Inventors
Tatyana Nikolaevna Vlasik, Armen Sergeevich Sadgyan, Igor Nikolaevich Rybalkin, and Alexander Yasenovich Shevelev
Assignees
- Tatyana Nikolaevna Vlasik
- Armen Sergeevich Sadgyan
- Igor Nikolaevich Rybalkin
- Alexander Yasenovich Shevelev
Abstract
The invention relates to the field of biotechnology, and specifically to methods and techniques for neutralizing the hepatitis C virus and specifically to antibodies against the hepatitis C virus, and can be used in medicine, the pharmaceutical industry and related areas of science and technology. Proposed is the use of fully human monoclonal antibodies – RYB1, RYB2, and RYB3 – and of a composition based thereon for the prevention and treatment of hepatitis C. Said antibodies are produced by cultivation using hybrid BIONA-RYB1, BIONA-RYB2 and BIONA-RYB3. The effectiveness of the antibodies is due to said antibodies binding epitopes, namely E1, E2 and E3 of an E2 protein of the hepatitis C viral envelope, respectively. The present invention has demonstrated a neutralizing activity of the antibodies in a model system of infection of human cells in a culture. It has been shown that use of the claimed group of inventions provides for more reliable antibody binding of the hepatitis C virus.
CovalX Technology Used
Outcomes
In order to obtain the nucleotide sequence that encodes for the E2 protein, the amino acid sequence of protein E2 NOSTA was reverse translated in silico. The E2 protein gene was created via chemical synthesis and then pET100/D-TOPO was used to produce plasmid rET100-E2. The regions that code Xpress-epitope and 6xHis-tag were removed. The rET100-E2 E. coli (BL21 (DE3)) cells were incubated at 37 °C in a shaker incubator. The expression of the recombinant product was induced by adding IPTG to 1 mM and then incubating the cells for an additional 150 minutes. The induced product was removed from the bacteria lysate using standard chromatographic procedures. To form antibody complexes, a mixture of 5 μL of E2 protein solution (4 μM) in PBS and 5 μL of solution of one antibody (RYB1, RYB2, or RYB3) (2 mM) was created. 9 μL of the mixture was removed and added to 1 μL of the CovalX K200 reagent before being incubated at room temperature for 3 hours. Following incubation, 1 μL of the crosslinked mixtures was sampled and added to 1 μL of matrix (recrystallized sinapic acid (10 mg/mL) in acetonitrile-water (1:1, v/v) with 0.1% TFA. 1 μL of this final mixture was plated on a MALDI target and allowed to crystallize at room temperature. The MALDI targets were analyzed using a mass spectrometer that had been modified with a CovalX HM3 high mass detection system. The use of the CovalX system gives the mass spectrometer the ability to detect molecular weights up to 2 MDa with increased sensitivity even into the nanomolar range. The data was analyzed using the CovalX Complex Tracker software.
In order to determine the epitopes of antibodies RYB1, RYB2, and RYB3, as well as antigen-antibody complexes, crosslinking and proteolysis were used. Crosslinking was performed by treating the samples with the CovalX K200 reagent to label them with deuterium. The crosslinked samples were then subjected to proteolysis. Using a combination of nano liquid chromatography and mass spectrometry, the epitopes were determined by analyzing the antigen spectrum proteolysis products for a change in labeled peptides.
To create the antibody-antigen complex, a ratio of 2:1 was used. 5 μL of recombinant protein solution E2 (4 M) was mixed with 5 μL of solution containing either RYB1, RYB2 or RYB3 antibodies (2 μM) and then incubated at 37 °C for 180 minutes. 10 μL of E2 protein solution (2 μM) was also incubated at 37 °C for 180 minutes. 1 μL of the complexes and pure antigen were individually mixed with 1 μL of premixed DSS-d0 and DSS-d12 (2 mg/mL) in dimethylformamide from the CovalX K200 stabilization kit. The samples were incubated at room temperature for 180 minutes to allow the crosslinking reaction to occur. Following incubation, 20 μL of 25 mM ammonium bicarbonate (pH 8.3) and 2.5 μL of 500 mM DTT were added to each sample and then the mixtures were incubated again at 55 °C for 60 minutes before adding 2.5 μL of 1 M iodoacetamide and incubating a third time at room temperature for 60 minutes. Using trypsin or α-chymotrypsin, the samples were proteolyzed.
- Trypsin
- Add 120 μL proteolysis buffer (100 mM Tris-HCl, pH 7.8, 10 mM SaS12)
- Add 2 μL trypsin solution (1 mg/ml)
- Incubate overnight at 37 °C
- α-chymotrypsin
- Add 120 μL proteolysis buffer (100 mM Tris-HCl, pH 7.8, 10 mM SaS12)
- Add 2 l of a solution of α-chymotrypsin (200 μM)
- Incubate overnight at 30 °C
The products of proteolysis were separated using a nano-liquid chromatograph with a pre-column and then analyzed using a mass spectrometer. From this analysis, the amino acid sequences of the peptides were determined using the CovalX database.
The spectra from the proteolyzed products was compared with the spectra from the pure E2 protein in order to determine the stoichiometry of the complexes. By determining the stoichiometry and taking into account the peptide proteolysis products that were lacking within the examined complexes, researchers were able to figure out which amino acids were in close proximity to one another and part of the peptide sequences of the epitope.