Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Kirkwood, J. M. et al. Immunotherapy of cancer in 2012. CA Cancer J. Clin. 62, 309–335 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Sahin, U. & Türeci, Ö. Personalized vaccines for cancer immunotherapy. Science 359, 1355–1360 (2018).

    Article  CAS  PubMed  Google Scholar 

  3. Mathias Vormehr, Ö. T. & Sahin, Ugur Harnessing tumour mutations for truly individualized cancer vaccines. Annu. Rev. Med. 70, 395–407 (2019).

    Article  PubMed  CAS  Google Scholar 

  4. Vela Ramirez, J. E., Sharpe, L. A. & Peppas, N. A. Current state and challenges in developing oral vaccines. Adv. Drug Deliv. Rev. 114, 116–131 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Qin, H. et al. Development of a cancer vaccine using in vivo click-chemistry-mediated active lymph node accumulation for improved immunotherapy. Adv. Mater. 33, e2006007 (2021).

    Article  PubMed  CAS  Google Scholar 

  6. Malik, B., Rath, G. & Goyal, A. K. Are the anatomical sites for vaccine administration selected judiciously? Int. Immunopharmacol. 19, 17–26 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Combadiere, B. et al. Preferential amplification of CD8 effector-T cells after transcutaneous application of an inactivated influenza vaccine: a randomized phase I trial. PLoS ONE 5, e10818 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Raz, E. et al. Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Proc. Natl Acad. Sci. USA 91, 9519–9523 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Vighi, G., Marcucci, F., Sensi, L., Di Cara, G. & Frati, F. Allergy and the gastrointestinal system. Clin. Exp. Immunol. 153, 3–6 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zimmermann, P. & Curtis, N. The influence of the intestinal microbiome on vaccine responses. Vaccine 36, 4433–4439 (2018).

    Article  CAS  PubMed  Google Scholar 

  11. Taddio, A. et al. Survey of the prevalence of immunization non-compliance due to needle fears in children and adults. Vaccine 30, 4807–4812 (2012).

    Article  PubMed  Google Scholar 

  12. Kim, S. H. & Jang, Y. S. The development of mucosal vaccines for both mucosal and systemic immune induction and the roles played by adjuvants. Clin. Exp. Vaccine Res. 6, 15–21 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. New, R. R. C. Formulation technologies for oral vaccines. Clin. Exp. Immunol. 198, 153–169 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Borges, O. et al. Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles. Eur. J. Pharm. Sci. 32, 278–290 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Mann, J. F. et al. Lipid vesicle size of an oral influenza vaccine delivery vehicle influences the Th1/Th2 bias in the immune response and protection against infection. Vaccine 27, 3643–3649 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Peek, L. J., Middaugh, C. R. & Berkland, C. Nanotechnology in vaccine delivery. Adv. Drug Deliv. Rev. 60, 915–928 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang, J., Li, Y. & Nie, G. Multifunctional biomolecule nanostructures for cancer therapy. Nat. Rev. Mater. 6, 766–783 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Din, M. O. et al. Synchronized cycles of bacterial lysis for in vivo delivery. Nature 536, 81–85 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Riglar, D. T. & Silver, P. A. Engineering bacteria for diagnostic and therapeutic applications. Nat. Rev. Microbiol. 16, 214–225 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Zhou, S., Gravekamp, C., Bermudes, D. & Liu, K. Tumour-targeting bacteria engineered to fight cancer. Nat. Rev. Cancer 18, 727–743 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Duan, F. & March, J. C. Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proc. Natl Acad. Sci. USA 107, 11260–11264 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hwang, I. Y. et al. Reprogramming microbes to be pathogen-seeking killers. ACS Synth. Biol. 3, 228–237 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Yang, C. et al. Upconversion optogenetic micro-nanosystem optically controls the secretion of light-responsive bacteria for systemic immunity regulation. Commun. Biol. 3, 561 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Steidler, L. et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289, 1352–1355 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Mimee, M., Tucker, A. C., Voigt, C. A. & Lu, T. K. Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in the murine gut microbiota. Cell Syst. 1, 62–71 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Isabella, V. M. et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 36, 857–864 (2018).

    Article  CAS  PubMed  Google Scholar 

  27. Zheng, D. W. et al. Prebiotics-encapsulated probiotic spores regulate gut microbiota and suppress colon cancer. Adv. Mater. 32, e2004529 (2020).

    Article  PubMed  CAS  Google Scholar 

  28. Ho, C. L. et al. Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nat. Biomed. Eng. 2, 27–37 (2018).

    Article  CAS  PubMed  Google Scholar 

  29. Shen, Y. et al. Outer membrane vesicles of a human commensal mediate immune regulation and disease protection. Cell Host Microbe 12, 509–520 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chu, H. et al. Gene–microbiota interactions contribute to the pathogenesis of inflammatory bowel disease. Science 352, 1116–1120 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Schwechheimer, C. & Kuehn, M. J. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat. Rev. Microbiol. 13, 605–619 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cheng, K. et al. Bioengineered bacteria-derived outer membrane vesicles as a versatile antigen display platform for tumour vaccination via Plug-and-Display technology. Nat. Commun. 12, 2041 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Li, Y. et al. Bacterial outer membrane vesicles presenting programmed death 1 for improved cancer immunotherapy via immune activation and checkpoint inhibition. ACS Nano. 14, 16698–16711 (2020).

