Native Capsular Polysaccharide Research Articles

A Fluorinated Sialic Acid Vaccine Lead Against Meningitis B and C.

Inspired by the specificity of α-(2,9)-sialyl epitopes in bacterial capsular polysaccharides (CPS), a doubly fluorinated disaccharide has been validated as a vaccine lead against Neisseria meningitidis serogroups C and/or B. Emulating the importance of fluorine in drug discovery, this molecular editing approach serves a multitude of purposes, which range from controlling α-selective chemical sialylation to mitigating competing elimination. Conjugation of the disialoside with two carrier proteins (CRM197 and PorA) enabled a semisynthetic vaccine to be generated; this was then investigated in six groups of six mice. The individual levels of antibodies formed were compared and classified as highly glycan-specific and protective. All glycoconjugates induced a stable and long-term IgG response and binding to the native CPS epitope was achieved. The generated antibodies were protective against MenC and/or MenB; this was validated in vitro by SBA and OPKA assays. By merging the fluorinated glycan epitope of MenC with an outer cell membrane protein of MenB, a bivalent vaccine against both serogroups was created. It is envisaged that validation of this synthetic, fluorinated disialoside bioisostere as a potent antigen will open new therapeutic avenues.

Epoxide-Mediated Trans-Thioglycosylation and Application to the Synthesis of Oligosaccharides Related to the Capsular Polysaccharides of C. jejuni HS:4.

The enzyme-resistant thioglycosides are highly valuable immunogens because of their enhanced metabolic stability. We report the first synthesis of a family of thiooligosaccharides related to the capsular polysaccharides (CPS) of Campylobacter jejuni HS:4 for potential use in conjugate vaccines. The native CPS structures of the pathogen consist of a challenging repeating disaccharide formed with β(1→4)-linked 6-deoxy-β-D-ido-heptopyranoside and N-acetyl-D-glucosamine; the rare 6-deoxy-ido-heptopyranosyl backbone and β-anomeric configuration of the former monosaccharide makes the synthesis of this family of antigens very challenging. So far, no synthesis of the thioanalogs of the CPS antigens have been reported. The unprecedented synthesis presented in this work is built on an elegant approach by using β-glycosylthiolate as a glycosyl donor to open the 2,3-epoxide functionality of pre-designed 6-deoxy-β-D-talo-heptopyranosides. Our results illustrated that this key trans-thioglycosylation can be designed in a modular and regio and stereo-selective manner. Built on the success of this novel approach, we succeeded the synthesis of a family of thiooligosaccharides including a thiohexasaccharide which is considered to be the desired antigen length and complexity for immunizations. We also report the first direct conversion of base-stable but acid-labile 2-trimethylsilylethyl glycosides to glycosyl-1-thioacetates in a one-pot manner.

Metabolic engineering of capsular polysaccharides.

With rising concerns about sustainable practices, environmental complications, and declining resources, metabolic engineers are transforming microorganisms into cellular factories for producing capsular polysaccharides (CPSs). This review provides an overview of strategies employed for the metabolic engineering of heparosan, chondroitin, hyaluronan, and polysialic acid - four CPSs that are of interest for manufacturing a variety of biomedical applications. Methods described include the exploitation of wild-type and engineered native CPS producers, as well as genetically engineered heterologous hosts developed through the improvement of naturally existing pathways or newly (de novo) designed ones. The implementation of methodologies like gene knockout, promoter engineering, and gene expression level control has resulted in multiple-fold improvements in CPS fermentation titers compared with wild-type strains, and substantial increases in productivity, reaching as high as 100% in some cases. Optimization of these biotechnological processes can permit the adoption of industrially competitive engineered microorganisms to replace traditional sources that are generally toxic, unreliable, and inconsistent in product quality.

