Engineered middle east respiratory syndrome proteins and related methods

Abstract

Disclosed herein are engineered Middle East Respiratory Syndrome (MERS) spike proteins and nucleic acids encoding the same. Also disclosed are fusion proteins comprising an engineered Middle East Respiratory Syndrome (MERS) spike protein and a binding partner, and nucleic acids encoding the same. Also disclosed are vaccine compositions comprising any of the aforementioned proteins or nucleic acids. Disclosed are methods of preventing a MERS infection or a disease associated with a MERS infection in a subject comprising administering to the subject an effect amount of said vaccine compositions.

Description

ENGINEERED MIDDLE EAST RESPIRATORY SYNDROME PROTEINS ANDRELATED METHODSRELATED APPLICATIONS

[0001] This application claims the priority and benefits of US. Provisional Application No. 63 / 587,130, filed October 1, 2023, US. Provisional Application No. 63 / 632,896, filed April 11, 2024, and US. Provisional Application No. 63 / 666,188, filed June 30, 2024, the contents of which are incorporated herein in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (File name: VCCN_005_02WO_SeqList_ST26.xml; Size: 903,613 bytes; and Date of Creation: September 27, 2024) are herein incorporated by reference in its entirety.BACKGROUND

[0003] Coronaviruses are a large family of medium sized, enveloped, positive-stranded ribonucleic acid (RNA) viruses that can cause mild to moderate upper-respiratory tract infection in humans. Three coronaviruses have notably caused life-threatening disease; severe acute respiratory syndrome coronavirus (SARS-CoV-1) that first emerged in late 2002, followed by Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and then SARS-CoV-2, circulating since 2019.

[0004] MERS-CoV was first identified in the Middle East in 2012. Since then, over 2,600 cases have been confirmed in over 28 countries. Although the source of the virus remains unknown, studies have linked primary transmission to from animals to human, with secondary human to human transmission accounting for a majority of documented infections. As seen with other coronavirus outbreaks, human to human transmission can occur in places with frequent human to human contact such as airports, shopping centers, sporting events, healthcare settings, households, and the like. It has been reported that approximately 35% of those diagnosed with MERS have died from the disease. Therefore there continues to be an urgent public health need for rapid development of durable vaccine interventions with significant breadth to address MERS should MERS emerge once again as a widespread public health problem. Disclosed herein are methods and compositions that address the same.SUMMARY

[0005] In one aspect, provided herein is an engineered Middle East Respiratory Syndrome (MERS) spike protein comprising at least 70% sequence identity to SEQ ID NO: 1 with a C- terminal truncation of at least 25 amino acids. In some embodiments, the engineered protein comprises a C-terminal truncation of at least 100 amino acids.

[0006] In some embodiments of the engineered MERS spike protein of the disclosure, the engineered protein comprises the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 70% sequence identity thereto.

[0007] In some embodiments of the engineered MERS spike protein of the disclosure, the engineered protein comprises one or more proline substitutions, including one or more of: A889P, S966P, A973P, T1014P, V1060P, and L1061P.

[0008] In some embodiments of the engineered MERS spike protein of the disclosure, the engineered protein is fused to a nanoparticle, thereby forming a fusion protein comprising the spike protein fused to a nanoparticle. In some embodiments, the nanoparticle comprises a ferritin subunit. In some embodiments, the fusion protein forms a self-assembling nanoparticle. In some embodiments, the ferritin subunit comprises H. pylori ferritin. In some embodiments, the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity thereto.

[0009] In some embodiments of the engineered MERS spike protein of the disclosure, the engineered protein comprises an amino acid sequence of SEQ ID NO: 285 or SEQ ID NO: 349, or an amino acid sequence having at least 70% sequence identity thereto.

[0010] Also provided herein is an engineered Middle East Respiratory Syndrome (MERS) spike protein comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1, comprising at least three proline substitutions. In some embodiments, the at least three proline substitutions comprise at least three of: A889P, S966P, A973P, T1014P, V1060P, or L1061P. In some embodiments, one of the at least three proline substitutions is selected from the group consisting of: A889P, V1060P, and L1061P. In some embodiments, one of the at least three proline substitutions is V1060P.

[0011] In some embodiments of the engineered MERS spike protein of the disclosure, the engineered protein consists of three proline substitutions. In some embodiments, the three prolinesubstitutions comprise A889P, V1060P, and L1061P. In some embodiments, the three proline substitutions are A889P, V1060P, and LI 06 IP.

[0012] In some embodiments of the engineered MERS spike protein of the disclosure, the engineered protein has a C terminal truncation. In some embodiments, the amino acid sequence of the engineered protein comprises SEQ ID NO: 157 or SEQ ID NO: 93, or an amino acid sequence having at least 70% sequence identity thereto.

[0013] In some embodiments, the engineered MERS spike protein is fused to a binding partner, thereby forming a fusion protein. In some embodiments, the binding partner is a ferritin subunit. In some embodiments, the fusion protein forms a nanoparticle.

