- Poster presentation
- Open Access
Isolation of broadly neutralizing HIV-1 antibodies from high-throughput single B cell culture
© Longo et al; licensee BioMed Central Ltd. 2012
- Published: 13 September 2012
- Affinity Maturation
- Human mAbs
- Vaccine Immunogen
- Repertoire Breadth
- Diverse Pathogen
The isolation of broadly neutralizing HIV-1 antibodies that arise during infection has provided insights into the design of vaccine immunogens capable of eliciting similar antibody response. The use of HIV-specific sorting probes resulted in the isolation of antibodies to vulnerable viral epitopes, such as the CD4-binding site, but the use of such probes does not explore the subject’s immunoglobulin repertoire breadth.
To identify new HIV-1 monoclonal antibodies (mAbs), we developed a B-cell culture system to isolate and screen thousands of B cells. Memory B cells were isolated by negative selection and individually cultured in 384 well plates with irradiated feeder cells expressing CD40 ligand. The addition of cytokines IL-2 and IL-21 stimulated proliferation and immunoglobulin secretion.
After 14 days in culture, approximately 35% of the B cell clones secreted >100 ng/ul of IgG which met the sensitivity threshold to screen each clone in an automated micro-neutralization assay. B cell clonal expansion allowed recovery of the immunoglobulin heavy and light chains by RT-PCR, with subsequent cloning into expression vectors and mAb testing against a large panel of viruses. In one experiment screening approximately 8600 B cells, 9 clones were identified as potential contributors to the neutralization detected in the patient’s serum. One of the isolated clones produced mAb VRC22 with 30% neutralization breadth and moderate potency. Further studies revealed that VRC22 was sensitive to JRCSF glycan mutants N332A and N301A but not N160K. VRC22 utilizes VH4-34 with a single amino acid CDR1 deletion and a mutation frequency of 7% which is a lower level of affinity maturation than observed for most known HIV-1 neutralizing antibodies.
This B-cell culture system allows efficient screening of thousands of individual B cells and the recovery of antigen specific mAbs. This approach can be used to isolate human mAbs to diverse pathogens.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.