A Human DPP4-Knockin Mouse's Susceptibility to Infection by Authentic and Pseudotyped MERS-CoV

Abstract

Infection by the Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory illness and has a high mortality rate (~35%). The requirement for the virus to be manipulated in a biosafety level three (BSL-3) facility has impeded development of urgently-needed antiviral agents. Here, we established anovel mouse model by inserting human dipeptidyl peptidase 4 (hDPP4) into the Rosa26 locus using CRISPR/Cas9, resulting in global expression of the transgene in a genetically stable mouse line. The mice were highly susceptible to infection by MERS-CoV clinical strain hCoV-EMC, which induced severe diffuse pulmonary disease in the animals, and could also be infected by an optimized pseudotyped MERS-CoV. Administration of the neutralizing monoclonal antibodies, H111-1 and m336, as well as a fusion inhibitor peptide, HR2P-M2, protected mice from challenge with authentic and pseudotyped MERS-CoV. These results confirmed that the hDPP4-knockin mouse is a novel model for studies of MERS-CoV pathogenesis and anti-MERS-CoV antiviral agents in BSL-3 and BSL-2facilities, respectively.

Keywords: MERS-CoV; authentic virus; hDPP4; mouse model; pseudotyped virus.

Figure 1

Establishment of a R26-hDPP4-knockin mouse model. (A) Schematic strategy for generation of R26-hDPP4-knockin mice via CRISPR/Cas9. (B) For the R26 probe, genomic DNA was digested with SpeI, and the expected sizes of wild-type and gene-targeted bands were 8.9 kb and 14.2 kb, respectively. For the WPRE probe, genomic DNA was digested with BglII, and the expected size of the gene-targeted band was 5.1 kb. Four representative results are shown. (C) PCR genotyping of R26-hDPP4 mice. The primer pair was designed to anneal in the coding region of hDPP4, and the expected PCR amplicon was 628 bp in length. (D) Quantitative reverse transcription PCR (RT-qPCR) of hDPP4 mRNA in R26-hDPP4 mice and wild-type C57BL/6 mice. Values are presented as means ± SEMs of three independent experiments and were normalized to GAPDH levels. No expression of R26-hDPP4 in wild-type mice was detected. (E) Detection of DPP4 protein by western blotting in brain and lung. Weaker blotting signal was detected in wild-type mice, indicating the anti-hDPP4 antibody could recognize mDPP4. (F) Bioluminescence imaging (BLI) of newborn R26-hDPP4-knockin mice showing hDPP4 expression in their organs. The imaged organs were: (1) thymus, (2) liver, (3) stomach, (4) kidney, (5) intestine, (6) spleen, (7) heart, (8) lung, (9) and (10) the whole bodies of wild-type mice and R26-hDPP4 mice, respectively.

Figure 2

R26-hDPP4-knockin mice were susceptible to infection by authentic MERS-CoV at low dose. (A) Weight loss in R26-hDPP4 mice challenged with hCoV-EMC at a dose of 1.5 × 10⁵ PFUs. (B) Viral titers in lungs of challenged R26-hDPP4 mice on day 5 p.i. LOD (limitation of detection): 0.85 PFU. (C) Quantitative analysis of pathological lesions in lungs. W. A. S. = widened alveolar septa; I. S. F. = inflammatory cells, serous and fibrinous exudation; D. N. B. = degeneration and necrosis of bronchial epithelial cells; P. I. I. = perivascular inflammatory cell infiltration; V. H. = vasodilator hyperemia. (D–O) Histopathological changes and viral loads in the lungs, brains, and kidneys of mice. R26-hDDP4 mice exhibited disease symptoms similar to those of MERS-CoV-infected human patients (D), while no or mild symptoms were observed in wild-type mice. (J). Perivascular gliosis in the cerebellum was observed in R26-hDPP4 mice (F) but not in wild-type mice (L). No pathological lesions were identified in the kidneys of either R26-hDPP4 or wild-type mice (I,O). IHC assays confirmed viral loads in lungs (E) and cerebella (G) of R26-hDPP4 mice; little or no virus was detected in the lungs (K) and cerebella (M) of wild-type mice. Four mice in each group were infected, and samples from all mice were subjected to tittering and histopathological analysis (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

MERS-CoV S-RBD-specific humanized neutralizing antibody H111-1 protected R26-hDPP4 mice from challenge with authentic MERS-CoV. Four-week-old mice were administered either PBS (control), 1 mg/kg mAb H111-1 or 5 mg/kg mAb H111-1 via the I.P. route, and 6 h later they were challenged I.N. with hCoV-EMC (1.5 × 10⁵ PFUs). On day 5 p.i., mice were sacrificed for virus titering and pathological analysis. (A) Treatment with mAb H111-1 significantly decreased viral titers in lungs. The dashed line indicates the LOD. (B) Efficacy of mAbH111-1 in abating pathological lesions caused by infection with authentic MERS-CoV. Explanation of pathological changes is given in Figure 2. (C➊–C➇) Histopathological changes and viral loads in lungs and brains of R26-hDPP4 mice administered 1 mg/kg mAb H111-1 (C➊,C➌,C➎,C➆) or 5 mg/kg mAb H111-1 (C➋,C➍,C➏,C➇), * p < 0.05.

