Copenhagen,
Denmark) and 5 ?g of 22F PS in a total volume of 1 ml of PBS containing 0.05%
Tween 20 for 30 min at room temperature (RT). The reference serum, pool
89-SF, from the Food and Drug Administration (Bethesda, MD), was absorbed
only with C-PS and was used as the standard. The preabsorbed serum samples
and 89-SF were serially diluted and added to the microtiter plates. After 2 h of
incubation at RT and after washing as described above, an alkaline phosphatase-
conjugated goat antibody specific for human IgA, IgG, or IgM (Southern Bio-
tech, Birmingham, AL) in PBS with 0.02% NaN3was added to the plates. After
2 h of incubation at RT, the plates were loaded with a p-nitrophenyl phosphate
substrate (Sigma-Aldrich, St. Louis, MO) in diethanolamine buffer and were
incubated for 2 h at RT. The reactions were stopped with NaOH, and the optical
density at 405 nm was measured using an ELISA microplate reader. The amount
of antibody was determined by comparing the optical densities for each of the
diluted serum samples with the optical density curve constructed with the stan-
dard sample (89-SF) at multiple dilutions.
MOPA. A multiplexed opsonophagocytic killing assay (MOPA) based on an-
tibiotic-resistant target bacteria was performed for three serotypes (serotypes 14,
18C, and 23F) as previously described (9). Briefly, frozen aliquots of each target
pneumococcus were thawed, washed twice, diluted to the proper bacterial den-
sity (?2 ? 105CFU/ml for each serotype), and pooled by mixing equal volumes.
All serum samples were incubated at 56°C for 30 min, and serial dilutions were
prepared. Serially diluted serum (20 ?l/well) was mixed with 10 ?l of the bac-
terial mixture in each well of round-bottom 96-well plates (Corning Inc., Corn-
ing, NY). After 30 min of incubation at RT with shaking, 10 ?l of complement
from 3- to 4-week-old rabbits (PelFreez Biologicals, Rogers, AR) and 40 ?l of
differentiated HL60 cells (4 ? 105cells) were added to each well. HL60 cells
were differentiated with dimethylformamide as previously described (9). After 45
min in an incubator (37°C, 5% CO2) with shaking, an aliquot of the final reaction
mixture (10 ?l) was spotted onto three different Todd-Hewitt yeast (THY) agar
plates (Todd-Hewitt broth with 0.5% yeast extract [THY broth] and 1.5% agar).
When the fluid was absorbed into the agar, an equal volume of an overlay agar
was applied to the THY agar plate. The overlay agar was THY broth with 0.75%
agar, 25 mg/liter of 2,3,5-triphenyltetrazolium chloride, and an antibiotic. Strep-
tomycin, optochin, or trimethoprim was chosen for the plates designed to detect
colonies expressing serotype 14, 18C, or 23F, respectively (9). After overnight
incubation at 37°C, the bacterial colonies on the agar plates were counted. The
opsonization index was defined as the serum dilution that kills 50% of bacteria.
A detailed protocol is posted at www.vaccine.uab.edu.
Affinity chromatography for absorption of IgA or IgM from immune sera. To
remove IgA or IgM antibodies from immune sera, each serum sample was mixed
with agarose beads conjugated with an antibody specific for anti-human IgA or
IgM (Sigma-Aldrich, St. Louis, MO) for 2 h at RT. For mock absorption,
unconjugated agarose beads were used instead of antibody-conjugated beads. To
recover the absorbed serum from the agarose beads, the mixture was centrifuged
in a Bio-Rad chromatography spin column (Bio-Rad Laboratories, Inc., Hercu-
les, CA) at 2,000 rpm for 1 min at 4°C. The completeness of absorption was
monitored by using a third-generation pneumococcal antibody ELISA to deter-
mine the level of anti-PPS antibody in the absorbed sera.
Statistical analysis. The geometric mean concentrations (GMC) of anti-PPS
IgG, IgM, and IgA antibodies were calculated for each group. The geometric
mean opsonophagocytic index (GMI) was also calculated for each group. Young
and old adults were compared using a two-sample, unpaired Student t test after
logarithmic transformation. P values less than 0.05 were considered to be signif-
icant. Statistical calculations were performed by use of JMP software, version 8.0
(SAS Institute Inc., Cary, NC).
PPV23 induces more-effective antibody responses in young
adults than in old adults. To confirm previous results indicat-
ing that young adults produce more-effective anti-capsule an-
tibodies than old adults (34), we obtained immune sera from
new groups of young and old vaccinees and then determined
their anti-PPS IgG antibody concentrations, opsonic indices
(OI), and antibody potency for serotypes 14, 18C, and 23F. As
was found previously, young and old adults showed similar
levels of IgG antibodies to serotypes 14, 18C, and 23F (P, 0.14,
0.8, and 0.47, respectively) (Table 1). However, young adults
had higher OI than did old adults for serotypes 14 and 23F
TABLE 1. IgA, IgG, and IgM antibodies against serotypes 14, 18C, and 23F among young and elderly adults
Concn of antibodyb
aValues in parentheses are 95% confidence intervals. ND, not determined.
bGeometric mean concentration, in micrograms per milliliter.
cGeometric mean opsonophagocytic index.
dGeometric mean antibody potency (OI/?IgG?).
