(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



Mouse-adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge

['Antonio Muruato', 'Departments Of Biochemistry', 'Molecular Biology', 'University Of Texas Medical Branch', 'Galveston', 'Texas', 'United States Of America', 'Departments Of Microbiology', 'Immunology', 'Michelle N. Vu']

Date: 2021-11

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2–infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease.

Funding: Research was supported by grants from NIAID of the NIH to (AI153602 and 1R21AI145400 to VDM to P-YS; R24AI120942 (WRCEVA) to SCW, P51OD011132, R56 AI147623 and U19AI090023 to MSS. AEM is supported by a Clinical and Translational Science Award NRSA (TL1) Training Core (TL1TR001440) from NIH. ALR was supported by an Institute of Human Infection and Immunity at UTMB COVID-19 Research Fund. Research was also supported by STARs Award provided by the University of Texas System to VDM, and trainee funding provided by the McLaughlin Fellowship Fund at UTMB. P-YS was also supported by CDC grant for the Western Gulf Center of Excellence for Vector-Borne Diseases, and awards from the Sealy & Smith Foundation, Kleberg Foundation, John S. Dunn Foundation, Amon G. Carter Foundation, Gilson Longenbaugh Foundation, and Summerfield Robert Foundation. MSS was also supported by the Emory Executive Vice President for Health Affairs Synergy Fund award, the Pediatric Research Alliance Center for Childhood Infections and Vaccines and Children’s Healthcare of Atlanta, COVID-Catalyst-I3 Funds from the Woodruff Health Sciences Center and Emory School of Medicine, Woodruff Health Sciences Center 2020 COVID-19 CURE Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

In order to alleviate these issues, we set out to develop a mouse-adapted strain of SARS-CoV-2 using standard laboratory strains. Building from our infectious clone system [ 12 ], we incorporated amino acid changes that facilitated replication in standard BALB/c mice and serially passaged the mutant to create a mouse-adapted strain (CMA3p20) that causes significant weight loss, disease, and lung damage following infection. Notably, virus replication in this model is limited to the respiratory system, thus recapitulating disease observed in most humans. Importantly, the SARS-CoV-2 CMA3p20 strain did not attenuate replication in primary human airway cultures or change the antigenicity of the mouse-adapted strain relative to WT control virus, making it suitable for vaccine and therapeutic studies. Finally, following prior infection with SARS-CoV-2 CMA3p20, mice were protected from lethal challenge with SARS-CoV despite the absence of sterilizing immunity. Adoptive serum transfer experiments indicated that protection was not mediated by antibody alone. Together, the results highlight the use of SARS-CoV-2 CMA3p20 to study infection and pathogenesis in standard mouse lines.

In responding to the outbreak, understanding the complexity of SARS-CoV-2 infection has been hampered by the limitations of small animal models [ 6 ]. Early on, wild-type (WT) SARS-CoV-2 was shown to be unable to use mouse angiotensin converting enzyme 2 (ACE2) for entry and infection [ 7 ]. Alternative models use receptor transgenic mice expressing human ACE2 or Syrian golden hamsters to evaluate SARS-CoV-2 infection and disease in vivo [ 6 ]. However, the transgenic models, while causing severe disease and lethality, have distinct infection tropism, leading to encephalitis in addition to lung disease [ 8 – 10 ]. Similarly, while the hamster model has provided use in studying disease and transmission [ 11 ], the absence of genetic knockout and immunological tools limits the types of studies that can be pursued. Without a robust mouse model, many of the resources used to study infection and the immune response are unavailable for SARS-CoV-2 experiments.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes Coronavirus Disease 2019 (COVID-19), emerged in late 2019 and has since caused an ongoing pandemic with over 153 million cases and over 3.2 million deaths in the last 17 months [ 1 , 2 ]. The novel coronavirus, similar to previous emergent Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), can produce severe respiratory disease characterized by fever, labored breathing, and pulmonary infiltration and inflammation [ 3 , 4 ]. In severe cases, SARS-CoV-2 can lead to acute respiratory distress and death. Unlike the earlier pandemic Coronaviruses (CoVs), SARS-CoV-2 maintains the ability to efficiently spread asymptomatically and causes a range of disease from mild to severe [ 5 ]. These factors have led to a pandemic that continues to rage over a year after its emergence.

