key: cord-0919236-matp1uqz
authors: Chaturvedi, Sonali; Vasen, Gustavo; Pablo, Michael; Chen, Xinyue; Beutler, Nathan; Kumar, Arjun; Tanner, Elizabeth; Illouz, Sylvia; Rahgoshay, Donna; Burnett, John; Holguin, Leo; Chen, Pei-Yi; Ndjamen, Blaise; Ott, Melanie; Rodick, Robert; Rogers, Thomas; Smith, David M.; Weinberger, Leor
title: Identification of a Therapeutic Interfering Particle — a single-administration SARS-CoV-2 antiviral intervention with a high barrier to resistance
date: 2021-11-10
journal: Cell
DOI: 10.1016/j.cell.2021.11.004
sha: 565422c7f8152a9fcf034899d5d56edfa683ade3
doc_id: 919236
cord_uid: matp1uqz

Viral-deletion mutants that conditionally replicate and inhibit wild-type virus (i.e., Defective Interfering Particles, DIPs) have long been proposed as single-administration interventions with high genetic barriers to resistance. However, theories predict that robust, therapeutic DIPs (i.e., Therapeutic Interfering Particles–TIPs) must conditionally spread between cells with R0>1. Here, we report engineering of TIPs that conditionally replicate with SARS-CoV-2, exhibit R0>1, and inhibit viral replication 10–100 fold. Inhibition occurs via competition for viral replication machinery and, a single administration of TIP RNA inhibits SARS-CoV-2 sustainably in continuous cultures. Strikingly, TIPs maintain efficacy against neutralization-resistant variants (e.g., B.1.351). In hamsters, both prophylactic and therapeutic intranasal administration of lipid-nanoparticle TIPs durably suppressed SARS-CoV-2 by 100 fold in the lungs, reduced pro-inflammatory cytokine expression, and prevented severe pulmonary edema. These data provide proof-of-concept for a class of single-administration antivirals that may circumvent current requirements to continually update medical countermeasures against new variants.

The evolution of resistance to both antimicrobials and vaccines is common across pathogens (Goldberg et al., 2012; Meylan et al., 2018; Petrova and Russell, 2018) . Evidence over the past year indicates that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unlikely to be an exception as viral genetic variability has resulted in variants with increasing resistance to antibody-mediated neutralization (Cele et al., 2021; Wang et al., 2021) . In particular, SARS-CoV-2 variants of concern (e.g., B.1.1.7, B.1.351, P.1, B.1.617.2) exhibit increased transmissibility and appear to circumvent natural convalescence and show diminution of vaccine efficacy (Wibmer et al., 2021; Wu et al., 2021) . The continued emergence of viral variants suggests parallels to seasonal influenza, where protracted arms races between immunity, waning immunity and less susceptible viruses occur with a considerable lag between variant emergence and redesigned vaccines.

SARS-CoV-2 is a beta coronavirus with a large ~30-kb positive-sense, single-stranded RNA genome that replicates with an ~8-hour intracellular replication cycle (Kim et al., 2020) . The large genome encodes a suite of nonstructural genes, including the RNA-dependent RNA polymerase (RdRp) within the ORF1ab gene, as well as structural genes-including spike (S), matrix (M), envelope (E), and nucleocapsid (N)-that are required for replication and packaging. In addition to these viral trans elements, the genome is flanked at the 5' and 3' termini by untranslated regions (UTRs) which encode regulatory cis elements, including the putative packaging element in the 5' UTR. Like many RNA viruses, beta coronaviruses also generate sub-genomic deletion mutants that are defective but if these RNAs retain obligate cis elements while carrying mutations in trans elements, they can act as defective interfering particles (DIPs) (Makino et al., 1990) .

DIPs-originally observed as "autointerference" for influenza virus in the 1940s by von Magnus (Von Magnus, 1954) , but since reported and studied for many viruses (Akpinar et al., 2016; Holland, 1990; Tapia et al., 2019) -were historically considered cell-culture artifacts with utility for molecular-mechanistic dissection of viruses. However, recent years have seen a renaissance in the study of DIPs following proposals that they could serve as chassis for a class of single-administration antivirals with a high barrier to the evolution of resistance (Metzger et al., 2011; Weinberger et al., 2003) . Since DIPs lack self-replication but conditionally replicate with their cognate virus, they have the potential to act as molecular J o u r n a l P r e -p r o o f Page 5 of 56 parasites of the wild-type virus within infected cells. Parasitism is mediated by competitive inhibition where viral cis elements encoded by DIPs interact with and 'steal' essential trans elements from wild-type virus (e.g., replication or packaging proteins). Consequently, DIPs suppress wild-type viral burst size (the number of viral particles released from an infected cell) and conditionally mobilize their own genomes, spreading their antiviral properties to new cells. Theoretical models predicted that DIPs engineered to have a basic reproductive ratio [R 0 ]>1 could act as durable therapeutics, termed Therapeutic Interfering particles ('TIPs') (Metzger et al., 2011; Weinberger et al., 2003) (Fig. 1a) . The predicted high genetic barrier to resistance arises from two considerations: (i) the nature of the cis-trans mechanism of inhibition (Rouzine and Weinberger, 2013) and (ii) the R 0 TIP >1 enabling TIPs to establish co-evolutionary arms races with wild-type virus (Metzger et al., 2011; Rouzine and Weinberger, 2013; Weinberger et al., 2003) .

Here, we first determined theoretical constraints for a TIP for SARS-CoV-2 and then built TIP candidates, using synthetic SARS-CoV-2 sub-genomic RNAs, that satisfy the constraints and inhibit viral replication in cell culture and donor-derived human lung organoids. The

RNAs conditionally propagated with R 0 >1-satisfying criteria for a TIP-and mechanistic analyses suggested this occurred via competitive inhibition. We then tested if TIP antiviral effects were recalcitrant to mutational escape, as predicted. Finally, to test efficacy in vivo, we used Syrian golden hamsters to determine if TIPs (administered pre-or post-infection)

propagate and durably suppress viral replication and associated disease pathology in the lungs. Overall, the data demonstrated that TIPs have robust antiviral efficacy, with a high genetic barrier to the evolution of resistance, indicating their potential as an antiviral countermeasure for respiratory viruses, including SARS-CoV-2.

We first determined if TIPs had the theoretical potential to suppress SARS-CoV-2 viral load in vivo. We used in silico modeling approaches that informed therapeutic regimens for other viral pathogens (Perelson, 2002; Perelson et al., 1997) and capitalized on a recent in silico patient-validated model of SARS-CoV-2 within-host viral dynamics (Ke et al., 2020) . The model tracks viral loads in two compartments (Fig. 1b) -upper respiratory tract (URT) and lower respiratory tract (LRT)-with parameter estimates determined by fitting to longitudinal J o u r n a l P r e -p r o o f

Page 6 of 56 viral titer data from individual infected patients. We expanded this model to include TIPs using a previously established modeling approach (Weinberger et al., 2003) and numerically solved (Fig. 1c) under a range of parameter values for individual patients (Fig. S1a) , a range of single-administration dose values (Fig. S1b) , and a range of viral inoculums (Fig. S1c) .

