5 research outputs found
Production and characterisation of a SARS-CoV-2 S-protein RBD homodimer with increased avidity for specific antibodies
Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies.The aim of the study was to obtain and characterise a SARS-CoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies.Materials and methods: the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved mono clonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones.Results: the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBDβs ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30β50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies.Conclusions: the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein
ΠΠΎΠ»ΡΡΠ΅Π½ΠΈΠ΅ ΠΈ Ρ Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΡΡΠΈΠΊΠ° Π³ΠΎΠΌΠΎΠ΄ΠΈΠΌΠ΅ΡΠ½ΠΎΠΉ ΡΠΎΡΠΌΡ RBD S-Π±Π΅Π»ΠΊΠ° SARS-CoV-2, ΠΎΠ±Π»Π°Π΄Π°ΡΡΠ΅ΠΉ ΠΏΠΎΠ²ΡΡΠ΅Π½Π½ΠΎΠΉ Π°Π²ΠΈΠ΄Π½ΠΎΡΡΡΡ ΠΊ ΡΠΏΠ΅ΡΠΈΡΠΈΡΠ΅ΡΠΊΠΈΠΌ Π°Π½ΡΠΈΡΠ΅Π»Π°ΠΌ
Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study w as t o obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Materials and methods: the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Results: the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBDβs ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30β50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusions: the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.ΠΠ°ΠΆΠ½ΡΠΌ ΠΏΠ°ΡΠ°ΠΌΠ΅ΡΡΠΎΠΌ, ΠΎΡΠ΅Π½ΠΈΠ²Π°Π΅ΠΌΡΠΌ ΠΏΡΠΈ ΠΌΠΎΠ½ΠΈΡΠΎΡΠΈΠ½Π³Π΅ ΠΈΠΌΠΌΡΠ½Π½ΠΎΠΉ ΠΏΡΠΎΡΠ»ΠΎΠΉΠΊΠΈ Ρ Π½Π°ΡΠ΅Π»Π΅Π½ΠΈΡ ΠΈ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π²Π°ΠΊΡΠΈΠ½Π°ΡΠΈΠΈ Π½Π°ΡΠ΅Π»Π΅Π½ΠΈΡ, ΡΠ²Π»ΡΠ΅ΡΡΡ ΡΡΠΎΠ²Π΅Π½Ρ Π²ΠΈΡΡΡΠ½Π΅ΠΉΡΡΠ°Π»ΠΈΠ·ΡΡΡΠΈΡ
Π°Π½ΡΠΈΡΠ΅Π». Π Π°Π·ΡΠ°Π±ΠΎΡΠΊΠ° ΠΏΠΎΠ΄Ρ
ΠΎΠ΄Π° ΠΊ Π²ΡΡΠ²Π»Π΅Π½ΠΈΡ Π²ΠΈΡΡΡΠ½Π΅ΠΉΡΡΠ°Π»ΠΈΠ·ΡΡΡΠΈΡ
Π°Π½ΡΠΈΡΠ΅Π» ΠΊ Π²ΠΈΡΡΡΡ SARS-CoV-2 Ρ ΠΏΠΎΠΌΠΎΡΡΡ Π±Π΅Π·ΠΎΠΏΠ°ΡΠ½ΠΎΠ³ΠΎ, ΠΏΡΠΎΡΡΠΎΠ³ΠΎ ΠΈ Π±ΡΡΡΡΠΎΠ³ΠΎ ΠΌΠ΅ΡΠΎΠ΄Π°, Π½Π΅ ΡΡΠ΅Π±ΡΡΡΠ΅Π³ΠΎ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΡ ΠΆΠΈΠ²ΡΡ
Π²ΠΈΡΡΡΠΎΠ², ΠΈΠΌΠ΅Π΅Ρ Π±ΠΎΠ»ΡΡΠΎΠ΅ Π·Π½Π°ΡΠ΅Π½ΠΈΠ΅ Π΄Π»Ρ Π±ΠΎΡΡΠ±Ρ Ρ ΠΏΠ°Π½Π΄Π΅ΠΌΠΈΠ΅ΠΉ COVID-19. ΠΠ»Ρ ΡΠ°Π·ΡΠ°Π±ΠΎΡΠΊΠΈ ΡΠ΅ΡΡ-ΡΠΈΡΡΠ΅ΠΌ Π΄Π»Ρ ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ΠΈΡ ΠΈΠΌΠΌΡΠ½ΠΎΡΠ΅ΡΠΌΠ΅Π½ΡΠ½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π° (ΠΠ€Π), Π΄Π΅ΡΠ΅ΠΊΡΠΈΡΡΡΡΠΈΡ
ΠΏΠΎΡΠ΅Π½ΡΠΈΠ°Π»ΡΠ½ΠΎ Π²ΠΈΡΡΡΠ½Π΅ΠΉΡΡΠ°Π»ΠΈΠ·ΡΡΡΠΈΠ΅ Π°Π½ΡΠΈΡΠ΅Π»Π°, Π½Π΅ΠΎΠ±Ρ
