Experiment
The humoral immune response protects against pathogens through mechanisms such as antibody-mediated neutralization, opsonization, antibody-dependent cellular cytotoxicity, and initiation of the classical complement pathway. The quality and/or magnitude of the antibody response is an important correlate of protection against many pathogens, including viruses such as influenza A virus (IAV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Host genetic factors play a fundamental role in regulating humoral immunity to viral infection. To study the role of genetic factors in driving the magnitude, kinetics, and composition of the antibody response to IAV, the authors measured IAV hemagglutinin (HA)-specific antibody levels (henceforth referred to as IAV-specific antibody) in a large population of F1 crosses between Collaborative Cross (CC) strains (N = 116).
CC mice show significant heritable variation in the magnitude, kinetics, and composition of IAV-specific antibody response. The authors map 23 significant and suggestive 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 (SARS-CoV and chikungunya virus). 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.
Mice from 64 CC strains were purchased from the UNC Systems Genetics Core Facility (SGCF) between July 2012 and July 2016. Female mice were generated from 116 CC/Unc-F1 crosses between these 64 CC/Unc strains. Female mice of 8-12 weeks of age at time of experiment were used.
Mice were infected intranasally with IAV A/CA/04/09 (H1N1) and sacrificed at multiple time points post-infection (3 mice per CC/Unc-F1 at each time point for a total of ~1,350 mice). IAV-specific antibody was measured from animals sacrificed at days 7, 10, 15, and 45 post-infection to capture both early and late humoral immune responses. IgM, IgG1, IgG2a+IgG2c (combined detection of subtypes that segregate across mouse strains (Zhang et al., 2012b)), IgG2b, IgG3, and total IgG were quantified from each animal (a total of ~8,000 antibody measurements across the 116 CC/Unc-F1s and 4 time points) to capture differential dynamics of individual antibody subtypes and isotypes across the infection time course.
IAV-specific antibody responses were significantly influenced by genetic factors across isotypes/subtypes and time points, with heritability estimates ranging from 26%72% (median 44%).
The authors performed genetic mapping on both total amount of antibody isotypes/subtypes at single time points (i.e., days 7, 10, 15, and 45 individually) in addition to the kinetics (slope) between adjacent time points (i.e., between days 7 and 10, 10 and 15, and 15 and 45) to identify genetic factors contributing to variation in the overall magnitude, composition, and kinetics of the antibody response.
Genetic mapping was performed using the DOQTL (1.18.0) (Gatti et al., 2014) package in the R statistical environment. Once loci were identified, haplotype groups for each QTL were manually determined based on visualization of haplotype effect plots. Additive haplotype scores for CC/Unc-F1s were computed by combining the haplotype scores for the dam and sire of the F1. All genome coordinates are relative to mm10 (Build 38.1).
Eight significant QTL were mapped for antibody response to influenza A:
Ari1 (antibody response to influenza 1, day 7, IgG2a+IgG2c) maps to Chr 17: 47 - 54.4 Mb with a peak p-val of 7.65E-02 at 52.6 Mb. NOD/ShiLtJ and WSB/EiJ alleles contribute lower antibody response at Ari1. Ari1 accounts for 4.5% of the total phenotypic variation (15.2% of the heritable variation). Unc5cl is the lead candidate gene for Ari1.
Ari2 (antibody response to influenza 2, day 10, IgG3) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 2.80E-02 at 71.7 Mb. WSB/EiJ alleles contribute lower antibody response at Ari2. Ari2 accounts for 9.8% of the total phenotypic variation (30.7% of the heritable variation). Nlrp1b, Wscd1, Rpain, Spns3, Mis12, and Nup88 are given as top-ranking candidate genes for Ari2.
Ari3 (antibody response to influenza 3, day 15, IgM) maps to Chr 8: 108.7 - 113.1 Mb with a peak p-val of 8.15E-02 at 109.4 Mb. WSB/EiJ alleles contribute higher antibody response at Ari3. Ari3 accounts for 5.8% of the total phenotypic variation (14.8% of the heritable variation). Bcar1, Hydin, Pkd1l3, Zfhx3, Kars, and Mlkl are given as top-ranking candidate genes for Ari3.
Ari4 (antibody response to influenza 4, day 15-45, IgG2b) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 3.95E-02 at 38.7 Mb. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ari4, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response. Cc2da2, Ldb2, Prom1, and Tapt1 are given as top-ranking candidate genes for Ari4.
Ari5 (antibody response to influenza 5, day 15, IgG1) maps to Chr 16: 40 - 44.7 Mb with a peak p-val of 8.00E-02 at 40.6 Mb. CAST/EiJ and PWK/PhJ alleles contribute higher antibody response at Ari5, while A/J alleles contribute lower antibody response.
Ari6 (antibody response to influenza 6, day 15, IgG2b) maps to Chr 7: 109.1 - 115.5 Mb with a peak p-val of 9.65E-02 at 114.3 Mb. NZO/HlLtJ alleles contribute higher antibody reponse at Ari6.
Ari7 (antibody response to influenza 7, day 45, IgG3) maps to Chr 9: 7.8 - 22.3 Mb with a peak p-val of 7.35E-02 at 13.5 Mb. 129S1/SvImJ, NZO/HlLtJ, and CAST/EiJ alleles contribute higher antibody reponse at Ari7, while A/J, C57BL/6J, NOD/ShiLtJ, PWK/PhJ alleles contribute lower antibody response.
Ari8 (antibody response to influenza 8, day 15-45, TotalG) maps to Chr 15: 51.6 - 55 Mb with a peak p-val of 9.45E-02 at 53.2 Mb. PWK/PhJ alleles contribute higher antibody reponse at Ari8, while CAST/EiJ alleles contribute lower antibody response.
Fifteen suggestive QTL were mapped for antibody response to influenza 1:
Ari9 (antibody response to influenza 9, day 7, IgG2a+IgG2c) maps to Chr 16: 3.1 - 26.7 Mb with a peak p-val of 1.97E-01 at 4.7 Mb.
Ari10 (antibody response to influenza 10, day 10, IgG1) maps to Chr 5: 52.2 - 53.5 Mb with a peak p-val of 1.34E-01 at 53.1 Mb.
Ari11 (antibody response to influenza 11, day 10, IgG2a+IgG2c) maps to Chr 5: 58.6 - 81.6 Mb with a peak p-val of 1.53E-01 at 73.7 Mb.
Ari12 (antibody response to influenza 12, day 10, IgG3) maps to Chr 12: 112.5 - 120 Mb with a peak p-val of 1.71E-01 at 117.7 Mb.
Ari13 (antibody response to influenza 13, day 15, Total IgG) maps to Chr 15: 28.9 - 36.6 Mb with a peak p-val of 2.10E-01 at 34.5 Mb.
Ari14 (antibody response to influenza 14, day 45, IgG1) maps to Chr 1: 160.1 - 165.4 Mb with a peak p-val of 1.85E-01 at 165 Mb.
Ari15 (antibody response to influenza 15, day 45, IgG2a+IgG2c) maps to Chr 5: 130.9 - 135.5 Mb with a peak p-val of 1.71E-01 at 132.6 Mb.
Ari16 (antibody response to influenza 16, day 45, IgG3) maps to Chr 5: 127.2 - 134.9 Mb with a peak p-val of 1.77E-01 at 133.6 Mb.
Ari17 (antibody response to influenza 17, day 45, Total IgG) maps to Chr 5: 130.9 - 148.7 Mb with a peak p-val of 1.09E-01 at 134.8 Mb.
Ari18 (antibody response to influenza 18, day 45, Total IgG) maps to Chr 14: 103.3 - 109.4 Mb with a peak p-val of 1.65E-01 at 105.6 Mb.
Ari19 (antibody response to influenza 19, day 10-15, IgG2b) maps to Chr 14: 114 - 118.6 Mb with a peak p-val of 1.66E-01 at 117.8 Mb.
Ari20 (antibody response to influenza 20, day 15-45, IgG1) maps to Chr 7: 41.7 - 56 Mb with a peak p-val of 1.55E-01 at 50.2 Mb.
Ari21 (antibody response to influenza 21, day 15-45, IgG2a+IgG2c) maps to Chr X: 45.2 - 48 Mb with a peak p-val of 1.04E-01 at 47.4 Mb.
Ari22 (antibody response to influenza 22, day 15-45, IgG2b) maps to Chr X: 139.1 - 141.2 Mb with a peak p-val of 1.42E-01 at 140 Mb.
