A series of group C meningococcal polysaccharide-tetanus toxoid (GCMP-TT) conjugates were prepared as vaccines with varying percentages of O-acetylation at the C-7 and C-8 positions of sialic acid residues in the polysaccharide (PS). The immune response in mice was highly dependent on the degree of O-acetylation. Less O-acetylation resulted in higher serum bactericidal activity (SBA) towards the O-acetylated (OA) meningococcal strain, C11. In addition, since an unconjugated de-O-acetylated (dOA) GCMP vaccine was previously shown to be highly immunogenic in humans, we had chosen this dOA form to couple with TT by reductive amination for clinical evaluation. This conjugate vaccine was shown to be well-tolerated and highly immunogenic in adults, children, and infants in the UK. To understand the nature of the GCMP protective epitope, a series of spectroscopic and serological studies were conducted, using high resolution H-NMR spectroscopy at 500 MHz and competitive inhibition SBA assays. The dOA GCMP was 10-1000 times better at inhibiting the SBA for an OA strain than the OA GCMP, suggesting that the GCMP-based protective epitope on the bacterium exists in a dOA form. In addition, SBA for an OA strain is highly correlated with dOA GCMP-specific IgG. NMR data on freshly isolated GCMP indicated that, on the surface of the organism, most of the O-acetylation exists at position C-8, with some regions containing dOA or OA C-7 sialic acid. After extraction of PS and storage in solution, most of the O-acetyl groups migrate to C-7, leaving an epitope that is conformationally related, but not quite identical (due to the presence of the O-acetyl group), to the one contained in the dOA PS. We speculate that the role of the O-acetyl group at the C-8 position of the PS on the organism is to form less immunogenic epitopes, or mask the protective epitope, and thus escape immune surveillance. The dOA form of the vaccine may therefore provide better protection against group C meningococcal disease than the OA form by eliciting a greater proportion of functional antibodies that are directly aimed at the protective epitope.