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 infections (52
). S. aureus
causes diverse infections such as endocarditis, septic arthritis, osteomyelitis, meningitis, skin infections, and abscesses (1
) and there appears to be an increase in the recognition of community-acquired S. aureus
infections, often involving methicillin-resistant S. aureus
). 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 targeted various virulence factors of this organism, including capsular polysaccharides (CP) (i.e., CP serotypes 5 and 8) (8
), cell wall-associated proteins (i.e., clumping factor A, fibronectin binding proteins, and collagen binding protein) (11
), toxins (i.e., alpha-toxin, enterotoxins, and toxic shock syndrome toxin 1) (6
), and the surface-associated polysaccharide, poly-N
-acetyl-β-(1-6)-glucosamine (PNAG) (25
). 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
), 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 chemically identical to PNAG has been isolated and characterized (51
The basic chemical constituents of PNAG were initially described by Mack et al. (24
) and referred to as the polysaccharide intercellular adhesin, but more recent studies showed differences between PNAG isolated from S. aureus
strain MN8m (18
) and the polysaccharide intercellular adhesin preparation 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 β-1-6-linked glucosamine residues that plays a key role in biofilm formation for both S. aureus
and CoNS (28
), as well as being a key virulence factor for S. epidermidis
in animal models of infection (38
). 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 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 μg/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 diphtheria 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 indicated 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
The S. aureus
MN8m strain that overproduces PNAG due to a 5-bp deletion in the ica
) 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
and Newman Δica
, 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.
Isopropyl β-d-thiogalactopyranose (IPTG) was obtained from Invitrogen 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 complement) Accurate Chemical and Scientific (Westbury, N.Y.) were used in the opsonophagocytic assay.
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 by 1
H-nuclear magnetic resonance as previously described (25
), and the resultant antigen was determined to have ∼15% of the amino groups substituted with acetate.
Purification of DT.
DT containing a six-His tag was expressed from the plasmid 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 chromatography on Sephacryl S-200. Endotoxin was removed using Detoxi-Gel (Pierce).
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 μl), 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 μl 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 subsequently 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-polymerization, 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 immunization with two 10-μg 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 as for the rabbits but increasing to 50 μg (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-μg 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.
PNAG- and dPNAG-specific antibodies were measured in sera obtained from immunized mice, rabbits, and a goat by ELISA as described previously (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 μg 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 incubated 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% NH4
Cl 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 × 106
WBC 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 ∼109
CFU of the target S. aureus
or S. epidermidis
strain. After adsorption, the complement solution was centrifuged and filter sterilized. The test sera were absorbed at 4°C for 30 min with bacteria resuspended from a pellet containing ∼109
CFU of the PNAG-negative S. aureus
) 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 OD650
of 0.4 (∼2 × 108
CFU/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 μl (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. Killing rates of >30% were considered biologically significant, as this level represents 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 μg/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 × 107 CFU 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 antibody injection maintained the highest levels of local antibody in the peritoneum. S. aureus strains were grown overnight on tryptic soy agar plates and resuspended in PBS to an OD650 of 1.2, corresponding to approximately 4 × 1010 to 8 × 1010 CFU/ml, depending on the strain. Mice were challenged i.p. with a dose of 2 × 109 to 5 × 109 CFU 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 Committee.
Bacteria use numerous strategies to avoid innate and acquired host defenses and maintain their capacity to cause serious 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 polysaccharides to protein carriers this immunologic barrier can be broken, and effective conjugate vaccines to Haemophilus influenzae
, Streptococcus pneumoniae
, and Neisseria meningitidis
have been developed (3
). 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 antibody isotype, or specificity for nonprotective epitopes (32
). 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 protective antibodies.
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 synergistic 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 bacteria 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 directed 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 version 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 antibody. 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 components of this polysaccharide (35
), and nonopsonic, nonprotective antibodies are directed to non-O-acetylated epitopes. Thus, there are examples with different bacterial capsular polysaccharides 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 glycoconjugate 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. Cyanlylation with CNBr is widely used as a method of activating hydroxyl groups. However, activation can be much more effective 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
). 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 contraindicated due to the potential of self-polymerization between activated 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. Glutaraldehyde-activated DT was subsequently reacted with dPNAG in the presence of NaCNBH3
for 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 polysaccharide to protein in the two conjugates both were nonetheless capable of eliciting high titers of antibodies to the polysaccharide contained in the conjugate. Indeed, the rabbit antisera raised to native PNAG-DT had higher titers to the immunizing 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 antibody to nonacetylated epitopes are effective at mediating opsonic 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 desirable immunogenic properties. Of note, Vuong et al. (49
) recently 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 became susceptible to antibody-independent killing by phagocytes 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 observations), 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 induction 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 significantly increased the immunogenicity of both of these antigens. 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 components, 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 recent 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
), the immunochemical properties of this antigen resulting in the immunodominance 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 antigen.