Hansaem Lee has completed her PhD from Ulsan University, Republic of Korea and Post-doctoral studies from Mayo Clinic, Minnesota, USA. She is a Staff Scientist at division of Emerging Infectious Diseases and Vector Research, Korea National Institute of Health, Korea Centers for Disease Control and Prevention, Republic of Korea.
Isolating human B cell cells is inevitable to investigate the development of protective antibodies, as vaccine or therapeutics candidates, against emerging infectious viruses. Single B cell sorting is a powerful tool for define ant-viral B cells. Thus, the acquisition of abundant B cells is essential to synthesize the broad-ranged antibody cDNAs for the screening of the broadly neutralizing monoclonal antibodies against the infectious viruses. In this study, we test and compare the human B cell isolation rates among the three human B cell isolation tools, a fluorescence-activated cell sorting (FACS), EasySep™ human B cell enrichment kit (STEMCELL Technologies), and human CD19 MciroBeads (MACS Miltenyi Biotec). We found that the method using FACS was more suitable to obtain more human B cells than the other methods. In addition, using a CMV tetramer probe or MERS-CoV spike protein probes, the antibody genes against CMV and MERS-CoV were amplified by heavy chain and light chain RT-PCR and nested PCR successfully. These genes were also analyzed by IMGT, resulting that the antibody sequences were proper IgG genes. The results demonstrate that human B cell isolation using FACS are a reliable tool for assisting in the rapid development of prophylactic or therapeutic antibodies neutralizing new emerging infectious diseases.
Hye-Min Woo has completed her PhD from Kookmin University, Republic of Korea. She is a Senior Researcher of division of Emerging Infectious Diseases & Vector Research, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Republic of Korea.
Middle East Respiratory Syndrome (MERS) is a severe respiratory infection caused by MERS Coronavirus (MERS-CoV). After the outbreak of MERS occurred in the Republic of Korea in 2015, it has been required to develop the efficient and safe therapeutics against MERS-CoV infection. The spike glycoproteins (S) of MERS-CoV bind cellular receptors, and mediate infection of target cells. Therefore, S protein is one of the most promising antigen candidates for the development of neutralizing antibodies of a newly emerging MERS-CoV. In this study, we employed the peripheral blood mononuclear cells (PBMCs) of Korean MERS recovered patients to obtain the memory B cells bearing immunoglobulin Gs (IgGs) against MERS S protein. The cells were isolated by using FACS. The cDNAs of IgGs were synthesized from the sorted B cell clones. The variable regions of heavy and light chains were amplified by PCR, and recombinant plasmids were cloned with pFUSE vectors containing signal peptides and the constant region of human IgG. The anti-MERS-CoV S IgGs were expressed in 293F cells and purified by a protein G affinity chromatography. Characteristics of anti-MERS-CoV S IgGs were analyzed by the S protein affinity assay and the neutralizing assays using a MERS-CoV pseudovirus and MERS-CoV strains. The antibodies generated in this study are expected to be applied as therapeutic agents or tools for diagnosis.