ΠΎΠ΄ΠΈΠΌΠΎ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΠ΅ Π²ΡΡΠΎΠΊΠΎΠΎΡΠΈΡΠ΅Π½Π½ΠΎΠ³ΠΎ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠ³ΠΎ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡ-ΡΠ²ΡΠ·ΡΠ²Π°ΡΡΠ΅Π³ΠΎ Π΄ΠΎΠΌΠ΅Π½Π° (RBD) S-Π±Π΅Π»ΠΊΠ°, ΠΎΠ±Π»Π°Π΄Π°ΡΡΠ΅Π³ΠΎ Π²ΡΡΠΎΠΊΠΎΠΉ Π°Π²ΠΈΠ΄Π½ΠΎΡΡΡΡ ΠΊ ΡΠΏΠ΅ΡΠΈΡΠΈΡΠ΅ΡΠΊΠΈΠΌ Π°Π½ΡΠΈΡΠ΅Π»Π°ΠΌ. Π¦Π΅Π»Ρ ΡΠ°Π±ΠΎΡΡ: ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΠ΅ ΠΈ Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΡΡΠΈΠΊΠ° Π³ΠΎΠΌΠΎΠ΄ΠΈΠΌΠ΅ΡΠ½ΠΎΠΉ ΡΠΎΡΠΌΡ RBD S-Π±Π΅Π»ΠΊΠ° Π²ΠΈΡΡΡΠ° SARS-CoV-2, Π° ΡΠ°ΠΊΠΆΠ΅ ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ Π»ΠΈΠ½ΠΈΠΈ, ΠΏΡΠΎΠ΄ΡΡΠΈΡΡΡΡΠ΅ΠΉ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΠΉ RBD, Π΄Π»Ρ ΡΠΎΠ·Π΄Π°Π½ΠΈΡ ΠΠ€Π ΡΠ΅ΡΡ-ΡΠΈΡΡΠ΅ΠΌΡ Π΄Π»Ρ Π²ΡΡΠ²Π»Π΅Π½ΠΈΡ ΠΏΠΎΡΠ΅Π½ΡΠΈΠ°Π»ΡΠ½ΠΎ Π²ΠΈΡΡΡΠ½Π΅ΠΉΡΡΠ°Π»ΠΈΠ·ΡΡΡΠΈΡ
Π°Π½ΡΠΈΡΠ΅Π». ΠΠ°ΡΠ΅ΡΠΈΠ°Π»Ρ ΠΈ ΠΌΠ΅ΡΠΎΠ΄Ρ: Π΄ΠΈΠ·Π°ΠΉΠ½ Π³Π΅Π½Π΅ΡΠΈΡΠ΅ΡΠΊΠΎΠΉ ΠΊΠΎΠ½ΡΡΡΡΠΊΡΠΈΠΈ ΠΏΡΠΎΠ²ΠΎΠ΄ΠΈΠ»ΠΈ in silico. Π‘ΡΠ°Π±ΠΈΠ»ΡΠ½ΡΡ ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ Π»ΠΈΠ½ΠΈΡ ΠΏΠΎΠ»ΡΡΠ°Π»ΠΈ ΠΏΡΠΈ ΠΏΠΎΠΌΠΎΡΠΈ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ CHO-S, ΡΠ΅Π»Π΅ΠΊΡΠΈΠΈ Π½Π° Π°Π½ΡΠΈΠ±ΠΈΠΎΡΠΈΠΊΠ΅ ΠΈ ΠΎΡΠ±ΠΎΡΠ° ΠΎΠΏΡΠΈΠΌΠ°Π»ΡΠ½ΠΎΠ³ΠΎ ΠΊΠ»ΠΎΠ½Π°. Π Π΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΠΉ RBD ΠΎΡΠΈΡΠ°Π»ΠΈ Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ Ρ
ΡΠΎΠΌΠ°ΡΠΎΠ³ΡΠ°ΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΌΠ΅ΡΠΎΠ΄ΠΎΠ², ΠΏΠΎΠ»ΡΡΠ°Π»ΠΈ ΠΌΠΎΠ½ΠΎΠΌΠ΅ΡΠ½ΡΡ ΠΈ Π³ΠΎΠΌΠΎΠ΄ΠΈΠΌΠ΅ΡΠ½ΡΡ ΡΠΎΡΠΌΡ RBD. ΠΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΠΏΠΎΠ»ΡΡΠ΅Π½Π½ΡΡ
ΡΠΎΡΠΌ Π°Π½Π°Π»ΠΈΠ·ΠΈΡΠΎΠ²Π°Π»ΠΈ Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ ΠΌΠ΅ΡΠΎΠ΄ΠΎΠ² ΠΠ΅ΡΡΠ΅ΡΠ½-Π±Π»ΠΎΡ, Π±ΠΈΠΎΡΠ»ΠΎΠΉΠ½ΠΎΠΉ ΠΈΠ½ΡΠ΅ΡΡΠ΅ΡΠΎΠΌΠ΅ΡΡΠΈΠΈ ΠΈ Π½Π΅ΠΏΡΡΠΌΠ³ΠΎ ΠΠ€Π. ΠΠ»Ρ Π°Π½Π°Π»ΠΈΠ·Π° ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π»ΠΈ ΠΌΠΎΠ½ΠΎΠΊΠ»ΠΎΠ½Π°Π»ΡΠ½ΡΠ΅ Π°Π½ΡΠΈΡΠ΅Π»Π° GamXRH19, GamP2C5 ΠΈ h6g3, Π° ΡΠ°ΠΊΠΆΠ΅ ΠΎΠ±ΡΠ°Π·ΡΡ ΡΡΠ²ΠΎΡΠΎΡΠΎΠΊ ΠΊΡΠΎΠ²ΠΈ Π΄ΠΎΠ±ΡΠΎΠ²ΠΎΠ»ΡΡΠ΅Π², Π²Π°ΠΊΡΠΈΠ½ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΎΠΌ ΠΠ°ΠΌ-ΠΠΠΠΠ-ΠΠ°ΠΊ, ΠΈ Π½Π΅Π²Π°ΠΊΡΠΈΠ½ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
