Generation of a cost-effective cell line for support of high-throughput isolation of primary human B cells and monoclonal neutralizing antibodies

AbstractA small number of HIV-1 infected individuals develop broadly neutralizing-antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1 - 3 years after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be editedin vitrousing CRISPR/Cas9 to express mature bNAbs from the endogenousIghlocus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization.One-sentence summaryB cells edited by CRISPR/Cas9 to produce antibodies participate in humoral immune reactions and secrete neutralizing serum titers of anti-HIV bNAbs.

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Anti-idiotypic antibodies elicit anti-HIV-1–specific B cell responses

Journal of Experimental Medicine ◽

10.1084/jem.20190446 ◽

2019 ◽

Vol 216 (10) ◽

pp. 2316-2330 ◽

Cited By ~ 5

Author(s):

Pia Dosenovic ◽

Anna-Klara Pettersson ◽

Abigail Wall ◽

Eddy S. Thientosapol ◽

Junli Feng ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Neutralizing Antibodies ◽

Target Cells ◽

Human B Cells ◽

Cd4 Binding Site ◽

Idiotypic Antibody ◽

Anti Hiv ◽

Hiv 1

Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5–amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)–specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1.

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Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells

Cell ◽

10.1016/j.cell.2020.05.025 ◽

2020 ◽

Vol 182 (1) ◽

pp. 73-84.e16 ◽

Cited By ~ 253

Author(s):

Yunlong Cao ◽

Bin Su ◽

Xianghua Guo ◽

Wenjie Sun ◽

Yongqiang Deng ◽

...

Keyword(s):

B Cells ◽

Single Cell ◽

High Throughput ◽

Neutralizing Antibodies ◽

Single Cell Sequencing

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Cultivation and Immortalization of Human B-Cells Producing a Human Monoclonal IgM Antibody Binding to MDA-LDL: Further Evidence for Formation of Atherogenic MDA-LDL Adducts in HumansIn Vivo

Oxidative Medicine and Cellular Longevity ◽

10.1155/2017/6047142 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Franz Tatzber ◽

Edith Pursch ◽

Ulrike Resch ◽

Roswitha Pfragner ◽

Sandra Holasek ◽

...

Keyword(s):

B Cells ◽

Cell Line ◽

Oxidized Ldl ◽

Malonic Dialdehyde ◽

Density Lipoprotein ◽

Ldl Oxidation ◽

Human Donor ◽

Human B Cells

Oxidatively modified low-density lipoprotein (oLDL) is firmly believed to play an important role in the initiation and development of atherosclerosis, and malonic dialdehyde (MDA) is one of the major lipid peroxidation breakdown products involved in this process. In recent decades, antibodies against MDA-LDL have been detected in human and animal sera. In our study, human B-cells from the peripheral blood of a healthy female donor were fused with the SP2/0 mouse myeloma cell line. Antibody-producing hybridomas were detected by MDA-LDL-IgG/IgM enzyme-linked immunosorbent assays (ELISA) and Cu++-oxidized LDL IgG/IgM (oLAb) ELISA. Cells with supernatants emitting positive signals for antibodies were then cloned and after sufficient multiplication frozen and stored under liquid nitrogen. Due to the loss of antibody-producing ability, we established an MDA-LDL-IgM-producing cell line by recloning. This allowed isolation and immortalization of several human B-cells. The human donor had not been immunized with MDA-modified proteins, thus obviously producing MDA-LDL antibodiesin vivo. Furthermore, using these antibodies forin vitroexperiments, we were able to demonstrate that MDA epitopes are among the epitopes generated during Cu++-LDL oxidation as well. Finally, these antibodies compete in ELISA and cell culture experiments with MDA as a challenging toxin or ligand.

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