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.