Results

The initial, emergent strains of SARS-CoV-2 had spike proteins unable to utilize mouse ACE2 and infect standard laboratory mice [7]. To overcome this barrier, we generated a series of mutations in the receptor-binding domain (RBD) of SARS-CoV-2 using our infectious clone [12]. Our initial efforts modeled the interaction between SARS-CoV-2 and mouse ACE2 and used previous mouse-adapted strains of SARS-CoV (MA15, MA20, and v2163) [13] to design mutants including changes at Y449H (MA1), Y449H/L455F (MA2), and F486L/Q498Y (MA4) (S1A–S1C Fig). We also generated a series of mutants based on a reported natural SARS-CoV-2 isolate (MASCP6) capable of infecting mice [14], which has spike change at N501Y and several additional mutations (S2A Fig). Given the capacity of the MASCP6 strain to replicate in mice, we generated mutants that had the spike mutation alone (CMA1), the spike/N protein mutation (CMA2), and all 4 changes (CMA3) (S2A Fig). For each of the 6 mutants, we used site-directed mutagenesis in the WA1 strain clone and rescued virus stocks on Vero E6 cells (S2B Fig). We subsequently infected 10-week-old female BALB/c mice with 105 plaque-forming units (PFUs) of each mutant virus and evaluated replication in the lung 2 days postinfection. For WT, MA1, and MA2, no evidence of viable infection was detected in mouse lung tissues (S2C Fig); however, MA4 and CMA1-3 had robust replication in mouse lung, suggesting that multiple combinations of RBD changes could provide compatibly with mouse ACE2 sufficient for replication in a standard laboratory mouse strain.

To further evaluate the mouse-adapted strains, we focused on SARS-CoV-2 CMA1, CMA2, and CMA3 mutants over a 4-day time course. In female 10-week-old BALB/c mice infected with 105 PFU, none of 3 mutants induced major disease (S3A Fig), although both CMA2 and CMA3 caused more weight loss than CMA1. Examining viral replication in the lung, all 3 mutants produced approximately 105 PFU per lobe at day 2 postinfection (S3B Fig). However, no virus was detected at day 4, suggesting rapid clearance by the host. To determine if type I interferon was the major factor blunting infection, IFNAR−/− SJV129 mice were infected with CMA1, CMA2, and CMA3 at 105 PFU. Following infection, all 3 CMA mutant strains caused significant disease, with both CMA2 and CMA3 peaking at approximately 10% weight loss (S3C Fig). However, despite increased disease, viral titers were only slightly higher at day 2 than immune competent BALB/c mice and still cleared by day 4 for all 3 strains (S3D Fig). Together, the results indicate that SARS-CoV-2 CMA1, CMA2, and CMA3 can replicate in both BALB/c and IFNAR−/− mice, but fail to sustain continued replication in vivo.

