Rapid generation of Shigella flexneri GMMA displaying natural or new and cross-reactive O-Antigens

Abstract

Generalized modules for membrane antigens (GMMA) are exosomes released from engineered Gram-negative bacteria and represent an attractive vaccine platform for the delivery of the O-Antigen (OAg), recognized as the key target for protective immunity against several pathogens such as Shigella. Shigella is a major cause of disease in Low- and Middle-Income countries and the development of a vaccine needs to deal with its large serotypic diversity. All S. flexneri serotypes, except serotype 6, share a conserved OAg backbone, corresponding to serotype Y. Here, a GMMA-producing S. flexneri scaffold strain displaying the OAg backbone was engineered with different OAg-modifying enzymes, either individually or in combinations. This strategy rapidly yielded GMMA displaying 12 natural serotypes and 16 novel serotypes expressing multiple epitopes combinations that do not occur in nature. Importantly, a candidate GMMA displaying a hybrid OAg elicited broadly cross-bactericidal antibodies against a large panel of S. flexneri serotypes.

Fig. 1. Conversion of serotype Y scaffold strain to different S. flexneri serotpes.

a Engineering strategy and resulting OAg structures as verified by ¹H-NMR. The numbers inside the blue circles refer to the position of the linkage of glucose to the RU. b FACS typing analysis of all strains with Denka Seiken monovalent antisera kit. Results are shown as a heat-map of log-transformed mean fluorescence intensities (MFI).

Fig. 2. Analytical and immunological comparison between GMMA from the converted scaffold strains and GMMA from the corresponding natural strains.

a ¹H-NMR spectra of OAg isolated from GMMA. Chemical shifts were assigned in the anomeric and O-acetyl regions (R = Rhamnose; GN = N-Acetyl Gucosamine; G = Glucose). b Bactericidal activity of sera raised in mice against the different GMMA. Serum dilutions able to kill 50% of bacteria in the assay (IC₅₀) are reported for individual mice (dots), error bars represent 95% confidence interval.

Fig. 3. Engineering of naturally non-occurring combinations of OAg-modifying enzymes.

a FACS typing analysis of all strains generated by transforming the serotype Y scaffold strain. b FACS typing analysis of all strains generated by transforming S. flexneri 3a. c Resulting OAg structures as verified by ¹H-NMR.

Fig. 4. Characterization and immunogenicity of GMMA displaying mixed or hybrid serotypes.

a Anomeric region of ¹H-NMR spectra of OAg isolated from GMMA (R = Rhamnose; GN = N-Acetyl Gucosamine; G = Glucose). Mixed or hybrid serotypes were compared to the corresponding individual serotypes. b Bactericidal activity of sera raised in mice by GMMA displaying mixed or hybrid serotypes compared to physical mixtures of GMMA displaying the corresponding individual serotypes. Serum dilutions able to kill 50% of bacteria in the assay (IC₅₀) are reported for individual mice (dots), error bars represent 95% confidence interval.

Fig. 5. Assessment of cross-reactivity induced by the hybrid 1+3 serotype.

Bactericidal activity of sera raised in mice by GMMA displaying hybrid 1+3 serotype against a panel of the most epidemiologically relevant S. flexneri serotypes. Pooled sera dilutions able to kill 50% of bacteria in the assay (IC50) are reported.