Discovery and characterization of SARS-CoV-2 reactive and neutralizing antibodies from humanized CAMouseHG mice through rapid hybridoma screening and high-throughput single-cell V(D)J sequencing

Abstract

The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective detection tools and therapeutic measures. During SARS-CoV-2 infection, a large number of SARS-CoV-2 reactive and neutralizing antibodies will be produced. Most SARS-CoV-2 reactive and neutralizing fully human antibodies are isolated from human and frequently encoded by convergent heavy-chain variable genes. However, SARS-CoV-2 viruses can mutate rapidly during replication and the resistant variants of neutralizing antibodies easily survive and evade the immune response, especially in the face of such focused antibody responses in humans. Therefore, additional tools are needed to develop different kinds of fully human antibodies to compensate for current deficiency. In this study, we utilized antibody humanized CAMouseHG mice to develop a rapid antibody discovery method and examine the antibody repertoire of SARS-CoV-2 RBD-reactive hybridoma cells derived from CAMouseHG mice by using high-throughput single-cell V(D)J sequencing analysis. CAMouseHG mice were immunized by 28-day rapid immunization method. After electrofusion and semi-solid medium screening on day 12 post-electrofusion, 171 hybridoma clones were generated based on the results of SARS-CoV-2 RBD binding activity assay. A rather obvious preferential usage of IGHV6-1 family was found in these hybridoma clones derived from CAMouseHG mice, which was significantly different from the antibodies found in patients with COVID-19. After further virus neutralization screening and antibody competition assays, we generated a noncompeting two-antibody cocktail, which showed a potent prophylactic protective efficacy against SARS-CoV-2 in cynomolgus macaques. These results indicate that humanized CAMouseHG mice not only provide a valuable platform to obtain fully human reactive and neutralizing antibodies but also have a different antibody repertoire from humans. Thus, humanized CAMouseHG mice can be used as a good complementary tool in discovery of fully human therapeutic and diagnostic antibodies.

A neutralizing bispecific single-chain antibody against SARS-CoV-2 Omicron variant produced based on CR3022

“…In brief, Vero cells were grown in 96-well plates 1 day prior to infection. Indicated concentrations of antibodies or mock-treated with Dulbecco’s Modified Eagle Medium (Corning Incorporated, NY, NY, USA) supplemented with 10% FBS and penicillin-streptomycin were mixed with SARS-CoV-2 at 100 TCID 50 ( Yang et al., 2022 ) per well and incubated on Vero cells at 37°C for 1 h under 5% CO 2 . After washing the cells with fresh medium, the mixture was incubated for 24 h at 37°C.…”

Differential detection workflows for multi-sample single-cell RNA-seq data

“…We here enumerate the genes within our subset of genes that have been identified previously as associated with COVID. ATP5F1A (Guarnieri et al, 2022), IGHV6-1 (Yang et al, 2022), ACTG1 (Yuka and Yilmaz, 2021), GDI2 (Lata et al, 2022), IGLV1-51 (Zhao et al, 2021) and PKM (Qi et al, 2021)) for the naïve B cells in moderately ill patients; IFITM1 (Arefinia et al, 2023), IGHV6-1 (Yang et al, 2022) and S100A8 (Mellett and Khader, 2022) for the naive B cells in critically ill patients; PSME2 (Alfaro et al, 2022), IGKC (Momeni et al, 2023), PMAIP1 (Park and Lee, 2020), DDX21 (Henriques-Pons et al, 2022), EIF2AK2 (Melnichuk et al, 2022) and CD27 (Wen et al, 2020) for the unswitched memory B cells in moderately ill patients; and DDX21 (Henriques-Pons et al, 2022), IGKC (Momeni et al, 2023), COX6A1 (Mukund et al, 2021), CD27 (Wen et al, 2020) and RHOA (Khatoon et al, 2020) for the unswitched memory B cells in critically ill patients. The significant number of genes with a potential link to the disease highlights the valuable biological insights gained from a DD analysis, beyond those obtained with a conventional DE analysis.…”

A rabies virus-vectored vaccine expressing two copies of the Marburg virus glycoprotein gene induced neutralizing antibodies against Marburg virus in humanized mice

“…To obtain humanized anti-MARV antibodies, we used transgenic mice (CAMouse) as an animal model to obtain anti-MARV monoclonal antibodies with neutralizing activity through hybridoma technology. The CAMouse is a transgenic mouse in which human immunoglobulin heavy chain and light chain genes have been inserted, while the endogenous mouse heavy chain and light chain genes have been deleted [ 45 ].…”