WO2024206843A9 - Compositions including modified coronavirus vaccines and uses thereof - Google Patents

Abstract

The present disclosure relates generally to compositions including attenuated modified coronavirus (CoV) vaccines and methods of using the same to prevent coronavirus infection in a subject in need thereof. In particular, the methods comprise administering an effective amount of one or more attenuated modified coronaviruses (e.g., SARS-CoV-2 and MERS-CoV) to the subject.

Description

COMPOSITIONS INCLUDING MODIFIED CORONAVIRUS VACCINES AND

USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to US Provisional Appl. No. 63/456,070, filed March 31, 2023, the contents of which are incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] The present technology relates generally to compositions including attenuated modified coronavirus (CoV) vaccines (e.g., SARS-CoV-2, MERS-CoV) and methods of using the same to prevent coronavirus infection in a subject in need thereof. In particular, the present disclosure provides prophylactic vaccine compositions against SARS-CoV-2 and MERS-CoV infection.

STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under grants GM113117, GM138029, GM103648, and AI134993 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Coronaviruses (CoVs) belong to the family coronaviridae and possess a large, positive-sense RNA genome. The subfamily coronavirinae is further subdivided into a, P, y and 5-CoVs, though only the a and P-CoVs include viruses that infect humans. Prior to the 21st century CoVs were predominantly known to cause mild respiratory disease in humans (1). However, with the emergence of SARS-CoV, MERS-CoV, and most recently SARS- CoV-2, it is now well-established that CoVs are implicated in severe human respiratory conditions and are a serious threat to human health. Coronavirus infectious disease (COVID- 19) caused by SARS-CoV-2 is responsible for the pandemic that has resulted in over 6 million deaths worldwide (WHO). In cases of severe COVID-19, SARS-CoV-2 induces a robust pro-inflammatory cytokine response, or cytokine storm, in the host leading to the development of acute respiratory distress syndrome (ARDS) and in some cases multiple organ pathologies (2). Introduction of SARS-CoV-2 mRNA vaccines have drastically increased antiviral immunity and has reduced the fatality caused by SARS-CoV-2 (CDC). However, many elderly or immunocompromised people have ineffective responses to vaccines (3), and with the rate of emergence of new SARS-CoV-2 variants like Omicron (BA.2, BA.4 and BA.5), there is an urgent need to identify effective therapeutic agents to prevent coronavirus infection.

SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides a composition comprising, consisting of, or consisting essentially of a recombinant modified SARS coronavirus (e.g., SARS-CoV-2), wherein the recombinant modified SARS coronavirus e.g., SARS-CoV-2) contains or has a deletion within Macrodomain 1 (Macl domain) of non- structural protein 3 (nsp3). In some embodiments, the amino acid sequence of the Macl domain of nsp3 is: IEVNSFSGYLKLTDNVYIKNADIVEEAKKVKPTVVVNAANVYLKHGGGVAGALNKA TNNAMQVESDDYIATNGPLKVGGSCVLSGHNLAKHCLHVVGPNVNKGEDIQLLKSA YENFNQHEVLLAPLLSAGIFGADPIHSLRVCVDTVRTNVYLAVFDKNLYDKLVSSFL E (SEQ ID NO: 2). In other embodiments, the amino acid sequence of the Macl domain of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0007] Additionally or alternatively, in some embodiments, the recombinant modified SARS coronavirus (e.g., SARS-CoV-2) contains a complete deletion of the Macl domain of nsp3. In certain embodiments, the recombinant modified SARS coronavirus (e.g., SARS- CoV-2) contains a partial deletion within the Macl domain of nsp3. The length of the deletion within the Macl domain of nsp3 may range from 20 amino acids to 170 amino acids. In certain embodiments, the deletion within the Macl domain of nsp3 is about 20-25, about 25-30, about 30-35, about 35-40, about 40-45, about 45-50, about 50-55, about 55-60, about 60-65, about 65-70, about 70-75, about 75-80, about 80-85, about 85-90, about 90-95, about 95-100, about 100-105, about 105-110, about 110-115, about 115-120, about 120-125, about 125-130, about 130-135, about 135-140, about 140-145, about 145-150, about 150-155, about 155-160, about 160-165 or about 165-170 amino acids in length. In some embodiments, the recombinant modified SARS-coronavirus (e.g., SARS-CoV-2) is derived from a SARS-CoV- 2 genetic variant selected from the group consisting of Alpha, Beta, Gamma, Delta, Epsilon, Eta, Iota, Kappa, Omicron, Zeta and Mu.

[0008] In one aspect, the present disclosure provides a composition comprising, consisting of, or consisting essentially of a recombinant modified SARS coronavirus (e.g., SARS-CoV-2), wherein the recombinant modified SARS coronavirus e.g., SARS-CoV-2) comprises one or more mutations within Macrodomain 1 (Macl domain) of non- structural protein 3 (nsp3), and wherein the one or more mutations correspond to one or more substitutions at N1062, H1067, D1044, G1152, 11153, or F1154 of SEQ ID NO: 1. In some embodiments, the one or more substitutions at N1062, H1067, D1044, G1152, 11153, or Fl 154 of SEQ ID NO: 1 are selected from the group consisting of N1062A, H1067A, D1044A, G1152V, Il 153A, and Fl 154A. Additionally or alternatively, in some embodiments, the amino acid sequence of nsp3 is identical to the amino acid sequence from position 818 to position 2763 of SEQ ID NO: 1. In certain embodiments, the amino acid sequence of the Macl domain of nsp3 is identical to the amino acid sequence from position 1023 to position 1192 of SEQ ID NO: 1. Additionally or alternatively, in some embodiments, the amino acid sequence of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence from position 818 to position 2763 of SEQ ID NO: 1. Additionally or alternatively, in some embodiments, the amino acid sequence of the Macl domain of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence from position 1023 to position 1192 of SEQ ID NO: 1.

[0009] In any and all of the preceding embodiments, the recombinant modified SARs- coronavirus (e.g., SARS-CoV-2) is derived from a SARS-CoV-2 genetic variant selected from the group consisting of Alpha, Beta, Gamma, Delta, Epsilon, Eta, Iota, Kappa, Omicron, Zeta and Mu. Additionally or alternatively, in some embodiments, the recombinant modified modified SARs-coronavirus (e.g., SARS-CoV-2) is formulated as a vaccine, and optionally comprises one or more adjuvants. [0010] In one aspect, the present disclosure provides a composition comprising, consisting of, or consisting essentially of a recombinant modified MERS coronavirus (e.g., MERS-CoV), wherein the recombinant modified MERS-CoV comprises one or more mutations within Macrodomain 1 (Macl domain) of non-structural protein 3 (nsp3), and wherein the one or more mutations correspond to one or more substitutions at DI 129, N1147, Hl 152, G1237, 11238, or F1239 of SEQ ID NO: 4. In some embodiments, the one or more substitutions at DI 129, N1147, Hl 152, G1237, 11238, or F1239 of SEQ ID NO: 4 are selected from the group consisting of DI 129A, N1147A, Hl 152A, G1237V, I1238A, and F1239A.

[0011] Additionally or alternatively, in some embodiments, the amino acid sequence of nsp3 is identical to the amino acid sequence from position 854 to position 2740 of SEQ ID NO: 4. In certain embodiments, the amino acid sequence of the Macl domain of nsp3 is identical to the amino acid sequence from position 1107 to position 1278 of SEQ ID NO: 4. Additionally or alternatively, in some embodiments, the amino acid sequence of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence from position 854 to position 2740 of SEQ ID NO: 4. Additionally or alternatively, in some embodiments, the amino acid sequence of the Macl domain of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence from position 1107 to position 1278 of SEQ ID NO: 4.

[0012] Additionally or alternatively, in some embodiments, the recombinant modified modified MERS-coronavirus (e.g., MERS-CoV) is formulated as a vaccine, and optionally comprises one or more adjuvants.

[0013] In any of the embodiments of the composition disclosed herein, the composition further comprises one or more pharmaceutically acceptable excipients, wherein the one or more excipients is selected from the group consisting of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and combinations of two or more of the foregoing.

[0014] In yet another aspect, the present disclosure provides a method for preventing a coronavirus infection (e.g., MERS-CoV, SAR-CoV2) in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) disclosed herein. In some embodiments, administration of the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) results in induction of an immune response to a coronavirus infection in the subject, maintains an immune response against a coronavirus infection in the subject, inhibits proliferation of a coronavirus within the subject, or eradicates coronavirus within the subject. In some embodiments, the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) is administered intranasally or intravenously. In yet another aspect, the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) is delivered at a dosage per administration within the range of about 10⁶- 10¹⁰ plaqueforming units (pfu), wherein the delivery is repeated at least twice.

[0015] Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is a human subject. The subject may be immunocompromised, a pediatric subject, a geriatric subject, or an adult subject. In some embodiments, administration of the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) prevents one or more signs or symptoms selected from among fatigue, tingling, tingling or numbness in the hands and feet, dizziness, confusion, brain fog, body ache, chills, loss of appetite, nausea, vomiting, abdominal pain or discomfort, loss of smell, inability to taste, muscle weakness, photophobia, adenopathy, headaches, cough, dry cough, shortness of breath, sore throat, lower extremity weakness/numbness, diarrhea, low blood O2, sneezing, runny nose or postnasal drip; severe symptoms, such as ventilatory use, high fever, severe cough, delirium, seizures, stroke, systematic inflammation, cytokine storm; and other symptoms, such as fever, swollen adenoids, pneumonia, bronchitis, and dyspnea.

[0016] Additionally or alternatively, in some embodiments, the methods further comprise separately, sequentially or simultaneously administering to the subject one or more additional therapeutic agents to the subject. Examples of the additional therapeutic agents include, but are not limited to an anti-viral agent, optionally remdesivir, lopinavir, ritonavir, ivermectin, tamiflu, or favipiravir; an anti-inflammatory agent, optionally dexamethasone, tocilizumab, kevzara, colcrys, hydroxychloroquine, chloroquine, or a kinase inhibitor; a covalescent plasma from a subject recovered from a SARS-CoV-2 infection; an antibody binding to SARS-CoV-2, optionally bamlanivimab, etesevimab, casirivimab, or imdevimab; or an antibiotic agent, optionally azithromycin.

[0017] Also disclosed herein is a kit comprising any and all embodiments of the compositions disclosed herein and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGs. 1A-1C: SARS-CoV-2 Macl deletion virus replicates normally in Vero E6 and A549-ACE2 cells. VeroE6 (FIGs. 1A-1B) and A549-ACE2 (FIG. 1C) cells were infected with SARS-CoV-2 WT and AMacl at an MOI of 0.1 (FIGs. 1A, 1C) and 1 (FIG. IB) PFU/cell. Both cell-associated and cell-free virus was collected at indicated time points and virus-titers were determined by plaque assays. Data shown is one experiment representative of three independent experiments, n = 3 per group for each experiment, ns - not significant.

[0019] FIGs. 2A-2F: SARS-CoV-2 has a mild replication defect in Calu-3 cells.

FIGs. 2A-2D: Calu-3 cells were infected with SARS-CoV-2 WT and AMacl viruses at both low (FIGs. 2A-2B) and high (FIGs. 2C-2D) MOI. Both cell-associated and cell-free virus was collected at indicated times and virus titers were determined by plaque assay. The data in FIGs. 2A and 2C are from one experiment representative of at least 3 independent experiments, n = 3 per group. The results of all combined experiments where the average WT values from each experiment were normalized to 1.0 at 24 and 48 hpi are shown in FIGs. 2B and 2D. Each point represents a separate biological replicate. FIGs. 2E-2F: Calu-3 cells were infected at an MOI of 1 PFU/cell as described above and cell lysates were collected, and viral protein levels were determined by immunoblotting (FIG. 2E) or cells fixed at 24 hpi were co-stained with DAPI and either anti-nsp3 or anti-N, and then analyzed by confocal microscopy at 20X magnification (FIG. 2F). The data in FIGs. 2E-2F shows data from one representative experiment of two independent experiments.

[0020] FIGs. 3A-3B: IFN-y, but not IFN- ?, pretreatment enhances replication defect of AMacl in Calu-3 cells. Calu-3 cells were pretreated for 18 h with increasing concentrations (0, 5, 50, and 500 units) of IFN-P (FIG. 3A) and IFN-y (FIG. 3B), then infected with either SARS-CoV-2 WT or AMacl at an MOI of 0.1 PFU/cell. Cells were collected at 48 hpi and titers were determined by plaque assay. Fold differences between WT and AMacl are indicated at each amount of IFN. The data shown are of one experiment representative of two (FIG. 3 A) and three (FIG. 3B) independent experiments, n = 3 for each group.

[0021] FIGs. 4A-4B: AMacl induces increased IFN and cytokines responses compared to WT SARS-CoV-2 in cell culture. Calu3 (FIG. 4A) and A549-ACE2 (FIG. 4B) cells were infected with SARS-CoV-2 WT and AMacl at an MOI of 0.1 PFU/cell and total RNA was collected 48 hpi. IFN-P, IFN-X, ISG15 and CXCL10 levels were determined by qPCR using ACt method with primers listed in Table S2 and normalized to HPRT mRNA levels. The data show one experimental representative of three independent experiments with n = 3 for each experiment.

[0022] FIGs. 5A-5F: AMacl is highly attenuated in K18-ACE2 mice. FIGs. 5A-5B: K18-ACE2 C57BL/6 mice were infected with 2.5 10⁴ PFU of WT or AMacl SARS-CoV-2 and survival and weight loss were measured over 12 days. FIG. 5C: Photomicrographs (hematoxylin and eosin stain) of lungs from WT and AMacl infected mice at 7 dpi demonstrating bronchointerstitial pneumonia (black arrow) and edema and fibrin (black asterisk). FIG. 5D: Mice were scored for bronchointerstitial pneumonia, inflammation, and edema/fibrin deposition. WT n=4; AMacl n=5. FIGs. 5E-5F: K18-ACE2 C57BL/6 mice were infected as described above and lung titers (FIG. 5E) and gRNA levels (FIG. 5F) were determined by plaque assay and RT-qPCR with primers specific for nspl2 and normalized to HPRT, respectively. Results are from one experiment representative of two independent experiments, n = 4-10 mice per group.

[0023] FIGs. 6A-6D: AMacl virus induces a robust innate immune response in the lungs following infection. FIG. 6A: K18-ACE2 C57BL/6 mice were infected with 2.5* 10⁴ PFU of indicated viruses and lungs were harvested at 1 dpi and total RNA was isolated. The relative levels of indicated transcripts were determined by qPCR using the ACt method with primers listed in Table S2 normalized to HPRT mRNA levels. The results are from one experiment representative of two independent experiments with an n = 4-8 mice per group. FIGs. 6B-6D: The total RNA from the samples in FIG. 6A were analyzed by RNAseq to determine the full transcriptome in the lung following infection. FIG. 6B: Volcano plot indicating differentially expressed genes (DEGs) between WT and AMacl infected mice. FIG. 6C: Functional enrichment analysis of biological processes enriched in the transcriptome of in mice infected with AMacl performed using DAVID functional annotation tool. FIG. 6D: Log2 fold change values of genes involved in innate immune response upregulated in mice infected with AMacl compared to WT virus.

[0024] FIGs. 7A-7B: AMacl virus infection results in reduced inflammatory monocytes and neutrophils. FIGs. 7A-7B: K18-ACE2 C57BL/6 mice were infected as described above and lungs were harvested at the indicated days post-infection, and the percentages and total numbers of infiltrating inflammatory monocytes (FIG. 7A) and neutrophils (FIG. 7B) were determined by flow cytometry. Data are derived from the results of 1 experiment representative of 2 independent experiments performed with 4-5 mice/group/experiment.

[0025] FIGs. 8A-8B: jff-CoV Macl deletion BAC clones differ in their ability to induce CPE following transfection. BHK-MVR cells (MHV-JHM) or Huh-7 cells (MERS- CoV & SARS-CoV-2) were transfected with WT or AMacl BAC DNA from the indicated virus Cells were analyzed for CPE from 3-5 days post-transfection by light microscopy with images taken using Capta Vision software (FIG. 8A) and the recovery rates for each virus were determined (FIG. 8B) Scale bars denote 220pm.

[0026] FIGs. 9A-9D: AMacl and WT virus have similar fitness in Calu-3 cells. (A-B) SARS-CoV-2 WT and AMacl BAC DNA was mixed at the indicated ratios and then PCR was performed using primers outside of Macl (Methods). PCR products were analyzed by gel electrophoresis (FIG. 9A) and the signal intensity of each band was quantitated using Image Studio software and the relative intensity of each band was calculated (FIG. 9B). FIGs. 9C-9D: Calu-3 cells were initially infected at an MOI of 0.1 PFU/cell with SARS- CoV-2 WT and AMacl at the indicated ratios for passage 1. For each subsequent passage 0.2 ml of the virus collected from each previous passaged was used to infect Calu-3 cells. Cells and supernatants were collected at 48 hpi following the first passage, and at 36 hpi for passages 2-4. At each passage total RNA was collected and converted into cDNA and then PCR was done to determine the relative ratios of each virus. PCR products were analyzed as described above. The image in FIG. 9C is from 1 experiment representative of 2 independent experiments, while the data in FIG. 9D is the combined results from 2 independent experiments.

[0027] FIG. 10: IFN-y and IFN- ? induce PARP expression in Calu-3 cells. Calu-3 cells were treated for 18 h with 500 units of IFN-P and IFN-y then total RNA was collected. Levels of PARP12 and PARP14 were determined by qPCR with primers listed in Table S2 and normalized to HPRT mRNA levels. Data are from one experiment representative of two independent experiments with n = 3 for each experiment.

[0028] FIGs. 11A-11B: SARS-CoV-2 WT and AMacl replication and pathogenesis in the brains of K18-ACE2 B6 mice. FIG. 11A: K18-ACE2 mice were infected with 2.5* 10⁴ PFU of WT or AMacl SARS-CoV-2 and brains were collected at indicated time points and viral loads were determined by plaque assay, n = 4 mice per group. FIG. 11B: Photomicrographs (hematoxylin and eosin stain) of brains from WT and AMacl infected mice at 7 dpi exhibiting neuronal necrosis (black arrows).

[0029] FIG. 12: RNA seq analysis of IFN, ISGs and viral RNA. Relative normalized counts (from the output of DESeq2) of reads mapped to transcripts in RNAseq of mice infected with SARS-CoV-2 WT and AMacl at 24 hpi.

[0030] FIGs. 13A-13D: Macl Mutant virus infection protects from future challenge with WT virus. FIGs. 13A-13B: K18-C57BL/6 mice were challenged with 1 xlO⁵ PFU of SARS-CoV-2 WT virus 5 weeks post infection with AMacl or PBS-treated control mice and mice were monitored for survival (FIG. 13A) or weight loss (FIG. 13B). N=S for both groups. FIGs. 13C-13D: hDPP4 knock-in C57BL/6 mice were infected with 750 PFU of MERS-CoV WT virus 5 weeks post infection with Macl mutant viruses (N1147A and DI 129A) or PBS-treated control mice and mice were monitored for survival (FIG. 13C) and weight loss (FIG. 13D). N=4 for PBS and N1147A groups, N=S for DI 129A.

[0031] FIG. 14 is a schematic drawing of ORF 1 AB protein, nsp3 and Macl domain in SARS-CoV-2 and MERS-CoV. All 170 amino acids of Macl domain was deleted in the attenuated SARS-CoV-2 virus.

[0032] FIG. 15 is a schematic illustration of SARS-CoV-2 Macl nucleotide sequence (SEQ ID NO: 53) and amino acid sequence (SEQ ID NO: 54). [0033] FIG. 16 is a schematic illustration of MERS-CoV Macl nucleotide sequence (SEQ ID NO: 55) and amino acid sequence (SEQ ID NO: 56).

[0034] FIGs. 17A-17D: SARS-CoV-1 point mutants are attenuated.

[0035] FIGs. 18A-18D: MERS-CoV point mutants are attenuated. MERS-CoV mutant virus vaccine protects animals from future challenge with WT MERS-COV virus.

