轻链单个氨基酸改变可增强新冠病毒嵌合抗体的中和能力

doi:10.3969/j.issn.1674-8115.2023.06.008

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (6): 718-727.doi: 10.3969/j.issn.1674-8115.2023.06.008

• 论著 · 基础研究 • 上一篇

轻链单个氨基酸改变可增强新冠病毒嵌合抗体的中和能力

殷姿(), 廉朝阳, 高波, 田莹, 郝茜(), 叶菱秀()

上海交通大学基础医学院免疫学与微生物学系，上海市免疫学研究所，上海 200025

收稿日期:2023-01-13 接受日期:2023-06-05 出版日期:2023-06-28 发布日期:2023-06-28
通讯作者: 郝茜,叶菱秀 E-mail:yinzi981030@sjtu.edu.cn;haoqian_2019@shsmu.edu.cn;yeaplengsiew@shsmu.edu.cn
作者简介:殷姿（1998—），女，硕士生；电子信箱：yinzi981030@sjtu.edu.cn。
基金资助:
国家自然科学基金(32000656);博士后创新人才支持计划(2020T130078ZX)

Enhancement of the neutralization ability resulting from a single amino acid change in the light chain of a chimeric antibody against SARS-CoV-2

YIN Zi(), LIAN Chaoyang, GAO Bo, TIAN Ying, HAO Qian(), YEAP Lengsiew()

Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China

Received:2023-01-13 Accepted:2023-06-05 Online:2023-06-28 Published:2023-06-28
Contact: HAO Qian,YEAP Lengsiew E-mail:yinzi981030@sjtu.edu.cn;haoqian_2019@shsmu.edu.cn;yeaplengsiew@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(32000656);China Postdoctoral Innovative Talent Support Program(2020T130078ZX)

摘要/Abstract

摘要：

目的·筛选对新冠病毒（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）具有中和能力的人鼠嵌合抗体，并分析其轻链上的关键氨基酸位点变化。方法·以SARS-CoV-2刺突蛋白作为免疫原，免疫抗体重链基因人源化的小鼠，通过酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）和假病毒中和实验检测小鼠血清抗体水平，应用流式细胞术分选小鼠脾脏和淋巴结中的浆细胞和生发中心（germinal center，GC）B细胞并分析各类B细胞比例。提取细胞的RNA构建鼠源抗体轻链基因文库，与固定的人源抗体重链基因配对表达抗体，并采用ELISA筛选高亲和力嵌合抗体。与鼠源胚系抗体轻链基因比对，分析高亲和力嵌合抗体轻链的氨基酸变化位点。分别构建并表达轻链单个氨基酸位点改变的嵌合抗体，通过ELISA和假病毒中和实验检测其亲和力及中和能力；比较上述位点改变前后嵌合抗体的亲和力及中和能力，并分析具有关键作用的氨基酸位点变化。结果·与未免疫小鼠相比，免疫后小鼠血清中针对SARS-CoV-2的特异性抗体的滴度增加，脾脏和淋巴结中的浆细胞和GC B细胞的比例亦有增加。ELISA的结果显示，从小鼠脾脏的GC B细胞中筛选到具有高亲和力的嵌合抗体。该抗体轻链的第76位苏氨酸替换成了异亮氨酸，且第98位苯丙氨酸发生缺失。随后，ELISA结果显示，该嵌合抗体具有较强的亲和力，轻链氨基酸没有变化的抗体和轻链仅携带第98位氨基酸缺失的抗体的亲和力较弱，而轻链仅携带第76位氨基酸替换的抗体的亲和力介于嵌合抗体和轻链氨基酸没有变化的抗体之间。假病毒中和实验的结果显示，该嵌合抗体的半数抑制浓度（half maximal inhibitory concentration，IC₅₀）为0.995 ng/μL；轻链仅携带第76位氨基酸替换的抗体的IC₅₀为1.724 ng/μL，中和能力有所减弱；轻链仅携带第98位氨基酸缺失的抗体的IC₅₀为71.05 ng/μL，轻链氨基酸没有变化的抗体的IC₅₀为42.06 ng/μL，中和能力均较弱。结论·成功筛选得到对SARS-CoV-2具有中和能力的人鼠嵌合抗体，且该抗体轻链上第76位苏氨酸替换为异亮氨酸是决定其中和能力的关键。

