A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

Abstract

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

Fig. 1. A modular platform for rapid self-assembly of OMV-based vaccine candidates.

a Schematic of AvidVax technology whereby ready-made OMVs displaying a synthetic antigen receptor (SNAP-OMVs) are remodeled with biotinylated antigens-of-interest. Using AvidVax, the surface of SNAP-OMVs can be remodeled with virtually any biomolecule that is amenable to biotinylation including peptides, proteins, carbohydrates, glycolipids, glycoproteins, haptens, lipids, and nucleic acids. Schematic created with BioRender.com. b Genetic architecture of SNAP constructs tested in this study. Numbers in parentheses denote amino acids of the scaffold that were fused to the biotin-binding eMA domain and used for membrane anchoring. Additional features include: export signal peptide from PelB (spPelB); c-Myc epitope tag (M); FLAG epitope tag (F), and NdeI, Xhol, SphI, and NcoI restriction enzyme sites used for cloning.

Fig. 2. Chimeric Lpp-OmpA-eMA SNAP enables controllable antigen loading on OMVs.

a Dose-response curve generated by loading biotin-GFP (b-GFP) or unmodified GFP (GFP) on SNAP-OMVs isolated from hypervesiculating E. coli strain KPM404 ΔnlpI expressing the Lpp-OmpA-eMA construct from plasmid pTrham (induced with 0.5 mM L-rhamnose). Blank OMVs were isolated from plasmid-free KPM404 ΔnlpI cells. Binding activity was determined by ELISA in which Lpp-OmpA-eMA SNAP-OMVs were immobilized on plates and subjected to varying amounts of biotin-GFP, after which plates were extensively washed prior to detection of bound biotin-GFP using anti-polyhistidine antibody to detect C-terminal 6xHis tag on GFP. Data were normalized to the maximum binding signal corresponding to Lpp-OmpA-eMA SNAP-OMVs in the presence of 3.3 nM biotin-GFP. Data are the mean ± SD with n = 2 biologically independent experiments. b Same OMVs as in (a) but dose-response was generated by first incubating OMVs with biotin-GFP or unmodified GFP in solution, washing to remove unbound protein, and determining GFP levels by ELISA-based detection using a standard curve with known amounts of GFP mixed with OMVs. Data are the mean ± SD with n = 3 biologically independent experiments. c Comparison of GFP levels on Lpp-OmpA-eMA SNAP-OMVs versus ClyA-GFP OMVs. ClyA-GFP OMVs were isolated from KPM404 ΔnlpI cells expressing ClyA-GFP fusion construct from plasmid pBAD18 as described in Kim et al.. Data are the mean ± SD with n = 6 biologically independent experiments for all cases except for ClyA-GFP OMVs where n = 3 biologically independent experiments. d Transmission electron micrograph of Lpp-OmpA-eMA SNAP-OMVs alone or following incubation with unmodified GFP or biotin-GFP as indicated. The scale bar represents 200 nm. The Z-average diameter of each formulation was measured by DLS and reported below each corresponding micrograph. Micrographs representative of two independently repeated experiments with similar results. Source data are provided as a Source Data file.

Fig. 3. Assembly of OMV vaccine candidates decorated with diverse biomolecular antigens.

a, b Dose–response curves generated by loading biotinylated or unbiotinylated antigens on SNAP-OMVs isolated from hypervesiculating KPM404 ΔnlpI cells expressing the Lpp-OmpA-eMA construct from plasmid pTrham (induced with 0.5 mM L-rhamnose). Blank OMVs were isolated from plasmid-free KPM404 ΔnlpI cells. Binding activity was determined by ELISA in which Lpp-OmpA-eMA SNAP-OMVs were immobilized on plates and subjected to varying amounts of unbiotinylated or biotinylated antigen, after which plates were extensively washed prior to detection of bound antigen using the antibodies indicated at top of each panel. Data were normalized to the maximum binding signal in each experiment. Data are the mean ± SD with n = 2 biologically independent experiments. Source data are provided as a Source Data file.

