Unreacted SBAP was taken out using a PD-10 column with a mixture of 0.1 M sodium phosphate, 0.15 M NaCl, and 10 mM EDTA, pH 8.0, and the eluate was concentrated to 400 l. be activated to produce sulfhydryl groups for conjugation to bromoacetyl groups introduced onto carrier proteins. Synthetic 5-mer GlcNH2(5GlcNH2) or 9GlcNH2conjugated to tetanus toxoid (TT) elicited mouse antibodies that mediated opsonic killing of multipleS. aureusstrains, while the antibodies that were produced in response to 5GlcNAc- or 9GlcNAc-TT did not mediate opsonic killing. Rabbit antibodies to 9GlcNH2-TT bound to PNAG and dPNAG antigens, mediated killing ofS. aureusandE. coli, and guarded againstS. aureusskin abscesses and lethalE. coliperitonitis. Chemical synthesis of a series of oligoglucosamine ligands with defined differences in N acetylation allowed us to identify a conjugate vaccine formulation that generated protective immune responses to two of the most challenging bacterial pathogens. This vaccine could potentially be used to engender protective immunity to the broad range of pathogens that produce surface PNAG. The continued threat from antibiotic-resistant microbial pathogens such as multidrug-resistantStaphylococcus aureusandEscherichia coliand the ongoing difficulty in adequately preventing and treating infectious diseases caused by such pathogens have driven the quest for more effective preventative and therapeutic approaches to contamination. Vaccination, when it works, not only dramatically decreases contamination and illness (35), but also has shown itself to be capable of eliminating endemic transmission of diseases such as polio, measles, and rubella from the United States and has eliminated smallpox worldwide (35). Bacterial surface or capsular antigens, which are commonly synthesized as polysaccharides and less commonly as proteins, represent the best-established targets for engendering protective immunity PF-06855800 by vaccination. Conjugating surface polysaccharides to carrier proteins greatly enhances the immunogenicity and effectiveness of the polysaccharides (40). Highly successful conjugate vaccines targeting the capsular polysaccharides (CPs) ofStreptococcus pneumoniae(5),Haemophilus influenzaetype b (38), andNeisseria meningitidis(40) have been produced and licensed for human use, with a major impact in reduction of disease due to these bacterial pathogens. Significant advances, including those from human trials, have been made for polysaccharide conjugate vaccines forSalmonella entericaserovar Typhi (20), group B streptococcus (4), andE. coliO157 (1). A promising target for vaccine development is a surface polysaccharide produced by a broad range of common pathogens and designated poly-N-acetylglucosamine (PNAG), a -(16)-linked polymer ofN-acetyl-d-glucosamine (GlcNAc) (22) with some proportion of the amino groups lacking acetate substituents. The basic chemical properties of PNAG were described by Mack et al. (21), who referred to the material as the polysaccharide intercellular adhesin. Among important bacterial pathogens, PNAG is known to be produced byS. aureusandStaphylococcus epidermidis(23,25,26),E. coli(13,42),Bordetella pertussisandBordetella parapertussis(29,36),Aggregatibacter actinomycetemcomitans(15),Acinetobacterspp. (8), andYersinia pestis(10,12). Based on genetic homology, loci likely encoding PNAG biosynthetic proteins are found inBurkholderia cenocepaciaandKlebsiella pneumoniae.Prior work has shown that antibodies to PNAG conjugated to a protein carrier can mediatein vitroopsonic killing and protect mice fromS. aureus(23,26) andE. coli(7) infections, but such immunity PF-06855800 can be engendered only by first removing the majority of the acetates from the PNAG polymer to produce deacetylated PNAG (dPNAG). These findings indicate that this immunodominant epitopes on native PNAG elicit nonopsonic, nonprotective antibodies and that antibodies to the core or backbone epitopes have superior opsonic and PF-06855800 protective properties, due likely to enhanced deposition of opsonically active fragments of the third component of complement, C3b (16). While conjugate vaccines comprising highly but not completely deacylated forms of PNAG appear to be effective at providing protective immunity in animal studies, the lack of definition of the chemical composition of dPNAG and the need to produce it by chemical deacetylation of highly acetylated PNAG, resulting in variability in the final composition, limit the conclusions that can be drawn about optimal vaccine formulation. Native PNAG (>90% acetylated) has a certain amount of deacetylated -(16)-d-glucosamine (GlcNH2) units but whether they are grouped together or interspersed throughout the molecule is not known, nor is it known if preparations of either native PNAG or dPNAG contain a proportion of molecules with low levels of acetylation among a greater MMP15 population of highly acetylated molecular species. To develop optimal vaccines that generate protective antibodies, the relative numbers of GlcNH2units and their spacing will need to be decided, and this will not be possible by chemical deacetylation, which would PF-06855800 randomly change GlcNAc units to GlcNH2units. To define more precisely the immune responses elicited by different epitopes around the PNAG molecule, oligoglucosamines made up of either.