Π΄ΠΎΠ±ΡΠΎΠ²ΠΎΠ»ΡΡΠ΅Π². Π Π΅Π·ΡΠ»ΡΡΠ°ΡΡ: ΠΏΠΎΠ»ΡΡΠ΅Π½Π° ΠΊΠ»Π΅ΡΠΎΡΠ½Π°Ρ Π»ΠΈΠ½ΠΈΡ CHO-S, ΡΡΠ°Π±ΠΈΠ»ΡΠ½ΠΎ ΠΏΡΠΎΠ΄ΡΡΠΈΡΡΡΡΠ°Ρ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΠΉ RBD S-Π±Π΅Π»ΠΊΠ° Π²ΠΈΡΡΡΠ° SARS-CoV-2. ΠΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ ΠΏΡΠΈ ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ Π΄Π°Π½Π½ΠΎΠΉ ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ Π»ΠΈΠ½ΠΈΠΈ Π² ΡΠ΅ΠΆΠΈΠΌΠ΅ fed-batch Π±ΠΎΠ»Π΅Π΅ 7 ΡΡΡΠΎΠΊ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΠΉ RBD ΡΠΏΠΎΡΠΎΠ±Π΅Π½ ΠΎΠ±ΡΠ°Π·ΠΎΠ²ΡΠ²Π°ΡΡ Π³ΠΎΠΌΠΎΠ΄ΠΈΠΌΠ΅ΡΡ Π·Π° ΡΡΠ΅Ρ Π½Π°Π»ΠΈΡΠΈΡ Π½Π΅ΡΠΏΠ°ΡΠ΅Π½Π½ΡΡ
ΡΠΈΡΡΠ΅ΠΈΠ½ΠΎΠ². ΠΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π΅Π½Π½ΡΠΉ Π²ΡΡ
ΠΎΠ΄ ΠΎΡΠΈΡΠ΅Π½Π½ΠΎΠ³ΠΎ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠ³ΠΎ RBD ΠΈΠ· ΠΊΡΠ»ΡΡΡΡΠ°Π»ΡΠ½ΠΎΠΉ ΠΆΠΈΠ΄ΠΊΠΎΡΡΠΈ ΡΠΎΡΡΠ°Π²ΠΈΠ» 30β50 ΠΌΠ³/Π». ΠΠΎΠ½ΠΎΠΌΠ΅ΡΠ½Π°Ρ ΠΈ Π³ΠΎΠΌΠΎΠ΄ΠΈΠΌΠ΅ΡΠ½Π°Ρ ΡΠΎΡΠΌΡ RBD Π±ΡΠ»ΠΈ ΡΠ°Π·Π΄Π΅Π»Π΅Π½Ρ ΠΏΡΠΈ ΠΏΠΎΠΌΠΎΡΠΈ Π³Π΅Π»Ρ-ΡΠΈΠ»ΡΡΡΠ°ΡΠΈΠΈ ΠΈ ΠΎΡ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΠ·ΠΎΠ²Π°Π½Ρ ΠΏΠΎ ΡΠΏΠΎΡΠΎΠ±Π½ΠΎΡΡΠΈ Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΎΠ²Π°ΡΡ ΡΠΎ ΡΠΏΠ΅ΡΠΈΡΠΈΡΠ΅ΡΠΊΠΈΠΌΠΈ ΠΌΠΎΠ½ΠΎΠΊΠ»ΠΎΠ½Π°Π»ΡΠ½ΡΠΌΠΈ Π°Π½ΡΠΈΡΠ΅Π»Π°ΠΌΠΈ, Π° ΡΠ°ΠΊΠΆΠ΅ ΡΡΠ²ΠΎΡΠΎΡΠΊΠ°ΠΌΠΈ ΠΊΡΠΎΠ²ΠΈ ΠΎΡ Π²Π°ΠΊΡΠΈΠ½ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
Π΄ΠΎΠ±ΡΠΎΠ²ΠΎΠ»ΡΡΠ΅Π². ΠΡΠΎΠ΄Π΅ΠΌΠΎΠ½ΡΡΡΠΈΡΠΎΠ²Π°Π½ΠΎ, ΡΡΠΎ ΠΈΠΌΠ΅Π½Π½ΠΎ Π³ΠΎΠΌΠΎΠ΄ΠΈΠΌΠ΅ΡΠ½Π°Ρ ΡΠΎΡΠΌΠ° ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠ³ΠΎ RBD ΠΎΠ±Π»Π°Π΄Π°Π΅Ρ ΠΏΠΎΠ²ΡΡΠ΅Π½Π½ΠΎΠΉ Π°Π²ΠΈΠ΄Π½ΠΎΡΡΡΡ ΠΊ ΠΌΠΎΠ½ΠΎΠΊΠ»ΠΎΠ½Π°Π»ΡΠ½ΡΠΌ Π°Π½ΡΠΈΡΠ΅Π»Π°ΠΌ ΠΈ Π°Π½ΡΠΈΡΠ΅Π»Π°ΠΌ Π² ΡΡΠ²ΠΎΡΠΎΡΠΊΠ΅ ΠΊΡΠΎΠ²ΠΈ Π²Π°ΠΊΡΠΈΠ½ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
. ΠΡΠ²ΠΎΠ΄Ρ: Π³ΠΎΠΌΠΎΠ΄ΠΈΠΌΠ΅ΡΠ½Π°Ρ ΡΠΎΡΠΌΠ° ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠ³ΠΎ RBD ΠΌΠΎΠΆΠ΅Ρ ΡΠ²Π»ΡΡΡΡΡ Π±ΠΎΠ»Π΅Π΅ ΠΏΡΠ΅Π΄ΠΏΠΎΡΡΠΈΡΠ΅Π»ΡΠ½ΠΎΠΉ Π΄Π»Ρ Π°Π½Π°Π»ΠΈΠ·Π° ΡΡΠΎΠ²Π½Ρ Π°Π½ΡΠΈΡΠ΅Π» ΠΊ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡ-ΡΠ²ΡΠ·ΡΠ²Π°ΡΡΠ΅ΠΌΡ Π΄ΠΎΠΌΠ΅Π½Ρ S-Π±Π΅Π»ΠΊΠ° Π²ΠΈΡΡΡΠ° SARS-CoV-2
rAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19
IntroductionNumerous agents for prophylaxis of SARS-CoV-2-induced diseases are currently registered for the clinical use. Formation of the immunity happens within several weeks following vaccine administration which is their key disadvantage. In contrast, drugs based on monoclonal antibodies, enable rapid passive immunization and therefore can be used for emergency pre- and post-exposure prophylaxis of COVID-19. However rapid elimination of antibody-based