INFECTION AND IMMUNITY, Oct. 2005, p. 6752–6762
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 10
Comparative Opsonic and Protective Activities of
Staphylococcus aureus Conjugate Vaccines Containing Native or
Deacetylated Staphylococcal Poly-N-Acetyl-?-(1-6)-Glucosamine
Toma ´s Maira-Litra ´n,1* Andrea Kropec,1Donald A. Goldmann,2and Gerald B. Pier1
Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital Harvard Medical School,
Boston, Massachusetts 02115,1and Division of Infectious Diseases, Department of Medicine,
Children’s Hospital, Harvard Medical School, Boston, Massachusetts 021152
Received 9 February 2005/Returned for modification 30 March 2005/Accepted 18 May 2005
Staphylococcus aureus and Staphylococcus epidermidis both synthesize the surface polysaccharide poly-N-
acetyl-?-(1-6)-glucosamine (PNAG), which is produced in vitro with a high level (>90%) of the amino groups
substituted by acetate. Here, we examined the role of the acetate substituents of PNAG in generating opsonic
and protective antibodies. PNAG and a deacetylated form of the antigen (dPNAG; 15% acetylation) were
conjugated to the carrier protein diphtheria toxoid (DT) and used to immunize animals. Mice responded in a
dose-dependent fashion to both conjugate vaccines, with maximum antibody titers observed at the highest dose
and 4 weeks after the last of three weekly immunizations. PNAG-DT and dPNAG-DT vaccines were also very
immunogenic in rabbits. Antibodies raised to the conjugate vaccines in rabbits mediated the opsonic killing of
various staphylococcal strains, but the specificity of the opsonic killing was primarily to dPNAG, as this
antigen inhibited the killing of S. aureus strains by both PNAG- and dPNAG-specific antibodies. Passive
immunization of mice with anti-dPNAG-DT rabbit sera showed significant levels of clearance of S. aureus from
the blood (54 to 91%) compared to control mice immunized with normal rabbit sera, whereas PNAG-specific
antibodies were ineffective at clearing S. aureus. Passive immunization of mice with a goat antiserum raised to
the dPNAG-DT vaccine protected against a lethal dose of three different S. aureus strains. Overall, these data
show that immunization of animals with a conjugate vaccine of dPNAG elicit antibodies that mediated opsonic
killing and protected against S. aureus infection, including capsular polysaccharide types 5 and 8 and an
Staphylococcus aureus and coagulase-negative staphylococci
(CoNS), principally Staphylococcus epidermidis, are the most
frequent causes of hospital acquired bloodstream infections
accounting for 40 to 60% of all nosocomial bloodstream infec-
tions (52). S. aureus causes diverse infections such as endocar-
ditis, septic arthritis, osteomyelitis, meningitis, skin infections,
and abscesses (1, 2, 7, 16, 23, 27) and there appears to be an
increase in the recognition of community-acquired S. aureus
infections, often involving methicillin-resistant S. aureus strains
(16, 27). It is now well appreciated that the emergence of
antibiotic resistance among staphylococcal isolates has made
the treatment of these infections increasingly difficult, which
has amplified the call for new approaches to treat and prevent
staphylococcal infections, such as immunotherapy.
Ongoing efforts to design vaccines for S. aureus have tar-
geted various virulence factors of this organism, including cap-
sular polysaccharides (CP) (i.e., CP serotypes 5 and 8) (8, 9),
cell wall-associated proteins (i.e., clumping factor A, fibronec-
tin binding proteins, and collagen binding protein) (11, 36, 48),
toxins (i.e., alpha-toxin, enterotoxins, and toxic shock syn-
drome toxin 1) (6, 14, 15, 21, 33), and the surface-associated
polysaccharide, poly-N-acetyl-?-(1-6)-glucosamine (PNAG)
(25, 26, 29). PNAG is synthesized by enzymes encoded in the
intercellular adhesin (ica) locus (12), which occurs not only in
most clinical isolates of S. aureus but also in the majority of
clinical isolates of CoNS (28, 31, 34, 53), making PNAG an
attractive vaccine candidate. Interestingly, a genetic locus
termed pga has been identified in a number of gram-negative
bacteria (51), and for Escherichia coli, a polysaccharide chem-
ically identical to PNAG has been isolated and characterized
The basic chemical constituents of PNAG were initially de-
scribed by Mack et al. (24) and referred to as the polysaccha-
ride intercellular adhesin, but more recent studies showed dif-
ferences between PNAG isolated from S. aureus strain MN8m
(18, 25) and the polysaccharide intercellular adhesin prepara-
tion of Mack et al. (24). PNAG is a high-molecular-weight
(high-MW) (100 to 500), highly acetylated (95 to 100% N-
acetylation) polymer of ?-1-6-linked glucosamine residues that
plays a key role in biofilm formation for both S. aureus and
CoNS (28, 47), as well as being a key virulence factor for S.
epidermidis in animal models of infection (38–40). Previous
work has demonstrated the protective efficacy of PNAG in
rabbits against S. epidermidis catheter-related infections (20)
and endocarditis (45), where it was referred to as the capsular
polysaccharide/adhesin. Purified PNAG (mistakenly identified
as poly-N-succinyl glucosamine) also elicited protective efficacy
in mice against renal infections due to S. aureus, following both
active and passive immunization (29). Further studies showed
that only the highest-molecular-weight forms of PNAG were
immunogenic in laboratory animals and that rabbit antibodies
* Corresponding author. Mailing address: Channing Laboratory,
181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-2667. Fax:
(617) 525-2510. E-mail: firstname.lastname@example.org.
specific to this surface-associated antigen were able to promote
the opsonophagocytosis of various staphylococcal strains in
vitro (25). However, it took fairly high doses (100 ?g/animal)
of purified PNAG to elicit antibodies in mice, and rabbits
responded to this antigen only when immunized along with
strong adjuvants (25).