VOL. 79, 2011 PNEUMOCOCCAL VACCINE WITH AGING315
(P, ?0.01 for both serotypes), and as a result, young adults had
higher antibody potency than did old adults for these serotypes
(P, ?0.01 for both serotypes) (Table 1). Even though the
difference was not statistically significant (P, 0.18), young
adults showed an approximately 2-fold higher GMI than old
adults for serotype 18C (1,149 [young] versus 698 [old])
A significant number of immune sera from young adults are
more opsonic than are other sera from young or old adults. To
investigate the observed difference in antibody potency be-
tween the two populations, the OI of sera were plotted against
their anti-PPS IgG antibody concentrations (Fig. 1). The slope
of the best-fit line was close to 1 for old adults but less than 1
for young adults. Also, the correlation between the OI and the
anti-PPS IgG antibody levels was significantly higher for old
adults than for young adults for all serotypes (0.58 [old] versus
0.35 [young] for serotype 14, 0.60 [old] versus 0.31 [young] for
serotype 23F, and 0.47 [old] versus 0.37 [young] for serotype
18C). Interestingly, for serotypes 14 and 23F, many samples
were away from the 45° trend line (shown by the dotted circles
in Fig. 1). For instance, in serotype 23F, a significant propor-
tion of sera from young adults (24 out of 55 [43.6%]) had high
opsonic activities even when their anti-PPS IgG levels were low
(less than 5.5 ?g/ml), whereas only a few such sera from old
adults (5 out of 44 [11.4%]) were observed (P, 0.001 by Fisher’s
exact test). The poor correlation and low slope for young
adults observed with serotypes 14 and 23F could be due to
those samples away from the trend line, which may have non-
IgG antibodies that are opsonic.
Anti-PPS IgM antibodies affect the functional difference
between young and old adults immunized with PPV23. To
directly investigate the contribution of anti-PPS IgM or IgA
antibodies to the opsonic capacity of the immune sera, we
selected four sera with low levels of anti-23F IgG antibody but
with high opsonic indices against serotype 23F (circled in Fig.
1C), selectively removed their IgM or IgA by affinity chroma-
tography, and determined their abilities to opsonize serotype
23F. The measured opsonic capacity was complement depen-
dent: the OI was reduced by more than 95% when the rabbit
serum (complement) was heat inactivated (data not shown).
Also, nonspecific removal of antibody during the absorption
was minimal (?10%), since mock absorption of serum samples
with plain agarose beads reduced antibody concentrations and
opsonic indices by less than 10%. Our affinity chromatography
selectively removed more than 80% of IgM or IgA from the
four immune sera but removed IgG minimally (less than
10%) and nonspecifically (Fig. 2A and B). When we deter-
mined the opsonophagocytic killing capacities of these ab-
sorbed sera, the opsonic capacities of the sera almost com-
pletely disappeared after IgM depletion (Fig. 2C) but
decreased only minimally (18 to 34%) after IgA depletion
(Fig. 2D). These studies strongly suggested that anti-PPS
IgM antibodies are responsible for the opsonic activity ob-
served for these four serum samples.
To extend this observation, we removed IgM from immune
sera from both young and old vaccinees and determined the
capacities of the sera to opsonize serotypes 14, 18C, and 23F
(Fig. 3) (see the table in the supplemental material). Since we
had technical difficulties with some samples, we could not
analyze some serum samples (see the table in the supplemental
material), and the numbers of samples analyzed are given in
the legend to Fig. 3. The removal of IgM increased the corre-
lation between the serum IgG concentration and the opsonic
indices for young adults for all three serotypes. Following the
removal, the correlation coefficient increased from 0.32 to 0.61
for serotype 14 (Fig. 3A) and from 0.34 to 0.62 for serotype
23F (Fig. 3C), but it did not change for serotype 18C (Fig. 3B).
The individual sera that showed higher opsonic titers than
expected on the basis of the levels of their anti-PPS IgG anti-
bodies (shown as circles in Fig. 1) were exactly matched with
the sera whose opsonic titers were decreased significantly after
IgM depletion (data not shown). For old adults, IgM absorp-
FIG. 1. Scatter plots evaluating associations between the anti-pneumococcal polysaccharide IgG concentration (in micrograms per milliliter)
and the opsonic index for serotypes 14 (A), 18C (B), and 23F (C). Circled points (A and C) represent subgroups of serum samples that have a
higher opsonic index with a lower IgG concentration. Solid and broken lines are the trend lines for each group.
