Viability of Escherichia coli K-12 as a multi-epitope delivery system
The current approach to immunizing individuals against COVID-19 involves an mRNA-based vaccine that engenders IgA and IgG antibodies against the RBD (Receptor Binding Domain) of the SARS-CoV-2 virus, effectively neutralizing viral attachment to ACE-2 receptors on the host cell’s surface. However, current research suggests that the antibodies originating from past infection or vaccination will bind with less affinity to SARS-CoV-2 Delta and Omicron variants, given the subtle changes in the RBD amino acid sequence and, therefore, structure.
In response to such concerns, we propose using Escherichia coli K-12 as a multi-epitope delivery system, namely against SARS-CoV-2 and enterotoxigenic E. coli (ETEC) Heat Stable Toxin (ST) epitopes. ETEC is a major cause of diarrheal disease and mortality in developing countries. The highly immunogenic E. coli Common Pilus (ECP), one of the many adhesive factors in E. coli, is made of EcpA dimers. The crystal structures of the EcpA dimers reveal 5 conformational loops protruding from the ECP’s tertiary structure. Based on past research in our lab, we believe that substitution of these conformational loop sequences within the EcpA gene with Omicron RBD or ST epitope sequences will yield expression of RBD and ST epitopes along the pili filaments. We hypothesize that: the immunization of mice with these engineered bacteria or purified pili expressing the foreign epitopes will yield anti-ST and anti-RBD antibodies.
Results: So far, we have successfully amplified custom EcpA-ST genes with ST Epitope Sequences in loops 1-5 in addition to EcpA-RBD genes with Omicron RBD sequences of varying length (485-505 bp) in loop 2. Confirmation was performed using gel electrophoresis. Furthermore, we have successfully ligated the EcpA-ST and EcpA-RBD genes into cloning plasmid pT3. Lastly, we are in the process of transforming an E. coli K-12 strain called DH5α with the newly ligated pT3-EcpA-ST and pT3-EcpA-RBD plasmids. Expression of ST and RBD epitopes at the site of the five separate conformational loops will be tested using indirect immunofluorescence, dot blot chemiluminescence, and commercial anti-ST and anti-RBD monoclonal antibodies. Our hope is to use the recombinant EcpA-ST and EcpA-RBD dimers and their 5 conformational loops as templates to express epidemiologically-relevant SARS-CoV-2 variant epitopes, thus prompting a mucosal IgA antibody response against circulating SARS-CoV-2 variants and the ETEC ST.