Mucosal IFNλ1 mRNA-based immunomodulation effectively reduces SARS-CoV-2 induced mortality in mice

Abstract

RNA vaccines elicit protective immunity against SARS-CoV-2, but the use of mRNA as an antiviral immunotherapeutic is unexplored. Here, we investigate the activity of lipidoid nanoparticle (LNP)-formulated mRNA encoding human IFNλ1 (ETH47), which is a critical driver of innate immunity at mucosal surfaces protecting from viral infections. IFNλ1 mRNA administration promotes dose-dependent protein translation, induction of interferon-stimulated genes without relevant signs of unspecific immune stimulation, and dose-dependent inhibition of SARS-CoV-2 replication in vitro. Pulmonary administration of IFNλ1 mRNA in mice results in a potent reduction of virus load, virus-induced body weight loss and significantly increased survival. These data support the development of inhaled administration of IFNλ1 mRNA as a potential prophylactic option for individuals exposed to SARS-CoV-2 or at risk suffering from COVID-19. Based on the broad antiviral activity of IFNλ1 regardless of virus or variant, this approach might also be utilized for other respiratory viral infections or pandemic preparedness.

Keywords: Immunology; Infectious Diseases; Interferon; Nucleic-acid Therapeutics; Viral Infection.

Figure 1. Overview of ETHRIS mRNA technology.

ETH47 (LNP-formulated IFNλ1 mRNA) drug product is intended for local delivery to the sites of virus entry and replication (nasal application via nasal spray or pulmonary application using a nebulizer).

Figure 2. Single-dose treatment of A549 cells and primary human air–liquid interface cultures with ETH47 leads to dose-dependent translation of target protein and target gene induction, with little to no cytokine induction.

(A–G) A549 cells were treated with indicated doses of ETH47 (LNP-formulated; n = 2 biological replicates). At 6 and 24 h post treatment, medium was exchanged. (A) hIFNλ1 protein expression in supernatants was measured via ELISA. (B–D) Target gene expression analysis via qPCR. (E–G) Cytokine expression analysis via qPCR. (H) Air–liquid interface cultures (n = 3 biological replicates) were treated with ETH47 (LNP-formulated mRNA) or Control-LNP (3 µg/insert each) or recombinant protein (100 ng/mL), both of which were removed at 6 h post treatment. hIFNλ1 levels were quantified 24 h post treatment in apical and basal compartments. Data Information: in (A–G) data are shown as single data points and mean. (A) All values for the lowest dose, 3 h values for the second lowest dose were BLQ, some values for highest dose were ALQ (see Source Data). (B–G) Missing data points reflect Cq values above the set cycle threshold. Dotted line at y = 2: fold changes below 2 are not considered as gene induction. (H) Single data points and mean +/− SD are shown. ns: P = 0.2038; ****P = 0.000046 (unpaired t test). Source data are available online for this figure.

Figure 3. ETH47 results in potent inhibition of SARS-CoV-2 virus replication in vitro.

Lipofectamine transfection of unformulated ETH47 mRNA in A549-ACE2 cells was done 24 h prior to infection with SARS-CoV-2 (MOI 3). Harvest was done at 48 h post infection (n = 2 biological replicates). (A) hIFNλ1 ELISA from supernatants. ALQ above upper limit of quantification (sample dilution resulted in a value above ULOQ, real concentration might be slightly different), BLQ below lower limit of quantification. (B) qPCR for virus load. Dotted line at y = 1 is untreated reference. Asterisk indicates transfection dose that results in approximately 100 ng/mL produced protein in the supernatant. Data Information: data are presented as individual data points and mean +/− SD. Source data are available online for this figure.

Figure 4. Single nasal administration of ETH47 induces pulmonary protein and target gene expression in mice.

Necropsy was done at 5, 24, and 48 h for all groups. In addition, 0.35 mg ETH47/kg bw group also included 72 and 96 h timepoints. Animals in vehicle group were sacrificed at 5 or 24 h post application. n = 3 biological replicates for each dose and timepoint. (A) hIFNλ1 protein expression in lung homogenates. One sample of 0.125 mg ETH47/kg bw group was excluded due to insufficient sample quality. (B) qPCR for target genes in lung homogenates. (C) Chemokine ELISA in plasma and lung homogenate. LLOQ lower limit of quantification, Bw body weight. Data Information: individual data points and mean are shown. Source data are available online for this figure.

Figure 5. ETH47 reduces virus replication, body weight reduction, and mortality in a hACE2-Transgenic mouse model challenged with SARS-CoV-2.

(A–C) Intranasal administration of 0.38 mg ETH47/kg bw 1 day before and 1 day after SARS-CoV-2 infection (2500 PFU). (D–F) Intranasal administration of 0.38 mg ETH47/kg bw one day before SARS-CoV-2 infection. (A, D) TCID₅₀ assay in lung homogenate (n = 10 individuals). Note that two animals of the vehicle group of panel (A) were excluded from the analysis due to humane reasons (being unable to eat due to elongated teeth) or processing issues during organ sampling, which led to high amounts of residual blood in the lung and precluded a proper downstream analysis. (B, E) Percentual weight change (n = 20 biological replicates until day 3, n = 10 from day 4 on). (C) Kaplan–Meier estimation (n = 20 biological replicates until day 3, n = 10 from day 4 on. (F) Kaplan–Meier estimation (n = 10 biological replicates). Bw body weight. Data Information: (A, D) Individual data points and mean are shown. *P = 0.0122, **P = 0.0031 (Mann–Whitney U test). (B, E) Data are shown as mean +/− SD. *P < 0.05; only significant comparisons are indicated, all other comparisons are nonsignificant (Mann–Whitney U test). (C, F) *P = 0.0113, **P = 0.003 (curve-comparison with Log-rank (Mantel–Cox) test). Source data are available online for this figure.

Figure EV1. Single-dose treatment of A549 cells with Control-LNP leads to no or very low target gene induction and no cytokine induction.

A549 cells were treated with Control-LNP (n = 2 biological replicates). At 6 and 24 h post treatment medium was exchanged (A) Target gene and (B) cytokine expression in A549 cells. Data information: data points show single values and mean. Dotted line at y = 2: fold changes below 2 are not considered as gene induction. Missing data points reflect Cq values above the set cycle threshold. Source data are available online for this figure.