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. 2013 Oct 22;110(43):E4088-97.
doi: 10.1073/pnas.1306262110. Epub 2013 Oct 8.

De novo identification of VRC01 class HIV-1-neutralizing antibodies by next-generation sequencing of B-cell transcripts

Jiang Zhu  1 , Xueling Wu, Baoshan Zhang, Krisha McKee, Sijy O'Dell, Cinque Soto, Tongqing Zhou, Joseph P Casazza; NISC Comparative Sequencing Program; James C Mullikin, Peter D Kwong, John R Mascola, Lawrence Shapiro
Collaborators, Affiliations

De novo identification of VRC01 class HIV-1-neutralizing antibodies by next-generation sequencing of B-cell transcripts

Jiang Zhu et al. Proc Natl Acad Sci U S A. .

Abstract

Next-generation sequencing of antibody transcripts provides a wealth of data, but the ability to identify function-specific antibodies solely on the basis of sequence has remained elusive. We previously characterized the VRC01 class of antibodies, which target the CD4-binding site on gp120, appear in multiple donors, and broadly neutralize HIV-1. Antibodies of this class have developmental commonalities, but typically share only ∼50% amino acid sequence identity among different donors. Here we apply next-generation sequencing to identify VRC01 class antibodies in a new donor, C38, directly from B cell transcript sequences. We first tested a lineage rank approach, but this was unsuccessful, likely because VRC01 class antibody sequences were not highly prevalent in this donor. We next identified VRC01 class heavy chains through a phylogenetic analysis that included thousands of sequences from C38 and a few known VRC01 class sequences from other donors. This "cross-donor analysis" yielded heavy chains with little sequence homology to previously identified VRC01 class heavy chains. Nonetheless, when reconstituted with the light chain from VRC01, half of the heavy chain chimeric antibodies showed substantial neutralization potency and breadth. We then identified VRC01 class light chains through a five-amino-acid sequence motif necessary for VRC01 light chain recognition. From over a million light chain sequences, we identified 13 candidate VRC01 class members. Pairing of these light chains with the phylogenetically identified C38 heavy chains yielded functional antibodies that effectively neutralized HIV-1. Bioinformatics analysis can thus directly identify functional HIV-1-neutralizing antibodies of the VRC01 class from a sequenced antibody repertoire.

