Akaluc bioluminescence offers superior sensitivity to track in vivo dynamics of SARS-CoV-2 infection

Abstract

Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. Because the gene size of RNA viruses is typically small, NanoLuc is the primary choice for accommodation within viral genome. However, NanoLuc/Furimazine and also the conventional firefly luciferase/D-luciferin are known to exhibit relatively low tissue permeability and thus less sensitivity for visualization of deep tissue including lungs. Here, we demonstrated in vivo sufficient visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using the pair of a codon-optimized Akaluc and AkaLumine. We engineered the codon-optimized Akaluc gene possessing the similar GC ratio of SARS-CoV-2. Using the SARS-CoV-2 recombinants carrying the codon-optimized Akaluc, we visualized in vivo infection of respiratory organs, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of antivirals by monitoring changes in Akaluc signals. Overall, we offer an effective technology for monitoring viral dynamics in live animals.

Keywords: Methodology in biological sciences; Virology.

Graphical abstract

Figure 1

The virological features of SARS-CoV-2 Akaluc in vitro (A) The gene structures of wild-type SARS-CoV-2 (B.1.1) and SARS-CoV-2 carrying either the wild type or codon-optimized Akaluc luciferase gene in place of ORFs 6–8. (B) Viral production was judged by observation of cytopathic effects upon transfection with the transfection of each CPER product for 3 respective genomes. Representative images are shown from 6 days post transfection. Scale bars: 100 μm. (C) Northern blot analysis of subgenomic RNAs (sgRNAs). RNA was extracted from VeroE6/TMPRSS2 cells infected with B.1.1 or B.1.1-Akaluc and subjected to northern blot analysis. Black arrows indicate the bands for each sgRNA with size in parentheses. (D) Growth kinetics of B.1.1 and B.1.1-Akaluc in vitro. VeroE6/TMPRSS2 cells were infected with either B.1.1 or B.1.1-Akaluc (MOI = 0.001). Infectious titers in the culture supernatants (left panel), the copy number of intracellular viral RNA (middle panel), and the luciferase activity (right panel) were determined at the indicated timepoints. The luminescence intensity was normalized with the signals acquired from uninfected VeroE6/TMPRSS2 cells. Asterisks indicate significant differences (∗, p < 0.05) with the results of the wild-type virus. The presented data are expressed as the average ±SEM. Assays were performed independently in duplicate.

Figure 2

The virological features of B.1.1-Akaluc in vivo (A) Syrian hamsters (n = 6 per group) were intranasally inoculated with saline, B.1.1, or B.1.1-Akaluc. Body weight was measured daily through 7 days post-infection (dpi). (B) In vivo bioluminescence imaging of hamsters inoculated with B.1.1-Akaluc was performed daily through 7 dpi and again at 10 dpi. A representative image from an infected hamster is shown. The image was acquired after intraperitoneal injection of AkaLumine-HCl (75 nmol/g). (C) Syrian hamsters (n = 6) were intranasally inoculated with B.1.1-Akaluc. Viral RNA copies in the oral swab (left panel) and the luminescence intensity of the nasal cavity of hamsters (middle panel) inoculated with B.1.1-Akaluc were measured every 24 h through 7 dpi and then again at 10 dpi. NA: Not applicable. ND: Not detected. The area of measurement of the luminescence intensity are shown in the representative image (right panel). (D) IHC of the viral N protein (stained brown) in the nasal cavity, olfactory bulb, trachea, and lungs of hamsters at 3 dpi with B.1.1-Akaluc. Representative figures are shown. Scale bars: 200 μm. HE, hematoxylin and eosin. (A and C) The presented data are expressed as the average ±SEM.

Figure 3

The virological features of B.1.1-NanoLuc in vivo (A and B) Syrian hamsters (n = 6) were inoculated with B.1.1 carrying the NanoLuc luciferase gene (B.1.1-NanoLuc). Bioluminescence imaging of hamsters was performed daily through 3 dpi and a representative image from an infected hamster is shown (A). Oral swabs were collected (B, left panel) and the luminescence intensity of the nasal cavity (B, right panel) measured daily through 3 dpi. The images were acquired after intraperitoneal injection of FFz (440 nmol/g). (C) The lung hilum was harvested at 3 dpi and for quantification of viral RNA. Two hamsters infected with B.1.1-Akaluc served as controls. (B and C) The presented data are expressed as the average ±SEM.

Figure 4

The virological features of SARS-CoV-2-Akaluc variants in vivo Syrian hamsters (n = 6 per group) were intranasally inoculated with B.1.351.1-Akaluc and BA.1-Akaluc. (A) Akaluc bioluminescence imaging of hamsters inoculated with the two viruses was performed daily through 3 dpi. Representative images are shown. The images were acquired after intraperitoneal injection of AkaLumine-HCl (75 nmol/g). (B) Viral RNA was quantified in oral swabs and the luminescence intensity of the nasal cavity of hamsters was measured daily through 3 dpi. (C) At 3 dpi, the lung hilum was harvested from hamsters and the viral RNA levels quantified. (D) IHC of the viral N protein (stained in brown) was assessed in the nasal cavity, trachea, and lungs at 3 dpi. Representative figures are shown. Scale bars: 100 μm. Percentage of N-positive cells in whole lung lobes (n = 3 per infection group) are shown (right panel). See Figure S3 showing the sections of all four lung lobes. (E) H&E staining of the areas in Figure 4D was shown. (B and C) The presented data are expressed as the average ±SEM.

Figure 5

Evaluation of the immunoprophylactic ability of SARS-CoV-2 monoclonal antibodies and mRNA vaccine (A) Schematic diagram of the experimental timeline for the evaluation of AZD7442 (Tixagevimab-Cilgavimab). One day before challenge, hamsters were immunized by AZD7442 (1 mg per dose) or isotype as control. Hamsters were inoculated with B.1.1-Akaluc (n = 6), or XBB.1.5-Akaluc (n = 6). One hamster in the XBB.1.5-Akaluc cohort unexpectedly died during the experiment. (B) Akaluc bioluminescence imaging was performed daily through 3 dpi. The images were acquired after intraperitoneal injection of AkaLumine-HCl (75 nmol/g). (C) The lung hilum was harvested at 3 dpi and subjected to viral RNA quantification. Four samples obtained from the B.1.1-Akaluc-infected hamsters administrated with AZD7442 were under detection limit of qPCR analysis. (D) Schematic diagram of experimental timeline for the evaluation of the mRNA vaccine mRNA-1273. BALB/c mice (n = 3) were vaccinated (4 μg per dose) twice at the indicated timepoints, and the other BALB/c mice (n = 3) were not vaccinated (were received PBS as a control). Fifty-six weeks after the first vaccination, mice were inoculated with MA10-Akaluc (carrying the S protein of BA.1). Neutralizing antibody titers are shown in Figure S2F. Bioluminescence imaging was performed at 3 dpi. (E) Representative images from infected mice (immunized and non-immunized) at 3 dpi. are shown. The images were acquired after intraperitoneal injection of AkaLumine-HCl (75 nmol/g). (F) The lung hilum was collected at 3 dpi and subjected to viral RNA quantification.