Bs 4482 Pdf
Kazuko Rizza
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The coronavirus disease 2019 (COVID-19) pandemic is having a catastrophic impact on human health1. Widespread community transmission has triggered stringent distancing measures with severe socio-economic consequences. Gaining control of the pandemic will depend on the interruption of transmission chains until vaccine-induced or naturally acquired protective herd immunity arises. However, approved antiviral treatments such as remdesivir and reconvalescent serum cannot be delivered orally2,3, making them poorly suitable for transmission control. We previously reported the development of an orally efficacious ribonucleoside analogue inhibitor of influenza viruses, MK-4482/EIDD-2801 (refs. 4,5), that was repurposed for use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently in phase II/III clinical trials (NCT04405570 and NCT04405739). Here, we explored the efficacy of therapeutically administered MK-4482/EIDD-2801 to mitigate SARS-CoV-2 infection and block transmission in the ferret model, given that ferrets and related members of the weasel genus transmit the virus efficiently with minimal clinical signs6,7,8,9, which resembles the spread in the human young-adult population. We demonstrate high SARS-CoV-2 burden in nasal tissues and secretions, which coincided with efficient transmission through direct contact. Therapeutic treatment of infected animals with MK-4482/EIDD-2801 twice a day significantly reduced the SARS-CoV-2 load in the upper respiratory tract and completely suppressed spread to untreated contact animals. This study identified oral MK-4482/EIDD-2801 as a promising antiviral countermeasure to break SARS-CoV-2 community transmission chains.
MK-4482/EIDD-2801 is the orally available pro-drug of the nucleoside analogue N4-hydroxycytidine (NHC), which has shown potent anti-influenza virus activity in mice, guinea pigs, ferrets and human airway epithelium organoids4,10,11. Acting through the induction of error catastrophe in virus replication4,12, NHC has broad-spectrum anti-RNA virus activity. In addition to ameliorating acute disease, we have demonstrated in a guinea pig transmission model that NHC effectively blocks the spread of influenza virus from infected animals to untreated contact animals11.
Several mouse models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been developed, some of which were also employed to confirm the in vivo efficacy of MK-4482/EIDD-2801 against betacoronaviruses13. However, human SARS-CoV-2 cannot productively infect mice without viral adaptation or introduction of human ACE2 into transgenic animals, and none of the mouse models support transmission to uninfected mice14. Spill-back of SARS-CoV-2 to farmed minks, subsequent large-scale mink-to-mink transmission and, in some cases, zoonotic transmission back to humans revealed efficient viral spread among members of the weasel genus without previous adaptation6,7,8,9. Although mink farms reported elevated animal mortality and gastrointestinal and respiratory clinical signs15, outbreak follow-up revealed continued intra-colony spread for extended periods of time9, suggesting that the acute clinical signs in the majority of infected animals may be mild. These mink field reports corroborated results obtained with experimentally infected ferrets showing that mustelids of the weasel genus transmit SARS-CoV-2 efficiently without strong clinical disease manifestation16,17. As this presentation of SARS-CoV-2 infection resembles the experience of frequently asymptomatic or mildly symptomatic SARS-CoV-2 spread in the human young-adult population18, ferrets represent a relevant model species to assess therapeutic impact on SARS-CoV-2 transmission.
Virus release from the upper respiratory tract peaked three days after infection and was undetectable by day seven in the animals of the high-inoculum group (Fig. 1a and Supplementary Table 1). Infection of animals in the low-inoculum group was less efficient. The shedding profiles correlated closely with the infectious particle load in nasal turbinates; a heavy virus tissue burden in the high-inoculum group was present on day four, which decreased substantially by approximately four orders of magnitude by day ten (Fig. 1b and Supplementary Table 2).
These results demonstrate the oral efficacy of therapeutic administration of MK-4482/EIDD-2801 against acute SARS-CoV-2 infection in the ferret model. Consistent with our previous pharmacokinetic and toxicology work-up of MK-4482/EIDD-2801 in ferrets, treatment did not cause any phenotypically overt adverse effects, and the white-blood-cell and platelet counts of the drug-experienced animals remained within the normal range (Extended Data Fig. 4).
