Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Identification of a gene essential for O-acetylation of the Staphylococcus aureus type 5 capsular polysaccharide

It has been hypothesized that the developmental toxicity of certain compounds is, in part, due to maternal toxicity resulting in alterations in zinc (Zn) metabolism that affects the developing conceptus. In the present work the effects of developmentally toxic doses of 2-ethylhexanoic acid (EHXA), 2-ethylhexanol (EHXO), and valproic acid (VPA) on Zn metabolism were investigated in the pregnant rat. In experiment 1, dams were intubated with EHXA (3.13, 6.25, 9.38 or 12.5 mmol/kg), EHXO (6.25, 9.38 or 12.5 mmol/kg), VPA (1.56, 3.13, 6.25 or 9.38 mmol/kg), or corn oil (control; 1.0 ml/kg) at 14:00 h on gestation day (GD) 11.5, intubated with 32 microCi 65Zn at 22:00 h, and then killed at 08:00 h on GD 12.5. At the higher dose levels of EHXA and EHXO, and at all dosages of VPA, the percentage of 65Zn retained in maternal liver was higher, while that in the embryos was lower, than in controls. Chemical-associated changes in 65Zn distribution were associated with increased maternal liver metallothionein (MT) concentrations. In experiment 2, dams were fed diets containing 1, 25 or 97 microg Zn/g from GD 0-16 and intubated with 3.5 mmol EHXA or 1.0 ml corn oil/kg/d from GD 8-15. Dams were killed on GD 16 or 19. High incidences of encephalocele and tail defects were noted in the GD 16 fetuses of EHXA-treated dams fed either the low or adequate Zn diet, the highest incidences being in the low Zn group. On GD 19 the incidence of tail defects tended to be higher in the EHXA groups than in oil-treated controls, the highest incidence occurring in the low Zn EHXA group. Encephalocele was only observed in the low Zn EHXA-treated group. Fetal weight and crown-rump lengths were decreased by EHXA treatment and low dietary Zn. The incidence of rib anomalies was higher in the EHXA-exposed groups than in their respective oil controls. In experiment 3, GD 10.5 embryos collected from control dams were cultured for 48 h in serum from control or EHXA-treated male rats fed 4.5 or 25.0 microg Zn/g diets. Embryos cultured in either EHXA or low Zn sera exhibited delayed development; the addition of Zn to these sera eliminated their developmental toxicity. These results support the hypothesis that certain chemicals which induce maternal toxicity act, in part, to influence embryonic Zn metabolism and trigger abnormal development. Importantly, the teratogenic effects of these chemicals can be modulated by dietary Zn intake.     

Regulation of Staphylococcus aureus capsular polysaccharide type 5: CO2 inhibition in vitro and in vivo

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats, and in vitro using ELISA and IFA. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1%-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1%-5% of CP5-positive cells were detectable in pouch exudates. CF and pouch isolates, however, reexpressed CP5 (70%-90% of cells) when grown in vitro with air. Addition of > or = 1% CO2 to air or to O2/N2 gas mixtures reduced CP5 expression significantly (P < .001) in a dose-dependent manner (6%-1% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal that regulates CP5 expression.     

Relationship of capsular type to biochemical and immunological properties of teichoic acid preparations from unencapsulated strains of Staphylococcus aureus

We investigated the biochemical and immunological characteristics of teichoic acid preparations (TAP) obtained from four unencapsulated strains of Staphylococcus aureus which nonetheless, according to the serum-soft agar technique, produced capsular type antigen and were representative of the four types A, B, C, and D. In the agar diffusion test, TAP of each strain produced a single precipitin line only against rabbit antisera corresponding to the homologous capsular type; no lines were observed against antisera to the heterologous capsular type. All TAP were ribitol type except one, glycerol, prepared from a capsular type D strain. Major acetylglucosaminyl residues of TAP from strains having capsular type A and C antigens were attached to the polyribitol phosphate by beta-linkage, whereas TAP from a type B antigen strain had an alpha-linkage; type D antigen was attached to the polyglycerol phosphate by the beta-linkage. Chemical analyses and infrared spectrograms of these TAP further confirmed their heterogeneous nature.     

