Decoration of outer membrane vesicles with multiple antigens by using an autotransporter approach

Abstract

Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

FIG 1

Hbp derivatives used for display at the OMV surface. (A) Wild-type Hbp (included for reference) is composed of three domains: (i) an N-terminal cleavable signal sequence (ss), (ii) a secreted passenger domain, and (iii) a C-terminal β-domain that becomes integrated into the OM. Side domains d1 to d5 and the autochaperone domain (ac) that is involved in OM translocation of the passenger domain are indicated, while the remainder of the passenger domain is black. The mutation that prevents autocatalytic intradomain cleavage and concomitant release of the passenger, resulting in a surface-exposed (display) version of Hbp, is marked X. Numbers above the diagrams correspond to the amino acid positions in wild-type Hbp. Insertion of a 9- to 11-aa flexible linker (FL) comprising Gly and Ser residues and insertions of the mycobacterial antigens ESAT6 (E-6), an N-terminal fragment (aa 1 to 126; 85_N) and a C-terminal (aa 118 to 285; 85_C) fragment of Ag85B, Rv2660c (26), and the internal fragments MOMPIV (aa 266 to 350; IV) and MOMPII (aa 155 to 190; II) of C. trachomatis MOMP are indicated. All inserts are flanked by short Gly/Ser linkers. (B) Schematic representation of Hbp-mediated antigen display on OMVs, exemplified by HbpD-Ag85B_C+N-ESAT6-Rv2660c. Ag85B, split into N-terminal and C-terminal fragments, ESAT6, and Rv2660c are fused to the Hbp passenger by replacement of side domains d2, d1, d4, and d5, respectively. Upon production in hypervesiculating E. coli or Salmonella strains, the Hbp chimera, which localizes in the bacterial outer membrane (OM), becomes incorporated in and displayed at the surface of the newly formed outer membrane vesicles (OMV). IM, inner membrane. The figure was made using Servier Medical Art and the crystal structures of the Hbp passenger (blue) and β-domain (red) (32, 66).

FIG 2

Targeting of (multiple) heterologous proteins to E. coli OMVs by fusion to the passenger of Hbp. (A) SDS-PAGE/Coomassie analysis of fractions containing an equivalent of 0.1 OD₆₆₀ unit of cells, culture medium, secreted soluble proteins (medium 2), and 3 OD₆₆₀ units of crude OMVs from E. coli JC8031 expressing HbpD-Δd1, HbpD-ESAT6, HbpD-Ag85B_C+N-ESAT6, and HbpD-Ag85B_C+N-ESAT6-Rv2660c in the presence of 50 μM IPTG or harboring the empty vector (EV). Hbp proteins (chimeras) are marked by asterisks, and arrowheads indicate the bands corresponding to PDH-E1, GroEL, OmpF/C, and OmpA. (B) The OMV samples from panel A were analyzed by immunoblotting using antibodies recognizing the β-domain of Hbp (αβ-dom), ESAT6, Ag85B, Rv2660c, and OmpA. (C and D) SDS-PAGE/Coomassie analysis of equal volumes of all fractions obtained after velocity density gradient centrifugation of crude OMVs containing HbpD-Δd1(C) or HbpD-ESAT6 (D). The concentration of OptiPrep (%) and bands corresponding to HbpD-Δd1, HbpD-ESAT6, PDH-E1, GroEL, OmpF/C, and OmpA are indicated. α, anti.

FIG 3

Surface labeling, shape, and size of E. coli OMVs containing Hbp (chimeras). (A and B) Intact OMVs isolated from JC8031 producing HbpD-Δd1 (A) or harboring the empty vector (B) were fixed and probed with anti-Hbp antibodies, followed by 10-nm gold-labeled secondary antibodies, and analyzed by electron microscopy. Scale bar, 50 nm. (C) Representative curves of the size distribution for OMVs isolated from JC8031 carrying the empty vector (EV) or producing HbpD-Ag85B_C+N-ESAT6-Rv2660c, analyzed by number. d, diameter.