Figure 4

Establishment of the R26-hDDP4 knockin model of infection with MERS-CoV pseudovirus. (A–C) Four-week-old R26-hDPP4 mice were inoculated with 1.27 × 10^7.5 TCID₅₀ (I.P. route) or 3.8 × 10^6.5 TCID₅₀ (I.T. route) of pHIV/MERSS/Fluc per animal. (B) Bioluminescent images (BLI) are shown at different days p.i. Relative bioluminescence intensity was shown in pseudocolor, with red representing the strongest and blue representing the weakest photon fluxes. Data are shown as means ± deviation (C). Organs were examined for Fluc expression using BLI: 1 = thymus; 2 = heart; 3 = liver; 4 = spleen; 5 = kidney; 6 = lung; 7 = lymph node; 8 = muscle; 9 = skin; 10 = ovary or testis; 11 = brain; 12 = intestine. (D) The copy number of pHIV/MERSS/Flucmeasured by RT-qPCR (I.P. challenge route). (E,F) Susceptibility tests for mice at different ages. Four- to 9- week-old mice could be infected, but younger mice were more susceptible, * p < 0.05. (G) Histopathological examination of organs of pseudovirus-infected mice; no histopathological changes were observed. (H) Pseudotypedvirions mainly infected the bronchi as shown by IHC and BLI. Bright spots indicate bronchi separated from lung (n = 4–6/group).

Figure 5

Relationship between pseudovirus and authentic virus models. (A–D) Pseudovirus and authentic virus infection in the lungs of R26-hDPP4 mice showed a similar pattern, as shown by IHC using mAb R723 against the RBD of the MERS-CoV S protein. (C,D) Both pseudovirus and authentic virus infected ciliated columnar epithelium of bronchi. (E) Dose conversion of pseudovirus and authentic virus. The full black line represented the inhibition rate (◆) of humanized mAb H111-1 (1 mg/kg) in vivo using different pseudovirus doses. The blue dashed line represents inhibition rate of H111-1 against authentic virus. When the dose of authentic virus was 1.5 × 10⁵ PFUs, we calculated that the inhibition rate of H111-1 at a dose of 1 mg/kg would be 60% (see main text). From the inhibition rate curve of pseudovirus, the corresponding pseudovirus dose was 3.25 × 10⁷ TCID₅₀. That is, 1 TCID₅₀ of pseudovirus corresponded to 0.0046 PFU of authentic virus (1.5 × 10⁵/3.25 × 10⁷). n = 4–6 mice per group.

Figure 6

Inhibition of pseudotyped MERS-CoV infection in R26-hDPP4 mice by the novel mAb H111-1 and the well-characterized mAb m336. For evaluation of H111-1, mice were administered 1 mg/kg of mAb either I.P. (A) or I.T. (B) and 6 h later, challenged with pseudovirus I.T. at a dose of 3.8 × 10^6.5 TCID₅₀. On day 11 p.i., BLI of the whole body or specific organs was conducted (D). To evaluate the efficacy of m336 (C), mice were administered mAb and challenged using the same doses as for H111-1, and typical images (E) are shown. (F) Bar of photo flux; for details see Figure 2. N = 4 mice in each group, * means p < 0.05, ** means p < 0.01.

Figure 7

Inhibition of pseudotyped MERS-CoV infection in R26-hDPP4-knockin mice by peptide HR2P-M2. Mice were administered HR2P-M2 peptide or phosphate-buffered saline (PBS) I.T., respectively. Thirty min later, mice were infected I.T. with pseudotyped MERS-CoV (3.8 × 10^6.5 TCID₅₀). BLI images were taken on day 11 p.i., and pseudovirus signals were recorded for the whole body or specific organs. (A) Flux value of pseudovirus for assessment of the protective efficacy of HR2P-M2. (B) BLI images of whole mice or their organs. (C) Bar of photo flux; for details see Figure 2. Four mice were used for each group, and representative images are shown.