Fig. 4. SNAP-OMVs decorated with biotin-GFP boost GFP-specific IgG titers.

a Mean GFP-specific IgG titers (horizontal black lines) in endpoint (day 56) serum of individual mice in each group (gray circles). Five groups of six-week-old BALB/c mice (seven mice per group) were immunized s.c. with the following: PBS, blank SNAP-OMVs, SNAP-OMVs mixed with 20 pmol unbiotinylated or biotinylated GFP, and ClyA-GFP OMVs displaying a ClyA-GFP fusion. All OMVs were isolated from KPM404 ΔnlpI cells with the appropriate expression plasmid. Mice received prime injections containing an equivalent amount of OMVs (20 μg total protein) on day 0 and were boosted on day 21 and 42 with the same doses. b Mean GFP-specific IgG1 and IgG2a titers in the same endpoint serum of individual mice in select groups from (a). c Mean GFP-specific IgG titers in mice receiving SNAP-OMVs formulated with escalating doses (2–200 pmol) of biotinylated GFP (physically associated, dark gray circles) or non-biotinylated GFP (non-associated mixtures, light gray circles). An equivalent amount of SNAP-OMVs was used in each case. Dashed black line indicates mean GFP-specific IgG titer of blank SNAP-OMV control group. Data are the mean titers of 6 mice in each group ± SD. Statistical significance was determined by two-tailed t test with Welch’s correction (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant). Actual p values in (a) from left-to-right: p < 0.0001, p < 0.0001, p = 0.0013, and p = 0.2751. Actual p values in (b) from left to right: p < 0.0001  and p = 0.5184 . Actual p values in (c) from left-to-right: p = 0.1401, p = 0.0001, p < 0.0001, p < 0.0001, p = 0.4719, p < 0.0001, p < 0.0001, p = 0.0413, p = 0.0022, p = 0.0072, p = 0.0654, p < 0.0001, p = 0.0004. Source data are provided as a Source Data file.

Fig. 5. SNAP-OMVs decorated with biotinylated Sx-Cm-MOMP elicit neutralizing IgGs.

a Schematic of SIMPLEx strategy for converting integral membrane proteins into water-soluble proteins that can be expressed at high titers in the cytoplasm of host cells. Here, the β-barrel outer membrane protein Cm-MOMP was fused at its N-terminus with E. coli maltose-binding protein (MBP) and at its C-terminus with truncated ApoAI (ApoAI*). Structural analysis indicates that ApoAI* adopts a belt-like conformation around the membrane helices of proteins to which it is fused, effectively shielding these highly hydrophobic segments from water. Schematic created with BioRender.com. (b, left blot) Antigenicity of Sx-Cm-MOMP construct evaluated by immunoblot analysis using mAb MoPn-40. Native Cm-MOMP (nCm-MOMP) and Sx-CtE-MOMP served as positive and negative controls, respectively. (b, right blot) The latter construct was detected with commercial antibody specific for CtE-MOMP, which did not react with Sx-Cm-MOMP or nMOMP. Expected location of full-length SIMPLEx fusion proteins are denoted by black arrows. Molecular weight (M_w) ladder is indicated at left. Blots representative of two independently repeated experiments with similar results. c Antigen-specific IgG titers against recombinant preparations of Cm-MOMP (rCm-MOMP; horizontal black lines) in endpoint (day 56) serum of individual mice in each group (gray circles). Three groups of six-week-old BALB/c mice (seven mice per group) were immunized s.c. with the following: PBS, blank SNAP-OMVs, and SNAP-OMVs mixed with biotinylated Sx-Cm-MOMP. Mice received prime injections containing an equivalent amount of OMVs (20 μg total protein) on day 0 and were boosted on day 21 and 42 with the same doses. d, e Same as in (c) but with either (d) a native preparation of Cm-MOMP (nCm-MOMP) or (e) elementary bodies (EBs) as immobilized antigens. f In vitro neutralizing titers in endpoint (day 56) serum of individual mice. Each dot corresponds to an individual mouse and horizontal line indicates mean titer. Statistical significance was determined by two-tailed t test with Welch’s correction (**p < 0.01, ****p < 0.0001). Actual p values: c p < 0.0001 and p < 0.0001; d p < 0.0001 and p < 0.0001; e p = 0.0024 and p = 0.0051; and f p = 0.0064 and p = 0.0069. Source data are provided as a Source Data file.