[0036] FIGs. 19A-19B: The GIF motif in loop 2 of Macl is highly conserved and is closely associated with both phosphate groups and the terminal ribose of ADP-ribose. FIG. 19A: Sequence alignment of Macl across viral and human macrodomains. GIF motif is boxed in Red. FIG. 19B: Overlay of the SARS-CoV-2 (purple) (6WOJ) and MERS-CoV (teal) (5HOL) Macl ADP-ribose binding domains with ADP-ribose, highlighting the GIF motif and conserved asparagine and aspartic acid residues discussed herein.

[0037] FIGs. 20A-20D: MHV F1441A mutation is attenuated in cell lines and in primary cells. DBTs (FIG. 20A), L929s (FIGs. 20B), and M2 macrophages (FIGs. 20C- 20D) were infected with JHMV at an MOI of 0.1 PFU/cell. Cells and supernatants were collected at indicated times and assayed for progeny infectious virus by plaque assay. The data in each panel show one experiment representative of three independent experiments with n = 3 for each experiment.

[0038] FIGs. 21A-21D: MHV F1441A, but not I1440A, is partially attenuated in in vivo. FIGs. 21A-21C: Male and female C57BL/6 mice were infected intranasally with WT, 11440 A, and Fl 441 A JHMV at 1 X 10⁴PFU. Mice were monitored for survival (FIG. 21 A), weight loss (FIG. 21B), and disease score (as described in Methods) (FIG. 21C) for 12 days post-infection (dpi). WT, n=4 mice; IA, n=8 mice; FA, n=8 mice. FIG. 21D: Brains were collected at 5 dpi and titers were determined by plaque assay. WT, n=6; IA, n=7; FA, n=8. The data show the combined results from two independent experiments.

[0039] FIGs. 22A-22E: MERS-CoV I1238A and F1239A Macl mutations have opposing effects on ADP-ribose binding and hydrolysis. FIG. 22A: Macl protein was incubated with free ADP-ribose and binding affinity was measured by isothermal calorimetry as described in Methods. FIG. 22B: An ADP-ribosylated peptide was incubated with indicated macrodomains at increasing concentrations and Alphacounts were measured as described in Methods. FIG. 22C: ADP-ribose (ADPr) competition assays were used to block the interaction between macrodomain proteins and ADP-ribosylation peptides in the AS assay. Data was analyzed as described in Methods. The data in FIGs. 22A-22C represent combined results of 2 independent experiments for each protein. FIG. 22D: WT, Il 153 A, and Fl 154 A MERS-CoV Macl proteins were incubated with MARylated PARP10 CD in vitro at an [E]/[S]molar ratio of 1 :5 for the indicated times at 37°C. ADP-ribosylated PARP10 CD was detected by IB with anti-ADP- ribose binding reagent (MAB1076; MilliporeSigma) while total PARP10 CD protein levels was detected by IB with GST antibody. The reaction with PARP10 CD incubated alone at 37°C was stopped at 0 or 30 min. The image in FIG. 22D is representative of 2-3 independent experiments. FIG. 22E: The level of de-MARylation was measured after 30 minutes by quantifying relative band intensity (ADP-ribose/GST-PARPlO) using Imaged software. Error bars represent standard deviations (SD). The results in FIG. 22E are the combined resulted of 2-3 independent experiments.

[0040] FIGs. 23A-23C: MERS-CoV 11238 A and Fl 239 A have similarly decreased replication in human and bat cell lines. Vero81 (FIG. 23 A), Calu3 (FIG. 23B), and AJK6 cells (FIG. 23C) were infected at an MOI of 0.1 PFU/cell. Cells and supernatants were collected at indicated times post-infection (hpi) and progeny virus was measured by plaque assay. The data in FIGs. 23A-23C show one experiment representative of three independent experiments with n = 3 for each experiment.

[0041] FIGs. 24A-24D: SARS-CoV-2 I1153A and F1154A have increased ADP- ribose binding. FIG. 24A: SARS-CoV-2 Macl protein was incubated with free ADP-ribose and binding affinity was measured by isothermal calorimetry as described in Methods. FIG. 24B: An ADP-ribosylated peptide was incubated with indicated macrodomains at increasing concentrations and Alphacounts were measured as described in Methods. FIG. 24C: WT, Il 153A, and Fl 154A SARS-CoV-2 Macl proteins were incubated with MARylated PARP10 CD in vitro at an [E]/[S]molar ratio of 1 :5 for the indicated times at 37°C. ADP-ribosylated PARP10 CD was detected by IB with anti-ADP- ribose binding reagent (MAB1076; MilliporeSigma) while total PARP10 CD protein levels were detected by IB with GST antibody. The reaction with PARP10 CD incubated alone at 37°C was stopped at 0 or 30 min. The data in FIGs. 24A-24C show one experiment representative of three independent experiments. FIG. 24D: The level of de-MARylation was measured by quantifying relative band intensity (ADP-ribose/GST-PARPIO) using ImageJ software. Intensity values were plotted and fitted to a nonlinear regression curve; error bars represent SD.

[0042] FIGs. 25A-25E: SARS-CoV-2 N1062A binds to ADP-ribose but is highly defective in ADP-ribosylhydrolase activity. FIG. 25A: ADP-ribosylated peptide was incubated with WT and N1062 A Macl proteins at increasing concentrations and Alphacounts were measured as described in Methods. FIG. 25B: ADP-ribose (ADPr) competition assays were used to block the interaction between WT and N1062 A Macl proteins and ADP- ribosylated peptides. Data was analyzed as described in Methods. The data represent the means ± SD of 2 independent experiments for each protein. FIG. 25C: WT and N1062 A Macl proteins (10 pM) were incubated with increasing concentrations of ADP-ribose and measured by DSF as described in Methods. FIG. 25D: WT and N1062A SARS-CoV-2 Macl proteins were incubated with MARylated PARP10 CD in vitro at an [E]/[S] molar ratio of 1 :5 for the indicated times at 37°C. ADP-ribosylated PARP10 CD was detected by IB with anti-ADP -ribose binding reagent (green) while total PARP10 CD protein levels were detected by IB with GST antibody (red). The reaction with PARP10 CD incubated alone at 37°C was stopped at 0 or 30 min. The data is representative of 2 independent experiments. FIG. 25E: The level of de-MARylation in D was measured by quantifying relative band intensity (ADP- ribose/GSTPARPlO) using ImageJ software. Intensity values were plotted and fitted to a nonlinear regression curve. The data represent the means ± SD of 2 independent experiments for each protein.

[0043] FIGs. 26A-26B: Increased binding has detrimental effects on SARS-CoV-2 replication the presence of IFNy. Calu3 (FIG. 26A) and A549-ACE2 (FIG. 26B) cells were pretreated with 500 units of IFNy for 18-20 hours prior to infection. Then cells were infected at an MOI of 0.1 PFU/cell. Cells and supernatants were collected at 48 hpi and progeny virus was measured by plaque assay. The data in FIGs. 26A-26B show one experiment representative of three independent experiments with n = 3 for each experiment.

[0044] FIGs. 27A-27G: SARS-CoV-2 I1153A and F1154A are highly attenuated and induce elevated innate immune responses in the lungs of infected mice. K18-ACE2 C57BL/6 mice were infected i.n. with 2.5 x 10⁴ PFU of virus. FIGs. 27A-27B: Survival (FIG. 27A) and weight loss (FIG. 27B) were monitored for 14 days. n=5 for survival and n=9 for weightloss for all groups. FIG. 27C: Lungs were harvested at 1 dpi and viral titers were determined by plaque assay. n=6 for all groups. FIG. 27D: Lungs were harvested at 8 dpi and viral titers were determined by plaque assay. Dotted line indicates limit of detection. n=3 for WT, n=4 for II 153 A and Fl 154A. FIG. 27E: Photomicrographs (hematoxylin and eosin stain) of lungs infected mice at 8 dpi demonstrating bronchointerstitial pneumonia (black arrow) and edema and fibrin (open arrow). FIG. 27F: Mice were scored for bronchointerstitial pneumonia, inflammation, and edema/fibrin deposition (each on a 0-5 scale). Bar graphs represent cumulative lung pathology score in WT n=3, Il 153 A n=4, Fl 154A n=4. FIG. 27G: Lungs were harvested at 1 dpi in Trizol and RNA was isolated. Transcripts levels were determined using qPCR with the ACT method. n=6 for all groups.

[0045] FIGs. 28A-28D: Models of isoleucine-to-alanine mutation on Macl structure and virus replication. FIG. 28A: Molecular simulation of the ADP-ribose binding domain of the SARSCoV-2 Macl protein was performed in absence and presence of ADP-ribose.

The 1 ns averaged LAI 153 to G1069 distance was measured through the course of four 25 ns MD simulations of ADP-ribose bound and unbound WT and II 153 A protein. FIGs. 28B- 28C: A representative image at 12 ns of the simulation demonstrating the distance between the 11153 and Al 153 residues and G1069 at 12 ns into the simulation without ADP-ribose in a space-filling (FIG. 28B) or stick model (FIG. 28C). FIG. 28D: (Left) In the presence of IFNy, the WT SARSCoV-2 Macl removes ADP-ribose from specific proteins (red and blue) that have an ADP-ribose on an acidic reside which enhances virus replication. (Right) Due to the open conformation of SARS-CoV-2 II 153 A Macl protein, it binds to ADP-ribose bound to non-acidic residues (gold and green). Since Macl cannot remove proteins from non-acidic residues, this limits its ability to interact with relevant substrate, and the ADP-ribose remains on its normal target proteins leading to poor virus replication.

[0046] FIG. 29: The absence of PARP12 enhances F1441A replication. BMDMs were harvested from PARP12+/+ mice and PARP12-/- mice and differentiated into M2 macrophages. Cells were infected at an MOI of 0.1 PFU/cell. Supernatants and cells were harvested at 20 hpi. Progeny infectious virus was determined by plaque assay. The data shows one experiment representative of three independent experiments with n = 3 for each experiment. [0047] FIGs. 30A-30B: Total protein shown by Coomassie blue staining for MERS-CoV WT, I1238A, and F1239A (FIG. 30A) and SARS-CoV-2 WT, I1153A, and F1154A (FIG.

30B)

[0048] FIGs. 31A-31E: SARS-CoV-2 N1062A binds to ADP-ribose but is highly defective in ADP-ribosylhydrolase activity. FIG. 31A: ADP-ribosylated peptide was incubated with WT and N1062 A Macl proteins at increasing concentrations and Alphacounts were measured as described in Methods. FIG. 31B: ADP-ribose (ADPr) competition assays were used to block the interaction between WT and N1062 A Macl proteins and ADP- ribosylated peptides in the AS assay. Data was analyzed as described in Methods. The data represent the means ± SD of 2 independent experiments for each protein. FIG. 31C: WT and N1062A Macl proteins (10 pM) were incubated with increasing concentrations of ADP- ribose and measured by DSF as described in Methods. FIG. 31D: WT and N1062A SARS- CoV-2 Macl proteins were incubated with MARylated PARP10 CD in vitro at an [E]/[S]molar ratio of 1 :5 for the indicated times at 37°C. ADP-ribosylated PARP10 CD was detected by IB with anti-ADP-ribose binding reagent (MAB1076; MilliporeSigma) while total PARP10 CD protein levels were detected by IB with GST antibody. The reaction with PARP10 CD incubated alone at 37°C was stopped at 0 or 30 min. FIG. 31E: The level of de-MARylation was measured by quantifying relative band intensity (ADP-ribose/GST- PARP10) using Imaged software. Intensity values were plotted and fitted to a nonlinear regression curve; error bars represent SD.

[0049] FIG. 32: SARS-CoV-2 I1153A and F1154A are cleared from K18-ACE2 mice by day 8 post-infection. K18-ACE2 C57BL/6 mice were infected intranasally with 2.5 x 10⁴ PFU of WT, Il 153A, and Fl 154A. Lungs were harvested at 8 dpi and viral titers were determined by plaque assay. Dotted line indicates limit of detection.

DETAILED DESCRIPTION

[0050] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0051] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al., eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al., (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson el al., (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al., eds (1996) Weir ’s Handbook of Experimental Immunology.

[0052] The present disclosure provides attenuated modified coronavirus vaccines (e.g., SARS-CoV-2, MERS) harboring a deletion or point mutations within the Macl domain of nsp3. The attenuated modified coronavirus (e.g., SARS-CoV-2, MERS) vaccines disclosed herein effectively protect subjects from subsequent infections from these viral pathogens.

Definitions

[0053] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. [0054] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0055] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0056] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (e.g., intravenously, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another. Administration of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.

[0057] The term administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The disclosure is not limited by the route of administration, the formulation or dosing schedule.

[0058] The term “adjuvant” refers to a substance or mixture that enhances the immune response to an antigen. As non-limiting example, the adjuvant can comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (See e.g., US 8,241,610). In another embodiment, the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant. As a non-limiting example, the synthetic nanocarrier comprising and adjuvant can be formulated by the methods described in WO201 1150240 and US20110293700, each of which is herein incorporated by reference in its entirety.

[0059] The terms “complementary” or “complementarity” as used herein with reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the base-pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association.” For example, the sequence “5'-A-G-T-3'” is complementary to the sequence “3'-T-C-A-5 ” Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. A complementary sequence can also be an RNA sequence complementary to the DNA sequence or its complementary sequence, and can also be a cDNA.

[0060] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease or condition, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0061] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. As used herein, a “therapeutically effective amount” of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0062] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.

[0063] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0064] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleobase or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;

Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non- redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0065] The term “hybridize” as used herein refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (T_m) of the formed hybrid. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. ; Ausubel, F. M. et al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a primer) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.

[0066] “Immune response”, as used herein, refers to the action of one or more of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of pathogens, etc. An immune response may include a cellular response, such as a T cell response that is an alteration (modulation, e.g., significant enhancement, stimulation, activation, impairment, or inhibition) of cellular, i.e., T cell function. A T cell response may include generation, proliferation or expansion, or stimulation of a particular type of T cell, or subset of T cells, for example, effector CD4+, CD4+ helper, effector CD8+, CD8+ cytotoxic, or natural killer (NK) cells. Such T cell subsets may be identified by detecting one or more cell receptors or cell surface molecules (e.g., CD or cluster of differentiation molecules). A T cell response may also include altered expression (statistically significant increase or decrease) of a cellular factor, such as a soluble mediator (e.g., a cytokine, lymphokine, cytokine binding protein, or interleukin) that influences the differentiation or proliferation of other cells. For example, Type I interferon (IFN-a/p) is a critical regulator of the innate immunity (52) (Huber et al. Immunology 132(4):466-474 (2011)). Animal and human studies have shown a role for IFN-a/p in directly influencing the fate of both CD4+ and CD8+ T cells during the initial phases of antigen recognition and immune response. IFN Type 1 is induced in response to activation of dendritic cells, in turn a sentinel of the innate immune system. An immune response may also include humoral (antibody) response.

[0067] As used herein, “oligonucleotide” refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. One or more bases of the oligonucleotide may also be modified to include a phosphorothioate bond (e.g., one of the two oxygen atoms in the phosphate backbone which is not involved in the internucleotide bridge, is replaced by a sulfur atom) to increase resistance to nuclease degradation. The exact size of the oligonucleotide will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof. The oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.

[0068] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20^th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0069] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0070] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0071] As used herein, an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence. Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST. In one aspect, equivalent polynucleotides, proteins and corresponding sequences can be determined using BLAST (accessible at blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on August 1, 2021).

[0072] The terms “polynucleotide”, and “nucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three dimensional structure and may perform any function, known or unknown. The following are non limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form.

[0073] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0074] In some embodiments, the term “engineered” or “recombinant” refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain or the parental host strain of the referenced species. In some embodiments, the term “engineered” or “recombinant” refers to being synthetized by human intervention. As used herein, the term “recombinant protein” refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.

[0075] As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample. As used herein, preventing a coronavirus infection, includes preventing or delaying the initiation of symptoms of a coronavirus infection. As used herein, prevention of a coronavirus infection also includes preventing a recurrence of one or more signs or symptoms of a coronavirus infection.

[0076] As used herein, the term “sample” refers to clinical samples obtained from a subject. Biological samples may include tissues, cells, protein or membrane extracts of cells, mucus, sputum, bone marrow, bronchial alveolar lavage (BAL), bronchial wash (BW), and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids (blood, plasma, saliva, urine, serum etc.) present within a subject.

[0077] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0078] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0079] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time. [0080] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to an individual organism, a vertebrate, a mammal, or a human. In certain embodiments, the individual, patient or subject is a human.

[0081] “Treating”, “treat”, or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0082] It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0083] As used herein, the term “vaccine” refers to a composition including an antigenic component for administration to a subject, which elicits an immune response to the antigenic component. In some embodiments a vaccine is a therapeutic. In some embodiments, a vaccine is prophylactic. In some embodiments a vaccine includes one or more adjuvants.

[0084] As used herein, "viral load", also known as "viral burden," "viral titer", "viral level" or "viral expression" in some embodiments, is a measure of the severity of a viral infection, and can be calculated by estimating the amount of virus in an infected organism, an involved body fluid, or a biological sample.

Coronavirus - SARS-CoV-2 and MERS-CoV

[0085] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also referred to as 2019 novel coronavirus (2019-nCoV) or human coronavirus 2019 (HCoV-19 or hCoV-19), is the virus that causes COVID-19 (coronavirus disease 2019), the respiratory illness responsible for the COVID-19 pandemic. [0086] Each SARS-CoV-2 virion is 50-200 nanometers in diameter, comprising a linear, positive-sense, single-stranded RNA genome (about 30,000 bases long) and four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. Coronavirus S proteins are glycoproteins that are divided into two functional parts (SI and S2). In SARS-CoV-2, the spike protein is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its SI subunit catalyzes attachment, the S2 subunit fusion. Studies have shown that SARS-CoV-2 has sufficient affinity to the receptor angiotensin converting enzyme 2 (ACE2) on human cells to use them as a mechanism of cell entry. Initial spike protein priming by transmembrane protease, serine 2 (TMPRSS2) is also shown as essential for entry of SARS-CoV-2. The host protein neuropilin 1 (NRP1) may aid the virus in host cell entry using ACE2. After a SARS-CoV-2 virion attaches to a target cell, the cell's TMPRSS2 cuts open the spike protein of the virus, exposing a fusion peptide in the S2 subunit, and the host receptor ACE2. After fusion, an endosome forms around the virion, separating it from the rest of the host cell. The virion escapes when the pH of the endosome drops or when cathepsin, a host cysteine protease, cleaves it. The virion then releases RNA into the cell and forces the cell to produce and disseminate copies of the virus, which infect more cells.

[0087] Genetic variants of SARS-CoV-2 have been emerging and circulating around the world throughout the COVID-19 pandemic. The B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and P.1 (Gamma) variants circulating in the United States are classified as variants of concern. Other variants are also present, such as B.1.526 (Iota), B.1.427 (Epsilon), B.1.429 (Epsilon), B.1.617 (Kappa, Delta), B.1.525 (Eta), and P.2 (Zeta). Accordingly, the term “SARS-CoV-2” as used herein can refer to any one or more of the variants. In some embodiments, SARS-CoV-2 as used herein refers to an omicron variant, which was first identified in South Africa. In further embodiments, SARS-CoV-2 omicron variant comprises mutations in the gene encoding the S protein.

[0088] Symptoms of coronavirus infection include, but are not limited to, fatigue, tingling, tingling or numbness in the hands and feet, dizziness, confusion, brain fog, body ache, chills, loss of appetite, nausea, vomiting, abdominal pain or discomfort, loss of smell, inability to taste, muscle weakness, photophobia, adenopathy, headaches, cough, dry cough, shortness of breath, sore throat, lower extremity weakness/numbness, diarrhea, low blood O2, sneezing, runny nose or post-nasal drip; severe symptoms, such as ventilatory use, high fever, severe cough, delirium, seizures, stroke, systematic inflammation, cytokine storm; and other symptoms, such as fever, swollen adenoids, pneumonia, bronchitis, and dyspnea.