Serial passage of SARS-CoV-2 CMA3 In order to generate a SARS-CoV-2 strain that produced significant disease in an immune competent mouse, we serially passaged SARS-CoV-2 CMA3 in 10-week-old BALB/c mice. A single mouse was infected with 105 PFU of CMA3 (p0); the mouse was subsequently euthanized at 1 day postinfection with half the lung lobes taken for viral RNA and the other lobes homogenized, clarified, and used to inoculate subsequent passages (Fig 1A); lung samples were titered by plaque assay to verify continued SARS-CoV-2 replication (Fig 1B). After passages (p) 10, p15, and p20, stock viruses were generated on Vero E6 cells, used to infect 10-week-old BALB/c mice, and compared to the disease caused by the original CMA3 p0 strain (Fig 1C). Following 105 PFU challenge, mice infected with p10 and p15 were found to have augmented weight loss compared to p0; however, mice infected with p20 showed 10% weight loss by day 3 and signs of disease including ruffled fur and hunched posture. We subsequently deep sequenced the passaged virus from the lung RNA and identified 2 additional spike mutations (K417N and H655Y) and a mutation in the E protein (E8V). Several other mutations were also found as minority variants in the spike and in other parts of the genome (Fig 1D). Modeling the RBD interaction (Fig 1E), K417N and N501Y likely improve binding to mouse ACE2 and facilitate increased in vivo disease. Similarly, H655Y may play a role in changes to proteolytic processing of the spike protein following receptor binding [15]. Together, mouse adaptation of SARS-CoV-2 CMA3 incorporated 3 additional fixed mutations that drive increased disease in mice. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Mouse adaptation of SARS-CoV-2. (a) Schematic of adaptation of SARS-CoV-2 CMA3p20. One 10-week-old female BALB/c mice was infected with SARS-CoV-2 CMA3 for 1 day, euthanized, and lung tissues harvested for viral RNA and viral titer determination. Lung tissues were homogenized, clarified, and 50 ul used to inoculate subsequent animals for 20 p. The figure was generated using BioRender software. (b) Viral replication of CMA3 p1-p20 from lung homogenates isolated from infected mice 1 day postinfection (n = 1). (c) Stock virus generated at p0, p10, p15, and p20 was used to infect female BALB/c mice at 105 PFU and evaluated for weight loss over a 4-day time course (n = 5). (d) Schematic of engineered (red stars) and passage-acquired (blue stars) mutations in CMA3p20 stock virus. Table includes Sanger equivalent accumulation of mutations over p5, p10, p15, p20, and final stock used for subsequent studies. (e) Modeling RBD spike mutations N501Y and K417N found in CMA3p20 with mouse ACE2. Model constructed using Pymol 2.4.2. Data presented as mean values +/− SEM in (c). Raw data are available in S1 Data. ACE2, angiotensin converting enzyme 2; p, passage; PFU, plaque-forming unit; RBD, receptor-binding domain; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi.org/10.1371/journal.pbio.3001284.g001

