title: Somatic Populations of PGT135-137 HIV-1-Neutralizing Antibodies Identified by 454 Pyrosequencing and Bioinformatics.
abstract: Select HIV-1-infected individuals develop sera capable of neutralizing diverse viral strains. The molecular basis of this neutralization is currently being deciphered by the isolation of HIV-1-neutralizing antibodies. In one infected donor, three neutralizing antibodies, PGT135-137, were identified by assessment of neutralization from individually sorted B cells and found to recognize an epitope containing an N-linked glycan at residue 332 on HIV-1 gp120. Here we use next-generation sequencing and bioinformatics methods to interrogate the B cell record of this donor to gain a more complete understanding of the humoral immune response. PGT135-137-gene family specific primers were used to amplify heavy-chain and light-chain variable-domain sequences. Pyrosequencing produced 141,298 heavy-chain sequences of IGHV4-39 origin and 87,229 light-chain sequences of IGKV3-15 origin. A number of heavy and light-chain sequences of ∼90% identity to PGT137, several to PGT136, and none of high identity to PGT135 were identified. After expansion of these sequences to include close phylogenetic relatives, a total of 202 heavy-chain sequences and 72 light-chain sequences were identified. These sequences were clustered into populations of 95% identity comprising 15 for heavy chain and 10 for light chain, and a select sequence from each population was synthesized and reconstituted with a PGT137-partner chain. Reconstituted antibodies showed varied neutralization phenotypes for HIV-1 clade A and D isolates. Sequence diversity of the antibody population represented by these tested sequences was notably higher than observed with a 454 pyrosequencing-control analysis on 10 antibodies of defined sequence, suggesting that this diversity results primarily from somatic maturation. Our results thus provide an example of how pathogens like HIV-1 are opposed by a varied humoral immune response, derived from intrinsic mechanisms of antibody development, and embodied by somatic populations of diverse antibodies.
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health Bethesda, MD, USA.
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhu
authors.forename: Sijy
authors.initials: S
authors.lastname: O'Dell
authors.forename: Gilad
authors.initials: G
authors.lastname: Ofek
authors.forename: Marie
authors.initials: M
authors.lastname: Pancera
authors.forename: Xueling
authors.initials: X
authors.lastname: Wu
authors.forename: Baoshan
authors.initials: B
authors.lastname: Zhang
authors.forename: Zhenhai
authors.initials: Z
authors.lastname: Zhang
authors.forename: James C
authors.initials: JC
authors.lastname: Mullikin
authors.forename: Melissa
authors.initials: M
authors.lastname: Simek
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Burton
authors.forename: Wayne C
authors.initials: WC
authors.lastname: Koff
authors.forename: Lawrence
authors.initials: L
authors.lastname: Shapiro
authors.forename: John R
authors.initials: JR
authors.lastname: Mascola
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong