Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency

Abstract

Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.

Figure 1. Use of CRISPR/Cas9 system to generate RAG2/IL2RG deficient pigs.

(a) Design of CRISPRs to target RAG2 and IL2RG. Sequences in blue indicate designed cRNA, letters in red reflect PAM sequences (NGG for coding and CCN for reverse strand), and arrows indicate the location of primers used to genotype embryos and piglets. (b) Schematic strategy. RNA form of sgRNA and Cas9 were injected into presumable zygotes at an optimized concentration. Then the injected embryos were transferred into surrogate sows. Piglets were derived by hysterectomy and maintained in Gn isolators. (c) Representative genotypes of RAG2/IL2RG deficient pigs. Bold letters indicate insertion or change of nucleotides, and ‘–’ indicates deletion of nucleotide. PAM sequences are highlighted in red.

Figure 2. SCID phenotype of RAG2/IL2RG deficient pigs at 34 days of age.

(a) Representative images showing thoracic thymus (black arrows) and cervical thymus (blue arrow) in WT pigs, while thoracic and cervical thymus were lacking in some RAG2/IL2RG pigs. (b) Representative images of the mesentery showing the lack of MLN in some RAG2/IL2RG pigs. Representative flow cytometry of MNC from ileum (c) and blood (d) showing a significant reduction of B cells (CD79⁺), T cells (CD3⁺), and NK cells (CD3⁻CD16⁺), as indicated by dot plots gated within lymphocytes. Cy7, cyanine 7; APC, allophycocyanin; PE, phycoerythrin. (e) Total number of MNC from 40 cm ileum and 70 ml blood were isolated and quantified for WT (n = 4) and RAG2/IL2RG deficient pigs (n = 6). (f) Lymphocytes in ileum and blood were quantified based on the total number of MNC and the proportion of cells within MNC (Supplementary Fig. 5). Data are presented as means ± s.e.m. with individual animal data points (e,f). Statistical significance was determined by Mann-Whitney test. NS, not significant, **P < 0.01.

Figure 3. Increased and prolonged fecal HuNoV shedding in RAG2/IL2RG deficient pigs.

(a) Daily virus shedding was monitored from PID1 to PID28 by rectal swab sampling of feces and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to quantify the HuNoV genomes. (b) Individual pigs’ cumulative virus shedding was presented as area under curve from (a). (c) Peak virus shedding titers from PID1 to PID3, PID4 to PID10, PID11 to PID17, and PID18 to PID28 in individual pigs. Sample sizes are shown in Table 1. Dashed line indicates limit of detection. Data are presented as mean ± s.e.m. Statistical significance was determined by two-way analysis of variance (ANOVA) (a) or Mann-Whitney test (b,c). NS, not significant, *P < 0.05, **P < 0.01.

Figure 4. HuNoV distribution in Gn pigs.

HuNoV genomes in stomach (Sto), duodenum (Duo), jejunum (Jej), ileum, and mesentery (Mes) from pigs euthanized on PID3 (a), PID10 (b), and PID28 (c) were measured by qRT-PCR. (d) Total HuNoV genomes in intestinal contents were measured by qRT-PCR. HuNoV genomes in plasma (e) and whole blood cells (f) were measured by qRT-PCR. (a–d) WT groups, PID3 n = 3, PID10 n = 3, PID n = 5; RAG2/IL2RG deficiency groups, PID3 n = 3, PID10 n = 5, PID n = 4. (e,f) Sample sizes are shown in Table 1. Dashed line indicates limit of detection. Data are presented as mean ± s.e.m. with individual animal data points. Statistical significance was determined by Student’s t-test (a) or Mann-Whitney test (b–f). *P < 0.05, **P < 0.01.