关键词: 嵌合抗体, 中和抗体, 新冠病毒, 轻链, 体细胞高频突变

Abstract:

Objective ·To screen the human-mouse chimeric antibodies that neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and analyse the key amino acid site changes in their light chain. Methods ·Antibody heavy chain gene-humanized mice were immunized with the spike protein of SARS-CoV-2. Enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assay were performed to analyse the antibody titer in serum level. Flow cytometry was used to sort plasma cells and germinal center (GC) B cells from the mouse spleen and lymph nodes, and the proportions of different B cells were analysed. RNA was extracted to construct a mouse-origin antibody light chain gene library, which was paired with the knocked-in human-origin antibody heavy chain gene for antibody expression. ELISA was employed to screen for high-affinity chimeric antibodies. Amino acid changes in the light chain of high-affinity chimeric antibodies were analysed by comparing them with mouse-origin germline antibody light chain genes. Chimeric antibodies with single amino acid changes in the light chain were constructed and expressed, and their affinity and neutralization abilities were tested through ELISA and pseudovirus neutralization experiments. By comparing the affinity and neutralization abilities of the chimeric antibodies with or without single amino acid changes in the light chain, the key single amino acid change was analysed. Results ·Compared to unimmunized mice, the immunized mice showed an increased titer of specific antibodies against SARS-CoV-2 on the serum level, along with an elevated proportion of plasma cells and GC B cells in the spleen and lymph nodes. ELISA showed that a high-affinity chimeric antibody was screened from GC B cells in the mouse spleen. The light chain of the antibody had a 76^th serine-to-isoleucine substitution and a 98^th phenylalanine deletion. Another ELISA showed that the chimeric antibody exhibited high affinity, the antibody without amino acid change on the light chain and the antibody with only the 98^th phenylalanine deletion showed low affinity, and the antibody with only the 76^th serine-to-isoleucine substitution demonstrated intermediate affinity between the chimeric antibody and the antibody without amino acid change on the light chain. Pseudovirus neutralization experiments revealed that the chimeric antibody had a half-maximal inhibitory concentration (IC₅₀) of 0.995 ng/μL; the antibody with only the 76^th serine-to-isoleucine substitution had an IC₅₀ of 1.724 ng/μL, indicating a slight decrease in neutralization ability; the antibody with only the 98^th phenylalanine deletion had an IC₅₀ of 71.05 ng/μL; the antibody without amino acid change on the light chain had an IC₅₀ of 42.06 ng/μL, suggesting weak neutralization ability. Conclusion ·For the screened human-mouse chimeric antibodies that neutralize SARS-CoV-2 in this study, the key amino acid change determining the neutralization ability of the chimeric antibody is the 76^th serine-to-isoleucine substitution.

Key words: chimeric antibody, neutralizing antibody, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), light chain, somatic hypermutation

中图分类号:

R392.11

殷姿, 廉朝阳, 高波, 田莹, 郝茜, 叶菱秀. 轻链单个氨基酸改变可增强新冠病毒嵌合抗体的中和能力[J]. 上海交通大学学报（医学版）, 2023, 43(6): 718-727.

YIN Zi, LIAN Chaoyang, GAO Bo, TIAN Ying, HAO Qian, YEAP Lengsiew. Enhancement of the neutralization ability resulting from a single amino acid change in the light chain of a chimeric antibody against SARS-CoV-2[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(6): 718-727.

图/表 10

图 1 小鼠免疫及血清抗体水平分析

Fig 1 Mice immunization and analysis of serum antibody levels

表 1 鼠源抗体轻链基因文库构建的第一轮巢式PCR的引物序列

Tab 1 Primer sequences of the first-round nested PCR for constructing mouse-origin antibody light chain gene library

