MEASLES VIRUS VACCINE EXPRESSING SARS-COV-2 PROTEIN(S)

Abstract

A recombinant measles viral vector comprising a nucleic acid sequence encoding a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike glycoprotein is provided. Polypeptides comprising the SARS-CoV-2 spike glycoprotein also are provided, as well as related nucleic acids, vectors, and compositions. The polypeptides, nucleic acids, vectors, and compositions can be used in methods of preventing, inhibiting, reducing, eliminating, protecting, or delaying the onset of an infection or an infectious clinical condition caused by coronavirus and methods for inducing an immune response against a coronavirus.

Description

SEQUENCE LISTING

Incorporated by reference in its entirety herein is a nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 337,215 Byte ASCII (Text) file named “754684_ST25,” created on Jun. 8, 2021.

BACKGROUND OF THE INVENTION

In December 2019, a novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2 or SARS-CoV-2) belonging to the betacoronavirus family emerged. All human betacoronaviruses are unique from one another, however, they do share a certain degree of genetic and structural homology. SARS-CoV-2 genome sequence homology with SARS-CoV and MERS-CoV is 77% and 50%, respectively.

In contrast to the relatively smaller outbreaks of SARS-CoV in 2002 and MERS-CoV in 2012, SARS-CoV-2 is exhibiting an unprecedented scale of infection, resulting in a global pandemic declaration of Coronavirus Infectious Disease (COVID-19) by the World Health Organization (WHO). COVID-19 has a high infection rate and long incubation period. Similar to influenza, COVID-19 has the potential to become a seasonal disease.

Therefore, there is a desire for a COVID-19 vaccine.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a recombinant measles viral vector (rMV) (e.g., Edmonston Zagreb (EZ) MV) comprising a nucleic acid sequence encoding a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein.

An embodiment of the invention provides a pharmaceutical composition comprising the recombinant measles viral vector, as well as methods for preventing, inhibiting, reducing, eliminating, protecting, or delaying the onset of an infection or an infectious clinical condition caused by coronavirus in a subject and methods for inducing an immune response against a coronavirus in a subject.

An embodiment of the invention also provides a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 8 (S6), SEQ ID NO: 9 (S-CO), SEQ ID NO: 10 (S-CO-AA), SEQ ID NO: 11 (S), SEQ ID NO: 12 (S-CO-AA-PP), SEQ ID NO: 13 (S-CO-AA-fneg-PP), and SEQ ID NO: 14 (S-CO-AA-fneg), as well as related nucleic acids, recombinant vectors, compositions, and methods.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-1C demonstrate the generation of rMV^EZ viruses expressing SARS-CoV-2 spike glycoprotein. FIG. 1A is a schematic representation of parental virus rMV^EZEGFP(3), the cDNA clone of which was used to generate cDNA plasmids for viruses rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-S-CO and rMV^EZSARS-CoV-2-S-COAA. Amino acid differences in the spike between the four viruses are shown, wherein underlined residues differ from wild-type spike sequence. Residues K and H in the cytoplasmic tail region were altered to A and A to disrupt a endoplasmic reticulum (ER) retention signal (KxHxx) sequence in viruses rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6 and rMV^EZSARS-CoV-2-S-COAA. RBD; receptor binding domain, TM; transmembrane domain. FIG. 1B is a phase image of Vero cell monolayer infected with rMV^EZSARS-CoV-2-S6 (shown as a representative image of a primary rescue). FIG. 1C are images of an immunoplaque assay of Vero cell monolayers infected with a 10-fold dilution of rescued rMV^EZSARS-CoV-2-S6. Plaques were stained using anti-SARS-CoV-2-S antibody. Dilutions 10⁻⁶ and 10⁻⁷ are shown as representatives.

FIG. 2 demonstrates that growth kinetics of rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-S-CO, and rMV^EZSARS-CoV-2-S-COAA compared to rMV^EZEGFP(3). Vero cells were infected at a multiplicity of infections (MOI) of 0.05. Samples were harvested at the indicated time points and titrated on Vero cells. Error bars indicate standard deviation (n=3).

FIGS. 3A-3B demonstrate an experimental set-up for mice immunized with recombinant viruses. FIG. 3A is a schematic describing the experiment. Groups of 5 mice were infected with either rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-S-CO, rMV^EZSARS-CoV-2-S-COAA, or rMV^EZEGFP(3). Serum was collected on days 21 and 42, and splenocytes were collected on day 42. FIG. 3B is table showing neutralization of SARS-CoV-2 using the harvested mice serum, indicating that SARS-CoV-2 neutralizing antibodies were produced in mice vaccinated with rMV^EZSARS-CoV-2-S-CO and rMV^EZSARS-CoV-2-S-COAA viruses.

FIG. 4 is a vector map of rMV^EZSARS-CoV-2-S6.

FIG. 5 is a vector map of rMV^EZSARS-CoV-2-S-CO.

FIG. 6 is a vector map of rMV^EZSARS-CoV-2-S-COAA.

FIG. 7 is a vector map of rMV^EZSARS-CoV-2-S.

FIG. 8 is a vector map of rMV^EZSARS-CoV-2-S-COAA-PP.

FIG. 9 is a vector map of rMV^EZSARS-CoV-2-S-COAA-fneg-PP.

FIG. 10 is a vector map of and rMV^EZSARS-CoV-2-S-COAA-fneg.

FIGS. 11A-B demonstrates an experimental set-up for non-human primates vaccinated (prime and/or boost) with any of the candidates. FIG. 11A is a schematic describing the experiment. Groups of 2 or 3 African green monkeys (AGMs) were vaccinated with rMV^EZSARS-CoV-2-S-CO or rMV^EZ and then challenged with SARS-CoV-2. FIG. 11B is a table with neutralization titers of serum against SARS-CoV-2, which were determined as described in Klimstra et al., 2020: PMID: 32821033, and MV as described in de Swart et al., 2017: PMID: 29263877. The results support that primates produce antibodies which neutralize both SARS-CoV-2 and measles virus following vaccination.

FIG. 12 is a graph demonstrating the secretion of IFN-γ in spleens from mice immunized with recombinant measles virus expressing SARS-CoV-2 codon-optimized spike protein. Splenocytes prepared from rMV^EZ, rMV^EZSARS-CoV-2-CO, rMV^EZSARS-CoV-2-S6, and rMV^EZSARS-CoV-2-COAA immunized mice were used in an ELISPOT assay to detect the secretion of proinflammatory cytokine IFN-γ. Mice splenocytes were re-stimulated for 24 hours with four pools of synthetic peptides (51, S2, S3 and S4) designed to span the entire SARS-CoV-2 spike protein. Unstimulated splenocytes (medium) served as negative controls and splenocytes treated with PMA/ionomycin confirmed splenocyte re-stimulation. Dots represent individual animals (n=5) for each vaccinated group. The number of cells expressing IFN-γ after re-stimulation are represented as 1×10⁵ cells.

FIGS. 13A and 13B demonstrate the T cell responses in immunized mice. Flow cytometry was used to determine the proportion of CD4⁺/CD44⁺ and CD8⁺/CD44⁺ T cells in the spleens of mice immunized with rMV_EZ, rMV^EZSARS-CoV-2-CO, rMV^EZSARS-CoV-2-S6, and rMV^EZSARS-CoV-2-COAA. To determine the optimal re-stimulation period splenocytes from two mice from each immunized group were first re-stimulated for 6 hours (FIG. 13A) while the remaining three mice spleens were re-stimulated for 12-hours (FIG. 13B). Cells were re-stimulated with spike specific peptide pools S1, S2, S3 and S4, or left unstimulated (negative control; medium). Re-stimulation was confirmed with a cell activation cocktail containing PMA/ionomycin and brefaldin-A. Intracellular cytokine staining for IFN-γ and IL-13 were then carried out to determine Th1 and Th2 responses, respectively. Dots represent individual animals.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides a recombinant measles viral vector that encodes one or more (e.g., two, three, four, or five) SARS-CoV-2 spike glycoproteins, wherein the SARS-CoV-2 spike glycoproteins may be the same or different. The recombinant measles viral vector or a composition thereof can be administered to a subject to prevent, inhibit, reduce, eliminate, protect, or delay the onset of an infection or an infectious clinical condition caused by coronavirus (e.g., SARS-CoV-2) in a subject. The recombinant measles viral vector or a composition thereof also can be administered to a subject to induce an immune response against a coronavirus (e.g., SARS-CoV-2) in a subject.

The recombinant measles viral vector can be any suitable recombinant measles viral vector. For example, the measles viral vector can be an Edmonston wild-type virus or vaccine strains of the Edmonston lineage, such as the AIK-C, Moraten, Rubeovax, Schwarz, or Zagreb strains (Bankamp et al., J. Infect. Dis., 204 Suppl. 1: 5533-5548 (2011)). Alternatively, the measles viral vector can be selected from the group consisting of CAM-70, Changchun-47, Leningrad-4, Shanghai-191 (Bankamp et al., J. Infect. Dis., 204 Suppl. 1: 5533-5548 (2011)), Leningrad-16, Moscow-5 (Sinitsyna et al., Res. Virol., 141(5): 517-31 (1990)), 9301B (Takeda et al., J Virol., 72(11): 8690-8696 (1998)), ATTENUVAX®, and those described in Schneider-Schaulies et al., PNAS, 92(2): 3943-7 (1995).

Measles viruses and recombinant measles viral vectors are described in WO 98/13501, which provides the sequence of a DNA copy of the positive strand (antigenomic) message sense RNA of various wild-type of vaccine measles strains, including Edmonston wild-type strain, Moraten strain, and Schwarz strain, and WO 97/06270, which discloses the production of recombinant measles vectors.

In one embodiment, the measles viral vector is an Edmonston-Zagreb (EZ) measles viral vector. Particular recombinant measles viral vectors of the Edmonston-Zagreb (EZ) strain include the following: rMV^EZSARS-CoV-2-S6 (FIG. 4), rMV^EZSARS-CoV-2-S-CO (FIG. 5), rMV^EZSARS-CoV-2-S-COAA (FIG. 6), rMV^EZSARS-CoV-2-S(FIG. 7), rMV^EZSARS-CoV-2-S-COAA-PP (FIG. 8), rMV^EZSARS-CoV-2-S-COAA-fneg-PP (FIG. 9), and rMV^EZSARS-CoV-2-S-COAA-fneg (FIG. 10).

The recombinant measles vector comprises a nucleic acid encoding the SARS-CoV-2 spike glycoprotein, wherein the nucleic acid sequence encoding the SARS-CoV-2 spike glycoprotein can be any suitable nucleic acid sequence.

In one embodiment, the nucleic acid sequence encoding the SARS-CoV-2 spike glycoprotein is codon optimized. Without being bound to a particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by ribosomes using tRNAs that are more readily available within a cell, thus increasing translation efficiency and overall protein production. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. rMV^EZSARS-CoV-2-S-CO, rMV^EZSARS-CoV-2-S-COAA, rMV^EZSARS-CoV-2-S-COAA-PP, rMV^EZSARS-CoV-2-S-COAA-fneg-PP, and rMV^EZSARS-CoV-2-S-COAA-fneg each contain codon optimized SARS-CoV-2 spike glycoprotein sequences. Techniques for codon optimization are known in the art.

The nucleic acid sequence encoding the SARS-CoV-2 spike glycoprotein can contain at least one modification that disrupts the endoplasmic reticulum (ER) retention sequence of the SARS-CoV-2 spike glycoprotein. For example, the modification can result in an ER retention sequence of the SARS-CoV-2 spike glycoprotein containing AxAxx rather than KxHxx in the cytoplasmic tail (as described in Case et al., bioRxiv (2020). doi:10.1101/2020.05.18.102038 and Example 2). rMV^EZSARS-CoV2-S6, rMV^EZSARS-CoV-2-S, rMV^EZSARS-CoV-2-S-COAA, rMV^EZSARS-CoV-2-S-COAA-PP, rMV^EZSARS-CoV-2-S-COAA-fneg-PP, and rMV^EZSARS-CoV-2-S-COAA-fneg each contain modifications to ablate the ER retention signal. In particular, the amino acid sequences of the SARS-CoV-2 spike glycoproteins encoded by rMV^EZSARS-CoV2-S6, rMV^EZSARS-CoV-2-S, rMV^EZSARS-CoV-2-S-COAA, rMV^EZSARS-CoV2-S, rMV^EZSARS-CoV-2-S-COAA-PP, rMV^EZSARS-CoV-2-S-COAA-fneg-PP, and rMV^EZSARS-CoV-2-S-COAA-fneg each contain two alanine substitutions at residues 1269 and 1271 of SEQ ID NOs: 8 and 10-14, respectively.

The nucleic acid encoding the SARS-CoV-2 spike glycoprotein can contain at least one modification that locks in the prefusion conformation. The spike (S) glycoprotein is a trimeric class I fusion protein that exists in a metastable prefusion conformation that undergoes a substantial structural rearrangement to fuse the viral membrane with the host cell membrane (Wrapp et al., Science, 13: 1260-1263 (2020)). This process is triggered when the 51 subunit binds to a host cell receptor. Receptor binding destabilizes the prefusion trimer, resulting in shedding of the 51 subunit and transition of the S2 subunit to a stable postfusion conformation. To engage a host cell receptor, the receptor-binding domain (RBD) of 51 undergoes hinge-like conformational movements that transiently hide or expose the determinants of receptor binding. The amino acid sequences of the SARS-CoV-2 spike glycoproteins encoded by rMV^EZSARS-CoV-2-S-COAA-PP and rMV^EZSARS-CoV-2-S-COAA-fneg-PP each contain two proline modifications (PP) at residues 986 and 987 of SEQ ID NOs: 9 and 10, respectively, which lock in the prefusion conformation.

The nucleic acid encoding the SARS-CoV-2 spike glycoprotein can contain at least one modification that ablates the furin cleavage signal, such as by modifying the furin cleavage site so that furin no longer cleaves the sequence. The furin cleavage site can be any polypeptide site cleavable by furin. The minimal cleavage site typically is, in the single letter code for amino acid residues, R-X-X-R, with cleavage occurring after the second “R” (Duckert et al., Protein Engineering, Design & Selection, 17(1):107-112 (2004); and WO 2009/032954). Whether or not any particular sequence is cleavable by furin can be determined by methods known in the art. For example, whether or not a sequence is cleavable by furin can be tested by incubating the sequence with furin.

rMV^EZSARS-CoV-2-S-COAA-fneg-PP and rMV^EZSARS-CoV-2-S-COAA-fneg each contain modifications to ablate the furin cleavage signal. In particular, the amino acid sequences of the SARS-CoV-2 spike glycoproteins encoded by rMV^EZSARS-CoV2-S-COAA-fneg-PP and rMV^EZSARS-CoV-2-S-COAA-fneg each contain four amino acid changes (ASVG; SEQ ID NO: 23) at residues 682-685 of SEQ ID NOs: 13 and 14, respectively, that ablate the furin cleavage signal.

In one embodiment, the nucleic acid encoding the SARS-CoV-2 spike glycoprotein encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (S6 spike glycoprotein), SEQ ID NO: 9 (S-CO spike glycoprotein), SEQ ID NO: 10 (S-CO-AA spike glycoprotein), SEQ ID NO: 11 (S spike glycoprotein), SEQ ID NO: 12 (S-CO-AA-PP spike glycoprotein), SEQ ID NO: 13 (S-CO-AA-fneg-PP spike glycoprotein), and SEQ ID NO: 14 (S-CO-AA-fneg spike glycoprotein).