This showed that TIPs would produce a median predicted knockdown in viral load of ~1-2 Log in the LRT and similar viral load reductions were predicted for the URT.

To understand if this reduction in viral load was specific to a particular parameter choice for TIPs, we explored the sensitivity to two molecular parameters previously found to be critical determinants of TIP R 0 and efficacy: (i) the TIP competitive efficiency () which describes the relative difference in production of TIP particles versus wild-type virus particles from a dually infected cell, and (ii) the interference efficiency () which describes the reduction in wild-type virus production by TIPs competing intracellularly for viral proteins. In terms of viral burst size,  reflects the relative burst size of TIPs compared to wild-type virus (from a dually infected cell) whereas  reflects the reduction in wild-type virus burst size between singly vs. dually infected cells.

The parameter sensitivity analysis showed a wide range of  and  values where TIPs generate a 1-Log reduction in peak viral load in the LRT but that >1-Log reductions in viral load were specific to larger values of  and moderate values of  (Fig. 1d) . The phenomenon of larger values of  having lesser viral load reduction has been previously predicted (Weinberger et al., 2003) and results from the TIP 'shooting itself in the molecular foot' and limiting efficient mobilization when interference is too strong. Consistent with previous analyses of TIPs for other viruses (Metzger et al., 2011) , larger values of  were the key factor enabling TIPs to generate larger viral load reductions (Fig. S1d) . Similar TIP-mediated reductions in URT viral load were also observed (Fig. S1e) . To be sure that knockdown of peak viral load reflected a true reduction in viral load, we also analyzed viral loads integrated over time (i.e., area under the curve) and found similar reductions by TIPs (Fig. S1f) .

The simulations showed that TIPs could yield on average 1-Log reductions in SARS-CoV-2 in a dose-dependent fashion, with substantially larger 6-Log reductions for some patientparameter estimates (Fig. S1a) . To be sure this was not model dependent we also analyzed a second patient-parameterized model of SARS-CoV-2 (Kim et al., 2021) and observed qualitatively similar effects of TIPs on viral load (Fig. S1g) . Based upon prior work linking SARS-CoV-2 viral load and infectiousness (Goyal et al., 2021; Jones et al., 2021) , we also analyzed how TIPs might affect SARS-CoV-2 transmission and found that a TIP-mediated reduction in viral load would generate a substantial reduction in SARS-CoV-2 secondary infections (from R=1.8 to R=0.07) (Fig. S1h) . In summary, these in silico analyses showed that TIPs have the theoretical capacity to suppress SARS-CoV-2 in vivo after a single administration, provided that  and  surpass specific thresholds.

Given the challenges of engineering TIPs for other viruses (Notton et al., 2021) we set out to construct a TIP with appropriate parameters. As a starting point we used previous highthroughput analyses of the cis genetic elements required for efficient propagation of subgenomic transcripts in other RNA viruses (Notton et al., 2021) and historical data from murine hepatitis virus (Baric et al., 1988; Makino et al., 1990) , both of which argued that at a minimum, the viral 5' and 3' UTRs were essential for conditional replication and construction of a TIP. Consequently, we designed two minimal sub-genomic synthetic constructs, encoding different lengths of the 5' and 3' regions of the viral genome and tested if they met the threshold values to act as TIPs. Both putative TIPs encompass stem loop 5 in the 5' UTR which encodes a predicted packaging signal (Chen and Olsthoorn, 2010; Rangan et al., 2020) , as well as the entirety of the 3'UTR and a 1280 nucleotide (nt) reporter cassette ( Fig. 2a) encoding an internal ribosome entry sequence (IRES) driving expression of a fluorescent reporter protein (mCherry). TIP1 (~2.1kb) encodes the first 450 nts of the 5' UTR plus part of polyprotein ORF1ab and the last 328 nts of the 3'UTR plus the reporter cassette, whereas TIP2 (~3.5kb) encodes 1540 nts encompassing the 5'UTR and part of ORF1ab and the last 713 nts of the genome containing part of N protein, ORF 10, and the 3'UTR, along with the reporter cassette. All TIP and control mRNAs were in vitro transcribed (Fig. 2b) and a 5' methyl cap and ~100-nt 3' polyA tail were added following in vitro transcription.

To evaluate the interference potential of the putative TIPs, virus yield-reduction assays were performed. Vero cells were transfected with the purified TIP mRNA, or a similarly-sized control mRNA encoding a luciferase-IRES-mCherry reporter cassette (hereafter referred to as 'Ctrl RNA') and then infected with SARS-CoV-2 virus (WA-1 isolate) at a multiplicity of J o u r n a l P r e -p r o o f hours using primers specific to SARS-CoV-2 genes not present in the TIPs-to assay at these time points, supernatant needed to be transferred to naïve target cells. The data show a significant 1.5-Log inhibition of SARS-CoV-2 replication at all time points for both TIP1 and TIP2 (Fig. 2c) . To verify that the observed interference was not due to off-target or cellmediated innate immune effects (e.g., cellular RNA interference), we examined an expanded panel of control RNAs encoding: (i) 5' UTR RNA alone; (ii) 3' UTR RNA alonewhich lacks the putative 5' UTR packaging signal; (iii) an RNA encoding SARS-CoV-2 Matrix (M) with 5' stop codons; and (iv) an RNA encoding the SARS-CoV-2 Spike (S) with 5' stop codons. Each of these control RNAs also encoded IRES-mCherry to match the TIP RNAs and were 5'-methyl capped and poly-A tailed and. None of the control RNAs generated a significant reduction in SARS-CoV-2 viral gene expression (Fig. 2d) .

To determine if innate antiviral mechanisms specific to the TIP RNA sequence were responsible for viral interference, we also analyzed expression of twelve common innateimmune responsive genes including IFN-stimulated genes (ISGs) in cells nucleofected with either TIP RNA or Ctrl RNA in the absence of infection (Fig. S2a) . These data show no significant upregulation of pro-inflammatory cytokines, downstream ISGs, or upstream RNA-sensing or signaling genes by TIP RNAs, inconsistent with an RNA-induced innate antiviral mechanism.

We next tested if TIP administration could exert an antiviral effect following infection in cell culture. Vero cells were first infected with SARS-CoV-2 (MOI=0.05) and TIP RNA then administered at either 8-hrs post infection and-16 hrs post infection and, as above, viral gene expression quantified by RT-qPCR at 48-hrs post infection using primers specific to SARS-CoV-2 genes not present in the TIPs. These post-infection TIP-administration data show a significant 1-Log inhibition of SARS-CoV-2 replication by TIPs (Fig. S2b) .

To confirm that TIP RNAs inhibit virus output, we used a plaque forming unit (PFU) assay, which detects and quantifies infectious virus produced from cells, to analyze at the same time points as in Fig. 2c (i.e., 24, 48 and 72 hours post infection). The PFU assay confirmed that

TIPs generate a 1.5-Log inhibition of infectious SARS-CoV-2 particles (Fig. 2e) .

J o u r n a l P r e -p r o o f (Fig. 2f) . Thus, for SARS-CoV-2, the initial TIP prototypes (i.e., starting points) appear to satisfy the requisite constraints.

