Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles.
Technical difficulties have severely limited the yield of methods for the generation of human antiviral monoclonal antibodies (Mabs) in the past. We describe here a novel method for the efficient development of human Mabs against viruses. Rotavirus (RV) is a major cause of gastroenteritis in infants and adults worldwide. We generated fluorescent virus-like particles (VLPs) to identify and physically sort single RV-specific B cells from healthy adult blood donors, or RV-infected infants or adults. We expanded the sorted single B cells in culture, tested for RV-specific antibody secretion, and cloned and sequenced the antibody heavy and light chain variable region (VH and VL) genes. The percentage of wells that produced antibodies after sorting and expanding RV-specific adult B cell clones was high at 23%. The overall efficiency of RV-specific antibody gene recovery after the isolation, confirmation, and cloning of RV-specific VH segments was 1.3% of sorted cells in adults. RV-specific variable gene segments also were obtained from acutely infected infants, although infant B cells did not proliferate and differentiate in culture as well as adult B cells. We expressed recombinant Fabs incorporating the VH and VL genes from RV-specific B cell clones using a new modified bacterial Fab expression vector that we describe. Finally, we demonstrated binding of purified Fabs to RV proteins by immunofluorescence and ELISA. This method for the generation of recombinant human Mabs to RV from single antigen-specific B cell clones selected with fluorescent VLPs could be used to generate human Mabs to many other viruses whose proteins can self-assemble into VLPs.
Weitkamp
Jörn Hendrik
JH
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232-2581, USA.
Kallewaard
Nicole
N
Kusuhara
Koichi
K
Feigelstock
Dino
D
Feng
Ninguo
N
Greenberg
Harry B
HB
Crowe
James E
JE
D000328:Adult; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D000914:Antibodies, Viral / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D000956:Antigens, Viral; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D036022:Capsid Proteins / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005759:Gastroenteritis / Q000276:immunology / Q000821:virology; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007223:Infant; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D012401:Rotavirus / Q000276:immunology*; D012400:Rotavirus Infections / Q000276:immunology / Q000821:virology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D004274:DNA, Recombinant; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; C061332:VP6 protein, Rotavirus; C050779:VP7 protein, Rotavirus