In another embodiment, the nucleic acid encoding the SARS-CoV-2 spike glycoprotein is selected from the group consisting of SEQ ID NO: 1 (S6), SEQ ID NO: 2 (S-CO), SEQ ID NO: 3 (S-CO-AA), SEQ ID NO: 4 (S), SEQ ID NO: 5 (S-CO-AA-PP), SEQ ID NO: 6 (S-CO-AA-fneg-PP), and SEQ ID NO: 7 (S-CO-AA-fneg).

The recombinant measles viral vector comprising a nucleic acid sequence encoding a SARS-CoV-2 spike glycoprotein can have any suitable nucleic acid sequence. For example, the recombinant measles viral vector can comprise, consist essentially of, or consist of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15 (rMV^EZSARS-CoV-2-S6), SEQ ID NO: 16 (rMV^EZSARS-CoV-2-S-CO), SEQ ID NO: 17 (rMV^EZSARS-CoV-2-S-COAA), SEQ ID NO: 18 (rMV^EZSARS-CoV-2-S), SEQ ID NO: 19 (rMV^EZSARS-CoV-2-S-COAA-PP), SEQ ID NO: 20 (rMV^EZSARS-CoV-2-S-COAA-fneg-PP), and SEQ ID NO: 21 (rMV^EZSARS-CoV-2-S-COAA-fneg). The vector sequences can comprise one or more nucleic acid sequences encoding the N, P, S, M, F, H, and L genes as described in GenBank Accession Nos. AY486083.1 and AY486084.1.

An embodiment of the invention also provides a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (S6), SEQ ID NO: 9 (S-CO), SEQ ID NO: 10 (S-CO-AA), SEQ ID NO: 11 (S), SEQ ID NO: 12 (S-CO-AA-PP), SEQ ID NO: 13 (S-CO-AA-fneg-PP), and SEQ ID NO: 14 (S-CO-AA-fneg), corresponding a SARS-CoV-2 spike glycoprotein.

The polypeptide can be prepared by any of a number of conventional techniques. In this respect, the polypeptide sequence can be synthetic, recombinant, isolated, and/or purified.

The polypeptide can be isolated or purified from a recombinant source. For instance, a DNA fragment encoding a desired polypeptide can be subcloned into an appropriate vector using well-known molecular genetic techniques. The fragment can be transcribed and the polypeptide subsequently translated in vitro. Commercially available kits also can be employed. The polymerase chain reaction optionally can be employed in the manipulation of nucleic acids.

The polypeptide also can be synthesized using an automated peptide synthesizer in accordance with methods known in the art. Alternately, the polypeptide can be synthesized using standard peptide synthesizing techniques well-known to those of skill in the art. In particular, the polypeptide can be synthesized using the procedure of solid-phase synthesis. If desired, this can be done using an automated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished by, for example, acid treatment at reduced temperature. The protein-containing mixture then can be extracted, for instance, with diethyl ether, to remove non-peptidic organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w/v acetic acid). Following the synthesis of the polypeptide, further purification (e.g., using HPLC) optionally can be performed in order to eliminate any incomplete proteins, polypeptides, peptides or free amino acids. Amino acid and/or HPLC analysis can be performed on the synthesized polypeptide to validate its identity. For other applications according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, either by chemical conjugation or through genetic means, such as are known to those skilled in the art. In this regard, an embodiment of the invention also provides a fusion protein comprising the polypeptide and one or more other protein(s) having any desired properties or functions, such as to facilitate isolation, purification, analysis, or stability of the fusion protein.

An embodiment of the invention also provides a nucleic acid encoding the polypeptide. In one embodiment, the nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 (S6), SEQ ID NO: 2 (S-CO), SEQ ID NO: 3 (S-CO-AA), SEQ ID NO: 4 (SARS-CoV2-S), SEQ ID NO: 5 (S-CO-AA-PP), SEQ ID NO: 6 (S-CO-AA-fneg-PP), and SEQ ID NO: 7 (S-CO-AA-fneg).

“Nucleic acid” as used herein includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.

In an embodiment, the nucleic acid is recombinant. As used herein, the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

The nucleic acid (e.g., DNA, RNA, cDNA, and the like) can be produced in any suitable matter including, but not limited to recombinant production and commercial synthesis. In this respect, the nucleic acid sequence can be synthetic, recombinant, isolated, and/or purified.

The nucleic acid can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green et al. (eds.), Molecular Cloning, A Laboratory Manual, 4^th Edition, Cold Spring Harbor Laboratory Press, New York (2012). For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston, Tex.).

The nucleic acid encoding the polypeptide can be provided as part of a construct comprising the nucleic acid and elements that enable delivery of the nucleic acid to a cell, and/or expression of the nucleic acid in a cell. For example, the polynucleotide sequence encoding the polypeptide can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG/AUG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

A large number of promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Representative sources of promoters include for example, virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3′ or 5′ direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system, the Ecdysone inducible system, the T-REX™ system (Invitrogen, Carlsbad, Calif.), LACSWITCH™ System (Stratagene, San Diego, Calif.), and the Cre-ERT tamoxifen inducible recombinase system.

The term “enhancer” as used herein, refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences. For example, the nucleic acid encoding the polypeptide can be operably linked to a CMV enhancer/chicken β-actin promoter (also referred to as a “CAG promoter”).

A nucleic acid encoding the polypeptide can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Qβ replicase amplification system (QB). For example, a polynucleotide encoding the polypeptide can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. A wide variety of cloning and in vitro amplification methodologies are well known to persons skilled in the art.

An embodiment of the invention also provides a recombinant vector comprising the nucleic acid. Examples of suitable vectors include plasmids (e.g., DNA plasmids), bacterial vectors, and viral vectors, such as poxvirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, poliovirus, alphavirus, baculovirus, measles virus, and Sindbis virus. When the vector is a plasmid (e.g., DNA plasmid), the plasmid can be complexed with chitosan.

The polypeptide, nucleic acid, or vector (e.g., recombinant measles virus vector) can be formulated as a composition (e.g., pharmaceutical composition) comprising the polypeptide, nucleic acid, or vector (e.g., recombinant measles virus vector) and a carrier (e.g., a pharmaceutically or physiologically acceptable carrier). Furthermore, the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition can be used in the methods described herein alone or as part of a pharmaceutical formulation.

The composition (e.g., pharmaceutical composition) can comprise more than one polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition of the invention. Alternatively, or in addition, the composition can comprise one or more (e.g., one, two, three, or more) additional pharmaceutically active agents or drugs, such as corticosteroids, antibiotics, and antivirals.

The carrier can be any of those conventionally used and is limited only by physio-chemical considerations, such as solubility and lack of reactivity with the active compound(s) and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particular polypeptide, nucleic acid, vector, or composition thereof of the invention and other active agents or drugs used, as well as by the particular method used to administer the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof.

The composition also can be formulated to enhance transduction efficiency. In addition, a person of ordinary skill in the art will appreciate that the one or more of the polypeptides, nucleic acids, or vectors (e.g., recombinant measles virus vectors) can be present in a composition with other therapeutic or biologically-active agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of one or more of the polypeptides, nucleic acids, or vectors (e.g., recombinant measles virus vector). Antibiotics, i.e., microbicides and fungicides, can be present to treat existing infection and/or reduce the risk of future infection.

The invention provides a method for preventing, inhibiting, reducing, eliminating, protecting, or delaying the onset of an infection or an infectious clinical condition caused by coronavirus in a subject comprising administering the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof to a subject. The invention also provides a method for inducing an immune response against a coronavirus in a subject comprising administering the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof to the subject.

