Anti-Ebola virus glycoprotein gp1 subunit monoclonal antibody 4f1 and its application

Abstract

The invention discloses an anti-Ebola virus glycoprotein GP1 subunit monoclonal antibody 4F1. The antibody has a unique CDR region and has specific binding activity with the EBOV GP1 subunit, and the binding is stable and is not subject to subunits. The influence of the low pH environment of endosomes provides the basis for its neutralization. Compared with the control antibody, 4F1 can effectively neutralize the EBOV pseudovirus in vitro. The neutralizing activity of 4F1 was enhanced with the increase of antibody concentration, and 100% protection against EBOV could be achieved at a concentration of 1 μg / mL. The monoclonal antibody 4F1 provided by the present invention and control antibodies such as MIL77‑3 all bind to the GP1 subunit and compete for binding, but 4F1 has neutralizing activity in vitro, while other antibodies do not have neutralizing activity, suggesting that 4F1 has the ability to bind to other Potential of a cocktail of neutralizing antibodies targeting GP2 subunits for combination therapy.

Description

technical field

[0001] The invention discloses an antibody and belongs to the technical field of polypeptides. Background technique

[0002] Ebola virus (EBOV) can cause acute severe hemorrhagic fever in humans and non-human primates. It is one of the viruses with the highest fatality rate found so far, with a fatality rate as high as 90%. It can be transmitted directly through contact , highly contagious and lethal. The glycoprotein (Glycoprotein, GP) spike on the surface of the Ebola virus envelope almost mediates all steps of the virus entering the cell, so Ebola GP is an important target of virus neutralizing antibodies. The Ebola virus GP gene is processed into two proteins, one is secreted non-structural GP (secreted glycoprotein, sGP); the other is structural GP. Ebola GP is first synthesized as a polypeptide, and then cleaved by Furin enzyme into GP1 (amino acids 1-501) and GP2 (amino acids 502-676) subunits. The two subunits are connected by disulfide bonds to for...

Examples

Embodiment 1

[0031] Example 1. Antibody Preparation

[0032] 1. Blood Sample Collection

[0033] After obtaining informed consent, 5 mL of blood samples were collected 28 days after the second immunization of the recombinant Ebola vaccine clinical trial subjects for subsequent experiments.

[0034] 2. FITC-labeled protein GPdM

[0035] Specific memory B cells need to be sorted by fluorescently labeled antigens. The method of FITC-labeled truncated antigen protein GPdM is as follows:

[0036] 1) Fluorescein Isothiocyanate_FITC (SIGMA, F4274) was dissolved in DMSO at a concentration of 20 mg / mL.

[0037] 2) Take 100 μL of GPdM (3.3 mg / mL), add buffer (pH9.6 carbonate buffer) to 400 μL.

[0038] 3) Add 8 μL FITC to the GPdM solution, and incubate at 4° C. in the dark for 3 hours.

[0039] 4) Use a 50kD centrifugal concentrator tube to replace the solution with PBS until the filtrate is transparent and colorless. Wrap the labeled protein in tinfoil and store at 4°C until use.

[0040] 3....

Embodiment 2

[0120] Example 2.ELISA detection of antibody binding activity

[0121] 1) The day before the experiment, 96-well enzyme-linked plates were coated with 1 μg / mL of EBOV GP, BDBV GP, SUDV GP and RESTV GP, 100 μL per well. Put the coated ELISA plate in a humid box overnight at 4°C.

[0122] 2) Wash 5 times with a plate washer on the day of the experiment.

[0123] 3) Add 100 μL of blocking solution to each well and let stand at room temperature for 1 hour.

[0124] 4) Wash the plates 5 times in a plate washer.

[0125] 5) Add 150 μL of 4F1 monoclonal antibody at a concentration of 10 μg / mL to the first well, and add 100 μL of diluent to the remaining wells. Aspirate 50 μL from the first well and add to the second well, and so on, dilute the solution in a 1:3 gradient to a final volume of 100 μL in each well. Let stand at room temperature for 1 hour.

[0126] 6) Wash the plates 5 times in a plate washer.

[0127] 7) Dilute the HPR-labeled goat anti-human IgG secondary antibod...

Embodiment 3

[0132] Embodiment 3. Pseudovirus neutralization experiment evaluates 4F1 neutralization activity

[0133] The EBOV pseudovirus packaged with the HIV backbone was used to evaluate the neutralizing activity of 4F1 in vitro, as follows:

[0134] 1) Dilute the 4F1 monoclonal antibody with DMEM medium, add 75 μL of antibody diluent with a concentration of 100 μg / mL to the first well of a 96-well cell culture plate, and add 50 μL of DMEM medium to the remaining wells.