[0014] In some embodiments of the engineered MERS spike protein of the disclosure, the engineered protein further comprises at least one insertion. In some embodiments, the insertion is at the C-terminal or N-terminal of the spike protein, or internal to the protein. In some embodiments, the insertion enables transit and extrusion of the spike protein in extracellular vesicles and / or promotes enveloped virus like particle (eVLP) production.

[0015] In some embodiments, the at least one amino acid insertion comprises insertion of an ESCRT-recruiting domain (ERD). In some embodiments, the amino acid sequence of the ERD comprises the sequence of any one of SEQ ID NOs: 421-432, or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments, the at least one amino acid insertion comprises insertion of a polypeptide that is ESCRT-independent.

[0016] Also provided here is a fusion protein comprising an engineered Middle East Respiratory Syndrome (MERS) spike protein and a binding partner. In some embodiments of the fusion protein of the disclosure, the spike protein comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments, the amino acid sequence of the spike protein comprises a C terminal truncation.

[0017] In some embodiments of the fusion protein of the disclosure, the spike protein is operably linked to the binding partner through a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a Gly-Ser linker. In some embodiments, the linker comprises a sequence selected from the group consisting of: SGG, GSG, SGG, GG, GSGG (SEQ ID NO: 418), NGTGGSG (SEQ ID NO: 419), and GGGGS (SEQ ID NO: 420).

[0018] In some embodiments of the fusion protein of the disclosure, the amino acid sequence of the spike protein comprises a sequence of SEQ ID NO: 66 or SEQ ID NO: 130, or an amino acid sequence having at least 70% sequence identity thereto.

[0019] In some embodiments of the fusion protein of the disclosure, the spike protein comprises one or more proline substitutions. In some embodiments, the one or more proline substitutions are selected from the group consisting of: A889P, S966P, A973P, T1014P, V1060P, and L1061P.

[0020] In some embodiments of the fusion protein of the disclosure, the spike protein comprise three proline substitutions, optionally wherein the protein consists of three proline substitutions. In some embodiments, the three proline substitutions are A889P, V1060P, and L1061P.

[0021] In some embodiments of the fusion protein of the disclosure, the amino acid sequence of the spike protein comprises an amino acid sequence of SEQ ID NO: 93 or SEQ ID NO: 157, or an amino acid sequence having at least 70% sequence identity thereto.

[0022] In some embodiments of the fusion protein comprising an engineered MERS spike protein and a binding partner of the disclosure, the binding partner comprises a ferritin subunit.

[0023] In some embodiments of the fusion protein of the disclosure, the amino acid sequence of the fusion protein comprises the amino acid sequence of: SEQ ID NO: 285 or SEQ ID NO: 349, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 285 or 349.

[0024] Also provided herein is an envelope eVLP comprising an engineered MERS spike protein of the disclosure or a fusion protein of the disclosure.

[0025] Also provided herein are nucleic acids that encode one or more engineered MERS spike proteins of the disclosure. In some embodiments, provided are DNAs that encode the engineered MERS spike proteins of the disclosure. In other embodiments, provided are RNAs, (e.g., mRNAs) that encode the engineered MERS spike proteins of the disclosure.

[0026] Also provided herein is a nucleotide sequence that encodes the amino acid sequence of an engineered MERS spike protein of the disclosure or a fusion protein of the disclosure.

[0027] In some embodiments of the nucleotide sequence of the disclosure, the nucleic acids of the nucleotide sequence comprise DNA.

[0028] In some embodiments of the nucleotide sequence of the disclosure, the nucleic acids of the nucleotide sequence comprise RNA. In some embodiments, the nucleic acids of the nucleotide sequence comprise mRNA.

[0029] Also provided herein is a vaccine composition comprising the engineered MERS spike protein of the disclosure, the fusion protein of the disclosure, the eVLP of the disclosure, or the nucleotide sequence of the disclosure. In some embodiments, the vaccine composition comprises one or more adjuvants.

[0030] In some embodiments of the vaccine composition of the disclosure which comprises a nucleotide sequence, the nucleic acids of the nucleotide sequence comprise DNA. In some embodiments, the nucleic acids of the nucleotide sequence comprise RNA. In some embodiments, the nucleic acids of the nucleotide sequence comprise mRNA.

[0031] Also provided herein is a method of preventing a Middle East Respiratory Syndrome (MERS) infection or a disease associated with a MERS infection in a subject, comprising administering to the subject an effective amount of one or more doses of the vaccine composition of the disclosure. In some embodiments, the subject is human.

[0032] In some embodiments of the method of preventing a MERS infection or a disease associated with a MERS infection, the route of administration is intramuscular, intravenous, intradermal, or subcutaneous.

