Sequencing of Ig genes from single circulating plasmablasts during acute SARS-CoV-2 infection; [Секвенирование генов Ig из единичных циркулирующих плазмабластов при острой SARS-CoV-2-инфекции]

Introduction. Despite the rapid development of high-throughput sequencing for Ig genes, the main method is still Sanger sequencing from single B lymphocytes. Using this method, a number of monoclonal antibodies have been generated for the treatment and prevention of COVID-19. Memory B cells or plasma cells usually act as a source of Ig genes. Another attractive object for sequencing Ig genes and the subsequent generation of antigen-specific monoclonal antibodies are circulating plasmablasts, which combine such properties as high mRNA expression and expression of the surface B cell receptor. The aim of the study is to optimize the method for sequencing Ig genes from single circulating plasmablasts. The study was carried out using the example of plasmablasts formed during acute SARS-CoV-2 infection. Material and methods. Blood was collected from a patient with a severe form of COVID-19 on day 10 from the onset of the first symptoms. Plasmablasts were isolated from the mononuclear fraction of blood cells using a flow cell sorter by the CD19+CD27++CD38++CD3–CD16–CD14–phenotype. IgG secretion was determined by enzyme-linked immunosorbent assay, and antibody-secreting cells were determined by ELISpot. For PCR, the cells were distributed one cell per tube. After cell lysis, cDNA synthesis was performed using the Invitrogen SuperScript III kit. The sample was then divided in half and semi-nested PCR was performed separately to amplify the variable genes of the heavy (VH) and light (VL) chains. The resulting PCR products were sequenced according to Sanger. Nucleotide sequences were analyzed using the Igblast online tool with the IMGT domain definition system and UGENE (version 50.0). PCR products obtained from one sample were cloned into the expression vectors AbVec-hIgG1-799 and AbVec-hIgKappa-798 for subsequent IgG production. HEK293 cells were co-transfected with plasmids encoding the Ig light and heavy chains. Results. In preliminary experiments, it was found that the isolation of B cells using a standard immunomagnetic separation kit resulted in a loss of the plasmablast population. Plasmablasts were then isolated by flow cytometric sorting. The percentage of plasmablasts in total B lymphocytes for the patient studied was 15 %. Antibody-secreting cells accounted for 38 % of the plasmablast population. Fifteen paired sequences of the VL and VH genes were obtained. 14 unique nucleotide sequences of the VL genes and 10 unique nucleotide sequences of the VH genes were identified. The obtained sequences differed from the germline configuration of VH and VL within 0 to 25 % (median 9 %) and from 0 to 11 % (median 7 %), respectively. The CDR3 regions of the VH genes differed significantly from each other, while the CDR3 regions of the VL genes were quite similar. Based on the determined nucleotide sequences, two IgG monoclones were obtained, which was secreted into the supernatant of HEK293 cells co-transfected with expression vectors carrying the genes of IgG heavy and light chains. Conclusion. The method of sequencing Ig genes from single circulating plasmablasts represents a promising approach for the creation of human monoclonal antibodies. This method, in combination with preliminary stimulation of B lymphocytes in vitro, sorting of antigen-specific cells, and the use of immunodeficient mice, opens up new possibilities for the development of targeted drugs. These approaches allow us to significantly accelerate the process of developing new therapeutic antibodies, which is especially important in the fight against emerging infectious diseases. © 2024 Geotar Media Publishing Group. All rights reserved.