Ari23 (antibody response to influenza 23, day 7-10, IgG2a+IgG2c) maps to Chr 2: 16 - 27 Mb with a peak p-val of 1.86E-01 at 24 Mb.
The authors interrogated the extent to which Ari1Ari4 haplotypes further correlated with other aspects of IAV-induced pathology or immune responses in the CC/Unc-F1 population. They reduced the number of states being analyzed from the 8 independent founder haplotypes used in the QTL mapping analysis to the 2 variant haplotype groups (e.g., high or low response) at the locus, thereby enhancing power to detect less strong phenotypic associations. None of the Ari loci showed correlations with gross IAV-induced disease as measured by weight loss, with control for Mx1 haplotype.
To further assess the role of Ari1Ari4 in virus-specific antibody responses, the authors tested to see if Ari1Ari4 haplotypes were associated with antibody responses to other viruses. For this analysis, they took advantage of existing datasets from related but independent studies with SARS-CoV, a respiratory virus, and chikungunya virus (CHIKV), an arbovirus. In the SARS-CoV study, mice from a similar panel of CC-F1s were infected with SARS-CoV, and antibody response to the spike (S) protein was measured at days 7, 15, and 29 post-infection. An additional measurement was taken 4 days following a secondary challenge ("rechallenge"), which was administered at day 28 post-primary infection. Ari1 haplotypes were not associated with SARS-CoVspecific antibody responses.
In contrast, Ari2 haplotype showed broad associations with antibody specific to SARS-CoV at days 7 and 29, and with IgM at all time points post-primary and secondary challenge.
Ari3 haplotype showed only a few correlations with SARS-CoV antibody, which were not indicative of trends either across isotypes or time points.
Ari4 showed the strongest correlations with the SARS-CoV-specific antibody response, most potently at day 7 but lasting through days 15 and 29.
In the CHIKV study, mice from a panel of 64 inbred CC strains were infected with CHIKV. Virus-specific IgM, IgG, and neutralizing antibody were measured at day 7 post-infection. While Ari1, Ari2, and Ari4 did not correlate with the CHIKV-specific antibody response (IgM, IgG, and neutralization titer) in CC-RI mice at day 7 post-infection, Ari3 to control viral replication. Notably, two highlighted CC-F1s with persistent IgM responses shared the parental strain CC032, suggesting that genetic contributions from CC032 (independent of Ari3, for which CC032 has the low-response haplotype) may be involved in longer lasting IgM responses. Such outlier strains can be used to model these aberrant responses and/or included in future targeted mapping studies (e.g., F2 crosses).
Ari1 showed broad effects on the early antibody response to IAV only and was not associated with antibody response to SARS-CoV or CHIKV, suggesting that it may be involved with specific responses to IAV such as innate immune recognition and response or antigen presentation. Ari2 showed broad early effects with IAV as well as SARS-CoV-specific responses, suggesting that the underlying gene is involved in early stages of infection or immune response for respiratory pathogens. Ari3 also showed broad early effects with IAV and a strong correlation with antibody response to CHIKV, though the haplotype effects were in opposing directions for these two different viruses. Ari4 was most relevant for day 15 antibody response to IAV and also showed strong associations with antibody response to SARS-CoV, suggesting that it may be widely relevant for the development of the humoral response. These broad associations highlight the utility of the CC as a resource to extend analyses into related datasets to gain further understanding of the role of loci across phenotypes while illustrating the need for future work to define the specific mechanisms by which each of these loci affect antiviral antibody response.
Ars1-11 overlap with Ari2 and are associated with variation in SARS-CoV-specific antibody response:
Ars1 (antibody response to SARS-CoV 1, day 7, IgG1) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 7.33E-03. WSB/EiJ alleles contribute lower antibody response at Ars1.
Ars2 (antibody response to SARS-CoV 2, day 7, IgG2a+IgG2c) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 1.40E-02. WSB/EiJ alleles contribute lower antibody response at Ars2.
Ars3 (antibody response to SARS-CoV 3, day 7, IgG2b) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 1.14E-02. WSB/EiJ alleles contribute lower antibody response at Ars3.
Ars4 (antibody response to SARS-CoV 4, day 7, IgG3) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 4.69E-04. WSB/EiJ alleles contribute lower antibody response at Ars4.
