Di-, tri-, and tetrasaccharides, synthesized according to the chemical structure of pneumococcal polysaccharide type 3 (PS3), were coupled to the cross-reactive material (CRM 197 ) of modified diphtheria toxin in different molar carbohydrate/protein ratios using the squarate coupling method. To study protective immunity, female BALB/c mice were subcutaneously immunized twice (with a 3-week interval) using the amount of conjugates corresponding to 2.5 g of oligosaccharide per mouse. The conjugates evoked PS3 binding immunoglobulin G antibodies that lasted for at least 7 weeks after the booster. Immunogenicity was not influenced by the carbohydrate/protein ratio. All mice with PS3-specific antibodies survived the intraperitoneal challenge with Streptococcus pneumoniae type 3. Therefore, synthetic oligosaccharide-protein conjugates might have potential as vaccines.The encapsulated bacterium Streptococcus pneumoniae is still a major cause of upper and lower respiratory tract infections. About 23 serotypes of the ca. 90 known serotypes cause the majority (ϳ90%) of pneumococcal infections, such as otitis media, pneumonia, and meningitis (3). S. pneumoniae type 3 strains are related to invasive pneumococcal infection in adults (1, 3) and are often used in experimental meningitis (7) and otitis media models (5) in rabbits and rats. Furthermore, because of its high virulence in mice, S. pneumoniae type 3 offers a good model to study protective immunogenic properties of candidate vaccines (6,12,13).Protection against encapsulated bacteria is primarily mediated by anticapsular antibodies. However, capsular polysaccharides are thymus-independent type 2 antigens and thus induce low-affinity antibodies that display a limited subclass distribution. These antigens evoke no B-cell memory, either. Vaccines consisting of polysaccharides coupled to a protein carrier can circumvent these disadvantages with an increased antibody response to capsular polysaccharides (2,9,14).Neoglycoprotein preparations consisting of polysaccharide or oligosaccharide fragments obtained by degradation of the polysaccharides are sometimes contaminated with other pneumococcal components. They also have an ill-defined structure due to multiple coupling sites or the use of oligosaccharide pools of different chain length, and they lose their reducing end upon conjugation to a carrier. The use of small chemically synthesized oligosaccharides results in a precisely defined conjugate, thereby offering a possibility to evaluate the immunogenic properties of a conjugate vaccine by varying its specific structural parameters, for example, the length of the saccharide fragment and the carbohydrate/protein ratio.Oligosaccharide-protein conjugates were prepared as follows. The pure synthetic monosaccharides Kamerling, and J. F. G. Vliegenthart, submitted for publication), representing fragments of the repeating unit of the type 3 polysaccharide (PS3), were conjugated to the cross-reactive material (CRM 197 ) of modified diphtheria toxin in different molar carbohydr...