In an effort to improve the immunogenicity of PNAG, we
investigated means to conjugate the polysaccharide to protein
carriers and the role of the acetate substituents in generating
protective antibody. In this work, we report the synthesis of
two conjugate vaccines using the S. aureus surface-associated
polysaccharide PNAG, as well as a deacetylated derivative of
PNAG termed dPNAG, conjugated to the carrier protein diph-
theria toxoid (DT) (10). PNAG and dPNAG are chemically
related but differ mainly in their degree of N-acetylation, 95
to 100% for PNAG and 15% for dPNAG, which also lacks
O-linked succinate due to the deacetylation procedure. We
compared the immunological properties of PNAG-DT and
dPNAG-DT conjugate vaccines in mice and rabbits and the
opsonophagocytic activity in rabbit antisera against several
staphylococcal strains in vitro. We also evaluated the ability of
rabbit antibodies raised to either PNAG-DT or dPNAG-DT to
reduce levels of S. aureus injected into the blood of mice,
following passive administration of antibodies raised to both of
the conjugate vaccines. Finally, as these initial studies indi-
cated that dPNAG-DT was more effective than PNAG-DT at
inducing opsonic killing and reductions in bacterial levels in
the blood of bacteremic mice, confirmatory immune protection
studies were carried out using a goat antiserum raised to
dPNAG-DT in a model of lethal S. aureus infection in mice.
MATERIALS AND METHODS
Bacterial strains. The S. aureus MN8m strain that overproduces PNAG due to
a 5-bp deletion in the ica promoter (17) was used for the preparation of PNAG.
The following strains were used in the in vitro opsonophagocytic assay: S. aureus
MN8 (capsular type 8), Reynolds (capsular type 5), and 502 (capsular type 5),
and S. epidermidis M187. Two S. aureus strains deleted for the ica locus,
MN8?ica and Newman ?ica, were also used in the opsonophagocytic assay; their
construction has been described previously (17). The murine bacteremia studies
were performed with S. aureus COL (capsular type 5), strain Becker (capsular
type 8), and strain 10833 (a derivative of strain Newman that does not produce
CP5). The recently sequenced S. aureus strain 476 (13), which does not produce
CP5 or CP8, was used for the murine lethality studies.
Reagents. Isopropyl ?-D-thiogalactopyranose (IPTG) was obtained from In-
vitrogen Corp. (Carlsbad, CA). Sephacryl S-200 and Superose 6 prep-grade gels
used to purify DT and conjugate vaccines, respectively, were purchased from
Amersham Pharmacia Biotech (Piscataway, NJ). The endotoxin removing gel
Detoxi-Gel was obtained from Pierce (Rockford, IL) and cyanylating agent
1-cyano-4-dimethylaminopyridinium tetrafluroborate (CDAP) was purchased
from Sigma Chemical Co. (St Louis, MO). Purified sodium cyanoborohydride
(NaCNBH3) was obtained from Matreya, Inc. (Pleasant Gap, PA). Fetal bovine
serum (FBS) HyClone, (Logan, Utah) and complement (baby rabbit comple-
ment) Accurate Chemical and Scientific (Westbury, N.Y.) were used in the
Purification of PNAG and preparation of dPNAG. The method of purification
of PNAG from the S. aureus MN8m strain and the chemical characterization of
the PNAG antigen used in this study have been previously described (25). One
fraction of this material with an average MW of ?100, corresponding to the
previously designated PNAG-II fraction (25), was found to have a degree of
substitution of the amino groups with acetate of ?95%. This material was then
used to prepare dPNAG. Native PNAG was dissolved to a concentration of 2
mg/ml in 5 M NaOH and incubated at 37°C for 18 h. After this time, the sample
was neutralized with an equal volume of 5 M HCl, dialyzed overnight against
distilled water, and lyophilized. Analysis of the size of the deacetylated polymer
showed no significant change in its elution profile when chromatography was
carried out with a molecular sieve column. The degree of residual acetylation was
measured by1H-nuclear magnetic resonance as previously described (25), and
the resultant antigen was determined to have ?15% of the amino groups sub-
stituted with acetate.
Purification of DT. DT containing a six-His tag was expressed from the plas-
mid pET22b-DT-51E148K carried by E. coli BL21 and kindly provided by John
Collier, Boston, MA (10). E. coli was grown in Luria-Bertani (LB) broth in an
8-liter fermentor at 37°C until the optical density at 650 nm (OD650) reached 1.
The temperature was reduced to 28°C, IPTG was added to 1 mM, and growth
was continued for 3 h. Periplasmic proteins were extracted by first resuspension
of the pelleted cells in 0.4 culture volumes of 20% sucrose, 1 mM EDTA, and 30
mM Tris-HCl, pH 8.0. After 10 min at room temperature, the mixture was
centrifuged, and the pelleted cells were resuspended in the same volume of
ice-cold 5 mM MgSO4. After 10 min on ice, cells were pelleted by centrifugation,
and DT was purified from the supernatant fluid by affinity chromatography on an
Ni2?-chelate column. The protein was further purified by size exclusion chro-
matography on Sephacryl S-200. Endotoxin was removed using Detoxi-Gel
Coupling of PNAG to DT. Native PNAG (average MW, ?100) containing
?95% acetate substituents was covalently coupled to purified DT with the
organic cyanylating agent CDAP to activate the polysaccharide hydroxyl groups.
CDAP-activated PNAG was subsequently coupled to DT without the need for
additional spacer molecules.
(i) Activation of PNAG with CDAP. Purified PNAG (10 mg) was first dissolved
in 5 M HCl (150 ?l), neutralized with an equal volume of 5 M NaOH, and then
diluted to 1 ml with 0.1 M borate buffer, pH 9.2. CDAP was made up at 100
mg/ml in acetonitrile and stored at ?20°C (stable for up to 1 month). A total of
200 ?l of the CDAP stock solution was slowly pipetted into the PNAG solution,
and the reaction was allowed to proceed for 2 min.
(ii) Coupling of CDAP-activated PNAG with DT. DT (5 mg) was immediately
added to the CDAP-activated PNAG, and the mixture was reacted at room
temperature for 3 h with stirring. After this time, the high-MW conjugate was
separated from uncoupled components by gel filtration chromatography with a
Superose 6 prep-grade column. Fractions containing PNAG-DT conjugate were
identified on the basis of the earlier elution of both polysaccharide and protein
compared with the elution of the nonconjugated components, pooled, dialyzed
against 20 mM HEPES buffer–50 mM NaCl (pH 8), and stored frozen at ?20°C.
Coupling of dPNAG to DT. DT was covalently coupled to purified dPNAG by
reductive amination. Aldehyde groups were first introduced onto the surface of
DT by treatment of this protein with glutaraldehyde. Activated DT was subse-
quently reacted with dPNAG through its free amino groups in the presence of
the reducing agent sodium NaCNBH3.