To test if TIPs interfered with SARS-CoV-2 in a more physiological setting, we employed a human lung organoid model ( Fig. 3a ) (Sachs et al., 2019; Zhou et al., 2018) . Previous analysis of SARS-CoV-2 infected lung organoids (Han et al., 2021) showed they are valuable for screening candidate COVID-19 therapeutics and revealed cytokine/chemokine and interferon signatures consistent with inflammatory changes observed in primary human COVID-19 pulmonary infections (Blanco-Melo et al., 2020) . We established and characterized organoids using primary human small-airway epithelial cells (Fig. 3b) , obtained from three donors (Fig. S2c) . The organoids were transfected with either TIP1, TIP2, or Ctrl RNA and then infected with SARS-CoV-2 virus at MOI=0.5 (as these cultures are known to be challenging to infect) 24 hours later. Viral titers in lung organoids were assayed by RT-qPCR ( Fig. 3c ) and PFU analysis (Fig. 3d ) 24 hours post infection and these assays both confirmed that TIPs reduced SARS-CoV-2 by ~1-Log compared to Ctrl RNA.

To understand the mechanism-of-action of TIP interference, we first determined if TIPs were restricting incoming virus infection, which might indicate interference via induction of innate cellular immune responses (e.g., interferon response) or similar cellular restrictions. Viral entry was analyzed by immunofluorescence staining for S protein at 2 hours post SARS-CoV-2 infection (MOI=20) and no significant effect of TIP RNA on viral entry was detected ( Fig. S3a) .

To test if TIPs affected the early events of SARS-CoV-2 infection, cells were infected with SARS-CoV-2 (MOI=0.05) and GFP positive cells were added (20% of total number of cells infected) at 2 hours post infection. Flow cytometry analysis of viral N expression was J o u r n a l P r e -p r o o f

Page 10 of 56 performed at eight hours following infection, within the first round of viral replication. The 8-hour time point was chosen to optimize the dynamic range for detection of early restrictions as 8 hours is near the end of the first round of the viral lifecycle, thereby allowing sufficient time for viral transcripts to accumulate, but is prior to substantial viral egress and 2 nd -round infection. We observed no significant impact of TIPs on SARS-CoV-2 viral N protein expression at 8 hours post infection in the GFP positive cells (Fig. S3b) , indicating that TIP interference could not be explained by early cellular restriction events and that interference occurred during later times in the viral life-cycle (e.g., viral packaging).

Next, to determine if TIP RNAs are packaged into VLPs we performed reconstitution assays ( Fig. 4a) . Cells were co-transfected with expression vectors each encoding a cDNA for the matrix (M), envelope (E), spike (S), or nucleocapsid (N) protein of SARS-CoV-2 together with TIP RNA, Ctrl RNA, or no RNA. Supernatant was concentrated (ultracentrifuged) and

imaged for presence of VLPs by transmission electron microscopy (EM) and, in parallel, analyzed for functional VLP transduction of naïve cells. EM analysis showed the presence of abundant ~100nm-diameter VLPs (Fig. S3c) . RT-qPCR for mCherry showed substantial TIP transduction of naïve cells when VLPs where reconstituted using TIP RNA but not Ctrl RNA ( Fig. 4a) .

To test if TIP mRNAs directly bind and compete for SARS-CoV-2 viral proteins, we performed electromobility shift assays (EMSA) on TIP mRNA and viral proteins. Since the RdRp complex and the N protein can directly interact with viral RNAs (Baric et al., 1988; Iserman et al., 2020) , we hypothesized that these proteins were the most likely to be competition substrates for the TIP. EMSA analysis of cell extracts expressing either RdRp complex or N protein, incubated with purified TIP1 or TIP2 RNA, show that TIP RNAs bind both RdRp complex and N proteins, whereas Ctrl RNA does not bind either of these proteins ( Fig. 4b) .

To quantify the R 0 of TIPs in the context of SARS-CoV-2 infection, we modified the supernatant-transfer assay into a '1 st round supernatant transfer assay'. TIP-transfected cells were infected at a low MOI (MOI=0.05), then thoroughly washed to remove virus, and at two hours post infection GFP+ reporter cells were introduced to the culture (at ~20% of total cells). TIP mobilization into reporter cells was quantified using the percentage of mCherry+ cells within the GFP+ population at 12hrs post infection. Infection-dependent mobilization Page 11 of 56 was confirmed by comparing to uninfected samples for all RNAs (Fig. 4c, d) and we verified that the control RNAs did not mobilize either in the absence or presence of virus (Fig S3d, e) , with the exception of 5'UTR, as expected, given that it carries the putative packaging signal (Iserman et al., 2020; Makino et al., 1990) . The fraction of TIP+ cells, approximately 8%, was corrected for background autofluorescence, to yield 6.3% TIP+ cells (as compared to approximately 5% infected cells for the original SARS-CoV-2 infection at MOI=0.05) and

this translated to 4% infected cells after accounting for the addition of 20% GFP+ cells in the assay. TIPs propagating into 6.3% of new cells from the initial wild-type infection of 4% of cells represents a roughly 50% increase or roughly an R 0 = 1.57; for comparison, R 0 =2 would require a doubling, from 4% to 8%, of cells being mCherry+. This R 0 >1 finding for TIPs is further verified below using a continuous serial-passage approach (see Fig. 5 ).

To verify that TIP RNA was packaged into virions at a high level, we quantified the relative fraction of TIP RNA versus SARS-CoV-2 genomic RNA in virions isolated from supernatant by RT-qPCR (Fig. 4e) . Analysis showed that the TIP RNA was significantly enriched (1.5-2

fold) compared to SARS-CoV-2 viral genomes (for standard curves see Fig. S3f ).

Overall, these data indicate the TIPs do not restrict viral entry or early viral expression (i.e., via induction of a cellular response), that TIP RNA generates functional TIP VLPs in the presence of M, N, E, and S, that TIP RNAs bind to, and may compete for SARS-CoV-2 proteins in cells, and that competition for packaging and replication resources is sufficient to quantitatively account for the measured TIP-mediated yield reduction.

Previous theoretical analyses for other rapidly evolving RNA viruses (e.g., HIV-1) (Metzger et al., 2011; Rast et al., 2016; Rouzine and Weinberger, 2013) predicted that TIPs would have a higher genetic barrier to the evolution of resistance. Based on modeling and comparative sequence analysis indicating that the SARS-CoV-2 UTRs are highly conserved and evolve relatively slowly (Chan et al., 2020; Rangan et al., 2020) SARS-CoV-2 replicative fitness was enhanced by ~1-Log over 3 weeks in the Ctrl RNA continuous culture (Fig. 5b) . This fitness increase was likely due to the furin cleavage mutation in the S gene, which we confirmed via sequencing was overrepresented in the day 20 culture (Fig. S3g) , and which has previously been reported to arise rapidly in Vero cells (Johnson et al., 2021) .

In contrast, the continuous cultures initiated in the presence of TIP RNA exhibited an immediate ~2-Log decrease in viral titer by PFU (Fig. 5b) , consistent with single-round yield reduction data (Figs. 2, 3) . This reduction in viral titer was sustained over the course of the 20-day culture.