Semisynthetic glycoconjugate vaccine candidate against Streptococcus pneumoniae serotype 5

Glycoconjugate vaccines based on isolated capsular polysaccharide (CPS) save millions of lives annually by preventing invasive pneumococcal disease caused by Streptococcus pneumoniae Some components of the S. pneumoniae glycoconjugate vaccine Prevnar13 that contains CPS antigens from 13 serotypes undergo modifications or degradation during isolation and conjugation, resulting in production problems and lower efficacy. We illustrate how stable, synthetic oligosaccharide analogs of labile CPS induce a specific protective immune response against native CPS using S. pneumoniae serotype 5 (ST-5), a problematic CPS component of Prevnar13. The rare aminosugar l-PneuNAc and a branched l-FucNAc present in the natural repeating unit (RU) are essential for antibody recognition and avidity. The epitope responsible for specificity differs from the part of the antigen that is stabilized by chemical modification. Glycoconjugates containing stable, monovalent synthetic oligosaccharide analogs of ST-5 CPS RU induced long-term memory and protective immune responses in rabbits superior to those elicited by the ST-5 CPS component in multivalent Prevnar13.

Structural characterisation of the capsular polysaccharide expressed by Burkholderia thailandensis strain E555:: wbiI (pKnock-KmR) and assessment of the significance of the 2-O-acetyl group in immune protection

Burkholderia pseudomallei and its close relative B. mallei are human pathogens that are classified as Tier 1 bio-threat agents. Both organisms have previously been shown to constitutively produce a capsular polysaccharide (CPS) that is both a virulence determinant and protective antigen. Extraction and purification of CPS for use as a potential vaccine candidate requires containment level 3 laboratories which is expensive and time-consuming. B. thailandensis strain E555 is closely related to B. pseudomallei and B. mallei, but is non-pathogenic to humans and based on immunological cross-reactivity has previously been shown to express a B. pseudomallei-like CPS. In this study, capsular polysaccharide isolated from an O-antigen deficient strain of B. thailandensis E555 was identified by 1H and 13C NMR spectroscopy as -3-)-2-O-acetyl-6-deoxy-β-d-manno-heptopyranose-(-1, and identical to that produced by B. pseudomallei. This was further substantiated by anti-CPS monoclonal antibody binding. In connection with the production of CPS fragments for use in glycoconjugate vaccines, we set out to assess the importance or otherwise of the CPS 2-OAc groups in immune protection. To this end conjugates of the native and de-O-acetylated CPS with the Hc fragment of tetanus toxin (TetHc) were used as vaccines in a mouse model of melioidosis. The level of protection provided by deacetylated CPS was significantly lower than that from native, acetylated CPS. In addition, sera from mice vaccinated with the deacetylated CPS conjugate did not recognise native CPS. This suggests that CPS extracted from B. thailandensis can be used as antigen and that the acetyl group is essential for protection.

Antibodies to Staphylococcus aureus capsular polysaccharides 5 and 8 perform similarly in vitro but are functionally distinct in vivo

ABSTRACTThe capsular polysaccharide (CP) produced by Staphylococcus aureus is a virulence factor that allows the organism to evade uptake and killing by host neutrophils. Polyclonal antibodies to the serotype 5 (CP5) and type 8 (CP8) capsular polysaccharides are opsonic and protect mice against experimental bacteremia provoked by encapsulated staphylococci. Thus, passive immunotherapy using CP antibodies has been considered for the prevention or treatment of invasive antibiotic-resistant S. aureus infections. In this report, we generated monoclonal antibodies (mAbs) against S. aureus CP5 or CP8. Backbone specific mAbs reacted with native and O-deacetylated CPs, whereas O-acetyl specific mAbs reacted only with native CPs. Reference strains of S. aureus and a selection of clinical isolates reacted by colony immunoblot with the CP5 and CP8 mAbs in a serotype-specific manner. The mAbs mediated in vitro CP type-specific opsonophagocytic killing of S. aureus strains, and mice passively immunized with CP5 mAbs were protected against S. aureus bacteremia. Neither CP8-specific mAbs or polyclonal antibodies protected mice against bacteremia provoked by serotype 8 S. aureus clinical isolates, although these same antibodies did protect against a serotype 5 S. aureus strain genetically engineered to produce CP8. We detected soluble CP8 in culture supernatants of serotype 8 clinical isolates and in the plasma of infected animals. Serotype 5 S. aureus released significantly less soluble CP5 in vitro and in vivo. The release of soluble CP8 by S. aureus may contribute to the inability of CP8 vaccines or antibodies to protect against serotype 8 staphylococcal infections.