(i) Activation of DT with glutaraldehyde. DT (10 mg) was dissolved in 0.1 M
carbonate buffer, pH 10, and glutaraldehyde was added to a final concentration
of 1.25% (vol/vol). This mixture was incubated at room temperature for 2 h, and
the glutaraldehyde-activated DT was dialyzed against phosphate-buffered saline
(PBS), pH 7.4, and concentrated to 10 mg/ml by ultrafiltration. Analysis of the
glutaraldehyde-treated DT after treatment showed no detectable self-polymer-
ization, following chromatography on a Superose 6 size-exclusion column.
(ii) Coupling of glutaraldehyde-activated DT to dPNAG. Purified dPNAG (10
mg) was dissolved in 5 M HCl (0.25 ml), neutralized with an equal volume of 5
M NaOH, and diluted to 2 ml with PBS. dPNAG (10 mg) was mixed with 1 ml
of a 10 mg/ml solution of activated DT in PBS, and the pH of the reaction was
adjusted to 7.5. Purified NaCNBH3(200 mg) was added to the mixture, and the
reaction was allowed to proceed in the dark for 14 h at 37°C with mixing. After
this time, the high-MW PNAG-DT conjugate was purified from uncoupled
reagents by gel filtration chromatography on a Superose 6 prep-grade column.
Fractions containing dPNAG-DT conjugate were identified as described above
for the PNAG-DT conjugate, pooled, dialyzed against 20 mM HEPES buffer–50
mM NaCl (pH 8), and stored frozen at ?20°C.
Chemical analysis of conjugate vaccines. Conjugate vaccines were analyzed
for their content of polysaccharide by the hexosamine assay described by Smith
and Gilkerson (44) with N-acetylglucosamine as the standard and for protein
with the Bradford assay (4) with DT as the standard.
Production of rabbit and goat antisera. Antibodies to purified PNAG-DT or
dPNAG-DT were raised in New Zealand white rabbits by subcutaneous immu-
nization with two 10-?g doses of conjugated polysaccharide emulsified for the
first dose in complete Freund’s adjuvant and for the second dose in incomplete
Freund’s adjuvant, followed 1 week later by three intravenous (i.v.) injections of
antigen in saline, each spaced 3 days apart. Rabbits were bled every 2 weeks, and
sera were tested by enzyme-linked immunosorbent assay (ELISA). In addition,
a goat was immunized with dPNAG-DT, following essentially the same protocol
VOL. 73, 2005 STAPHYLOCOCCAL PNAG AND dPNAG CONJUGATE VACCINES6753
as for the rabbits but increasing to 50 ?g (each) the doses of conjugated dPNAG
used per injection.
Immunization of mice. To study the immunogenicity of PNAG and dPNAG-DT
conjugates, groups of 10 mice (Swiss-Webster; female; 5 to 7 weeks of age) were
immunized subcutaneously at day 0 and boosted at weeks 1 and 2 with 0.15-,
0.75-, or 1.5-?g doses of conjugated PNAG or dPNAG in PBS. Control groups
received a mixture of unconjugated polysaccharide and protein in PBS in the
same ratio as in the conjugate vaccine. Blood was withdrawn weekly for a month,
and specific antibody titers were determined by ELISA.
ELISA. PNAG- and dPNAG-specific antibodies were measured in sera ob-
tained from immunized mice, rabbits, and a goat by ELISA as described previ-
ously (25). PNAG fraction II was used to sensitize the ELISA plate. A similar
protocol was used to detect the responses to DT, using plates sensitized with 5 ?g
DT/ml in phosphate buffer, pH 7.4.
Phagocyte-dependent killing assays. White blood cells (WBC) were prepared
from fresh human blood collected from healthy adult volunteers. Twenty-five
milliliters were mixed with an equal volume of dextran-heparin buffer and incu-
bated at 37°C for 1 h. The upper layer containing the leukocytes was collected,
the cells were pelleted by centrifugation, and hypotonic lysis of the remaining
erythrocytes was accomplished by resuspension of the cell pellet in 1% NH4Cl
and incubation for 10 min at room temperature. WBC were then washed and
resuspended with RPMI with 15% FBS (RPMI-FBS). With trypan blue staining
to differentiate dead from live leukocytes, the final WBC count was adjusted to
5 ? 106WBC per ml. The complement source (1 ml of baby rabbit serum diluted
1:15 in RPMI-FBS) was adsorbed at 4°C for 30 min with continual mixing with
bacteria resuspended from a pellet containing ?109CFU of the target S. aureus
or S. epidermidis strain. After adsorption, the complement solution was centri-
fuged and filter sterilized. The test sera were absorbed at 4°C for 30 min with
bacteria resuspended from a pellet containing ?109CFU of the PNAG-negative
S. aureus MN8 ?ica strain (17) to remove antibodies not directed to the PNAG
antigen. The bacteria were removed by centrifugation, and the test sera were
filter sterilized. The bacterial strains to be evaluated for phagocyte-dependent
killing activity of antibody in immune sera were grown overnight in tryptic soy
broth (TSB) with 1% glucose, adjusted to an OD650of 0.4 (?2 ? 108CFU/ml),
and a 1:100 dilution in RPMI-FBS was made for use in the killing assay.
The actual phagocytic killing assay was performed by mixing 100 ?l (each) of
the WBC suspension, target bacteria, dilutions of test sera, and the complement
source. The reaction mixture was incubated on a rotor rack at 37°C for 90 min;
samples were taken at time zero and after 90 min. A 10-fold dilution was made
in TSB with 0.5% Tween to inhibit bacterial aggregation, and samples were
plated onto tryptic soy agar plates. Tubes lacking any serum and tubes with
normal rabbit serum (NRS) were used as controls, as were tubes containing
serum and complement but lacking WBC to control for potential aggregation of
bacteria by the antibody, which would reduce the apparent CFU counts at the
end of the assay. At the concentrations of antisera used in the opsonic killing
assay, there was no reduction in CFU of ?10% in samples lacking phagocytes but
containing antibody and complement, indicating little agglutinating activity of
the antibody to PNAG or dPNAG antigens. The percentage of killing was
calculated by determining the ratio of the number of CFU surviving in the tubes
with bacteria, leukocytes, complement, and sera to the number of CFU surviving
in tubes lacking sera but containing bacteria, complement, and leukocytes. Kill-
ing rates of ?30% were considered biologically significant, as this level repre-
sents the upper limit of the percentage reduction in CFU that occurs in the
opsonic killing assay with normal sera lacking antibody to S. aureus, due to
normal variation in CFU counts in this assay.
