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. 2015 Nov 3;6(6):e01375-15.
doi: 10.1128/mBio.01375-15.

Rhesus Macaque B-Cell Responses to an HIV-1 Trimer Vaccine Revealed by Unbiased Longitudinal Repertoire Analysis

Kaifan Dai  1 , Linling He  1 , Salar N Khan  1 , Sijy O'Dell  2 , Krisha McKee  2 , Karen Tran  3 , Yuxing Li  4 , Christopher Sundling  5 , Charles D Morris  1 , John R Mascola  2 , Gunilla B Karlsson Hedestam  5 , Richard T Wyatt  6 , Jiang Zhu  7
Affiliations

Rhesus Macaque B-Cell Responses to an HIV-1 Trimer Vaccine Revealed by Unbiased Longitudinal Repertoire Analysis

Kaifan Dai et al. mBio. .

Abstract

Next-generation sequencing (NGS) has been used to investigate the diversity and maturation of broadly neutralizing antibodies (bNAbs) in HIV-1-infected individuals. However, the application of NGS to the preclinical assessment of human vaccines, particularly the monitoring of vaccine-induced B-cell responses in a nonhuman primate (NHP) model, has not been reported. Here, we present a longitudinal NGS analysis of memory B-cell responses to an HIV-1 trimer vaccine in a macaque that has been extensively studied by single B-cell sorting and antibody characterization. We first established an NHP antibodyomics pipeline using the available 454 pyrosequencing data from this macaque and developed a protocol to sequence the NHP antibody repertoire in an unbiased manner. Using these methods, we then analyzed memory B-cell repertoires at four time points of NHP immunization and traced the lineages of seven CD4-binding site (CD4bs)-directed monoclonal antibodies previously isolated from this macaque. Longitudinal analysis revealed distinct patterns of B-cell lineage development in response to an HIV-1 trimer vaccine. While the temporal B-cell repertoire profiles and lineage patterns provide a baseline for comparison with forthcoming HIV-1 trimer vaccines, the newly developed NHP antibody NGS technologies and antibodyomics tools will facilitate future evaluation of human vaccine candidates.

Importance: The nonhuman primate model has been widely used in the preclinical assessment of human vaccines. Next-generation sequencing of B-cell repertoires provides a quantitative tool to analyze B-cell responses to a vaccine. In this study, the longitudinal B-cell repertoire analysis of a rhesus macaque immunized with an HIV-1 trimer vaccine revealed complex B-cell lineage patterns and showed the potential to facilitate the evaluation of future HIV-1 vaccines. The repertoire sequencing technologies and antibodyomics methods reported here can be extended to vaccine development for other human pathogens utilizing the nonhuman primate model.

