Shigella infections are one of the top causes of moderate and severe diarrhea throughout the world and no vaccine is widely available. The serotype-specific O-antigen (OAg) moiety of Shigella lipopolysaccharide has been recognized as a key target for protective immunity and different OAg-based vaccines are under development. Here we present our glycoconjugate approach for the development of an OAg-based candidate vaccine against S. flexneri 6. In particular, we focused on the impact of the OAg length on the induced immune response.
The OAg biosynthesis involves a first step of sugar polymerization during which the OAg chain length is regulated to achieve specific modal lengths. Indeed, analysis of different S. flexneri 6 strains have confirmed the display of a low molecular mass (LMM) OAg, a medium molecular mass (MMM) OAg and a high molecular mass OAg capsule (CPS), with average size of 1, 23 and 175 kDa respectively. The single OAg populations were isolated and separately conjugated to CRM197. All polysaccharide populations shared the expected structure, consisting of a tetrasaccharide repeat containing one N-acetylgalactosamine (GalNAc), one galacturonic acid (GalA) and two rhamnose residues (RhaII-RhaIII), with RhaIII being variably O-acetylated on C3 or C4 position.
CPS was conjugated to CRM197 through a random chemistry targeting multiple points along the saccharide chain. On the contrary, a selective approach was used for terminal linkage of LMM OAg to CRM197, not to alter OAg epitopes. Both chemistries were used to conjugate MMM OAg to CRM197 in order to verify no impact of conjugation chemistry on the immune response induced. The CPS was also de-Oacetylated before conjugation, to verify the role that the O-acetyl groups can have on the immune response induced by S. flexneri 6 OAg. A panel of analytical tools was optimized to fully characterize all the resulting conjugates.
Independently from chain length, O-acetylation and conjugation chemistry, all the conjugates were able to induce high levels of anti-OAg specific IgG titers with bactericidal activity against the homologous strain in mice. However, compared to the other candidate vaccines, four weeks after the first dose, LMM OAg conjugate induced significantly lower IgG titers, which were significantly boosted two weeks after the second injection, reaching levels similar to those induced by the other conjugates. Furthermore, MMM and LMM OAg, but not CPS OAg, induced a pure T-dependent response, as verified in T-cell knockout mice, with possible positive impact on memory and persistency of the antibody response.
This work contributes to identify optimal design of a glycoconjugate vaccine against S. flexneri 6 and confirms the critical role that sugar length can have on the immune response induced by glycoconjugate vaccines.
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