key: cord-0689306-hcop0ry7
authors: Abdelnabi, Rana; Foo, Caroline S.; Kaptein, Suzanne J. F.; Boudewijns, Robbert; Vangeel, Laura; De Jonghe, Steven; Jochamns, Dirk; Weynand, Birgit; Neyts, Johan
title: A SCID mouse model to evaluate the efficacy of antivirals against SARS-CoV-2 infection
date: 2022-05-16
journal: bioRxiv
DOI: 10.1101/2022.05.13.491916
sha: 41541bbd81f971d9787372a92f99ff1984ee3460
doc_id: 689306
cord_uid: hcop0ry7

Ancestral SARS-CoV-2 lacks the intrinsic ability to bind to the mouse ACE2 receptor and therefore establishment of SARS-CoV-2 mouse models has been limited to the use of mouse-adapted viruses or genetically modified mice. Interestingly, some of the variants of concern, such as the beta B.1.351 variant, show an improved binding to the mouse receptor and hence better replication in different Wild type (WT) mice species. Here, we desribe the establishment of SARS-CoV-2 beta B.1.351 variant infection model in male SCID mice as a tool to assess the antiviral efficacy of potential SARS-CoV-2 small molecule inhibitors. Intranasal infection of male SCID mice with 105 TCID50 of the beta B.1.351 variant resulted in high viral loads in the lungs and moderate signs of lung pathology on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir (200 mg/kg, BID) or Nirmatrelvir (300 mg/kg, BID) for 3 consecutive days significantly reduced the infectious virus titers in the lungs by 1.9 and 3.8 log10 TCID50/mg tissue, respectively and significantly improved lung pathology. Together, these data demonstrate the validity of this SCID mice/beta B.1.351 variant infection model as a convenient preclinical model for assessment of potential activity of antivirals against SARS-CoV-2. Importance Unlike the ancestral SARS-CoV-2 strain, the beta (B.1.351) VoC has been reported to replicate to some extent in WT mice (species C57BL/6 and BALB/c). We here demonstrate that infection of SCID mice with SARS-CoV-2 beta variant results in high viral loads in the lungs on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir or Nirmatrelvir for 3 consecutive days markedly reduced the infectious virus titers in the lungs and improved lung pathology. The advantages of using this mouse model over the standard hamster infection models to assess the in vivo efficacy of small molecule antiviral drugs are (i) the use of a clinical isolate without the need to use mouse-adapted strains or genetically modified animals (ii) lower amount of the test drug is needed and (ii) more convenient housing conditions compared to bigger rodents such as hamsters.

CoV-2) has resulted in a global pandemic with officially >517 million cases (as of May 10, 2022) and 48 around 15 million deaths as estimated by WHO (1) . Several SARS-CoV-2 variants of concern (VoC), that 49 result in immune escape and/or enhanced viral transmission have since then emerged (2, 3). Small 50 animal models are necessary to study the virus-induced pathogenesis as well as to serve as preclinical 51 tool to assess the efficacy of vaccine and therapeutics against the viral infection. Similar to SARS-CoV, 52 SARS-CoV-2 enters the host cells through attachment to the cellular angiotensin-converting enzyme 2 53 (ACE2) (4). Since SARS-CoV-2 binds effeciently to the hamster ACE2 (5), Syrian hamsters are considered 54 so far one of the best small animal models avaliable for SARS-CoV-2. On the other hand, the spike of 55 the ancestral SARS-CoV-2 lacks the intrinsic ability to efficiently bind to the murine ACE2 (5) and hence 56 this strain has a limited replication in WT mice. Consequently, alternative strategies have been 57 developed to allow the establishment of mouse models for SARS-CoV-2. One of these strategies is 58 adaptation of the virus in murine lung tissues to enhance the binding capacity to the murine ACE2 (6, 59 7). Other strategies focused on introduction of human ACE2 in wild-type mice either by transduction 60 adenovirus or adeno-associated virus that expresses human ACE2 (8) or using genetically modified 61 human ACE2 transgenic (9) or humanized mice (10). Unlike the ancestral strain, some of the evolved 62 SARS-CoV-2 VoCs proved to carry spike protein mutations, mainly the N501Y, that enable efficient 63 binding to the murine ACE2 and hence better replication in WT mice (11, 12) . Besides the N501Y 64 mutation, the spike of the beta B.1.135 variant carries the K417N mutation that was previously 65 reported in a virulent mouse-adapted SARS-CoV-2 variant (13). Several studies have shown the ability 66 of the beta variant to replicate to some extent in WT mice species such as C57BL/6 (11, 12) and BALB/c 67 (14, 15) . Here, we wanted to explore whether the beta SARS-CoV-2 variant replicates more efficiently 68 in severe combined immune deficient (SCID) mice than in the reported wild type mice and whether in 69 such case, SCID mice can be used to develop a suficienlty robust infection model to study the efficacy 70 of small molecules inhibitors of SARS-CoV-2 infection. 71 virus titers. As expected, the infectious virus titer in the lungs of infected SCID mice (median TCID50/mg 78 lung of 3.28x10 4 ) was significantly higher than that observed in the lungs of infected BALB/c mice 79 (median TCID50/mg lung of 3.26x10 3 , p=0.047) and C57BL/6 mice (median TCID50/mg lung of 2.05x10 3 , 80 p=0.0054), supplementary Figure S1 . 81 Next, we explored the kinetics of replication of the beta variant in SCID mice. To that end, 7-9 weeks 82 old male SCID mice were infected intranasally with with 10 5 TCID50 of the beta variant. At days 3 83 through 7 post-infection (pi), 10 animals were euthanized and lungs were collected to quantify the 84 infectious virus titers. The highest infectious virus titers were observed at day 3 pi (Fig. 1A) . From day 85 4 pi onwards, the infectious virus titers in the lungs were significantly lower than those observed at 86 day 3 pi (Fig. 1A) . A minor weight loss was observed on day 3 pi (average %body weight change of -87 0.8) after which animals started to gain weight normally (average %body weight change of 4 on day 4 88 pi) (Fig. 1B) . When a group of 5 infected mice were monitored up to 14 days pi, no weight loss or any 89 signs of morbidity were observed in this group (average %body weight change of 13 on day 14 pi). 90

