Complex Genetic Architecture Underlies Regulation of Influenza-A-Virus-Specific Antibody Responses in the Collaborative Cross

Abstract

Host genetic factors play a fundamental role in regulating humoral immunity to viral infection, including influenza A virus (IAV). Here, we utilize the Collaborative Cross (CC), a mouse genetic reference population, to study genetic regulation of variation in antibody response following IAV infection. CC mice show significant heritable variation in the magnitude, kinetics, and composition of IAV-specific antibody response. We map 23 genetic loci associated with this variation. Analysis of a subset of these loci finds that they broadly affect the antibody response to IAV as well as other viruses. Candidate genes are identified based on predicted variant consequences and haplotype-specific expression patterns, and several show overlap with genes identified in human mapping studies. These findings demonstrate that the host antibody response to IAV infection is under complex genetic control and highlight the utility of the CC in modeling and identifying genetic factors with translational relevance to human health and disease.

Keywords: Collaborative Cross; antibody; complex trait; genetic architecture; genetic mapping; genetic reference population; host genetics; humoral immunity; influenza; influenza virus.

Graphical abstract

Figure 1

The CC-F1 Population Exhibits Broad Between-Strain Variation in the Magnitude, Kinetics, and Composition of IAV-Specific Antibody Responses (A) The CC-F1 population generally exhibited an overall pattern of antibody responses that is consistent with canonical antibody maturation (e.g., IgM peaking early and then waning, concurrent with a continual expansion of IgG isotypes). Bar heights represent mean raw area under the curve (AUC) values across all CC-F1s. (B) The correlation structure of antibody isotypes and subtype responses in the F1s over time was evaluated to determine the relationship between the development of various antibody types across the population. (C and D) Representative examples of variation in the magnitude and kinetics of IgG (C) and IgM (D), with some exceptionally notable outliers for IgM. Each point represents an individual mouse, and bars represent mean values for F1s. (D). This variation was independent of Mx1 haplotype (for both panel sets red = Mx1 −/−, blue = Mx1 +/−). See also Figures S1–S3.

Figure 2

Multiple Loci Drive Antibody Responses to IAV QTL mapping allowed us to identify 23 loci contributing to the antibody response composition, magnitude, and kinetics. We summarize these loci (Ari1–Ari23) in a chromosomal ideogram showing their positions, as well as the antibody type and the time point for which they were mapped on their corresponding genomic loci.

Figure 3

Representative Ari QTLs Associated with Variation in IAV-Specific Antibody Responses (A–C) show Ari1, D–F show Ari2, G–I show Ari3, and J–L show Ari4. (A, D, G, and J) Phenotypic distributions for the traits mapped to Ari1–Ari4, respectively. Each plot is independently ordered by the CC-F1 means for that trait (black points) and also shows the individual mice (purple points). The exception is (J), which only shows mean values, as the ratios of antibody response between time points were calculated using the mean value for each F1 at the relevant individual time points. (B, E, H, and K) show the associated QTL LOD plots (significance score across the genome) for Ari1–Ari4. Significance thresholds are shown in red (genome-wide p = 0.05), blue (p = 0.1), and green (p = 0.2). Following identification of QTLs, we determined the causal haplotypes driving these responses (C, F, I, and L). Each plot is zoomed in to the relevant QTL loci on the x axis. The lower black line shows the LOD score for that region, and the colored lines display the estimated effect of each of the 8 CC founder haplotypes (A/J = yellow, C57BL/6J = gray, 129S1/SvImJ = pink, NOD/ShiLtJ = dark blue, NZO/HlLtJ = light blue, CAST/EiJ = green, PWK/PhJ = red, and WSB/EiJ = purple). Causal haplotype groups are determined based on direction and distance from mean effect and the largest split distance between lines (e.g., in I, the WSB line is furthest away from all others).

Figure 4

Ari2 Shows Broad Effects on IAV-Specific Antibody Responses in the CC-F1 Population We assessed the impact that a WSB/EiJ haplotype (x axis; 1 = one WSB haplotype; 2 = two non-WSB haplotypes) have on other IAV antibody responses in this study (y axis: CC-F1 mean levels of transformed AUC levels for given isotypes/time points). Points represent mean values for CC-F1s, with ~3 mice per CC-F1. Annotations refer to transformations applied to datasets (^∗p < 0.1, ^∗∗p < 0.05, ^∗∗∗p < 0.01). See also Table S1.

Figure 5

Pipeline to Narrow Down Candidate Genes under Ari1–Ari4 Variants under each QTL were considered if they were above the association testing threshold and were contained in a protein-coding gene. Protein-coding genes were then evaluated based on whether they were expressed in the lung or immune tissue. Using a separate transcriptional dataset from 11 CC strains infected with H3N2 influenza, IAV-specific and haplotype-specific differential expression was evaluated. Genes were considered if they had non-synonymous coding variants or splice region variants or showed haplotype-specific expression. See also Figure S4 and Table S7.