For inhibition studies, rabbit antiserum was diluted 1:10 and incubated for 90
min at 4°C with an equal volume of a solution containing 0.8 to 100 ?g/ml of
either native PNAG or deacetylated PNAG. Subsequently, the antiserum was
centrifuged, and the supernatant was used in the opsonophagocytic assay as
described above. Inhibition of ?40% was considered biologically significant, as
this represented twice the upper limit of nonspecific inhibition seen in control
tubes containing irrelevant polysaccharide antigens as inhibitors.
Murine bacteremia model. Groups of eight mice (Swiss-Webster; female; 5 to
7 weeks of age) were immunized intraperitoneally (i.p.) with 0.4 ml of NRS or
immune sera raised to PNAG-DT or dPNAG-DT. In preliminary studies, we
determined that serum titers peaked 48 h after the i.p. injection (data not
shown). Thus, after 48 h, each group was challenged i.v. with a dose of 3.1 ? 105
to 2.5 ? 107CFU of S. aureus (depending on the strain) in 0.2 ml of PBS. The
strains used had not been recovered from previously infected mice to promote
animal adaptation. Mice were sacrificed 2 h after bacterial challenge, blood
samples were withdrawn, and the number of bacteria were expressed as the
number of CFU per milliliter of blood. Comparisons between immune and
control groups were by unpaired t tests.
Murine intraperitoneal infection and dissemination. Groups of eight mice
(Swiss-Webster; female; 5 to 7 weeks of age) were immunized i.p. 24 h before
and 24 h after infection with 0.2 ml of normal goat serum (NGS) or immune goat
serum raised to dPNAG-DT. Preliminary studies indicated this regimen of an-
tibody injection maintained the highest levels of local antibody in the perito-
neum. S. aureus strains were grown overnight on tryptic soy agar plates and
resuspended in PBS to an OD650of 1.2, corresponding to approximately 4 ? 1010
to 8 ? 1010CFU/ml, depending on the strain. Mice were challenged i.p. with a
dose of 2 ? 109to 5 ? 109CFU in 0.2 ml of PBS and monitored twice daily.
Moribund mice were sacrificed as per criteria described in an approved animal
studies protocol and counted as dead along with any animal deaths that occurred
between monitoring periods. Survival was compared by chi-square analysis with
the continuity correction. Animal maintenance and experimental protocols were
approved by the Harvard Medical Area Institutional Animal Care and Use
Preparation of PNAG and dPNAG-DT conjugate vaccines.
PNAG and dPNAG were coupled to DT in two steps using two
different conjugation chemistries that were needed due to the
individual chemical properties of each antigen. As most of the
amino groups on PNAG were acetylated and not available for
conjugation, a small proportion (?5%) of the free hydroxyl
groups on the PNAG molecule was instead used for coupling
to the protein carrier. Limited activation of the hydroxyl
groups on PNAG was achieved with the organic cyanylating
reagent CDAP, after which the activated polysaccharide was
immediately conjugated to free amino groups on the carrier
Use of CDAP with the dPNAG molecule was contraindi-
cated, as the large number of free amino groups on this mol-
ecule would react with activated hydroxyl groups, resulting in
self-polymerization of the dPNAG. Therefore, we chose to use
the free amino groups on the dPNAG molecule to conjugate
the polysaccharide to DT by the classic reaction of reductive
amination. DT was first derivatized with glutaraldehyde, and
then the activated DT was allowed to react overnight with
dPNAG in the presence of the reducing agent NaCNBH3. This
coupling method uses mild pH and temperature, conditions
required to preserve the structure of both protein and poly-
The polysaccharide (OD650) and protein (OD595) profiles of
the conjugates chromatographed over a Superose 6 column
readily allowed for separation of the conjugated PNAG-DT
(Fig. 1A) and dPNAG-DT (Fig. 1B) from nonconjugated com-
ponents. As can be seen from the elution profiles, the conju-
gated protein and polysaccharide start to coelute in the void
volume of the column (void volume ? 40 ml). This is indicative
of the formation of high-MW conjugates. Fractions were
pooled, which contained material reactive in both the protein
and hexosamine assays and which eluted from the column
earlier than any fraction predetermined to contain the free
polysaccharide or DT protein. To be sure that the high-MW
fractions contained conjugated protein, we performed sodium
dodecyl sulfate-polyacrylamide gel electrophoresis analysis on
individual fractions from the Superose 6 column. Fractions
containing polysaccharide-conjugated protein did not enter the
polyacrylamide gel, due to their high MW (fractions, 40 to 80
ml); neither free polysaccharide nor free DT was detected in
the gel after silver staining. On the other hand, fractions con-
taining unconjugated DT were easily identified and excluded
6754MAIRA-LITRA ´N ET AL.INFECT. IMMUN.
from the pools of conjugate vaccines. The ratio of polysaccha-
ride to protein was about 4:1 for the PNAG-DT conjugate
(80% polysaccharide, 20% protein) and almost 1:1 in the case
of the dPNAG-DT conjugate (44% polysaccharide, 56% pro-
Immunogenicity and IgG isotype distribution in sera of mice
immunized with PNAG-DT and dPNAG-DT. Mice vaccinated
with the PNAG-DT conjugate developed high antibody titers
to the immunizing polysaccharide as determined by ELISA
(Fig. 2A). Mice responded in a dose-dependent fashion with
immunoglobulin G (IgG) titers that increased during the
weeks postimmunization. Maximum IgG titers (ca. 5,500) were
seen in mice vaccinated with 1.5 ?g of conjugated PNAG and
achieved 4 weeks after the last immunization. Serum samples
beyond 4 weeks postimmunization were not obtained. A sim-
ilar dose-dependent response was observed in mice immunized
with the dPNAG-DT vaccine. The highest anti-dPNAG titers
(ca. 10,000) were again found in the final serum sample ob-
tained 4 weeks after immunization with 1.5 ?g of dPNAG-DT
vaccine (Fig. 2B). Control mice, which were immunized with
the same doses of a mixture of unconjugated polysaccharide
and protein, did not develop any detectable titers to the poly-
saccharides (not shown). Interestingly, the titers to DT in the
mice were rather low, ranging from 0 in animals given the
protein and polysaccharide as a mixture to a maximum of 1,741
in mice immunized with 1.5 ?g of the dPNAG-DT conjugate.
This finding is consistent with previously described results
showing low immunogenicity of carrier proteins such as DT
when used in polysaccharide-protein conjugate vaccines (41).
Overall, these results demonstrate that the conjugation of
PNAG and dPNAG to the carrier protein DT significantly
increased the immunogenicity of these polysaccharide antigens
and induced modest amounts of antibody to DT.
