key: cord-0316163-bdkpap6z
authors: Dijkman, Ronald; Selvaraj, Muneeswaran; Gad, Hans Hendrik; Hartmann, Rune; More, Sunil; Perlman, Stanley; Thiel, Volker; Channappanavar, Rudragouda
title: Effective interferon (IFN)-λ treatment regimen to control lethal MERS-CoV infection in mice
date: 2021-05-27
journal: bioRxiv
DOI: 10.1101/2021.05.26.445685
sha: 540a89f2204f333b80dd09bae609ea80afb53c11
doc_id: 316163
cord_uid: bdkpap6z

Effective broad-spectrum antivirals are critical to prevent and control emerging human coronavirus (hCoV) infections. Despite considerable progress made towards identifying and evaluating several synthetic broad-spectrum antivirals against hCoV infections, a narrow therapeutic window has limited their success. Enhancing the endogenous interferon (IFN) and interferon-stimulated gene (ISG) response is another antiviral strategy known for decades. However, the side effects of pegylated type-I IFNs (IFN-Is) and the pro-inflammatory response detected after delayed IFN-I therapy have discouraged their clinical use. In contrast to IFN-Is, IFN-λ, a dominant IFN at the epithelial surface, is shown to be less pro-inflammatory. Consequently, we evaluated the prophylactic and therapeutic efficacy of IFN-λ in hCoV infected airway epithelial cells and mice. Human primary airway epithelial cells treated with a single dose of IFN-I (IFN-α) and IFN-λ showed similar ISG expression, whereas cells treated with two doses of IFN-λ expressed elevated levels of ISG compared to IFN-a treated cells. Similarly, mice treated with two dose IFN-λ were better protected compared to mice receiving a single dose, and a combination of prophylactic and delayed therapeutic regimens completely protected mice from lethal MERS-CoV-infection. A two dose IFN-λ regimen significantly reduced lung viral RNA and inflammatory cytokine levels with marked improvement in lung inflammation. Collectively, we identify an ideal regimen for IFN-λ use and demonstrate the protective efficacy of IFN-λ in MERS-CoV infected mice.

spectrum and CoV-specific antivirals. The broad-spectrum anti-CoV drugs that completed clinical trials 71 or those in advanced stage of clinical trials include remdesivir (nucleoside analog), EIDD-2810 72 (nucleoside analog), lopinavir/ritonavir (protease inhibitor), and camostat mesylate (TMPRSS2 inhibitor) 73 (16, 17) (NCT04392219 and NCT0445739) although none of this has demonstrated substantial clinical 74 efficacy. CoV-specific antivirals such as CoV 3CLPro/PLpro protease inhibitors and monoclonal 75 antibodies against spike protein (S) or receptor binding domain of the S protein, are also being 76 investigated for their preclinical and clinical antiviral efficacy (18) (19) (20) . Several of these broad-spectrum 77 and CoV-specific antivirals effectively reduced viral load in in vitro studies (18, (21) (22) (23) (24) . Preclinical studies 78 in animal models and recent human clinical trials also showed the therapeutic efficacy of these antiviral 79 agents when used in the early stages of infection (21, (24) (25) (26) (27) . However, so far, antivirals used at later (42, 43) . IFN-λ on the contrary is less pro-90 inflammatory, and peg-IFN-λ has relatively fewer side effects compared peg- IFNa/b (44, 45) . 91 Consequently, peg-IFN-λ is in clinical trials to treat chronic virus infection and, more recently COVID19 92 (44-46). Despite recent advances, the prophylactic use of IFN-λ, ideal route administration, and an 93 optimal regimen of IFN-λ to prevent and treat hCoV infections are not well established. 94 Here, we report that a two dose IFN-λ regimen induces robust ISG expression compared to IFN-95 a/b as opposed to single-dose IFN-a/band IFN-λ regimens in primary human airway epithelial cells. We Human airway epithelial cells: 106 Primary human airway epithelial cells were procured from patients who underwent surgical lung 107 resection in their diagnostic pathway for any pulmonary disease and who gave informed consent. This 108 was done in accordance with our ethical approval (EKSG 11/044, EKSG 11/103 and KEK-BE 302/2015) .

Pathologically examined tracheobronchial segments were used as starting material for isolation, 110 propagation, and establishment of well-differentiated primary human airway cell (hAEC) cultures (47) Animals were anesthetized and transcardially perfused with 10 mL PBS followed by 5 mL zinc formalin.