To verify that this viral load reduction in the continuous culture was due to TIP interference and not a cellular peculiarity, supernatant from a parallel control culture after day 20 was used to infect cells in the presence of TIP RNA, and the 2-Log decrease in viral titer was recapitulated (Fig. 5c) . RT-qPCR analysis of the culture supernatants indicated that TIP RNA exhibited a 4-fold increase relative to SARS-CoV-2 RNA on day 20 (Fig. 5d) . These continuous culture data indicate conditional amplification and sustained transmission of the TIP, i.e., R 0 >1, since the TIP RNA was only added to the infected culture once (i.e., a single administration on day 0). The data are not consistent with recombination hypotheses (e.g., of the TIP reporter into the wild-type virus). Indeed, recombination leading to extinction of TIPs would result in a fitness increase in the TIP continuous culture (i.e., rescue), whereas a recombinant virus with reduced fitness would be selectively outcompeted by a wild type with a 2-3 Log replicative advantage. We further analyze recombination potential below.

Given the TIP mechanism of action and relatively high barrier to resistance, we hypothesized that TIPs would robustly inhibit SARS-CoV-2 variants of concern, including variants that are resistant to antibody neutralization . To test this hypothesis, we performed yield-reduction assays for the South African variant of concern (501Y.V2, a.k.a., B.1.351) as well as the UK variant of concern (B.1.1.7) (Fig. 6a) . TIP RNA generated ~2-Log reduction in viral titers (PFU/ml) for both variants and RT-qPCR showed significant yield reductions.

Notably, the TIP-mediated reduction in viral RNA was reduced compared to the effect on J o u r n a l P r e -p r o o f

Page 13 of 56 PFU, likely due to known complications of excess sub-genomic RNA (Sia et al., 2020) .

Remarkably, dose-response analysis showed that the half maximal inhibitory concentrations (IC 50 s) of TIP1 for the WA-1 historical isolate and neutralization-resistant B.1.351 variant were indistinguishable (1.50.510 -7 M versus 1.20.310 -7 M) (Fig. 6b) , indicating no loss in TIP interference potential. This IC 50 of ~100 nM is similar to a recently reported broadspectrum SARS-CoV-2 antiviral advancing to clinical trials (Sheahan et al., 2020) .

To further determine limits for TIP robustness to mutational escape, we tested if TIP RNA could efficiently bind to proteins of the evolutionarily distinct beta coronavirus lineage SARS-CoV. Analyses indicate that the SARS-CoV virus (Guan et al., 2003) and SARS-CoV-2 diverge by about 20% in sequence (Kaur et al., 2021) . Given that the mechanism of TIP interference for SARS-CoV-2 involves competition for N protein, we assayed if TIP RNA could efficiently bind to SARS-CoV N protein. The N protein from both SARS-CoV-2 and SARS-CoV were expressed from cDNAs (Gordon et al., 2020a) in cell culture and cell extracts were co-incubated with purified TIP RNA and analyzed by EMSA (Fig. 6c) . The

EMSAs show that TIP RNA binds SARS-CoV N protein about as efficiently as SARS-CoV-2 N protein, suggesting that the virus would need to evolve farther away in sequence space than SARS-CoV to escape competitive inhibition by the TIP.

To assay the in vivo efficacy of TIPs, we utilized the Syrian Golden Hamster model of SARS-CoV-2 infection (Sia et al., 2020 ). First, we tested intranasal administration of various RNA delivery approaches for their ability to efficiently deliver RNA to the respiratory tract of rodents. Using an in vitro transcribed luciferase-expressing RNA, we tested purified RNA alone ('naked RNA'), RNA encapsulated into cationic polymer nanocarriers (i.e., polyethylenimine), and RNA encapsulated in lipid nanoparticles (LNPs) .

LNPs exhibited efficient in vivo RNA delivery to the lungs after intranasal administration ( Fig. S4a) . We generated LNPs containing either TIP1 RNA or Ctrl RNA, characterized them and confirmed that LNP-encapsulated TIP RNA retained antiviral efficacy using yieldreduction assays in Vero cells (Fig. S4b ).

Page 14 of 56

Next, we administered the TIP or Ctrl RNA LNPs intranasally to Syrian Golden hamsters and then challenged them with SARS-CoV-2 (10 6 PFUs) (Fig. 7a) . As expected, control-treated hamsters showed weight loss following infection, but this was significantly ameliorated by TIP treatment (Fig. S4c) . Previous studies indicated that the observed weight retention would quantitatively correlate with a 2-3 Log reduction in SARS-CoV-2 viral load in the lungs (Rogers et al., 2020) . Analysis of infectious virus in lung tissue harvested on day 5 from hamsters confirmed a significant ~2-Log reduction in SARS-CoV-2 viral load in TIP-treated animals ( Fig. 7b) . One animal did not exhibit a reduction in viral load which may be consistent with inefficient TIP dosing/delivery. RT-qPCR analysis of viral transcripts in the lung exhibited a correlated, but lesser, 1-Log reduction in viral load for TIP-treated animals ( Fig. 7c) , consistent with previous studies in SARS-CoV-2 infected hamsters (Sia et al., 2020) .

To determine if conditional propagation of TIPs correlated with SARS-CoV-2 inhibition in vivo, we also analyzed TIP expression in the lungs on day 5 by RT-qPCR, and observed high levels of TIP RNA (Fig. 7d) , whereas Ctrl RNA on day 5 was present at substantially lower levels ( Fig. 7e) . Moreover, to confirm that presence of SARS-CoV-2 infection is obligatory for conditional propagation of TIPs, we compared the amount of TIP or Ctrl RNA in the presence vs. absence of virus on day 5 in hamster lungs. Ctrl RNA levels in the lungs were unaffected by SARS-CoV-2 infection, which starkly contrasted with TIP RNA that was significantly amplified by 4-Log in the presence of SARS-CoV-2 infection (Fig. S4d) . All RT-qPCR threshold cycle (Ct) values for luciferase in the TIP-treated animals and mCherry in the control animals were >30, indicating negligible non-specific amplification;

nevertheless, by convention, normalized RNA values for these samples were reported.

Since inflammation has been implicated in SARS-CoV-2 pathogenesis (Lucas et al., 2020) ,

we assessed cytokine and interferon responses in the lungs of infected animals by performing RNA sequencing (RNAseq). Analysis of hamster lung samples showed that TIP-treated animals could be clearly differentiated from control-treated animals, with 206 upregulated genes and 233 downregulated genes (Fig. S5a) . These differentially expressed genes (DEGs) form four clusters when analyzed together with uninfected hamster lung samples (Fig. S5a ).

The majority of downregulated genes in TIP-treated animals were interferon-stimulated genes (ISGs) (157 out of 233; Fig. S5b ), especially for genes in cluster III (97 out of 121; 2020)-were significantly reduced in TIP-treated animals ( Fig. 7g and Fig. S5d) .

Importantly, DEGs that can distinguish TIP-treated from Ctrl-treated in infected animals cannot separate TIP from control in uninfected animals ( Fig. S5a vs. Fig. S4f ), indicating the alleviated proinflammatory immune response is infection-dependent and not solely due to TIP RNAs.