316PARK AND NAHMINFECT. IMMUN.
tion did not increase the correlation coefficient for any of the
three serotypes (Fig. 3). IgM antibody depletion also reduced
the differences in the geometric means of OI observed between
young and old adults (Fig. 4). When the OI was compared with
the IgM antibody concentration, they showed a modest corre-
lation (r, 0.4 to 0.7) (see the figure in the supplemental mate-
rial). We also analyzed the effect of IgA absorption on the
opsonic titers of young and old adults for serotype 23F, but no
changes were observed (Fig. 5). Thus, anti-PPS IgM antibodies
are responsible for the high potency of the antibodies from
Anti-PPS IgM antibody levels are small compared with anti-
PPS IgG antibody levels. In view of the large impact of anti-
PPS IgM antibodies on opsonization indices, we studied the
amounts of those antibodies in the immune sera from young
and old vaccinees. The level of anti-PPS IgM antibody was
relatively low (only about 3 to 10%) compared with the level
of anti-PPS IgG antibody (Table 1). Thus, although anti-
PPS IgM antibodies are present in small quantities, they
make an important contribution to the opsonic capacity of
Consistent with the results of previous studies (32, 34, 37),
we found higher opsonic capacities in young adults than in old
adults for serotypes 14 and 23F, with no significant difference
in IgG levels between the two groups. However, anti-PPS IgA
and IgM antibody levels, in contrast to IgG, are lower in older
adults than in young adults. Furthermore, the opsonic capacity
differences between the two groups disappeared after the re-
moval of IgM (but not IgA) antibodies from immune sera for
all three serotypes. The anti-PPS IgA antibody levels of the
immune sera studied here are likely too low to influence the
opsonic activity of IgG antibodies (16). In contrast, low levels
of anti-PPS IgM antibodies can account for the functional
difference observed between young and old adults immunized
with PPV23, since IgM is more efficient at fixing complement
FIG. 2. Absorption of IgA or IgM from sera immunized with PPV23. Four samples (S1, S2, S3, and S4) were selected from the subgroup
circled in Fig. 1C. Pooled control sera (P22) obtained from several healthy persons either left unimmunized or immunized with PPV23 were
also used. The levels of IgA and IgM were measured at an 80-fold sample dilution, and the level of IgG was measured at a 400-fold sample
dilution. (A) Absorption of IgM from immune sera. IgA, IgM, or IgG levels are shown for four immune sera and P22 after mock absorption
(Mock-abs) and IgM absorption (Post-abs). (B) Absorption of IgA from immune sera. IgA, IgM, or IgG levels are shown for four immune
sera and P22 after mock absorption and IgA absorption. (C) Opsonic indices of IgM-absorbed fractions for four immune sera and P22 after
mock absorption and IgM absorption. (D) Opsonic indices of IgA-absorbed fractions for four immune sera and P22 after mock absorption
and IgA absorption.
VOL. 79, 2011 PNEUMOCOCCAL VACCINE WITH AGING 317
than IgG. Indeed, studies with human monoclonal antibodies
have shown that IgM antibodies can be 10- to 100-fold more
effective than IgG antibodies at opsonizing bacteria or protect-
ing animals from infections (30, 39).
Some IgM antibodies that are protective against bacterial
infections bind many unrelated antigens, such as single-
stranded DNA, thyroglobulin, and ?-galactosidase (27). Such
IgM antibodies, which are called polyreactive or natural anti-
bodies, are found in preimmune sera (3, 27, 49, 50) and have
been isolated as hybridomas producing anti-PPS antibody (6).
Also, the level of natural antibodies has been shown to de-
crease with age (10). Nevertheless, the absorption of several
postimmune sera that are rich in IgM antibody with a mixture
of single-stranded DNA, thyroglobulin, and ?-galactosidase
did not reduce the opsonic activities of the sera (unpublished
observation). Thus, the highly opsonic anti-PPS IgM antibody
present in our postimmune sera is not likely to be polyreactive
but specific for PPS produced in response to vaccination.