Structure of the capsular polysaccharide from the Klebsiella K8 reference strain 1015

The structure of the capsular polysaccharide from the Klebsiella K8 reference strain 1015 has been elucidated. The structure was deduced from sugar analysis, different methylation analyses, a uronic acid degradation, and NMR spectroscopy. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with the structure: [formula: see text] The structure differs from that of the previously published structure of the capsular polysaccharide from Klebsiella K8, which originates from another strain and has the following structure: [formula: see text] The serological similarity between the two strains is most likely derived from a common tetrasaccharide which is substituted in different ways in the two strains. Since the strain in the present investigation originates from the Klebsiella K reference strain collection of the International Escherichia and Klebsiella Centre, Copenhagen, Denmark, it is suggested that it should keep the designation K8. The other polysaccharide with Klebsiella K8 specificity should be renamed as K8,52,59 based on the cross-reactivity of the strain (I. Orskov, unpublished).     

Peptides that mimic the group B streptococcal type III capsular polysaccharide antigen

In some but not all arterial beds, smooth muscle cell calcium-activated K+ channels (KCa channels) play a central role in the mediation of the vasodilator response to nitric oxide (NO) and other nitrates. We investigated the effect of nitrates on KCa channels in the relaxation of human coronary arteries by means of isometric contraction experiments in arterial rings. We also measured whole-cell currents in freshly isolated human coronary artery vascular smooth muscle cells via the patch-clamp technique. Sodium nitroprusside, diethylamine-nitric oxide complex sodium salt and isosorbide mononitratre completely relaxed rings preconstricted with 5 microM serotonin and produced dose-dependent relaxations of 5 microM serotonin-preconstricted human rings. The relaxations were inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-oxyl 3-oxide (10 microM), which neutralizes nitric oxide. The KCa channel blockers iberiotoxin (100 nM) and tetraethylammonium ions (1 mM) significantly inhibited SNP-induced relaxations of human coronary arteries. Moreover, in the patch-clamp experiments, SNP (1 microM) stimulated KCa currents and spontaneous transient outward K+ currents carried by Ca spark activated KCa channels. The SNP-induced (1 microM) KCa current was strongly inhibited by iberiotoxin (100 nM). These data show that activation of KCa channels in smooth muscle cells contributes to the vasodilating actions of nitrates and nitric oxide in human coronary arteries. This finding may have unique clinical significance for the development of antianginal and antihypertensive drugs that selectively target K+ channels and Ca sparks.     

Genetic analysis of the cap5 locus of Staphylococcus aureus

BACKGROUND: Fifteen patients with femoral shaft fractures complicated by infected nonunions were treated with a two-stage protocol. METHODS: In the first stage, radical debridement was performed along with antibiotic bead chains local therapy and external skeletal fixation. In the second stage, the debrided nonunion site was repaired with bone grafting and the external skeletal fixator was used until bony union was achieved. The time between the first and second stages of treatment was 2 to 6 weeks. The debrided bone defects ranged from 0.5 to 15 cm. Autogenous iliac cancellous bone grafting was performed in 11 patients, and microvascularized osteoseptocutaneous fibular transfer was performed in 4 patients. RESULTS: Wound healing and bone union were achieved in all 15 cases. The duration of external fixation of these patients ranged from 7 to 15 months, with an average of 9 months. Minor pin-track infection was seen in seven patients. Postoperative infection after the second-stage bone grafting occurred in three patients. These three infections were arrested by limited debridement along with 2 to 4 weeks of parenteral antibiotic therapy. In one case, stress fracture occurred at 11 months after microvascularized fibular transfer; this was managed with another 5 months of external skeletal fixation. With an aggressive physical therapy program, 10 patients achieved nearly full range of knee motion and 5 patients had relevant knee flexion deficits. The follow-up averaged 58 months (range, 40-76 months); no recurrence of osteomyelitis was observed even at 76 months. CONCLUSION: We have found that our two-stage treatment with antibiotic beads local therapy, definitive external skeletal fixation, and staged bone grafting is an acceptable treatment protocol for the management of femoral diaphyseal infected nonunion. It results in rapid recovery from osteomyelitis and a predictable recovery from nonunion.     

Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease

The amount of cell surface fibronectin (Fn)-binding protein (FnBP) adhesin expressed by Staphylococcus aureus is maximal during exponential growth but disappears rapidly as the culture progresses into stationary phase. To identify factors responsible for the loss of cell surface FnBP, a culture of S. aureus L170, which shows high levels of Fn binding, was supplemented at the time of inoculation with concentrated stationary-phase supernatant from S. aureus L530, a strain which binds Fn poorly. The resulting exponential-phase cells were devoid of FnBP. The factor responsible for this activity was purified from the culture supernatant and identified as V8 protease. When cultured with 375 ng of exogenous V8 protease ml(-1), exponential-phase cells of S. aureus L170 were devoid of cell surface FnBP, and concentrations as low as 23 ng x ml(-1) resulted in reduced amounts of FnBP. Addition of the protease inhibitor alpha2-macroglobulin to the culture medium prevented the growth-phase-dependent loss of cell surface FnBP, whereas growth with exogenous V8 protease resulted in reduced adherence to the solid-phase N-terminal fragment of Fn and to the extracellular matrix synthesized by fetal rabbit lung fibroblasts. Although FnBP was extremely sensitive to V8 protease, exogenous protease did not exert a significant influence on the amount of cell surface protein A. However, a limited number of other high-molecular-weight cell surface proteins were also sensitive to V8 protease. Therefore, both the adhesive phenotype and cell surface protein profile of S. aureus can be modified by V8 protease activity.     

Vaccination with protein-conjugated and native type 3 capsular polysaccharide in an ethanol-fed rat model of pneumococcal pneumonia

A chronic ethanol-fed rat model was used to determine the effect of alcohol ingestion on production of antibody to type 3 pneumococcal capsular polysaccharide. Sprague-Dawley rats were fed a liquid diet containing 36% of calories as ethanol (ethanol-fed), an isocaloric diet containing dextrin-maltose (pair-fed) or standard rat chow (chow-fed). After 7 days of feeding, the rats were vaccinated subcutaneously with placebo or with either 25 microg of type 3 pneumococcal capsular polysaccharide (SpnCP) or 5 microg of SpnCP linked to the protein carrier CRM197 (SpnCP/CRM197). Rats given the conjugated vaccine received a booster injection 14 days later. Maximum antibody titers were observed six days postvaccination for rats given SpnCP alone and 21 days postvaccination for rats given SpnCP/CRM197. All rats were infected transtracheally with 2-3 times the expected lethal dose50 for each feeding group of type 3 Streptococcus pneumoniae on the day of peak antibody titers. Mortality was recorded for a 10-day period. Vaccination with SpnCP increased survival of ethanol- and chow-fed, but not pair-fed rats. This protection was only statistically significant in the chow-fed group (p < 0.01). Vaccination with SpnCP/CRM197 moderately increased survival of rats in all three feeding groups, but this was not statistically significant in any of them.     

Streptococcus pneumoniae type 14 polysaccharide-conjugate vaccines: length stabilization of opsonophagocytic conformational polysaccharide epitopes

