Bioengineered bacteria-derived outer membrane vesicles as a versatile antigen display platform for tumor vaccination via Plug-and-Display technology

Abstract

An effective tumor vaccine vector that can rapidly display neoantigens is urgently needed. Outer membrane vesicles (OMVs) can strongly activate the innate immune system and are qualified as immunoadjuvants. Here, we describe a versatile OMV-based vaccine platform to elicit a specific anti-tumor immune response via specifically presenting antigens onto OMV surface. We first display tumor antigens on the OMVs surface by fusing with ClyA protein, and then simplify the antigen display process by employing a Plug-and-Display system comprising the tag/catcher protein pairs. OMVs decorated with different protein catchers can simultaneously display multiple, distinct tumor antigens to elicit a synergistic antitumour immune response. In addition, the bioengineered OMVs loaded with different tumor antigens can abrogate lung melanoma metastasis and inhibit subcutaneous colorectal cancer growth. The ability of the bioengineered OMV-based platform to rapidly and simultaneously display antigens may facilitate the development of these agents for personalized tumour vaccines.

Fig. 1. Engineered display of heterologous proteins or antigen peptides on the OMVs surface.

a Schematic representation of the pET28a-Luc and pET28a-ClyA-Luc construct, and western blot analysis of ClyA-none (CN), Luc, and ClyA-Luc (CL) expression in E. coli Rosetta (DE3), and OMVs derived from the bacteria. b In vitro bioluminescence images to demonstrate luciferase enzyme activity following expression in Rosetta (DE3) and OMVs. c Schematic representation of the pET28a-ClyA-OVA-3HA construct, and western blot analysis of 3× HA-tagged ClyA-OVA (ClyA-OVA-3HA, CO-3HA) expression in E. coli Rosetta (DE3) and OMVs. pET28a-ClyA-OVA-3HA was the expression plasmid, and IPTG was the expression inducer. d TEM image and DLS analysis of ClyA-OVA OMVs (CO OMVs). Scale bar, 100 nm. Source data are provided as a Source Data file.

Fig. 2. Innate immune response and antigen-specific T-cell-mediated anti-tumor immunity induced by tumor antigen peptide-displayed OMVs.

a, b Maturation of BMDCs following treatment with OMVs preparations or controls. Flow cytometry was used to measure the percentage of CD80⁺ (a) or CD86⁺ (b) cells in CD11c⁺ BMDCs (n = 4). c The expression of the MHCI-OVA complex on the surface of BMDCs was measured by flow cytometry (n = 3). d–f TNF-α (d), IL-6 (e), and IL-1β (f) levels in the BMDC-conditioned medium after the indicated treatments (n = 3). g Schema showing the mouse B16-OVA melanoma model used to study the effects of OMVs vaccination (Vacc.) on lung metastasis. C57BL/6 mice were inoculated with B16-OVA melanoma cells (n = 4, 2 × 10⁵ cells/mouse, i.v.), then immunized with the following vaccines: saline, OVA_257–264, CN OMVs (ClyA-none OMVs), OVA_257–264 + CN OMVs or CO OMVs 3, 6, and 11 days later. Lung metastasis and immune responses were analyzed on day 17. h, i The maturation status of DCs in inguinal lymph nodes on days 17 post immunization. The percentage of CD80⁺ (h) or CD86⁺ (i) cells in CD11c⁺ cells was assessed by flow cytometry. j, k Lung metastasis was assessed on days 17 after tumor cell administration and following the indicated vaccine treatments. The lungs were photographed (j), and the tumor nodules in the lungs were counted (k). l Flow cytometry analysis of the percentage of IFNγ⁺ cytotoxic T lymphocytes (CD3⁺CD8⁺IFNγ⁺ T cells) in splenocytes re-stimulated with OVA_257–264 antigen. m, n IFNγ secretion, as measured by the ELISPOT assay, from splenocytes which had been re-stimulated with OVA_257-264 (m). Quantitative analysis of the ELISPOT assay for IFNγ secretion is shown in (n). g–n, n = 4. The data (a–f, h, i, k, l, n) are shown as mean ± SD. Statistical analysis was performed by a two-tailed unpaired t test. Source data are provided as a Source Data file.