A previous study reported that following immunization with
PPV23, less anti-PPS IgM antibody appeared in old adults than
in young adults (38). Another study reported that naturally
acquired (in nonvaccinated persons) anti-PPS IgM antibody
levels decreased with aging (40) for five out of six different
capsule types. However, these studies used a nonspecific
ELISA and did not investigate the functional significance of
the reduced anti-PPS IgM antibodies. Consequently, to our
knowledge, our report may be the first linking anti-PPS IgM
antibodies with the observed functional difference between
sera from young and old adults immunized with PPV23. Nev-
ertheless, one must be aware that there could be additional
explanations, since our studies are limited to several pneumo-
FIG. 3. Correlation between IgG concentration (x axis) and op-
sonic index (y axis) after mock absorption (filled symbols) and IgM
absorption (open symbols) from immune sera from young (circles) or
old (triangles) adults immunized with PPV23 for three serotypes: se-
rotypes 14 (A), 18C (B), and 23F (C). For each serotype, samples from
young versus old adults were compared: 34 young and 29 old adults for
serotype 14, 33 young and 27 old adults for serotype 18C, and 47 young
and 40 old adults for serotype 23F. Straight lines represent immune
sera after mock absorption (solid lines) or IgM absorption (broken
FIG. 4. Comparisons of geometric means of the opsonic index
(GMI) between young (filled bars) and old (open bars) adults after
mock absorption (Mock-abs) and IgM absorption (Post-abs) for sero-
types 14, 18C, and 23F. ?, P ? 0.05 by the Student t test. Error bars
represent 2 standard deviations of the mean of the logarithm of the
FIG. 5. Comparisons of geometric means of the opsonic index
(GMI) between young (filled bars) and old (open bars) adults after
mock absorption (Mock-abs) and IgA absorption (Post-abs) for sero-
type 23F. ?, P ? 0.05 by the Student t test.
318 PARK AND NAHMINFECT. IMMUN.
coccal capsule types, and immune responses to some pneumo-
coccal PSs may differ.
The relative deficiency of anti-PPS IgM antibodies observed
among older adults may be a result of their deficient IgM
memory B cells. Shi et al. reported a significant reduction in
the number of IgM?CD27?B cells with aging (38). Even
though Moens et al. reported that IgM?CD27?B lympho-
cytes are also involved in IgG antibody production in response
to PPV23, IgM?CD27?B lymphocytes are generally accepted
to be IgM memory B cells and to produce anti-PPS IgM anti-
body in response to TI-2 antigens, including PPS (25). Also,
common variable immunodeficiency (CVID) patients with
an IgM?CD27?B cell deficiency are poorly responsive to
pneumococcal vaccines and thus suffer from frequent pneumo-
coccal infections (21). Therefore, future work should further
investigate the impact of aging on IgM?CD27?B cell popu-
lations and their role in pneumococcal vaccine responses.
The deficiency in anti-PPS IgM antibody responses may ex-
plain the reduced effectiveness of pneumococcal vaccines in
populations other than old adults. For instance, HIV?persons
and splenectomized patients have deficient IgM memory B
cells (13, 21). Interestingly, HIV?persons are known to have
normal levels of IgG antibodies against pneumococcal capsular
PS but to have reduced OPA titers (23). Similar observations
have been made for patients with certain forms of CVID (21).
Thus, the immunogenicity of pneumococcal vaccines for these
populations should be reassessed by investigating the impact of
anti-PPS IgM antibodies. Also, it is possible that a new pneu-
mococcal vaccine may elicit anti-PPS IgM antibodies better
than current vaccines.
Despite our observation, the deficiency of anti-PPS IgM
antibodies is not likely to be the only aging- or HIV-related
immune change. Patients with HIV infections have fewer B
cells expressing the VH3 subtype (7). Studies of old adults have
suggested an aging-associated reduction in isotype switching
and somatic hypermutation and a loss of antibody oligoclonal-
ity (20). Some studies with human antibodies suggested age-
related changes in the V regions of pneumococcal antibodies
(20, 41, 46, 47), but another study failed to find such changes
(19). In our study, an OPA showed no evidence that anti-PPS
IgG antibodies from older adults are less effective than those
from young adults. This finding may suggest that the V regions
of anti-PPS IgG antibodies may be similar in young and old
adults. However, our studies were not designed to investigate
V regions and thus should not be interpreted to mean that
there are no aging-associated V region differences.
In conclusion, we show that anti-PPS IgM antibodies can
provide significant immune protection despite their low levels
of expression. In addition to differences in fixing complement,
IgM and IgG antibody responses have different time courses;
consequently, their relative levels may differ during the course
of a vaccinee’s response to a vaccination. For instance, IgM
levels were several times higher than IgG levels and correlated
with opsonic capacity better than IgG levels among toddlers
who received only one dose of a 9-valent pneumococcal con-
jugate vaccine (B. Simell, B. A. Nurkka, K. Jousimies, S. Gron-
holm, N. Givon-Lavi, H. Kayhty, and R. Dagan, presented at
ISPPD-7, Tel Aviv, Israel, 2010). We propose that both anti-
PPS IgM and IgG levels should be used in the future to mon-
itor responses to pneumococcal vaccines as well as to vaccines
that are similar to pneumococcal vaccines (e.g., meningococcus
This work was supported by National Institutes of Health grant
R01-AI-69509 from the National Institute of Allergy and Infectious
Diseases to M.H.N.
The University of Alabama at Birmingham has applied for the
intellectual property rights for some of the methods and reagent used
in this research.
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