Given the reduced inflammatory profile in TIP-treated animals, we performed histological analysis of day 5 hamster lung tissue samples. This revealed dramatic differences in the lungs of Ctrl vs. TIP-treated animals ( Fig. 7h) , with control animals exhibiting signs of severe pulmonary edema not present in TIP-treated animals. Specifically, despite all animals exhibiting some signs of inflammation consistent with infection, control animals evidenced pronounced alveolar edema and conspicuous cell infiltrates in alveolar spaces (Fig. 7i) ,

indicating severe vascular leakage. In stark contrast, lungs of TIP-treated animals showed substantially less edema and cell infiltration, which is linked to heart failure (Cotter et al., 2001) . Histopathological scoring of the images (Fig. S6a ) indicated significant reductions in alveolar edema and cell infiltrates in the TIP-treated hamsters (Fig. 7i) . Uninfected hamsters treated with either TIP or Ctrl RNA LNPs were used as control and showed non-significant difference in the alveolar edema and infiltrates, confirming the severe vascular leakage is due to viral infection (Fig. S4e) .

To test the efficacy of TIPs in a post-exposure therapeutic setting, hamsters were inoculated with SARS-CoV-2 (10 6 PFUs) and then given a single intranasal administration of LNP TIP or LNP Ctrl RNA at 12 hrs post infection ( Fig. 7j )based on previous post-exposure timing used for other therapeutic antivirals now in clinical development (Sheahan et al., 2020) , and in vitro analysis that a 12 hrs post-infection administration could yield viral knockdown (Fig. S2b ). In agreement with the above results, we observed a significant reduction in SARS-CoV-2 viral load ( Fig. 7k) as well as reduced pathogenesis in the lungs of animals at day 5 ( Fig. 7l-m, S6b ).

Together, these data demonstrate that a synthetic sub-genomic viral-deletion mutant can conditionally replicate to durably suppress a virus infection (i.e., SARS-CoV-2) in vivo, thereby constituting a Therapeutic Interfering Particle (TIP). If successfully translated to the clinic, TIPs could represent a class of single-administration antiviral with a high genetic barrier to the evolution of resistance. Below, we discuss potential clinical translational paths for this class of antiviral intervention in comparison to other interventions, the longer-term evolutionary considerations, and the limitations of the present study.

The therapeutic and vaccine landscape has migrated significantly throughout the course of the COVID-19 pandemic with the emergence of variants that appear to have increased ability to evade immunity and vaccines. Whereas early after the introduction of vaccines the perceived unmet need was largely for post-infection hospital administration therapeutics for unvaccinated individuals, the recent emergence of vaccine-resistant escape variants coupled with the increased availability of at-home rapid tests has highlighted the unmet need for preand post-exposure prophylactics. Previously, this unmet clinical need focused primarily on certain individuals (i.e., immunosuppressed from chemotherapy, biologics, transplants, etc.) who could not mount an immune response to the vaccines, and thus required a prophylactic.

However, the general consensus now is that this need has expanded to the general population at large and active development of such prophylactic treatments for people once they have been exposed remains a goal as there is no agent to prevent infection after someone has been exposed to SARS-CoV-2.

Monoclonal antibodies (mAbs) (e.g., Bamlanivimab and the REGN COV cocktail) are being tested as prophylactic agents in nursing home patients (Sheahan et al., 2020) .

Relative to these interventions, TIPs exhibit substantial reduction in SARS-CoV-2 viral load per dose delivered. Specifically, for a 0.44 mg/kg dose in hamsters, TIPs exhibit a ~2-Log reduction in infectious SARS-CoV-2 viral load (Fig. 7b) . In comparison, mAb studies in mice show that at a 2 mg/kg dose can produce a 4-Log viral-load decrease , or that a 10 mg/kg dose can produce a 3-Log reduction (Martinez et al., 2021) . In hamsters, mAb studies have reported that a 18 mg/kg dose can produce a 1-Log reduction (Kreye et al., 2020) , a 16.5 mg/kg dose can produce a 2.5-Log reduction (Rogers et al., 2020) , or that a 0.5-50 mg/kg dose produces no significant effect on viral load (Baum et al., 2020) . Similarly, hamster studies of small-molecule inhibitors of SARS-CoV-2 (i.e.,

Molnupiravir) have reported that a 250 mg/kg 12hr-repeated dose generates a 2-Log viralload reduction (Rosenke et al., 2021) , or a 200 mg/kg dose generates a 2-Log reduction (Abdelnabi et al., 2021a )-with similar results for Favipiravir (Abdelnabi et al., 2021b) .

Notably, these studies in SARS-CoV-2 infected rodents consistently show that RT-qPCR analysis generates a lesser (by 1-Log) reduction in viral load, likely due to residual noninfectious RNA fragments-hence the focus on infectious viral-load reduction as measured by PFU. Overall, relative to mAbs and small-molecule antivirals, TIPs appear to have a comparable antiviral effect at a substantially reduced dose (i.e., 0.44 mg/kg for TIPs versus 4-250 mg/kg for mAbs and small molecules).

Like these other antivirals under development, TIPs could serve as similar pre/post-exposure prophylactic therapies based on the current precedent of Oseltamivir (Tamiflu) pre/postexposure prophylaxis for influenza in at-risk household-exposure settings (Hayden et al., 2004 ) and the data above show that intranasal TIP delivery could act as a singleadministration intervention. Notably, TIP-mediated reduction in SARS-CoV-2 viral load, like mAbs and small-molecule inhibitors, could generate long-term protection to re-infection akin to protection from natural infection, which may offer more durable protection against SARS-CoV-2 reinfection than current vaccines (Gazit et al., 2021) . Historically, natural immunity and live-attenuated vaccines provide more durable and effective protection than subunit vaccines for diverse viruses including influenza, rubella and others (Christenson and Bottiger, 1994; Cox et al., 2004; Horstmann et al., 1985; Johnson et al., 1986) .

It remains unclear whether TIP-like deletions of SARS-CoV-2 have spontaneously arisen and spread. From an evolutionary perspective, it is also not immediately obvious why endogenous TIPs have not naturally evolved to limit virus infections and the considerations are complex, particularly in a virus's natural hosts (Daugherty and Malik, 2012) . For lentiviruses such as HIV, the barrier appears more straightforward, as R 0 >1 variants appear to require recombination 'acrobatics' since a cis element necessary for efficient transmission is within the TIP deletion and must be recovered and repositioned outside the deleted region (Notton et al., 2021) . However, for flaviviruses there is evidence that natural DIPs arise and transmit through host populations (Aaskov et al., 2006) and historical hypotheses have asserted that such DIPs have biological fitness roles aiding the parent wild-type virus. For example, it has been postulated that such DIPs may enhance virus persistence, serve as immunological decoys, or reduce pathogenicity of wild-type virus to enable transmission (Vignuzzi and Lopez, 2019) , though our analysis ( Fig. S1g) indicates that TIPs would reduce transmission of SARS-CoV-2 and bring the R value below 1, leading to contraction of epidemic spread.

