key: cord-0951984-8v7150vv
authors: Marx, Samira; Kümmerer, Beate M.; Grützner, Christian; Kato, Hiroki; Schlee, Martin; Renn, Marcel; Bartok, Eva; Hartmann, Gunther
title: RIG-I-induced innate antiviral immunity protects mice from lethal SARS-CoV-2 infection
date: 2022-02-13
journal: Mol Ther Nucleic Acids
DOI: 10.1016/j.omtn.2022.02.008
sha: 79a7070c20505c30932c4a5a0ec0fb6b8b6bddf9
doc_id: 951984
cord_uid: 8v7150vv

The SARS-CoV-2 pandemic has underscored the need for rapidly employable prophylactic and antiviral treatments against emerging viruses. Targeted stimulation of antiviral innate immune receptors can trigger a broad antiviral response that also acts against new, unknown viruses. Here, we utilized the K18-hACE2 mouse model of COVID-19 to examine whether activation of the antiviral RNA receptor RIG-I protects mice from lethal SARS-CoV-2 infection and reduces disease severity. We found that prophylactic, systemic treatment of mice with the specific RIG-I ligand 3pRNA, but not type-I interferon, one to seven days before viral challenge, improved survival of mice by up to 50 %. Survival was also improved with therapeutic 3pRNA treatment starting one day after viral challenge. This improved outcome was associated with lower viral load in oropharyngeal swabs and in the lungs and brain of 3pRNA-treated mice. Moreover, 3pRNA-treated mice exhibited reduced lung inflammation and developed a SARS-CoV-2-specific neutralizing antibody response. These results demonstrate that systemic RIG-I activation by therapeutic RNA oligonucleotide agonists is a promising strategy to convey effective, short-term antiviral protection against SARS-CoV-2 infection, as well as its potential as a broad-spectrum approach to constrain the spread of newly emerging viruses until virus-specific therapies and vaccines become available.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has called to attention 34 the vital importance of rapid, effective strategies for limiting the spread of emerging viruses. SARS-35

CoV-2 is the etiological agent of Coronavirus Disease 2019 (COVID-19) 1,2 , which infects the upper and 36 lower airways of patients but also can cause neurological symptoms, in particular anosmia 3,4 . The 37 clinical course of COVID-19 is extremely variable between individuals, from mild symptoms to severe 38 interstitial pneumonia requiring mechanical ventilation. Since its initial outbreak in 2019 in Wuhan, 39 SARS-CoV-2 virus infection has resulted in over 290 million confirmed COVID-19 cases and over 5.4 40 million deaths (https://COVID19.who.int/, Status: 06 January 2022). Moreover, we are only beginning 41 to understand the extent of its socioeconomic repercussions, including the impact of chronic disease 5 , 42 loss of primary caregivers 6 , unemployment and school closures. While tremendous efforts are being 43 made to control the virus, the development of vaccines has progressed more rapidly than the 44 development of direct antiviral treatments 7 . Repurposed small-molecule antiviral drugs like 45 remdesivir, molnupiravir or monoclonal antibody cocktails have shown only modest efficacy with 46 moderately improved survival rates vs placebo in hospitalized patients [8] [9] [10] . Hence, there is still a great 47 6 post-infection when left untreated (Fig. 1C-D) . Some mice exhibited severe intestinal symptoms like 106 bowel obstructions or neurological symptoms marked by progressive motor deficits. A single injection 107 of 3pRNA administered one day (d-1) prior to inoculation with the virus improved the survival rate 108 from 0% (treatment with control RNA) to 50% (Fig. 1C ). Injection at three days (d-3) or seven days (d-109 7) days prior to infection still improved the survival rate by 25-30%. Some of the 3pRNA-treated mice 110 that eventually succumbed to SARS-CoV-2 infection still showed slower weight loss and a delayed 111 onset of other symptoms resulting in a right shift of the Kaplan-Meier curve (Fig. 1C) . Surviving animals 112 maintained or increased their weight and did not show any signs of disease for the duration of the 113 experiment (Fig. 1E) . 114

