Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA)

Abstract

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.

Long-Term Anti-Bacterial Immunity against Systemic Infection by Salmonella enterica Serovar Typhimurium Elicited by a GMMA-Based Vaccine

“…Additionally, the OAg on the surface of bacteria or GMMA provides a shield which protects the bacteria from outer membrane protein specific antigens (unpublished data). A similar study has been done looking at the role of OAg and protein in S. sonnei GMMA [ 56 ].…”

GMMA-Based Vaccines: The Known and The Unknown

“…Indeed, the selection of the modifications on the lipid A acylation status are critical, as most of the genes implicated in LPS biosynthesis are necessary to preserve bacterial membrane stability and therefore viability. Genes that encode for acyltransferases such as HtrB or MsbB in Shigella ( 4 , 5 , 7 , 9 ), MsbB and PagP in Salmonella ( 8 ), or LpxL1 in N. meningitidis ( 16 , 17 , 35 ) have been mutated to generate GMMA with different penta-acylated lipid A forms.…”

“…The low reactogenicity was also confirmed by two in vivo models: a modified rabbit pyrogenicity test based on the intramuscular administration of the full human dose and a multiple dose toxicology study in rabbits using intramuscular, intranasal, and intradermal administration routes ( 5 ). Moreover, Shigella sonnei GMMA elicited in the animal model antibodies not only against the key antigen LPS, but also against GMMA proteins ( 9 ). In phase I and II clinical studies, the vaccine was well tolerated up to 100 μg following intramuscular (two or three doses), intradermal or intranasal administrations ( 48 ).…”

“…However, bacteria naturally release OMV but in relatively low amounts, and contain endotoxins in the natural form, which might cause systemic reactogenicity in humans depending on the vaccine dose used. Hence, in order to overcome issues of limited yield and to reduce the levels of endotoxicity, bacterial strains have been genetically modified to increase outer membrane vesiculation (4)(5)(6) and reduce LPS endotoxicity (7)(8)(9). We named the resulting vesicles, deriving from the over-vesiculating strain and with mutations in the LPS genes, Generalized Modules for Membrane Antigens (GMMA) whereas others called them mutant-derived or genetically detoxified OMV.…”

“…Indeed, the entire production process from fermentation to final purified GMMA lasts 3 days and thus, depending on the size of the vaccine dose, a relatively small manufacture facility with a 500 L fermenter could produce 100 000 000 doses of vaccines per year at a manufacturing cost of approximately $1 per dose (10). GMMA from Shigella (4,5,7,9,(11)(12)(13), Salmonella (8,14,15), and Neisseria (16,18) species have been already generated using this approach, which is shown to be flexible enough to be potentially extended with minimal adjustments to any Gram-negative bacterial species. Indeed multiple industrial (17)(18)(19) and research (20)(21)(22) approaches based on genetic engineering of bacteria for hyper-vesiculating and surface-expression of a variety of homologous and heterologous antigens, including bacterial (20)(21)(22), viral (23), parasitic (24) and even cancer antigens (25) have been described.…”