S2). Anti-OAg IgM were detected only at day 42 for OAg-oxTEMPO conjugates (Fig. S3). After two doses, anti-CRM197 IgG responses obtained with OAg-oxTEMPO-CRM197 conjugates were higher than for the other groups, likely the result of the higher proportion of carrier protein present in these vaccines compared with the others (Table 1). After three doses, differences were significant only between OAg-oxTEMPO2h-CRM197, and both OAg-NH2-SIDEA-CRM197 and OAg-ADH-SIDEA-CRM197 (p = 0.0025) ( Fig. 4b). Sera collected at day 42 were pooled Adriamycin solubility dmso and tested for SBA against S. Typhimurium D23580, an invasive Malawian clinical

isolate [31]. All conjugates induced bactericidal antibodies with complete killing achieved with as little as 0.1 anti-OAg IgG ELISA units/mL ( Fig. 5a). PLX3397 cell line Bactericidal activity of sera from mice immunized with selective OAg-KDO conjugates was similar, regardless of the length of the spacer used, while all the random conjugates induced sera with greater bacterial growth inhibition per anti-OAg IgG ELISA unit than the selective conjugates. There was a trend for less bactericidal activity with increasing degree of OAg chain derivatization

of the random conjugates: the least derivatized OAg-oxTEMPO2h-CRM197 conjugate produced sera with the highest bactericidal activity. To evaluate possible differences in cell-surface binding, pooled sera at day 42 were tested by FACS against two S. Typhimurium invasive clinical isolates D23580 and Ke238. DNA ligase As shown in Fig. 5b, all sera could bind both strains, and greater antibody binding was found with random conjugates-sera. There is increasing awareness of the significance of NTS as a major public health concern in the developing world [1], [32] and [33]. While responsible for gastroenteritis in high-income countries, NTS is a common cause of fatal invasive disease in Africa. Currently no vaccines are available against this disease and glycoconjugation is a promising approach for vaccine development [34]. The conjugation chemistry used to synthesize a glycoconjugate vaccine can impact on its immunogenicity [15]. Here S. Typhimurium OAg-CRM197

conjugates obtained by random derivatization along the sugar chain were compared with conjugates obtained by one-site linkage at the terminus of the core region. For the random approach, a milder oxidation by TEMPO was compared to oxidation with NaIO4 which opens the sugar units with corresponding likely greater impact on OAg epitopes and conformation. Regarding the selective approach, two different lengths of the spacer present between the sugar and the protein were compared. From a process perspective, all conjugation methods resulted in no residual free protein, which is the most expensive component of the vaccine. The carrier protein did not need to be derivatized for both type of chemistries, but the production of random conjugates required one step less compared with the selective ones.