Similar to gene drives (Burt, 2003) , there are understandable fears that TIPs may drive evolution of increased wild-type virus virulence. This theoretical possibility has been previously addressed at both the host and population scales (Rast et al., 2016) and the hypothesis of TIPs driving increased virulence does not appear consistent with selection theory, but additional in vivo empirical testing will be the best way to address this concern.

There is also the possibility that the virus will evolve to escape from TIP. For example, one proposed escape mechanism is upregulation of viral packaging proteins in order to outcompete the TIP and diminish interference; however, this particular compensation mechanism was previously found to be evolutionarily non-robust as it also provides an equivalent excess of trans elements for TIP packaging (Rouzine and Weinberger, 2013) .

Whereas the current TIPs generate a significant protection from disease via a 2-Log reduction in SARS-CoV-2 viral load (Fig. 7b) , computational models ( Fig. 1) argue that this viral-load reduction could be enhanced by engineering to optimize TIP transmission (ρ) and

interference (ψ) parameters. Specifically, the parameter ρ helps reduce the viral load by spreading the TIP to more cells in the tissue. This is the reason for the seemingly counterintuitive effect that moderate values of ψ result in the largest reduction in patient viral load (Fig. 1e) . Since the TIP requires wild-type virus to mobilize, if ψ is too large (generating too much inhibition) the TIP essentially 'shoots itself in the molecular foot' and less virus is available to mobilize the TIP. This phenomenon was previously predicted for HIV (Metzger et al., 2011; Rouzine and Weinberger, 2013; Weinberger et al., 2003) . In essence ρ and ψ generate a type of synergistic effect at the whole tissue scale. As such, optimization of TIPs by enhancing ρ via addition of specific packaging signals (Iserman et al., 2020) could generate more effective TIPs.

As with all models, the computational analysis herein is a relatively simple representation of a complex system and necessarily makes certain assumptions. Nevertheless, despite these limitations, this model of SARS-CoV-2 replication in the human airway generates predictions of TIP efficacy ( Fig. 1 ) that appear roughly in line with the in vivo hamster qRT-PCR data from challenge experiments (Fig. 7c )-we note that the human model is calibrated on patient RT-PCR data, not PFU data, so the comparison to PFU data is arguably less relevant.

While our cell-culture measurements show that mobilization of TIPs in the presence of SARS-CoV-2 infection exhibit an R 0 >1 (Fig. 4c) , different cell types and contexts outside of tissue culture may alter this result, though the hamster data appear to show substantial mobilization of TIPs in animal lungs in vivo (Fig. 7d) . The use of reporter expression (mCherry) to quantify transmission does raise concerns regarding recombination of the reporter into the wild-type virus, thereby causing the reporter to be an indicator of virus rather than TIP transmission, but we have not seen evidence of recombination even in vivo ( Fig. S7a-d) .

Moreover, persistent TIP-mediated knockdown of viral load in continuous cultures (Fig. 5ac) is not parsimonious with recombination of mCherry, which could only occur if a less fit recombinant was carrying mCherry. Such a less-fit recombinant would be selected against, and a higher fitness non-mCherry virus would dominate, but this was not observed in our system.

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One concern is that the continuous culture may not have been run for sufficient time to enable selection of escape mutants, although for many viruses (Coffin, 1995; Schnipper and Crumpacker, 1980; Turner and Chao, 1999) , the time frames we used are sufficient for evolution of resistance. Further, the 1-Log increase in fitness in the control (and the emergence of the furin cleavage mutation; Fig. S3g ) argues that 20 days is sufficient for selection. Moreover, the binding of TIP RNA to the N protein from SARS-CoV (Fig. 6c) argues that there may be an extremely high barrier to the evolution of viral escape and a correspondingly long time frame needed to select for such putative escape variants.

While clinical translation of this technology would still need to overcome significant regulatory and other challenges, the data above demonstrate the potential of intranasal lipidnanoparticle TIPs, and mRNA technology faced similar challenges and skepticism until it was successfully adopted as the basis for SARS-CoV-2 vaccines. These proinflammatory cytokines (Ccl7, Ccr1, Cxcl10, Cxcl11) were previously reported to be upregulated in COVID-19 patients but are significantly reduced in TIP-treated animals.

[For all panels: * denotes p<0.05 from Student's t test]. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Leor Weinberger (leor.weinberger@gladstone.ucsf.edu).

All unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer agreement. All the cells were cultured under 5% CO2 in a humidified incubator at 37 o C.

A previous ordinary differential equation model of within-host SARS-CoV-2 infection (Ke et al., 2020) was extended to include TIPs, by introducing two new parameters (burst size of TIP particles relative to SARS-CoV-2 particles from dually infected cells, i.e., infected with both TIP and wild-type virus) and (burst size of SARS-CoV-2 from dually infected cells, compared to the burst size of SARS-CoV-2 from cells infected with only wild-type virus).

Similar to previous approaches (Rouzine and Weinberger, 2013; Weinberger et al., 2003) , the model was also expanded to include state variables for TIP infected cells, dually infected cells, and TIP particles. To simulate treatment with TIPs, a fraction of target cells 1 and 2 in the upper and lower respiratory tracts are converted into TIP-carrier cells 1, and 2, , as in previous TIP models. TIP entry into cells, clearance of TIPs and TIP-containing cells, and transport between the upper and lower respiratory tract are assumed to be equivalent for TIP and wild-type virus. The model equations are provided below; parameter descriptions and values are described in Table S2 . For analytical expediency, a minor modification is made to the initial conditions from (Ke et al., 2020) , setting the initial virus in the URT ( 1 ( = 0)) rather than the initial infected cells in the URT ( 1 ( = 0)). Since viral entry is fast (minutes) relative to the timescales of patient infection (days-weeks), the initial exposure is well-captured by specifying initial virions instead of infected cells. This expediency simplified our sensitivity analysis for dose regimens. That is, we do not have to vary both ( = 0) and 1, ( = 0). Importantly, this modification does not change our overall results: the no-TIP predictions match well to the original model, and the efficacy of TIPs are robust to the initial viral inoculum.

We assessed the robustness of the within-host model predictions by incorporating TIPs into a second simpler within-host model, also validated on clinical samples (Kim et al., 2021 ). This simpler model only has four state variables:

where denotes the fraction of target cells remaining, denotes the fraction of TIP-carrier target cells remaining, is SARS-CoV-2 per ml, and is TIPs per ml. First, and set TIP efficacy as described above. This model assumes SARS-CoV-2 (and TIPs) are in quasisteady-state with respect to the number of infected cells. As a result, we do not explicitly model the infected cell state, and instead have the aggregated parameter ≡ 0 / where is the per cell viral production rate, 0 is the number of initial target cells, and is the rate of viral clearance. The other parameters and represent infectivity and infected cell clearance. Sensitivity analysis allowed and to vary over several orders of magnitude, though we focus on = 1.5 and = 0.02. Half the target cells were converted to TIP carriers prior to virion exposure ( 0 = ,0 = 0.5). These simulations were performed for thirty individual parameter sets calibrated to patient samples from Singapore, China, Germany, and Korea by (Kim et al., 2021) , reproduced in Table S3 for completeness.

