Medical academic journalMedical academic journal1608-41012687-1378Eco-Vector10867110.17816/MAJ108671Conference Report, Theses of ReportSurface expression of SARS-CoV-2 epitopes in <i>Enterococcus faecium</i> L3 for live oral vaccine against new coronavirus infectionKoptevaOlga S.<p>Research Associate of the World-class Research Center “Center for Personalized Medicine”; Postgraduate Student</p>olga.s.kopteva@yandex.ruhttps://orcid.org/0000-0002-2645-3433DeshevaYulia A.<p>MD, Dr. Sci. (Med.), Leading Research Associate of the World-class Research Center “Center for Personalized Medicine” and Leading Research Associate of the Virology Department; Professor of the Department of Fundamental Problems of Medicine and Medical Technologies of the Faculty of Dentistry and Medical Technologies</p>desheva@mail.ruhttps://orcid.org/0000-0001-9794-3520IvanovaAlexandra N.<p>Cand. Sci. (Biol.), Specialist in Sample preparation for Transmission Electron Microscopy of the Resource Center “Development of Molecular and Cellular Technologies”</p>alexandra.ivanova@spbu.ruVorobyovMaxim G.<p>Specialist in Transmission and scanning electron microscopy of the Resource Center “Development of Molecular and Cellular Technologies”</p>vorobiev.maxim@spbu.ruLeontievaGalina F.<p>Cand. Sci. (Biol.), Senior Research Associate of the World-class Research Center “Center for Personalized Medicine” and Senior Research Associate of the Molecular Microbiology Department</p>galeonte@yandex.ruGupalovaTatiana V.<p>Dr. Sci. (Biol.), Leading Research Associate of the World-class Research Center “Center for Personalized Medicine” and Leading Research Associate of the Molecular Microbiology Department</p>tvgupalova@rambler.ruBormotovaElena A.<p>Cand. Sci. (Biol.), Research Associate of the World-class Research Center “Center for Personalized Medicine” and Research Associate of the Molecular Microbiology Department</p>bormotovae@rambler.ruSuvorovAlexander N.<p>MD, Dr. Sci. (Med.), Professor, Corresponding Member of the Russian Academy of Sciences, Head of the Microbial Therapy Department of the World-class Research Center “Center for Personalized Medicine” and the Head of the Molecular Microbiology Department; Head of the Department of Fundamental Problems of Medicine and Medical Technologies of the Faculty of Dentistry and Medical Technologies</p>suvorov.an@iemspb.ruInstitute of Experimental MedicineSaint Petersburg State University061120222221972021606202217072022Copyright © 2022, Eco-Vector2022<p><strong><em>BACKGROUND:</em></strong> 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.</p>
<p><strong><em>AIM:</em></strong> The aim was to study expression of mRNA fragment of <em>S1</em> SARS-CoV-2 gene in <em>Enterococcus faecium </em>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 (<em>E. faecium</em> L3) and genetically modified strain (L3-SARS) with human sera obtained from patients with SARS-CoV-2.</p>
<p><strong><em>MATERIALS AND METHODS:</em></strong> 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 <em>E. faecium</em> 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.</p>
<p><strong><em>RESULTS:</em></strong> A sharp increase in mRNA amplification of inserted genetic sequence of <em>S1</em> SARS-CoV-2 gene fragment relatively to the control was demonstrated. These results confirmed that DNA of <em>S1</em> gene in <em>E. faecium</em> L3 genome is transcribed together with the target pili gene in <em>E. faecium</em> 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.</p>
<p><strong><em>CONCLUSIONS:</em></strong> Evaluation in expression of SARS-CoV-2 S1 protein after introduction of the corresponding genetic elements into genome of probiotic strain <em>E. faecium</em> 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 <em>E. faecium</em> L3-SARS.</p>probioticenterococcusprobiotic-based vaccinesSARS-CoV-2S proteinпробиотикэнтерококкпробиотические вакциныSARS-CoV-2S-белок[Mohseni AH, Taghinezhad-SS, Keyvani H. The first clinical use of a recombinant lactococcus lactis expressing human papillomavirus type 16 E7 oncogene oral vaccine: a phase i safety and immunogenicity trial in healthy women volunteers. Mol Cancer Ther. 2020;19(2):717–727. DOI: 10.1158/1535-7163.MCT-19-0375][Suvorov A, Gupalova T, Desheva Y, et al. Construction of the enterococcal strain expressing immunogenic fragment of SARS-CoV-2 virus. Front Pharmacol. 2022;12:807256. DOI: 10.3389/fphar.2021.807256][Gupalova T, Leontieva G, Kramskaya T, et al. Development of experimental pneumococcal vaccine for mucosal immunization. PloS one. 2019;14(6):e0218679. DOI: 10.1371/journal.pone.0218679][Khare B, Narayana SVL. Pilus biogenesis of gram-positivebacteria: roles of sortases and implications for assembly. Protein Sci. 2017;26(8):1458–1473. DOI: 10.1002/pro.3191][Montealegre MC, Singh KV, Somarajan SR, et al. Role of the Emp pilus subunits of Enterococcus faecium in biofilm formation, adherence to host extracellular matrix components, and experimental infection. Infect Immun. 2016;84(5):1491–1500. DOI: 10.1128/IAI.01396-15]