A simple and convenient method was developed for the preparation of Streptococcus pneumoniae type 14 polysaccharide (Pn14PS)-tetanus toxoid (TT) conjugate vaccines, using terminally linked Pn14PS fragments of different lengths. Native Pn14PS was simultaneously depolymerized and activated for conjugation by partial N-deacetylation followed by nitrous acid deamination which yielded fragments (1.4 to 150.0 kDa) having a free aldehyde at the reducing end. These were then conjugated to TT through their terminal aldehydic groups, using the reductive amination procedure. All of the above conjugates, when injected in rabbits, induced anti-Pn14PS antibodies, whereas the native Pn14PS did not. The amounts of anti-Pn14PS antibodies elicited by these conjugates, as determined by enzyme-linked immunosorbent assay, followed a trend with conjugates containing the highest-molecular-weight Pn14PS eliciting the highest titers. The same trend was also observed in the ability of the antibodies to opsonize and kill live type 14 pneumococci, although the increase in opsonophagocytic activity was more pronounced and did not correlate linearly with increases in antibody titer. Competitive inhibition of the binding of different conjugate antisera to the native Pn14PS, using Pn14PS fragments as inhibitors, established that the conjugates induced antibodies with specificities for different lengths of Pn14PS beginning at 2 repeating units (RU). It was also established, both immunologically and antigenically, that at least 4 RU of Pn14PS were required to form an extended conformational epitope and that approximately 22 RU of Pn14PS were required to duplicate the same epitope on the same saccharide chain. The conformational epitope was found to be essential for the induction of antibodies with high opsonophagocytic activity and that augmentation of opsonophagocytic activity was also dependent on further chain extension.     

Potential and limitations of polysaccharide vaccines in infancy

Of infections caused by encapsulated bacteria, those due to Haemophilus influenzae b (Hib) are among the most restricted to infancy and require very early immunisation. Hib capsular polysaccharide (CPS) has the most typical T-cell independent profile. The absence of efficacy of this vaccine in infants triggered development of conjugate vaccines which are so effective that there is now no room for plain polysaccharide Hib vaccines. Pneumococcal infections pose similar problems to Hib, but are more complex. The immunogenicity of the different pneumococcal serotypes varies considerably in infancy. Although the current CPS vaccine provides limited protection in infancy, the burden of pneumococcal infection is so high that its use could be reconsidered should conjugate vaccines be available later than expected. Meningococcal infections are less a specific problem for infants. Again, serogroup immunogenicity varies widely. Group B meningococcal CPS is not immunogenic even in adults, Group C behaves as Hib CPS, whereas Group A is immunogenic as early as 6 months of age. Group A CPS may prove of interest for an infant vaccine, especially in epidemic situations. Typhoid fever is uncommon in infancy; Vi CPS is poorly immunogenic in infancy and is, therefore, of limited interest for use as an infant vaccine.     

Epitopic overload at the site of injection may result in suppression of the immune response to combined capsular polysaccharide conjugate vaccines

RATIONALE AND OBJECTIVES: The therapeutic gain of neutron capture therapy with a neutral macrocyclic gadolinium (Gd) complex (Gadobutrol) was evaluated through in vitro and in vivo studies in a beam of low-energy neutrons. METHODS: Neutron irradiation for both the in vitro and in vivo studies was performed in a beam of low-energy neutrons produced by the research reactor of the Hahn-Meitner-Institut, Berlin. Malignant melanoma cells of human origin were irradiated in the presence or absence of Gadobutrol. In vivo irradiation was performed on tumor-bearing nude mice. The tumor site was irradiated subsequent to intratumoral injection of Gadobutrol and compared with irradiation in the absence of the Gd complex. RESULTS: In vitro studies showed a Gd-dependent delay of cell proliferation as a consequence of neutron irradiation. In animals, intratumoral administration of the Gd complex at a dose of 1.2 mmol Gd/kg before neutron irradiation results in a significant delay in tumor growth with respect to the control groups. CONCLUSIONS: In vitro and in vivo studies showed a therapeutic benefit with the neutral Gd complex and suggest Gd-containing magnetic resonance contrast media are potential candidates for neutron capture therapy. The Gd dose used in the irradiation experiments was four times the presently accepted high dose in clinical magnetic resonance imaging.     

NMR assignment and conformational analysis of the antigenic capsular polysaccharide from Streptococcus pneumoniae type 9N in aqueous solution

Complete 1H and 13C NMR assignments, determined by one- and two-dimensional homo- and hetero-nuclear experiments, are reported for the antigenic capsular polysaccharide (CPS) from Streptococcus pneumoniae serotype 9N (S9 in American nomenclature). Distance constraints derived from 1D NOE difference experiments were combined with energy minimisation (simulated annealing) and molecular dynamics (MD) calculations to determine the most favoured conformation of S9 in aqueous solution at 70 degrees C. NOE values simulated for several static conformational models using the NOEMOL program did not correlate well with experimental values, whereas time averaged interproton distances calculated from 500 ps of restrained MD (using the Tropp formalism to account for rapid internal mobility) were in close agreement with experimentally derived distance estimates.     