Primer	Sequence (5'→3')
1mFK_Ⅰ	RGTGCAGATTTTCAGCTTCCTGCT
1mFK_Ⅱ	TGGACATGAGGGCYCCTGCTCAGT
1mFK_Ⅲ	CTSTGGTTGTCTGGTGTTGAYGGA
1mFK_Ⅳ	GTTGCTGCTGCTGTGGCTTACA
1mFK_Ⅴ	GTATCTGGTACCTGTGG
1mFK_Ⅵ	TGCCTGTTAGGCTGTTGGTGCT
1mFK_Ⅶ	GCTCAGTTCCTTGGTCTCCTGTTGC
1mFK_Ⅷ	TGGGTGCTGCTGCTCTGGGT
1mFK_Ⅸ	CAGTTCCTGTTTCTGTTARTGCTCTGG
1mFK_Ⅹ	TGCTCTGGTTATATGGTGCTGATGGG
1mFK_Ⅺ	GCTGTTTTGTATACCTGGG
1mFK_Ⅻ	CTCAGCTCCTGTTGCTGTGGC
1mFK_􀃼	CCTCATATTTTTGCTGCTATGGG
1mFK_ⅩⅣ	TGCTTTTCTGGATTTCAGCCTCCAG
1mFK_ⅩⅤ	TCAACTTCTGCTCTTCCTGCTGTTC
1mFK_ⅩⅥ	CTAGCTCYTCTCCTCAGYCTTCTTCTCCTC
1mFK_ⅩⅦ	GTGTMTGGTGCTBRTGGG
1mFK_ⅩⅧ	STGYTGHTGYTCTGG
1mFK_ⅩⅨ	GGGTATCTGGTACCTGTGG
1mRK	ACTGAGGCACCTCCAGATGTT
1mFL_Ⅰ	ACTTATACTCTCTCTCCTGGCTCTC
1mFL_Ⅱ	CTCTTCTTCTTCTTTGTTCTTCATTGCT
1mRL	GTACCATYTGCCTTCCAGKCCACT

表 2 鼠源抗体轻链基因文库构建的第二轮巢式PCR的引物序列

Tab 2 Primer sequences of the second-round nested PCR for constructing mouse-origin antibody light chain gene library

Primer	Sequence (5'→3')
2mFK_Ⅰ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGAAAWTGTGCTCACCCAGTC
2mFK_Ⅱ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCCAAATTGTTCTCACCCAGTC
2mFK_Ⅲ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCRACATTGTGCTGACCCAATC
2mFK_Ⅳ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGAAACAACTGTGACCCAGTC
2mFK_Ⅴ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGATATTGTGATGACSCAGGC
2mFK_Ⅵ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCRRTRTTGTGATGACCCARAC
2mFK_Ⅶ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGATATCCAGATGACACAGAC
2mFK_Ⅷ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGACATTGTGATGACMCAGTC
2mFK_Ⅸ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGACATCCAGATGACHCAGTC
2mFK_Ⅹ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGACATCTTGCTGACTCAGTC
2mFK_Ⅺ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCACACAGGCTGCACTCTCCAA
2mFK_Ⅻ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCACTGGAGAAACAACACAGGC
2mFK_􀃼	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGATGTCCAGATAACCCAG
2mFK_ⅩⅣ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGACATCCTGATGACCCAATC
2mFK_ⅩⅤ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCGATGTTGTGGTGACTCAAAC
2mFK_ⅩⅥ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCAATATCCAGGTGATCCAG
2mRK_Ⅰ	GATGAAGACAGATGGCGCCGCCACAGTTCGTTTCAGCTCCAGCTTGGTCCC
2mRK_Ⅱ	GATGAAGACAGATGGCGCCGCCACAGTTCGTTTTATTTCCAGTCTGGTCCC
2mRK_Ⅲ	GATGAAGACAGATGGCGCCGCCACAGTTCGTTTKATTTCCARCTTKGTSCC
2mFL_Ⅰ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCTTACTCAGCCAAGCTCT
2mFL_Ⅱ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCCAGGCTGTTGTGACTCAG
2mFL_Ⅲ	CTGCTGCTCTGGTTCCCAGGTTCCAGATGCCAACYTGTGCTCACTCAG
2mRL_Ⅰ	AGTGACCGTGGGGTTGGCCTTGGGCTGACCTAGGACAGTCAGTTTGGTTCC
2mRL_Ⅱ	AGTGACCGTGGGGTTGGCCTTGGGCTGACCTAGGACAGTGACCTTGGTTCC

图 2 鼠源抗体轻链基因文库构建及高亲和力人鼠嵌合抗体筛选流程

Fig 2 Construction of mouse-origin antibody light chain gene library and screening workflow of high-affinity human-mouse chimeric antibody

表 3 鼠源抗体轻链单个氨基酸改变的质粒构建的引物序列

Tab 3 Primer sequences for plasmid construction with single amino acid mutation in mouse-origin antibody light chain