Deciphering Antigenic Determinants of Streptococcus pneumoniae Serotype 4 Capsular Polysaccharide using Synthetic Oligosaccharides.

Streptococcus pneumoniae is a major cause of mortality and morbidity worldwide. More than 90 S. pneumoniae serotypes are distinguished based on the structure of their primary targets to the human immune system, the capsular polysaccharides (CPSs). The CPS of the prevalent serotype 4 (ST4) is composed of tetrasaccharide repeating units and is included in existing pneumococcal vaccines. Still, the structural antigenic determinants that are essential for protective immunity, including the role of the rare and labile cyclic trans-(2,3) pyruvate ketal modification, remain largely unknown. Molecular insights will support the design of synthetic subunit oligosaccharide vaccines. Here, we identified the key antigenic determinants of ST4 CPS with the help of pyruvated and nonpyruvated synthetic repeating unit glycans. Glycan arrays revealed oligosaccharide antigens recognized by antibodies in the human reference serum. Selected depyruvated ST4 oligosaccharides were used to formulate neoglycoconjugates and immunologically evaluated in mice. These oligosaccharides were highly immunogenic, but the resulting antiglycan antibodies showed only limited binding to the natural CPS present on the bacterial surface. Glycan array and surface plasmon resonance analysis of murine polyclonal serum antibodies as well as monoclonal antibodies revealed that terminal sugars are important in directing the immune responses. The pyruvate modification on the oligosaccharide is needed for cross-reactivity with the native CPS. These findings are an important step toward the design of oligosaccharide-based vaccines against S. pneumoniae ST4.

Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis.

Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections.

Structural determination of Streptococcus pneumoniae repeat units in serotype 41A and 41F capsular polysaccharides to probe gene functions in the corresponding capsular biosynthetic loci

We report the repeating unit structures of the native capsular polysaccharides of Streptococcus pneumoniae serotypes 41A and 41F. Structural determinations yielded six carbohydrate units in the doubly branched repeating unit to give the following structure for serotype 41A: [Display omitted] The structure determinations were motivated (1) by an ambition to help close the remaining gaps in S. pneumoniae capsular polysaccharide structures, and (2) by the attempt to derive functional annotations of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides from the determined structures. An activity present in 41F but not 41A is identified as an acetyltransferase acting on the rhamnopyranosyl sidechain E. The genes encoding the formation of the six glycosidic bonds in serogroup 41 were determined from the capsular polysaccharide structures of serotype 41A, 41F, and genetically related serotypes, in conjunction with corresponding genomic information and computational homology searches. In combination with complementary information, NMR spectroscopy considerably simplifies the functional annotation of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides.

Determination of native capsular polysaccharide structures of Streptococcus pneumoniae serotypes 39, 42, and 47F and comparison to genetically or serologically related strains

The diversity of capsular polysaccharides of the bacterial pathogen Streptococcus pneumoniae leads to at least 91 different serotypes. While the genetic loci for capsular biosynthesis have been characterized for all serotypes, the determination of resultant polysaccharide structures remains incomplete. Here, we report the chemical structures of the capsular polysaccharides of serotypes 39, 42, and 47F from the genetic cluster 4, and discuss the structures in the context of structures from serologically and genetically related serotypes. Antigenic determinants can be approximated in this manner.The structure of the serotype 39 capsular polysaccharide is [Display omitted] and has identical composition to the capsular polysaccharide 10A, but two different linkages.The serotype 42 structure [Display omitted] closely resembles the genetically related serotype 35A, which does not contain residue A.The structure of the serotype 47F capsular polysaccharide [Display omitted] is somewhat different from a recently determined structure from the same serogroup, while containing a structural motif that is reflected in serotype 35A and 42 capsular polysaccharide structures, thus explaining the cross-reactivity of serotype 47F with the typing serum 35a.