Characterization of CMA3p20 Having observed significant disease in mice infected with CMA3p20 relative to the initial strain of CMA3, we next evaluated weight loss, viral replication, and histopathology in BALB/c mice. First, we tested CMA3p20 for a dose-dependent impact on weight loss (S4A Fig); both 106 and 105 PFU caused significant, dose-dependent weight loss with minimal disease observed in the 104 challenge. We also compared CMA3p20 infection associated weight loss to a B.1.1.7 SARS-CoV-2 variant (United Kingdom) that contains the N501Y mutation that permits virus replication in mice (S3A–S3D Fig). After challenge with the 106 PFU of the B.1.1.7 variant, female BALB/c mice lost approximately 10% of their starting weight by day 2 and recovered (S4B Fig). Together, the results indicate more severe disease with the mouse-adapted CMA3p20 than the B.1.1.7 variant. We subsequently used the 105 PFU dose of CMA3p20 to examine infection compared to SARS-CoV-2 CMA3 over a 7-day time course. Following infection, 10-week-old female BALB/c mice CMA3p20-infected mice lost significant weight over the first 4 days, peaking at day 3 with >10% weight loss (Fig 2A). By contrast, the original CMA3 caused minimal weight loss over the course of the 7-day infection. We next examined viral replication in the lung at days 2, 4, and 7 postinfection (Fig 2B). CMA3p20 infection had a significant 0.5 log increase in viral load over CMA3 in the lung at day 2; this difference was diminished at day 4 (0.25 log increase, not statically significant), and both virus strains were cleared by day 7 in the lung. We also observed day 2 replication in the trachea of mice that was cleared by day 4 in both CMA3 and CMA3p20 infection (Fig 2C). Together, the data demonstrate robust weight loss and clear replication in the mouse respiratory tract. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. SARS-CoV-2 CMA3p20 induces disease restricted to the lung. (a–f) Ten-week-old BALB/c mice were infected with 105 PFU of SARS-CoV-2 CMA3 (black triangle, n = 20) or CMA3p20 (blue circles, n = 12) and followed for (a) weight loss and viral titer in the (b) lung, (c) trachea, (d) heart, (e) brain, and (f) blood (n = 4). (g–k) Histology from CMA3p20-infected mice showed viral antigen (N-protein) staining in the (g) airways and (h) parenchyma at day 2 with high magnification in the insets. Significant lung infiltration, inflammation, and damage was observed at (i and j) day 2 and (k) day 4 postinfection. Images representative of most significant damage observed in lung sections observed in CMA3p20. Data presented as mean values +/− SEM in (a). P values based on a 2-tailed Student t test. Magnification at 10× for (g and i). Raw data are available in S2 Data. PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi.org/10.1371/journal.pbio.3001284.g002 We next evaluated SARS-CoV-2 replication in nonrespiratory tissues. Following infection, we noted replication in the heart tissue of a subset of animals at day 2 (Fig 2D). However, infection was transient and not uniform in all animals, and no virus was detected in the heart at later time points. We subsequently evaluated viral load in the brain and blood and found no evidence for CMA3 or CMA3p20 infection by plaque assay (Fig 2E and 2F). To further verify viral replication, we also examined viral RNA expression in the lung, heart, brain, spleen, and liver (S4C Fig). While robust viral RNA was observed in the lung, the other tissues had minimal evidence for CMA3p20 replication. Together, the data indicate that the SARS-CoV-2 CMA3p20 strain is primarily restricted to and disease driven by virus replication in the respiratory tract.

CMA3p20 induces significant immune infiltration and lung damage Further examining lung tissue, histopathology analysis of CMA3p20 infection indicated robust virus replication, immune infiltration, and tissue damage. Using antigen staining against the N protein, we saw evidence for viral replication primarily in the bronchioles with additional staining in the lung parenchyma at day 2 postinfection (Fig 2G and 2H). We also observed lung infiltration and inflammation following CMA3p20 challenge characterized by peribronchioloitis, perivascular cuffing, and perivasculitis by day 2 postinfection (Fig 2I and 2J, S5A and S5B Fig). Similarly, at day 4, we noted collapsed airways and interstitial pneumonia (Fig 2K). Some portions of the day 4 lungs infected with CMA3p20 also had virus induced damage including enlarged and multinucleated alveolar type II cells (S5C Fig) and loss of cellular polarity (S5D Fig). Together, the histopathology results demonstrated significant damage, inflammation, and disease in the lung following infection with SARS-CoV-2 CMA3p20.

