Procedure for the conjugation of the Streptococcus pneumoniae serotype 6B capsular polysaccharide to the tetanus toxoid

Streptococcus pneumoniae causes annually 826 000 deaths in children under fi ve years. The serotype 6B, one of higher incidence, is targeted by the Cuban research and development project to develop a conjugate vaccine. There is limited data on how modifi cations caused by conjugation affect the physicochemical and antigenic characteristics of polysaccharides, particularly for serotype 6B capsular polysaccharide (PS6B), despite being the least immunogenic among S. pneumoniae polysaccharides. In this work, a conjugation procedure was established for PS6B comprising: fragmentation by acid hydrolysis, activation by periodate oxidation, and conjugation to tetanus toxoid (TT) by reductive amination to increase its immunogenicity. Reaction conditions were set to obtain the polysaccharide in three molecular size ranges (1-10, 10-30, 30-100 kDa) and levels of oxidation. PS6B fragmentation below 10 kDa and oxidation above 24 % of the repetitive units implied the loss of antigenicity. Polysaccharide length but not oxidation level had an impact on the physicochemical characteristics of the conjugates in the tested conditions. Unlike the native polysaccharide, conjugated 10-30 kDa and 30-100 kDa PS6B were immunogenic in rabbits, with evidence of thymus-dependent response. The procedure described supports obtaining PS6B-TT conjugates reproducibly in the 30100 kDa and 10-30 kDa molecular size ranges and with 8-18 % oxidized repeat units, which are immunogenic.