Administration of the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof to the subject can be used to protect against one or more strains of coronavirus (e.g., SARS-CoV-2), thereby treating, preventing, and/or protecting against coronavirus-based pathologies.

Administration of the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof can significantly induce an immune response of a subject administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof, thereby protecting against and treating coronavirus (e.g., SARS-CoV-2) infection.

Administration of the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof can induce a humoral immune response in a subject. The induced humoral immune response can be specific for the SARS-CoV-2 spike glycoprotein. The humoral immune response can be induced in the subject administered the vaccine by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 10.5-fold, at least about 11-fold, at least about 11.5-fold, at least about 12-fold, at least about 12.5-fold, at least about 13-fold, at least about 13.5-fold, at least about 14-fold, at least about 14.5-fold, at least about 15-fold, at least about 15.5-fold, at least about 16-fold, at least about 16.5-fold, at least about 17-fold, at least about 17.5-fold, at least about 18-fold, at least about 18.5-fold, at least about 19-fold, at least about 19.5-fold, at least about 20-fold, or ranges of any combination of these values as compared to a subject not administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof.

The induced humoral immune response can include an increased level of neutralizing antibodies as compared to a subject not administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof. The neutralizing antibodies can be specific for the SARS-CoV-2 spike glycoprotein. The neutralizing antibodies can provide protection against and/or treatment of SARS-CoV-2 infection and its associated pathologies in the subject administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof.

The induced humoral immune response can include an increased level of IgG antibodies as compared to a subject not administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof. These IgG antibodies can be specific for the SARS-CoV-2 antigens. The level of IgG antibody can be increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 10.5-fold, at least about 11-fold, at least about 11.5-fold, at least about 12-fold, at least about 12.5-fold, at least about 13-fold, at least about 13.5-fold, at least about 14-fold, at least about 14.5-fold, at least about 15-fold, at least about 15.5-fold, at least about 16-fold, at least about 16.5-fold, at least about 17-fold, at least about 17.5-fold, at least about 18-fold, at least about 18.5-fold, at least about 19-fold, at least about 19.5-fold, at least about 20-fold, or ranges of any combination of these values as compared to a subject not administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof.

The induced humoral immune response can include an increased level of IgM and/or IgA antibodies as compared to a subject not administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof. These IgM and or IgA antibodies can be specific for the SARS-CoV-2 antigen. The level of IgM/IgA antibody can be increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 10.5-fold, at least about 11-fold, at least about 11.5-fold, at least about 12-fold, at least about 12.5-fold, at least about 13-fold, at least about 13.5-fold, at least about 14-fold, at least about 14.5-fold, at least about 15-fold, at least about 15.5-fold, at least about 16-fold, at least about 16.5-fold, at least about 17-fold, at least about 17.5-fold, at least about 18-fold, at least about 18.5-fold, at least about 19-fold, at least about 19.5-fold, at least about 20-fold, or ranges of any combination of these values as compared to a subject not administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof.

Administration of the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof can induce a cellular immune response in the subject. The induced cellular immune response can be specific for the SARS-CoV-2 antigen.

The induced cellular immune response can include eliciting a CD8+ T cell response, which can include eliciting a CD8+ T cell response in which the CD8+ T cells produce interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), or a combination thereof (e.g., a combination of IFN-γ and TNF-α).

The CD8+ T cell response can be increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 10.5-fold, at least about 11-fold, at least about 11.5-fold, at least about 12-fold, at least about 12.5-fold, at least about 13-fold, at least about 13.5-fold, at least about 14-fold, at least about 14.5-fold, at least about 15-fold, at least about 15.5-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, or ranges of any combination of these values as compared to the subject not administered the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof.

Administration of the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof can also include eliciting a CD4+ T cell response, which can include eliciting a CD4+ T cell response in which the CD4+ T cells produce IFN-γ, TNF-α, IL-2, or a combination thereof (e.g., a combination of IFN-γ and TNF-α).

The polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof can be administered to the subject by various routes including, but not limited to, oral, sublingual, buccal, intradermal, topical, parenteral (using single or arrays of dissolvable and hybrid microneedles, in lyophilized or solution), subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, interperitoneal, intranasal, large and/or small particle aerosol, dry-powder aerosols or intratracheal administration, or subretinal injection or intravitreal injection.

The invention includes a prime and boost protocol. In particular, the protocol includes an initial “prime” with the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof, followed by one or preferably multiple “boosts” with the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof. The boosts can be administered 1-3 times (e.g., 1, 2, or 3 times) at any suitable time period (e.g., every 3-4 weeks, every six months, or once a year) for any suitable length of time.

The polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The subject can be a mammal, such as a human, a horse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.

The polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof can be administered prophylactically or therapeutically. In prophylactic administration, the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof are administered in an amount sufficient to induce an immune response. In therapeutic applications, the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the polypeptide, nucleic acid, vector (e.g., recombinant measles virus vector), or composition thereof administered to a subject, the manner of administration, the stage and severity of the infection, the general state of health of the patient, and the judgment of the prescribing physician.

The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

Example 1

This example provides materials and methods for the studies described in Example 2.

Cell Lines, Viruses and Plasmids

Vero cells (African green monkey kidney cells; ATCC CCL-81) purchased from ATCC (Manassas, Va., USA) were grown in advanced Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 10% (V/V) fetal bovine serum (HI FBS heat inactivated, Gibco) and GlutaMAX-I (Gibco). Cells were grown and maintained at 37° C. and 5% (V/V) CO₂, and tested negative for mycoplasma contamination prior to use in virus rescue experiments. MV plasmids expressing N, P and L, pMV_EZEGFP(3), as well as rMV_EZEGFP(3) virus have been described previously (Rennick et al., J. Virol. (2015). doi:10.1128/jvi.02924-14).

Construction of MV^EZ cDNA Plasmids Expressing SARS-CoV-2 Spike Glycoprotein

As described in Rennick et al., J. Virol. (2015). doi:10.1128/jvi.02924-14), three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMV_EZEGFP(1), rMV^EZEGFP(3), and rMV_EZEGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were compared in vitro by growth curves, which indicated that rMV^EZEGFP(1) was over-attenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMV^EZEGFP(1) and rMV^EZEGFP(6) did not induce satisfactory serum antibody responses, whereas both in vitro and in vivo rMV^EZEGFP(3) was functionally equivalent to the commercial MV^EZ-containing vaccine. Intramuscular vaccination of macaques with rMV^EZEGFP(3) resulted in the identification of EGFP⁺ cells in the muscle at days 3, 5, and 7 post-vaccination. Therefore, rMV^EZEGFP(3) was selected for further experiments.

pMV^EZ versions expressing SARS-CoV-2 spike glycoprotein (Accession number MN908947) were generated by replacing the open reading frame of EGFP in pMV^EZEGFP(3) (Rennick et al., J. Virol. (2015). doi:10.1128/jvi.02924-14).

Plasmid pMV^EZEGFP(3) was linearized using restriction sites Asc I at genome position 3439 and Aat II at genome position 4176. These restriction sites were originally designed into pMV^EZEGFP(3) to allow easy replacement of foreign genes in place of EGFP. SARS-CoV-2 spike with mutations in the endoplasmic reticulum (ER) retention signal sequence as previously described (Case et al., bioRxiv (2020). doi:10.1101/2020.05.18.102038) were obtained in two gene fragments. These fragments were ligated into the linearized pMV^EZEGFP(3) using Gibson Assembly (NEBuilder® HiFi DNA assembly, NEB). This generated two versions pMV_EZSARS-CoV-2-S and pMV^EZSARS-CoV-2-S6.