Histological examination of the lungs from infected mice at day 3 pi revealed mild signs of peri-91 bronchial inflammation, significant peri-vascular inflammation and intra-alveolar hemorrhage (Fig. 1C ) 92 with median cumulative pathology score of (4.5). 93

In case the infectious virus detected at day 3 post infection represents actively replicating virus, it 94 should be possible to suppress replication by treating the animals with antiviral drugs. We therefore 95 assessed the potential antiviral efficacy of two clinically relevant SARS-CoV-2 inhibitors i.e. 96

Molnupiravir (EIDD-2801) and Nirmatrelvir (PF-332) against beta variant replication in SCID mice. 97 (p<0.0001) log10 TCID50/mg tissue, respectively compared to the vehicle-treated group (Fig. 2C) . No 105 infectious virus titers were detected in four (out of 14) and eight (out of 14) animals in the Molnupiravir 106 and Nirmatrelvir-treated groups, respectively (Fig. 2C) . A significant improvement of lung 107 histopathology scores was also observed in both the Molnupiravir (p=0.025) and Nirmatrelvir 108 (p=0.0007)-treated groups (Fig. 2D) . No significant weight loss or clinical signs of adverse effects were 109 observed in the compounds-treated groups (Fig. 2E) . 110 to escape vaccine-induced or naturally acquired immunity and to transmit faster than the ancestral 113 strains. Besides, some of these variants have acquired certain mutations in the spike protein that 114 allowed them to expand their host species (2, 12). The beta variant (B.1.351 or 501Y.V2) has been first 115 reported in South Africa in October 2020 (16). The beta variant has acquired three mutations in the 116 receptor binding domain (RBD) namely N501Y, K417N and E484K, in addition to other mutations in the 117 spike and non-structural proteins (2). Among these mutations, the N501Y mutation (also presents in 118 alpha variant) has been previously described in mouse-adapted viruses and proven to play an 119 important role in increasing the affinity to the mouse ACE2 receptor (6). The K417N mutation has also 120 previously been reported in a virulent mouse-adapted SARS-CoV-2 variant (13). In a pseudotype-based 121 entry assay, the pseudoviruses carrying the beta variant spike attached more efficiently to the mouse 122 ACE2 receptor than the alpha variants, suggesting that the K417N and E484K mutations in the RBD of 123 beta variant may further enhance the binding to the mouse recptor (11). Recently, a comparative 124 infection study in BALB/c mice revealed that the beta variant replicates more efficiently than the alpha 125 and delta variant (15). 126

