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Isolation of mAbs from sorted single B cells by RT/PCR for analysis of B cell responses to HIV-1 MPER epitope in vaccinated rhesus macaques

Isolation of mAbs from sorted single B cells by RT/PCR for analysis of B cell responses to HIV-1 MPER epitope in vaccinated rhesus macaques
R-J Zhang1, M.A. Moody1, M.S Alam1, J-S Yu1, S. Santra2, G. Kelsoe1, R. Bhaskarabhatla3, M-Y. Wang1, F. Jaeger1, S.M. Dennison1,K. Anasti1, T.C. Gurley1, A.A. Allen1, L.C. Armand1, D.J. Marshall1, J.F. Whitesides1, L. Sutherland1, R.M. Scearce1, A. Foulger1, M. Cooper1, J. Pritchett1, A. Hogan1, R. De1, C. Perckels1, C. Stolarchuk1, E. Solomon1, E. Friberg1, Y. Yang1, F. Gao1, J.E. Schmitz2, T.B. Kepler3, B.F. Haynes1 and H-X. Liao1.
1Duke Human Vaccine Institute, Duke University, Durham, NC, USA; 2Beth Israel Deaconess Med. Ctr. Boston, MA, USA; 3Department of Microbiology, Boston University, Boston, MA 02215, USA.
Contact: Ruijun.zhang@duke.edu and  hliao@duke.edu
Background: Rhesus macaques are important models for evaluation of HIV-1 vaccines. Analysis of immunoglobulin (Ig) VH and VL genes derived from single B cells is a powerful technology for definition of Ig repertoires to viral infections and vaccination. We have produced a large panel of recombinant mAbs from immunized rhesus macaques to ask if a HIV-1 gp41 immunogen could induce an antibody lineage with binding characteristics of a broadly neutralizing antibody (BnAb).
Methods: Primers were designed for amplification of rhesus Ig VH and VL gene segments from sorted single memory B cells by RT/PCR. The isolated VH and VL gene segments were analyzed using a newly assembled and annotated database of rhesus germline gene segments, and used to produce mAbs using linear antibody gene expression constructs generated by PCR without cloning. PBMC from rhesus macaques that were immunized with HIV-1 JRFL Env followed by boosts with MPER 2F5 epitope peptide in liposomes were used for sorting HIV-1 antigen-specific single memory B cells.
Results: A total of 416 unique VH and VL gene pairs were isolated from MPER epitope sorted single memory B cells and expressed as IgG1, of which, 238 mAbs reacted with MPER03. We found 68.9% of these antibodies belonged to 25 clonal lineages. Antibodies in the Tr900364 clonal lineage (15 members) have VH mutation frequencies ranging from 4.48%-12.96% and bound to HIV-1 gp41 inter, MPER epitope, KYNU/W, but not KYNU/M, with a footprint involving the 2F5-nominal epitope, DKW. MAbs bound to MPER peptide-liposomes in a 2F5-like 2-step binding mechanism.
Conclusion A streamlined strategy for amplification and expression of rhesus Ig VH and VL genes has been developed for study of the rhesus macaque antibody repertoire in response to immunization with HIV-1 vaccines. Our study showed that immunization with HIV-1 JRFL Env and MPER peptide-liposomes induced an antibody clonal lineage with characteristics of precursors of an MPER BnAb.
Supported by the Center for HIV/AIDS Vaccine Immunology NIAID grant AI0678501 and Collaboration for HIV Vaccine Discovery grant from the Bill and Melinda Gates Foundation.