A probabilistic model of SARS-CoV-2 transmissions using viral dynamics (Goyal et al., 2021) to calculate transmission risk was extended and recalibrated to predict TIP efficacy and TIP transmission. Transmission model calibration was done as follows: SARS-CoV-2

infections were simulated using a patient-validated model, producing viral load dynamics of 10,000 untreated infections based on per-patient parameter uncertainty (Ke et al., 2020) .

Viral load dynamics were converted to transmission risk dynamics using = /( + ), with time-varying log10 viral load and parameters = 10.18 and = 7.165 set based on . Each individual had simulated daily contacts drawn from the Gamma distribution ( / , ), whose parameters were fit by minimizing the error between measured and predicted secondary transmission distributions (resulting in =10, =10). New secondary transmission events were estimated using a one-day timestep.

After calibration to untreated transmissions, we simulated 10,000 TIP-treated index patients and predicted changes in both SARS-CoV-2 secondary transmissions and TIP secondary transmissions (TIP parameters =1.5 and =0.02 as above). We assumed identical transmission risk parameters for TIP and SARS-CoV-2, so transmission differences were purely driven by changes in viral load.

To model direct administration of TIP to patients, we assumed that half the target cells in the upper and lower respiratory tract received TIP. To model indirect administration of TIP (i.e., due to person-to-person transmission) we assume an equivalent dose of TIP as to SARS-CoV-2.

All simulations were performed in Python. NumPy (v1.19.4) and SciPy ( 

All gene fragments and PCR primers used (see Table S1 ) were obtained from Integrated DNA Technology and assembled using standard molecular cloning techniques. RNA was in vitro transcribed from 1g of agarose gel-purified band corresponding to the intended size J o u r n a l P r e -p r o o f England Biolabs Inc.) and a poly-A tail added using E.coli Poly(A) polymerase (cat#M0276S, New England Biolabs Inc.). Transcribed RNA was purified using phenolchloroform extraction. Briefly, an equal amount of phenol:chloroform was added to the RNA, followed by vortexing for 10 seconds and centrifugation at 10,000 r.p.m. for 10 minutes at room temperature. The aqueous phase was harvested and one volume of isopropanol was added to the RNA, incubated for 5 minutes at room temperature, centrifuged for 10 minutes at 10,000 r.p.m, washed twice with ice-cold 70% ethanol and the pellet was resuspended in nuclease free water. Naïve Vero cells were transfected with either Ctrl or TIP RNAs at a concentration of 1 g RNA per 1 million cells using 4D-Nucleofector (cat# AAF-1002B, Lonza Inc.) and the SE cell-line 4D nucleofector kit (cat#V4XC-1012, Lonza Inc).

For post-infection therapy experiment, we transfected SARS-CoV-2 infected cells using

Lipofectamine 3000 transfection reagent (cat# L3000001, Thermofisher Scientific) at 8 or 16

hrs post infection at the concentration of (1 g RNA per 1 million cells). mix (cat#4309155, Thermofisher Scientific) with sequence specific primers. All the RT-qPCR measurements were normalized to GAPDH or beta-actin (Table S1 ). For quantification of relative packaging of RNA in virions, Vero cells were nucleofected with TIP1 or TIP2 RNA (1g RNA for 1 million cells), and infected with SARS-CoV-2 (MOI=0.05) at 24 hours post nucleofection. Supernatants were harvested at 24 hours post infection, followed by qPCR using mCherry and E gene primers. TIP fold enrichment was quantified relative to viral genome.

Infectious virus titers were quantified by plaque forming unit (PFU/ml) assay on Vero cells.

Briefly, Vero cells were prepared by plating as a confluent monolayer in 12-or 24-well plates 24 hours before performing the plaque assay. On the day of the plaque assay, media was aspirated, cells were washed with 2ml of PBS, 250l of diluted virus in modified DMEM media (DMEM, 2%FBS, L-glut, P/S) was added to confluent monolayer followed by incubation at 37 o C for 1 hour with gentle rocking every 15 minutes. After one hour of incubation, 2 ml of overlay media (1.2% Avicel in 1X MEM) was added to each well. At 72h post infection, overlay media was aspirated, monolayer was washed with PBS and fixed with 10% formalin for 1 hour. Plaques were stained with 0.1% crystal violet for 10 minutes and washed with cell culture grade water three times, followed by enumeration of plaques and viral titer calculation to pfu/ml.

Virus yield-reduction assays were performed by transfecting Vero cells with TIP or Ctrl

RNAs ( stain was added for 10 minutes, followed by two washes in PBS. High-throughput microscopy was performed on an ImageXpress-Confocal Microscope, and images were analyzed using MetaXpress software.

Organoids were generated as described previously (Sachs et al., 2019; Zhou et al., 2018) (cat#100-19, Peprotech), 20ng/ml FGF-10 (cat#100-26, Peprotech), 100g/ml primocin (antpm-1) and 5nM heregulin beta-1 (cat#100-03, Peprotech).

Medium was changed every 4 days and organoids were passaged when drops became too dense. Media was carefully aspirated and BME drops were collected in with ice-cold AdV+++ media, and transferred to 15 ml tubes. Drops were manually dissociated using sterile 1ml pipette tip and organoids were pelleted at 500g, 4°C for 5 min. Cells were washed with ice-cold AdV+++ to remove any remaining BME and centrifuged again. Then, the pellet was dissociated with 10x TrypLE (Invitrogen, A1217701) and incubated in a 37°C

incubator for up to 10 min. AdV+++ was added, cells were centrifuged and resuspended in ice cold 3:1 BME in AdV+++. Finally, 50 L drops were formed and covered in AO medium as described. Organoids were imaged every other day to observe their morphology using transmission microscopy. For transfecting organoids, organoids were trypsinized and cells spun at 800g for 5 minutes, supernatant was removed and cells were plated at high density in organoid media, followed by preparation of RNA-lipofectamine 3000 complexes using standard lipofectamine protocol. 4ml of Lipofectamine 3000 reagent (in Opti-MEM media) and 1g of RNA were mixed together, incubated for 5 minutes, and added to cells (50 l per well). Plates were centrifuged at 600g at 32 o C for 1 hour, and incubated for 4 hours in 37 o C incubator with 5% CO 2 , followed by resuspending in AO medium with ice-cold BME diluted 1:500 (2%) to a final concentration of 0.5 million cells/ml. Then, 200 l of cell suspension/well was added to 8-well chamber slides pre-coated with BME 1:100. Cells were allowed to attach for at least 1 day. In BSL3, media was removed and fresh media with SARS-CoV-2 virus was added at MOI=0.5. Cells were incubated at 37°C for 2 hours and then media was replaced with warm AO medium without virus. Slides were incubated for one day until harvested for measuring viral load by RT-qPCR and plaque assay.