To monitor viral replication, oropharyngeal swabs were taken one-to-three days post infection. CoV-2 viral antigens were quantified by ELISA (Fig. 1F) , and viral RNA was quantified by qPCR (Supp. 116 Fig. 1) . Pretreatment with 3pRNA one (d-1) and three (d-3) days (trend for d-7) prior to infection led to 117 a significant reduction of viral burden on day 1 and day 2 after viral challenge (Fig. 1 F; Supp. Fig. 1 ). In 118 contrast, pre-treatment with control RNA had no effect compared to untreated animals. At 3 days after 119 viral challenge, viral antigen was no longer detectable in most of the mice (Fig. 1F) . We then examined 120 the levels of anti-SARS-CoV-2 specific IgG antibody titers in the sera of all animals at the time of death 121 (non-surviving mice, marked in black) or at the end of the observation period (day 13, surviving mice, 122 marked in green). 3pRNA-treated mice showed significantly higher anti-SARS-CoV-2 specific IgG 123 antibodies titers post-infection than untreated infected control mice (Fig. 1G ). SARS-CoV-2 specific 124 antibodies also conferred neutralizing activity by blocking the interaction between ACE2 and the spike 125 protein in vitro (Fig. 1H ). Of note, the fact that sera were obtained at the time of death or at day 13 126 after infection (surviving mice) limits a comparative analysis of the data. 127 128

We then tested whether RIG-I stimulation was beneficial after viral infection had already occurred. 130 K18-hACE2 mice were infected with 5x10 4 PFU SARS-CoV-2, and 20µg 3pRNA or control RNA were 131 injected i.v. 24 hours later and repeated on days 4, 7 and 10 post infection ( Fig. 2A) . In contrast to mice 132 treated with control RNA and untreated control animals, 25% of mice treated with 3pRNA recovered 133 from the initial weight loss and survived (Fig. 2 B, C) . Moreover, the surviving mice developed a 134 neutralizing SARS-CoV-2 specific antibody response (Fig. 2D, E) . 135 136 3pRNA treatment reduces viral load and inflammation 137

Next, we analyzed the viral load in the lungs and the brain of mice i.v. injected with 20µg 3pRNA either 138 one day before (prophylactic) or one day after (therapeutic) i.n. inoculation with SARS-CoV-2. Mice 139 were sacrificed and their lungs and brain were prepared on day four after viral challenge (Fig. 3A) . 140

Prophylactic 3pRNA treatment significantly reduced the viral burden (Fig. 3B ,C) in the lungs and brain 141 as well as inflammation in lung tissue as indicated by a diminished expression of the chemokines Cxcl10 142 and Ccl2 and the pro-inflammatory cytokine Il6 (Fig. 3D ). The number of viral RNA copies as well as the 143 expression of pro-inflammatory cytokines in the brain (Fig. 3C , E) of the mice were overall lower than 144 in the lungs (Fig. 3B ,D) at four days after infection. 145

We also analyzed the viral burden in the mice shown in Figure 1 from which organs were taken at the 146 time of death or at the end of the observation period. Surviving animals from all 3pRNA-treated groups 147 and non-surviving mice from the 3pRNA treated d-1 group showed a significant reduction of viral RNA 148 in the lungs (Supp. Fig. 2 A) . However, in the brain, this was only seen for surviving animals from the 149 3pRNA-treated d-1 group, with a similar tendency for the 3pRNA-treated d-3 and d-7 animals (Supp. 150 Immunohistochemical staining of SARS-CoV-2 nucleocapsid in lung and brain sections correlated with 155 the results of qPCR analysis when scored by three independent scientists in a blinded fashion (suppl. 156 Fig. 2C,D) . Histological scores for the nucleocapsid staining were lower in the lungs of all 3pRNA-157 treated animals and in the brains of the surviving 3pRNA-treated mice, but did not reach significance 158 (supp. Fig. 2 E,F) . 159 160 3pRNA confers superior protection compared to recombinant type I interferon 161

