Development of a Heat-Stable and Orally Delivered Recombinant M2e-expressing B. Subtilis Spore-Based Influenza Vaccine

Hum Vaccin Immunother. 2014;10(12):3649-58. doi: 10.4161/hv.36122.


Highly conserved ectodomain of influenza virus M2 protein (M2e) is an important target for the development of universal influenza vaccines. Today, the use of chemical or genetic fusion constructs have been undertaken to overcome the low immunogenicity of M2e in vaccine formulation. However, current M2e vaccines are neither orally delivered nor heat-stable. In this study, we evaluated the immune efficacy of an orally delivered recombinant M2e vaccine containing 3 molcules of M2e consensus sequence of influenza A viruses, termed RSM2e3. To accomplish this, CotB, a spore coat of Bacillus subtilis (B. subtilis), was used as a fusion partner, and heat-stable nonpathogenic B. subtilis spores were used as the carrier. Our results showed that CotB-M2e3 fusion had no effect on spore structure or function in the resultant recombinant RSM2e3 strain and that heterologous influenza virus M2e protein was successfully displayed on the surface of the recombinant RSM2e3 spore. Importantly, recombinant RSM2e3 spores elicited strong and long-term M2e-specific systemic and mucosal immune responses, completely protecting immunized mice from lethal challenge of A/PR/8/34(H1N1) influenza virus. Taken together, our study forms a solid basis for the development of a novel orally delivered and heat-stable influenza vaccine based on B. subtilis spore surface display.

Keywords: ADCC, antibody-dependent cell-mediated cytotoxicity; CDC, complement-dependent cytotoxicity; CPE, cytopathic effect; IAVs, influenza A viruses; M2e; M2e, ectodomain of influenza virus M2 protein; bacillus subtilis; influenza virus; spores; surface display.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Administration, Oral
  • Animals
  • Bacillus subtilis / genetics*
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / immunology
  • Female
  • Influenza A Virus, H1N1 Subtype / immunology*
  • Influenza Vaccines / biosynthesis*
  • Influenza Vaccines / immunology
  • Mice
  • Mice, Inbred BALB C
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / immunology
  • Spores, Bacterial
  • Vaccines, Synthetic / biosynthesis
  • Vaccines, Synthetic / immunology
  • Viral Matrix Proteins / genetics*
  • Viral Matrix Proteins / immunology


  • Bacterial Proteins
  • CotB protein, Bacillus subtilis
  • Influenza Vaccines
  • M2 protein, Influenza A virus
  • Recombinant Fusion Proteins
  • Vaccines, Synthetic
  • Viral Matrix Proteins