Systems analysis of adaptive immunity by utilization of high-throughput technologies

Reddy, Sai T.; Georgiou, George

doi:10.1016/j.copbio.2011.04.015

Cited by 27 publications

(23 citation statements)

References 48 publications

(49 reference statements)

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“…Because variable region transcripts of antibodies are over 300 nucleotides in length and because the high similarity between different antibody transcripts precludes assembly of full sequences from fragments, we used 454 pyrosequencing, which is currently one of the few next-generation sequencing technologies to provide reads of sufficient length (Reddy et al, 2009; Reddy and Georgiou, 2011; Wu et al, 2011). However, 454 pyrosequencing is known to suffer from high error rates (Prabakaran et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

“…Advances in genomics and computational science have the potential for an equally profound impact on our understanding of the immune response. Here we focus on the application of new genomic and computational techniques, particularly 454 pyrosequencing of B cell transcripts (Reddy et al, 2009; Reddy and Georgiou, 2011; Wu et al, 2011) and systems-level bioinformatics (Kitano, 2002), to understand the antibody response to infection.…”

Section: Introductionmentioning

confidence: 99%

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Somatic populations of PGT135–137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatic

et al. 2012

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Somatic populations of PGT135–137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatic

et al. 2012

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“…Generally, only a few monoclonal antibodies from each donor have been identified, although it is possible to identify substantially more (6,12). Indeed, next-generation sequencing technologies (13)(14)(15)(16)(17)(18) seem to offer an efficient means for identifying thousands of somatic variants (19). The massively parallel sequencing that is used by such technologies, however, leads to the loss of information on individual pairings of heavy and light chain and as such, has made it a challenge to discern native antibodies (with naturally paired heavy and light chains) in next-generation sequencing data.…”

mentioning

confidence: 99%

Mining the antibodyome for HIV-1–neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains

Zhu

Ofek

Yang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

140

165

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Next-generation sequencing of antibody transcripts from HIV-1-infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy-and lightchain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy-and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141-145. Heavy-and light-chain phylogenetic trees of PGT141-145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141-145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings.antibody-affinity maturation | antibodyomics | B-cell ontogeny | DNA sequencing | immunological tolerance

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“…Next-generation sequencing of immunoglobulin variable region and T-cell receptor repertoires is providing information that is key to understanding adaptive immune responses and to diagnostic and therapeutic applications [1][2][3][4][5][6] . However, existing immune repertoire sequencing technologies yield data on only one of the two chains of immune receptors and thus cannot provide information about the identity of immune receptor pairs encoded by individual B or T lymphocytes 2,7,8 .…”

mentioning

confidence: 99%

High-Throughput Sequencing of the Paired Human Immunoglobulin Heavy and Light Chain Repertoire

DeKosky¹

2017

Decoding the Antibody Repertoire

118

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Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy-and light-chain variable regions (V H and V L ) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10 4 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V H :V L linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V H :V L pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG + B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

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Systems analysis of adaptive immunity by utilization of high-throughput technologies

Cited by 27 publications

References 48 publications

Somatic populations of PGT135–137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatic

Somatic populations of PGT135–137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatic

Mining the antibodyome for HIV-1–neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains

High-Throughput Sequencing of the Paired Human Immunoglobulin Heavy and Light Chain Repertoire

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