Primer	Sequence (5 ́→3 ́)
Adapter forward primer	TTCCCAGGTTCCAGATGCCAGGCTGTTGTGACTCAG
F98-deletion reverse primer	TGACCTTGGTTCCTCCACCGAACACCCAATGGTTGCTGTA
T76-mutation reverse primer	CCTTGGTTCCTCCACCGAACACCCAGAAATGGTTGCTGTACCATAG
Adapter reverse primer	GGTTGGCCTTGGGCTGACCTAGGACAGTGACCTTGG

图 3 鼠源抗体轻链单个氨基酸改变的质粒的构建

Fig 3 Construction of plasmids for mouse-origin antibody light chain with single amino acid change

图 4 2组小鼠的血清抗体水平及各类B细胞水平分析Note：A. Detection of serum antibody titer using ELISA with SARS-CoV-2 RBD antigen. B. Detection of serum antibody titer using ELISA with SARS-CoV-2 S-trimer antigen. C. Detection of the proportions of different types of B cells in the spleen and lymph nodes of the two groups by flow cytometry.

Fig 4 Analysis of serum antibody levels and different types of B cell levels in the two groups of mice

图 5 ELISA筛选人鼠嵌合抗体的亲和力Note：A. First-round screening of human-mouse chimeric antibodies. B. Second-round screening of human-mouse chimeric antibodies.

Fig 5 Screening for affinity of human-mouse chimeric antibodies by ELISA

图 6 高亲和力人鼠嵌合轻链上单个氨基酸位点的改变分析以及单个氨基酸改变的抗体的纯化Note：A. Display of amino acid sequences of the screened chimeric antibodies and antibodies with single amino acid changes in light chains. B. SDS-PAGE results for chimeric antibodies purification.

Fig 6 Analysis of single amino acid site changes in the light chain of high-affinity human-mouse chimeric antibody and purification of antibodies with single amino acid changes

图 7 单个氨基酸改变对高亲和力的人鼠嵌合抗体的亲和力及中和能力的影响Note： A. Analysis of antibody binding affinity by ELISA. B. Analysis of antibody neutralizing ability by pseudovirus neutralization assay.

Fig 7 Effect of single amino acid changes on the affinity and neutralization ability of high-affinity human-mouse chimeric antibodies

参考文献 25

1	OOSTINDIE S C, LAZAR G A, SCHUURMAN J, et al. Avidity in antibody effector functions and biotherapeutic drug design[J]. Nat Rev Drug Discov, 2022, 21(10): 715-735.
2	ROBINSON W H. Sequencing the functional antibody repertoire: diagnostic and therapeutic discovery[J]. Nat Rev Rheumatol, 2015, 11(3): 171-182.
3	YU X C, TSIBANE T, MCGRAW P A, et al. Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors[J]. Nature, 2008, 455(7212): 532-536.
4	LI T T, CHEN J Y, ZHENG Q B, et al. Identification of a cross-neutralizing antibody that targets the receptor binding site of H1N1 and H5N1 influenza viruses[J]. Nat Commun, 2022, 13(1): 5182.
5	DUFLOO J, PLANCHAIS C, FRÉMONT S, et al. Broadly neutralizing anti-HIV-1 antibodies tether viral particles at the surface of infected cells[J]. Nat Commun, 2022, 13(1): 630.
6	WALKER L M, HUBER M, DOORES K J, et al. Broad neutralization coverage of HIV by multiple highly potent antibodies[J]. Nature, 2011, 477(7365): 466-470.
7	WU Y C, KIPLING D, LEONG H S, et al. High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations[J]. Blood, 2010, 116(7): 1070-1078.
8	ROGERS T F, ZHAO F Z, HUANG D L, et al. Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model[J]. Science, 2020, 369(6506): 956-963.
9	LIANG W C, YIN J P, LUPARDUS P, et al. Dramatic activation of an antibody by a single amino acid change in framework[J]. Sci Rep, 2021, 11(1): 22365.
10	RAPP M, GUO Y C, REDDEM E R, et al. Modular basis for potent SARS-CoV-2 neutralization by a prevalent VH1-2-derived antibody class[J]. Cell Rep, 2021, 35(1): 108950.
11	TORTORICI M A, BELTRAMELLO M, LEMPP F A, et al. Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms[J]. Science, 2020, 370(6519): 950-957.
12	LIU L H, WANG P F, NAIR M S, et al. Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike[J]. Nature, 2020, 584(7821): 450-456.
13	VON BOEHMER L, LIU C, ACKERMAN S, et al. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells[J]. Nat Protoc, 2016, 11(10): 1908-1923.
14	MASCOLA J R, HAYNES B F. HIV-1 neutralizing antibodies: understanding nature's pathways[J]. Immunol Rev, 2013, 254(1): 225-244.
15	CORTI D, LANZAVECCHIA A. Broadly neutralizing antiviral antibodies[J]. Annu Rev Immunol, 2013, 31: 705-742.
16	MUECKSCH F, WEISBLUM Y, BARNES C O, et al. Affinity maturation of SARS-CoV-2 neutralizing antibodies confers potency, breadth, and resilience to viral escape mutations[J]. Immunity, 2021, 54(8): 1853-1868.e7.
17	KREER C, ZEHNER M, WEBER T, et al. Longitudinal isolation of potent near-germline SARS-CoV-2-neutralizing antibodies from COVID-19 patients[J]. Cell, 2020, 182(4): 843-854.e12.
18	CHEN X J, ZHOU T Q, SCHMIDT S D, et al. Vaccination induces maturation in a mouse model of diverse unmutated VRC01-class precursors to HIV-neutralizing antibodies with >50% breadth[J]. Immunity, 2021, 54(2): 324-339.e8.
19	TIAN M, MCGOVERN K, CHENG H L, et al. Conditional antibody expression to avoid central B cell deletion in humanized HIV-1 vaccine mouse models[J]. Proc Natl Acad Sci U S A, 2020, 117(14): 7929-7940.
20	ZHOU J Q, KLEINSTEIN S H. Cutting edge: Ig H chains are sufficient to determine most B cell clonal relationships[J]. J Immunol, 2019, 203(7): 1687-1692.
21	JAFFE D B, SHAHI P, ADAMS B A, et al. Functional antibodies exhibit light chain coherence[J]. Nature, 2022, 611(7935): 352-357.
22	WANG L T, HURLBURT N K, SCHÖN A, et al. The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria[J]. Cell Rep, 2022, 38(7): 110367.
23	BARNES C O, WEST A P Jr, HUEY-TUBMAN K E, et al. Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies[J]. Cell, 2020, 182(4): 828-842.e16.
24	LOMBANA T N, DILLON M, BEVERS J 3rd, et al. Optimizing antibody expression by using the naturally occurring framework diversity in a live bacterial antibody display system[J]. Sci Rep, 2015, 5: 17488.
25	KLEIN F, DISKIN R, SCHEID J F, et al. Somatic mutations of the immunoglobulin framework are generally required for broad and potent HIV-1 neutralization[J]. Cell, 2013, 153(1): 126-138.

[1]	朱啸巍, 钟平, 曹立, 栾兴华. 腓骨肌萎缩症合并小脑性共济失调的临床及遗传学特点[J]. 上海交通大学学报（医学版）, 2023, 43(3): 350-357.
[2]	张剑, 宋菲, 王西樵. 成纤维细胞的自噬性溶解死亡在增生性瘢痕消退过程中的作用[J]. 上海交通大学学报(医学版), 2022, 42(1): 44-50.
[3]	黄聪华，高婷，陆晓晔，朱长清. κ轻链型多发性骨髓瘤合并可逆性后部脑病综合征 1例[J]. 上海交通大学学报（医学版）, 2019, 39(3): 336-.
[4]	胡珊，吴钢 . 肌球蛋白调节性轻链磷酸化在慢性心力衰竭发生机制中的作用[J]. 上海交通大学学报（医学版）, 2017, 37(8): 1165-.
[5]	张珍珍, 尹延青, 周嘉伟, 等. GST-p43/AIMP1融合蛋白表达和纯化以及与NF-L的相互作用[J]. , 2012, 32(5): 580-.

轻链单个氨基酸改变可增强新冠病毒嵌合抗体的中和能力

Enhancement of the neutralization ability resulting from a single amino acid change in the light chain of a chimeric antibody against SARS-CoV-2

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 25

相关文章 5

编辑推荐

Metrics