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Figures

FIG 1
FIG 1
Analysis of reported 454 sequencing data of F128 heavy chains. (A) General repertoire properties determined from the 454-derived F128 heavy chain population, including germline gene usage, germline gene divergence, and the length of heavy chain complementarity determining region 3 (HCDR3). The 454 sequencing data were reanalyzed by the NHP antibodyomics pipeline using either the CS germline gene database (black histogram) or the IMGT germline gene database (gray histogram). (B) Two-dimensional (2D) identity-divergence analysis of the 454-derived F128 heavy chain population. Seven CD4-binding site (CD4bs)-directed monoclonal antibodies (MAbs) isolated from F128 were used as the template in the sequence identity calculation. The heavy chains are plotted as a function of sequence identity to the template and sequence divergence from putative germline V genes. The template and the somatic variants identified by an HCDR3 identity cutoff of 95% or greater are shown as black and magenta dots, respectively, with the number of somatic variants provided on the 2D plots. For contour plots derived from the CS and IMGT germline gene databases, the density of sequence distribution is indicated by color coding and darkness of the shading, respectively.
FIG 2
FIG 2
Experimental strategies for unbiased longitudinal B-cell repertoire analysis of NHP immunization. (A) Schematic flow of an NGS-based vaccine strategy consisting of vaccine immunogen design, NHP immunization, and sample collection, unbiased repertoire capture and library preparation, NGS profiling of B-cell response, and antibodyomics analysis. (B) F128 immunization regimen and time points selected for longitudinal analysis of memory B-cell response. Four time points—3 weeks (w3) after 2 immunizations and 2 weeks (w2) after 3, 4, and 5 immunizations—were selected for NGS profiling and are labeled with “NGS.” (C) Schematic flow of unbiased capture of NHP antibody repertoire starting from peripheral blood mononuclear cells (PBMCs) to loaded sequencing chip. (D) Comparison of PCR products of human (left) and NHP (right) antibody transcripts obtained from gene-specific primers and 5′ RACE PCR. The PCR products are characterized with gel electrophoresis (top) and PGM sequencing (bottom). For the purpose of formatting, the gel of human antibody libraries has been rearranged with splicing (labeled with orange lines). The average read length from PGM sequencing, without the default 3′ trimming in the base-calling process, is provided on the read length distribution.
FIG 3
FIG 3
Unbiased memory B-cell repertoires at four time points of F128 immunization. For each time point, unbiased heavy (H) and light chain (κ and λ) libraries were obtained using a 5′ RACE PCR protocol. PGM sequencing was performed using Ion 318 (or 316) v2 chips, and the sequencing data were processed with the NHP antibodyomics pipeline (Tables 1 and 2). The processed antibody chain sequences were used to determine general repertoire properties such as germline gene usage (A), germline gene divergence (B), and complementarity determining region 3 (CDR3) length (C) for H, κ, and λ chains.
FIG 4
FIG 4
Longitudinal analysis of the GE136 lineage development. (A) Identity-divergence analysis of the unbiased heavy (H) and light (λ) chain repertoires. The sequences are plotted as a function of sequence identity to GE136 and sequence divergence from putative germline V genes. Color coding denotes sequence density. (B) Maximum-likelihood (ML) tree of selected GE136 heavy chain variants rooted by the putative germline V gene, VH4.11. The bar represents a 0.001 change per nucleotide site. A bioinformatics procedure consisting of HCDR3 comparison at an identity cutoff of 95%, motif filtering, and clustering at a full-length identity cutoff of 99% was used to select sequences for phylogenetic analysis and experimental validation. Four selected sequences are displayed in the ML tree. Reconstituted antibodies are labeled with blue dots if expressed and red asterisks if reactive with the HIV-1 trimer vaccine antigen. (C) (Left) Enzyme-linked immunosorbent assay (ELISA) analysis of antibody binding to the vaccine antigen, with standard deviation (SD) bars. (Right) Averaged EC50s.
FIG 5
FIG 5
Longitudinal analysis of the GE140 lineage development. (A) Identity-divergence analysis of the unbiased heavy (H) and light (λ) chain repertoires. The sequences are plotted as a function of sequence identity to GE140 and of sequence divergence from putative germline V genes. Color coding denotes sequence density. The VH4.11 heavy chain family at each time point is visualized by black contour lines (middle row). The GE140-like heavy chains with an HCDR3 identity of over 95% are shown as red dots on the plots; the numbers are numbers of sequences. (B) ML tree of selected GE140 heavy chain variants rooted by the putative germline VH4.11 gene (see Fig. 4 for sequence selection and labeling). The bar represents a 0.001 change per nucleotide site. (C) ELISA analysis of antigen binding is shown for six antibodies reconstituted from GE140 heavy chain variants with SD bars. (D) Analysis of GE140-like light chains. (Left) The VL2.7 light chain family at the last time point is visualized with black contours. Light chains with an LCDR3 identity of 95% or greater are shown as red dots on the 2D plot. (Right) ELISA analysis of HIV-1 trimer vaccine antigen binding (right) is shown for three antibodies reconstituted from GE140 light chain variants, with SD bars.
FIG 6
FIG 6
Longitudinal analysis of the GE143 lineage development. (A) Identity-divergence analysis of the unbiased heavy (H) and light (λ) chain repertoires. The sequences are plotted as a function of sequence identity to GE143 and sequence divergence from putative germline V genes. Color coding denotes sequence density. The VH4.34 and VH4.40 families are visualized by black contour lines (second and third rows). Heavy chains with an HCDR3 identity of over 92% and 95% to GE143 are shown as blue and red dots, respectively. (B) ML tree of selected GE143-like heavy chains of VH4.34 origin (left). ELISA analysis of HIV-1 vaccine antigen binding is shown for 12 antibodies reconstituted from GE143-like heavy chains of VH4.34 origin with SD bars (right). (C) (Left) ML tree of selected GE143-like heavy chains of VH4.40 origin. (Right) ELISA analysis of HIV-1 vaccine antigen binding for four antibodies reconstituted from GE143-like heavy chains of VH4.40 origin, with SD bars. The procedure used for sequence selection and labeling of ML tree is described in the legend to Fig 4. The bar represents a 0.001 change per nucleotide site.
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