CMA3p20 retains replication capacity in primary human respiratory cells Altering SARS-CoV-2 to be permissive in mice can impact its replication capacity in human cells [16]. Therefore, we examined the ability of CMA3p20 to replicate in primary human airway epithelial (HAE) cultures compared with the WT SARS-CoV-2 WA1 strain. Grown on an air–liquid interface, primary HAEs represent a useful in vitro model of the human airway [17]. Following infection, CMA3p20 had equal replication to WT SARS-CoV-2 over a 72-hour time course in primary HAE cultures (Fig 3A). Similarly, viral RNA levels at 72 hours postinfection were equivalent between CMA3p20 and SARS-CoV-2 WA1 strain (Fig 3B). Together, the results indicate that mouse adaption resulted in no significant replication attenuation of CMA3p20 in primary human airway cells. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. CMA3p20 strain maintains human replication capacity and antigenicity. (a and b) Primary human airway cultures were infected with SARS-CoV-2 WT (black square) or CMA3p20 (blue circles) at an MOI of 0.01 and evaluated for (a) viral titer and (b) viral RNA (n = 3). (c) Sera collected from female BALB/c mice 28 days postinfection with 106 PFU of SARS-CoV-2 CMA3p20 were evaluated for capacity to neutralize WT SARS-CoV-2 (WA1), B.1.1.7, and B.1.351 via PRNT 50 assay (n = 12). Log10 value of dilution used to plot points on the x-axis. (d) PRNT 50 values from COVID-19 patient sera plotted against WT virus (y-axis) versus CMA3p20 virus (x-axis). (e and f) Ten-week-old female BALB/c mice were treated intraperitoneally with 100 ul of human COVID-19 sera (n = 10) or control (PBS, n = 10) 1 day prior to infection. Mice were subsequently challenged with 105 PFU of SARS-CoV-2 CMA3p20 and evaluated for (e) weight loss and (f) viral titer in the lung (n = 5). Data presented as mean values +/− SD in (a–c) and +/− SEM in (e). P values based on a 2-tailed Student t test. Raw data are available in S3 Data. COVID-19, Coronavirus Disease 2019; MOI, multiplicity of infection; PFU, plaque-forming unit; PRNT, plaque reduction neuralization titer; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001284.g003

CMA3p20 retains antigenicity similar to WT SARS-CoV-2 In addition to differences in replication in human cells, spike changes in SARS-CoV-2 could alter the overall antigenicity of CMA3p20 as compared to SARS-CoV-2 derived from humans; this result would make it more difficult to interpret vaccine and protection studies derived from mice. Therefore, to evaluate antigenicity, we infected 10-week-old female BALB/c mice with 106 PFU of CMA3p20 and then euthanized and harvested sera 28 days postinfection. We subsequently used the mouse sera to measure plaque reduction neuralization titer (PRNT 50 ) against the WT SARS-CoV-2 WA1 strain as well as 2 variants of concern (B.1.1.7 and B.1.351) (Fig 3C). Mouse sera from mice (n = 12) infected with CMA3p20 neutralized WT SARS-CoV-2 (WA1) and the other SARS-CoV-2 variants of concern with a PRNT 50 value ranging from 776 for WT, 1312 for B.1.1.7, and 1091 for B.1.351. To further evaluate CMA3p20 antigenicity, we examined PRNT 50 assays using sera from acutely infected, hospitalized COVID-19 patients (Fig 3D). Performing neutralization assays in parallel, we found that CMA3p20 had PRNT 50 values similar to WT SARS-CoV-2 with each COVID-19 patient serum tested. With a R2 value of 0.8651 over the 13 samples, the results indicated that CMA3p20 retains similar antigenicity to the WT SARS-CoV-2 and has potential use for vaccine and protection studies. To further demonstrate the use of CMA3p20 to understand in vivo protection, we performed a passive transfer experiment with a COVID-19 patient serum. One day prior to infection, 10-week-old female BALB/c mice were pretreated intraperitoneally with either control (PBS) or 100 ul of serum from a COVID-19 patient with a neutralization titer of 1,230. Mice were subsequently challenged with 105 PFU of CMA3p20 and monitored for weight loss and viral titer. Mice treated with acutely infected COVID-19 patient serum had significantly reduced weight loss at day 3 and 4 postinfection as compared to control mice (Fig 3E). Similarly, viral titers in the lung were reduced at both day 2 and day 4 in mice receiving COVID-19 patient serum as compared to control. Consistent with the PRNT 50 titer (Fig 3D), the results from the passive transfer experiment demonstrate that antibody-based immunity generated following human infection can effectively neutralize SARS-CoV-2 CMA3p20. Together, the results confirm similar antigenicity of CMA3p20 and WT SARS-CoV-2.

[END]

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001284

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.