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Antigen-bearing outer membrane vesicles as tumour vaccines produced in situ by ingested genetically engineered bacteria

Nature Biomedical Engineering volume 6, pages 898–909 (2022)Cite this article

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Abstract

The complex gastrointestinal environment and the intestinal epithelial barrier constrain the design and effectiveness of orally administered tumour vaccines. Here we show that outer membrane vesicles (OMVs) fused to a tumour antigen and produced in the intestine by ingested genetically engineered bacteria function as effective tumour vaccines in mice. We modified Escherichia coli to express, under the control of a promoter induced by the monosaccharide arabinose, a specific tumour antigen fused with the protein cytolysin A on the surface of OMVs released by the commensal bacteria. In mice, oral administration of arabinose and the genetically engineered E. coli led to the production of OMVs that crossed the intestinal epithelium into the lamina propria, where they stimulated dendritic cell maturation. In a mouse model of pulmonary metastatic melanoma and in mice bearing subcutaneous colon tumours, the antigen-bearing OMVs inhibited tumour growth and protected the animals against tumour re-challenge. The in situ production of OMVs by genetically modified commensal bacteria for the delivery of stimulatory molecules could be leveraged for the development of other oral vaccines and therapeutics.

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Fig. 1: Genetically engineered bacteria-derived-OMV-based oral tumour vaccine.
Fig. 2: Biodistribution of the engineered E. coli after oral administration.
Fig. 3: Epithelial penetration and immune stimulation analysis of the oral vaccines.
Fig. 4: Evaluation of the antitumour effects of the oral vaccines in a lung metastatic melanoma model.
Fig. 5: Evaluation of the antitumour effects of the oral vaccines in a subcutaneous colon cancer model.
Fig. 6: Evaluation of long-term immune memory elicited by the oral vaccines.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. All the raw and analysed data generated during the study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Key R&D Program of China (2018YFA0208900 to G.N. and 2021YFA0909900 to X.Z.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB36000000 to G.N.), the CAS Project for Young Scientists in Basic Research (YSBR-010 to X.Z.) and the National Natural Science Foundation of China (31820103004 to G.N.).

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Author notes

  1. These authors contributed equally: Yale Yue, Jiaqi Xu, Yao Li.

Authors and Affiliations

  1. CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, China

    Yale Yue, Jiaqi Xu, Yao Li, Keman Cheng, Qingqing Feng, Xiaotu Ma, Nana Ma, Tianjiao Zhang, Xinwei Wang, Xiao Zhao & Guangjun Nie

  2. Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing, China

    Yale Yue, Jiaqi Xu & Guangjun Nie

  3. School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China

    Yale Yue

  4. IGDB-NCNST Joint Research Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Xiao Zhao

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Contributions

Y.Y., J.X. and Y.L. contributed equally to this work. Y.Y., J.X., Y.L., X.Z. and G.N. designed the research. Y.Y., J X., Y.L., K.C., Q.F., X.M., N.M., T.Z. and X.W. performed the research. All authors analysed and interpreted the data. Y.Y., J.X., Y.L., X.Z. and G.N. wrote the paper.

Corresponding authors

Correspondence to Xiao Zhao or Guangjun Nie.

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Competing interests

G.N., X.Z. and Y.Y. are inventors on a filed provisional application patent (PCT/CN2021/135329) submitted in China by the National Center for Nanoscience and Technology that covers the potential diagnostic and therapeutic uses of the oral vaccine for cancer immunotherapy. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Xiawei Wei and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Antitumor effect in the lung metastatic melanoma model.

Mice were intravenously injected with 2 × 105 B16-OVA cells on day 0 and then randomly divided into five groups for different treatments on days 3, 6 and 11. Mice were orally administrated with PBS in group 1 (G1), ClyA-OVA-mFc OMVs (G2, 50 μg protein per mouse, a commonly used dose in OMV-based vaccine) or oral ClyA-OVA-mFc vaccine (G5). In addition, mice received intracolonic administration with the lysis of engineered ClyA-OVA-mFc bacteria obtained via repeated freezing and thawing after arabinose-induced expression (G3, 50 μg protein per mouse) or the ClyA-OVA-mFc OMVs (G4, 50 μg protein per mouse). All the mice were sacrificed on day 17 for further analysis. The operation of intracolonic administration is as follows: mice were depilated on the abdomen and anesthetized. After disinfection with iodine, the abdominal cavity of mouse was opened by 1-2 cm. Then, 20 μL of OMVs or bacterial fragments was injected into the colonic intestine. a, The experimental groups. b, Image of lungs collected at the end of experiment (day 17); scale bar, 1 cm. c, Quantitative analysis of lung metastasis (n = 5). d–g, Antigen-specific immune response analysis. The splenocytes were collected on day 17 and stimulated with OVA peptide. The percentages of IFN-γ+ in CD3+CD8+ cells in the splenocytes were analyzed by flow cytometry (d,e) (n = 5). The IFN-γ secretion by splenocytes after re-stimulation was determined by the ELISPOT assay (f,g) (n = 3). h–i, The cytotoxic effects of splenocytes on B16-OVA cells (with OVA antigen, h) and MC38 cells that lack the OVA antigen (i) analyzed using the CCK-8 assay (n = 5). The data are presented as the mean ± SD and were analyzed by one-way analysis of variance (ANOVA) with GraphPad Prism software. N.S., no significance; *, P < 0.05; ****, P < 0.0001.

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Supplementary data

Unprocessed western blot for Supplementary Fig. 1a.

Supplementary Table 1

Source data for Supplementary Fig. 1c.

Supplementary Table 2

Source data for Supplementary Fig. 2b.

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Supplementary Table 4

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Supplementary Table 5

Source data for Supplementary Fig. 8.

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Source Data Fig. 2

Unprocessed western blot.

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Source Data Extended Data Fig. 1

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Yue, Y., Xu, J., Li, Y. et al. Antigen-bearing outer membrane vesicles as tumour vaccines produced in situ by ingested genetically engineered bacteria. Nat. Biomed. Eng 6, 898–909 (2022). https://doi.org/10.1038/s41551-022-00886-2

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