Fig. 3. Design and characterization of the flexible OMV-based antigen display platform-antigen presentation on BMDCs and enrichment of antigens and adjuvants in lymph nodes.

a Schematic illustration of ClyA-Catcher (CC) OMVs system for antigen display. SpyCatcher (SpC) and SnoopCatcher (SnC) were expressed as fusion proteins with ClyA (ClyA-Catcher, CC) on the OMVs surface. SpyTag (SpT) or SnoopTag (SnT)-labeled antigens bind to CC OMVs through isopeptide bond formation between the tag and catcher. b TEM and DLS analysis of CC OMVs. Scale bar, 100 nm. c, d The conjugation of SpT-HA (c) or SnT-HA (d) to ClyA-Catchers on the CC OMVs surface was verified by western blot analysis using an anti-HA antibody. e The simultaneous display of two Catcher/Tag pairs by the same OMVs. SpT-Cys- and SnT-Cys labeled with 5 (white arrow) and 10 nm (red arrow) gold nanoparticles, respectively, were used to identify SpC and SnC on CC OMVs. Scale bar, 20 nm. f Confocal microscopy images of antigen presentation by BMDCs incubated with the indicated formulations for 12 h. The cell nuclei were stained blue (DAPI), and MHCI-OVA complexes were stained green (PE-anti-mouse H-2Kb bound to SIINFEKL) (n = 3). BF bright field. Scale bar, 50 µm. g Confocal microscopy images of antigen uptake by BMDCs after incubation with the indicated OMVs formulations for 12 h. The cell nuclei were stained blue (DAPI), and the antigen was labeled with Cy5.5 (red) (n = 3). Scale bar, 50 µm. h, i Lymph node accumulation of OMVs in vivo (n = 3). Various organs and the inguinal draining lymph nodes of mice were collected 12 h after s.c. immunization with the indicated OMVs formulations to examine the accumulation of Cy5.5 fluorescence (h). Frozen sections of the lymph nodes were prepared and examined by fluorescence microscopy (i). Cell nuclei were stained blue (DAPI), and the antigen was labeled with Cy5.5 (red). Scale bar, 1 mm. Source data are provided as a Source Data file.

Fig. 4. Single-tumor antigen (TRP2_180–188) display by catcher-decorated OMVs.

a Schema showing the B16-F10 melanoma lung metastasis mouse model and the OMVs vaccination (Vacc.) timeline. C57BL/6 mice were inoculated with B16-F10 melanoma cells (n = 4, 2 × 10⁵ cells/mouse, i.v.), then immunized with the following vaccines or controls: saline; SnT-TRP2; CN OMVs; SnT-TRP2 + CN OMVs; or CC-SnT-TRP2 OMVs on days 3, 6, and 11 after inoculation of the tumor cells. Lung metastasis and immune responses were analyzed on day 17. TRP2: tyrosinase-related protein 2. b, c Analysis of DC maturation in inguinal lymph nodes at the end of the treatment period (day 17). The percentage of CD80⁺ (b) or CD86⁺ (c) cells in CD11c⁺ cells was determined by flow cytometry. d, e Lungs were collected at the end of the treatment period and photographed (d), then the tumor nodules in the lung were counted (e). f Flow cytometry analysis of IFNγ⁺ cytotoxic T lymphocytes in splenocytes re-stimulated with TRP2_180-188 antigen. The percentage of IFNγ⁺ cells in the CD3⁺CD8⁺ T-cell subpopulation is shown. g, h IFNγ secretion from splenocytes (as determined by the ELISPOT assay) which had been re-stimulated with TRP2_180–188 (g). Quantitative analysis of the ELISPOT data is shown in (h). The data (b, c, e, f, h) are shown as mean ± SD. Statistical analysis was performed by a two-tailed unpaired t test. N.S. no significance. Source data are provided as a Source Data file.

Fig. 5. Dual-tumor antigen (OVA_257–264 and OVA_223–339) display by catcher-decorated OMVs triggers CD4⁺ and CD8⁺ T-cell-mediated synthetic anti-tumor immunity.

SpT-OTI and SnT-OTII were displayed either singly (CC-SpT-OTI OMVs or CC-SnT-OTII OMVs) or simultaneously (CC-SpT-OTI/SnT-OTII OMVs) by CC OMVs. Mixed formulations (SpT-OTI + CN OMVs, SnT-OTII + CN OMVs, and SpT-OTI + SnT-OTII + CN OMVs) were used as controls. OTI: OVA_257–264, OTII: OVA_223–339. a, b Lungs were collected on day 17 at the end of the treatment period and photographed (a); the tumor nodules were counted (b). c, d IFNγ secretion by splenocytes isolated from the various treatment groups was determined by the ELISPOT assay after re-stimulation of the cells with OVA_257–264 and OVA_223–339 (c). Quantitative data derived from the ELISPOT assays are shown in (d). e, f Flow cytometry analysis of IFNγ⁺ cells in the CD3⁺CD8⁺ T-cell subpopulation (e) or the CD3⁺CD4⁺ T-cell subpopulation (f) in splenocytes re-stimulated with OVA_257–264 and OVA_223–339 antigens. The data (b, d–f) are shown as mean ± SD (n = 4). Statistical analysis was performed by a two-tailed unpaired t test. Source data are provided as a Source Data file.