[0089] Viral infection of a coronavirus, such as SARS-COV-2, can be detected via a commercially available test known in the art, for example via polymerase chain reaction (PCR) or immunoassay may be used. In some embodiments, a method as disclosed herein further comprises detecting a coronavirus using a test known in the art. In one embodiment, active viral infection refers to an ongoing infection wherein the virus is replicating and producing new virus. Such active viral infection may be detected using polymerase chain reaction (PCR). Non-limiting examples of primers and probes suitable for use in the PCR include 2019-nCoV CDC Probe and Primer Kit for SARS-CoV-2 (BioSearch Technologies, Catalog No. KIT-nCoV-PPl- 1000), 2019-nCoV Kit, 500 rxn (Integrated DNA Technologies (IDT), Catalog No. 10006606) and 2019-nCoV Kit, 1000 rxn (Integrated DNA Technologies (IDT), Catalog No. 10006770). Also see, www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr- panel-primer-probes.html and www.cdc.gov/coronavirus/2019-ncov/downloads/List-of- Acceptable-Commercial-Primers-Probes.pdf. Suitable protocols for performing such tests can be found at www.cdc.gov/coronavirus/2019-ncov/lab/virus-requests.html, www.fda.gov/media/134922/download, www.cdc.gov/coronavirus/2019- ncov/downloads/processing-sputum-specimens.pdf, www.fda.gov/media/134922/download, www.fda.gov/media/134919/download, www.fda.gov/media/134922/download, last accessed on August 10, 2021. In some embodiments, diagnostic assays for COVID-19 based on detecting antibodies is combined with those disclosed herein, such as those discussed by Lisboa Bastos M et al. (Diagnostic accuracy of serological tests for covid- 19: systematic review and meta-analysis. BMJ. 2020 Jul l;370:m2516. doi: 10.1136/bmj.m2516).

[0090] Other commercially available tests include, but not limited to those listed in the Table below.

Commercially available tests for SARS-CoV-2 and COVID-19

Macl Domains of Coronaviruses

[0091] All CoVs encode a conserved set of 15-16 non- structural proteins that direct the formation of the replication transcription complex (RTC) and carryout the process of RNA transcription and replication, making these proteins important targets for antiviral therapies. While much progress has been made in identifying the functions of many of the non- structural proteins, a complete understanding of how these proteins contribute to RNA replication and evasion of the host immune response is still lacking. Non- structural protein 3 (nsp3) is the largest non-structural protein encoded in the CoV genome and consists of several modular protein domains, such as the papain-like protease (PLP) domain. Included in these domains of nsp3 are 3 tandem macrodomains (Macl, Mac2 and Mac3). Macl is conserved throughout all Co Vs unlike Mac2 and Mac3 (7-12). Structurally, macrodomains are characterized by the presence of a conserved three-layered a/p/a fold. Biochemically, the conserved viral macrodomain binds to ADP-ribose moi eties with high affinity (15, 16) and in some cases can hydrolyze the bond between ADP-ribose and proteins, reversing ADP- ribosylation, a common post-translational modification (15, 17-20).

[0092] Exemplary coronavirus replicase polyprotein sequences are provided below:

[0093] YP 009724389.1 ORFlab polyprotein [Severe acute respiratory syndrome coronavirus 2] (SEQ ID NO: 1).

1 meslvpgfne kthvqlslpv Iqvrdvlvrg fgdsveevls earqhlkdgt cglvevekgv

61 Ipqleqpyvf ikrsdartap hghvmvelva elegiqygrs getlgvlvph vgeipvayrk 121 vllrkngnkg agghsygadl ks fdlgdelg tdpyedfqen wntkhs sgvt relmrelngg 181 aytryvdnnf cgpdgyplec ikdllaragk as ctlseqld fidtkrgvyc creheheiaw 241 yterseksye Iqtpfeikla kkfdtfngec pnfvfplnsi iktiqprvek kkldgfmgri 301 rsvypvaspn ecnqmclstl mkcdhcgets wqtgdfvkat cefcgtenlt kegattcgyl 361 pqnavvkiyc pachnsevgp ehslaeyhne sglktilrkg grtiafggcv fsyvgchnkc 421 aywvprasan igcnhtgvvg egseglndnl leilqkekvn inivgdfkln eeiaiilas f 481 sastsafvet vkgldykafk qives cgnfk vtkgkakkga wnigeqksil splyafasea 541 arvvrsi fs r tletaqnsvr vlqkaaitil dgisqyslrl idammftsdl atnnlvvmay 601 itggvvqlts qwltni fgtv yeklkpvldw leekfkegve flrdgweivk fistcaceiv 661 ggqivtcake ikesvqtffk Ivnkflalca dsiiiggakl kalnlgetfv ths kglyrkc 721 vks reetgll mplkapkeii flegetlpte vlteevvlkt gdlqpleqpt seaveaplvg 781 tpvcinglml leikdtekyc alapnmmvtn ntftlkggap tkvtfgddtv ievqgyksvn 841 itfelderid kvlnekcsay tvelgtevne facvvadavi ktlqpvsell tplgidldew 901 smatyyl fde sgefklashm ycs fyppded eeegdceeee fepstqyeyg teddyqgkpl 961 efgatsaalq peeeqeedwl dddsqqtvgq qdgsednqtt tiqtivevqp qlemeltpvv 1021 qtievnsfsg ylkltdnvyi knadiveeak kvkptvwna anvylkhggg vagalnkatn

1081 namqvesddy iatngplkvg gscvlsghnl akhclhwgp nvnkgediql Iksayenfnq 1141 hevllaplls agifgadpih slrvcvdtvr tnvylavfdk nlydklvssf lemksekqve 1201 qkiaeipkee vkpfites kp sveqrkqddk kikacveevt ttleetkflt enlllyidin 1261 gnlhpdsatl vsdiditflk kdapyivgdv vqegvltavv iptkkaggtt emlakalrkv 1321 ptdnyittyp gqglngytve eaktvlkkck safyilpsii snekqeilgt vswnlremla 1381 haeetrklmp vcvetkaivs tiqrkykgik iqegvvdyga rfyfyts ktt vaslintlnd 1441 Inetlvtmpl gyvthglnle eaarymrslk vpatvsvs sp davtayngyl ts s s ktpeeh 1501 fietislags ykdwsysgqs tqlgieflkr gdksvyytsn pttfhldgev itfdnlktll 1561 slrevrtikv fttvdninlh tqvvdmsmty gqqfgptyld gadvtkikph nshegktfyv 1621 Ipnddtlrve afeyyhttdp s flgrymsal nhtkkwkypq vngltsikwa dnncylatal 1681 Itlqqielkf nppalqdayy rarageaanf calilaycnk tvgelgdvre tmsyl fqhan 1741 Ids ckrvlnv vcktcgqqqt tlkgveavmy mgtlsyeqfk kgvqipctcg kqatkylvqq 1801 espfvmmsap paqyelkhgt ftcaseytgn yqcghykhit s ketlycidg alltks seyk 1861 gpitdvfyke nsytttikpv tykldgvvct eidpkldnyy kkdnsyfteq pidlvpnqpy 1921 pnas fdnfkf vcdnikfadd Inqltgykkp as relkvtff pdlngdvvai dykhytps fk 1981 kgakllhkpi vwhvnnatnk atykpntwci rclwstkpve tsns fdvlks edaqgmdnla

2041 cedlkpvsee vvenptiqkd vlecnvktte vvgdiilkpa nnslkiteev ghtdlmaayv

2101 dns sltikkp nels rvlglk tlathglaav nsvpwdtian yakpf Inkvv stttnivtrc

2161 Inrvctnymp yf f tlllqlc tftrstns ri kasmpttiak ntvksvgkf c leas fnylks

2221 pnf s klinii iwf lllsvcl gsliystaal gvlmsnlgmp syctgyregy Instnvtiat

2281 yctgsipcsv clsgldsldt ypsletiqit is s fkwdlta f glvaewf la yil ftrf fyv

2341 Iglaaimql f f syf avhf is nswlmwliin Ivqmapisam vrmyi f fas t yyvwksyvhv

2401 vdgcns stcm mcykrnratr vecttivngv rrs f yvyang gkgf cklhnw nevnedtf ca

2461 gstf isdeva rdlslqf krp inptdqs syi vdsvtvkngs ihlyfdkagq ktyerhslsh

2521 fvnldnlran ntkgslpinv ivfdgks kce es saksasvy ysqlmcqpil lldqalvsdv

2581 gdsaevavkm fdayvntf s s tfnvpmeklk tlvataeael aknvsldnvl stf isaarqg

2641 fvdsdvetkd vveclklshq sdievtgds c nnymltynkv enmtprdlga cidcsarhin

2701 aqvakshnia liwnvkdfms Iseqlrkqir saakknnlpf kltcattrqv vnvvttkial

2761 kggkivnnwl kqlikvtlvf I fvaai fyli tpvhvms kht df s seiigyk aidggvtrdi

2821 astdtcf ank hadfdtwf sq rggsytndka cpliaavitr evgfvvpglp gtilrttngd

2881 f Ihf Iprvf s avgnicytps klieytdf at sacvlaaect i f kdasgkpv pycydtnvle

2941 gsvayeslrp dtryvlmdgs iiqfpntyle gsvrvvttfd seycrhgtce rseagvcvst

3001 sgrwvlnndy yrslpgvf eg vdavnlltnm f tpliqpiga Idisasivag givaivvtcl

3061 ayyfmrf rra f geyshvvaf ntll flms ft vlcltpvys f Ipgvysviyl yltf yltndv

3121 s f lahiqwmv mf tplvpfwi tiayiicist khf ywf f sny Ikrrvvfngv s f stf eeaal

3181 ctf llnkemy iklrsdvllp Itqynrylal ynkykyf sga mdttsyreaa cchlakalnd

3241 f snsgsdvly qppqtsitsa vlqsgf rkma fpsgkvegcm vqvtcgtttl nglwlddvvy

3301 cprhvictse dmlnpnyedl lirksnhnf 1 vqagnvqlrv ighsmqncvl klkvdtanpk

3361 tpkykfvriq pgqtf svlac yngspsgvyq camrpnf tik gs flngs cgs vgfnidydcv

3421 s f cymhhmel ptgvhagtdl egnf ygpfvd rqtaqaagtd ttitvnvlaw lyaavingdr

3481 wf Inrf tttl ndfnlvamky nyepltqdhv dilgplsaqt giavldmcas Ikellqngmn

3541 grtilgsall edef tpfdvv rqcsgvtf qs avkrtikgth hwllltilts llvlvqstqw

3601 sl f f flyena f ipf amgiia msaf ammfvk hkhaflcl fl Ipslatvayf nmvympaswv

3661 mrimtwldmv dtslsgf klk dcvmyasavv llilmtartv yddgarrvwt Imnvltlvyk

3721 vyygnaldqa ismwaliisv tsnysgvvtt vmf largivf mcveycpi f f itgntlqcim

3781 Ivycf Igyf c tcyfgl f ell nryf rltlgv ydylvstqef rymnsqgllp pknsidaf kl

3841 nikllgvggk pcikvatvqs kmsdvkctsv vllsvlqqlr ves s s klwaq cvqlhndill

3901 akdtteaf ek mvsllsvlls mqgavdinkl ceemldnrat Iqaiasef s s Ipsyaaf ata

3961 qeayeqavan gdsevvlkkl kkslnvakse fdrdaamqrk lekmadqamt qmykqarsed

4021 krakvtsamq tml ftmlrkl dndalnniin nardgcvpln iiplttaakl mvvipdynty

4081 kntcdgttf t yasalweiqq vvdads kivq Iseismdnsp nlawplivta Iransavklq

4141 nnelspvalr qms caagttq tactddnala yynttkggrf vlallsdlqd Ikwarfpksd

4201 gtgtiytele ppcrfvtdtp kgpkvkylyf ikglnnlnrg mvlgslaatv rlqagnatev

4261 panstvls f c af avdaakay kdylasggqp itncvkmlct htgtgqaitv tpeanmdqes

4321 fggas cclyc rchidhpnpk gf cdlkgkyv qipttcandp vgf tlkntvc tvcgmwkgyg

4381 cs cdqlrepm iqsadaqs fl nrvcgvsaar Itpcgtgtst dvvyrafdiy ndkvagf akf

4441 Iktnccrf qe kdeddnlids yfvvkrhtf s nyqheetiyn llkdcpavak hdf f kf ridg

4501 dmvphis rqr Itkytmadlv yalrhfdegn cdtlkeilvt ynccdddyfn kkdwydfven

4561 pdilrvyanl gervrqallk tvqf cdamrn agivgvltld nqdlngnwyd f gdf iqttpg

4621 sgvpvvdsyy sllmpiltlt raltaeshvd tdltkpyikw dllkydf tee rlkl fdryf k

4681 ywdqtyhpnc vnclddrcil hcanfnvl f s tvfppts f gp Ivrki fvdgv pfvvstgyhf

4741 relgvvhnqd vnlhs s rls f kellvyaadp amhaasgnll Idkrttcf sv aaltnnvaf q

4801 tvkpgnfnkd fydfavs kgf f kegs svelk hf f f aqdgna aisdydyyry nlptmcdirq

4861 ll fvvevvdk yfdcydggci nanqvivnnl dksagfpfnk wgkarlyyds msyedqdal f

4921 aytkrnvipt itqmnlkyai saknrartva gvsicstmtn rqfhqkllks iaatrgatvv

4981 igts kfyggw hnmlktvysd venphlmgwd ypkcdrampn mlrimaslvl arkhttccsl

5041 shrf yrlane caqvlsemvm cggslyvkpg gts sgdatta yansvfnicq avtanvnall

5101 stdgnkiadk yvrnlqhrly eclyrnrdvd tdfvnef yay Irkhf smmil sddavvcfns

5161 tyasqglvas iknf ksvlyy qnnvfmseak cwtetdltkg phef csqhtm Ivkqgddyvy

5221 Ipypdps ril gagcfvddiv ktdgtlmier fvslaidayp Itkhpnqeya dvfhlylqyi

5281 rklhdeltgh mldmysvmlt ndnts rywep ef yeamytph tvlqavgacv lensqtslrc

5341 gacirrpf 1c ckccydhvis tshklvlsvn pyvenapged vtdvtqlylg gmsyyckshk 5401 ppis fplcan gqvfglyknt cvgsdnvtdf naiatcdwtn agdyilantc terlkl faae

5461 tlkateetfk Isygiatvre vlsdrelhls wevgkprppl nrnyvftgyr vtkns kvqig

5521 eytfekgdyg davvyrgttt yklnvgdyfv Itshtvmpls aptlvpqehy vritglyptl

5581 nisdefs snv anyqkvgmqk ystlqgppgt gkshfaigla lyypsarivy tacshaavda

5641 Icekalkylp idkcs riipa rarvecfdkf kvnstleqyv fctvnalpet tadivvfdei

5701 smatnydlsv vnarlrakhy vyigdpaqlp aprtlltkgt lepeyfnsvc rlmktigpdm

5761 flgtcrrcpa eivdtvsalv ydnklkahkd ksaqcfkmfy kgvithdvs s ainrpqigvv

5821 refltrnpaw rkavfispyn sqnavas kil glptqtvds s qgseydyvi f tqttetahs c

5881 nvnrfnvait rakvgilcim sdrdlydklq ftsleiprrn vatlqaenvt gl fkdcs kvi

5941 tglhptqapt hlsvdtkfkt eglcvdipgi pkdmtyrrli smmgfkmnyq vngypnmfit

6001 reeairhvra wigfdvegch atreavgtnl plqlgfstgv nlvavptgyv dtpnntdfs r

6061 vsakpppgdq fkhliplmyk glpwnvvrik ivqmlsdtlk nlsdrvvfvl wahgfeltsm

6121 kyfvkigper tcclcdrrat cfstasdtya cwhhsigfdy vynpfmidvq qwgftgnlqs

6181 nhdlycqvhg nahvascdai mtrclavhec fvkrvdwtie ypiigdelki naacrkvqhm

6241 vvkaalladk fpvlhdignp kaikcvpqad vewkfydaqp csdkaykiee I fysyathsd

6301 kftdgvcl fw ncnvdrypan sivcrfdtrv Isnlnlpgcd ggslyvnkha fhtpafdksa

6361 fvnlkqlpff yysdspcesh gkqvvsdidy vplksatcit rcnlggavcr hhaneyrlyl

6421 daynmmisag fslwvykqfd tynlwntftr Iqslenvafn vvnkghfdgq qgevpvsiin

6481 ntvytkvdgv dvel fenktt Ipvnvafelw akrnikpvpe vkilnnlgvd iaantviwdy

6541 krdapahist igvcsmtdia kkpteticap Itvffdgrvd gqvdl frnar ngvlitegsv

6601 kglqpsvgpk qaslngvtli geavktqfny ykkvdgvvqq Ipetyftqs r nlqefkprsq

6661 meidflelam defierykle gyafehivyg dfshsqlggl hlliglakrf kespfeledf

6721 ipmdstvkny fitdaqtgs s kcvcsvidll Iddfveiiks qdlsvvs kvv kvtidyteis

6781 fmlwckdghv etfypklqs s qawqpgvamp nlykmqrmll ekcdlqnygd satlpkgimm

6841 nvakytqlcq ylntltlavp ynmrvihfga gsdkgvapgt avlrqwlptg tllvdsdlnd

6901 fvsdadstli gdcatvhtan kwdliisdmy dpktknvtke nds kegffty icgfiqqkla

6961 Iggsvaikit ehswnadlyk Imghfawwta fvtnvnass s eafligcnyl gkpreqidgy

7021 vmhanyi fwr ntnpiqls sy sl fdms kfpl klrgtavmsl kegqindmil slls kgrlii

7081 rennrvvis s dvlvnn

Macl domain =Boldface

[0094] ORF lab, partial [Middle East respiratory syndrome-related coronavirus]

GenBank: QKF93417.1 (SEQ ID NO: 4)

