5 research outputs found
Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale
Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product
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
Π‘ΡΠ°Π²Π½Π΅Π½ΠΈΠ΅ ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ ΡΠ΅Ρ Π½ΠΎΠ»ΠΎΠ³ΠΈΠΉ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠ³ΠΎ Π°Π΄Π΅Π½ΠΎΠ°ΡΡΠΎΡΠΈΠΈΡΠΎΠ²Π°Π½Π½ΠΎΠ³ΠΎ Π²ΠΈΡΡΡΠ° Π² Π»Π°Π±ΠΎΡΠ°ΡΠΎΡΠ½ΠΎΠΌ ΠΌΠ°ΡΡΡΠ°Π±Π΅
Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.ΠΠ΅ΠΊΡΠΎΡΡ Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ Π°Π΄Π΅Π½ΠΎΠ°ΡΡΠΎΡΠΈΠΈΡΠΎΠ²Π°Π½Π½ΠΎΠ³ΠΎ Π²ΠΈΡΡΡΠ° ΡΠ²Π»ΡΡΡΡΡ ΠΎΠ΄Π½ΠΈΠΌΠΈ ΠΈΠ· Π½Π°ΠΈΠ±ΠΎΠ»Π΅Π΅ ΠΏΠ΅ΡΡΠΏΠ΅ΠΊΡΠΈΠ²Π½ΡΡ
Π΄Π»Ρ Π΄ΠΎΡΡΠ°Π²ΠΊΠΈ ΡΡΠ°Π½ΡΠ³Π΅Π½ΠΎΠ² Π² ΡΠ°Π·Π»ΠΈΡΠ½ΡΠ΅ ΠΎΡΠ³Π°Π½Ρ ΠΈ ΡΠΊΠ°Π½ΠΈ. Π Π΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΠΉ Π°Π΄Π΅Π½ΠΎΠ°ΡΡΠΎΡΠΈΠΈΡΠΎΠ²Π°Π½Π½ΡΠΉ Π²ΠΈΡΡΡ (rAAV) ΡΠΏΠΎΡΠΎΠ±Π΅Π½ ΡΡΠ°Π½ΡΠ΄ΡΡΠΈΡΠΎΠ²Π°ΡΡ ΠΊΠ°ΠΊ Π΄Π΅Π»ΡΡΠΈΠ΅ΡΡ, ΡΠ°ΠΊ ΠΈ Π½Π΅Π΄Π΅Π»ΡΡΠΈΠ΅ΡΡ ΠΊΠ»Π΅ΡΠΊΠΈ, ΠΎΠ±Π»Π°Π΄Π°Π΅Ρ Π½ΠΈΠ·ΠΊΠΎΠΉ ΠΈΠΌΠΌΡΠ½ΠΎΠ³Π΅Π½Π½ΠΎΡΡΡΡ ΠΈ ΡΠΏΠΎΡΠΎΠ±Π΅Π½ ΠΎΠ±Π΅ΡΠΏΠ΅ΡΠΈΠ²Π°ΡΡ Π΄ΠΎΠ»Π³ΠΎΡΡΠΎΡΠ½ΡΡ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡ ΡΡΠ°Π½ΡΠ³Π΅Π½ΠΎΠ². ΠΠ° ΡΠ΅Π³ΠΎΠ΄Π½ΡΡΠ½ΠΈΠΉ Π΄Π΅Π½Ρ ΡΡΡΠ΅ΡΡΠ²ΡΡΡ ΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΠΈΠΈ, ΠΏΠΎΠ·Π²ΠΎΠ»ΡΡΡΠΈΠ΅ ΠΏΠΎΠ»ΡΡΠ°ΡΡ rAAV Π΄Π»Ρ ΠΏΡΠΈΠΌΠ΅Π½Π΅Π½ΠΈΡ in vivo, ΠΎΠ΄Π½Π°ΠΊΠΎ ΠΎΠ½ΠΈ Π½Π΅ Π»ΠΈΡΠ΅Π½Ρ Π½Π΅Π΄ΠΎΡΡΠ°ΡΠΊΠΎΠ², ΡΠ²ΡΠ·Π°Π½Π½ΡΡ
Ρ ΡΡΡΠ΄ΠΎΠ΅ΠΌΠΊΠΎΡΡΡΡ, ΡΠ»ΠΎΠΆΠ½ΠΎΡΡΡΠΌΠΈ ΠΌΠ°ΡΡΡΠ°Π±ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΠΈ Π²ΡΡΠΎΠΊΠΎΠΉ ΡΡΠΎΠΈΠΌΠΎΡΡΡΡ, ΠΏΠΎΡΡΠΎΠΌΡ Π²ΠΎΠΏΡΠΎΡ ΠΎΠ± ΡΡΠΎΠ²Π΅ΡΡΠ΅Π½ΡΡΠ²ΠΎΠ²Π°Π½ΠΈΠΈ ΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΡ
Π΅ΠΌ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ rAAV ΡΠ²Π»ΡΠ΅ΡΡΡ Π°ΠΊΡΡΠ°Π»ΡΠ½ΡΠΌ.Β Π¦Π΅Π»Ρ ΡΠ°Π±ΠΎΡΡ: ΡΡΠ°Π²Π½Π΅Π½ΠΈΠ΅ ΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΏΠΎΠ΄Ρ
