Guinea pig immunoglobulin VH and VL naïve repertoire analysis

Abstract

The guinea pig has been used as a model to study various human infectious diseases because of its similarity to humans regarding symptoms and immune response, but little is known about the humoral immune response. To better understand the mechanism underlying the generation of the antibody repertoire in guinea pigs, we performed deep sequencing of full-length immunoglobulin variable chains from naïve B and plasma cells. We gathered and analyzed nearly 16,000 full-length VH, Vκ and Vλ genes and analyzed V and J gene segment usage profiles and mutation statuses by annotating recently reported genome data of guinea pig immunoglobulin genes. We found that approximately 70% of heavy, 73% of kappa and 81% of lambda functional germline V gene segments are integrated into the actual V(D)J recombination events. We also found preferential use of a particular V gene segment and accumulated mutation in CDRs 1 and 2 in antigen-specific plasma cells. Our study represents the first attempt to characterize sequence diversity in the expressed guinea pig antibody repertoire and provides significant insight into antibody repertoire generation and Ig-based immunity of guinea pigs.

Fig 1. Workflow of NGS-based guinea pig naïve V gene analysis.

(A) Naïve B cells in spleens and lymph nodes were isolated from two individual naïve guinea pigs by FACS. Each of the V_H(μ), V_κ and V_λ gene libraries was prepared by 5’ RACE PCR. Multiplex identifier (MID), which identifies the sample origin, is added with the sequencing adaptor. (B) The libraries are sequenced with the 454 GS-FLX Plus system. Raw data are first processed using the sequence quality, and in-frame reads with lengths greater than 400 bp are pooled. After partitioning each V(D)J combinatorial match into its own subset, IgBLAST alignments are performed using custom IgBLAST databases for guinea pigs. Output data are sorted by MID and analyzed for V gene usage frequency, CDR analysis, and mutation frequency.

Fig 2. V and J gene segment usages in naïve B cells from individual guinea pigs.

The x-axis represents the germline V gene segment, and the y-axis represents the germline J gene segment. Each point in the x-y axis represents all possible V-J gene segment combinations. The bubble size at each point corresponds to the percent frequency of reads from each sample matching a particular V-J gene segment combination. The bubble color indicates the sample origin, spleen (red) and lymph node (blue) from guinea pig no. one and spleen (green) and lymph node (orange) from guinea pig no. two.

Fig 3. CDR3 length distributions of V_H, V_κ and V_λ in naïve B cells.

The relative frequencies of the CDR3 amino acid lengths of the V_H (A), V_κ (B) and V_λ (C) genes are shown. Data are from four MID samples (N = 5675 for V_H, 9360 for V_κ and 1230 for V_λ). CDR3s are defined according to IMGT unique numbering.

Fig 4. V and J gene segment usage in plasma cells from immunized guinea pigs.

The x-axis represents the germline V gene segment, and the y-axis represents the germline J gene segment. Each point in the x-y axis represents all possible V-J gene segment combinations. The bubble size corresponds to the percent frequency of the V-J gene segment combination. Black bubbles represent total V and J gene segment usage in naïve B cells from four MID samples in Fig 2. Red bubbles represent the frequencies of V and J gene segment usage in plasma cells (N = 86 for V_H, 68 for V_κ and 25 for V_λ).

Fig 5. Distribution of mutational status in naïve and immunized guinea pigs.

The x-axis represents nucleotide position defined according to IMGT unique numbering. The mutation frequencies in V gene segments (VH3-183-54, VH3-157-75, VK4-95, VK4-45, VL11-35 and VL4-82) of B cells in naïve guinea pigs (black) and plasma cells in immunized guinea pigs (red) are determined. Analyzed Ig sequence counts: VH3-183-54, naïve = 100, PC = 14; VH3-157-75, naïve = 88, PC = 8; VK4-95, naïve = 100, PC = 23; VK4-45, naïve = 100, PC = 16; VL11-35, naïve = 30, PC = 10; VL4-82, naïve = 44, PC = 6.