The selected lymphocyte antibody method : a novel approach for the generation and genetic analysis of human cytomegalovirus (HCMV) neutralizing monoclonal IgG antibodies

• Main Author: Olsen, Ole Andrew
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/11986

The generation of human monoclonal antibodies that may be clinically useful is hampered by the lack of an efficient, applicable technology. In this thesis I report the development of the Selected Lymphocyte Antibody Method (SLAM) as an alternative approach to generate monoclonal antibodies and to facilitate the analysis of the human humoral immune response. SLAM technology is based upon the molecular cloning and expression of immunoglobulin heavy and light chain variable region cDNAs from single B lymphocytes that have been identified in a modified hemolytic plaque assay as secreting immunoglobulin which is specific for a target antigen. SLAM technology was first developed in a murine model. Immunoglobulin variable region cDNAs were RT-PCR amplified from single murine B lymphocytes that secreted immunoglobulin which is specific for an epitope found in gpll6 of the Human Cytomegalovirus (HCMV) or in gpl20 of HIV-1. These immunoglobulin variable regions were then cloned and expressed in the context of human immunoglobulin constant regions. The resulting chimeric immunoglobulins were shown to reproduce the same specificity as the "parental" immunoglobulin. To generate human monoclonal antibodies that were specific for gpl 16, peripheral blood B lymphocytes from a single HCMV immune donor were cultured at limiting dilution in the "EL4.B5" B-cell culture system. 107 single B lymphocytes, corrresponding to 34 clones that secreted gpll6-specific immunoglobulin, were identified by hemolytic plaque assay and then isolated by micromanipulation. Immunoglobulin variable region cDNAs were successfully RT-PCR amplified from 29 of the 34 clones. Sequence analysis of the V region cDNAs showed a remarkable restriction in the use of heavy chain variable and joining gene segments as well as restriction in the use of kappa light chain variable and joining gene segments in the associated light chains. Two independent B cell clonotypes were identified based on the utilization of different diversity gene segments in the heavy chain variable regions. Two recombinant monoclonal antibodies (mAbs) that represent each clonotype were expressed and were shown to neutralize HCMV in vitro. Thus, SLAM technology facilitated the generation of two potentially clinically useful antibodies and may be used in the future to generate mAbs against a wide variety of pathogens.