Development of pneumococcal polysaccharide conjugate vaccine with long spacer arm

Streptococcus pneumoniae is a serious Gram-positive pathogen responsible for several life-threatening pneumococcal diseases. Pneumococcal capsular polysaccharide (CPS) is a key virulence determinant of S. pneumoniae and its immunogenicity can be improved by conjugation with a carrier protein. Reductive amination, the most widely used approach for pneumococcal CPS conjugate vaccine (PCV), suffers from low conjugation efficiency and the problem of steric hindrance. Here, copper-catalyzed azide-alkyne cycloaddition was used for development of PCV with long spacer arm (L-PCV). Tetanus toxoid (TT) was used as the carrier protein. The long spacer arm in L-PCV can minimize the problem of steric hindrance between CPS and TT, thereby improving the CPS-specific antibody titers in the mice model. L-PCV can also induce high avidity functional antibody and elicit immunological memory in response to the native CPS.

Structure determination ofStreptococcus suisserotype 14 capsular polysaccharide

The capsular polysaccharide (CPS) of Streptococcus suis serotype 14 was purified, chemically modified, and characterized. Sugar and absolute configuration analyses gave the following CPS composition: d-Gal, 3; d-Glc, 1; d-GlcNAc, 1; d-Neu5Ac, 1. The Sambucus nigra lectin, which recognizes the Neu5Ac(α2–6)Gal/GalNAc sequence, showed binding to the native CPS. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. It was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analyses,1H and13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [6)[Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)]Gal(β1–3)Gal(β1–4)Glc(β1–]n. S. suis serotype 14 CPS has an identical sialic acid-containing side chain as serotype 2 CPS, but differs by the absence of rhamnose in its composition. The same side chain is also present in group B Streptococcus type Ia CPS, except that in the latter sialic acid is 2,3- rather than 2,6-linked to the following galactose. A correlation between the S. suis CPS sequence and genes of the serotype 14 cps locus encoding putative glycosyltransferases and polymerase responsible for the biosynthesis of the repeating unit is proposed.

Sialylation of Streptococcus suis serotype 2 is essential for capsule expression but is not responsible for the main capsular epitope

The capsular polysaccharide is a critical virulence factor of the swine and zoonotic pathogen Streptococcus suis serotype 2. The capsule of this bacterium is composed of five different sugars, including terminal sialic acid. To evaluate the role of sialic acid in the pathogenesis of the infection, the neuC gene, encoding for an enzyme essential for sialic acid biosynthesis, was inactivated in a highly virulent S. suis serotype 2 strain. Using transmission electron microscopy, it was shown that inactivation of neuC resulted in loss of expression of the whole capsule. Compared to the parent strain, the ΔneuC mutant strain was more phagocytosed by macrophages and was also severely impaired in virulence in a mouse infection model. Both native and desialylated S. suis serotype 2 purified capsular polysaccharides were recognized by a polyclonal anti-whole cell S. suis serotype 2 serum and a monospecific polyclonal anti-capsule serotype 2 serum. In contrast, only the native capsular polysaccharide was recognized by a monoclonal antibody specific for the sialic acid moiety of the serotype 2 capsule. Together, our results infer that sialylation of S. suis serotype 2 may be essential for capsule expression, but that this sugar is not the main epitope of this serotype.

Structure determination ofStreptococcus suisserotype 2 capsular polysaccharide

The capsular polysaccharide (CPS) of Streptococcus suis serotype 2 was isolated, purified, chemically modified, and characterized. Sugar and absolute configuration analyses of the CPS gave the following composition: D-Gal, 3; D-Glc, 1; D-GlcNAc, 1; D-Neu5Ac, 1; L-Rha, 1. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. The CPS was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analysis, 1H and 13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [4)[Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)]Gal(beta1-4)[Gal(alpha1-3)]Rha(beta1-4)Glc(beta1-]n. The backbone sequence was found to be identical to that of Streptococcus agalactiae or group B Streptococcus (GBS) type VIII and Streptococcus pneumoniae type 23F. The S. suis CPS shares the sequence Neu5Ac-Gal-GlcNAc-Gal in common with GBS types Ia, Ib, II, III, and IV CPSs but differs from them by the presence of rhamnose and the fact that sialic acid is 2,6- rather than 2,3-linked to the following Gal. A correlation between the S. suis CPS sequence and genes of the serotype 2 cps locus encoding putative enzymes responsible for the biosynthesis of the repeating unit was tentatively established.

