Comparison of Shigella GMMA and glycoconjugate four-component formulations in animals

Abstract

Shigellosis is leading bacterial cause of diarrhea with high prevalence in children younger than 5 years in low- and middle-income countries, and increasing number of reports of Shigella cases associated to anti-microbial resistance. No vaccines against Shigella are still licensed, but different candidates based on the O-antigen portion of lipopolysaccharides are in clinic. Generalized Modules for Membrane Antigens (GMMA) have been proposed as an alternative delivery system for the O-antigen, and a 4-component vaccine candidate (altSonflex1-2-3), containing GMMA from S. sonnei and S. flexneri 1b, 2a and 3a is being tested in a phase 1/2 clinical trial, with the aim to elicit broad protection against the most prevalent Shigella serotypes. Here, the 4-component GMMA vaccine candidate has been compared to a more traditional glycoconjugate formulation for the ability to induce functional antibodies in mice and rabbits. In mice, in the absence of Alhydrogel, GMMA induce higher IgG antibodies than glycoconjugates and stronger bactericidal titers against all Shigella serotypes. In the presence of Alhydrogel, GMMA induce O-antigen specific IgG levels similar to traditional glycoconjugates, but with a broader range of IgG subclasses, resulting in stronger bactericidal activity. In rabbits, GMMA elicit higher functional antibodies than glycoconjugates against S. sonnei, and similar responses to S. flexneri 1b, 2a and 3a, independently from the presence of Alhydrogel. Different O-antigen based vaccines against Shigella are now in clinical stage and it will be of particular interest to understand how the preclinical findings in the different animal models translate in humans.

Keywords: GMMA; O-antigen; Shigella; glycoconjugate; multicomponent; vaccine.

FIGURE 1

Purified S. sonnei, S. flexneri 1b, 2a and 3a OAg extracted from GMMA by acetic acid hydrolysis were conjugated to CRM₁₉₇ through random activation of hydroxyl groups along the polysaccharide chain using the cyanilating agent CDAP, followed by linkage with lysines on the carrier protein.

FIGURE 2

Comparison of GMMA and glycoconjugate 4-component formulations in mice in the absence (A,B) or presence (C,D) of Alhydrogel. CD1 mice were immunized intraperitoneally (i.p.) at day 0 and 28 with 9 ng, 37 ng, 150 ng and 600 ng total OAg dose. If present, concentration of Alhydrogel was 0.7 mg/ml (Al3+). Results at 150 ng total OAg dose are reported here as representative of the other doses. Sera were analyzed by (A,C) ELISA for LPS-specific (S. sonnei) or OAg-specific (S. flexneri) total IgG (EU/mL) and (B,D) serum bactericidal activity (SBA) assay for bactericidal titers expressed as IC50. Summary graphs of geometric mean units (bars) and individual levels (dots) are reported. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Characterization of the quality of the humoral response elicited by GMMA and glycoconjugates in mice. CD1 mice were immunized i. p. at day 0 and 28 with 150 ng total OAg dose in absence or presence of Alhydrogel. Anti-OAg-specific IgG subclasses have been evaluated at day 42. Relative percentage of each specific IgG subclass in respect to total IgG (ratio of geometric means) is reported in different colors in the bar plot for each formulation tested.

FIGURE 4

Comparison of GMMA and glycoconjugates 4-component formulations in rabbits in the absence (A,B) or presence (C,D) of Alhydrogel. New Zealand rabbits were immunized intramuscolarly (i.m.) at day 0 and 28 with 600 ng total OAg per dose. If present, concentration of Alhydrogel was 0.7 mg/ml (Al3+). Sera were analyzed by (A,C) ELISA for LPS-specific (S. sonnei) or OAg-specific (S. flexneri) total IgG (EU/mL) and (B,D) SBA for bactericidal titers expressed as IC50. Summary graphs of geometric mean units (bars) and individual levels (dots) are reported. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

Comparison of two different immunization schemes for GMMA and glycoconjugate 4-component formulations in rabbits. New Zealand rabbits were immunized i. m. at day 0 and 28 (0–1 month) or 0 and 84 (0–3 months) with 600 ng total OAg per dose in presence of 0.7 mg/ml of Alhydrogel (Al3+). Sera were analyzed by (A,C) ELISA for LPS-specific (S. sonnei) or OAg-specific (S. flexneri) total IgG (EU/mL) pre second vaccination (at day 27 or 83) and post second vaccination (at day 42 or 98) and by (B,D) SBA for bactericidal titers expressed as IC50 post second vaccination only. Summary graphs of geometric mean units (bars) and individual levels (dots) are reported. *p < 0.05; **p < 0.01; ***p < 0.001.