[0135] 2) Pipette 25 μL of liquid from the first well into the second well, mix well, and so on, dilute at a ratio of 1:3, and the final volume of each well is 50 μL.

[0136] 3) Dilute the pseudovirus 1:5 with DMEM medium (control the fluorescein reading in the control well between 20,000 and 100,000), add to each antibody well, 50 μL per well. Mix well and incubate at 37°C for 1h.

[0137] 4) 293T cell count, 2×10 5 cells / mL, add 100 μL to each well.

[0138] 5) Put the 96-well cell culture plate into a 37°C th...

technical field

[0001] The invention discloses an antibody and belongs to the technical field of polypeptides. Background technique

[0002] Ebola virus (EBOV) can cause acute severe hemorrhagic fever in humans and non-human primates. It is one of the viruses with the highest fatality rate found so far, with a fatality rate as high as 90%. It can be transmitted directly through contact , highly contagious and lethal. The glycoprotein (Glycoprotein, GP) spike on the surface of the Ebola virus envelope almost mediates all steps of the virus entering the cell, so Ebola GP is an important target of virus neutralizing antibodies. The Ebola virus GP gene is processed into two proteins, one is secreted non-structural GP (secreted glycoprotein, sGP); the other is structural GP. Ebola GP is first synthesized as a polypeptide, and then cleaved by Furin enzyme into GP1 (amino acids 1-501) and GP2 (amino acids 502-676) subunits. The two subunits are connected by disulfide bonds to for...

Examples

Embodiment 1

[0031] Example 1. Antibody Preparation

[0032] 1. Blood Sample Collection

[0033] After obtaining informed consent, 5 mL of blood samples were collected 28 days after the second immunization of the recombinant Ebola vaccine clinical trial subjects for subsequent experiments.

[0034] 2. FITC-labeled protein GPdM

[0035] Specific memory B cells need to be sorted by fluorescently labeled antigens. The method of FITC-labeled truncated antigen protein GPdM is as follows:

[0036] 1) Fluorescein Isothiocyanate_FITC (SIGMA, F4274) was dissolved in DMSO at a concentration of 20 mg / mL.

[0037] 2) Take 100 μL of GPdM (3.3 mg / mL), add buffer (pH9.6 carbonate buffer) to 400 μL.

[0038] 3) Add 8 μL FITC to the GPdM solution, and incubate at 4° C. in the dark for 3 hours.

[0039] 4) Use a 50kD centrifugal concentrator tube to replace the solution with PBS until the filtrate is transparent and colorless. Wrap the labeled protein in tinfoil and store at 4°C until use.

[0040] 3....

Embodiment 2

[0120] Example 2.ELISA detection of antibody binding activity

[0121] 1) The day before the experiment, 96-well enzyme-linked plates were coated with 1 μg / mL of EBOV GP, BDBV GP, SUDV GP and RESTV GP, 100 μL per well. Put the coated ELISA plate in a humid box overnight at 4°C.

[0122] 2) Wash 5 times with a plate washer on the day of the experiment.

[0123] 3) Add 100 μL of blocking solution to each well and let stand at room temperature for 1 hour.

[0124] 4) Wash the plates 5 times in a plate washer.

[0125] 5) Add 150 μL of 4F1 monoclonal antibody at a concentration of 10 μg / mL to the first well, and add 100 μL of diluent to the remaining wells. Aspirate 50 μL from the first well and add to the second well, and so on, dilute the solution in a 1:3 gradient to a final volume of 100 μL in each well. Let stand at room temperature for 1 hour.

[0126] 6) Wash the plates 5 times in a plate washer.

[0127] 7) Dilute the HPR-labeled goat anti-human IgG secondary antibod...

Embodiment 3

[0132] Embodiment 3. Pseudovirus neutralization experiment evaluates 4F1 neutralization activity

[0133] The EBOV pseudovirus packaged with the HIV backbone was used to evaluate the neutralizing activity of 4F1 in vitro, as follows:

[0134] 1) Dilute the 4F1 monoclonal antibody with DMEM medium, add 75 μL of antibody diluent with a concentration of 100 μg / mL to the first well of a 96-well cell culture plate, and add 50 μL of DMEM medium to the remaining wells.

[0135] 2) Pipette 25 μL of liquid from the first well into the second well, mix well, and so on, dilute at a ratio of 1:3, and the final volume of each well is 50 μL.

[0136] 3) Dilute the pseudovirus 1:5 with DMEM medium (control the fluorescein reading in the control well between 20,000 and 100,000), add to each antibody well, 50 μL per well. Mix well and incubate at 37°C for 1h.

[0137] 4) 293T cell count, 2×10 5 cells / mL, add 100 μL to each well.

[0138] 5) Put the 96-well cell culture plate into a 37°C th...

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