drugs from the circulation limits their usage for prolonged pre-exposure prophylaxis.MethodsIn current work we developed a recombinant adeno-associated viral vector (rAAV), expressing a SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibody P2C5 fused with a human IgG1 Fc fragment (P2C5-Fc) using methods of molecular biotechnology and bioprocessing.Results and discussionsA P2C5-Fc antibody expressed by a proposed rAAV (rAAV-P2C5-Fc) was shown to circulate within more than 300 days in blood of transduced mice and protect animals from lethal SARS-CoV-2 virus (B.1.1.1 and Omicron BA.5 variants) lethal dose of 105 TCID50. In addition, rAAV-P2C5-Fc demonstrated 100% protective activity as emergency prevention and long-term prophylaxis, respectively. It was also demonstrated that high titers of neutralizing antibodies to the SARS-CoV-2 virus were detected in the blood serum of animals that received rAAV-P2C5-Fc for more than 10 months from the moment of administration.Our data therefore indicate applicability of an rAAV for passive immunization and induction of a rapid long-term protection against various SARS-CoV-2 variants
Safety and immunogenicity of rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine against SARS-CoV-2 in healthy adolescents: an open-label, non-randomized, multicenter, phase 1/2, dose-escalation study
To protect young individuals against SARS-CoV-2 infection, we conducted an open-label, prospective, non-randomised dose-escalation Phase 1/2 clinical trial to evaluate the immunogenicity and safety of the prime-boost βSputnik Vβ vaccine administered at 1/10 and 1/5 doses to adolescents aged 12β17 years. The study began with the vaccination of the older cohort (15-to-17-year-old participants) with the lower (1/10) dose of vaccine and then expanded to the whole group (12-to-17-year-old participants). Next, 1/5 dose was used according to the same scheme. Both doses were well tolerated by all age groups. No serious or severe adverse events were detected. Most of the solicited adverse reactions were mild. No significant differences in total frequencies of adverse events were registered between low and high doses in age-pooled groups (69.6% versus 66.7%). In contrast, the 1/5 dose induced significantly higher humoral and T cell-mediated immune responses than the 1/10 dose. The 1/5 vaccine dose elicited