In mice, IgG3 is the predominant isotype made to unconju-
gated polysaccharide antigens, whereas IgG1 is often the pre-
dominant isotype responding to polysaccharide conjugated to
protein (3). We investigated the IgG isotype distribution of
mice vaccinated with either native or deacetylated conjugate,
by determining titers of the antisera with ELISA plates coated
with an excess of antigen relative to the amount of binding
antibody in the most concentrated sample to ensure that no
isotype blocked binding of another. We found that IgG1 was
the predominant isotype elicited by both vaccines, followed by
a lower titer of IgG3 and still lower and comparable levels of
IgG2a and IgG2b (Fig. 3).
Immunogenicity of conjugate vaccines and antibody speci-
ficity in rabbits. To investigate further the immunological
properties of native and deacetylated conjugate vaccines, two
rabbits were immunized with each conjugate, and the antibody
titers were determined by ELISA. Both conjugates were very
immunogenic in rabbits, which responded with high IgG titers
to the immunizing polysaccharide antigens (Table 1). As with
the mice, titers to DT were quite low, ranging between 452 and
831 in the four immune rabbit sera (data not shown). Since
PNAG and dPNAG differed mainly in the degree of N-acetyl-
FIG. 1. Gel filtration elution profiles of PNAG-DT (A) and dPNAG-
DT conjugate vaccines (B) on a Superose 6 prep-grade column. The
presence of polysaccharide (open circles) in fractions was monitored
by the hexosamine assay (OD650) and the presence of protein (closed
circles) in the fractions was monitored by the Bradford assay (OD595).
Double-headed arrows indicate the fractions containing clearly conju-
gated protein and polysaccharide, as well as where the unconjugated
polysaccharide (PNAG or dPNAG) and protein (DT) eluted.
FIG. 2. Mean titers of IgG antibodies in sera pooled from 10 mice
immunized three times at weekly intervals with the indicated dose of
PNAG-DT (A) or dPNAG (B) conjugate vaccines. Sera were collected
weekly for 4 weeks starting 1 week after the last dose of conjugate
vaccine and were tested by ELISA with the homologous immunizing
antigen, either PNAG (A) or dPNAG (B) as a coating antigen. Bars
represent means and error bars indicate standard deviations. All pre-
immune titers were ?25.
VOL. 73, 2005STAPHYLOCOCCAL PNAG AND dPNAG CONJUGATE VACCINES 6755
ation (?95% in PNAG versus 15% in dPNAG), we deter-
mined the epitope specificity of the rabbit antibodies raised to
each conjugate by measuring ELISA titers in the different sera
to both the native and deacetylated antigens (Table 1). Rabbits
immunized with the highly acetylated PNAG conjugate vaccine
had significantly higher antibody titers to PNAG than to dPNAG.
On the other hand, rabbits vaccinated with poorly acetylated
dPNAG-DT had comparable antibody titers to PNAG and
dPNAG (Table 1). These results indicate that antibodies to
PNAG-DT mainly react with acetyl-containing epitopes,
whereas antibodies in the dPNAG-DT vaccinated rabbits are
primarily directed to epitopes not dependent on the presence
of acetate but expressed comparably on both highly and poorly
Phagocyte-dependent killing activity of vaccine-induced an-
tibodies. We used a phagocytosis assay to compare the phago-
cyte-dependent killing ability of rabbit antibodies induced by
the PNAG-DT conjugate, which were primarily reactive with
the highly acetylated molecule, with antibodies elicited by the
dPNAG-DT conjugate that primarily recognized nonacetyl-
ated or backbone epitopes. As the opsonic killing activity of the
two rabbits immunized with PNAG-DT were virtually identical
to each other, as was the opsonic killing activity of the two
rabbits immunized with dPNAG-DT, results from the individ-
ual sera from the two rabbits immunized with the same vaccine
were averaged. Additionally, since we grew the bacteria in TSB
with 1% glucose to promote in vitro PNAG expression, it is
important to emphasize that accurate determinations of
phagocyte-dependent killing of S. aureus and S. epidermidis
required the incorporation of 0.5% Tween into TSB that was
used for the dilutions to be plated for bacterial enumeration.
This prevented the otherwise highly adherent S. aureus and S.
epidermidis strains from rapidly adhering (within seconds) to
the plastic or glass dilution vessels and pipettes, which would
have distorted the measurement of the actual CFU surviving in
the assay. As can be seen in Fig. 4A to C, S. epidermidis strain
M187, S. aureus strain 502, and S. aureus strain MN8 were
killed more efficiently when dPNAG-specific antibodies were
used than with the antibodies specific to the native PNAG. On
the other hand, in the case of the S. aureus strain Reynolds, we
found low but comparable levels of killing by antibodies raised
by both the PNAG-DT and dPNAG-DT vaccines (Fig. 4D). As
we know from other assays that strain Reynolds makes very
low levels of PNAG in vitro (unpublished observations), this
may account for the overall low level of killing and lack of
difference in efficacy between the two antibody preparations.
In no case did preimmune rabbit antisera at a dilution of 1:10
elicit killing rates of ?10% of any of the test strains (not
shown), nor was there any level of killing of ?10% in control
tubes which contained bacteria but lacked one or more of the
other three components of the opsonic killing assay (not
To show the specificity of the opsonic killing antibody for the
PNAG antigen, we evaluated antibody-dependent killing using
two strains of S. aureus deleted for the ica locus (17), strains
MN8?ica and Newman ?ica. Of note, the loss of ica in S.
aureus leads to increased killing by complement and white cells
alone, as has been previously shown for S. epidermidis (42, 43,
50) and confirmed by us for S. aureus (A. Kropec, T. Maira-
Litra ´n, K. Jefferson, M. Grout, S. E. Cramton, F. Goetz, D. A.
Goldmann, and G. B. Pier, submitted for publication). Thus,
for use with strains MN8?ica and Newman ?ica, the level of
the complement source had to be reduced to 5% final concen-
tration; we could not use strain 10833?ica, as this strain was
killed at a rate of ?90% at complement levels that were ?3%
of final concentration, which is too low for antibody-mediated
opsonic killing (Kropec et al., submitted). Notably, strains
MN8?ica and Newman ?ica express either the CP8 or CP5
capsule, whereas 10833?ica does not, which may account for
its high susceptibility to antibody-independent killing. None-
theless, even at a level of 5% complement, wild-type S. aureus
strains were clearly killed by antibody in sera raised to dPNAG
and PNAG while there was only minimal killing in NRS,
whereas the ica-deleted strains were not killed any better by
antisera raised to either conjugate vaccine when compared to
background killing by NRS (Fig. 5). Thus, in the absence of an
ica locus, the antibodies to native or deacetylated PNAG are
ineffective at mediating opsonophagocytic killing.