1 ms fvagviaq gargtyraal nsekhqdhvs Itvplcgsgn Iveklspwfm dgenayevvk

61 amllkkepll yvpirlaght rhlpgprvyl verliacenp fmvnqlays s sangslvgtt

121 Iqgkpigmff pydielvtgk qnillrkygr ggyhytpvhy erdnts cpew mddfeadpkg

181 kyaqnllkkl iggdvtpvdq ymcgvdgkpi sayaflmakd gitkladvea dvaaraddeg

241 fitlknnlyr Ivwhverkdv pypkqsi fti nsvvqkdgve ntpphyftlg ckiltltprn

301 kwsgvsdlsl kqkllytfyg keslenptyi yhsafiecgs cgndswltgn aiqgfacgcg

361 asytandvev qs sgmikpna llcatcpfak gds cs snckh svaqlvsyls ercnviadsk

421 s ftli fggva yayfgceegt myfvpraksv vs rigdsi ft gctgswnkvt qianmfleqt

481 qhslnfvgef vvndvvlail sgtttnvdki rqllkgvtid klrdyladyd vavtagpfmd

541 nainvggtgl qyaaitapyv vltglges fk kvatipykvc nsvkdtltyy ahsvlyrvfp

601 ydmdsgvs s f sell fdcvdl svastyflvr llqdktgdfm stiits cqta vs klldtcfe

661 ateatfnfll dlagl fri fl rnayvytsqg fvvvngkvst Ivkqvldlln kgmqllhtkv

721 swagsnisav iysgresli f psgtyycvtt kaksvqqdld vilpgefs kk qlgllqptdn

781 sttvsvtvs s nmvetvvgql eqtnmhspdv ivgdyviise kl fvrs keed gfafypactn

841 ghavptl frl kggapvkkva fggdqvheva avrsvtveyn ihavldtlla s s slrtfvvd

901 kslsieefad vvkeqvsdll vkllrgmpip dfdlddfida pcycfnaegd asws stmifs

961 Ihpvecdeec seveasglee gesecisets teqvdvshev sddewaaavd eafpldeaed

1021 vtesvqeeaq pvevpvedia qvviadtlqe tpvvsdtvev ppqvvklpse pqtiqpevke

1081 vapvyeadte qtqsvtvkpk rlrkkrnvdp Isnfehkvit ecvtivlgda iqvakcyges

1141 vlvnaanthl khgggiagai naaskgavqk esdeyilakg plqvgdsvll qghslaknil

1201 hwgpdarak qdvsllskcy kamnayplw tplvstgifg vkpavsfdyl ireaktrvlv

1261 wnsqdvyks Itivdipqsl tfsydglrga irkakdygft vfvctdnsan tkvlrnkgvd 1321 ytkkf Itvdg vqyycyts kd tlddilqqan ksvgiismpl gyvshgldli qagsvvrrvn 1381 vpyvcllank eqeailmsed vklnpsedf i khvrtnggyn swhlvegell vqdlrlnkll 1441 hwsdqticyk dsvf yvvkns tafpf etlsa craylds rtt qqltievlvt vdgvnf rtvv 1501 Innkntyrsq Igcvf fngad isdtipdekq nghslyladn Itadetkalk elygpvdptf 1561 Ihrf yslkaa vhkwkmvvcd kvrslklsdn ncylnavimt Idllkdikfv ipalqhafmk 1621 hkggdstdf i alimaygnct fgapddas rl Ihtvlakael ccsarmvwre wcnvcgikdv 1681 vlqglkaccy vgvqtvedlr armtyvcqcg gerhrqiveh ttpwlllsgt pneklvttst 1741 apdfvafnvf qgietavghy vhaclkggli Ikfdsgtvs k tsdwkckvtd vl fpgqkys s 1801 dcnvvrysld gnf rtevdpd Isaf yvkdgk yf tseppvty spatilagsv ytns clvs sd 1861 gqpggdaisl s fnnllgfds s kpvtkkyty s f Ipkedgdv llaefdtydp iykngamykg 1921 kpilwvnkas ydtnlnkf r aslrqi fdva pielenkf tp Isvastpvep ptvdvvalqq 1981 emtivkckgl nkpfvkdnvs fvvddsgtpv veyls kedlh tlyvdpkyqv ivlkdnvls s 2041 mlrlhtvesg dinvvaasgs Itrkvkll f r as f yf kef at rtf tattavg s ciksvvrhl 2101 gvtkgiltgc fs fvkml fil playf sds kl gttevkvsal ktagvvtgnv vkqcctaavd 2161 Ismdklrrvd wkstlrlllm Icttmvlls s vyhlyvfnqv Is sdvmfeda qglkkf ykev 2221 raylgis sac dglasayran s fdvptf can rsamcnwcli sqdsithypa Ikmvqthlsh 2281 yvlnidwlwf af etglayml ytsafnwlll agtlhyf f aq tsi fvdwrsy nyavs safwl 2341 f thipmaglv rmynllaclw llrkf yqhvi ngckdtacll cykrnrltrv eastvvcggk 2401 rtf yitangg is f crrhnwn cvdcdiagvg ntf iceevan dlttalrspi natdrshyyv 2461 dsvtvketvv qfnyrrdgqp f yerfplcaf tnldklkf ke vcktttgipe ynfiiyds sd 2521 rgqeslarsa cvyysqvlck sillvds slv tsvgds seia tkmfds fvns fvslynvtrd 2581 kleklistar dgvrrgdnfh svlttf idaa rgpagvesdv etneivdsvq yahkhdiqit 2641 nesynnyvps yvkpdsvsts dlgslidcna asvnqivlrn sngaciwnaa aymklsdalk 2701 rqiriacrkc nlaf rltts k Irandnilsv rf tankivgg aptwfnvlrd f tlkgyvlat 2761 iivf Icavlm ylclptf smv pvef yedril df kvldngii rdvnpddkcf ankhrs f tqw 2821 yhehvggvyd nsitcpltva viagvagari pdvpttlawv nnqii f fvs r vf antgsvcy 2881 tpideipyks f sdsgcilps ectmf rdaeg rmtpychdpt vlpgaf aysq mrphvrydly 2941 dgnmf ikfpe vvf estlrit rtlstqycrf gs ceyaqegv cittngswai fndhhlnrpg 3001 vycgsdf idi vrrlavsl fq pityf qltts Ivlgiglcaf Itll fyyink vkraf adytq 3061 caviavvaav Inslcicfva siplcivpyt alyyyatf yf tnepaf imhv swyimf gpiv 3121 piwmtcvytv amcf rhf fwv layf s kkhve vf tdgklncs fqdaasni fv inkdtyaalr 3181 nsltndays r flgl fnkyky f sgametaay reaaachlak alqtysetgs dllyqppncs 3241 itsgvlqsgl vkmshpsgdv eacmvqvtcg smtlnglwld ntvwcprhvm cpadqlsdpn 3301 ydallismtn hs f svqkhig apanlrvvgh amqgtllklt vdvanpstpa ytf ttvkpga 3361 af svlacyng rptgtf tvvm rpnytikgs f Icgs cgsvgy tkegsvinf c ymhqmelang 3421 thtgsafdgt mygafmdkqv hqvqltdkyc svnvvawlya ailngcawfv kpnrtsvvs f 3481 newalanqf t efvgtqsvdm lavktgvaie qllyaiqqly tgf qgkqilg stmledef tp 3541 edvnmqimgv vmqsgvrkvt ygtahwl fat Ivstyviilq atkf tlwnyl fetistql fp 3601 ll fvtmafvm llvkhkhtf 1 tl fllpvaic Ityanivyep ttpis salia vanwlaptna 3661 ymrtthtdig vyismslvlv ivvkrlynps Isnf alalcs gvmwlytysi geas spiayl 3721 vfvttltsdy titvfvtvnl akvctyai fa yspqltlvfp evkmilllyt clgfmctcyf 3781 gvf s f Inlkl rapmgvydf k vstqef rfmt annltaprns weamalnf kl igiggtpcik 3841 vaamqs kltd Ikctsvvlls vlqqlhlean s rawaf cvkc hndilaatdp seaf ekfvsl 3901 f atlmtf sgn vdldalasdi fdtpsvlqat Isef shlatf aeleaaqkay qeamdsgdts 3961 pqvlkalqka vniaknayek dkavarkler madqamtsmy kqaraedkka kivsamqtml 4021 f gmikkldnd vlngiisnar ngciplsvip Icasnklrvv ipdf tvwnqv vtypslnyag 4081 alwditvinn vdneivks sd vvdsnenltw plvlectras tsavklqnne ikpsglktmv 4141 vsagqeqtnc nts slayyep vqgrkmlmal Isdnaylkwa rvegkdgfvs velqppckf 1 4201 iagpkgpeir ylyfvknlnn Ihrgqvlghi aatvrlqags ntefasns sv Islvnf tvdp 4261 qkayldfvna ggapltncvk mltpktgtgi aisvkpesta dqetyggasv clycrahieh 4321 pdvsgvckyk gkfvqipaqc vrdpvgf cis ntpcnvcqyw igygcncdsl rqvalpqs kd 4381 snf Inrvrgs ivnariepcs sglstdvvf r afdicnykak vagigkyykt ntcrfveldd 4441 qghhldsyfv vkrhtmenye lekhcydllr dcdavaphdf fi fdvdkvkt phivrqrlte 4501 ytmmdlvyal rhfdqnsevl kailvkygcc dvtyf enklw fdfvenpsvi gvyhklgerv 4561 rqailntvkf cdhmvkaglv gvltldnqdl ngkwydf gdf vitqpgsgva ivdsyysylm 4621 pvlsmtdcla aethrdcdfn kpliewplte ydf tdykvql f ekyf kywdq tyhancvnct 4681 ddrcvlhcan fnvl famtmp ktcf gpivrk i fvdgvpfvv s cgyhykelg Ivmnmdvslh 4741 rhrlslkelm myaadpamhi as snafldlr ts cf svaalt tgltf qtvrp gnfnqdf ydf

4801 vvs kgf f keg s svtlkhf f f aqdgnaaitd ynyysynlpt mcdikqml f c mevvnkyf ei

4861 ydggclnase vvvnnldksa ghpfnkf gka rvyyesmsyq eqdel famtk rnviptmtqm

4921 nlkyaisakn rartvagvsi Istmtnrqyh qkmlksmaat rgatcvigtt kf yggwdfml

4981 ktlykdvdnp hlmgwdypkc dramp nmcri f aslilarkh gtccttrdrf yrlanecaqv

5041 Iseyvlcggg yyvkpggts s gdattayans vfnilqatta nvsalmgang nkivdkevkd

5101 mqfdlyvnvy rstspdpkfv dkyyaf Inkh f smmilsddg vvcynsdyaa kgyiagiqnf

5161 ketlyyqnnv fmseakcwve tdlkkgphef csqhtlyikd gddgyf Ipyp dps rilsagc

5221 fvddivktdg tlmverfvsl aidaypltkh edieyqnvfw vylqyiekly kdltghmlds

5281 ysvmlcgdns akfweeaf yr dlys spttlq avgs cvvchs qtslrcgtci rrpf Icckcc

5341 ydhviatphk mvlsvspyvc napgcgvsdv tklylggmsy f cvdhrpvcs fplcanglvf

5401 glyknmctgs psivefnrla tcdwtesgdy tlantttepl kl faaetlra teeas kqsya

5461 iatikeivge rqlllvweag ks kpplnrny vf tgyhitkn s kvqlgeyi f eridysdavs

5521 yks sttyklt vgdi fvltsh svatltapti vnqeryvkit glyptitvpe ef ashvanf q

5581 ksgys kyvtv qgppgtgksh f aiglaiyyp tarvvytacs haavdalcek af kylniakc

5641 s riipakarv ecydrf kvne tnsqyl f sti nalpetsadi Ivvdevsmct nydlsiinar

5701 ikakhivyvg dpaqlpaprt lltrgtlepe nfnsvtrlmc nlgpdi flsm cyrcpkeivs

5761 tvsalvynnk llakkelsgq cf kilykgnv thdas sainr pqltfvknf i tanpaws kav

5821 f ispynsqna varsmlgltt qtvds sqgse yqyvi f cqta dtahanninr fnvaitraqk

5881 gilcvmtsqa I fesleftel s f tnyklqsq ivtgl f kdcs retsglspay aptyvsvddk

5941 yktsdelcvn Inlpanipys rvis rmgf kl datvpgypkl f itreeavrq vrswigfdve

6001 gahas rnacg tnvplqlgf s tgvnfvvqpv gvvdtewgnm Itgiaarppp geqf khlvpl

6061 mhkgaawpiv rrrivqmlsd tldklsdyct fvcwahgf el tsasyf ckig keqkccmcnr

6121 raaays splq syacwths cg ydyvynpf fv dvqqwgyvgn latnhdrycs vhqgahvasn

6181 daimtrclai hs cfiervdw dieypyishe kklns ccriv ernvvraall ags fdkvydi

6241 gnpkgipivd dpvvdwhyfd aqpltrkvqq I fytedmas r fadglcl fwn cnvpkypnna

6301 ivcrfdtrvh sefnlpgcdg gslyvnkhaf htpaydvsaf rdlkplpf f y ysttpcevhg

6361 ngsmiedidy vplksavcit acnlggavcr khateyreym eaynlvsasg f rlwcyktfd

6421 iynlwstf tk vqgleniafn vvkqghf igv egelpvavvn dki ftksgvn dicmf enktt

6481 Iptniaf ely akravrshpd f kllhnlqad icykfvlwdy ersniygtat igvckytdid

6541 vnsalnicfd irdngslekf mstpnai fis drkikkypci vgpdyayfng aiirdsdvvk

6601 qpvkf ylykk vnnef idpte ciytqs rs cs df Iplsdmek df Is fdsdvf ikkyglenya

6661 f ehvvygdf s httlgglhll iglykkqqeg hiimeemlkg s stihnyfit etntaaf kav

6721 csvidlkldd fvmilksqdl gvvs kvvkvp idltmiefml wckdgqvqtf yprlqasadw

6781 kpghampsl f kvqnvnlerc elanykqsip mprgvhmnia kymqlcqyln tctlavpanm

6841 rvihf gagsd kgiapgtsvl rqwlptdaii idndlnefvs daditl fgdc vtvrvsqqvd

6901 Ivisdmydpt tknvtgsnes kal f ftylcn linnnlalgg svaikitehs wsvelyelmg

6961 kf awwtvf ct nanas s segf llginylgti kenidggamh anyi fwrnst pmnlstysl f

7021 dls kfqlklk gtpvlqlkes qinelvisll sqgkllirdn dtlsvstdvl vntyrklr

Macl domain =Boldface

[0095] The sequence of the Macl domains of NSP3 in SARS-CoV-2 is:

IEVNSFSGYLKLTDNVYIKNADIVEEAKKVKPTVVVNAANVYLKHGGGVAGALNKA

TNNAMQVESDDYIATNGPLKVGGSCVLSGHNLAKHCLHVVGPNVNKGEDIQLLKSA

YENFNQHEVLLAPLLSAGIFGADPIHSLRVCVDTVRTNVYLAVFDKNLYDKLVSSFL E (SEQ ID NO: 2).

[0096] The sequence of the Macl domains of NSP3 in MERS-CoV is:

NVDPLSNFEHKVITECVTIVLGDAIQVAKCYGESVLVNAANTHLKHGGGIAGAINAA

SKGAVQKESDEYILAKGPLQVGDSVLLQGHSLAKNILHVVGPDARAKQDVSLLSKC YKAMNAYPLVVTPLVSAGIFGVKPAVSFDYLIREAKTRVLVVVNSQDVYKSLTIVDI

PQ (SEQ ID NO: 3).

Pharmaceutical Compositions of the Present Technology

[0097] In one aspect, the present disclosure provides a composition comprising, consisting of, or consisting essentially of a recombinant modified SARS coronavirus (e.g., SARS-CoV-2), wherein the recombinant modified SARS coronavirus e.g., SARS-CoV-2) contains or has a deletion within Macrodomain 1 (Macl domain) of non- structural protein 3 (nsp3). In some embodiments, the amino acid sequence of the Macl domain of nsp3 is: IEVNSFSGYLKLTDNVYIKNADIVEEAKKVKPTVVVNAANVYLKHGGGVAGALNKA TNNAMQVESDDYIATNGPLKVGGSCVLSGHNLAKHCLHVVGPNVNKGEDIQLLKSA YENFNQHEVLLAPLLSAGIFGADPIHSLRVCVDTVRTNVYLAVFDKNLYDKLVSSFL E (SEQ ID NO: 2). In other embodiments, the amino acid sequence of the Macl domain of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0098] Additionally or alternatively, in some embodiments, the recombinant modified SARS coronavirus (e.g., SARS-CoV-2) contains a complete deletion of the Macl domain of nsp3. In certain embodiments, the recombinant modified SARS coronavirus (e.g., SARS- CoV-2) contains a partial deletion within the Macl domain of nsp3. The length of the deletion within the Macl domain of nsp3 may range from 20 amino acids to 170 amino acids. In certain embodiments, the deletion within the Macl domain of nsp3 is about 20-25, about 25-30, about 30-35, about 35-40, about 40-45, about 45-50, about 50-55, about 55-60, about 60-65, about 65-70, about 70-75, about 75-80, about 80-85, about 85-90, about 90-95, about 95-100, about 100-105, about 105-110, about 110-115, about 115-120, about 120-125, about 125-130, about 130-135, about 135-140, about 140-145, about 145-150, about 150-155, about 155-160, about 160-165 or about 165-170 amino acids in length.

[0099] In one aspect, the present disclosure provides a composition comprising, consisting of, or consisting essentially of a recombinant modified SARS coronavirus (e.g., SARS-CoV-2), wherein the recombinant modified SARS coronavirus (e.g., SARS-CoV-2) comprises one or more mutations within Macrodomain 1 (Macl domain) of non- structural protein 3 (nsp3), and wherein the one or more mutations correspond to one or more substitutions at N1062, H1067, D1044, G1152, 11153, or F1154 of SEQ ID NO: 1. In some embodiments, the one or more substitutions at N1062, H1067, D1044, G1152, 11153, or Fl 154 of SEQ ID NO: 1 are selected from the group consisting of N1062A, H1067A, D1044A, G1152V, Il 153A, and Fl 154A. Additionally or alternatively, in some embodiments, the amino acid sequence of nsp3 is identical to the amino acid sequence from position 818 to position 2763 of SEQ ID NO: 1. In certain embodiments, the amino acid sequence of the Macl domain of nsp3 is identical to the amino acid sequence from position 1023 to position 1192 of SEQ ID NO: 1. Additionally or alternatively, in some embodiments, the amino acid sequence of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence from position 818 to position 2763 of SEQ ID NO: 1. Additionally or alternatively, in some embodiments, the amino acid sequence of the Macl domain of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence from position 1023 to position 1192 of SEQ ID NO: 1.

[00100] In any and all of the preceding embodiments, the recombinant modified SARs- coronavirus (e.g., SARS-CoV-2) is derived from a SARS-CoV-2 genetic variant selected from the group consisting of Alpha, Beta, Gamma, Delta, Epsilon, Eta, Iota, Kappa, Omicron, Zeta and Mu. Additionally or alternatively, in some embodiments, the recombinant modified modified SARs-coronavirus (e.g., SARS-CoV-2) is formulated as a vaccine, and optionally comprises one or more adjuvants.

[00101] In one aspect, the present disclosure provides a composition comprising, consisting of, or consisting essentially of a recombinant modified MERS coronavirus (e.g., MERS-CoV), wherein the recombinant modified MERS-CoV comprises one or more mutations within Macrodomain 1 (Macl domain) of non-structural protein 3 (nsp3), and wherein the one or more mutations correspond to one or more substitutions at DI 129, N1147, Hl 152, G1237, 11238, or F1239 of SEQ ID NO: 4. In some embodiments, the one or more substitutions at DI 129, N1147, Hl 152, G1237, 11238, or F1239 of SEQ ID NO: 4 are selected from the group consisting of DI 129A, N1147A, Hl 152A, G1237V, I1238A, and F1239A. Additionally or alternatively, in some embodiments, the amino acid sequence of nsp3 is identical to the amino acid sequence from position 854 to position 2740 of SEQ ID NO: 4. In certain embodiments, the amino acid sequence of the Macl domain of nsp3 is identical to the amino acid sequence from position 1107 to position 1278 of SEQ ID NO: 4. Additionally or alternatively, in some embodiments, the amino acid sequence of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence from position 854 to position 2740 of SEQ ID NO: 4. Additionally or alternatively, in some embodiments, the amino acid sequence of the Macl domain of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence from position 1107 to position 1278 of SEQ ID NO: 4.

[00102] Additionally or alternatively, in some embodiments, the recombinant modified modified MERS-coronavirus (e.g., MERS-CoV) is formulated as a vaccine, and optionally comprises one or more adjuvants.

[00103] In any of the embodiments the composition disclosed herein, the composition further comprises, consists of, or consists essentially of one or more pharmaceutically acceptable excipients, wherein the one or more excipients is selected from the group consisting of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and combinations of two or more of the foregoing.

[00104] Pharmaceutical compositions of the present technology, suitable for inoculation or for parenteral, intranasal, or oral administration, comprise attenuated or inactivated coronavirus (e.g., SARS-CoV-2, MERS-CoV), and optionally sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions can further comprise auxiliary agents or excipients, as known in the art. The compositions of the present technology are generally presented in the form of individual doses (unit doses).

[00105] The vaccine forming the main constituent of the vaccine composition of the present technology may comprise a virus of type A, B or C, or any combination thereof, for example, at least two of the three types, at least two of different subtypes, at least two of the same type, at least two of the same subtype, or a different isolate(s) or reassortant(s), at least two of different variants etc. [00106] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.

Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

[00107] When a composition of the present technology is used for administration to an individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. For vaccines, adjuvants, substances which can augment a specific immune response, can be used. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the organism being immunized. Examples of materials suitable for use in vaccine compositions are provided in Osol (1980).

[00108] Heterogeneity in a vaccine may be provided by mixing replicated coronaviruses (e.g., SARS-CoV-2, MERS-CoV) for at least two attenuated Macl deficient coronavirus strains (e.g., SARS-CoV-2, MERS-CoV), such as 2-50 strains or any range or value therein. According to the present technology, vaccines can be provided for variations in a single strain of a coronavirus (e.g., SARS-CoV-2, MERS-CoV), using techniques known in the art.