A human codon optimized SARS-CoV-2 spike glycoprotein expressing plasmid was obtained from GenScript (Piscataway, N.J., USA). Spike was amplified from the plasmid using oligonucleotides that contained a 35 nucleotide homology (lower case sequence) to the linearized pMV^EZEGFP(3) (Forward primer: 5′caaagtgattgcctcccaagttccacaggcgcgccATGTTCGTCTTCCTGGTC3′ (SEQ ID NO: 24) and reverse primer: 5′gttggcaggtaagttgagctgtaggacgtcgcgcgTTAGGTGTAATGCAGCTTCAC3′ (SEQ ID NO: 25)). The amplified product was then ligated into linearized pMV^EZEGFP(3) using Gibson Assembly (NEBuilder® HiFi DNA assembly, NEB). This generated pMV_EZSARSCoV2-S-CO. pMV_EZSARS-CoV-2-S-COAA was generated the same way, but using a reverse primer (5′ggttggcaggtaagttgagctgtaggacgtcgcgcgTTAGGTGTAAGCCAGCGCCACGCC3′ (SEQ ID NO: 26) with nucleotide changes (in bold) to disrupt the ER retention signal.

The spike sequence in all plasmids were sequence confirmed via Sanger sequencing (Genewiz, N.J., USA).

Generation of Recombinant rMV_EZ-SARS-CoV-2-Spike Glycoprotein Viruses

Vero cell monolayers in 6-well trays were infected with a recombinant vaccinia virus expressing T7 polymerase (MVA-T7) in Opti-MEM (Gibco) for 30 mins at 37° C. and then spinoculated at room temperature for another 30 mins. Virus inoculum was removed and 1 ml fresh Opti-MEM was added onto cells. Cells were then transfected with 5 μg of pMV^EZSARS-CoV-2-S, pMV^EZSARS-CoV-2-S6, pMV^EZSARS-CoV-2-S-CO, or pMV^EZSARS-CoV-2-S-COAA. MV plasmids expressing nucleoprotein (N), phosphoprotein (P) and polymerase (L) at 1 μg, 0.6 μg and 0.4 μg respectively, were also transfected into the cells. 24 hours post-transfection (h.p.t.), medium was removed from the cells and DMEM/2% (V/V) fetal bovine serum (FBS) was added. Cells were monitored daily for approximately 14 days post-transfection (d.p.t.) for syncytium formation. Plaque picked viruses were then grown in Vero cells and harvested by free-thaw when complete cytopathic effect was visible. Virus titers were determined by endpoint titration and expressed as 50% tissue culture infectious (TCID₅₀) units.

Reverse Transcription Polymerase Chain Reaction (RT/PCR) and Sequencing

Total RNA from working virus stocks was extracted using TRIzol LS reagent (ThermoFisher) according to manufacturer's recommendations and RNA pellet resuspended in 40 μl nuclease-free water (Invitrogen). cDNA was generated with 5 μl of resuspended RNA using SuperScript™ III First-strand synthesis system (Thermo Fisher Scientific) and random primers. 3 μl of the resultant cDNA was then used to amplify MV-spike fragments with primers using Phusion high-fidelity DNA polymerase (NEB) in a total volume of 50 μl (using a touch-down PCR amplification protocol). Amplified PCR products were analyzed on a 1% agarose gel and bands gel purified using QIAquick gel extraction kit (Qiagen). Products were sequenced confirmed via Sanger sequencing (Genewiz, N.J., USA).

Immunofluorescence

Confluent Vero cells in 24-well trays were infected with rMV^EZSARS-CoV2-S6 or rMV^EZSARS-CoV-2-S-CO at a multiplicity of infection (MOI) of 0.01. At 2 days post infection, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells were washed twice in PBS and permeabilized (0.1% Triton-X in PBS) at 37° C. for 30 minutes before incubating with primary antibody (Rabbit anti-SARS2-S, Sino biologicals 40150-R007; 1:500) made in PBS with 0.1% (V/V) Triton-X at 37° C. for 1 hour. Cells were then washed three times in PBS before incubating with secondary antibody (Chicken anti-rabbit Alexa Fluor 488, Invitrogen; 1:400) at 37° C. for 1 hour. Cells were washed three times in PBS and stained with DAPI nuclei stain (Invitrogen; 300 nM DAPI stain solution) for 10 minutes at room temperature in the dark. Images were obtained using a fluorescent microscope (Leica).

Virus Growth Kinetics

Vero cell monolayers at 2×10⁵ cells in 24-well trays were infected with rMV^EZEGFP(3), rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-S-CO, or rMV^EZSARS-CoV-2-S-COAA at a 0.05 MOI. Cell monolayers were infected for 1 hour at 37° C. after which virus inoculum was removed and cell monolayers washed twice using phosphate-buffered saline (PBS; Gibco). DMEM/2% (V/V) FBS was added onto the cells and at the desired time points cells were scraped into culture medium and placed at −80° C. After freeze-thawing the cells and medium, cell debris were clarified and total virus measured by TCID₅₀.

Immunoplaque Assay

Confluent Vero cell monolayers in 6-well trays were infected with a 10-fold serial dilution of virus prepared in Opti-MEM. Cells were incubated for 1 h at 37° C. and then overlaid with 0.6% Avicel (FMC Biopolymer) supplemented with 2×MEM (10×MEM, no glutamine, Gibco) and 2% FBS. Cells were incubated for 5 days and then fixed using 4% paraformaldehyde for 30 minutes at room temperature. Cells were permeabilized (0.5% Triton X-100, 20 mM sucrose in PBS; 1 ml) for 30 minutes at room temperature and then washed (0.1% Tween-20 in PBS; 1 ml) once before incubating with primary antibody (Rabbit anti-SARS2-S, Sino biologicals 40150-R007; 1:1000) in blocking buffer (4% dried milk/0.1% Tween-20 in PBS) for 1 hour at room temperature. Cells were washed three times and incubated with secondary antibody (Goat anti-rabbit HRP, Abcam, ab6721; 1:1000) for 1 hour at room temperature. Plaques were visualized using KPL TrueBlue Peroxidase Substrate solution (Sera Care, 5510-0050). Plates were digitalized using a scanner.

Animal Study Design

All animal experiments were conducted in compliance with all applicable U.S. Federal policies and regulations and AAALAC International standards for the humane care and use of animals. All protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC).

Twenty-five IFNar1 knockout mice in groups of five were infected with 10⁴ TCID₅₀ of rMV^EZEGFP(3), rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-S-CO, or rMV^EZSARS-CoV-2-S-COAA viruses via the intraperitoneal (IP) route. 21 days post-infection, mice were bled for serum, and then boosted with 10⁵ TCID₅₀ of the respective virus.

African green monkeys were immunized with either measles vaccine or rMV^EZSARS-CoV-2-S-CO (in groups of 2 or 3) with 10⁵ TCID₅₀ of candidate vaccine. In our proof of principle study we focused on a prime/boost (day 0 and 21 days). Animals were challenged 42 days after immunization with 10⁶ plaque forming units of SARS-CoV-2.

All animals seroconverted generating antibodies to both measles and SARS-CoV-2. These antibodies neutralized both viruses, some at levels higher than what is seen in human convalescent serum.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism software (La Jolla, Calif.).

Vector Maps

Vector maps are provided in FIGS. 4-10 for each of rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-S-CO, rMV^EZSARS-CoV-2-S-COAA, rMV^EZSARS-CoV-2-S, rMV^EZSARS-CoV-2-S-COAA-PP, rMV^EZSARS-CoV-2-S-COAA-fneg-PP, and rMV^EZSARS-CoV-2-S-COAA-fneg, wherein the spike glycoprotein is inserted after the measles virus N and P genes (in the third (3) position).

Example 2

This example demonstrates the generation and characterization of recombinant MV vaccine strain viruses expressing SARS-CoV-2 spike glycoprotein.