ΠΎΠ΄ΠΎΠ² ΠΊ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ rAAV, ΠΎΡΠ½ΠΎΠ²Π°Π½Π½ΡΡ
Π½Π° ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΡΡΠ»ΠΎΠ²ΠΈΡΡ
ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΡΡΠ°Π½ΡΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΠΎΠΉ ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ Π»ΠΈΠ½ΠΈΠΈ HEK293 Π² Π»Π°Π±ΠΎΡΠ°ΡΠΎΡΠ½ΠΎΠΌ ΠΌΠ°ΡΡΡΠ°Π±Π΅.Β ΠΠ°ΡΠ΅ΡΠΈΠ°Π»Ρ ΠΈ ΠΌΠ΅ΡΠΎΠ΄Ρ: Π² ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠΈ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π»ΠΈ ΠΊΡΠ»ΡΡΡΡΡ ΠΊΠ»Π΅ΡΠΎΠΊ HEK293, ΠΏΠ»Π°Π·ΠΌΠΈΠ΄Π½ΡΡ ΡΠΈΡΡΠ΅ΠΌΡ AAV-DJ Packaging System, ΡΠΈΡΡΠ΅ΠΌΡ PlasmidSelect Xtra Starter Kit. Π ΠΊΠ°ΡΠ΅ΡΡΠ²Π΅ ΠΌΠΎΠ΄Π΅Π»ΠΈ Π΄Π»Ρ ΡΡΠ°Π²Π½Π΅Π½ΠΈΡ ΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΠΈΠΉ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π»ΠΈ Π²Π΅ΠΊΡΠΎΡ rAAV Ρ ΡΡΠ°Π½ΡΠ³Π΅Π½ΠΎΠΌ ΠΎΠ΄Π½ΠΎΠ΄ΠΎΠΌΠ΅Π½Π½ΠΎΠ³ΠΎ Π°Π½ΡΠΈΡΠ΅Π»Π°, ΡΠ»ΠΈΡΠΎΠ³ΠΎ Ρ Fc-ΡΡΠ°Π³ΠΌΠ΅Π½ΡΠΎΠΌ IgG1, ΡΠΏΠ΅ΡΠΈΡΠΈΡΠ½ΠΎΠ³ΠΎ ΠΊ Π±ΠΎΡΡΠ»ΠΎΡΠΎΠΊΡΠΈΠ½Ρ. ΠΡΠΈΠΌΠ΅Π½ΡΠ»ΠΈ ΠΌΠ΅ΡΠΎΠ΄ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ HEK293 ΡΡΠΏΠ΅ΡΡΠΊΡΡΡΠ΅Π½Π½ΠΎΠΉ ΠΏΠ»Π°Π·ΠΌΠΈΠ΄Π½ΠΎΠΉ ΠΠΠ, Π²ΡΠ΄Π΅Π»Π΅Π½Π½ΠΎΠΉ ΠΏΡΠΈ ΠΏΠΎΠΌΠΎΡΠΈ ΡΡΠ΅Ρ
ΡΡΡΠΏΠ΅Π½ΡΠ°ΡΠΎΠΉ Ρ
ΡΠΎΠΌΠ°ΡΠΎΠ³ΡΠ°ΡΠΈΡΠ΅ΡΠΊΠΎΠΉ ΠΎΡΠΈΡΡΠΊΠΈ. ΠΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΠ΅ ΠΏΠΎΠ΄Π»ΠΈΠ½Π½ΠΎΡΡΠΈ ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠ° rAAV ΠΏΡΠΎΠ²ΠΎΠ΄ΠΈΠ»ΠΈ ΠΌΠ΅ΡΠΎΠ΄Π°ΠΌΠΈ ΡΠ»Π΅ΠΊΡΡΠΎΡΠΎΡΠ΅Π·Π°, ΠΈΠΌΠΌΡΠ½ΠΎΠ±Π»ΠΎΡΡΠΈΠ½Π³Π° ΠΈ ΠΏΠΎΠ»ΠΈΠΌΠ΅ΡΠ°Π·Π½ΠΎΠΉ ΡΠ΅ΠΏΠ½ΠΎΠΉ ΡΠ΅Π°ΠΊΡΠΈΠΈ Π² ΡΠ΅ΠΆΠΈΠΌΠ΅ ΡΠ΅Π°Π»ΡΠ½ΠΎΠ³ΠΎ Π²ΡΠ΅ΠΌΠ΅Π½ΠΈ.