Recombinant Escherichia coli-derived outer membrane vesicles as an antigen delivery platform for heterologous protein

Sikha, V T R (2023) Recombinant Escherichia coli-derived outer membrane vesicles as an antigen delivery platform for heterologous protein. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS dissertation of Sikha V T R (18MS037)) Thesis_18MS037.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have potential adjuvant properties due to bacterial antigens and lipopolysaccharides (LPS) on their surface. In general, OMVs are nanosized vesicles capable of entering both phagocytic and non-phagocytic cells. Together with these features, OMVs produced from bioengineered or recombinant bacteria can also carry heterologous proteins, making them an attractive candidate for antigen delivery platforms. The present study explored the possible advantages of bioengineered OMVs produced by a non-pathogenic laboratory strain of E. coli, Rosetta 2. The E. coli Rosetta 2 cells were bioengineered (rE. coli) to express the ectodomain of Type A influenza virus Matrix protein 2 (M2e). The plasmid construct encoding a four tandem repeat of M2e (4xM2e) sequence was fused downstream of the OmpA protein (to localize the protein in the outer membrane) and upstream to a reporter gene, mCherry. Transformed E. coli Rosetta 2 cells were used for OMV production. The OMVs were isolated from the culture supernatant of rE. coli and subjected to a range of morphological, biophysical, and immunological analyses. Next, to see the immunogenicity of 4xM2e-OMVs, 5–6-week-old BALB/c mice were injected, and the serum samples were collected on day 7 post-last immunization. The data from in vitro characterization and in vivo immunization studies confirm 4xM2e-OMVs’ potential in carrying 4xM2e protein and retaining its ability to induce antigen (4xM2e) specific antibodies in mice. However, further study is needed to investigate whether the immune response induced by bioengineered E. coli OMVs can translate into protective immunity against target pathogens.

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