Synthesis, Conjugation, and Immunological Evaluation of the Serogroup 6 Pneumococcal Oligosaccharides

The first synthesis of the newly discovered oligosaccharide of pneumococcal serotype 6C and its spacer-containing analogue is reported. Conjugation of the spacer-containing oligosaccharides of pneumococcal saccharides 6A, 6B, 6C and derivatives thereof with bovine serum albumin (BSA) protein carrier was carried out by using squaric-acid approach to obtain the oligosaccharide-protein conjugates in excellent yields. The conjugates have been tested with a rabbit antiserum pool (Pool B) used for pneumococcal serotyping. The results showed that synthetic carbohydrate conjugates express epitopes found in native capsular polysaccharides of serotypes 6A, 6B, and 6C.

Functional Characterization of PmHS1, a Pasteurella multocida Heparosan Synthase

Heparosan synthase 1 (PmHS1) from Pasteurella multocida Type D is a dual action glycosyltransferase enzyme that transfers monosaccharide units from uridine diphospho (UDP) sugar precursors to form the polysaccharide heparosan (N-acetylheparosan), which is composed of alternating (-alpha4-GlcNAc-beta1,4-GlcUA-1-) repeats. We have used molecular genetic means to remove regions nonessential for catalytic activity from the amino- and the carboxyl-terminal regions as well as characterized the functional regions involved in GlcUA-transferase activity and in GlcNAc-transferase activity. Mutation of either one of the two regions containing aspartate-X-aspartate (DXD) residue-containing motifs resulted in complete or substantial loss of heparosan polymerizing activity. However, certain mutant proteins retained only GlcUA-transferase activity while some constructs possessed only GlcNAc-transferase activity. Therefore, it appears that the PmHS1 polypeptide is composed of two types of glycosyltransferases in a single polypeptide as was found for the Pasteurella multocida Type A PmHAS, the hyaluronan synthase that makes the alternating (-beta3-GlcNAc-beta1,4-GlcUA-1-) polymer. However, there is low amino acid similarity between the PmHAS and PmHS1 enzymes, and the relative placement of the GlcUA-transferase and GlcNAc-transferase domains within the two polypeptides is reversed. Even though the monosaccharide compositions of hyaluronan and heparosan are identical, such differences in the sequences of the catalysts are expected because the PmHAS employs only inverting sugar transfer mechanisms whereas PmHS1 requires both retaining and inverting mechanisms.

Conjugates of group A and W135 capsular polysaccharides of neisseria meningitidis bound to recombinant Staphylococcus aureus enterotoxin C1: preparation, physicochemical characterization, and immunological properties in mice.

Neisseria meningitidis groups A (GAM) and W135 capsular polysaccharides (CPs) were bound to recombinant Staphylococcus aureus enterotoxin C1 (rSEC). The CPs were activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate and then bound to adipic acid dihydrazide derivatives of rSEC. Syntheses were conducted with native GAM CP (GAMP), W135 CP (W135P), and ultrasonicated or hydrazine-treated W135P at various concentrations of reactants, pHs, and ionic strengths. The conjugates were characterized by compositional and serologic analyses, high-performance size-exclusion chromatography with multi-angle laser light scattering detection, and immunogenicity in 5- to 6-week-old mice. Conjugates injected subcutaneously in phosphate-buffered saline elicited immunoglobulin G (IgG) responses against their respective CPs and rSEC, whereas GAMP and W135P alone did not induce detectable CP antibodies. The O-acetyl content of W135P was low, and its removal had no adverse effect upon the conjugate's immunogenicity. Reduction of the molecular size of W135P by treatment with hydrazine improved the immunogenicity of W135P-rSEC. IgG anti-CP elicited by the conjugates showed complement-dependent bactericidal activity against their respective organisms, and IgG anti-rSEC neutralized the T-cell proliferative activity of native SEC. A bivalent formulation of GAMP-rSEC and W135P-rSEC elicited IgG anti-CP at comparable levels to those induced by the conjugates administered separately.