Inhibitory capacity of PNAG and dPNAG in the opsonopha-
gocytic assay. Since there were low but nonetheless measur-
FIG. 3. IgG subclass distributions in mouse sera obtained 4 weeks
after the third booster immunizations with PNAG-DT and dPNAG-
DT conjugate vaccines.
TABLE 1. Antibody titers determined by ELISA to purified
PNAG and dPNAG antigens in sera of two animals immunized
with PNAG-DT or two different animals immunized
with dPNAG-DT conjugate vaccines
Animal Immunogen Coating antigenTiter
Rabbit 1 PNAG-DT PNAG
6756MAIRA-LITRA ´N ET AL.INFECT. IMMUN.
able amounts of antibodies to dPNAG epitopes present in the
antisera raised to PNAG-DT, we used an inhibition assay to
determine if phagocytic killing in the PNAG-DT antisera in-
volved the antibodies that were specific to the dPNAG
epitopes. Figure 6 shows the comparative ability of PNAG and
dPNAG (concentrations of 0.8 to 100 ?g/ml in the assay) to
inhibit the opsonic killing of four staphylococcal strains by
serum raised to PNAG-DT. Our results indicated that for all
strains and at every concentration tested, dPNAG was either as
effective as PNAG or more effective than PNAG at inhibiting
killing mediated by antibodies raised to PNAG. Thus, inhibit-
ing binding of antibodies with specificity for the nonacetylated
epitopes on PNAG with dPNAG eliminated almost all phago-
cytic killing in the antisera raised to PNAG-DT. dPNAG and
PNAG antigens both showed the same pattern in inhibiting the
phagocyte-dependent killing of S. aureus mediated by antibody
to dPNAG (Fig. 7), further validating that the dPNAG-specific
epitopes were available on the acetylated PNAG antigen for
binding to antibody raised to dPNAG.
Protective efficacy of rabbit PNAG-DT and dPNAG-DT an-
tibodies against bacteremia due to S. aureus in mice. We next
compared the protective activity of antibodies specific to
PNAG and dPNAG in vivo using a mouse bacteremia model.
Groups of mice were passively immunized i.p. with either NRS
or immune sera raised to PNAG or dPNAG conjugates. Two
days later, when maximum antibody levels were achieved in
blood (data not shown), animals were infected i.v. with S.
aureus. Two hours after infection, mice were sacrificed and the
levels of bacteria were measured by serial dilution and plating
FIG. 4. Opsonophagocytic killing of various Staphylococcal strains indicated in each panel by rabbit sera raised to PNAG-DT conjugate vaccine
(open symbols) or dPNAG-DT conjugate vaccines (solid symbols). Points represent mean of quadruplicate determinations, two for each of the two
rabbits immunized with each vaccine, and error bars indicate standard deviations. Opsonic killing of ?30% is considered biologically significant
and is statistically significant at P ? 0.01; analysis of variance (ANOVA) and Fisher probable least squares difference (PLSD) posthoc, pairwise analysis.
FIG. 5. Opsonic killing of native and ?ica S. aureus strains MN8
and Newman by 1:10 dilutions of either preimmune serum or anti-
serum raised to native PNAG-DT or dPNAG DT in the presence of
5% final concentration of complement. Bars represent mean percent-
ages of killing and error bars indicate the standard deviation. Killing of
wild-type strains by antisera to either native PNAG or dPNAG was
significant at P values of ?0.05, compared to killing by NRS.
VOL. 73, 2005STAPHYLOCOCCAL PNAG AND dPNAG CONJUGATE VACCINES6757
of blood samples; results were expressed as the geometric
mean number of bacteria per milliliter of blood.
As shown in Table 2, mice passively immunized with rabbit
sera raised to dPNAG-DT were able to clear 54% to 91% of
the CFU of four different S. aureus strains compared to the
control groups given NRS. We then evaluated the clearance
activity of antibody raised to the PNAG-DT conjugate vaccine
against S. aureus strains MN8 and 10833, for which we
achieved the greatest reductions in CFU/ml blood with anti-
body to dPNAG-DT. Our results showed that in comparison to
CFU levels in the blood of mice given NRS, the number of
CFU per milliliter of blood in mice given antibody to PNAG
was slightly, but not significantly, increased (120% for 10883
[P ? 0.1, t test]; 106% for strain MN8 [P ? 0.1, t test]). We
could not test the strains deleted for the ica locus in this or any
animal model to show specificity of the antibodies, because loss
of ica greatly compromises bacterial virulence and there are
virtually no PNAG-deficient bacteria in the blood or tissues of
nonimmune mice by 2 h post-i.v. injection (Kropec et al.,
submitted). Overall, the results indicated that antibody to
epitopes encompassing acetate residues that are present on the
native PNAG was ineffective at promoting clearance of S.
aureus from the blood of mice. In addition, the inability of
antibody raised to PNAG-DT to reduce bloodstream bacterial
levels indicated no protective effect of antibodies present in
these sera, due to use of complete Freund’s adjuvant in the
Protective efficacy of goat antibodies raised to dPNAG-DT
in a murine model of lethal S. aureus infection. Since dPNAG-
DT appeared to elicit killing and protective antibodies whereas
antibodies elicited by native PNAG were not protective, we
used this conjugate vaccine to immunize a goat to obtain large
amounts of dPNAG-specific antibodies. Three bleeds were
obtained from the goat at 2, 6, and 10 weeks after the final
immunization, and the antisera were tested for opsonic killing
activity. We found the three serum samples mediated an aver-
age killing rate of 48%, 60%, and 50% of S. aureus MN8,
whereas preimmune NGS had no phagocyte-dependent killing
activity. We next tested the protective efficacy of goat antibody
to dPNAG against a lethal i.p. challenge of mice with several S.
aureus strains. Two of the strains used in the above-described
bacteremia model, COL and Becker, displayed insufficient vir-
ulence in the mice after injection by the i.p. route to yield a
lethal outcome (?25% lethality at a dose of ?5 ? 109CFU/
mouse), so we instead used strain M476, a recently sequenced
S. aureus isolate that was found to have a lethal effect in 100%
of mice following injection of 5 ? 109CFU. Mice were pas-
sively immunized i.p. with NGS or anti-dPNAG goat sera 24 h
before and 24 h after a high dose of S. aureus was administered
i.p.; morbidity and mortality were recorded for 5 days. As can
be seen in Table 3, dPNAG-vaccinated mice were highly pro-
tected (62.5 to 100% survival) against a lethal dose of three S.