higher antigen-binding (both S and RBD-specific) as well as virus-neutralising antibody titres at the maximum of response (day 42), also resulting in a statistically significant difference at a distanced timepoint (day 180) compared to the 1/10 vaccine dose. Higher dose resulted in increased cross-neutralization of Delta and Omicron variants.;Clinical Trial RegistrationClinicalTrials.gov, NCT04954092, LP-007632
DataSheet_1_Estimation of anti-orthopoxvirus immunity in Moscow residents and potential risks of spreading Monkeypox virus.docx
WHO has declared the outbreak of monkeypox as a public health emergency of international concern. In less than three months, monkeypox was detected in more than 30 000 people and spread to more than 80 countries around the world. It is believed that the immunity formed to smallpox vaccine can protect from monkeypox infection with high efficiency. The widespread use of Vaccinia virus has not been carried out since the 1980s, which raises the question of the level of residual immunity among the population and the identification of groups requiring priority vaccination. We conducted a cross-sectional serological study of remaining immunity among Moscow residents. To do this, a collection of blood serum samples of age group over 30 years old was formed, an in-house ELISA test system was developed, and a virus neutralization protocol was set up. Serum samples were examined for the presence of IgG antibodies against Vaccinia virus (n=2908), as well as for the ability to neutralize plaque formation with a Vaccinia virus MNIIVP-10 strain (n=299). The results indicate the presence of neutralizing antibody titer of 1/20 or more in 33.3 to 53.2% of people older than 45 years. Among people 30-45 years old who probably have not been vaccinated, the proportion with virus neutralizing antibodies ranged from 3.2 to 6.7%. Despite the higher level of antibodies in age group older than 66 years, the proportion of positive samples in this group was slightly lower than in people aged 46-65 years. The results indicate the priority of vaccination in groups younger than 45, and possibly older than 66 years to ensure the protection of the population in case of spread of monkeypox among Moscow residents. The herd immunity level needed to stop the circulation of the virus should be at least 50.25 β 65.28%.</p