Β Π Π΅Π·ΡΠ»ΡΡΠ°ΡΡ: ΠΏΡΠΎΠ΄Π΅ΠΌΠΎΠ½ΡΡΡΠΈΡΠΎΠ²Π°Π½Π° ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΡΡΠΏΠ΅ΡΡΠΊΡΡΡΠ΅Π½Π½ΠΎΠΉ ΡΠΎΡΠΌΡ ΠΏΠ»Π°Π·ΠΌΠΈΠ΄Π½ΠΎΠΉ ΠΠΠ, ΠΏΡΠΈΠΌΠ΅Π½ΠΈΠΌΠΎΠΉ Π΄Π»Ρ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΠΉ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ Ρ ΡΠ΅Π»ΡΡ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ rAAV. ΠΡΠΎΠ²Π΅Π΄Π΅Π½ΠΎ ΡΡΠ°Π²Π½Π΅Π½ΠΈΠ΅ ΠΏΡΠΎΡΠ΅ΡΡΠ° ΡΡΠ°Π½Π·ΠΈΠ΅Π½ΡΠ½ΠΎΠΉ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΈ ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΡΡΠ°Π½ΡΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ HEK293 Π² ΡΡΠ»ΠΎΠ²ΠΈΡΡ
ΡΡΡΠΏΠ΅Π½Π·ΠΈΠΈ Π² ΠΊΠΎΠ»Π±Π°Ρ
, Π°Π΄Π³Π΅Π·ΠΈΠΈ Π² ΠΊΡΠ»ΡΡΡΡΠ°Π»ΡΠ½ΡΡ
ΡΠ»Π°ΠΊΠΎΠ½Π°Ρ
ΠΈ Π°Π΄Π³Π΅Π·ΠΈΠΈ Π² Π±ΠΈΠΎΡΠ΅Π°ΠΊΡΠΎΡΠ΅ BioBLU 5p Π½Π° ΠΌΠ°ΡΡΠΈΡΠ΅ ΠΈΠ· Π΄ΠΈΡΠΊΠΎΠ² Fibra-Cel Ρ ΡΠ΅Π»ΡΡ ΠΏΡΠΎΠ΄ΡΠΊΡΠΈΠΈ rAAV.Β ΠΡΠ²ΠΎΠ΄Ρ: ΠΏΠΎΠΊΠ°Π·Π°Π½Π° Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ ΠΏΡΠΈΠΌΠ΅Π½Π΅Π½ΠΈΡ ΠΎΠΏΠΈΡΠ°Π½Π½ΡΡ
ΠΏΠΎΠ΄Ρ
ΠΎΠ΄ΠΎΠ² ΠΊ ΠΎΡΠΈΡΡΠΊΠ΅ ΠΏΠ»Π°Π·ΠΌΠΈΠ΄Π½ΠΎΠΉ ΠΠΠ, ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΈ ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΡΡΠ°Π½ΡΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ Π² ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΡΡΠ»ΠΎΠ²ΠΈΡΡ
Π΄Π»Ρ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠ° rAAV, ΡΡΠΏΡΠ΅ΡΡΠΈΡΡΡΡΠ΅Π³ΠΎ Π³Π΅Π½ Π°Π½ΡΠΈΡΠ΅Π»Π°. Π Π΅Π°ΠΊΡΠΎΡ BioBLU 5p Ρ Π΄ΠΈΡΠΊΠ°ΠΌΠΈ Fibra-Cel Π±ΡΠ» Π²ΠΏΠ΅ΡΠ²ΡΠ΅ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ Π΄Π»Ρ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΈΠ²Π½ΡΡ
ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ² rAAV Π² Π»Π°Π±ΠΎΡΠ°ΡΠΎΡΠ½ΠΎΠΌ ΠΌΠ°ΡΡΡΠ°Π±Π΅, ΡΡΠΎ ΠΏΠΎΠ·Π²ΠΎΠ»ΠΈΠ»ΠΎ ΡΠ²Π΅Π»ΠΈΡΠΈΡΡ ΠΏΠ»ΠΎΡΠ°Π΄Ρ ΠΏΠΎΠ²Π΅ΡΡ
Π½ΠΎΡΡΠΈ Π°Π΄Π³Π΅Π·ΠΈΠΈ ΠΏΡΠΈ ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ ΠΈ ΡΡΠ°Π½ΡΡΠ΅ΠΊΡΠΈΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ ΠΈ, ΠΊΠ°ΠΊ ΡΠ»Π΅Π΄ΡΡΠ²ΠΈΠ΅, ΡΠ²Π΅Π»ΠΈΡΠΈΡΡ Π²ΡΡ
ΠΎΠ΄ ΡΠ΅Π»Π΅Π²ΠΎΠ³ΠΎ ΠΏΡΠΎΠ΄ΡΠΊΡΠ°
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