aureus strains compared with control groups given NGS (0 to
18.7% survival [P ? 0.01, chi-square]). To ensure that antibod-
FIG. 6. Inhibitory capacity of PNAG and dPNAG in the opsonophagocytic assay. Inhibition of opsonic killing of the staphylococcal strain
indicated in each panel by rabbit serum raised to PNAG-DT conjugate mixed with purified PNAG (solid bars) or dPNAG (open bars) antigens
at the indicated concentration. Bars represent means of quadruplicate determinations and error bars indicate the standard deviations. Inhibition
of ?40% is considered biologically significant and is statistically significant at P values of ?0.01; ANOVA and Fisher PLSD posthoc, pairwise
6758 MAIRA-LITRA ´N ET AL.INFECT. IMMUN.
ies present in the goat immune serum that were potentially
cross-reactive with S. aureus antigens due to the use of com-
plete Freund’s adjuvant during immunization did not account
for the protection seen, we tested the protective efficacy
against challenge with S. aureus MN8 of a goat antiserum
raised to an irrelevant peptide antigen. This serum was pro-
duced using the same immunization schedule with the same
adjuvants as was used for the dPNAG-DT conjugate. All
(100%) of the animals in groups given either NGS or a com-
parable dose of the irrelevant immune sera became moribund
or died within 72 h of infection, indicating no contribution to
protection from antibody to adjuvant components.
Bacteria use numerous strategies to avoid innate and ac-
quired host defenses and maintain their capacity to cause se-
rious infections. One well-known strategy is illuminated by the
poor immune response of human infants and young children to
polysaccharide antigens, which are major protective antigens
for many bacterial pathogens. However, by conjugating poly-
saccharides to protein carriers this immunologic barrier can be
broken, and effective conjugate vaccines to Haemophilus influ-
enzae, Streptococcus pneumoniae, and Neisseria meningitidis
have been developed (3, 19, 54). Another strategy pathogens
use to avoid host immune effectors is to elicit high levels of
poorly protective antibodies, which can have this property
based on low antibody affinity, production of an inappropriate
FIG. 7. Inhibitory capacity of PNAG and dPNAG in the opsonophagocytic assay against antisera raised to dPNAG-DT. Inhibition of opsonic
killing of the staphylococcal strain indicated in each panel by rabbit serum raised to dPNAG-DT conjugate mixed with purified PNAG (solid bars)
or dPNAG (open bars) antigens at the indicated concentration. Bars represent means of quadruplicate determinations, and error bars indicate the
standard deviations. Inhibition of ?40% is considered biologically significant and is statistically significant at P values of ?0.01; ANOVA and
Fisher PLSD posthoc, pairwise analysis.
TABLE 2. Protection elicited by rabbit dPNAG-DT sera against
various S. aureus strains in the murine bacteremia model
in comparison to NRS
Mean CFU/ml blood ? SEMa
2.5 ? 106
1.8 ? 106
3.1 ? 105
2.5 ? 107
1,925 ? 588
184 ? 3.5
229 ? 2.1
9,821 ? 3,545
446 ? 52
82 ? 2.5
105 ? 2
914 ? 167
aP value determined by unpaired t test. SEM, standard error of the mean.
TABLE 3. Protection against lethality caused by various S. aureus
strains by a goat antiserum raised to the dPNAG-DT conjugate
vaccine in comparison to NGS
No. of survivors/total (%)
NGS dPNAG-DT serum
2 ? 109
5 ? 109
5 ? 109
aP value determined by chi-square analysis with continuity correction.
VOL. 73, 2005 STAPHYLOCOCCAL PNAG AND dPNAG CONJUGATE VACCINES6759
antibody isotype, or specificity for nonprotective epitopes (32,
37, 46). In the case of the PNAG antigen, it appears that both
poor overall immunogenicity of the native polysaccharide and
a preferential induction of poorly opsonic, poorly protective
antibody induced by highly acetylated, native PNAG could
contribute to ineffective immune responses to this antigen. By
both deacetylating PNAG and conjugating this polysaccharide
to DT we were able to elicit phagocyte-dependent killing and
For three of four staphylococcal strains, the antisera raised
to dPNAG-DT mediated higher levels of phagocyte-dependent
killing than antisera raised to PNAG-DT. However, the killing
activity in the antisera to PNAG-DT for all four staphylococcal
strains evaluated was completely inhibited by purified dPNAG
antigen, indicating that antibodies with specificity to the
deacetylated form of the antigen are key for mediating killing,
even when the native form of PNAG is used to elicit the
antibodies. Since the titers to dPNAG in the rabbit antiserum
raised to native PNAG were quite low, it may be that the
killing achieved with antisera to PNAG-DT was a result of a
synergistic interaction between the antibodies specific to the
native and deacetylated epitopes and that inhibiting the
dPNAG-specific antibodies was sufficient to abrogate the syn-
ergistic effect leading to phagocyte-dependent killing activity in
this serum. The remaining antibody specific to the native
PNAG could not mediate phagocyte-dependent killing on its
own. Overall, it is clear that the dPNAG antigen contained
epitopes against which phagocyte-dependent opsonic killing
antibody was directed.
The protection assays also supported the conclusion that
antibodies reactive with the nonacetylated, backbone portion
of the PNAG antigen were able to mediate clearance of bac-
teria from the blood and protection against a high-dose lethal
infection. Evaluation of the contribution of both O- and N-
linked acetyl groups to opsonic and protective antibody di-
rected to other bacterial polysaccharide antigens has led to the
conclusion that the role of acetylation in inducing protective
antibodies is antigen specific. For example, Fattom et al. (9)
found that antibodies to native and de-O-acetylated CP5 were
both effective at mediating opsonic killing of a variety of S.
aureus strains, and that native, O-acetylated CP5 conjugated to
Pseudomonas aeruginosa exotoxin A elicited populations of
antibodies directed to both highly and poorly O-acetylated
CP5. In contrast, and more consistent with our findings using
PNAG, Michon et al. (30) showed that a de-O-acetylated ver-
sion of N. meningitides group C capsular polysaccharide was
immunogenic in humans and superior to a highly O-acetylated
group C capsular polysaccharide at inducing bactericidal anti-
body. In the case of P. aeruginosa alginate (also called mucoid
exopolysaccharide) opsonic, protective antibodies are directed
to the O-acetylated residues on the mannuronic acid compo-
nents of this polysaccharide (35), and nonopsonic, nonprotec-
tive antibodies are directed to non-O-acetylated epitopes.