[00109] The composition may also contain variable but small quantities of endotoxin-free formaldehyde, and preservatives, which have been found safe and not contributing to undesirable effects in the organism to which the composition is administered.

[00110] A vaccine of the present technology may comprise immunogenic proteins or carriers. In one embodiment, the coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines of the present technology may be vaccine vectors. [00111] A complete virion vaccine may be concentrated by ultrafiltration and then purified by zonal centrifugation or by chromatography. It is inactivated before or after purification using formalin or beta-propiolactone, for instance. Alternatively, the compositions of the present technology may be formulated as subunit vaccines or split vaccines using methods known in the art.

[00112] Inactivated Vaccines. Inactivated coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines of the present technology are provided by inactivating replicated virus of the present technology using known methods, such as, but not limited to, formalin or p-propiolactone treatment. Inactivated vaccine types that can be used in the present technology can include whole-virus (WV) vaccines or subvirion (SV) (split) vaccines. The WV vaccine contains intact, inactivated virus, while the SV vaccine contains purified virus disrupted with detergents that solubilize the lipid-containing viral envelope, followed by chemical inactivation of residual virus.

[00113] In addition, vaccines that can be used include those containing the isolated HA and NA surface proteins, which are referred to as surface antigen or subunit vaccines. In general, the responses to SV and surface antigen (i.e., purified HA or NA) vaccines are similar. An experimental inactivated WV vaccine containing an NA antigen immunologically related to the epidemic virus and an unrelated HA appears to be less effective than conventional vaccines (Ogra et al., 1977). Inactivated vaccines containing both relevant surface antigens are generally employed.

[00114] Live Attenuated Virus Vaccines. Live, attenuated coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines, can also be used for preventing or treating coronavirus infection, according to known methods. Attenuation may be achieved in a single step by transfer of attenuated genes from an attenuated donor virus to a replicated isolate or reassorted virus according to known methods (see, e.g., Murphy, 1993). The attenuated genes are derived from the attenuated parent. In this approach, genes that confer attenuation do not code for the NSP3 proteins. Otherwise, these genes could not be transferred to reassortants bearing the surface antigens of the clinical virus isolate. Other attenuating mutations can be introduced into coronavirus genes by site-directed mutagenesis to rescue infectious viruses bearing these mutant genes. Attenuating mutations can be introduced into non-coding regions of the genome, as well as into coding regions.

[00115] In one embodiment, such attenuated viruses maintain the genes from the virus that encode antigenic determinants substantially similar to those of the original clinical isolates. This is because the purpose of the attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, while at the same time lacking infectivity to the degree that the vaccine causes minimal change of inducing a serious pathogenic condition in the vaccinated mammal.

[00116] The virus can thus be attenuated or inactivated, formulated and administered, according to known methods, as a vaccine to induce an immune response in an animal, e.g., a mammal. Methods are well-known in the art for determining whether such attenuated or inactivated vaccines have maintained similar antigenicity to that of the clinical isolate or high growth strain derived therefrom. Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantidine); NSP3 activity and inhibition; and DNA screening (such as probe hybridization or PCR) to confirm that donor genes encoding the antigenic determinants (e.g., NSP3 genes) are not present in the attenuated viruses.

[00117] The pharmaceutical compositions of the present disclosure may be prepared by any of the methods known in the pharmaceutical arts. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, the amount of active compound will be in the range of about 0.1 to 99 percent, more typically, about 5 to 70 percent, and more typically, about 10 to 30 percent.

[00118] In some embodiments, pharmaceutical compositions of the present technology may contain one or more pharmaceutically-acceptable carriers, which as used herein, generally refers to a pharmaceutically-acceptable composition, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, useful for introducing the active agent into the body.

[00119] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the present technology include, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[00120] In some embodiments, the formulations may include one or more of sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; alginic acid; buffering agents, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; preservatives; glidants; fillers; and other non-toxic compatible substances employed in pharmaceutical formulations.

[00121] Various auxiliary agents, such as wetting agents, emulsifiers, lubricants (e.g., sodium lauryl sulfate and magnesium stearate), coloring agents, release agents, coating agents, sweetening agents, flavoring agents, preservative agents, and antioxidants can also be included in the pharmaceutical composition of the present technology. Some examples of pharmaceutically-acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. In some embodiments, the pharmaceutical formulation includes an excipient selected from, for example, celluloses, liposomes, lipid nanoparticles, micelle-forming agents (e.g., bile acids), and polymeric carriers, e.g., polyesters and polyanhydrides. Suspensions, in addition to the active compounds, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Prevention of the action of microorganisms on the active compounds may be ensured by the inclusion of various antibacterial and antifungal agents, such as, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

Methods of the Present Technology

[00122] The administration of the compositions of the present technology may be for either a “prophylactic” or “therapeutic” purpose. When provided prophylactically, the compositions of the present technology, are provided before any symptom of a disease becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate one or more symptoms associated with the disease.

[00123] When provided therapeutically, an attenuated or inactivated viral vaccine is provided upon the detection of a symptom of actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. Thus, an attenuated or inactivated vaccine composition of the present technology may thus be provided either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection.

[00124] A composition is said to be “pharmacologically acceptable” if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a therapeutically effective amount if the amount administered is physiologically significant. A composition of the present technology is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular immune response against at least one infectious strain of SARS-CoV-2 or MERS-CoV.

[00125] The “protection” provided need not be absolute, i.e., the viral infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of patients. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of the coronavirus infection.

[00126] In one aspect, the present disclosure provides a method for preventing a coronavirus infection (e.g., SARS-CoV-2 or MERS-CoV) in a subject in need thereof, comprising administering to the subject an effective amount of any and all embodiments of the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) disclosed herein. In some embodiments, administration of the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) results in induction of an immune response to a coronavirus infection in the subject, maintains an immune response against a coronavirus infection in the subject, inhibits proliferation of a coronavirus within the subject, or eradicates coronavirus within the subject. In some embodiments, the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) is administered intranasaly or intravenously. In yet another aspect, the recombinant modified coronavirus composition (e.g., SARS-CoV-2, MERS) is delivered at a dosage per administration within the range of about 10⁶- 10¹⁰ plaqueforming units (pfu), wherein the delivery is repeated at least twice.

[00127] Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is a human subject. The subject may be immunocompromised, a pediatric subject, a geriatric subject, or an adult subject.

[00128] In some embodiments, administration of the recombinant modified coronavirus (e.g., SARS-CoV-2, MERS) prevents one or more signs or symptoms selected from among fatigue, tingling, tingling or numbness in the hands and feet, dizziness, confusion, brain fog, body ache, chills, loss of appetite, nausea, vomiting, abdominal pain or discomfort, loss of smell, inability to taste, muscle weakness, photophobia, adenopathy, headaches, cough, dry cough, shortness of breath, sore throat, lower extremity weakness/numbness, diarrhea, low blood O2, sneezing, runny nose or post-nasal drip; severe symptoms, such as ventilatory use, high fever, severe cough, delirium, seizures, stroke, systematic inflammation, cytokine storm; and other symptoms, such as fever, swollen adenoids, pneumonia, bronchitis, and dyspnea.

Modes of Administration and Effective Dosages

[00129] Any method known to those in the art for contacting a cell, organ or tissue with one or more of the compositions disclosed herein may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccine compositions of the present technology to a mammal, suitably a human. When used in vivo for therapy, the one or more coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines described herein are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the pharmaceutical composition used, e.g., its therapeutic index, and the subject’s history.

[00130] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of the one or more coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines of the present technology useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines of the present technology may be administered systemically or locally.

[00131] The one or more coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of a coronavirus infection. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[00132] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment). Parenteral administration can be by bolus injection or by gradual perfusion over time.

[00133] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[00134] The pharmaceutical compositions comprising one or more coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[00135] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00136] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[00137] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798. [00138] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

[00139] A therapeutic agent can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle, or a lipid nanoparticle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity. One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.

[00140] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother ., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[00141] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy, et al.), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO 96/40073 (Zale, et al.), and PCT publication WO 00/38651 (Shah, et al.). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[00142] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[00143] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro. [00144] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00145] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00146] Typically, an effective amount of the one or more coronavirus (e.g., SARS-CoV- 2, MERS-CoV) vaccines disclosed herein sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, one or more coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccine concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime. The dosage of an attenuated virus vaccine for a mammalian (e.g., human) adult organism can be from about 10³- 10⁷ plaque forming units (PFU)/kg, or any range or value therein. However, the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point.

[00147] In some embodiments, a therapeutically effective amount of one or more of the coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines disclosed herein may be defined as a concentration of vaccine at the target tissue of 10'³² to 10'⁶ molar, e.g., approximately 10'⁷ molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).

[00148] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[00149] A vaccine composition of the present technology may confer resistance to one or more pathogens, e.g., one or more coronavirus (e.g., SARS-CoV-2, MERS-CoV) strains, by either passive immunization or active immunization. In active immunization, an inactivated or attenuated live vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host's immune response to the administration protects against infection. For passive immunization, the elicited antisera can be recovered and administered to a recipient suspected of having an infection caused by at least one coronavirus strain. [00150] In one embodiment, the vaccine is provided to a mammalian female (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of an immune response which serves to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta or in the mother's milk).

[00151] A typical regimen for preventing, suppressing, or treating coronavirus infection comprises administration of an effective amount of a coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccine composition as described herein, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months, or any range or value therein.

[00152] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.

Combination Therapy

[00153] In some embodiments, the one or more coronavirus (e.g., SARS-CoV-2, MERS- CoV) vaccine compositions of the present technology may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent. Thus, in some embodiments, one or more of the coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccines disclosed herein may be combined with one or more additional therapies for the prevention of a coronavirus infection. Additional therapeutic agents include, but are not limited to, an antiviral agent (e.g., remdesivir, lopinavir, ritonavir, ivermectin, tamiflu, or favipiravir); an antiinflammatory agent (e.g., dexamethasone, tocilizumab, kevzara, colcrys, hydroxychloroquine, chloroquine, or a kinase inhibitor); covalescent plasma from a subject recovered from a SARS-CoV-2 or MERS-CoV infection; an antibody binding to SARS-CoV- 2 or MERS-CoV (e.g., bamlanivimab, etesevimab, casirivimab, or imdevimab); or an antibiotic (e.g., azithromycin), or a combination thereof.

[00154] In any case, the multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.

Kits

[00155] The present disclosure also provides kits for the prevention of a coronavirus infection, comprising one or more coronavirus (e.g., SARS-CoV-2, MERS-CoV) vaccine compositions of the present technology. Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for the prevention of coronavirus infection.

[00156] The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts. The kits may optionally include instructions customarily included in commercial packages of therapeutic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products. [00157] The kit can also comprise, e.g., a buffering agent, a preservative or a stabilizing agent. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. In certain embodiments, the use of the reagents can be according to the methods of the present technology.

EXAMPLES

[00158] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compositions and systems of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology. The following Examples demonstrate the preparation, characterization, and use of illustrative compositions of the present technology for vaccine compositions in the treatment of coronaviruses.

Example 1: Experimental methods for Examples 2-3

[00159] Cell culture and reagents. Vero E6, Huh-7, Baby Hamster Kidney cells expressing the mouse virus receptor CEACAM1 (BHK-MVR) (gifts from Stanley Perlman, University of Iowa), and A549-ACE2 cells (a gift from Susan Weiss, University of Pennsylvania), were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Calu-3 cells (ATCC) were grown in MEM supplemented with 20% FBS. Human IFN- ? and IFN-y were purchased from R&D Systems. Cells were transfected with either Polyjet (Amgen) or Lipofectamine 3000 (Fisher Scientific) per the instructions of the manufacturers. [00160] Mice. Pathogen-free K18-ACE2 C57BL/6 mice were purchased from Jackson Laboratories. Mice were bred and maintained in the animal resources facility at the Oklahoma State University. Animal studies were approved by the University of Oklahoma State Institutional Animal Care and Use Committee (IACUC) and met stipulations of the Guide for the Care and Use of Laboratory Animals.

[00161] Generation of recombinant pBAC-SARS-CoV-2, pBAC-MERS-CoV, and pBAC- JHMV constructs. All recombinant pBAC constructs were created using Red recombination (58) with several previously described CoV bacterial artificial chromosomes (BACs). These include the WT-SARS-CoV-2 BAC based off the Wuhan-Hu- 1 isolate provided by Sonia Zuniga, Li Wang, Isabel Sola and Luis Enjuanes (CNB-CSIC, Madrid, Spain) (59), a MERS- CoV BAC based of the EMC isolate (48), and an MHV BAC based on the JHMV isolate (26). All constructs were engineered using a Kanr-I-Scel marker cassette for dual positive and negative selection as previously described (see primers in Table SI) (60). Both forward and reverse primers were designed to include a 40bp region upstream of Mac 1 to facilitate the deletion of Macl by recombination (Table SI). Final BAC DNA constructs were confirmed by restriction enzyme digestion, PCR, and direct sequencing for the identification of correct clones.

[00162] Reconstitution of recombinant pBAC-SARS-CoV-2-derived virus. All work with SARS-CoV-2 and MERS-CoV was conducted in either the University of Kansas or the Oklahoma State University EHS-approved BSL-3 facilities. To generate SARS-CoV-2 or MERS-CoV viruses, approximately 5* 10⁵ Huh-7 cells were transfected with 2 pg of purified BAC DNA using Lipofectamine 3000 (Fisher Scientific) as a transfection reagent. SARS- CoV-2 generated from these transfections (pO) was then passaged in Vero E6 cells to great viral stocks (pl). All pl stocks were again sequenced to confirm that they retained the correct Macl deletion and to ensure the furin cleavage site had not been lost (for primers see Table S2). To generate MHV-JHM and MERS-CoV virus, approximately 5* 10⁵ BHK-MVR cells were transfected with 1 pg of purified BAC DNA using PolyJet™ Transfection Reagent (SignaGen). In the case of MHV-JHM, an additional 1 pg of N protein-expressing plasmid was co-transfected with genomic BAC DNA. [00163] Virus infection. Vero-E6, A549-ACE2, or Calu-3 cells were infected at the indicated MOIs. For Calu-3 cells, trypsin-TPCK (1 g/ml) was added to the medium at the time of infection. All infections included a 1-hour adsorption phase, except for Calu-3 cells where the adsorption phase was increased to 2 hrs. Infected cells and supernatants were collected at indicated time points and titers were determined on Vero E6 cells. For IFN pretreatment experiments, human IFN-/? and IFN-y were added to Calu-3 cells 18-20 hours prior to infection and were maintained in the culture media throughout the infection. For animal infections, 12-16-week-old K18-ACE2 C57BL/6 female mice were lightly anesthetized using isoflurane and were intranasally infected with 2.5* 10⁴ PFU in 50 //I DMEM. To obtain tissue for virus titers, mice were euthanized at different days post challenge, lungs or brains were removed and homogenized in phosphate buffered saline (PBS) and titers were determined on Vero E6 cells.

[00164] Immunoblotting. Total cell extracts were lysed in sample buffer containing SDS, protease and phosphatase inhibitors (Roche), /?-mercaptoethanol, and a universal nuclease (Fisher Scientific). Proteins were resolved on an SDS polyacrylamide gel, transferred to a polyvinylidene difluoride (PVDF) membrane, hybridized with a primary antibody, reacted with an infrared (IR) dye-conjugated secondary antibody, visualized using a Li-COR Odyssey Imager (Li-COR), and analyzed using Image Studio software. Primary antibodies used for immunoblotting included anti-SARS-CoV-2 N (SinoBiological 40143-R001) and GAPDH (Millipore- Sigma G8795) monoclonal antibodies. Secondary IR antibodies were purchased from Li-COR.

[00165] Confocal Immunofluorescence . Calu-3 cells were cultured with approximately 1.4* 10⁵ cells per well in 8-well, removable chamber slides (ibidi 80841) and infected with SARS-CoV-2 at an MOI of 1 PFU/cell. At 24 hpi, monolayers were fixed for 20 minutes with ice cold methanol then 10 minutes with 2% paraformaldehyde in HBSS + 0.01% Sucrose (HBSS/Su). Permeabilization with 0.1% Saponin in HBSS/Su was then performed, followed by overnight blocking at 4°C using 3% goat serum in HBSS/Su + Saponin. Primary antibody incubation was conducted for 3 hours at room temperature (1 :2,000 a-N protein, Sino Biological 40143-R001; 1 :500 a-nsp3, abeam ab283958) followed by a 1 hour, room temperature secondary antibody incubation (1 :200 AlexaFluor 555 Goat a-rabbit, Invitrogen A32732). Nuclear stain with 300nM DAPI was performed at room temperature for 30 minutes followed by mounting in Vectashield Vibrance Mounting Medium (Vector Labs H- 1700) and storage at 4°C. Images were acquired using an Olympus F VI 000 laser- scanning confocal microscope equipped with Fluoview software. Images were z-projected using maximum intensity.

[00166] Semi-quantitative PCR analysis. B AC DNA or infection-derived cDNA was PCR amplified by primers that bind outside of the Macl coding sequence. PCR products were analyzed by gel electrophoresis using a LICOR M imager and bands were quantified using Image Studio software and the relative intensity of each band was determined by adding the overall intensity of both bands together and then diving the intensity of each individual band by the total intensity.

[00167] Real-time quantitative PCR (RT-qPCR) analysis. RNA was isolated from cells using Trizol (Invitrogen). Lungs from K18-ACE2 C57BL/6 mice infected with virus were collected at indicated time points and were homogenized in Trizol (Invitrogen) and RNA was isolated using manufacturer’s instructions. cDNA was prepared using MMLV-reverse transcriptase per the manufacturer’s instructions (Thermo Fisher Scientific). qPCR was performed using PowerUp SYBR green master mix (Applied Biosystems) and primers listed in Table S3. Cycle threshold (CT) values were normalized to hypoxanthine phosphoribosyltransferase (HPRT) levels by using the ACt method.

[00168] RNAseq. RNA was isolated from K18-ACE2 mice as described above. Library preparation was performed by the University of Kansas Genome Sequencing core facility, using the NEB Next RNA Library kit (NEB) with indexing. RNA-seq was performed using an Illumina NextSeq2000 high-output system with a paired-end reads of 50 bp each. RNAseq data quality was checked using FastQC analysis pipeline. Samples had a minimum of 16 million reads and a mean quality score (PF) >33. The mouse (C57BL6) transcriptome reference sequence (GCF_000001635.27_GRCm39) and SARS-CoV-2 genome (Accession number - NC 045512.2) were downloaded from NCBI genome collections and appended into a single sequence and used as the reference sequence. RNAseq reads were mapped to the indexed reference sequence using kallisto v0.44.0. Transcripts per kilobase per million mapped reads (TPM) and read counts per transcript were extracted from the kallisto output. TPM values and read counts for all transcripts from each gene were summed to obtain gene- level expression estimates, and the counts per gene were then rounded to the nearest integer. For a given sample, only genes with at least 50 mapped reads total across all replicates from the samples were considered. DESeq2 was used to identify DEGs between the SARS-CoV-2 WT and AMacl infected samples using simply “treatment” as a factor. DEGs were identified based on the false-discovery rate corrected P-value (PAD J) and log2-fold-change of (log2FC) between the samples. Genes were considered upregulated in a SARS-CoV-2-infected sample if PADJ < 0.05 and log2FC > 0.6, which is nearly equivalent to a 1.5-fold increase. Similarly, genes were considered downregulated if PADJ < 0.05 and log2FC < -0.6, or a 1.5-fold decrease. DEGs were subjected to gene ontology analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID: https://david.ncifcrf.gov/). Gene lists were analyzed for biological processes that were significantly enriched with P < 0.05 and displayed as a clustered bar graph.