Vero cells were co-transfected with 0.5 g of expression plasmids for nucleocapsid (N), matrix (M), and envelop (E) proteins plus 25 ng of expression plasmid for spike (S) protein (all kindly provided by Nevan Krogan and as described previously (Gordon et al., 2020b) and Mice were 4-6 weeks old at time of experiments. A total of 9 mice were injected either with lipid nano particle (LNP), naked RNA, or saline control. n=3 mice were intranasally instilled with 20 l (10ul each nostril) LNP, n=3 mice were intranasally instilled with 20 l (10 l each nostril) naked RNA, and n=3 mice were intranasally instilled with 20 l (10 l each nostril) saline control. All mice were injected or instilled within 30 mins and bioluminescence was analyzed at 6 hours post injection. The in vivo imaging system (Lago in vivo Imaging System, Spectral Instruments Imaging) was used for bioluminescence. D-Luciferin potassium salt (PerkinElmer), the substrate for firefly luciferase, was dissolved in phosphate-buffered saline at a concentration of 30 g/ml and filtered through a 0.22-mpore-size filter before use. Mice were injected with 100 l of luciferin (3g) and

immediately anesthetized in an oxygen-rich induction chamber with 3-5% isoflurane. The mice were transferred to imaging box and positioned in ventrodorsal and lateral positions for imaging. Mice were maintained for at least 5 min so that there was adequate dissemination of the injected substrate and so that the animals were fully anesthetized. Images were taken using Aura imaging software (Spectral Instruments Imaging) using the following settings:

120-180s acquisition time and heavy binding. Imaging analysis was done using the Aura imaging software; quantitation of signal was done using regions of interest (ROI) over mice and recorded as photons per second (flux).

8-week old Syrian golden hamsters were infected through intranasal installation of 10 6 PFU per animal of SARS-CoV-2 (USA-WA1/2020) in 100 l of DMEM, as described (Rogers et al., 2020) . imaging. Briefly, formalin-fixed lung from each animal group were processed and paraffin embedded, and tissue sections were stained with hematoxylin and eosin (H&E) as described (Fischer et al., 2008) , imaged, and images were analyzed using Leica Aperio ImageScope software. To assess the statistical significance of the observed variation between the scoring of control and therapeutic histopathology microscopy, we performed a permutation test (Manly, 2006; Neuhauser and Manly, 2004) , an extension of the Fisher exact test for multinomial variables via random sampling. We randomly partitioned concatenated control and variable observations 100,000 times, then tabulated the null hypothesis probability that

 SARS-CoV-2 does not evolve to escape TIPs  In hamsters, a single intranasal administration of TIPs reduces viral load in lungs  TIPs suppress inflammation and severe disease when given pre-or post-infection A defective viral particle derived from SARS-CoV-2 competes with the full virus for resources to replicate, showing therapeutic potential by inhibiting viral proliferation in culture, and reducing viral load and pathology in animal models for infection.

J o u r n a l P r e -p r o o f 

Ctrl TIP1

Ctrl TIP1

Ctrl TIP1 Uninf.

Long-term transmission of defective RNA viruses in humans and Aedes mosquitoes

Molnupiravir Inhibits Replication of the Emerging SARS-CoV-2 Variants of Concern in a Hamster Infection Model

The combined treatment of Molnupiravir and Favipiravir results in a potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection model

High-Throughput Single-Cell Kinetics of Virus Infections in the Presence of Defective Interfering Particles

Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription

REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Site-specific selfish genes as tools for the control and genetic engineering of natural populations

Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma

Conserved Genomic Terminals of SARS-CoV-2 as Co-evolving Functional Elements and Potential Therapeutic Targets. bioRxiv

In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains

Group-specific structural features of the 5'-proximal sequences of coronavirus genomic RNAs

Measles antibody: comparison of long-term vaccination titres, early vaccination titres and naturally acquired immunity to and booster effects on the measles virus

HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy

Bamlanivimab prevents COVID-19 morbidity and mortality in nursing-home setting

Pulmonary edema: new insight on pathogenesis and treatment

Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines

Rules of engagement: molecular insights from host-virus arms races

Hematoxylin and eosin staining of tissue and cell sections

Comparing SARS-CoV-2 natural immunity to vaccine-induced immunity: reinfections versus breakthrough infections. medRxiv

Outwitting evolution: fighting drug-resistant TB, malaria, and HIV

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Viral load and contact heterogeneity predict SARS-CoV-2 transmission and super-spreading events

Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China

Identification of SARS-CoV-2 inhibitors using lung and colonic organoids

Management of influenza in households: a prospective, randomized comparison of oseltamivir treatment with or without postexposure prophylaxis

Generation and replication of defective viral genomes

Persistence of vaccine-induced immune responses to rubella: comparison with natural infection

Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis

Immunity to influenza A virus infection in young children: a comparison of natural infection, live cold-adapted vaccine, and inactivated vaccine

Estimating infectiousness throughout SARS-CoV-2 infection course

Genetic comparison among various coronavirus strains for the identification of potential vaccine targets of SARS-CoV2

Kinetics of SARS-CoV-2 infection in the human upper and lower respiratory tracts and their relationship with infectiousness. medRxiv

The Architecture of SARS-CoV-2 Transcriptome

A quantitative model used to compare within-host SARS-CoV-2, MERS-CoV, and SARS-CoV dynamics provides insights into the pathogenesis and treatment of SARS-CoV-2

A Therapeutic Non-self-reactive SARS-CoV-2 Antibody Protects from Lung Pathology in a COVID-19 Hamster Model

Longitudinal analyses reveal immunological misfiring in severe COVID-19

Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal

Randomization, bootstrap and Monte Carlo methods in biology

Prevention and therapy of SARS-CoV-2 and the B.1.351 variant in mice

Autonomous targeting of infectious superspreaders using engineered transmissible therapies

Targeting Antibiotic Tolerance, Pathogen by Pathogen

Impact of mRNA chemistry and manufacturing process on innate immune activation

The Fisher-Pitman permutation test when testing for differences in mean and variance

High-Throughput Method to Map Viral cis-and trans-Acting Elements

Casirivimab with imdevimab antibody cocktail for COVID-19 prevention: Interim results

Modelling viral and immune system dynamics

Dynamics of HIV-1 and CD4+ lymphocytes in vivo

The evolution of seasonal influenza viruses

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look

Conflicting Selection Pressures Will Constrain Viral Escape from Interfering Particles: Principles for Designing Resistance-Proof Antivirals

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

Orally delivered MK-4482 inhibits SARS-CoV-2 replication in the Syrian hamster model

Design requirements for interfering particles to maintain coadaptive stability with HIV-1

Long-term expanding human airway organoids for disease modeling

AI-guided discovery of the invariant host response to viral pandemics

Resistance of herpes simplex virus to acycloguanosine: role of viral thymidine kinase and DNA polymerase loci

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

Pathogenesis and transmission of SARS-CoV-2 in golden hamsters

Production of Defective Interfering Particles of Influenza A Virus in Parallel Continuous Cultures at Two Residence Times-Insights From qPCR Measurements and Viral Dynamics Modeling

Prisoner's dilemma in an RNA virus

Defective viral genomes are key drivers of the virushost interaction

Incomplete forms of influenza virus

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7

Theoretical design of a gene therapy to prevent AIDS but not human immunodeficiency virus type 1 infection

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants. bioRxiv

A Thermostable mRNA Vaccine against COVID-19

Differentiated human airway organoids to assess infectivity of emerging influenza virus

Heightened Innate Immune Responses in the Respiratory Tract of COVID-19 Patients