Thus, there are examples with different bacterial capsular poly-
saccharides indicating that the acetate substituents either are
of no consequence, interfere with eliciting protective antibody,
or are required for generating protective antibody.
A key problem associated with preparation of glycoconju-
gate vaccines is choosing an appropriate, simple, and clinically
acceptable method of conjugation chemistry that will introduce
neither toxic components or neoepitopes likely to cross-react
with host antigens. Native PNAG is a large, almost fully N-
acetylated glucosamine polymer in which the only functional
groups available for conjugation are the hydroxyl groups. Cy-
anlylation with CNBr is widely used as a method of activating
hydroxyl groups. However, activation can be much more effec-
tive if performed with other cyanylating reagents such as
CDAP. In comparison to CNBr, CDAP is easier to use, can be
employed at a milder pH, and has fewer side reactions (5, 22).
We used CDAP to activate PNAG before it was reacted with
the carrier protein DT. Both reactions were carried out at
room temperature in 0.1 M borate buffer, pH 9.2, for a total
time of 3 h. These conjugation conditions were mild enough to
preserve the N-acetyl groups on PNAG, which require much
stronger conditions (high alkalinity, high temperature, and
longer periods of incubation) to be removed. On the other
hand, dPNAG was covalently linked to DT by using the classic
reductive amination reaction, as cyanylation was contraindi-
cated due to the potential of self-polymerization between ac-
tivated hydroxyls and free amino groups. To conjugate the
dPNAG molecule to the DT carrier, aldehyde groups were first
introduced onto the surface of DT by incubation of the protein
with glutaraldehyde, which was added in excess to the protein
solution to prevent the self-polymerization of DT. Glutaralde-
hyde-activated DT was subsequently reacted with dPNAG in
the presence of NaCNBH3for 14 h in PBS at pH 7.5. Although
the reductive amination reaction has been reported to be more
efficient at higher pHs, we carried out this reaction at several
pHs (10, 9, 8, and 7.5) and found pH 7.5 to be optimal (data
not shown). Additionally, increasing the incubation time from
14 h to 3 days or 6 days did not improve the yield of this
reaction (data not shown). Overall, we have defined clearly
acceptable and useful means to conjugate both native PNAG
and dPNAG to carrier proteins for immunologic studies.
Although we had somewhat different ratios of polysaccha-
ride to protein in the two conjugates both were nonetheless
capable of eliciting high titers of antibodies to the polysaccha-
ride contained in the conjugate. Indeed, the rabbit antisera
raised to native PNAG-DT had higher titers to the immuniz-
ing antigen than to dPNAG in animals immunized with the
dPNAG-DT conjugate vaccine. As the primary goal of this
study was to compare the killing and protective antibodies with
specificities to different epitopes on PNAG, the conjugates we
produced clearly achieved this purpose and showed that anti-
body to nonacetylated epitopes are effective at mediating op-
sonic killing and protection. We cannot exclude the possibility
that a different ratio of native PNAG to protein might have
induced a different population of antibodies that could have
killing and protective properties comparable to those elicited
by the dPNAG-DT vaccine. Also, we did not explore whether
a PNAG molecule with an intermediate amount of acetylation
between native PNAG and dPNAG might have highly desir-
able immunogenic properties. Of note, Vuong et al. (49) re-
cently identified the product of the icaB gene in the ica locus
of S. epidermidis as an N-deacetylase, removing the N-linked
acetates from fully acetylated PNAG. In the absence of IcaB-
dependent partial deacetylation of PNAG (referred to as PIA)
there was no PNAG retained on the S. epidermidis cell surface,
and all of the highly acetylated antigen was found in culture
supernates. As a result, the S. epidermidis strain studied be-
6760MAIRA-LITRA ´N ET AL.INFECT. IMMUN.
came susceptible to antibody-independent killing by phago-
cytes and complement and had significantly reduced virulence
in a foreign-body infection model (50). We have found that
icaB of S. aureus provides an identical function in this species
(N. Cerca, K. Jefferson, and G. B. Pier, unpublished obser-
vations), suggesting that one reason that antibodies to the
dPNAG epitopes are effective mediators of protection is that
they bind preferentially to the low-acetate PNAG molecules
most closely associated with the cell surface. Overall, while
additional studies may be indicated to determine the optimal
ratio of either PNAG or dPNAG to carrier protein for induc-
tion of protective antibody in humans, our study indicates that
poorly acetylated forms of the vaccine are clearly capable of
inducing the desired antibody.
In summary, conjugation of PNAG and dPNAG to DT sig-
nificantly increased the immunogenicity of both of these anti-
gens. Mice injected with either PNAG-DT or dPNAG-DT
conjugate vaccines responded with high titers of antibodies to
the immunizing polysaccharides in a dose-dependent fashion,
as opposed to control groups given mixtures of these compo-
nents, wherein antibody titers to the polysaccharide antigens
did not develop. Critically important, we showed that de-N-
acetylation of PNAG to a level of ?15% was needed to induce
the most effective killing and protective antibodies. As highly
acetylated PNAG is likely the form of this antigen naturally
expressed by most isolates of S. aureus and CoNS, it appears
that the immunodominance of the nonprotective epitopes on
this molecule may contribute to the avoidance of host immune
effectors by PNAG-expressing bacteria. Furthermore, as re-
cent evidence suggests that not only staphylococci, but a variety
of gram-negative pathogens including E. coli, Yersinia pestis,
Bordetella pertussis, and others have a genetic locus related to
the ica locus and may be capable of producing PNAG, which
has been definitively shown for E. coli (51), the immunochemi-
cal properties of this antigen resulting in the immunodomi-
nance of nonprotective antibodies may be beneficial to the
virulence of a variety of pathogens. It will be of great interest
to pursue further the role of antibodies to different epitopes on
the PNAG molecule in mediating immunity to the multitude of
bacterial pathogens that appear to be able to synthesize this
This work was supported by National Institutes of Health grant R01
We thank Martha Grout for conducting opsonic killing assays, Sarah
Cramton for provision of the S. aureus MN8?ica strain used to adsorb
the antisera in the opsonic assays, Robin Ross for first isolating the S.
aureus MN8m PNAG-overproducing strain, John Collier for provision
of the recombinant plasmid expressing the DT carrier protein, and
Arthur Tzianabos for providing the goat antiserum raised to a control
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