Neisseria meningitidis Factor H Binding Protein Surface Exposure on Salmonella Typhimurium GMMA Is Critical to Induce an Effective Immune Response against Both Diseases

Abstract

GMMA, outer membrane vesicles resulting from hyperblebbing mutated bacterial strains, are a versatile vaccine platform for displaying both homologous and heterologous antigens. Periplasmic expression is a popular technique for protein expression in the lumen of the blebs. However, the ability of internalized antigens to induce antibody responses has not been extensively investigated. Herein, the Neisseria meningitidis factor H binding protein (fHbp) was heterologously expressed in the lumen of O-antigen positive (OAg+) and O-antigen negative (OAg−) Salmonella Typhimurium GMMA. Only the OAg− GMMA induced an anti-fHbp IgG response in mice if formulated on Alum, although it was weak and much lower compared to the recombinant fHbp. The OAg− GMMA on Alum showed partial instability, with possible exposure of fHbp to the immune system. When we chemically conjugated fHbp to the surface of both OAg+ and OAg− GMMA, these constructs induced a stronger functional response compared to the fHbp immunization alone. Moreover, the OAg+ GMMA construct elicited a strong response against both the target antigens (fHbp and OAg), with no immune interference observed. This result suggests that antigen localization on GMMA surface can play a critical role in the induction of an effective immune response and can encourage the development of GMMA based vaccines delivering key protective antigens on their surface.

Novel Simple Conjugation Chemistries for Decoration of GMMA with Heterologous Antigens

“…For Pfs25 and R06C, the amount of linked protein antigen was determined via cELISA [ 70 , 71 ] as detailed below. For FdEc and SslE, quantification through amino acid analysis was performed, as described in [ 18 ].…”

“…cELISA assay was performed as previously described [ 70 , 71 ]. A standard curve was prepared by 10 sequential 2-fold or 1.4-fold dilution steps of protein antigens for Pfs25 or R06C respectively, starting from a known concentration (1.2 µg/mL for R06C and 120 µg/mL for Pfs25).…”

“…Percentage of active ester groups introduced was calculated using the following equation, with sulfo-NHS and total NH 2 concentrations expressed as µmol/mL and GMMA concentrations expressed as µg/mL: cELISA assay was performed as previously described [70,71]. A standard curve was prepared by 10 sequential 2-fold or 1.4-fold dilution steps of protein antigens for Pfs25 or R06C respectively, starting from a known concentration (1.2 µg/mL for R06C and 120 µg/mL for Pfs25).…”

Investigating the Role of Antigen Orientation on the Immune Response Elicited by Neisseria meningitidis Factor H Binding Protein on GMMA

“…The amount of fHbp linked to the GMMA surface in the two conjugates was estimated by competitive ELISA [ 34 ], working with anti-fHbp polyclonal mouse serum and using a GMMA naturally expressing fHbp and with a known amount of fHbp quantified by SRM mass spectroscopy for building the standard curve ( Figure S2 ). The amount of conjugated fHbp was 3.4% and 3.6% with respect to the total protein in the random and selective conjugates, respectively.…”

Immunobiology of Carbohydrates: Implications for Novel Vaccine and Adjuvant Design Against Infectious Diseases

“…GMMA platforms have been used for several O-antigen-based vaccine candidates, including Shigella sonnei , which have been tested in Phase 1 and 2 clinical trials showing to be well tolerated and immunogenic ( Launay et al., 2017 ; Obiero et al., 2017 ). GMMA can also be carriers for heterologous antigens, introduced either by genetic engineering ( Micoli and MacLennan, 2020 ) or by chemical conjugation ( Micoli et al., 2020a ; Necchi et al., 2021 ). To sum up, OMVs can be manipulated to express glycans or proteins from a pathogen of interest and their use allows simplified manufacturing processes, lower analytical controls, and overall reduced cost of production ( Kis et al., 2019 ).…”