Surface expression of SARS-CoV-2 epitopes in <i>Enterococcus faecium</i> L3 for live oral vaccine against new coronavirus infection

Abstract

BACKGROUND: Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing viral or bacterial antigens. Probiotic-based mucosal vaccines are easy to produce in large quantities, they have a low cost, provide a fairly long T-cell memory. AIM: The aim was to study expression of mRNA fragment of S1 SARS-CoV-2 gene in Enterococcus faecium L3 culture and to confirm the insertion of S1 SARS-CoV-2 protein fragment into the pili of this bacterial strain by immunoelectron microscopy of original (E. faecium L3) and genetically modified strain (L3-SARS) with human sera obtained from patients with SARS-CoV-2. MATERIALS AND METHODS: mRNA expression was studied by real-time PCR with reverse transcription using primers specific to S1 protein. Immunoelectron microscopy was aimed to study the structure of E. faecium L3 pili with the expression of viral protein SARS-CoV-2. Bacteria were washed three times in PBS by centrifugation at 3500 rpm for 20 min and suspended in 0.1 M NaCl. A 10-fold bacterial concentrate was used. The source of the primary antibodies was a set of polyclonal human sera containing IgG. Labeling was performed using goat IgG conjugated with 18 nm gold particles. RESULTS: A sharp increase in mRNA amplification of inserted genetic sequence of S1 SARS-CoV-2 gene fragment relatively to the control was demonstrated. These results confirmed that DNA of S1 gene in E. faecium L3 genome is transcribed together with the target pili gene in E. faecium genome. Specific antigens of SARS-CoV-2 on the surface of L3-SARS were determined using electron microscopy, which demonstrated the correct assembly of chimeric molecules of pili on the surface of bacteria. CONCLUSIONS: Evaluation in expression of SARS-CoV-2 S1 protein after introduction of the corresponding genetic elements into genome of probiotic strain E. faecium L3 allows us to conclude that selected DNA fragments of SARS-CoV-2 were able to direct the synthesis of immunogenic protein S1 that was expressed by the strain E. faecium L3-SARS.