Based on previously generated reverse genetics system for live-attenuated measles virus (MV) vaccine strain Edmonston-Zagreb (EZ) that expresses an enhanced green fluorescent protein (EGFP; rMV^EZ EGFP(3)), recombinant MV vaccine viruses encoding SARS-CoV-2 spike glycoprotein were generated and rescued. The open reading frame for the spike glycoprotein lies within an additional transcriptional unit at position 3 in the MV genome in place of EGFP. These viruses were compared in vitro for replication and expression of the spike glycoprotein. The growth kinetics of all tested viruses were equivalent to rMV_EZEGFP(3).

pMV^EZEGFP(3) (Rennick et al., J. Virol. (2015). doi:10.1128/jvi.02924-14) was modified to express SARS-CoV-2 spike glycoprotein in place of EGFP. In a manner similar to that described in Case et al, bioRxiv (2020). doi:10.1101/2020.05.18.102038, an endoplasmic reticulum (ER) retention signal sequence present in the cytoplasmic tail (CT) of the spike was altered from KxHxx to AxAxx-COOH, and anti-genomic plasmids expressing a non-codon and human codon optimized version (pMV^EZSARS-CoV2-S and pMV^EZSARS-CoV-2-COAA, respectively) were generated. An additional human codon optimized version with the authentic KxHxx-COOH sequence was also generated (pMV^EZSARS-CoV-2-CO).

Recovery of a plasmid with the correct spike sequence proved challenging for the non-codon optimized version, and as a result two plasmids were generated: one with the authentic sequence (pMV^EZSARS-CoV-2-S) and one with three nucleotide changes, one of which does not cause an amino acid change (pMV^EZSARS-CoV-2-S6).

To recover recombinant viruses, Vero cells were transfected with either pMV^EZSARS-CoV-2-S, pMV^EZSARS-CoV-2-S6, pMV^EZSARS-CoV-2-CO or pMV^EZSARS-CoV-2-COAA along with expression plasmids for nucleoprotein (N), phosphoprotein (P) and polymerase (L) proteins. Sequence confirmation of the virus generated from clone pMV^EZSARS-CoV-2-S revealed an amino acid change in the CT tail. This virus was subsequently named rMV^EZSARS-CoV-2-S3 (FIG. 1A).

The spike glycoprotein sequences in rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-CO, and rMV^EZSARS-CoV-2-COAA correspond to their cDNA clones (FIG. 1A). Rescue experiments were reproducible with syncytium formation about 14 days post transfection (FIG. 1B).

To test if the spike glycoprotein was expressed, Vero cells were infected and fixed and stained at 48 hours post-infection using an anti-SARS2-S antibody. Substantial amounts of spike expression were detectable in the infected cells, especially for rMV^EZSARS-CoV-2-CO (FIG. 1C). Expression was further confirmed by staining viral plaques with anti-SARS2-S antibody (FIG. 1D).

Growth analysis of rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-CO, and rMV^EZSARS-CoV-2-COAA were compared to rMV^EZEGFP(3) in Vero cells over a 3 day period. Viruses replicated similar to rMV^EZEGFP(3) by reaching a titers close to 10⁷ TCID₅₀/ml (FIG. 2).

Additional vectors were generated with modifications to ablate the furin cleavage signal (rMV^EZSARS-CoV-2-S-COAA-fneg and rMV^EZSARS-CoV-2-S-COAA-fneg-PP), and to lock in the prefusion conformation by substitution of two proline residues (rMV^EZSARS-CoV-2-S-COAA-PP and S-CO-AA-fneg-PP).

rMV^EZSARS-CoV-2-S3 and rMV^EZSARS-CoV-2-S6 encode a spike glycoprotein with nucleotide changes that arose during virus rescue and plasmid generation, respectively.

rMV^EZSARS-CoV-2-S-CO, rMV^EZSARS-CoV-2-S-COAA, rMV^EZSARS-CoV-2-S-COAA-PP, rMV^EZSARS-CoV-2-S-COAA-fneg, and rMV^EZSARS-CoV-2-S-COAA-fneg-PP encode a human codon optimized spike glycoprotein.

rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-S, rMV^EZSARS-CoV-2-S-COAA, rMV^EZSARS-CoV-2-S-COAA-PP, rMV^EZSARS-CoV-2-S-COAA-fneg-PP, and rMV^EZSARS-CoV-2-S-COAA-fneg contain mutations in the spike glycoprotein to disrupt the endoplasmic reticulum (ER) retention sequence that has been described as essential for SARS-CoV-2 virion assembly via the ER-Golgi compartment.

The nucleic acid sequence encoding the spike glycoprotein of rMV^EZSARS-CoV-2-S3 corresponds to SEQ ID NO: 22.

The following table summarizes the sequence information for each of the vectors and the corresponding nucleic acid and amino acid sequences of the spike glycoprotein.

	Nucleic Acid	Amino Acid
	Sequence of	Sequence of	Nucleic Acid
	Spike	Spike	Sequence of
Vector Designation	Glycoprotein	Glycoprotein	Vector	Modifications
rMVEZSARS-COV-2-S6	1	8	15	modifications to ablate ER retention signal
rMVEZSARS-COV-2-S-CO	2	9	16	codon optimized
rMVEZSARS-COV-2-S-COAA	3	10	17	codon optimized;
				modifications to ablate ER retention signal
rMVEZSARS-COV-2-S	4	11	18	modifications to ablate ER retention signal
rMVEZSARS-COV-2-S-COAA-PP	5	12	19	codon optimized;
				modifications to ablate ER retention signal;
				modifications to lock in prefusion conformation
rMVEZSARS-COV-2-S-COAA-	6	13	20	codon optimized;
fneg-PP				modifications to ablate ER retention signal;
				modifications to lock in prefusion conformation;
				modifications to ablate furin cleavage signal
rMVEZSARS-COV-2-S-COAA-fneg	7	14	21	codon optimized;
				modifications to ablate ER retention signal;
				modifications to ablate furin cleavage signal
rMVEZSARS-COV-2-S3	22			modifications to ablate ER retention signal

Example 3

This example describes vaccination/challenge studies with recombinant MV vaccine strain viruses expressing SARS-CoV-2 spike glycoprotein.

Mice

The experimental set-up for mice immunized with recombinant viruses is described in FIG. 3A. Groups of 5 mice were infected with either rMV^EZSARS-CoV-2-S3, rMV^EZSARS-CoV-2-S6, rMV^EZSARS-CoV-2-S-CO, rMV^EZSARS-CoV-2-S-COAA, or rMV^EZEGFP(3). Serum was collected on days 21 and 42, and splenocytes were collected on day 42. Neutralization of SARS-CoV-2 using the harvested mice serum indicates that SARS-CoV-2 neutralizing antibodies were produced in mice vaccinated with rMV^EZSARS-CoV-2-S-CO and rMV^EZSARS-CoV-2-S-COAA viruses (FIG. 3B).

Splenocytes prepared from rMV^EZ, rMV^EZSARS-CoV-2-CO, rMV^EZSARS-CoV-2-S6, and rMV^EZSARS-CoV-2-COAA immunized mice were used in an ELISPOT assay to detect the secretion of proinflammatory cytokine IFN-γ. Mice splenocytes were re-stimulated for 24 hours with four pools of synthetic peptides (51, S2, S3 and S4) designed to span the entire SARS-CoV-2 spike protein. Unstimulated splenocytes (medium) served as negative controls and splenocytes treated with PMA/ionomycin confirmed splenocyte re-stimulation. The number of cells expressing IFN-γ after re-stimulation are represented as 1×10⁵ cells in FIG. 12. The results demonstrate that IFN-γ is secreted in spleens from mice immunized with recombinant measles virus expressing SARS-CoV-2 codon-optimized spike protein.

