期刊
FRONTIERS IN MICROBIOLOGY
卷 3, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2012.00315
关键词
antibody bioinformatics; high-throughput sequencing; HIV-1; immunity; N-linked glycan
类别
资金
- Intramural Research Program of the Vaccine Research Center
- National Institute of Allergy and Infectious Diseases
- National Human Genome Research Institute
- National Institutes of Health
- International AIDS Vaccine Initiative's Neutralizing Antibody Consortium
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [ZIAHG200330, ZIBHG000196] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [ZIAAI005095, ZIAAI005022] Funding Source: NIH RePORTER
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 similar to 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.
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