Lungs were removed, fixed in zinc formalin, and paraffin embedded. Tissue sections were stained with 167 hematoxylin and eosin (H & E) and examined and scored by light microscopy in a blinded fashion by 168 board certified veterinary pathologist as described previously (52). Control and rIFN-λ-treated lungs were 169 scored for edema with scores of 0, 1, 2, 3, and 4 representing lung areas with 0%, less than 3%, 6%-170 33%, 33%-66%, and more than 66% detectable proliferation, respectively. Lungs were also scored for 171 inflammatory cell infiltration, with scores of 0, 1, 2, 3, and 4 representing areas with 0%, less than 3%, 172 6%-33%, 33%-66%, and more than 66% of perivascular poly/mononuclear cell distribution, respectively. Two dose IFN-λ treatment reduces hCoV titers in human airway epithelial cells. 198 We next examined whether increased ISG expression in dual IFN-λ treatment groups correlates 199 with reduced virus titers. Well-differentiated hAEC cultures were treated with single or two doses of rIFN- two-dose IFN-λ treatment further reduced hCoV-229E RNA levels ( Figure 2B ) demonstrating that two-206 dose regimen is much more effective than the single-dose treatment. We then evaluated the efficacy of 207 rhIFN-λ in suppressing MERS-CoV replication. As shown in Figure 2C -D, MERS-CoV RNA levels were 208 reduced upon IFN-λ administration compared to mock treatment, with two-dose regimen being more 209 effective than one dose IFN-λ in reducing viral RNA levels (albeit not statistically significant).

Nonetheless, we show that IFN-λ treatment reduced hCoV RNA levels compared to controls, and hCoV-211 229E was highly sensitive to IFN-λ-treatment compared to MERS-CoV in hAEC cultures. (43). IFN-λ on the contrary is shown to be less pro-inflammatory due to limited 295 expression of IFN-λR on inflammatory cells (65, 66) and, as a result, is believed to provide better 296 protection as opposed to IFN-a/b following therapeutic administration. Our results, however, showed that 297 delayed IFN-λ administration is pathogenic (Figures 3 and 6) , which is similar to outcomes resulting from 298 delayed IFN-a/b treatment. As shown in Fig 3, a combination of prophylactic and delayed therapeutic 299 antiviral therapy will overcome these detrimental effects. However, given that IFN-λ is less pro-300 inflammatory, whether disease exacerbation following delayed IFN-λ administration occurs due to robust 301 inflammation (43) or epithelial cell apoptosis (70, 71) resulting in impaired tissue repair requires further 302 investigation. Similar to results from the timing of IFN-a/b response, we observed efficient virus clearance 303 upon early but not delayed IFN-λ treatment ( Figure 5 ). Such an impaired virus clearance is also observed 304 upon delayed administration of other anti-CoV antivirals such as remdesivir, and anti-S and anti-S-RBD 305 monoclonal antibodies (17, 33, 34, (72) (73) (74) , suggesting that early antiviral therapy is critical to effectively 306 reduce virus burden and protect the host from lethal disease.

In addition to an optimal IFN-λ regimen to protect the host from lethal virus infection, route of 308 administration likely plays a key role in the disease outcome. IFNs are mainly administer via the 309 subcutaneous (s/c) or intravenous route to treat virus infections and other immunoinflammatory 310 conditions. Considering hCoV replication is largely limited to airways and lungs, and IFN-λR is mainly 311 expressed on epithelial cells, IFN-λ intranasal spray will likely be more effective than the s/c or systemic Well-differentiated hAEC cultures were treated with a single or double dose of IFN-aA/D (100IU/ml) or hIFN-L3 (100 ng/ml) at indicated time points prior to expression analysis of MxA mRNA. For the two-dose regime hAEC cultures were stimulated 6 days prior to readout, followed by a restimulation at the indicated time points. The fold change in MxA mRNA expression over mock is shown for the individual A) single or B) two dose IFN-lambda regimes. In addition to the duration of the antiviral response, the epithelial barrier integrity among the C) single and D) two dose regimes was assessed by measuring the transepithelial electrical resistance (TEER) of each regime prior to whole cell lysis. Data is shown as mean and standard deviation from 3 biological donors with two technical replicates. Well-differentiated hAEC cultures were treated with a single or two doses of IFN-aA/D (100IU/ml) or hIFN-L3 (100 ng/ml) at indicated time points (minus (-) symbol refers to day-before infection, and plus (+) to days-after infection). The hAEC cultures were challenged with 4000 PFU of HCoV-229E or MERS-CoV and 72 hours post-infection the viral RNA load (Genome Equivalents (EQ) / uL) was measured by qPCR. Bar graphs represent HCoV-229E (A, B) or MERS-CoV (C, D) genomic RNA levels in control and IFN-L treated hAEC cultures during a single or double dose regime, respectively. Data is shown as mean and standard deviation from 3 biological donors with two technical replicates. Statistical significance was determined using Student's t test with * P<0.05 and ** P<0.01, *** P<0.001, and **** P<0.0001. Male hDPP4-KI mice were treated with a single or two doses of mIFNL (1.5 to 2.0µg/mouse, vial intranasal route) at indicated time points (minus (-) symbol refers to day-before infection and plus (+) to days-after infection). Mice were challenged with 1000 PFU of mouse-adapted MERS-CoV and mice were monitored for morbidity and mortality for 14 days. A) Bar graphs represent MERS-CoV genomic RNA levels in control and IFNL treated lungs at 4 DPI, with each scatter points representing a mouse. B) Bar graphs represent mRNA levels of ISGs and inflammatory cytokines in control and IFNL treated lungs at 4 DPI, with each scatter points representing a mouse. Data are pooled from 2 experiments with 3-4 mice per group per experiment. Statistical significance was determined using Student's t test with * P<0.05, ** P<0.01, and ***P<0.001. 

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