Structural elucidation of type III group B Streptococcus capsular polysaccharide using molecular dynamics simulations: the role of sialic acid

The conformational properties of the capsular polysaccharide (CPS) from group B Streptococcus serotype III (GBS III) are derived from 50 ns explicitly solvated molecular dynamics simulations of a 25-residue fragment of the CPS. The results from the simulations are shown to be consistent with experimental NMR homo- and heteronuclear J-coupling and NOE data for both the sialylated native CPS and for the chemically desialylated polysaccharide. A helical structure is predicted with a diameter of 29.3 Å and a pitch 89.5 Å, in which the sialylated side chains are arrayed on the exterior surface of the helix. The results provide an explanation for the observation that CPS antigenicity varies with carbohydrate chain length up to approximately 4 pentasaccharide repeat units. The conformation of the immunodominant region is established and shown to be independent of the presence of sialic acid. The data provide an explanation for the observation that the specificity of the determinant, associated with the major population of antibodies raised upon immunization of rabbits with GBS III, is dependent on the presence of sialic acid. In the sialylated native CPS, the antibody response is largely directed against the immunodominant core of the helix. From simulations of the desialylated CPS, a model emerges which suggests that the minor population of antibodies, whose determinant is not sialic acid dependent, recognizes the same immunodominant region, but that in the disordered CPS this region is not presented in a regular repeating motif.

Effect of O acetylation of Neisseria meningitidis serogroup A capsular polysaccharide on development of functional immune responses.

The importance of O-acetyl groups to the immunogenicity of Neisseria meningitidis serogroup A polysaccharide (PS) was examined in studies using human sera and mouse immunization. In 17 of 18 postimmunization human sera, inhibition enzyme-linked immunosorbent assay indicated that the majority of antibodies binding to serogroup A PS were specific for epitopes involving O-acetyl groups. Studies with mice also showed an essential role for O-acetyl groups, where serum bactericidal titers following immunization with de-O-acetylated (de-O-Ac) conjugate vaccine were at least 32-fold lower than those following immunization with O-Ac PS-conjugate vaccine and 4-fold lower than those following immunization with native capsular PS. Inhibition studies using native and de-O-Ac PS confirmed the specificity of murine antibodies to native PS. The dramatic reduction in immunogenicity associated with removal of O-acetyl groups indicates that O acetylation is essential to the immunogenic epitopes of serogroup A PS. Since levels of bactericidal antibodies are correlated with protection against disease, O-acetyl groups appear to be important in protection.

Antigenic Determinants of Staphylococcus aureus Type 5 and Type 8 Capsular Polysaccharide Vaccines

Bacterial capsular polysaccharides (CP) are carbohydrate polymers comprised of repeating saccharide units. Several of these CP have side chains attached to their backbone structures. The side chains may include O-acetyl, phosphate, sialic acid, and other moieties. Those moieties represent the immunodominant epitopes and the most functional ones. The clinically significant Staphylococcus aureus type 5 CP (CP 5) and type 8 CP (CP 8) are comprised of a trisaccharide repeat unit with one O-acetyl group attached to each repeat unit. The immunogenicity of these CP and the functionality of antibodies to the backbone and the O-acetyl moieties were investigated. Immunization with the native CP conjugates (CP with 75% O-acetylation) elicited a high proportion of antibodies directed against the O-acetyl moiety. Nonetheless, all of the vaccinees produced antibodies to the backbone moieties as well. Conjugate vaccines made of de-O-acetylated CP elicited backbone antibodies only. Antibodies to both backbone and O-acetyl groups were found to be opsonic against S. aureus strains which varied in their O-acetyl content. Absorption studies with O-acetylated and de-O-acetylated CP showed that (i) native CP conjugates generated antibodies to both backbone and O-acetyl groups and (ii) O-acetylated isolates were opsonized by both populations of antibodies while the non-O-acetylated strains were predominantly opsonized by the backbone antibodies. These results suggest that S. aureus CP conjugate vaccines elicit multiple populations of antibodies with diverse specificities. Moreover, the antibodies of different specificities (backbone or O-acetyl) are all functional and efficient against the variations in bacterial CP that may occur among clinically significant S. aureus pathogenic isolates.