Novel Simple Conjugation Chemistries for Decoration of GMMA with Heterologous Antigens

Abstract

Outer Membrane Vesicles (OMV) constitute a promising platform for the development of efficient vaccines. OMV can be decorated with heterologous antigens (proteins or polysaccharides), becoming attractive novel carriers for the development of multicomponent vaccines. Chemical conjugation represents a tool for linking antigens, also from phylogenetically distant pathogens, to OMV. Here we develop two simple and widely applicable conjugation chemistries targeting proteins or lipopolysaccharides on the surface of Generalized Modules for Membrane Antigens (GMMA), OMV spontaneously released from Gram-negative bacteria mutated to increase vesicle yield and reduce potential reactogenicity. A Design of Experiment approach was used to identify optimal conditions for GMMA activation before conjugation, resulting in consistent processes and ensuring conjugation efficiency. Conjugates produced by both chemistries induced strong humoral response against the heterologous antigen and GMMA. Additionally, the use of the two orthogonal chemistries allowed to control the linkage of two different antigens on the same GMMA particle. This work supports the further advancement of this novel platform with great potential for the design of effective vaccines.

Keywords: GMMA; OMV; carrier protein; conjugation chemistry; glycoconjugate; vaccine.

Figure 1

Two novel simple conjugation chemistries developed for decoration of GMMA with heterologous antigens, using STm GMMA as models: (a) oxidation of LPS/LOS molecules on GMMA followed by reductive amination with the antigen of interest, (b) functionalization of proteins on GMMA with the BS3 linker followed by immediate reaction with the antigen of interest.

Figure 2

Identification of optimal conditions for GMMA oxidation: 2D surface contour plot for % GMMA oxidation (a–c) and OAg size (d–f) responses at pH of 5 (a,d), 6.5 (b,e) and 8 (c,f).

Figure 3

Impact of GMMA oxidation degree on GMMA immunogenicity in mice and conjugation efficiency. (a) Immunogenicity of oxidized STm GMMA compared to starting GMMA. CD1 mice were immunized subcutaneously at day 0 and 28 with 0.5 μg mannose dose. All constructs were formulated in saline. Sera were analyzed at days −1, 14, 27 and 42 by enzyme-linked immunosorbent assay (ELISA) using as coating antigen STm OAg. Summary graph of anti-antigen specific IgG geometric mean units (bars) and individual antibody levels (dots) is reported. (b) Characterization by WB analysis of the purified conjugates in comparison to corresponding unconjugated Pfs25. Ten µg of conjugates and 2 µg of protein were loaded per well. Lane 1: marker, lane 2: Pfs25, lane 3: conjugate with 26% oxidized GMMA, lane 4: conjugate with 14% oxidized GMMA, lane 5: conjugate with 60% oxidized GMMA.

Figure 4

Identification of optimal conditions for GMMA derivatization with BS3: 2D surface contour plot for % NH₂ activation (a–c) and % active ester groups (d–f) responses at pH 6 (a,d), 7.5 (b,e) and 9 (c,f).

Figure 5

Impact of GMMA-BS3 activation degree on conjugation efficiency. Conjugate characterization. (a) WB analysis of the purified conjugates in comparison to corresponding unconjugated protein. Ten µg of conjugates and 2 µg of protein were loaded per well. Lane 1: marker, lane 2: Pfs25, lane 3: conjugate with 32% activated GMMA-BS3, lane 4: conjugate with 11% activated GMMA-BS3. (b) dls analysis of GMMA (red line, Z average r of 52.0 nm, PdI of 0.209); GMMA-BS3 (32% activation) (green line, Z average r of 47.1 nm, PdI of 0.245) and GMMA-BS3-Pfs25 conjugate (blue line, Z average r of 49.8 nm, PdI of 0.329 nm), confirming no crosslinking after GMMA derivatization with BS3 linker and conjugation to Pfs25.

Figure 6

Immunogenicity of Pfs25 GMMA conjugates produced by reductive amination or BS3 chemistry in mice. CD1 mice were immunized subcutaneously at day 0 and 28 with 2.5 μg total protein dose. All constructs were formulated with 0.7 mg/mL Alhydrogel. Sera were analyzed at days −1, 14, 27 and 42 by ELISA using as coating antigens Pfs25 (a) or STm OAg (b). Summary graphs of anti-antigen specific IgG geometric mean units (bars) and individual antibody levels (dots) are reported.

Scheme 1

Comparison of the two conjugation approaches developed for linkage of heterologous protein antigens to GMMA surface.

Figure 7

Further simplification of the reductive amination chemistry by introducing a NaIO₄ quenching step. (a) Characterization by WB analysis of the conjugates in comparison to corresponding unconjugated Pfs25. Ten µg of conjugates and 2 µg of protein were loaded per well. Lane 1: marker, lane 2: Pfs25, lane 3: STm GMMAox-Pfs25, lane 4: STm GMMAox-Pfs25 quenched. (b,c) Comparison in mice of GMMAox-Pfs25 conjugates prepared with or without the quenching step. CD1 female mice were immunized subcutaneously at days 0 and 28 with 2.5 μg total protein dose. Both conjugates were formulated with 0.7 mg/mL Alhydrogel. Sera were analyzed at days −1, 14, 27 and 42 by ELISA using as coating antigens Pfs25 (b) or STm OAg (c). Summary graphs of anti-antigen specific IgG geometric mean units (bars) and individual antibody levels (dots) are reported.

Figure 8

GMMA conjugated to additional heterologous antigens (R06C and fHbp v2) and possibility of recycling the unconjugated protein verified: immunogenicity of R06C conjugates (reductive amination) (a) and fHbp v2 conjugates (BS3) (b) in mice. CD1 mice were immunized subcutaneously at day 0 and 28 with 4 μg R06C/dose (a), or 2.5 μg total protein/dose (b). All constructs were formulated with 0.7 mg/mL Alhydrogel. Sera were analyzed at days −1, 14, 27 and 42 by ELISA using as coating antigens R06C (a) or fHbp v2 (b). Summary graphs of anti-antigen specific IgG geometric mean units (bars) and individual antibody levels (dots) are reported.

Figure 9

Conjugation strategy used for generation of a bivalent conjugate: conjugation of FdeC on S. sonnei GMMA through BS3 chemistry, followed by oxidation and linkage to SslE by reductive amination.

Figure 10

SslE and FdeC presented on the same S. sonnei GMMA particle compared to proteins alone and to the corresponding monovalent conjugates mixed together. CD1 mice were immunized intramuscularly at day 0 and 28, with 10 µg E. coli protein/dose (group with proteins alone), 5 µg total protein/dose (groups with conjugates). All constructs were formulated with 0.7 mg/mL Alhydrogel. Sera were collected at days −1, 27 and 42 and analyzed by ELISA for anti-E. coli antigen-specific (a,b) and anti-S. sonnei LPS (c) IgG response. Summary graphs of anti-antigen specific IgG geometric mean units (bars) and individual antibody levels (dots) are reported.