[00169] Lung cell preparation and flow cytometry. For phenotypic analyses of lung infiltrating immune cells, lungs collected at different days post-infection, PBS perfused lungs (left lobe were cut into small pieces, treated with collagenase-D and DNAse 1 for 30 minutes at room temperature, followed by homogenization of lung pieces using a 3ml syringe plunger flang/thumb rest. Homogenized cells were passed through 70pM strainer to obtain single cell suspension. Isolated single cell suspension was surface immunolabelled for neutrophil (CD45+ CD1 lb+ Ly6Ghi) and inflammatory monocyte (CD45+ CD1 lb+ Ly6chi) markers by flow cytometry. For cell surface staining, lung cells were labelled with the following fluorochrome-conjugated monoclonal antibodies: PECy7 a-CD45 (clone: 30-F11); FITC a- Ly6G (clone: 1A8); PE/PerCp-Cy5.5 a-Ly6C (clone: HK1.4); V450 a-CDl lb (clone: MI/70); APC a-F4/80 (clone: BM8) (all procured from Biolegend). A detailed cell surface and intracellular immunolabelling protocol for flow cytometry studies are described in our recent publication (61). All fluorochrome-conjugated antibodies were used at a final concentration of 1 :200 (antibody: FACS buffer), except for FITC labeled antibodies used at 1 : 100 concentration.

[00170] Histopathology. The lung lobes were perfused and placed in 10% of formalin. Brain samples were fixed in 10% formalin. The lung lobes and brain were then processed for hematoxylin and eosin (H & E). The lung lesions were blindly scored by an American College of Veterinary Pathology Board-certified pathologist. The lesions were scored on a scale of 0-10% (score 1), 10-40% (score 2), 40-70% (score 3) and >70% (score 4) and cumulative scores were obtained for each mouse. The lesions scored were bronchiointerstitial pneumonia, peribronchial inflammation, edema/fibrin, necrosis, and perivascular inflammation.

[00171] Statistics. A Student’s t test was used to analyze differences in mean values between groups. All results are expressed as means ± standard errors of the means (SEM). Differences in survival were calculated using a Kaplan-Meier log-rank test. P values of <0.05 were considered statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, p <0.0001; n.s., not significant).

[00172] Data and materials availability. All the RNAseq reads data are deposited in NCBI under the BioProject ID PRJNA928501 and BioSample ID SAMN32942656 and SAMN32942675 and will be made public upon publication or August 31 2023, whichever comes first.

[00173] The primers used in this study are provided below:

Table SI. Linear Recombination Primers for Engineering Macl Deletion BACs

Letters in Bold indicate Kar -I sequence (SEQ ID NOs: 5-10 respectively, in order of appearance)

F ACCTCAATTAGAGATGGAACTTACACCAGTTGTTCAGACTAGGATGACGACGATAAGTAGGG r 1/ 7 1 R GCGATCTTTTGTTCAACTTGCTTTTCACTCTTCATAGTCTGAACAACTGGTGTAAGTTCCATCTCTAATTGAGGTGCCAGTGTT CQI/-Z K ACAACCAATTAACC

1 ^F GGTATGACATTTAGTATGTCTCCCTTTGAGATCGCCCAGAGGATGACGACGATAAGTAGGG

/MM 1 R ATTGAAATGATAGCGCTTTGGTACCAGCAACGGAGGTCTGGGCGATCTCAAAGGGAGACATACTAAATGTCATACCAGCCAG JH/W K TGTTACAACCAATTAACC AATGTTGACCCTTTGTCCAATTTTGAACATAAGGTTATTAGGATGACGACGATAAGTAGGG TAGCCTCCCTAATAAGATAATCAAAAGACACAGCTGGTTTAATAACCTTATGTTCAAAATTGGACAAAGGGTCAACATTGCCAG

TGTTACACCAATTAACC

Table S2. Sequencing Primers (SEQ ID NOs: 11-18 respectively, in order of appearance)

Table S3. qPCR Primers (SEQ ID NOs: 19-52, respectively in order of appearance)

Example 2: Macl is required for SARS-CoV-2 to block the immune response during

SARS-CoV-2 infection in vivo

[00174] SARS-CoV-2 Macl deletion virus infectious virus was easily recovered while Macl deletion viruses in other /?-CoVs were not recovered. Several Macl mutations in murine hepatitis virus strain JHM (MHV-JHM) that were unrecoverable from a bacterial artificial chromosome (BAC) based reverse genetic system were recently identified (33). These results indicated that Macl may be critical for MHV replication. As point mutations could result in toxic unfolded proteins, created an MHV-JHM Macl deletion BAC to confirm our prior results. No infectious virus (FIGs. 8A-8B) was recovered from the Macl deletion BAC, further indicating that Macl is critical for MHV-JHM replication. A complete deletion of Macl was created in MERS-CoV, and again no infectious virus was recovered, indicating that Macl is also critical for MERS-CoV replication (FIGs. 8A-8B). To determine whether Macl might be essential for the replication of all CoVs, a Macl deletion ( Macl) was engineered into a SARS-CoV-2 BAC (Wuhan strain). However, unlike MHV-JHM or MERS-CoV, this virus was easily recoverable (FIGs. 8A-8B). This result indicates that there are stark differences in the requirement for Macl between SARS-CoV-2 and other ?-CoVs.

[00175] SARS-CoV-2 dMacl replicates like WT virus in most cell types. Next, the ability of SARS-CoV-2 Macl to replicate in several cell types susceptible to SARS-CoV-2 was assessed. In Vero E6 cells Macl replicated like WT virus cells at both low (FIG. 1A) and high (FIG. IB) multiplicity of infection (MOI), indicating that Macl is not required for general virus replication. Vero E6 cells lack the ability to produce IFN-I, and MHV-JHM Macl mutant viruses are more attenuated in cells that maintain the ability to produce IFN-I (32). Without wishing to be bound by theory, it is believed that SARS-CoV-2 Macl may be attenuated in either A549-ACE2 (alveolar epithelial cells) or Calu-3 cells (bronchial epithelial cells) that have a functional IFN system. Macl replicated equally to WT virus in A549-ACE2 cells (FIG. 1C), however there was a mild, ~2-3-fold reduction in Macl titers in Calu-3 cells compared to WT virus at both low (FIGs. 2A-2B) and high MOI (FIGs. 2C- 2D). Only mild, if any, reduction in viral N protein was observed when analyzed by immunoblotting and roughly equal levels of both N protein and nsp3 staining was observed by confocal microscopy (FIGs. 2E-2F) in Calu-3 cells infected by WT and Macl. To evaluate the relative fitness of Macl compared to WT virus, a competition experiment was performed where co-infected Calu-3 cells with WT and Macl at ratios of 1 : 1 and 1 :9, respectively, and followed these viruses over the course of 4 passages. Virus was collected at approximately 36 hpi after each passage to isolate virus during active replication, and not after peak replication has been reached. To distinguish between WT and Macl viruses, semi -quantitative RT-PCR with primer sets outside of Mac 1 that produce different sized PCR products from each virus was performed. First, using B AC DNA, it was found that the ratio of these bands correlated with the ratio of input BAC (FIGs. 9A-9B), indicating that this method could faithfully define the relative abundance of each virus following passaging. It was found that after 4 rounds of passaging Macl had not been outcompeted by WT virus as the ratios of these two viruses stayed relatively stable over the entire experiment (FIGs. 9C- 9D), though WT virus was starting to increase in abundance in the 9: 1 ( Macl :WT) sample at passage 4. In total, these results indicate that Macl generally replicates like WT virus but has a mild replication defect in Calu-3 cells, though it has similar fitness as WT virus in Calu- 3 cells.

[00176] SARS-CoV-2 dMacl induces increased IFN and cytokine responses in cell culture. Next, SARS-CoV-2 Macl was tested for its ability to induce IFNs and pro- inflammatory cytokines in cell culture, as has previously been shown for Macl mutants in SARS-CoV and MHV (18, 32). In both Calu-3 (FIG. 4A) and A549-ACE2 cells, Macl infection induced greater levels of both IFN-I and IFN-III transcript levels, and of ISGs such as ISG15 and CXCL-10 (FIG. 4B). However, the increase in IFN/ISG transcript levels, ~2-3 fold, is somewhat reduced compared to those prior results with SARS-CoV-1 and MHV-JHM Macl mutant viruses (26, 32). This differences in IFN induction between WT and Macl deleted/mutant viruses between different CoVs could be due to alterations in the functions or abundance of other CoV-encoded IFN repressing proteins expressed by SARS-CoV-2.

[00177] SARS-CoV-2 AMacl is highly attenuated in K18-ACE2 mice. The ability of SARS-CoV-2 WT and Macl to cause disease in K18-ACE2 C57BL/6 mice, a lethal animal model of SARS-CoV-2 infection was tested. Following intranasally inoculation of 2.5* 10⁴ PFU WT SARS-CoV-2, 100% morbidity and mortality was observed. In contrast, SARS- CoV-2 Macl infection did not cause any weight loss or lethality, indicating extreme attenuation (FIGs. 5A-5B). When analyzing the infected lungs by hematoxylin and eosin staining, significantly higher levels of bronchointerstitial pneumonia, inflammation, and edema and fibrin were observed in WT SARS-CoV-2 infected lungs compared to Macl virus infected lungs (FIG. 5C). The WT and Macl SARS-CoV-2 loads in infected lungs were compared. By day 1 post-infection there was a significant reduction in viral titers (FIG. 5E) and viral genomic RNA (gRNA) (FIG. 5F) of ~ 1 log in the lungs of Macl infected mice compared to WT SARS-Cov-2 infected lungs. The difference in viral load between WT and Macl increased to 2.5 logs by day 3, and by day 7 Macl was effectively cleared from the lungs while WT virus was still present at about 10⁵ PFU in the lung (FIGs. 5E-5F). In contrast, viral loads in the brain were very low until after day 3 post-infection, though WT virus was present at low levels in most mice by day 7, whereas Macl titers were below the detection limit at all days tested. (FIG. 11 A). Further, there was no significant difference in brain pathology between WT and Macl infected mice (FIG. 11B), indicating that brain infection and pathology did not significantly contribute to the weight loss and mortality of WT virus infected mice.

[00178] SARS-CoV-2 dMacl induces a robust innate immune response in the lungs of K18-ACE2 mice. The rapid clearance of Macl in the lungs of infected mice and prior results with Macl SARS-CoV mutant viruses (18) suggested that Macl would induce a strong innate immune response in mice. To test this possibility, the transcripts of a small panel of IFN and ISGs were measured for their expression following infection of WT and Macl at 1 day post infection (FIG. 6A). IFN-/? and IFN-A were upregulated by more than 10-fold in Macl infected lungs, while IFN-y was not detected. A ~2-3-fold increase in several ISGs, such as OAS, ISG15, CXCL10, IL-6, PARP12, and PARP14 was observed. These results suggest that the attenuation of Macl virus could, at least in part, be due to a robust IFN response at the early stages of infection. To get a global view of all the transcriptional changes occurring in the absence of Macl, RNAseq of whole lung samples collected at day 1 post-infection was performed. Differentially expressed genes were define as having at least 1.5-fold increased expression in either WT or Macl infected lungs with an adjusted p value of <0.05. In total, 645 genes were increased following infection with Macl, and another 230 were increased following WT infection, including viral gRNA, for a total of 875 differentially regulated genes (FIG. 6B). A gene ontology analysis was performed, and it was found that genes related to immunity, innate immunity, and antiviral defense were the pathways that were most significantly upregulated in Macl infected lungs (FIG. 6C). In addition, genes in the categories of adaptive immunity, ubiquitin conjugation, inflammatory responses, peptide transport, cytolysis, and apoptosis were also significantly upregulated in Macl infected lungs (FIG. 6C). The individual expression of a panel of ISGs was assessed and it was found that most ISGs were increased between 2 and 4-fold in Macl when compared to WT virus infection, while IFN-/? and IFN-A were increased more than 10-fold (FIG. 6D, FIG. 12). In total, Macl is required for SARS-CoV-2 to block the innate immune response during SARS-CoV-2 infection in mice.

[00179] SARS-CoV-2 dMacl infection results in reduced myeloid cell accumulation in the lungs. Next, the impact of WT and Macl virus infection on the recruitment of innate immune cells was assessed, specifically inflammatory monocytes and neutrophils, into the lung that might contribute differential lung inflammation and disease severity. Inflammatory monocytes were found to contribute to disease severity in SARS-CoV-1 and MERS-CoV infected mice by promoting the production of TNFa and increased T cell apoptosis (44, 45). Previously, IFN-I was shown to enhance inflammatory monocyte accumulation in the lung, though this was due to IFN-I production in the later stages of SARS-CoV-1 replication (44). However, earlier exogenous addition of IFN-I reduced inflammatory monocyte infiltration following MERS-CoV infection was shown to reduce the number of inflammatory monocytes (45). Without wishing to be bound by theory, it is believed that the early IFN-I and IFN-III induction by Macl would result in fewer infiltrating inflammatory immune cells. Following infection with Macl, a substantial reduction in both the percentage and total number of inflammatory monocytes was observed at both 3 and 7 days after infection (FIG. 7A), which could also play a role in the attenuation of the disease severity. Neutrophils were slightly increased in percentage in Macl infected lungs at day 3 but had similar total numbers when compared to WT virus infection (FIG. 7B). However, by day 7 there was a significant reduction in the total number of neutrophils in Macl infected lungs (FIG. 7B). Overall, our results indicate that the absence of Macl promotes a strong IFN response with a reduction in inflammatory cell types that may both play a role in reducing viral loads and preventing disease following infection. [00180] It was discovered that Macl is critical for the replication of MHV-JHM, as at least two Macl mutant recombinant BACs failed to produce infectious virus (33). However, one of these mutations, G1439V, did replicate after acquiring a second site mutation in the residue immediately preceding it, A1438T. To confirm these results, a complete deletion of Macl was created in the MHV-JHM BAC and again no infectious virus was recovered (FIGs. 8A- 8B) To determine if a Macl deletion may be detrimental across CoVs, MERS-CoV and SARS-CoV-2 Macl recombinant BACs were created. Surprisingly, no infectious virus from the MERS-CoV Macl BAC was recovered whereas infectious SARS-CoV-2 Macl was easily recovered (FIGs. 8A-8B).

[00181] It was discovered there was no defect in the replication of Macl in Vero E6 and A549 cells and only a modest defect in Calu-3 cells. Given these results, along with modularity of the various domains of nsp3, it is highly unlikely that the complete deletion of Macl had a significant effect on the overall structure of nsp3. Despite the lack of a large replication defect of Macl under normal growth conditions, it was found that Macl had a >1 log defect in IFN-y, but not IFN-/? treated Calu-3 cells (FIG. 5). IFN-y induces a small number of ISGs compared to IFN-/? (43). Without wishing to be bound by theory, it is believed that while the PARP enzymes that inhibit Macl mutant MHV are upregulated by both types of IFN, other more potent anti-SARS-CoV-2 ISGs are only upregulated by IFN-/?, or at least upregulated to a much higher level by IFN-/?. These ISGs might limit viral entry, mitigating the effect of PARP enzymes to specifically target the Macl mutant virus during later stages of the viral lifecycle.

[00182] Similar to SARS-CoV-1, it was discovered that SARS-CoV-2 Macl induces a robust innate immune response both in cell culture and in mice (FIGs. 4A-4B, FIGs. 6A- 6D), further confirming that Macl is one of the many potent IFN repressing proteins expressed by CoVs. This innate immune response occurred within one day of infection, and likely before peak replication of the virus. Whole lung RNAseq data identified over 100 genes involved in immunity to virus infection, demonstrating the breadth of the immune response that is triggered following Macl infection (FIGs. 6B-6D).

[00183] These results demonstrate that the immune response triggered following Macl infection at least partially protects mice from coronavirus infection. Example 3: Methods for Viral Challenge Experiments

[00184] SARS-CoV-2. K18-C57BL/6 mice were initially infected with 2.5* 10⁴ PFU of SARS-CoV-2 Macl or given PBS intranasally (i.n.). 5 weeks post-infection mice were challenged with 1 * 10⁵ PFU of WT virus i.n. and mice were monitored for survival or weight loss. N=5 for both PBS and Macl groups.

[00185] MERS-CoV. hDPP4 knock-in C57BL/6 mice were either treated with PBS or infected i.n. with 250 PFU of mouse-adapted MERS-CoV N1147 A or DI 129A. At 5 weeks post-infection mice were challenged with 750 PFU of WT virus i.n. and mice were monitored for survival and weight loss. N=4 for PBS and N 1147A groups, N=5 for DI 129A.

[00186] As shown in FIGs. 13A-13D and FIGs. 18A-18D, the modified Macl domain deficient coronavirus (e.g., SARS-CoV-2, MERS) vaccines disclosed herein effectively protect subjects from subsequent infections from these viral pathogens. Likewise, it is expected that modified SARS-CoV-2 harboring point mutations within the Macl domain (e.g., N1062, H1067, D1044, G1152, 11153, Fl 154) will also effectively protect subjects from subsequent infection.

Example 4: Experimental Methods for Examples 5-10

[00187] Plasmids. MERS-CoV Macl (residues 1110-1273 of ppla) and mutations were cloned into pET21a+ with a C-terminal His tag. SARS-CoV-2 Macl (residues 1023-1197 of ppla) was cloned into the pET30a+ expression vector with an N-terminal His tag and a TEV cleavage site (Synbio).

[00188] Protein Expression and Purification. A single colony of E. coli cells BL21 C41 (DE3) or pRARE (DE3) containing plasmids harboring the constructs of the macrodomain proteins was inoculated into 10 mL LB media and grown overnight at 37°C with shaking at 250 rpm. For most proteins, the overnight culture was transferred to a shaker flask containing TB media at 37°C until the OD600 reached 0.7. The proteins were either induced with either 0.4 mM (SARS-CoV-2 proteins) or 0.05 mM (MERS-CoV proteins) IPTG at 17°C for 20 hours. Cells were pelleted at 3500 x g for 10 min and frozen at -80°C. Frozen cells were thawed at room temperature, resuspended in 50 mM Tris (pH 7.6), 150 mM NaCl, and sonicated using the following cycle parameters: Amplitude: 50%, Pulse length: 30 seconds, Number of pulses: 12, while incubating on ice for >lmin between pulses. The soluble fraction was obtained by centrifuging the cell lysate at 45,450 x g for 30 minutes at 4°C. The expressed soluble proteins were purified by affinity chromatography using a 5 ml prepacked HisTrap HP column on an AKTA Pure protein purification system (GE Healthcare). The fractions were further purified by size-exclusion chromatography (SEC) with a Superdex 75 10/300 GL column equilibrated with 20mM Tris (pH 8.0), 150 mM NaCl and the protein sized as a monomer relative to the column calibration standards. For the SARS-CoV-2 N1062A protein several modifications to this protocol were made to obtain stable soluble protein. First, the overnight culture was transferred to LB instead of TB and grown to OD600 0.5 before the protein was induced with 0.05 mM IPTG at 17°C for 20 hours. Cells were resuspended in water prior to sonication. Tris and NaCl were added after sonication. The cell lysate was then incubated with HIS-select HF Nickel Affinity Gel (Millipore-Sigma) overnight, rotating at 4°C. The lysate was then passed into gravity flow chromatography. Columns were washed with 0.5M NaCl and 50 mM Tris-Cl pH 8 and eluted with 0.5 ml of elution buffer with 0.1 M of Imidazole. Following elution, the protein was immediately purified by size-exclusion chromatography as described above.

[00189] Isothermal Titration Calorimetry. All ITC titrations were performed on a MicroCai PEAQ-ITC instrument (Malvern Pananalytical Inc., MA). All reactions were performed in 20 mM Tris pH 7.5, 150 mM NaCl using 100 pM of all macrodomain proteins at 25°C. Titration of 2 mM ADP-ribose or ATP (MilliporeSigma) contained in the stirring syringe included a single 0.4 pL injection, followed by 18 consecutive injections of 2 pL. Data analysis of thermograms was analyzed using one set of binding sites model of the MicroCai ITC software to obtain all fitting model parameters for the experiments.