The structure of the capsular polysaccharide from Klebsiella type 52, using the computerised approach CASPER and NMR spectroscopy

In swine, distinct centromeric satellite DNA families have been described that correspond to either all the metacentric chromosomes except the Y (Mc1) or all the acrocentric chromosomes (Ac2). Using primed in situ (PRINS) labeling, we show here that primers derived from various sequences specifically label the centromeres of different subgroups of chromosomes. Among five primers derived from centromeric sequences of acrocentric chromosomes reported to be very homogeneous, four recognize all the acrocentric chromosomes, whereas one labels prominently chromosome 17. For the metacentric chromosomes, six primers have been derived from several divergent sequences. Among these primers, two recognize all the metacentric chromosomes except 5, 10, and 12. Three other primers label small subsets of metacentric chromosomes, including the X and one or two additional chromosomes. The last primer is specific to chromosome 1. These preliminary results suggest that it should be possible to define specific primers for almost every swine chromosome. Already, some of the primers reported here permit a distinction between swine chromosomes difficult to differentiate without banding, such as the X chromosome and chromosome 9. Therefore, the PRINS technique using centromeric motifs constitutes an additional tool for cytogenetic studies in swine.     

Laboratory and preliminary clinical characterization of Vi capsular polysaccharide-protein conjugate vaccines

To improve its immunogenicity for children and adults and to make it suitable for routine immunization of infants against typhoid fever, the capsular polysaccharide of Salmonella typhi (Vi) was bound to the B subunit of the heat-labile toxin (LT-B) of Escherichia coli or the recombinant exoprotein A (rEPA) of Pseudomonas aeruginosa. The conjugates elicited higher levels of antibodies (micrograms per milliliter of serum) in mice and in guinea pigs than did Vi and, unlike Vi alone, elicited booster antibody responses in both species. In adult volunteers, Vi-LT-B and Vi-rEPA, respectively, elicited higher levels of antibodies than Vi alone after the first injection (4.74 versus 1.77 and 4.91 versus 1.77; P < 0.005) and 26 weeks later (2.32 and 2.69 versus 0.54; P < 0.04); a second injection of the conjugates did not elicit a booster response of Vi antibodies. None of the 51 vaccinees had fever or significant local reactions. Vi-rEPA elicited slightly higher levels of Vi antibodies than did Vi-LT-B at all intervals after injection, but these differences were not significant. Each conjugate elicited antibodies to its carrier protein. The antibody responses elicited in adults by Vi bound to LT-B and rEPA are similar to those of other polysaccharide-protein conjugates. These conjugates promise to be an improved Vi vaccine. Studies of Vi conjugates with adults and infants in areas where typhoid is endemic are planned.     

Characteristics and treatment of Staphylococcus aureus endocarditis

In order to evaluate whether there were changes of the clinical features of Staphylococcus (Staph.) aureus endocarditis in recent years, the data of 21 cases of Staph. aureus endocarditis diagnosed from 1977 to 1994 were analyzed and compared with those of 11 cases of Staph. aureus endocarditis from 1957 to 1977. The results showed the following changes in recent years. The incidence of Staph. aureus endocarditis cases has been increasing. Cases of right-sided endocarditis increased in the recent two decades and this increase was related to intravenous drug abuse and increased use of vascular intervention. Right-sided endocarditis was different from left-sided endocarditis in their risk factors, underlying heart diseases, clinical manifestations and prognosis. The clinical manifestations of ventricular-wall endocarditis were atypical and it could be definitely diagnosed only with echocardiogram. Complications of Staph. aureus endocarditis became more common and serious, in recent decades but hospital mortality decreased markedly due to effective antibiotic management. The authors believe that sound knowledge of Staph. aureus endocarditis is essential for the proper diagnosis and treatment of Staph. aureus endocarditis.     

