Synergistic Protective Activity of Tumor-Specific Epitopes Engineered in Bacterial Outer Membrane Vesicles

Abstract

Introduction: Bacterial outer membrane vesicles (OMVs) are naturally produced by all Gram-negative bacteria and, thanks to their plasticity and unique adjuvanticity, are emerging as an attractive vaccine platform. To test the applicability of OMVs in cancer immunotherapy, we decorated them with either one or two protective epitopes present in the B16F10EGFRvIII cell line and tested the protective activity of OMV immunization in C57BL/6 mice challenged with B16F10EGFRvIII.

Materials and methods: The 14 amino acid B cell epitope of human epidermal growth factor receptor variant III (EGFRvIII) and the mutation-derived CD4+ T cell neo-epitope of kif18b gene (B16-M30) were used to decorate OMVs either alone or in combination. C57BL/6 were immunized with the OMVs and then challenged with B16F10EGFRvIII cells. Immunogenicity and protective activity was followed by measuring anti-EGFRvIII antibodies, M30-specific T cells, tumor-infiltrating cell population, and tumor growth.

Results: Immunization with engineered EGFRvIII-OMVs induced a strong inhibition of tumor growth after B16F10EGFRvIII challenge. Furthermore, mice immunized with engineered OMVs carrying both EGFRvIII and M30 epitopes were completely protected from tumor challenge. Immunization was accompanied by induction of high anti-EGFRvIII antibody titers, M30-specific T cells, and infiltration of CD4+ and CD8+ T cells at the tumor site.

Conclusion: OMVs can be decorated with tumor antigens and can elicit antigen-specific, protective antitumor responses in immunocompetent mice. The synergistic protective activity of multiple epitopes simultaneously administered with OMVs makes the OMV platform particularly attractive for cancer immunotherapy.

Keywords: BALB/c-CT26 cancer mouse model; EGRRvIII; bacterial outer membrane vesicles; cancer immunotherapy; cancer neoepitopes; precision medicine.

Figure 1

Expression and surface localization of EGFRvIII epitope in BL21ΔompA(pET-Nm-fHbpvIII) strain and in its derived outer membrane vesicles (OMVs). (A) Schematic representation of pET-Nm-fHbpvIII plasmid encoding three copies of EGFRvIIIpep fused to the C-terminus of Neisseria meningitidis fHbp. (B) SDS-PAGE and Western Blot analyses of OMVs. OMVs were purified from BL21ΔompA(pET21b+) (“Empty” OMVs) and BL21ΔompA(pET-Nm-fHbpvIII) strains and loaded on SDS-polyacrylamide gels for SDS-PAGE analysis (20 µg OMVs) and Western Blot analysis (1 µg OMVs). After proteins transfer to the nitrocellulose membrane, Nm-fHbp-vIII fusion was visualized using rabbit anti-EGFRvIIIpep antibodies and peroxidase-conjugated anti-rabbit immunoglobulins. (C) Flow cytometry analysis of BL21ΔompA(pET21b+) and BL21ΔompA(pET-Nm-fHbpvIII) strains. Bacterial cells were incubated first with anti-EGFRvIIIIpep rabbit antibodies and subsequently with FITC-labeled anti-rabbit secondary antibodies. Fluorescence was measured by flow cytometry. Gray areas represent the background fluorescence signals obtained incubating the cells with the secondary antibody only. (D) Confocal microscopy analysis of BL21ΔompA(pET21b+) (“Empty” OMVs) and BL21ΔompA(pET-Nm-fHbpvIII) strains. After induction of protein expression with IPTG, bacterial cells were fixed in 4% formaldehyde solution and incubated first with rabbit anti-EGFRvIIIpep polyclonal antibodies and mouse anti-LPS mAb, and subsequently with goat anti-rabbit IgG, Alexa Fluor 594 conjugated-antibodies (red), and goat anti-mouse IgG, Alexa Fluor 488 conjugated-antibodies (green). (E) Immuno Transmission Electron Microscopy (TEM) analysis of OMVs purified from BL21ΔompA(pET-Nm-fHbpvIII) strain using primary anti-EGFRvIIIpep rabbit antibodies and 5-nm gold-labeled anti-rabbit secondary antibody (see Materials and Methods for details).

Figure 2

Immunogenicity and protective activity of Nm-fHbpvIII-outer membrane vesicles (OMVs). (A) Schematic representation of immunization and challenge schedules in C57BL/6 mice. (B) Anti-EGFRvIIIpep antibody titers in C57BL/6 mice immunized with “Empty” OMVs and with Nm-fHbpvIII-OMVs. Sera from mice immunized as reported in (A) were pooled and total IgGs, IgG1, and IgG2a were measured by ELISA, coating the plates with synthetic EGFRvIIIpep (0.5 μg/well). (C) Analysis of tumor development in C57BL/6 mice immunized with “Empty” OMVs and with Nm-fHbpvIII-OMVs. The figure reports the tumor size in each mouse as measured at day 30 after challenge with 0.5 × 10⁵ B16F10EGFRvIII cells. *** indicates a statistically significant difference of P < 0.001. (D) Analysis of tumor-infiltrating cell populations. At the end of the challenge experiment, two tumors/group were randomly selected and the percentage of infiltrating CD4+ T cells, CD8+ T cells, MDSCs, and Tregs was determined by flow cytometry, as described in Section “Materials and Methods” (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3

Synergistic protective activity of Nm-fHbpvIII-OMVs/M30 peptide combination. (A) Analysis of tumor development in C57BL/6 mice immunized as shown in Figure 2A. The figure reports the tumor size in each mouse as measured at day 30 after the challenge with 0.5 × 10⁵ B16F10EGFRvIII cells. *** indicates that the difference in tumor size between each group and control group is statistically significant with P < 0.001. (B) Analysis of M30pep-specific CD4+ T cells in immunized mice. At the end of the challenge experiment, spleens from two animals were collected. Splenocytes were stimulated with M30pep and IFNγ-producing CD4+ T cells were analyzed by flow cytometry (**P < 0.01).

Figure 4

Immunogenicity and protective activity of outer membrane vesicles (OMVs) decorated with Nm-fHbp-M30-vIII fusion. (A) Schematic representation of pET-Nm-fHbp-M30-vIII plasmid. The DNA sequence refers to the 3′ end of the gene fusion encoding three copies of both M30 and EGFRvIII epitopes. (B) Mice were immunized with (i) “Empty” OMVs, (ii) Nm-fHbpvIII OMVs, and (iii) Nm-fHbpvIII OMVs + M30pep, and (iv) Nm-fHbp-M30-vIII-OMVs and subsequently challenged with 0.5 × 10⁵ B16F10EGFRvIII cells following the schedule indicated in Figure 2A. Tumor size in each mouse was measured at day 30 post challenge. Seven days after the last immunization, serum samples were also collected from mice immunized with (i) “Empty” OMVs, (ii) Nm-fHbpvIII OMVs, and (iii) Nm-fHbp-M30-vIII-OMVs, and total anti-EGFRvIII IgGs were measured by ELISA (**P < 0.01; ***P < 0.001). (C) M30-specific CD4+ T cells induced in mice immunized with OMVs decorated with M30 peptide. Mice were immunized twice i.p. at days 0 and 7 with either 20 µg “Empty” OMVs + 100 mg M30pep or 20 µg Nm-fHbp-M30-vIII-OMVs. Five days after the second immunization, splenocytes were stimulated with either an irrelevant peptide or with M30 peptide and IFNγ-positive CD4+ T cells were counted by FACS (**P < 0.01).