We here wanted to assess the infectivity of the beta SARS-CoV-2 variant in an immunodeficient mouse 127 model i.e. SCID mice, with the aim to develop a robust SARS-CoV-2 mouse infection model for 128 preclinical evaluation of potential antivirals. So far, the hamster SARS-CoV-2 infection model has been 129 regarded as the best model to study the effect of antiviral agents, yet use of mice would facilitate such 130 studies. We selected SCID mice as these animals are severely deficient in functional B and T 131 lymphocytes and therefore they are believed to be more susceptible to viral infections than 132 immunocompetent mice. Indeed, in our pilot infection study, the infectious virus titers of the beta 133 variant in the lungs of infected SCID mice on d3 pi were significantly higher than that observed in the 134 lungs of the immunocompetent BALB/c (1 log10 higher) and C57BL/6 (1.2 log10 higher) mice that were 135 2801, Merck) is the orally bioavailable prodrug of the ribonucleoside analogue N4-hydroxycytidine 142 (NHC, EIDD-1931) , which was initially developed for influenza (18) and has now also been approved by 143 several countries/regions for the treatment of We have previously shown that Molnupiravir (EIDD-2801) and Nirmatrelvir (PF-332) significantly inhibit 145 the replication of the beta variant in Syrian hamsters (19, 20) . Therefore, we used these two antiviral should have equipotent activity against all variants this is not of concern for studies with such drugs. 168

However, for testing of therapeutic antibodies, infection models (in hamsters) with the different VoC 169 will still be needed. Likewise, for vaccine studies, fully immunocompetent animals are needed, hence 170 SCID mice are not useful for this purpose. Therefore, this SCID mice/beta variant infection model will 171 be mainly advantageous for the evaluation of small molecule inhibitors of SARS-CoV-2 replication. 172

Virus 174

The SARS-CoV-2 strain used in this study, the beta variant B.1.351 (hCoV-19/Belgium/rega-1920/2021; 175 EPI_ISL_896474, 2021-01-11), was recovered from a nasopharyngeal swab taken from a patient with 176 respiratory symptoms returning to Belgium in January 2021 (24). A passage two virus on Vero E6 cells 177 was used for the study described here. Live virus-related work was conducted in the high-containment 178 

Lung tissues were collected after sacrifice and were homogenized using bead disruption (Precellys) 202 in TRK lysis buffer (E.Z.N.A. ® Total RNA Kit, Omega Bio-tek) and centrifuged (10.000 rpm, 5 min) to 203 pellet the cell debris. RNA was extracted according to the manufacturer's instructions. RT-qPCR was 204 performed on a LightCycler96 platform (Roche) using the iTaq Universal Probes One-Step RT-205 qPCR kit (BioRad) with N2 primers and probes targeting the nucleocapsid (25). Standards of SARS-CoV-206 2 cDNA (IDT) were used to express viral genome copies per mg tissue. 207

Lung tissues were homogenized using bead disruption (Precellys) in minimal essential medium and 209 centrifuged (10,000 rpm, 5min, 4°C) to pellet the cell debris. To quantify infectious SARS-CoV-2 210 blindly for lung damage by an expert pathologist. The scored parameters, to which a cumulative score 217 of 1 to 3 was attributed, were the following: congestion, intra-alveolar hemorrhagic, apoptotic bodies 218 in bronchus wall, necrotizing bronchiolitis, perivascular edema, bronchopneumonia, perivascular 219 inflammation, peribronchial inflammation and vasculitis. 220 Bao L, Deng W, Huang B, Gao H, Liu J, Ren L, Wei Q, Yu P, Xu Y, Qi F, Qu Y, Li F, Lv Q, Wang W, 280 Xue J, Gong S, Liu M, Wang G, Wang S, Song Z, Zhao L, Liu P, Zhao L, Ye F, Wang H, Zhou W, 281 Zhu N, Zhen W, Yu H, Zhang X, Guo L, Chen L, Wang C, Wang Y, Wang X, Xiao Y, Sun Q, Liu H, 282 Zhu , EIDD-2801-treated and PF-332-treated beta SARS-CoV-2−infected SCID mice at day 3 pi are expressed as log10 TCID50 per mg lung tissue. Individual data and median values are presented. (D) Cumulative severity score from H&E stained slides of lungs from control (vehicle-treated), EIDD-2801-treated (200 mg/kg, BID) and PF-332-treated (300 mg/kg, BID) SARS-CoV-2−infected SCID mice at day 3 pi. Individual data are presented and bars represent means ± SD. The dotted line represents the median score of untreated noninfected hamsters. (E) Weight change at day 3 pi in percentage, normalized to the body weight at the time of infection. Bars represent means ± SD. Data were analyzed with the Mann−Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns=non-significant. All data (panels B, C, D) are from two independent experiments with 14 animals per group except for vehicle group (n=20). 

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