    Article  CAS  Google Scholar 

  34. Fabrega, M. J. et al. Intestinal anti-inflammatory effects of outer membrane vesicles from Escherichia coli Nissle 1917 in DSS-experimental colitis in mice. Front. Microbiol. 8, 1274 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Camacho, A. I., Irache, J. M., de Souza, J., Sanchez-Gomez, S. & Gamazo, C. Nanoparticle-based vaccine for mucosal protection against Shigella flexneri in mice. Vaccine 31, 3288–3294 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Kaparakis-Liaskos, M. & Ferrero, R. L. Immune modulation by bacterial outer membrane vesicles. Nat. Rev. Immunol. 15, 375–387 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Stentz, R., Carvalho, A. L., Jones, E. J. & Carding, S. R. Fantastic voyage: the journey of intestinal microbiota-derived microvesicles through the body. Biochem. Soc. Trans. 46, 1021–1027 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Akilesh, S., Christianson, G. J., Roopenian, D. C. & Shaw, A. S. Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism. J. Immunol. 179, 4580–4588 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Sockolosky, J. T. & Szoka, F. C. The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy. Adv. Drug Deliv. Rev. 91, 109–124 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Baker, K. et al. Neonatal Fc receptor expression in dendritic cells mediates protective immunity against colorectal cancer. Immunity 39, 1095–1107 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nguyen, V. H. et al. Genetically engineered Salmonella typhimurium as an imageable therapeutic probe for cancer. Cancer Res. 70, 18–23 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Zheng, J. H. Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin. Sci. Transl. Med. 9, 9537–9637 (2017).

    Article  CAS  Google Scholar 

  43. Hilgendorf, C. et al. Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport. J. Pharm. Sci. 89, 63–75 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Li, Y. et al. Gut microbiota dependent anti-tumour immunity restricts melanoma growth in Rnf5-/- mice. Nat. Commun. 10, 1492 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Chen, Q. et al. A hybrid eukaryotic-prokaryotic nanoplatform with photothermal modality for enhanced antitumour vaccination. Adv. Mater. 32, e1908185 (2020).

    Article  PubMed  CAS  Google Scholar 

  46. Márquez-Rodas, Iván Intratumoural nanoplexed poly I:C BO-112 in combination with systemic anti-PD-1 for patients with anti-PD-1-refractory tumours. Multicent. Study 12, eabb0391 (2020).

    Google Scholar 

  47. Hafner, A. M., Corthesy, B. & Merkle, H. P. Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant. Adv. Drug Deliv. Rev. 65, 1386–1399 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. Flentie, K. et al. A bioluminescent transposon reporter-trap identifies tumour-specific microenvironment-induced promoters in Salmonella for conditional bacterial-based tumour therapy. Cancer Discov. 2, 624–637 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Duong, M. T., Qin, Y., You, S. H. & Min, J. J. Bacteria–cancer interactions: bacteria-based cancer therapy. Exp. Mol. Med. 51, 1–15 (2019).

    Article  CAS  PubMed  Google Scholar 

  50. Forbes, N. S. Engineering the perfect (bacterial) cancer therapy. Nat. Rev. Cancer 10, 785–794 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ryan, R. M. et al. Bacterial delivery of a novel cytolysin to hypoxic areas of solid tumours. Gene Ther. 16, 329–339 (2009).

    Article  CAS  PubMed  Google Scholar 

  52. Chien, T., Doshi, A. & Danino, T. Advances in bacterial cancer therapies using synthetic biology. Curr. Opin. Syst. Biol. 5, 1–8 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Hong, H. et al. Targeted deletion of the ara operon of Salmonella typhimurium enhances l-arabinose accumulation and drives PBAD-promoted expression of anti-cancer toxins and imaging agents. Cell Cycle 13, 3112–3120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Weigmann, B. et al. Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nat. Protoc. 2, 2307–2311 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

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.).

Author information

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

Authors
  1. Yale Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiaqi Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Keman Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qingqing Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiaotu Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nana Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tianjiao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xinwei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xiao Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Guangjun Nie
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

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.

Peer review

Peer review information

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

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

Supplementary information

Supplementary Information

Supplementary figures and tables.

Reporting Summary

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.

Supplementary Table 3

Source data for Supplementary Fig. 6.

Supplementary Table 4

Source data for Supplementary Fig. 7.

Supplementary Table 5

Source data for Supplementary Fig. 8.

Source data

Source Data Fig. 2

Unprocessed western blot.

Source Data Fig. 2

Source data.

Source Data Fig. 3

Source data.

Source Data Fig. 4

Source data.

Source Data Fig. 5

Source data.

Source Data Fig. 6

Source data.

Source Data Extended Data Fig. 1

Source data.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-022-00886-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research