Intravenous injection of 3pRNA induces significant amounts of IL-6, CXCL10, and type-I (α/β) and type-162 II (γ) IFN (suppl. Fig. 3 ). Since treatment of COVID-19 with recombinant type-I IFN has already been 163 studied in clinical trials, we compared the antiviral efficacy of 3pRNA to high-dose recombinant 164 universal IFNα (IFN-a/d; 2x10 5 U, 877ng) which is equivalent to the IFN levels observed after 3pRNA 165 treatment and has been used by others 39 While vaccines will likely continue to be the most important weapon against SARS-CoV-2, the capability 236 of RIG-I agonists to induce protection in immunocompromised hosts and their effectiveness against 237 variants of concern 39 would help to fill important niches, such as antiviral prophylaxis in organ 238 transplant recipients or the treatment of front-line health care workers exposed to emerging variants. 239

In our study, the reduced morbidity and mortality after prophylaxis, even when administered seven 240 days prior to infection, show that RIG-I agonists induce a relatively long-lasting antiviral state which 241 would allow for a clinically feasible weekly pre-exposure prophylaxis in high exposure environments. 242

Moreover, the ability of RIG-I ligands to reduce viral load and thereby the inflammation in the lungs in 243 response to therapeutic treatment could be used to treat high risk COVID-19 patients immediately 244 after a positive qPCR diagnosis in order to reduce the likelihood of hospitalization or death. Because 245 double-stranded RIG-I agonists have already been tested in phase I/II clinical studies for oncologic 246 indications (NCT03739138, NCT0306502) 33 , trials for the prophylaxis or treatment of COVID-19 could 247 swiftly be initiated. In conclusion, our study demonstrates that RIG-I agonist-mediated antiviral 248

prophylaxis has great potential in the context of SARS-CoV-2 but is also a promising approach against 249 newly emerging viruses, where it could be employed to limit outbreaks and prevent pandemic spread. Hospital Bonn. K18-hACE2 transgenic mice were lightly anesthetized with ketamine/xylazine, before 281 5x10 4 PFU of SARS-CoV-2 virus was pipetted onto the nose and subsequently inhaled by the animal. 282

On day 1 to day 3, oropharyngeal swabs were obtained using minitips and placed in 1 ml UTM medium 283 (360C, COPAN, Hain Lifescience GmbH, Nehren, Germany). Viral antigen in the oropharyngeal swabs 284 was quantified with ELISA according to the manufacturer's protocol (SARS-CoV-2-Antigen-ELISA, 285

Euroimmun, Lübeck, Germany). Following infection, weight loss and survival were monitored up to 286 twice daily for 13 days. Endpoint criteria were ≥20% weight loss, lethargy, motor deficits and high 287 respiratory rates. 288

Total RNA was extracted from mouse lung and brain tissues using TRIzol (ThermoFisherScientific, 291 dpi. For B-E, expression was quantified by qPCR relative to murine gapdh expression Plotted are the 552 mean + SEM (n=6, uninfected n=3). Statistical significance was calculated by one-way ANOVA (Welch) 553

with Dunnett's T3 multiple testing, when the data were lognormally distributed and otherwise a non-554

parametric Kruskal-Wallis test with Dunn's multiple testing was applied. * p<0.05, ** p<0.01, *** 555 p<0.001, **** p<0.0001. 556 

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MTNA-D-21-01134 RIG-I-induced innate antiviral immunity protects mice from lethal SARS-CoV-2 infection eTOC Synopsis Nucleic acid receptors, such as RIG-I, are essential to the antiviral innate immune response. In this study, Marx and colleagues report that activation of RIG-I by a specific 3pRNA ligand protects hACE2-transgenic mice from otherwise lethal SARS-CoV-2 infection