Rift valley fever virus humanized monoclonal antibodies and application thereof

A technology of antibodies and antigens, applied in the field of medicine, can solve the problems of insufficient clinical data, cost problems, and the impossibility of large-scale vaccination, and achieve high application value and the effect of preventing infection

Active Publication Date: 2019-05-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, high-risk groups in Africa can be vaccinated with inactivated formalin vaccine, but due to cost issues, it is impossible to

Method used

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  • Rift valley fever virus humanized monoclonal antibodies and application thereof
  • Rift valley fever virus humanized monoclonal antibodies and application thereof
  • Rift valley fever virus humanized monoclonal antibodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Expression and purification of RVFV Gn and Gc proteins

[0070] The genes encoding the RVFV Gn (amino acid sequence shown in SEQ ID NO:23) and Gc (amino acid sequence shown in SEQ ID NO:24) proteins were respectively connected to the pFastBac1 vector, wherein Gn was connected to the N-terminal of the Gc protein gene There is an insect cell membrane protein Gp67 signal peptide sequence for antibody secretion; a His tag is attached to the C-terminus, which is convenient for purification and staining of B cells; all coding genes have been optimized for insect cell codons.

[0071] Transform Escherichia coli DH10Bac competent cells with the pFastBac1 vector containing the target gene, and perform blue-white screening of recombinant baculoviruses on a plate containing ampicillin, kanamycin, tetracycline and Blue-gal. After the white plaques were identified by PCR with the upstream and downstream primers of M13, the positive clones were shaken, and the recombinant ...

Embodiment 2

[0075] Example 2: Isolation of RVFV Gn and Gc protein-specific memory B cells

[0076] With the patient's informed consent, 30 mL of blood was collected and PBMCs were isolated.

[0077] Separated PBMCs in 10 7 Individual / mL density and RVFV Gn and RVFV Gc proteins with a final concentration of 100nM were incubated on ice for half an hour, then washed twice with PBS, and then incubated with the following antibodies on ice for half an hour: anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti-human IgG / FITC and anti -His / PE, then washed 2 times with PBS.

[0078] The PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+ cells were collected by FACSAria III sorting, and directly collected into a 96-well plate, 1 cell / well.

Embodiment 3

[0079] Example 3: Single B cell PCR and sequence analysis

[0080] The cells obtained in Example 2 were reverse-transcribed by Superscript III reverse transcriptase (Invitrogen), and reacted at 55° C. for 60 min.

[0081] Using this reverse transcription product as a template, PCR was performed with HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa). The reaction conditions were as follows: 95°C, 5min; 95°C, 30s, 55°C (heavy chain / κ chain) / 50°C (λ chain), 30s, 72°C, 90s, 35 cycles, 72°C, 7min.

[0082] Use this as a template for another round of PCR (PCRb), the conditions are as follows: 95°C, 5min; 95°C, 30s, 58°C (heavy chain) / 60°C (κ chain) / 64°C (λ chain), 30s, 72 ℃, 90s, 35 cycles, 72℃, 7min.

[0083] 1.2% agarose gel electrophoresis to separate the PCR products. The bands with a size of 400-500 bp were recovered and sent to the sequencing company for sequencing. The sequencing results were analyzed with IMGT online software.

[00...

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Abstract

The invention discloses rift valley fever virus humanized monoclonal antibodies and application thereof and belongs to the technical field of medicine. According to the antibodies, RVFV glycoproteinsGn and Gc expressed by baculovirus are adopted as antigens, memory B cells which can be combined with the RVFV glycoproteins Gn and Gc are screened from PBMCs of a patient suffering from rift valley fever, and through experiments such as single B cell sequencing, in-vitro neutralization and in-vivo protection, eight Gn humanized monoclonal antibodies capable of efficiently neutralizing RVFV infection and one Gc humanized monoclonal antibody are identified. The humanized monoclonal antibodies have very high in-vitro neutralization activity (IC50 can be as low as 1.93 +/- 0.6 pM and reaches a pMlevel), can effectively treat mice infected with RVFV, prevents the mice from being infected with RVFV, and has an extremely high application value on clinical treatment and prevention of the infection of RVFV.

Description

technical field [0001] The invention relates to a Rift Valley fever virus human monoclonal antibody and application thereof, belonging to the technical field of medicine. Background technique [0002] Rift Valley fever virus (RVFV) is one of the arboviruses that seriously threaten human and animal health. It can cause the zoonotic Rift Valley fever (Rift Valley fever, RVF), causing livestock death and abortion. Its surface is covered with envelopes and glycoprotein protrusions, and the viral genome (S, M, and L) is composed of 3 segments of negative-strand RNA. Among them, the M segment encodes surface glycoproteins Gn and Gc, and encodes non-structural proteins at the 5' end of Gn Protein (NSm), and Gn and Gc are the main envelope proteins of the virus, which are the key proteins for virus invasion and membrane fusion; its spread is very wide, and sheep, goats, cattle, buffaloes, camels and other livestock can be infected with the virus ; Its lethality rate is very high, ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCC07K16/10A61P31/14C07K2317/56C07K2317/52A61K2039/505C07K2317/24C07K2317/92C07K2317/76Y02A50/30
Inventor 严景华王奇慧马桐杨化冰高福马素芳
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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