Fig. 6. Dual-tumor antigen (OVA_257–264 and TRP2_180–188) display by catcher-decorated OMVs stimulates CD8⁺ T-cell-mediated, synergistic immune therapeutic effects.

SpT-OTI and SnT-TRP2 were displayed either singly (CC-SpT-OTI OMVs or CC-SnT-TRP2 OMVs) or simultaneously (CC-SpT-OTI/SnT-TRP2 OMVs) by CC OMVs. Mixed formulations (SpT-OTI + CN OMVs, SnT-TRP2 + CN OMVs, and SpT-OTI + SnT-TRP2 + CN OMVs) were used as controls. a, b Lungs were collected on day 17 at the end of the treatment period and photographed (a); the tumor nodules were counted (b). c, d IFNγ secretion by splenocytes isolated from the various treatment groups was determined by the ELISPOT assay after re-stimulation of the cells with OVA_257–264 and TRP2_180–188 (c). Quantitative data derived from the ELISPOT assays are shown in (d). e Flow cytometry analysis of IFNγ⁺ cytotoxic T lymphocytes in splenocytes isolated from the various treatment groups and re-stimulated with OVA_257–264 and TRP2_180–188 antigens. Data are presented as the percentage of IFNγ⁺ cells in the CD3⁺CD8⁺ T-cell subpopulation. The data (b, e (n = 6), d (n = 5)) are shown as mean ± SD. Statistical analysis was performed by a two-tailed unpaired t test. Source data are provided as a Source Data file.

Fig. 7. The anti-tumor immunity of the antigen-displayed OMVs in the subcutaneous MC38 tumor model.

The SpT-labeled Adpgk (a neoantigen in MC38 cells), SpT-Adpgk was displayed by CC OMVs (CC-SpT-Adpgk OMVs). The adjuvant Poly (I:C) and CN OMVs were used as a control to mix with SpT-Adpgk, respectively (Poly (I:C) + SpT-Adpgk and SpT-Adpgk + CN OMVs). a Schema showing the subcutaneous tumor model utilizing MC38 cells and the timing of vaccination (Vacc.) with different formulations. C57BL/6 mice were inoculated with MC38 cells (1 × 10⁶ cells/mouse, s.c.) and immunized with the indicated formulations on days 3, 7, and 11. b–d Tumor volumes were recorded, and survival was monitored. The data are shown as mean ± SD (n = 10). e In another set of MC38 tumor-bearing animals, tumors were harvested on day 29 for flow cytometry analysis (n = 4) of the following immune cells: CD3⁺, CD3⁺CD8⁺, CD3⁺CD4⁺, CD3⁺CD4⁺Foxp3⁺ T lymphocytes, activated neutrophils (CD11b⁺Ly6G⁺ cells), macrophages (F4/80⁺ cells), dendritic cells (CD11c⁺ cells), and MDSCs (CD11b⁺Gr1⁺ cells). The data are shown as mean ± SD. Statistical analysis was performed by two-tailed unpaired t test (b, e) and two-sided log-rank test (c). N.S. no significance. Source data are provided as a Source Data file.

Fig. 8. The long-term immune memory in vivo elicited by antigen-loaded CC OMVs.

a Schema of immune memory analysis. C57BL/6 mice were immunized with the formulations shown in (b) on days 0, 3, and 8. Immune responses were evaluated, and the mice were challenged with B16-OVA cells (2 × 10⁵ cells/mouse, i.v.) on days 60. Lung metastasis was analyzed on day 80. c, d Specific killing ability of splenocytes collected on day 60 toward B16-OVA cells with OVA antigen (c) and MC38 cells without OVA antigen (d) analyzed by CCK-8 assay. e, f Quantitative analysis of tetramer⁺ T cells in splenocytes (e) and blood (f) on day 60 through flow cytometry. g Flow cytometry analysis of IFNγ⁺ cytotoxic T lymphocytes in splenocytes re-stimulated with OVA_257–264. h The proportion of T_em cells (CD8⁺CD44⁺CD62L⁻) in splenocytes on day 60 (n = 5). I, j Lungs were collected on day 80 and photographed (n = 10). k Schema of tumor rechallenge model. The mice were inoculated with B16-OVA cells and treated with CC-SpT-OVA OMVs vaccine. Then, the survived animals (complete tumor regression) were rechallenged with s.c. injection of B16-F10 and B16-OVA cells on day 60. l B16-OVA or B16-F10 tumor growth curve (B16-OVA (Control), n = 8; B16-OVA (CC-SpT-OVA OMVs), n = 8; B16-F10 (Control), n = 6; B16-F10 (CC-SpT-OVA OMVs), n = 8). The controls were healthy mice without tumor burden. The data are shown as mean ± SD. Statistical analysis was performed by a two-tailed unpaired t test. Source data are provided as a Source Data file.