Importantly, treatment suppressed all transmission to the untreated direct contacts despite a prolonged direct proximity of the source and contact animals as well as detectable virus shedding from the source animals at the beginning of the co-housing phase. This complete block may indicate a bottom threshold of the shed SARS-CoV-2 load for successful spread. In addition, the genome integrity of some EIDD-2801-experienced virions shed from the treated animals may have been only partially compromised. Rather than being chain-terminating when incorporated by the viral polymerase, NHC undergoes spontaneous tautomeric interconversions, leading to base pairing either as cytosine or uracil25. The resulting randomly positioned transition mutations induce viral error catastrophe26, causing a collapse of the virus population. This mechanism of antiviral activity of NHC was demonstrated for alphaviruses12, pneumoviruses11 and orthomyxoviruses4, and confirmed to equally apply to betacoronaviruses19 and specifically SARS-CoVs13. In our study, a limited presence of the analogue in the viral genomes generated shortly after the start of treatment may have had a greater impact on natural invasion of an immune-competent host in vivo than on virus replication in type I interferon-deficient cultured cells such as the Vero E6 used for titration27. This view is consistent with the frequent observation (such as in ref. 28) that many mutant viruses can be propagated in cell culture but are attenuated in vivo and incapable of productive host invasion.
Consistent with the conserved antiviral mechanism of action of NHC across diverse viral targets, several previous attempts to induce robust resistance to the compound in alphaviruses12, orthomyxoviruses4 and betacoronaviruses19 were unsuccessful, indicating a high genetic barrier against viral escape. For betacoronaviruses specifically, a very moderate twofold increase in the EC90 concentration was reported after 30 passages in the presence of inhibitor19. Given that these mutations delayed viral replication and thus posed a fitness penalty, it is unlikely that clinical use of MK-4482/EIDD-2801 will result in the emergence of pre-existing resistance in circulating virus populations or trigger the appearance of viral variants with enhanced pathogenicity.
The relative expression of interferon, ISGs and cytokines was determined by real-time PCR analysis. RNA was extracted from PBMCs that were harvested at various time points after infection. Complementary DNA was reverse transcribed with SuperScript III (Invitrogen) using oligo-dT primers and analysed by real-time PCR using Fast SYBR Green master mix (Applied Biosystems). The signals were normalized to glyceraldehyde-3-phosphate dehydrogenase messenger RNA, analysed using the comparative threshold cycle (ΔΔCt) method and expressed relative to day 0 of infection for each respective animal. The sequences of the primers used for the analyses are shown in Supplementary Table 13.
All animal work was performed in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act Code of Federal Regulations. Experiments with SARS-CoV-2 involving ferrets were approved by the Georgia State Institutional Animal Care and Use Committee under protocol A20031. All experiments using infectious SARS-CoV-2 were approved by the Georgia State Institutional Biosafety Committee under protocol B20016 and performed in BSL-3/ABSL-3 facilities at the Georgia State University.
We thank M. Kumar for providing an aliquot of 2019-nCoV/USA-WA1/2020 stock, members of the GSU High Containment Core and the Department for Animal Research for support, and J. Sourimant and A. L. Hammond for critical reading of the manuscript. This work was supported, in part, by Public Health Service grant nos AI071002 (to R.K.P.) and AI141222 (to R.K.P.) from the NIH/NIAID. The funders had no role in the study design, data collection and interpretation or the decision to submit the work for publication.
NGC 4482 is a dwarf elliptical galaxy located about 60 million light-years away[3] in the constellation Virgo.[4] NGC 4482 was discovered by astronomer William Herschel on March 15, 1784. It was rediscovered by astronomer Arnold Schwassmann on September 6, 1900 and was listed as IC 3427.[5] It is a member of the Virgo Cluster.[6]
Due to SCP-4482's ability to manifest anywhere within the Foundation communication network, it has proven beneficial as an aid in alerting of and preventing dangerous events from occurring. Also, it has been proven impossible to isolate SCP-4482 from any Foundation communication network. It has been noted that SCP-4482 spends a majority of its time exploring the different communication networks, although it has gone to a great deal of effort not to eavesdrop on secure or classified calls.
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