that are compatible with normal B cell maturation. It will be of in-
terest to apply our de novo recombination strategy to the expres-
sion of other broadly neutralizing antibodies and test whether our
approach could alleviate negative selection against certain poly-
reactive or autoreactive antibodies.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
dKEY RESOURCES TABLE
dCONTACT FOR REAGENT AND RESOURCE SHARING
dEXPERIMENTAL MODEL AND SUBJECT DETAILS
BMice
dMETHODS DETAILS
BGeneration and Characterization of VH1-2 and VH1-2/
LC Mouse Models
BHTGTS-Rep-Seq Analysis of B Cell Repertoire
BSingle-Cell RT-PCR
BImmunizations
BImmunogen Amino Acid Sequences
BProtein Production
BELISA
BTZM-bl Neutralization Assay
BFlow Cytometry and B Cell Sorting
BSingle B Cell RT-PCR, Gene Amplification, and Cloning
BBio-Layer Interferometry Antigenicity Analysis
BMutation Profile Construction and Statistical Test for
Enriched and Depleted Mutations
BHigh-Throughput Paired Heavy-Light Chain RT-PCR
dQUANTIFICATION AND STATISTICAL ANALYSIS
dDATA AND SOFTWARE AVAILABILITY
BData ResourcesSUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures and seven tables and can be
found with this article online athttp://dx.doi.org/10.1016/j.cell.2016.07.029.
AUTHOR CONTRIBUTIONS
M.T., C.C., X.C., H.D., H.-L.C., P.D.K., B.H., J.M., and F.W.A. designed the
study and analyzed and interpreted data. M.T., C.C., X.C., H.D., H.-L.C.,
M.D., Z.S., M.K., L.W., S.L., S.S., M.G.J., Y.C., B.D., Y.C., E.N., E.C., R.C.,
N.A.D.-R., Y.Z., W.S., W.-P.K., M.C., A.H., F.L., I.G., P.-Y.H., and S.J. per-
formed experiments. A.M., W.S., D.D., and L.S. (FHCRC) provided reagents.
Z.S., B.D., and L.S. (Columbia University) performed computational analyses.
M.T., J.M., and F.W.A. drafted the manuscript. B.H., L.S. (FHCRC), W.S., A.M.,
C.C., X.C., and H.D. helped revise and polish the manuscript. M.T., C.C., X.C.,
H.D., M.D., M.K., S.L., Z.S., B.D., and S.J. generated figures and tables.
ACKNOWLEDGMENTS
We thank Norman Letvin and Connie Gee for stimulating this collaborative
study. We also thank Sam Darko for assistance with data analysis. This
work was supported by NIH grants R01AI077595 and AI020047 (to F.W.A.);
P01 AI094419 and U19AI109632 (to L.S., FHCRC); P01-AI104722 (to L.S.,
Columbia University); NIAID, Division of AIDS, Center for HIV/AIDS Vaccine
Immunology- Immunogen Discovery (CHAVI-ID) 5UM1 AI100645 (to B.F.H.
and F.W.A.); and NIH Center for HIV/AIDS Vaccine Immunology and Immu-
nogen Discovery (CHAVI-ID) 1UM1 AI100663 (to W.R.S.). This work was
also supported by the intramural research program of the Vaccine Research
Center, NIAID, NIH and the International AIDS Vaccine Initiative Neutralizing
Antibody Consortium and Center (to W.R.S.); CAVD funding for the IAVI NAC
Center (W.R.S.); and the Ragon Institute of MGH, MIT and Harvard (W.R.S.).
M.D. was supported by an HHMI Medical Student Fellowship. F.W.A. is an
investigator and Z.D. is a postdoctoral fellow of the Howard Hughes Medical
Institute. W.R.S. is a co-founder and stockholder in Compuvax, Inc., which
has programs in non-HIV vaccine design that might benefit indirectly from
this research.Received: May 25, 2016
Revised: July 11, 2016
Accepted: July 20, 2016
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