Ars5 (antibody response to SARS-CoV 5, day 7, IgM) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 2.29E-02. WSB/EiJ alleles contribute lower antibody response at Ars5.
Ars6 (antibody response to SARS-CoV 6, day 15, IgM) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 4.80E-02. WSB/EiJ alleles contribute lower antibody response at Ars6.
Ars7 (antibody response to SARS-CoV 7, day 29, IgG1) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 4.73E-02. WSB/EiJ alleles contribute lower antibody response at Ars7.
Ars8 (antibody response to SARS-CoV 8, day 29, IgG2a+IgG2c) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 1.76E-02. WSB/EiJ alleles contribute lower antibody response at Ars8.
Ars9 (antibody response to SARS-CoV 9, day 29, IgM) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 9.40E-03. WSB/EiJ alleles contribute lower antibody response at Ars9.
Ars10 (antibody response to SARS-CoV 10, 4 days post-rechallenge, IgG2a+IgG2c) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 1.17E-02. WSB/EiJ alleles contribute lower antibody response at Ars10.
Ars11 (antibody response to SARS-CoV 11, 4 days post-rechallenge, IgM) maps to Chr 11: 69.1 - 72.6 Mb with a peak p-val of 8.45E-03. WSB/EiJ alleles contribute lower antibody response at Ars11.
Ars12 overlaps with Ari3 and is associated with variation in SARS-CoV-specific antibody response:
Ars12 (antibody response to SARS-CoV 12, 4 days post-rechallenge, IgM) maps to Chr 8: 108.7 - 113.1 Mb with a peak p-val of 2.91E-02. WSB/EiJ alleles contribute higher antibody response at Ars12.
Arc1-3 overlap with Ari3 and are associated with variation in CHIKV-specific antibody response:
Arc1 (antibody response to CHIKV 1, day 7, IgG) maps to Chr 8: 108.7 - 113.1 Mb with a peak p-val of 3.13E-03. WSB/EiJ alleles contribute higher antibody response at Arc1.
Arc2 (antibody response to CHIKV 2, day 7, IgM) maps to Chr 8: 108.7 - 113.1 Mb with a peak p-val of 1.52E-02. WSB/EiJ alleles contribute higher antibody response at Arc2.
Arc3 (antibody response to CHIKV 3, day 7, FRNT50) maps to Chr 8: 108.7 - 113.1 Mb with a peak p-val of 1.17E-03. WSB/EiJ alleles contribute higher antibody response at Arc3.
Ars13-24 overlap with Ari4 and are associated with variation in SARS-CoV-specific antibody response:
Ars13 (antibody response to SARS-CoV 13, day 7, IgG2a+IgG2c) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 9.46E-06. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars13, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars14 (antibody response to SARS-CoV 14, day 7, IgG3) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 1.69E-03. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars14, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars15 (antibody response to SARS-CoV 15, day 7, IgM) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 1.37E-04. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars15, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars16 (antibody response to SARS-CoV 16, day 7, Total IgG) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 3.77E-04. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars16, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars17 (antibody response to SARS-CoV 17, day 15, IgG2a+IgG2c) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 3.76E-03. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars17, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars18 (antibody response to SARS-CoV 18, day 15, IgG3) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 2.95E-03. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars18, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars19 (antibody response to SARS-CoV 19, day 15, IgM) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 2.56E-02. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars19, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars20 (antibody response to SARS-CoV 20, day 15, Total IgG) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 7.62E-03. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars20, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars21 (antibody response to SARS-CoV 21, day 29, IgG2a+IgG2c) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 1.85E-02. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars21, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars22 (antibody response to SARS-CoV 22, day 29, IgG3) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 4.47E-02. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars22, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars23 (antibody response to SARS-CoV 23, day 29, Total IgG) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 3.52E-02. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars23, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
Ars24 (antibody response to SARS-CoV 24, 4 days post-rechallenge, IgG3) maps to Chr 5: 36.8 - 45.3 Mb with a peak p-val of 2.05E-02. C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ alleles contribute higher antibody response at Ars24, while A/J, 129S1/SvImJ, CAST/EiJ, and WSB/EiJ alleles contribute lower antibody response.
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