Coronaviruses, vaccines comprising the same, and methods for preventing disease

#The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 6, 2018, is named B8-5129_SL.txt and is 43,750 bytes in size.

#The present invention generally relates to coronaviruses. The invention particularly relates to vaccines and methods of producing the same for existing and emerging coronaviruses.

#Coronaviruses are species of viruses belonging to the subfamily Coronavirinae in the family Coronaviridae, and are positive-sense RNA viruses that infect humans and animals and cause respiratory, gastrointestinal or neurologic disease. Coronaviruses can emerge from animal reservoirs to cause significant epidemics in humans, exemplified by Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2002-2003 and Middle East Respiratory Syndrome coronavirus (MERS-CoV), which was recognized as an emerging virus in 2012. Remarkably, these viruses can replicate in the cytoplasm of macrophages, a cell type considered to be a critical innate sentinel for detecting and eliminating invading pathogens. Coronaviruses encode multiple interferon antagonists that likely function to impede and delay the activation of type I interferon (IFN) and interferon stimulated genes (ISGs) and that expression of a constellation of antagonists contributes to pathogenesis. A recent study using SARS-CoV infection of mice documented the delayed and limited production of interferon that contributes to disease.

#FIGS. 1A through 1D include a schematic diagram of murine coronavirus genome highlighting nonstructural protein 15 and conserved residue threonine 98. FIG. 1A is a schematic diagram of the mouse hepatitis virus (MHV) A59 (MHV-A59) genome. PLP1/2: papain-like protease 1/2; ADRP, ADP-ribose-1′-monophosphatase; 3CLpro, 3C-like protease; RDRP, RNA-dependent RNA polymerase; Hel, helicase; ExoN, 3′→5′ exonuclease; NendoU, Nidovirus uridylate-specific endoribonuclease; 2′OMT, ribose-2′-O-methyltransferase. FIG. 1 B represents the crystal structure of nsp15, wherein the N domain, M domain, C domain are indicated at 90° C. rotation. T98 is shown in relation to L57 (N domain) and the catalytic residues (C domain). Protein Data Base ID: 2GTH. FIG. 1C indicates sites of mutation(s) for each coronavirus genotype and indication of induction of type I interferon in virus-infected bone marrow derived macrophages (BMDMs). FIG. 1C discloses SEQ ID NOS 1-6, respectively, in order of appearance. FIG. 1D indicates the sequence alignment of nsp15 T98 region from representative strains of coronavirus sub-groups using Clustal W. FIG. 1D discloses SEQ ID NOS 7-13, respectively, in order of appearance.

#FIGS. 2A through 2D indicate that MHV nsp15 mutant virus activates expression of type I interferon and is impaired for replication in BMDMs. FIG. 2A indicates IFN-α11 mRNA levels in BMDMs infected with Wild-Type (WT) MHV or N15m1 by quantitative RT-PCR. Values were normalized to β-actin and analyzed using unpaired T test. FIG. 2B indicates secreted IFNα protein levels in supernatant of infected BMDMs detected by quantitative ELISA. Values were analyzed using 2way-Anova test. FIGS. 2C and 2D indicate growth kinetics of WT and N15m1 in B6 BMDMs and ifnar−/− BMDMs, respectively (MOI of 0.1). Viral titers of supernatants were collected at indicated hpi and titers were determined by plaque assay using 17Cl-1 cells. Values were analyzed using unpaired T test. Data are representative of 2-3 independent experiments and presented as Mean±SD.

#FIGS. 3A through 3D indicate MHV nsp15 mutant virus induces a rapid apoptotic cell death in BMDMs. FIG. 3A indicates BMDMs were infected with WT, N15m1, or UV-inactivated N15m1 (UV-N15m1) MHV. At 24 hpi, cell viability of viral infected-BMDMs were quantified using CellTiter Glo assay. Values were analyzed using unpaired T test. FIG. 3B indicates BMDMs were infected with WT, or N15m1 MHV and subsequently treated with either DMSO, zVAD (20 μM), Necrostatin-1 (Nec-1) (25 μM), or VX-765 (20 μM). Cell viability was measured at 24 hpi by CellTiter Glo assay. Values were analyzed using unpaired T test. FIG. 3C indicates BMDMs were inoculated with WT or N15m1 MHV and zVAD (20 μM) was added to the media. At 24 hpi, caspase 3/7 activity was determined by a caspase 3/7-Glo activity assay. Values were displayed in relative light units (RLU) and analyzed using 2way-Anova test. FIG. 3D indicates BMDMs were infected with WT or N15m1 MHV and cell lysates were collected at indicated time points. Western blot detection of cleaved-caspase 3 and N protein. All infections are at an MOI of 0.1. Data are representative of 2-3 independent experiments and presented as Mean±SD.

#The present disclosure provides mutant coronaviruses, vaccines comprising mutant coronaviruses, methods of producing vaccines, and methods of preventing disease.

#RNA viruses that replicate via dsRNA intermediates can be detected as “non-self” by host dsRNA sensors, including cytoplasmic RIG-like receptors (RLRs). Activation of RLRs stimulate the production of interferon, which upregulates additional dsRNA sensors, such as PKR and the OAS/RNase L system, and hundreds of antiviral interferon-stimulated genes (ISGs). Additionally, interferon secreted from virus-infected cells that successfully sense the dsRNA can induce an antiviral state in neighboring cells and limit replication of potentially invading RNA viruses. Thus, many viruses have evolved strategies to sequester dsRNA to escape detection by host sensors. This disclosure presents a previously unrecognized role for coronavirus nsp15 in blocking the activation of dsRNA sensors in macrophages, thus enabling viral replication and the dissemination of the progeny virus.

#To investigate coronavirus antagonism of the interferon response, investigations leading to the present invention tested mouse hepatitis virus strain A59 (MHV-A59), a model coronavirus that replicates in multiple murine cell types, including macrophages, and can cause acute hepatitis or lethal encephalitis, depending on the site of injection. The viral genomic RNA is thirty-two kilobases and two-third of the genome encodes a large replicase polyprotein, while the remainder of the genome codes for structural proteins and strain-specific accessory proteins (FIG. 1A). The replicase polyprotein is processed by viral proteases into sixteen nonstructural proteins (nsp's). The viral nsp's assemble together with host endoplasmic reticulum to generate convoluted membranes and double membrane vesicles (DMVs), which are the sites of viral RNA synthesis. Coronavirus RNA replication proceeds via the generation of a nested-set of negative-strand RNAs that serve as the templates for synthesis of new positive-strand genomes and mRNAs. Double-stranded RNA (dsRNA) intermediates, potent stimulators of cytoplasmic innate sensors, are produced during this process and visualized in association with the DMVs. A potential function for coronavirus DMVs may be to sequester viral dsRNA away from host dsRNA sensors. However, it is unclear if DMVs alone are sufficient to prevent activation of the host innate immune response. Investigations reported herein surprisingly showed that coronavirus nonstructural protein 15 (nsp15), a highly conserved Nidovirus (coronaviruses and arterivuses) component with endoribonuclease activity, acts in conjunction with the viral replication complex to limit the exposure of viral dsRNA to host dsRNA sensors.