[00190] Differential Scanning Fluorimetry (DSF). Thermal shift assay with DSF involved use of LightCycler® 480 Instrument (Roche Diagnostics). In total, a 15 pL mixture containing 8X SYPRO Orange (Invitrogen), and 10 pM macrodomain protein in buffer containing 20 mM Hepes, NaOH, pH 7.5 and various concentrations of ADP-ribose were mixed on ice in 384-well PCR plate (Roche). Fluorescent signals were measured from 25 to 95°C in 0.2°C/30-s steps (excitation, 470-505 nm; detection, 540-700 nm). Data evaluation and Tm determination involved use of the Roche LightCycler® 480 Protein Melting Analysis software, and data fitting calculations involved the use of single site binding curve analysis on Graphpad Prism.

[00191] AlphaScreen (AS) Assay. The AlphaScreen reactions were carried out in 384- well plates (Alphaplate, PerkinElmer, Waltham, MA) in a total volume of 40 pL in buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 0.5 mM TCEP, 0.1% BSA, and 0.05% CHAPS. All reagents were prepared as 4X stocks and 10 pL volume of each reagent was added to a final volume of 40 pL. All compounds were transferred acoustically using ECHO 555 (Beckman Inc) and preincubated after mixing with purified His-tagged macrodomain protein (250 nM) for 30 min at RT, followed by addition of a 10 amino acid biotinylated and ADP-ribosylated peptide [ARTK(Bio)QTARK(Aoa-RADP)S] (Cambridge peptides) (625 nM). After Ih incubation at RT, streptavidin-coated donor beads (7.5 pg/mL) and nickel chelate acceptor beads (7.5 pg/mL); (PerkinElmer AlphaScreen Histidine Detection Kit) were added under low light conditions, and plates were shaken at 400 rpm for 60 min at RT protected from light. Plates were kept covered and protected from light at all steps and read on BioTek plate reader using an AlphaScreen 680 excitation/570 emission filter set. For data analysis, the percent inhibition was normalized to positive (DMSO + labeled peptide) and negative (DMSO + macrodomain + peptide, no ADPr) controls. The ICso values were calculated via four-parametric non-linear regression analysis constraining bottom (=0), top (=100), & Hillslope (=1) for all curves.

[00192] MAR Hydrolase Assays. First, a 10 pM solution of purified PAPR10-CD protein was incubated for 20 minutes at 37°C with 1 mM final concentration of P- Nicotinamide Adenine Dinucleotide (P NAD⁺) (Millipore-Sigma) in a reaction buffer (50 mM HEPES, 150 mM NaCl, 0.2 mM DTT, and 0.02% NP-40). MARylated PARP10 was aliquoted and stored at -80°C. Next, a 0.5 (I-A/F-A) or 5 (N/A) pM solution of MARylated PARP10-CD and 0.1 (I-A/F-A) or 1 (N-A) pM purified Macl protein was added in the reaction buffer (50 mM HEPES, 150 mM NaCl, 0.2 mM DTT, and 0.02% NP-40) and incubated at 37°C for indicated times. The reaction was stopped with addition of 2X Laemmli sample buffer containing 10% P-mercaptoethanol. Protein samples were heated at 95°C for 5 minutes before loading and separated onto SDS-PAGE cassette (Thermo Fisher Scientific Bolt™ 4-12% Bis-Tris Plus Gels) in MES running buffer. For direct protein detection, the SDS-PAGE gel was stained using InstantBlue® Protein Stain (Expedeon). For immunoblotting, the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membrane using iBlot™ 2 Dry Blotting System (ThermoFisher Scientific). The blot was blocked with 5% skim milk in PBS containing 0.05% Tween-20 and probed with antimono ADP-ribose binding reagent MABE1076 (a-MAR) (Millipore-Sigma) and anti-GST tag monoclonal antibody MA4-004 (ThermoFisher Scientific). The primary antibodies were detected with secondary infrared anti-rabbit and anti-mouse antibodies (LI-COR Biosciences). All immunoblots were visualized using Odyssey® CLx Imaging System (LI- COR Biosciences). The images were quantitated using Image J (National Institutes for Health (NIH)) or Image Studio software.

[00193] Molecular Dynamics (MD) Simulations. 25 ns simulations were performed for WT and II 153 A protein in the presence and absence of ADP-ribose using GROMACS 2019.4 [Van Der Spoel etal., J Comput Chem. 2005;26(16): 1701-18], Protein structures used were ADP -ribose-bound SARS-2-CoV Macl, PDB 6W02 [Schuller et al., Sci Adv. 2021;7(16). Epub 20210414], and unbound SARS-2-CoV Macl, PDB 7KQO [Michalska et al., lUCrJ. 2020;7(Pt 5):814-24], The simulations were prepared, including virtual mutagenesis, using CHARMM-GUFs Solution Builder [Jo et al., J Comput Chem.

2008;29(l 1): 1859-65], which was used to build a solvated, rectangular box around one protein, parameterize the ligand, add ions to neutralize the system, set up periodic boundary conditions, and generate the files to perform a gradient based minimization, 100 ps equilibration with a NVT ensemble, and then a 25 ns production run with an NPT ensemble at 303.15 K.

[00194] Cell Culture and Reagents. Vero E6, Huh-7, Vero81, DBT, L929, HeLa cells expressing the MHV receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) (HeLa-MHVR), Baby Hamster Kidney cells expressing the mouse virus receptor CEACAM1 (BHK-MVR) (all gifts from Stanley Perlman, University of Iowa), AJK6, and A549-ACE2 cells (both gifts from Susan Weiss, University of Pennsylvania), were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Calu-3 cells (ATCC) were grown in MEM supplemented with 20% FBS. Bone marrow-derived macrophages (BMDMs) sourced from PARP12^+/+ and PARP12' ^A mice were differentiated into M0 macrophages by incubating cells in Roswell Park

Memorial Institute (RPMI) media supplemented with 10% FBS, sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin, L-glutamine, M-CSF (Genscript) for six days. Then to differentiate into M2 macrophages, IL-4 (Peprotech Inc.) was added for 1 day. Cells were washed and replaced with fresh media every other day after the 4^th day. Human IFN-y was purchased from R&D Systems. Cells were transfected with either Polyjet (Amgen) or Lipofectamine 3,000 (Fisher Scientific) per the instructions of the manufacturers.

[00195] Generation of Recombinant pBAC-JHMV, pBAC-MERS-CoV, and pBAC- SARS-CoV-2 Constructs. All recombinant pBAC constructs were created using Red recombination with several previously described CoV BACs as previously described [Fehr et al., Methods Mol Biol. 2020;2099:53-68], These include the WT-SARS-CoV-2 BAC based off the Wuhan-Hu-1 isolate provided by Sonia Zuniga, Li Wang, Isabel Sola and Luis Enjuanes (CNB-CSIC, Madrid, Spain) [Roy et al, bioRxiv. 2021. Epub 2021/04/29], a MERS-CoV mouse-adapated BAC (a gift from Dr. Stanley Perlman) with GFP inserted into ORF5 [Almazan et al., mBio. 2013;4(5):e00650-13, Li et al., mBio. 2020; 11(2). Epub 20200407], and an MHV BAC based off of the JHMV isolate [Fehr et al., J Virol.

2015;89(3): 1523-36], Primers used to create each mutation are listed in Table 2 (represented as SEQ ID NOs: 57-76 in order of appearance).

Table 2. Primers for generating recombinant CoV BACs

Viral sequences are indicated in uppercase; marker sequences are indicated in lowercase. [00196] Reconstitution of Recombinant pBAC-JHMV, pBAC-MERS-CoV, and pBAC-SARS-CoV-2-Derived Virus. All work with SARS-CoV-2 and MERS-CoV was conducted in either the University of Kansas or the Oklahoma State University EHS- approved BSL-3 facilities. To generate SARS-CoV-2 or MERS-CoV, approximately 5 x io⁵ Huh-7 cells were transfected with 2 pg of purified BAC DNA using Lipofectamine 3,000 (Fisher Scientific) as a transfection reagent. SARS-CoV-2 generated from these transfections (pO) was then passaged in Vero E6 (SARS-CoV-2) or Vero 81 (MERS-CoV) cells to generate viral stocks (pl). All pl stocks were again sequenced by Sanger sequencing to confirm that they retained the correct mutations. To generate MHV-JHM, approximately 5 x 10⁵ BHK- MVR cells were transfected with 1 pg of purified BAC DNA and 1 pg of N-protein expressing plasmid using PolyJetTM Transfection Reagent (SignaGen).

[00197] Mice. Pathogen-free C57BL/6NJ (B6) and KI 8-ACE2 C57BL/6 mice were originally purchased from Jackson Laboratories and mice were bred and maintained in the animal care facilities at the University of Kansas and Oklahoma State University. Animal studies were approved by the Oklahoma State University and University of Kansas Institutional Animal Care and Use Committees (IACUC) following guidelines set forth in the Guide for the Care and Use of Laboratory Animals.

[00198] Virus Infection. Cells were infected at the indicated MOIs. All infections included a 1 hr adsorption phase. Infected cells and supernatants were collected at indicated time points and titers were determined. For IFN pretreatment experiments, human IFN-y was added to Calu-3 or A549-ACE2 cells 18 to 20 h prior to infection and was maintained in the culture media throughout the infection. For MHV mouse infections, 5-8 week-old male and female mice were anesthetized with isoflurane and inoculated intranasally with 1 x 10⁴ PFU recombinant MHV in a total volume of 12pl DMEM. MHV infected mice were scored for disease based on the following scale: 0: normal, 0-5% weight loss with normal movement and normal behavior; 1 : mild disease, 6-12% weight loss, slightly slower movement, and mild neurological issues including circling, sporadic and sudden jumping/hyperreaactivity; 2: moderate disease, 13-20% weight loss, slow movement with notable difficulty, moderate neurological issues including occasional circling or head pressing; 3 : severe, >20% decrease in weight, severely reduced mobility, and severe neurological symptoms. Mice were euthanized if any of the conditions for a score of 3 were met. For SARS-CoV-2 mouse infections, 12 to 16-wk-old K18-ACE2 C57BL/6 female mice were lightly anesthetized using isoflurane and were intranasally infected with 2.5 x 10⁴ PFU in 50pL DMEM. To obtain tissue for virus titers, mice were euthanized on different days post challenge, lungs or brains were removed and homogenized in phosphate buffered saline (PBS), and titers were determined by plaque assay on either Hela-MVR (MHV) or VeroE6 (SARS-CoV-2) cells.

[00199] Histopathology. The lung lobes were perfused and placed in 10% formalin. The lung lobes were then processed for H&E staining. The lung lesions were blindly scored by an American College of Veterinary Pathology Board-certified pathologist. The lesions were scored on a scale of 0 to 10% (score 1), 10 to 40% (score 2), 40 to 70% (score 3), and >70% (score 4), and cumulative scores were obtained for each mouse. The lesions scored were bronchointerstitial pneumonia, perivascular inflammation, edema/fibrin, and necrosis.

[00200] Real-time qPCR analysis. RNA was isolated from cells and lungs using TRIzol (Invitrogen) and cDNA was prepared using MMLV-reverse transcriptase as per manufacturer’s instructions (Thermo Fisher Scientific). Quantitative real-time PCR (qRT- PCR) was performed on a QuantStudio3 real-time PCR system using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). Primers used for qPCR were previously described [Alhammad et al., Proc Natl Acad Sci USA. 2023; 120(35):e2302083120], Cycle threshold (CT) values were normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) by the following equation: CT = CT(_gene of interest) - CT(HPRT) Results are shown as a ratio to HPRT calculated as 2'^ACT.

[00201] Statistics. A Student’s t test was used to analyze differences in mean values between 2 groups, for multiple group comparisons, a one-way ANOVA was used. All results are expressed as means ± standard errors of the means (SEM) unless stated as standard differentiation (SD). P values of <0.05 were considered statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, p <0.0001; ns, not significant).

Example 5: Murine hepatitis strain JHM (MHV) F1441A, but not I1440A, has decreased replication in cell culture and in mice

[00202] To uncover the relative contributions of the residues in the highly conserved GIF loop of Macl in CoV replication and pathogenesis, we first compared the replication of the Embecovirus MHV I1440A and F1441 A mutations to WT and a previously characterized mutant virus, N1347A. Previously, we found that N1347A replicates normally in most cell lines susceptible to MHV but replicates poorly in primary macrophages and in mice [Fehr et al., J Virol. 2015;89(3): 1523-36, Grunewald et al., PLoS Pathog. 2019;15(5):el007756], Here, we tested the replication of recombinant viruses in several different cell types that are susceptible to MHV including: a mouse astrocyte cell line (DBT), a mouse fibroblast cell line (L929), and primary bone-marrow cells differentiated into M2 macrophages. As expected, the Fl 441 A mutation had decreased replication in all cell types and in mice, with replication defects seen at peak titers ranging from 2.7-fold in DBT cells, to 4.3-fold in L929 cells, and 21.7-fold in M2 macrophages (FIGs. 20A-20C). We previously found that a knockout (KO) of PARP12 can restore the replication of N1347A, therefore we also tested the ability of F1441 A to replicate in PARP12 KO M2 BMDMs (FIG. 20D). In the absence of PARP12, F1441A replication increased by 9.3-fold, indicating that PARP12 contributes to the restriction of this virus, much like N1347A. However, it was not fully rescued, indicating that F1441 may also be important for ADP -ribose binding. In contrast, I1440A replicated at WT levels in all cell types and at all time points (FIGs. 20A-20D).

[00203] We hypothesized that the importance of the isoleucine residue may become more apparent in vivo, so we tested the ability of these mutant MHV to cause severe encephalitis in mice. C57BL/6 mice were infected intranasally with 10⁴ PFU of each virus and were monitored for weight loss and survival over 12 days, and viral loads in the brain were measured at day 5 post-infection. The F1441 A mutant was attenuated in mice as only 50% of the mice succumbed to infection, while the other half recovered after losing -10% of their body weight (FIGs. 21A-21B). This attenuation of F1441A was also demonstrated in the disease scores of the F1441 A virus, as disease scores started to reverse by day 8 for F1441 A infected mice (FIG. 21C). Furthermore, F1441 A virus infected mice had -7.5 fold lower viral loads in mice than WT virus infected mice (FIG. 21D). These titers were highly variable, reflecting the fact that 50% of the mice survived. In contrast, I1440A infected mice all succumbed to disease by 9 dpi, and much like the cell culture results, the I1440A viral loads were equivalent to WT virus in mice (FIGs. 21A-21D). Taken together, this indicates that F1441 is required for efficient virus replication and disease progression in JHMV, while the 11440 residue does not impact JHMV replication or pathogenesis. The lack of any impact of the I1440A mutation on MHV replication or pathogenesis was surprising, considering the extreme conservation of this residue through all CoVs [Leung et al., PLoS Pathog. 2018;14(3):el006864],

Example 6: MERS-CoV I238A has increased binding activity

[00204] To determine how these mutations impact the biochemical functions of Mac 1, we aimed to purify Macl protein with these mutations and utilize in vitro assays to measure ADP-ribosylhydrolase and ADP-ribose binding activity of each mutant protein. Multiple attempts to produce MHV Macl protein failed, so we engineered these mutations into the Merbecovirus MERS-CoV and the Sarbecovirus SARS-CoV-2 Macl recombinant proteins, as we’ve previously produced WT Macl proteins from each virus [Alhammad et al., J Virol. 2021 ;95(3)] . We first produced soluble I1238A and F1239A MERS-CoV Macl proteins and performed isothermal titration calorimetry (ITC) to measure Macl -ADP-ribose binding. ITC measures the release or absorption of energy during a binding reaction and has been used extensively to measure macrodomain-ADP-ribose interactions [Alhammad et al., J Virol. 2021;95(3), Abraham et al., mBio. 2020; 11(1), McPherson et al., Proc Natl Acad Sci USA. 2017; 114(7): 1666-71, Karras et al., EMBO J. 2005;24(l l): 1911-20, Neuvonen et al., J Mol Biol. 2009;385(l):212-25], Compared to WT protein, the F1239A protein bound to free ADP-ribose with a substantially higher KD value (60 pM vs. 7.2 pM) indicating reduced binding ability. In contrast, the I1238A protein bound to ADP-ribose with a KD nearly equivalent to that of WT (12.7 pM vs 7.2 pM) (FIG. 22A). In addition to ITC, we also performed an AlphaScreen assay, as previously described [Roy et al., Antiviral Res.

2022;203: 105344, Schuller et al, ACS Chem Biol. 2017; 12(11):2866-74, Ekblad et al., SLAS Discov. 2018;23(4):353-62], to determine the ability of each protein to bind to an ADP- ribosylated peptide. Similar to the ITC assay, the F1239A Macl had substantially reduced AlphaScreen counts at all concentrations of protein tested as compared to WT protein, indicating poor binding to the ADP-ribosylated peptide (FIG. 22B). Remarkably, the 11238 A Macl protein had dramatically increased AlphaScreen counts at all concentrations of protein tested, indicating that this mutation has enhanced binding to an ADP-ribosylated peptide (FIG. 22B). To further test this observation, we performed a competition assay by adding increasing amounts of ADP-ribose to the reaction. ADP-ribose inhibited the peptide- ADP-ribose interaction of WT MERS-CoV protein with an average ICso value, of 0.155 gM, while it had a much higher ICso value of 1.6 gM for the I1238A protein. These results

-n- demonstrate that 11238 A had a stronger interaction with the ADP-ribosylated peptide than WT protein (FIG. 22C).

[00205] We next tested the ability of the MERS-CoV I1238A and F1238A Macl proteins to hydrolyze mono- ADP -ribose (MAR) from protein as previously described [Alhammad et al., J Virol. 2021 ;95(3)]. The WT, I1238A, and F1239A Macl proteins were incubated with MARylated PARP10 at a 1 :5 enzyme to substrate ([E]/[S]) ratio, and the reaction was stopped at several timepoints to determine the ability of each protein to hydrolyze MAR. As a control, MARylated PARP10 was collected at the first (0 min) and the final (30 min) timepoints. Over the course of 30 minutes, the MERS-CoV I1238A Macl protein decreased the level of MARylated PARP10 to similar levels of the MERS-CoV WT Macl protein, while the MERS-CoV Fl 239 A Macl protein did not efficiently remove the MARylation from P ARP 10 (FIGs. 22D-22E). Taken together, we conclude that the MERS-CoV 11238 A and Fl 239 A mutations had somewhat opposing effects on the activity of Macl. While F1239A mutant Macl protein has decreased ADP -ribose binding and hydrolysis activity, the 11238 A Macl has increased ADP-ribose binding with only a modest reduction in enzyme activity compared to the MERS-CoV WT Macl (Table 1).

Table 1. Summary of Macl ADP-ribose binding, hydrolysis, and replication activity

nd, not determined

*SARS-CoV-2 replication is defined in the presence of IFNy

Example 7: MERS-CoV I1238A and F1239A viruses have decreased replication in human and bat cell lines

[00206] With biochemical results in hand, we next tested MERS-CoV 11238 A and F1239A viruses for their ability to replicate in multiple cell types. First, using recombination, we inserted a GFP cassette in place of ORF5 in the MERS-CoV-MA BAC, as ORF5 quickly mutates in cell culture, which could complicate our results [Fehr et al., Methods Mol Biol.

2020;2099:53-68, Li et al., 4S2017;114(15):E3119-E28], Considering that the 11238 A had equivalent or enhanced biochemical activities compared to WT protein, we hypothesized that only the F1239A virus would impact MERS-CoV replication, similar to results seen with MHV (FIGs. 20-21). Both mutant viruses replicated near WT levels at all time points in Vero81 cells, which are unable to produce interferon (IFN) (FIG. 23 A). Next, we tested the replication of these viruses in IFN-competent Calu3 cells, human bronchial epithelial cells, and AJK6 cells, a Jamaican bat kidney cell line previously shown to be susceptible to MERS- CoV. To our surprise, both viruses replicated poorly in these cells. In Calu3 cells F1239A replicated at 4.7 and 46.7-fold lower than WT virus at 48 and 72 hours, respectively, while I1238A replication was reduced 5.3 and 34.1-fold at 48 and 72 hpi (FIG. 23B). In the AJK6 cells, the F1239A virus had replication defects of 2.5 and 12-fold at 24 and 48 hours respectively, while the I1238A virus replicated was reduced 18.5-fold at 48 hpi (FIG. 23C). We conclude that each of these residues is critical for MERS-CoV replication in cell culture, and that the defect of the I1238A virus could be due to enhanced ADP -ribose binding (Table 1).

