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

Abstract

GMMA are outer membrane vesicles (OMVs) released from Gram-negative bacteria genetically modified to enhance OMVs formation that have been shown to be optimal systems to enhance immunogenicity of protein antigens. Here, we selected Neisseria meningitidis factor H binding protein (fHbp) and used the conjugation chemistry as a tool to alter antigen orientation on GMMA. Indeed, fHbp was randomly linked to GMMA or selectively attached via the N-terminus to mimic native presentation of the protein on the bacterial surface. Interestingly, protein and peptide array analyses confirmed that antibodies induced by the selective and the random conjugates showed a pattern very similar to fHbp natively expressed on bacterial surfaces or to the recombinant protein mixed with GMMA, respectively. However, the two conjugates elicited antibodies with similar serum bactericidal activity against meningococcal strains, superior to the protein alone or physically mixed with GMMA. Presentation of fHbp on GMMA strongly enhances the functional immune response elicited by the protein but its orientation on the bacterial surface does not have an impact. This study demonstrates the flexibility of the GMMA platform as a display and delivery system for enhancing antigen immunogenicity and further supports the use of such promising technology for the development of effective vaccines.

Keywords: GMMA; Neisseria meningitidis; conjugation; outer membrane vesicles.

Figure 1

(a) Conjugation scheme for random linkage of fHbp to GMMA: fHbp was derivatized with EMCS linker followed by chemical conjugation to GMMA functionalized with SH linker; (b) MALDI-MS analysis of fHbp-maleimido compared to starting fHbp; (c) DSC analysis of fHbp-maleimido compared to starting fHbp; (d) fHbp structure highlighting lysine residues involved in the derivatization with EMCS linker as identified by peptide mapping analysis.

Figure 2

(a) Conjugation scheme for random linkage of fHbp to GMMA (fHbp derivatized with SH linker and chemically conjugated to GMMA functionalized with maleimido linker) or selective linkage of fHbp terminally mutated to have a Cys residue to GMMA-maleimido; (b) SDS-PAGE (left panel)/WB (right panel) analysis of fHbp randomly derivatized with -SH or terminally mutated with a Cys residue; (c) WB analysis of fHbp, random and selective conjugates and a physical mixture of GMMA with fHbp-NCys.

Figure 3

Protein microarray reactivity profile of pooled sera collected two weeks after the third immunization (day 35) with fHbp constructs, normalized according to ELISA titers. Each horizontal bar represents a single protein or protein fragment spotted in the microarray for fHbp v3.28 and color changes from light gray to dark red according to increasing mean fluorescence intensity (MFI) values, as shown in the vertical bar.

Figure 4

Immunogenicity study of random and selective GMMA-fHbp conjugates compared to GMMA OE fHbp, fHbp alone or physically mixed with GMMA 4KO. Eight 6-week-old female CD1 mice per group were i.p. immunized three times at days 0, 21 and 35. Summary graphs of anti-fHbp IgG response 3 weeks post first immunization (a) and 2 weeks after the third immunization (b): geometric mean units (bars) and individual antibody levels are reported together with corresponding serum bactericidal assay (SBA) titers of pooled sera for each group against M01-240320 MenB strain, expressing fHbp v3.45; (c) SBA titers of individual sera two weeks after the third immunization measured for selected groups against M01-240320 and M1239 MenB strains, expressing fHbp v3.45 and fHbp v3.28, respectively.