Keywords: DNA sequencing; antibodyomics; cross-donor phylogenetic analysis; humoral immune response; sequence signature.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Donor C38 serum analysis and identification of HIV-1–neutralizing antibodies based on sequence identity. (A) Reduction in neutralization ID50 resulting from RSC3 vs. ΔRSC3 competition against eight HIV-1 strains for a 2008 serum sample of donor C38. Sera from donors 45, 74, and 0219 are included in this analysis for comparison. Red bars indicate the mean reduction. (B) Divergence/identity analysis of donor C38 heavy chain sequences generated from 454 pyrosequencing with H1 (Left) and G1 (Right) primers. Heavy chain sequences are plotted as a function of maximal sequence identity to the heavy chains of 13 template VRC01 class antibodies (VRC01, VRC02, VRC03, NIH45-46, VRC-PG04, VRC-PG04b, VRC-CH30, VRC-CH31, VRC-CH32, 3BNC60, 3BNC117, 12A12, and 12A21) and of sequence divergence from inferred germ-line alleles.
Fig. 2.
Fig. 2.
Identification of functional VRC01 class antibody heavy chains by frequency-based method, lineage rank. (A) Flowchart of lineage rank method. Starting from the heavy and light chain antibodyomes, after a primary analysis to calculate parameters such as germ-line divergence, a CDR H3 or L3-specific lineage analysis is carried out to identify all heavy and light chain lineages, which are then ranked by their prevalence (the number of sequences in a lineage), and representative heavy and light chains from the top-ranking lineages are paired for experimental assessment, e.g., neutralization. Because the VRC01 light chain can complement all VRC01 class antibody heavy chains, here lineage rank is only tested with the C38 heavy chains. (B) Sequence distribution of IGHV1 germ-line genes for G1 primer–derived data set. Blue bars (and percentage values above each bar) indicate the sequences with 20% or greater divergence from inferred germ line, which are considered heavy chains of mature antibodies and subjected to the lineage rank analysis. (C) Prevalent CDR H3 lineages identified within the five major IGHV1 germ-line gene families: IGHV1-18, IGHV1-2, IGHV1-46, IGHV1-69, and IGHV1-8. None of the 35 heavy chain sequences selected from these 22 lineages shows HIV-1 neutralization when paired with VRC01 light chain.
Fig. 3.
Fig. 3.
Identification of VRC01 class antibody heavy chains from donor C38 antibodyome by cross-donor phylogenetic analysis. Cross-donor phylogenetic trees of C38 heavy chain sequences generated by H1 (A) and G1 (B) primer sets. For both data sets, maximum-likelihood trees of variable domain sequences of the IGHV1-2*02 origin from donor C38, along with 13 known VRC01 class antibody heavy chain sequences from five other donors, are rooted by the germ-line gene sequence. VRC01-like donor sequences segregate with these known VRC01 class antibodies. Bars representing 0.1 changes per nucleotide site are shown. Two and 10 heavy chain sequences were selected from H1 and G1 primer data sets, respectively, for functional assessment and identity/divergence analysis. The neutralization profiles of selected heavy chain sequences reconstituted with the VRC01 light chain [named gVRC-H(n)dC38] are depicted with 20 isolate neutralization dendrograms. Explicit neutralization IC50s are provided in SI Appendix, Table S10. In the rightmost panels, the repertoire of heavy chain sequences is plotted as a function of sequence identity to a selected heavy chain sequence and of sequence divergence from inferred germ-line alleles. The number of sequences >75% (or 80%) identical to the selected sequence, which are likely the somatic variants of the reference heavy chain sequence, is provided for each plot. Notably, the identified heavy chains from donor C38 do not display significant sequence similarity to previously identified VRC01 class antibodies. Color coding indicates the number of sequences.
Fig. 4.
Fig. 4.
Sequence comparison of C38 heavy chains identified by cross-donor phylogenetic analysis. (A) Sequence alignment of 12 expressed C38 heavy chains from amplifications using H1 and G1 primers, inferred germ-line gene (IGHV1-2*02), and the heavy chains of 13 template VRC01 class antibodies (VRC01, VRC02, VRC03, VRC-PG04, VRC-PG04b, VRC-CH30, VRC-CH31, VRC-CH32, 3BNC60, 3BNC117, 12A12, 12A21, and NIH45-46). Amino acids in V genes that differ from IGHV1-2*02 are highlighted in red. The CDR H3 regions are circled in blue dash line. The nomenclature gVRC-HndC38 is used for neutralizing heavy chains, and the indexes of two nonneutralizing heavy chains, 304943 and 240171, are shown in italic. Contact residues between VRC01 V-gene segment and gp120 are labeled above the germ-line gene: ●, backbone and side chain contacts; ○, backbone contacts only; *, side chains contacts only. (B) Identity/divergence analysis of 10 neutralizing C38 heavy chains. Heavy chain sequences obtained from H1 (Left) and G1 (Right) primers are plotted as a function of maximal sequence identity to the heavy chains of 13 template VRC01 class antibodies and of sequence divergence from putative germ-line genes. The 10 neutralizing heavy chains are shown as black dots on the plots.
Fig. 5.
Fig. 5.
Identification of VRC01 class antibody light chains from donor C38 antibodyome. (A) Thirteen VRC01-like light chains identified from two sequencing experiments are aligned to the putative germ-line gene allele IgKV3-20*01. All 13 light chains possess the same signature: CDR L3 loop of five amino acids and a glutamine or glutamate acid at position 96 (Kabat numbering). Amino acids mutated from the germ-line sequence are colored in red. Light chain sequences that show neutralization when reconstituted with VRC01 heavy chain and gVRC-H3dC38 (Fig. 6) are named gVRC-L(n)dC38. (B) Divergence/identity analysis of donor C38 κ-light chain sequences generated from 454 pyrosequencing with both κ and λ primers (Left) and κ primers only (Right), with the 13 VRC01-like light chains shown as black dots. Light chain sequences are plotted as a function of maximal sequence identity to the light chains of 13 template VRC01 class antibodies (VRC01, VRC02, VRC03, NIH45-46, VRC-PG04, VRC-PG04b, VRC-CH30, VRC-CH31, VRC-CH32, 3BNC60, 3BNC117, 12A12, and 12A21) and of sequence divergence from inferred germ-line alleles.
Fig. 6.
Fig. 6.
Identification of unique VRC01 class antibodies by pairing heavy and light chains. (A) Neutralization of six reconstituted antibodies by pairing VRC01 heavy chain with C38 light chains. (B) Neutralization screening of functional partner chains for C38 heavy chain, gVRC-H3dC38, and C38 light chain, gVRC-L1dC38. IC50 values listed in B and C represent antibody concentration in µg/mL required to achieve 50% neutralization (IC50), and color coding indicates the potency, with IC50 < 1 µg/mL in red shading, 1 µg/mL < IC50 <50 µg/mL in green shading, and no shading for IC50 >50 µg/mL. (C) Maximum-likelihood trees of functional C38 heavy chains and light chains rooted in their respective germ-line genes, with the most potent heavy/light chain pair highlighted in red shading. Bars representing 0.1 changes per nucleotide site are shown.
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