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S. pneumoniae (SP) serotypes 15B and 15C are frequent causative agents of invasive pneumococcal disease (IPD), otitis media, and nasopharyngeal colonization in the post PCV13 era. The principal difference between serotypes 15B and 15C is the presence of an O-acetyl group on the pentasaccharide repeating unit of 15B polysaccharide. It remains unclear if antibodies against SP15B polysaccharide demonstrate functional cross reaction with SP15C strains. We compared functional activity of polyclonal rabbit anti-capsular 15B sera against SP15B and SP15C isolates. Using flow cytometry we measured complement factors C3c and C4d deposition on SP15B and 15C in the presence of polyclonal rabbit anti capsular 15B sera. We measured the binding of C3c, common to all complement pathways, and C4d, specific to classical pathway, on SP serotypes 15B and 15C when co-incubated with polyclonal rabbit anti capsular 15B sera and antibody depleted complement. Both the proportion of bacteria with complement deposition and the fluorescence intensity were measured. We also measured agglutination as the increase in forward and side scatter. Polyclonal rabbit anti-capsular 15B sera activated classical pathway resulting in deposition of C4d and C3c at high intensity on all SP15B cells but only achieved limited deposition and intensity of C4 with SP15C. Similarly, polyclonal rabbit anti-capsular 15B sera induced agglutination of SP15B strains in a dose dependent manner and limited agglutination of SP15C. Anti-capsular 15B sera induce limited C4d and C3c deposition, and minimal agglutination with SP15C strains, reflecting lower classical pathway activation in contrast to high C4d and C3c deposition and agglutination of SP15B. These observations support limited functional cross reactivity of anti-15B to SP15C strains and are consistent with the reduction in opsonophagocytic killing of SP15C reported following immunization with vaccines containing 15B polysaccharide. •Antibodies against SP serotype 15B activated both classical and alternative pathway of complement activation on the surface of SP15B.•15B antibodies showed relatively less complement binding and agglutination with 15C compared with 15B.•Study suggest antibodies against SP15B may not confer protection against SP 15C by complement dependent killing.

•A correlate of protection for prevention of colonization in children for serotypes 15B and 15C.•Antibody levels correlated poorly with opsonophagocytic titers.•Low functional cross-reactivity between serotypes 15B and 15C in child sera was observed. Serotypes 15B and 15C have been added to new different pneumococcal-conjugate vaccines (PCV20 and V116, respectively). We determined a serum anti-15B antibody level that would be a correlate of protection (COP) against nasopharyngeal colonization and assessed functional cross-reactivity against serotype 15B and 15C in children following natural immunization. IgG-antibody to serotype 15B polysaccharide was measured by ELISA in 341 sera from 6 to 36 month old children collected before, at the time of, and after pneumococcal colonization caused by serotypes 15B and 15C. 155 age-matched controls who had no detected colonization caused by serotype 15B or 15C strains were used as controls. A two-step method was used for construction of COP models: a generalized estimating equation followed by logistic-regression. Opsonophagocytic (OPA) assays assessed functional cross-reactivity between serotypes 15B and 15C. The derived COP for prevention of colonization was 1.18 µg/ml for serotype 15B and 0.63 µg/ml for serotype 15C, with a predictive probability of 80 %. Antibody levels did not correlate with OPA titers. 30 % of child samples, with moderate to high amounts of ELISA-measured antibody, showed no OPA titer against either serotype 15B or 15C. For remaining samples, very low or no functional cross-reactivity between serotypes 15B and 15C was measured. A COP for prevention of colonization in young children based on naturally-induced antibody levels was derived for serotypes 15B and 15C that differed. Antibody levels correlated poorly with OPA titers and low functional cross-reactivity between serotypes 15B and 15C in child sera was observed.

Background/Objectives: Polysaccharide-protein conjugate vaccines have proven highly effective, yet they remain limited by manufacturing complexity, cost, and variable performance across serotypes, while carrier proteins can add unwanted immunological and production burdens. To address these constraints, we explored a carrier-protein-free conjugate vaccine concept in which a broadly MHC class II-binding helper epitope (PADRE) replaces the conventional protein carrier to provide T-cell help for a pneumococcal capsular polysaccharide antigen. Methods: Using serotype 15C CPS as a model, we generated CPS–PADRE conjugates and compared designs with or without a putative cleavable motif (RR) at the junction, alongside a conventional protein conjugate as a benchmark. Results: In mice, the CPS–protein conjugate induced the strongest CPS-specific IgG response, whereas CPS–PADRE conjugates elicited clear but overall lower antibody levels. Notably, incorporation of the cleavable motif did not improve immunogenicity and instead reduced humoral responses relative to the non-cleavable design. Conclusion: These findings support the feasibility of carrier-protein-free polysaccharide-peptide conjugate vaccines, while highlighting that cleavable junctions are not universally advantageous and must be empirically optimized for polysaccharide-helper epitope architectures.

Forty three isoniazid (INH)-resistant Mycobacterium tuberculosis isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA -15C → T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity). Typing for resistance mutations was performed by Multiplex Allele-Specific PCR and sequencing reaction. Mutations at either codon were detected in 67.5% of isolates: katG315 in 37.2, mabA -15C → T in 27.9 and both of them in 2.4%, respectively. katG sequencing showed a G insertion at codon 325 detected in 2 strains and leading to amino acid change T326D which has not been previously reported. Distribution of each mutation, among the investigated strains, showed that katG S315T was associated with multiple-drug profile, high-level INH resistance and loss or decreased catalase activity; whereas the mabA -15C → T was more prevalent in mono-INH resistant isolates, but it was not only associated with a low-level INH resistance. It seems that determination of catalase activity aids in the detection of isolates for which MICs are high and could, in conjunction with molecular methods, provide rapid detection of most clinical INH-resistant strains.