Flow cytometry was used to determine the proportion of CD4⁺/CD44⁺ and CD8⁺/CD44⁺ T cells in the spleens of mice immunized with rMV^EZ, rMV^EZSARS-CoV-2-CO, rMV^EZSARS-CoV-2-S6, and rMV^EZSARS-CoV-2-COAA. To determine the optimal re-stimulation period splenocytes from two mice from each immunized group were first re-stimulated for 6 hours (FIG. 13A) while the remaining three mice spleens were re-stimulated for 12-hours (FIG. 13B). Cells were re-stimulated with spike specific peptide pools S1, S2, S3 and S4, or left unstimulated (negative control; medium). Re-stimulation was confirmed with a cell activation cocktail containing PMA/ionomycin and Brefaldin-A. Intracellular cytokine staining for IFN-γ and IL-13 were then carried out to determine Th1 and Th2 responses, respectively. The results in FIGS. 13A and 13B demonstrate the T cell responses in immunized mice.

Non-Human Primates

Multi-route delivery and challenge testing for natural measles is described in de Swart et al., NPJ vaccines, 2(1), pp. 1-11 (2017). Specifically, infection of rhesus macaques with rMV^EZEGFP(3) vaccine via the intramuscular, intranasal, and aerosol routes, along with the intratracheal route as a control matching the usual experimental inoculation route, has been described. Serum antibody responses were detected by enzyme linked immunosorbent assay (ELISA), virus neutralization, or indirect immunofluorescence for MV fusion (MV-F) or hemagglutinin (MV-H) glycoprotein-specific antibodies. Animals vaccinated by the intramuscular (IM) route produced similar antibody responses to those inoculated via the aerosol route. In all assays, lowest serum antibody levels were consistently observed in animals immunized by intranasal instillation. All animals were protected from challenge after intratracheal instillation of a wild-type strain of MV.

For this study, African green monkeys (AGMs) were used since a SARS-CoV-2 challenge model has been established (Hartman et al., doi: https://doi.org/10.1101/2020.06.20.137687 (2020)—in press PLoS Pathogens). In this challenge model, AGMs were inoculated via a multi-route mucosal (oral, nasal, and ocular) exposure with a low passage, clinical isolate of SARS-CoV-2. The experimental design is detailed in FIGS. 11A and 11B.

All AGMs develop mild disease with pulmonary lesions detectable by PET/CT in the acute phase which subsequently resolve. All AGMs exhibit prolonged shedding of infectious virus from oral, nasal, conjunctival, and rectal mucosal surfaces with viral RNA (vRNA) detectable throughout the respiratory and gastrointestinal tissues at later timepoints in the absence of replication-competent virus.

Animals (maximum group size n=6) were vaccinated (IM) with the recombinant measles viral vector of the invention. Two animals were vaccinated with the standard measles virus Edmonston Zagreb vaccine strain. Some animals received a boost at 3 or 4 weeks post-vaccination. Animals were sampled before vaccination and then weekly over 6 or 8 weeks for the development of SARS-CoV-2 and MV serum antibody responses (IgG, IgM, IgA and neutralizing antibodies and T cell responses).

Development of MV-specific immune response will be compared to historical controls using banked samples from the studies analyzing intramuscular, aerosol, intranasal, and intratracheal administration of MV vaccine (de Swart et al., NPJ vaccines, 2(1), pp. 1-11 (2017)). EDTA blood samples will be collected in Vacuette tubes containing K₃EDTA as an anticoagulant. A sample of whole blood will be used directly for hematology analysis. Plasma will be separated from blood cells by low speed centrifugation and used in a commercial ELISA classic Measles Virus IgG assay (Serion) to assess anti-MV IgG alongside an in house MV-N-specific IgG ELISA.

Development of anti SARS-CoV-2 specific IgG and IgM will be determined using an in house ELISA that detects antibodies against the SARS-CoV-2 spike protein receptor binding domain (RBD). ELISA plates will be coated with 50 ng/well of SARS-CoV-2 RBD and subsequently blocked in 5% (V/V) FBS, 5% (W/V) skim milk in PBS with 0.1% (V/V) Tween-20 (PBS T) for 1 hour at 37° C. Serial dilutions of plasma will be made in block solution and incubated on the blocked plates for 2 h at 37° C. After washing with PBS T, bound antibodies will be detected by incubation with goat-anti-monkey IgM(p)-HRP (Seracare/KPL #5220-0334) or goat-anti-rhesus IgG (H+L)-HRP (Southern Biotech #6200-05), both used at a 1:5,000 dilution in blocking solution for 1 hour at 37° C. After washing with PBS T the assay will be developed by incubation with TMB (Seracare) for 7 min prior to the addition of TMB stop solution (Seracare). Absorbance values will be determined at 450 nm.

PBMC are isolated from the residual blood cell pellet by layering diluted whole blood onto Lymphoprep and subsequent density gradient centrifugation. A sample of PBMC will be used for MV virus isolation by plating dilutions of PBMC with Vero cells expressing human CD150 (Vero hCD150 cells). Assays are incubated at 37° C. for 3-5 days and then scored for cytopathic effect. A portion of PBMC will also be used for RNA extraction and subsequent RT/PCR analysis for detection of viral genome.

Blood samples for serum will be collected in Vacuette tubes (FIG. 11B). After coagulation, low speed centrifugation will be used to remove the clot from the serum supernatant. A sample will be used for serum biochemistry and the rest will be available for neutralizing antibody analysis. Virus neutralizing antibodies will be detected using a fluorescent focus reduction neutralization test (FRNT) for MV and a plaque reduction neutralization test (PRNT) for SARS-CoV-2. The FRNT uses a MV that expresses EGFP during replication; this facilitates rapid screening of assays. Detection of fluorescence indicates virus replication and seronegativity while lack of fluorescence indicates the presence of neutralizing antibodies which prevent virus infection. Serum dilutions will be mixed with 100 plaque forming units (p.f.u.) of MV and incubated at 37° C. for 1 hour. After addition of Vero cells expressing the MV receptor human CD150, assays will be incubated at 37° C. for 3-5 days before screening for fluorescence as a measure of virus replication. For the PRNT, serum dilutions will be mixed with 100 plu. of SARS-CoV-2 and incubated at 37° C. for 1 hour after which they will be added to confluent Vero E6 cell monolayers. After incubation at 37° C. for 1 hour, medium will be replaced by immunodiffusion agarose. After incubation at 37° C. for 72 hours, the agarose overlay will be removed and the cell monolayer fixed and stained with crystal violet. Plaques will be enumerated and the PRNT₈₀ calculated. The SARS-CoV-2 molecular, virological and immunological assays are described in Klimstra et al., J. Gen. Virol., doi: 10.1099/jgv.0.001481 (2020).

The vaccinated and sham vaccinated animals will be challenged via multi route mucosal exposure with 10⁶ plu. of SARS-CoV-2. Protection will be assessed by sampling oral, ocular, rectal and nasal swabs for decreases in infectious virus and viral RNA copies following challenge, decreased PET/CT signals in lungs and lymph nodes and secondary immune responses. Brushes will be used to collect throat and rectal samples and swabs will be used to collect nasal and ocular samples into virus transport medium. After vortexing the brush/swab is removed and the remaining liquid is used directly for SARS-CoV-2 virus isolation by adding to VeroE6 cell monolayers, and for RNA isolation for RTqPCR analysis. For virus isolations, after incubation at 37° C. for 1 hour, medium will be replaced by immunodiffusion agarose. After incubation at 37° C. for 72 hours, the agarose overlay will be removed and the cell monolayer fixed and stained with crystal violet to allow visualization of plaques. After RNA isolation, one-step RT-qPCR will be performed using a One-Step Multiplex RT-qPCR Supermix (BioRad), and primers and probe targeting the SARS-CoV-2 N gene sequence. Quantitation of virus genome copies will be determined by comparing the cycle threshold values from the unknown samples to cycle threshold values from a positive-sense SARS-CoV-2 vRNA standard curve generated from 10-fold serial dilutions of in house synthesized template.