Diversity of capsular polysaccharide synthesis gene clusters in Streptococcus pneumoniae

Echoplanar functional magnetic resonance imaging (fMRI) was used in normal human subjects to investigate the role of the amygdala in conditioned fear acquisition and extinction. A simple discrimination procedure was employed in which activation to a visual cue predicting shock (CS+) was compared with activation to another cue presented alone (CS-). CS+ and CS- trial types were intermixed in a pseudorandom order. Functional images were acquired with an asymmetric spin echo pulse sequence from three coronal slices centered on the amygdala. Activation of the amygdala/periamygdaloid cortex was observed during conditioned fear acquisition and extinction. The extent of activation during acquisition was significantly correlated with autonomic indices of conditioning in individual subjects. Consistent with a recent electrophysiological recording study in the rat (Quirk et al., 1997), the profile of the amygdala response was temporally graded, although this dynamic was only statistically reliable during extinction. These results provide further evidence for the conservation of amygdala function across species and implicate an amygdalar contribution to both acquisition and extinction processes during associative emotional learning tasks.     

A new typhoid vaccine composed of the Vi capsular polysaccharide

Typhoid is still prevalent in many parts of the world. We reviewed all published and unpublished studies of a newly licensed vaccine composed of the Vi capsular polysaccharide of Salmonella typhi, the causative agent of the disease, which had been licensed previously outside the United States. These included observational studies and double-blind randomized studies done in the United States, Europe, and the developing world in which children and adults unexposed to typhoid or those living in endemic areas were enrolled. A single dose of 25 micrograms of the purified polysaccharide was given by intramuscular injection. The vaccine was well tolerated, inducing only minor reactions in fewer than 10% of subjects. An antibody response occurred in about 90% of subjects and lasted about 3 years. Seroconversion was shown in children as young as 2 years. Protective efficacy was evaluated in two studies conducted in areas in which typhoid is endemic; the efficacy was 55% and 75%, respectively, in adults and in children older than 5 years. The Vi vaccine compares favorably with other typhoid vaccines in regard to safety, patient compliance, immunogenicity, and efficacy. Vi polysaccharide is a well-standardized antigen that is effective in a single parenteral dose, is safer than whole-cell vaccine, and may be used in children 2 years of age or older.     

Antibody responses to capsular polysaccharide backbone and O-acetate side groups of Streptococcus pneumoniae type 9V in humans and rhesus macaques

Streptococcus pneumoniae is responsible for high rates of pneumococcal bacteremia, meningitis, pneumonia, and acute otitis media worldwide. Protection from disease is conferred by antibodies specific for the polysaccharide (Ps) capsule of the bacteria. Of the four types of group 9 pneumococci, types 9N and 9V cause the most disease, and both types are included in the polyvalent pneumococcal vaccine. The type 9V capsule consists of repeating pentasaccharide units linearly arranged, with an average of 1 to 2 mol of O-acetate side chains per mol of repeat units, added in a complex pattern in which not all repeat units are alike. alpha-GlcA residues may be O-acetylated in the 2 (17%) or 3 (25%) position and beta-ManNAc residues may be O-acetylated in the 4 (6%) or 6 (55%) position. Under certain conditions, the O-acetate side chains are subject to oxidation, which results in subsequent de-O-acetylation of a significant number of the repeat units. This de-O-acetylation could adversely affect the efficacy of a vaccine containing the 9V Ps. A study was undertaken to compare the relative contributions of O-acetate and Ps backbone epitopes in the immune response to S. pneumoniae 9V type-specific Ps. In both an infant rhesus monkey model and humans, antibodies against the non-O-acetylated 9V backbone as well as against O-acetylated 9V Ps were detected. Functional (opsonophagocytic) activity was observed in antisera in which the predominant species of antibody recognized de-O-acetylated 9V Ps. We concluded that the O-acetate side groups, while recognized, are not essential to the ability of the 9V Ps to induce functional antibody responses.