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The disaccharide (β-D-glucopyranosyluronic acid)-(1→4)-β-D-glucopyranoside represents a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 3. A conjugate of the disaccharide with BSA (di-BSA conjugate) adjuvanted with aluminum hydroxide induced - in contrast to the non-adjuvanted conjugate - IgG1 antibody production and protected mice against S. pneumoniae serotype 3 infection after intraperitoneal prime-boost immunization. Adjuvanted and non-adjuvanted conjugates induced production of Th1 (IFNγ, TNFα); Th2 (IL-5, IL-13); Th17 (IL-17A), Th1/Th17 (IL-22), and Th2/Th17 cytokines (IL-21) after immunization. The concentration of cytokines in mice sera was higher in response to the adjuvanted conjugate, with the highest level of IL-17A production after the prime and boost immunizations. In contrast, the non-adjuvanted conjugate elicited only weak production of IL-17A, which gradually decreased after the second immunization. After boost immunization of mice with the adjuvanted di-BSA conjugate, there was a significant increase in the number of CD45+/CD19+ B cells, TCR+ γδ T cell, CD5+ В1 cells, and activated cells with MHC II+ expression in the spleens of the mice. IL-17A, TCR+ γδ T cells, and CD5+ В1 cells play a crucial role in preventing pneumococcal infection, but can also contribute to autoimmune diseases. Immunization with the adjuvanted and non-adjuvanted di-BSA conjugate did not elicit autoantibodies against double-stranded DNA targeting cell nuclei in mice. Thus, the molecular and cellular markers associated with antibody production and protective activity in response to immunization with the di-BSA conjugate adjuvanted with aluminum hydroxide are IL-17A, TCR+ γδ T cells, and CD5+ В1 cells against the background of increasing MHC II+ expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Akhmatova, Kurbatova, Zaytsev, Akhmatova, Yastrebova, Sukhova, Yashunsky, Tsvetkov and Nifantiev.)

A number of studies have demonstrated the limited efficacy of S. pneumoniae type 3 capsular polysaccharide (CP) in the 13-valent pneumococcal conjugate vaccine against serotype 3 invasive pneumococcal diseases and carriage. Synthetic oligosaccharides (OSs) may provide an alternative to CPs for development of novel conjugated pneumococcal vaccines and diagnostic test systems. A comparative immunological study of di-, tri-, and tetra-bovine serum albumin (BSA) conjugates was performed. All oligosaccharides conjugated with biotin and immobilized on streptavidin-coated plates stimulated production of IL-1 α , IL-2, IL-4, IL-5, IL-10, IFN γ , IL-17A, and TNFα, but not IL-6 and GM-CSF in monocultured mice splenocytes. The tetrasaccharide-biotin conjugate stimulated the highest levels of IL-4, IL-5, IL-10, and IFN γ , which regulate expression of specific immunoglobulin isotypes. The tetra-BSA conjugate adjuvanted with aluminum hydroxide elicited high levels of IgM, IgG1, IgG2a, and IgG2b antibodies (Abs). Anti-CP-induced Abs could only be measured using the biotinylated tetrasaccharide. The tetrasaccharide ligand possessed the highest binding capacity for anti-OS and antibacterial IgG Abs in immune sera. Sera to the tetra-BSA conjugate promoted greater phagocytosis of bacteria by neutrophils and monocytes than the CRM 197 -CP-antisera. Sera of mice immunized with the tetra-BSA conjugate exhibited the highest titer of anti-CP IgG1 Abs compared with sera of mice inoculated with the same doses of di- and tri-BSA conjugates. Upon intraperitoneal challenge with lethal doses of S. pneumoniae type 3, the tri- and tetra-BSA conjugates protected mice more significantly than the di-BSA conjugate. Therefore, it may be concluded that the tetrasaccharide ligand is an optimal candidate for development of a semi-synthetic vaccine against S. pneumoniae type 3 and diagnostic test systems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Kurbatova, Akhmatova, Zaytsev, Akhmatova, Egorova, Yastrebova, Sukhova, Yashunsky, Tsvetkov and Nifantiev.)