Example 8: SARS-CoV-2 I1153A and F1154A have increased ADP-ribose binding activity

[00207] The MERS-CoV data indicated that increased ADP-ribose binding activity may lead to replication defects in culture. However, each of the MERS-CoV mutants had at least a modest defect in enzyme activity, which could account for the poor replication of each virus (Table 1). To further test the hypothesis that increased ADP-ribose binding could be detrimental to infection, we engineered these mutations in SARS-CoV-2 to analyze their impact on Macl biochemical functions and viral replication. We produced soluble II 153 A and the Fl 154A SARS-CoV-2 Macl proteins and first performed ITC to determine the ADP- ribose binding ability of each Macl mutant protein. Interestingly, both the SARS-CoV-2 Il 153A and the Fl 154A Macl proteins had increased binding to free ADP-ribose, with KD values of 5.49 pM and 5.11 pM, respectively, compared to the KD value of 16.8 pM for WT protein (FIG. 24A). Next, we tested the ability of each protein to bind to the ADP- ribosylated peptide in the AlphaScreen assay. Again, we observed that both the II 153 A and the Fl 154 A Macl proteins had increased binding to the ADP-ribosylated peptide compared to the WT Macl protein (FIG. 24B). [00208] Next, we tested the ability of each SARS-CoV-2 protein to remove MAR from MARylated PARP10, again at a 1 :5 [E]/[S] ratio to account for defects in enzyme turnover. Like MERS-CoV F1239A, SARS-CoV-2 Fl 154A had only modest hydrolysis activity. In contrast, Il 153 A Macl protein had robust enzymatic activity, which was virtually indistinguishable from WT protein (FIGs. 24C-24D), which is consistent with previously published results [Rack etal., Open Biol. 2020; 10(11):200237], These results demonstrate that the II 153 A and Fl 154A both have enhanced ADP-ribose binding, but that only Fl 154A has reduced enzymatic activity (Table 1).

[00209] As many previous studies of Macl in virus replication include the highly conserved asparagine-to-alanine mutation, we also generated an N1062A SARS-CoV-2 Macl protein. This protein is highly unstable, so several modifications to the normal protocol were made to create a small amount of soluble protein. While the small amount of protein did not allow for ITC measurements, this protein had similar ADP-ribose binding properties as WT protein, as determined by the alphascreen, ADP-ribose competition, and differential scanning fluorimetry assays (FIGs. 25A-25C). In contrast, this protein had substantially reduced ADP-ribosylhydrolase activity (FIG. 25D-25E), indicating that this mutation primarily impacts the enzyme activity of Macl. Previously published biochemical data also supports the hypothesis that this mutation primarily impacts the enzyme activity of Macl [Egloff et al., J Virol. 2006;80(17):8493-502, Fehr et al., mBio. 2016;7(6), Abraham et al., mBio.

2020; 11(1), Taha et al., PLoS Pathog. 2023; 19(8):el011614, Putics et al., J Virol. 2005;79(20): 12721-31, Karras et al., EMBO J. 2005;24(l l): 1911-20], This mutation can be directly compared with Fl 154 A to determine the impact of increased ADP-ribose binding on virus replication and pathogenesis, as both Fl 154 A and N1062 A have similar defects in enzyme activity but only Fl 154A has increased ADP-ribose binding (Table 1).

Example 9: Increased Macl ADP-ribose binding increases the sensitivity of SARS-CoV- 2 to IFNy

[00210] We previously reported that a Macl deleted SARS-CoV-2 is highly sensitive to IFNy pretreatment in Calu-3 cells. After demonstrating that both the SARS-CoV-2 II 153 A and Fl 154 A Macl proteins have increased ADP-ribose binding, we next tested the ability of these recombinant viruses to replicate in the presence of IFNy. Without IFNy pre-treatment at 48 hpi, both II 153 A and Fl 154A replicate at WT levels (FIG. 26A). In contrast, there is a substantial decrease in both II 153 A and Fl 154A replication in both cell lines compared to WT SARS-CoV-2 in the presence of IFNy (FIGs. 26A-26B). Furthermore, mutation at the N1062 residue, which primarily reduces ADP-ribosylhydrolase activity, has increased sensitivity to IFNY ^as well, though not as severe as the II 153 A and Fl 154A mutant viruses [Alhammad et al., Proc Natl Acad Sci USA. 2023; 120(35):e2302083120], These results support a model where Macl enzyme activity is primarily responsible for its ability to counter IFNy-mediated antiviral responses, and based on results with II 153 A, that increased ADP-ribose binding may negatively affect Macl’s ability to hydrolyze protein substrates in cells. We conclude that increased ADP-ribose binding by Macl is detrimental for the ability of SARS-CoV-2 to replicate efficiently in the presence of IFNy.

Example 10: SARS-CoV-2 I1153A and F1154A are attenuated in K18-ACE2 mice

[00211] Next, we tested whether enhanced ADP-ribose binding activity would be detrimental to SARS-CoV-2 infection in mice. Previously, a SARS-CoV-2 Macl deletion virus was shown to be extremely attenuated in K18-ACE2 mice, while the N1062 A mutant was mildly attenuated, with approximately 50% of mice surviving the infection [Alhammad et al, Proc Natl Acad Sci USA. 2023; 120(35):e2302083120, Taha et al., PLoS Pathog.

2023 ; 19(8):el 011614] . We hypothesized that like the SARS-CoV-2 N1062A mutant, there would be at least partial attenuation of II 153 A and Fl 154A viruses in mice. Following an intranasal infection, we were surprised to see that both the SARS-CoV-2 II 153 A and Fl 154A viruses were extremely attenuated in mice, as they did not cause any weight loss or lethal disease in mice, similar to Macl, whereas WT SARS-CoV-2 causes 100% mortality by 9 dpi (FIGs. 27A-27B). Viral titers were reduced by ~4-5 fold at 1 dpi (FIG. 27C), and by 8 dpi both the II 153 A and Fl 154A viruses were cleared from the lungs of mice (FIG. 27D). Furthermore, mice infected with these viruses had reduced signs of disease, such as bronchointerstitial pneumonia, edema, or fibrin, as measured by H&E staining (FIGs. 27E- 27F). Finally, both II 153 A and Fl 154A infected mice had significantly increased levels of IFN-I, IFN-III, ISG15, and CXCL-10 mRNA, similar to Macl infection levels (FIG. 27G). These results are consistent with the idea that enhanced ADP-ribose binding likely leads to severe defects in deMARylation during infection, as increased cytokine levels were previously demonstrated shown to be due to reduced deMARylating activity of Mac 1 mutant viruses [Taha et al., PLoS Pathog. 2023; 19(8):el011614, Voth et al., J Virol.

2021;95(15):e0076621], Taken together, these results demonstrate that increased ADP- ribose binding by Macl is detrimental to SARS-CoV-2 replication and pathogenesis in vivo.

[00212] In total, while the highly conserved isoleucine and phenylalanine mutations in MERS-CoV and SARS-CoV-2 have different effects on Macl biochemical activities in vitro, their impact on virus replication and pathogenesis were remarkably similar (Table 1). The simplest explanation is that enhanced ADP-ribose binding has a severe effect on Macl’s ability to hydrolyze specific substrates during infection, and that the conserved isoleucine residue acts to control ADP-ribose binding to allow for optimal ADP-ribosylhydrolase activity. But how does this isoleucine residue control ADP-ribose binding? Prior NMR data from the Venezuelan equine encephalitis virus (VEEV) macrodomain indicated that prior to ADP-ribose binding there is a significant transition that increases the distance between loop 1 and loop 2 from 7 to 10 A to accommodate ADP-ribose as a substrate [Makrynitsa et al., J Struct Biol. 2019;206(l): 119-27], We hypothesized that the II 153A protein has increased binding because the protein no longer requires this transition to bind ADP-ribose. To support this hypothesis, we performed a molecular dynamic (MD) simulation of the II 153 A and WT proteins in the presence and absence of ADP-ribose and measured the 1 ns running average distance between the I or A 1153 residue and G1069 (FIGs. 28A-28C). In the presence of ADP-ribose, these residues were nearly the same distance apart, -7.5-8 A. However, in the absence of ADP-ribose, the mutant protein (Al 153) consistently sampled conformations containing a larger distance between these residues, around or longer than -7.5 A, which results in a largely open crevice between the two loops (FIG. 28A). In contrast, the distance between these residues for the WT protein (11153) was more often less, even sampling distances below 5 A (FIG. 28A), and the crevice appears mostly in a closed state, only occasionally opening wide enough to allow for ADP-ribose binding (FIGs. 28B-28C). These results indicate that the isoleucine residue controls the ability of ADP-ribose to enter the ADP-ribose binding domain.

[00213] Conclusion. Here we created an N1062A Macl protein from SARS-CoV-2 and found that it had a severe defect in enzymatic activity, but only had a mild reduction in ADP- ribose binding compared to WT protein (FIG. 25). This confirms that this residue plays a large role in ADP -ribosylhydrolase activity but only minimally impacts ADP-ribose binding. These results further indicate that phenotypes associated with this mutation, including increased IFN production and enhanced sensitivity to IFN-I and IFN-II, are likely due to the loss of ADP-ribosylhydrolase activity.

[00214] This study focused on the isoleucine and phenylalanine residues located in loop 2 of Macl, near this asparagine residue. The isoleucine in loop 2 of the CoV Macl protein has been described as a bridge that extends from loop 2 to loop 1 that covers the phosphate binding domain of Macl, forming a narrow channel that might impact binding or hydrolysis. Furthermore, this residue participates in the transition of these loops from the apo form to the ADP-ribose bound form, again indicating that this residue may impact ADP-ribose binding. Somewhat surprisingly, we found that an I-A mutation instead led to enhanced ADP-ribose binding based a peptide- ADP-ribose binding assay for both the MERS-CoV and SARS-CoV- 2 Macl proteins (FIGs. 22B-22C, 24A-24B). Modeling data indicates that with this mutation, the distance between the two loops is consistently large enough that Macl can likely accept substrates at any time, as opposed to Macl with the isoleucine (FIGs. 28A- 28C). Following ADP-ribose binding, the I-A mutation does not appear to impact the distance between the loops, perhaps explaining why the hydrolysis activity of Macl was not affected for either Macl protein. These results suggest that the isoleucine residue serves as a gate to control ADP-ribose binding levels.

[00215] In contrast, the phenylalanine forms van der Waals interactions with the distal ribose, and similar to the nearby asparagine residue, appears to help position the ribose for hydrolysis. Biochemical data has supported those predictions, as mutations of this residue generally result in substantial loss of hydrolysis activity, which we observed here for both the MERS-CoV and SARS-CoV-2 Macl proteins (FIGs. 22D-22E, 24C-24D). Interestingly, the F-A mutation had diverse roles in ADP-ribose binding. For MERS-CoV Macl, this mutation led to reduced binding, while for SARS-CoV-2 this mutation enhanced both free and peptide- conjugated ADP-ribose binding. As the phenylalanine residue resides just outside the terminal ribose, it’s conceivable that in some cases this residue may occlude ADP-ribose binding during its transitions, while in others it may be just far enough away to not impact the ability of ADP-ribose to enter the binding pocket. While it’s unclear how these identical mutations had opposing effects on ADP-ribose binding, it highlights the difficulty in attributing specific biochemical roles for individual residues from one macrodomain to another.

[00216] Both MERS-CoV and SARS-CoV-2 I-A and F-A mutations were equally attenuated in both cell culture and in mice (FIGs. 23, and 26-27) despite having somewhat distinct biochemical properties. The MERS-CoV mutant viruses replicated normally in Vero81 cells but replicated poorly in Calu-3 and AJK6 bat kidney cells, at levels similar to the N1147A virus. This demonstrates that bats also utilize ADP-ribosylation to restrict CoV replication and indicates that loss of enzyme activity during infection may lead to the observed reduction in virus replication. Furthermore, both F-A and I-A SARS-CoV-2 mutant viruses replicated poorly following IFN-y treatment and induced high levels of IFN and ISG levels following infection in mice, again indicating that increased ADP-ribose binding might lead to poor ADP-ribosylhydrolase activity during infection (FIGs. 25, 27). Why might an increase in ADP-ribose binding lead to reduced enzyme activity during infection? One hypothesis would be that enhanced binding would negatively affect enzyme turnover. However, our in vitro enzyme assays were performed at an [E]/[S] ratio of 1 :5, indicating that the mutant protein has largely normal enzyme turnover. ADP-ribose can be covalently attached to several different amino acids, including cysteine, serine, arginine, glutamic and aspartic acid, but the MacroD2 class of macrodomains primarily removes ADP-ribose from acidic residues. Therefore, a second hypothesis is that enhancing the ADP-ribose binding abilities of Mac 1 may cause it to bind to proteins with ADP-ribose attached at non-acidic residues that it can’t remove and are not relevant for virus infection. Based on this hypothesis, we propose the following model for both SARS-CoV-2 and MERS-CoV. During infection WT Macl primarily engages with either anti- or pro-viral proteins that are MARylated on an acidic residue. Macl removes these modifications, which promotes virus replication and pathogenesis. In contrast, Macl I-A binds non-specifically to proteins MARylated at non-acidic residues, such as serine or cysteine, reducing its ability to engage with its primary targets. Macl becomes stuck to irrelevant targets, while its main target proteins remain ADP-ribosylated, leading to reduced virus replication and increased IFN production (FIG. 28D). Additional experiments will need to be designed to demonstrate that Macl I-A hydrolysis activity is reduced in a pool of ADP-ribosylated proteins during infection. [00217] The function of the isoleucine residue on the MHV Macl protein appears to be unique, as the mutation of I-A had little-to-no impact on virus replication. As we have been unable to purify the WT MHV Macl protein in bacteria, we can only speculate as to how this mutation impacts ADP-ribose binding and hydrolysis. The simplest hypothesis is that this mutation does not enhance ADP-ribose binding as it did for MERS-CoV or SARS-CoV-2. Alternatively, as MHV appears to be highly dependent on the ADP-ribose binding function of Macl, an increase in ADP-ribose binding may have some beneficial outcome that counteracts the negative effects of reduced enzyme activity, resulting in a virus that replicates much like WT. Conversely, the F1441 A mutant virus replicates poorly in all cells tested and was partially attenuated in mice. It was partially, but not fully, rescued in PARP12 KO cells, which we previously found fully rescued N1347A, but had no effect on D1329A, a mutation predicted to largely impact ADP-ribose binding. Thus, based on these and prior results with N1347A and D1329A, we hypothesize that this mutation reduces both enzyme and binding activity, like the MERS-CoV Fl 239 A Macl protein.

EQUIVALENTS

[00218] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[00219] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. [00220] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

[00221] Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth. All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

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Claims

WHAT IS CLAIMED IS:

1. A composition comprising a recombinant modified SARS-CoV-2, wherein the recombinant modified SARS-CoV-2 contains or has a deletion within Macrodomain 1 (Macl domain) of non- structural protein 3 (nsp3).

2. The composition of claim 1, wherein the amino acid sequence of the Macl domain of nsp3 is: IEVNSFSGYLKLTDNVYIKNADIVEEAKKVKPTVVVNAANVYLKHGGGVAG ALNKATNNAMQVESDDYIATNGPLKVGGSCVLSGHNLAKHCLHVVGPNVN KGEDIQLLKSAYENFNQHEVLLAPLLSAGIFGADPIHSLRVCVDTVRTNVYLA VFDKNLYDKLVSSFLE (SEQ ID NO: 2).

3. The composition of claim 1, wherein the amino acid sequence of the Macl domain of nsp3 is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

4. The composition of any one of claims 1-3, wherein the recombinant modified SARS- CoV-2 contains a complete deletion of the Macl domain of nsp3.

5. The composition of any one of claims 1-3, wherein the recombinant modified SARS- CoV-2 contains a partial deletion within the Macl domain of nsp3.

6. The composition of any one of claims 1-5, wherein the deletion within the Macl domain of nsp3 is about 20 amino acids to about 170 amino acids in length.

7. A composition comprising a recombinant modified SARS-CoV-2, wherein the recombinant modified SARS-CoV-2 comprises one or more mutations within Macrodomain 1 (Macl domain) of non- structural protein 3 (nsp3), wherein the one or more mutations correspond to one or more substitutions at N1062, Hl 067, DI 044, G1152, 11153, or Fl 154 of SEQ ID NO: 1.

8. The composition of claim 7, wherein the one or more substitutions at N1062, H1067, D1044, G1152, 11153, or Fl 154 of SEQ ID NO: 1 are selected from the group consisting ofN1062A, H1067A, D1044A, G1152V, I1153A, and F1154A.

9. The composition of any one of claims 1-8, wherein the recombinant modified SARS- CoV-2 is derived from a SARS-CoV-2 genetic variant selected from the group consisting of Alpha, Beta, Gamma, Delta, Epsilon, Eta, Iota, Kappa, Omicron, Zeta and Mu.

10. The composition of any one of claims 1-9, wherein the recombinant modified SARS- CoV-2 is formulated as a vaccine, and optionally comprises one or more adjuvants.

11. A composition comprising a recombinant modified MERS-CoV, wherein the recombinant modified MERS-CoV comprises one or more mutations within Macrodomain 1 (Macl domain) of non- structural protein 3 (nsp3), wherein the one or more mutations correspond to one or more substitutions at DI 129, N1147, Hl 152, G1237, 11238, or F1239 of SEQ ID NO: 4.

12. The composition of claim 11, wherein the one or more substitutions at DI 129, N1147, Hl 152, G1237, 11238, or F1239 of SEQ ID NO: 4 are selected from the group consisting of D1129A, N1147A, H1152A, G1237V, I1238A, and F1239A.

13. The composition of any one of claims 11-12, wherein the recombinant modified MERS-CoV is formulated as a vaccine, and optionally comprises one or more adjuvants.

14. The composition of any one of claims 1-13, wherein the composition further comprises one or more pharmaceutically acceptable excipients, wherein the one or more excipients is selected from the group consisting of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and any combination thereof.

15. A method for preventing a coronavirus infection in a subject in need thereof comprising administering to the subject an effective amount of the composition of any one of claims 1-14.

16. The method of claim 15, wherein the subject is an immunocompromised subject, a pediatric subject, a geriatric subject, or an adult subject.

17. The method of claim 15 or 16, wherein administration of the composition results in induction of an immune response to a coronavirus infection in the subject, maintains an immune response against a coronavirus infection in the subject, inhibits proliferation of a coronavirus within the subject, or eradicates coronavirus within the subject.

18. The method of any one of claims 15-17, wherein the composition is administered intravenously or intranasally.

19. The method of any one of claims 15-18, wherein administration of the composition prevents one or more signs or symptoms selected from among fatigue, tingling, tingling or numbness in the hands and feet, dizziness, confusion, brain fog, body ache, chills, loss of appetite, nausea, vomiting, abdominal pain or discomfort, loss of smell, inability to taste, muscle weakness, photophobia, adenopathy, headaches, cough, dry cough, shortness of breath, sore throat, lower extremity weakness/numbness, diarrhea, low blood O2, sneezing, runny nose or post-nasal drip; severe symptoms, such as ventilatory use, high fever, severe cough, delirium, seizures, stroke, systematic inflammation, cytokine storm; and other symptoms, such as fever, swollen adenoids, pneumonia, bronchitis, and Dyspnea.

20. The method of any one of claims 15-19, further comprising separately, sequentially or simultaneously administering one or more additional therapeutic agents to the subject.

21. The method of claim 20, wherein the one or more additional therapeutic agents are selected from the group consisting of remdesivir, lopinavir, ritonavir, ivermectin, tamiflu, favipiravir, dexamethasone, tocilizumab, kevzara, colcrys, hydroxychloroquine, chloroquine, a kinase inhibitor; covalescent plasma therapy, bamlanivimab, etesevimab, casirivimab, or imdevimab, and azithromycin.

22. A kit comprising the composition of any one of claims 1-14 and instructions for use.