Serial PET/CT images will be acquired pre-infection and at 3 or 4 and 10 or 11 dpi. The scans will be performed on a MultiScan LFER 150 (Mediso Medical Imaging Systems). CT acquisition will be performed using the following parameters: Semi-circular single field-of-view, 360 projections, 80 kVp, 670 μA, exposure time 90 ms, binning 1:4, voxel size of final image: 500×500 μm. PET acquisition will be performed 55 min after intravenous injection of 18F-fluoro-2-deoxy-D-glucose (FDG) with the following parameters: 10 min acquisition, single field-of-view, 1-9 coincidence mode, 5 ns coincidence time window. PET images will be reconstructed with the following parameters: Tera-Tomo 3D reconstruction, 400-600 keV energy window, 1-9 coincidence mode, median filter on, spike filter on, voxel size 0.7 mm, 8 iterations, 9 subsets, scatter correction on, attenuation correction based on CT material map segmentation. Images will be analyzed using OsiriX MD or 64-bit (v.11, Pixmeo, Geneva, Switzerland). Before analysis, PET images will be Gaussian smoothed in OsiriX and smoothing will be applied to raw data with a 3×3 matrix size and a matrix normalization value of 24. Whole lung FDG uptake will be measured by first creating a whole lung region-of-interest (ROI) on the lung in the CT scan by creating a 3D growing region highlighting every voxel in the lungs between −1024 and −500 Hounsfield units. This whole lung ROI will be copied and pasted to the PET scan and gaps within the ROI will be filled in using a closing ROI brush tool with a structuring element radius of 3. All voxels within the lung ROI with a standard uptake value (SUV) below 1.5 will be set to zero and the SUVs of the remaining voxels will be summed for a total lung FDG uptake (total inflammation) value. Thoracic lymph nodes will be analyzed by measuring the maximum SUV within each lymph node using an oval drawing tool. Both total FDG uptake and lymph node uptake values will be normalized to back muscle FDG uptake measured by drawing cylinder ROIs on the back muscles adjacent to the spine at the same axial level as the carina (SUVCMR; cylinder-muscle-ratio). PET quantification values will be organized in Microsoft Excel and graphed using GraphPad Prism.

Blood samples will be collected and processed as for the vaccination phase of the study and the secondary immune response to SARS-CoV-2 will be measured as before using the SARS-CoV-2 spike protein receptor binding domain ELISA for detection of IgG and IgM, and the PRNT for detection of neutralizing antibodies and the T cells assays already established in the IFNAR mice (FIGS. 13A and 13B) to assess cellular immune responses.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A recombinant measles viral vector comprising a nucleic acid sequence encoding a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike glycoprotein.

2. The recombinant measles viral vector of claim 1, wherein the recombinant measles viral vector is a recombinant Edmonston-Zagreb (EZ) measles viral vector.

3. The recombinant measles viral vector of claim 1, wherein the nucleic acid sequence has been codon optimized.

4. The recombinant measles viral vector of claim 2, wherein the nucleic acid sequence has been codon optimized.

5-6. (canceled)

7. The recombinant measles viral vector of claim 2, wherein the nucleic acid sequence contains at least one modification that disrupts the endoplasmic reticulum (ER) retention sequence of the SARS-CoV-2 spike glycoprotein.

8. The recombinant measles viral vector of claim 7, wherein the ER retention sequence of the SARS-CoV-2 spike glycoprotein contains AxAxx rather than KxHxx in the cytoplasmic tail.

9. The recombinant measles viral vector of claim 3, wherein the nucleic acid sequence contains at least one modification that disrupts the endoplasmic reticulum (ER) retention sequence of the SARS-CoV-2 spike glycoprotein.

10. The recombinant measles viral vector of claim 9, wherein the ER retention sequence of the SARS-CoV-2 spike glycoprotein contains AxAxx rather than KxHxx in the cytoplasmic tail.

11. The recombinant measles viral vector of claim 4, wherein the nucleic acid sequence contains at least one modification that disrupts the endoplasmic reticulum (ER) retention sequence of the SARS-CoV-2 spike glycoprotein.

12. The recombinant measles viral vector of claim 11, wherein the ER retention sequence of the SARS-CoV-2 spike glycoprotein contains AxAxx rather than KxHxx in the cytoplasmic tail.

13. The recombinant measles viral vector of claim 1, wherein the nucleic acid sequence encoding the SARS-CoV-2 spike glycoprotein is selected from the group consisting of SEQ ID NO: 1 (S6), SEQ ID NO: 2 (S-CO), SEQ ID NO: 3 (S-CO-AA), SEQ ID NO: 4 (S), SEQ ID NO: 5 (S-CO-AA-PP), SEQ ID NO: 6 (S-CO-AA-fneg-PP), and SEQ ID NO: 7 (S-CO-AA-fneg).

14. The recombinant measles viral vector of claim 1, wherein the amino acid sequence of the SARS-CoV-2 spike glycoprotein is selected from the group consisting of SEQ ID NO: 8 (S6), SEQ ID NO: 9 (S-CO), SEQ ID NO: 10 (S-CO-AA), SEQ ID NO: 11 (CoV2-S), SEQ ID NO: 12 (S-CO-AA-PP), SEQ ID NO: 13 (CO-AA-fneg-PP), and SEQ ID NO: 14 (S-CO-AA-fneg).

15. The recombinant measles viral vector of claim 1 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 15 (pSARS-CoV2-S6), SEQ ID NO: 16 (SARS-CoV-2-S-CO), SEQ ID NO: 17 (SARS-CoV-2-S-CO-AA), SEQ ID NO: 18 (SARS-CoV-2-S), SEQ ID NO: 19 (SARS-CoV-2-S-CO-AA-PP), SEQ ID NO: 20 (SARS-CoV-2-S-CO-AA-fneg-PP), and SEQ ID NO: 21 (SARS-CoV-2-S-CO-AA-fneg).

16. A pharmaceutical composition comprising the recombinant measles viral vector of claim 1 and a pharmaceutically acceptable carrier.

17. A method for preventing, inhibiting, reducing, eliminating, protecting, or delaying the onset of an infection or an infectious clinical condition caused by coronavirus in a subject comprising administering to the subject (i) the recombinant measles viral vector of claim 1 or (ii) a pharmaceutical composition comprising the vector and a pharmaceutically acceptable carrier.

18-20. (canceled)

21. A method for inducing an immune response against a coronavirus in a subject comprising administering to the subject (i) the recombinant measles viral vector of claim 1 or the pharmaceutical composition comprising the vector and a pharmaceutically acceptable carrier.

22-24. (canceled)

25. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (S6), SEQ ID NO: 9 (S-CO), SEQ ID NO: 10 (S-CO-AA), SEQ ID NO: 11 (S), SEQ ID NO: 12 (S-CO-AA-PP), SEQ ID NO: 13 (S-CO-AA-fneg-PP), and SEQ ID NO: 14 (S-CO-AA-fneg).

26. A nucleic acid encoding the polypeptide of claim 25.

27. A nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 (S6), SEQ ID NO: 2 (S-CO), SEQ ID NO: 3 (S-CO-AA), SEQ ID NO: 4 (SARS-CoV-2-S), SEQ ID NO: 5 (S-CO-AA-PP), SEQ ID NO: 6 (S-CO-AA-fneg-PP), and SEQ ID NO: 7 (S-CO-AA-fneg).

28-29. (canceled)

30. A pharmaceutical composition comprising (i) the polypeptide of claim 25, a nucleic acid encoding the polypeptide, or a recombinant vector comprising the nucleic acid and (ii) a pharmaceutically acceptable carrier.

31-32. (canceled)