FIG 4

Surface localization and effect on vesicle protein composition of Hbp (chimeras) in E. coli OMVs. (A and B) Equal amounts of intact OMVs (−tx) or OMVs permeabilized with Triton X-100 (+tx) from E. coli JC8031 harboring the empty vector (EV) or producing Hbp display constructs (described in the legend of Fig. 1) in the presence of 50 μM (A) or 1 mM (B) IPTG were incubated with proteinase K (+pk) or mock treated (−pk), separated by SDS-PAGE, and stained with Coomassie. Asterisks, Hbp (chimeras); filled circles, PDH-E1; open circles, GroEL, arrowheads, OmpF/C, OmpA, and proteinase K. (C) Immunoblot analysis of fractions containing an equivalent of 0.1 OD unit of whole cells, 0.1 OD unit of soluble material (medium 2), or 3 OD units of insoluble material (OMVs) obtained after ultracentrifugation of cell-free culture supernatants from E. coli JC8031 cells harboring the empty vector or producing HbpD-Δd1 or HbpD-ESAT6. The periplasmic proteins SurA and Skp, the inner membrane proteins Lep and SecG, and the cytoplasmic proteins TF, DnaJ, and RNA polymerase were detected with specific antibodies.

FIG 5

Display of M. tuberculosis antigens at the surface of OMVs derived from an OMV-overproducing strain of S. Typhimurium. (A) SDS-PAGE/Coomassie analysis of an equivalent of 0.05 OD₆₆₀ unit of cell pellets (P) and 10-fold more culture supernatants (S) from S. Typhimurium SL3261 (wt) and an isogenic OMV-overproducing mutant constructed by deletion of tolR and tolA (ΔtolRA). (B and C) Crude OMVs isolated from SL3261 ΔtolRA producing Hbp (chimeras) in the presence of 100 μM IPTG (HbpD-Δd1 and HbpD-ESAT6) or 50 μM IPTG (HbpD-Ag85B_C+N-ESAT6 and HbpD-Ag85B_C+N-ESAT6-Rv2660c) or harboring the empty vector (EV) were separated by SDS-PAGE and analyzed by Coomassie-staining (B) and immunoblotting using antibodies specific for the β-domain of Hbp (αβ-dom) and ESAT6 (C). (D) Proteinase K accessibility of OMVs described in panels B and C. Equivalent amounts of intact OMVs (−tx) or OMVs permeabilized with Triton X-100 (+tx), were incubated with proteinase K (+pk) or mock treated (−pk) and analyzed by SDS-PAGE/Coomassie staining. Full-length Hbp species are marked with asterisks, and OmpF/C, OmpA, and proteinase K are indicated with arrowheads.

FIG 6

Display of C. trachomatis MOMP fragments at the surface of Salmonella OMVs. Equivalent amounts of OMVs isolated from S. Typhimurium SL3261 ΔtolRA producing HbpD-Δd1 or HbpD-MOMPIV-MOMPII in the presence of 100 μM IPTG or carrying the empty vector (EV) were left intact (−tx) or permeabilized with Triton X-100 (+tx), prior to incubation with proteinase K (+pk) or mock treatment (−pk). Samples were analyzed by SDS-PAGE/Coomassie staining (A) and immunoblotting using antiserum recognizing the β-domain of Hbp (αβ-dom), MOMP, and OmpA (B). Full-length Hbp species are marked with asterisks, and OmpF/C, OmpA, and proteinase K are indicated with arrowheads.

FIG 7

Processing and presentation of M. tuberculosis antigens displayed at the surface of S. Typhimurium OMVs to the MHC class II pathway. C57BL/6 (H-2^b) BMDCs were incubated with OMVs isolated from S. Typhimurium SL3261 ΔtolRA producing Hbp (chimeras) or harboring the empty vector (EV). The OMVs were added at different dilutions, expressed as the concentration of Hbp-antigen fusion (Ag) in the vesicle preparations. The BMDCs were washed and incubated with I-A^b-restricted Ag85B_281–300-specific DE10 T cell hybridoma cells. The production of IL-2 was measured by enzyme-linked immunosorbent assay. The results are presented as means of two replicates ± standard deviations (error bars) and are representative of three experiments.