[0033] In some embodiments, the method comprises preventing a MERS infection or a disease associated with a MERS infection in a subject by a MERS virus strain from MERS clade A, MERS clade B, or MERS clade C. In some embodiments, the MERS virus strain from MERS clade A comprises the MERS strain EMC. In some embodiments, the MERS virus strain from MERS clade B comprises a MERS strain selected from the group consisting of: Englandl, Hasal4B, Bishal, and KOR / NIH / 002. In some embodiments, the MERS virus strain from MERS clade C comprises a MERS virus strain selected from the group consisting of: Nigl657, NRCE-HKU270, C1272, and MRCE-HKU205.

[0034] In some embodiments, the method comprises preventing a MERS infection or a disease associated with a MERS infection in a subject by a MERS virus strain including a merbecovirus. In some embodiments, the merbecovirus comprises a merbecovirus strain of MjHKU4r-CoV.

[0035] Also provided herein is the vaccine composition of the disclosure, for use in a method of preventing a Middle East Respiratory Syndrome (“MERS”) infection or a disease associated with a MERS infection in a subject; wherein the method comprises administering to the subject an effective amount of one or more doses of the vaccine composition.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 depicts a schematic of a Middle East Respiratory Syndrome (“MERS”) spike protein.

[0037] FIG. 2A depicts a schematic of an exemplary fusion protein comprising a nanoparticle displaying a plurality of spike trimers formed by a fusion protein of MERS-1227 and a ferritin subunit (SEQ ID NO: 285).

[0038] FIG. 2B depicts a schematic of an exemplary fusion protein comprising a nanoparticle displaying a plurality of spike trimers formed by a fusion protein of MERS-1236 and a ferritin subunit (SEQ ID NO: 349).

[0039] FIG. 2C depicts a schematic of the exemplary fusion protein of FIG. 2A, comprising a MERS-1227 Spike protein in fusion with a ferritin subunit. Non-limiting mutations to the spike protein are provided as exemplary.

[0040] FIG. 2D depicts a schematic of the exemplary fusion protein of FIG. 2B, comprising a MERS-1236 Spike protein coupled to a ferritin subunit. Non-limiting mutations to the spike protein are provided as exemplary.

[0041] FIG. 3A depicts an SDS-PAGE gel of MERS-1227 fusion protein and MERS-1236 fusion protein supernatants, to analyze protein expression levels according to the methods of Example 1.

[0042] FIG. 3B depicts an SDS-PAGE gel of the MERS-1227 fusion protein and the MERS- 1236 fusion protein after purification, under non -reducing (“NR”) or reducing (“R”) conditions.

[0043] FIG. 4A depicts a graph of Dynamic Light Scattering of each of the MERS-1227 fusion protein and the MERS-1236 fusion protein.

[0044] FIG. 4B depicts a graph of Differential Scanning Fluorimetry of each of the MERS- 1227 fusion protein and the MERS-1236 fusion protein.

[0045] FIG. 5 depicts a graph of Analytical Size-Exclusion Chromatography and Light Scattering Analysis of each of the MERS-1227 fusion protein and MERS-1236 fusion protein.

[0046] FIGS. 6A-6B depict graphs of Biolayer Interferometry to each of MERS-1227 fusion protein and MERS-1236 fusion protein.

[0047] FIG. 7A depicts representative images of Transmission Electron 7CMicroscopy and 2D class averaging of purified MERS-1227 fusion protein.

[0048] FIG. 7B depicts representative images of Transmission Electron Microscopy and 2D class averaging of purified MERS-1236 fusion protein.

[0049] FIG. 7C depicts representative images of Transmission Electron microscopy of MERS- 1227.

[0050] FIG. 7D depicts representative images of Transmission Electron microscopy of MERS- 1236

[0051] FIG. 8A depicts a graph of neutralizing antibody Titers for fusion proteins comprising MERS-1227 Spike protein coupled to a ferritin nanoparticle or MERS-1236 Spike protein coupled to a ferritin nanoparticle, 21 days after a first dose in mice.

[0052] FIGS. 8B-8C depict graphs of neutralizing antibody titers 21 days after a first dose in mice, for fusion proteins comprising MERS-1227 Spike protein coupled to a ferritin nanoparticle or MERS-1236 Spike protein coupled to a ferritin nanoparticle, wherein each fusion protein was subjected to different stability conditions.

[0053] FIG. 9 depicts a graph of neutralizing antibody titers in mice measured at Day 56, wherein the mice were administered a dose of a composition comprising a SARS-CoV-2 nanoparticle (designated as DCFHP) followed by a boost with either the MERS-1227 fusion protein or MERS-1236 fusion protein on Day 49.

[0054] FIG. 10 depicts a graph of neutralizing antibody titers in Non-Human Primates (“NHPs”) 21 days after the 1stdose, wherein the NHPs were administered a dose of a vaccine composition comprising either the MERS-1227 fusion protein and an adjuvant or the MERS-1236 fusion protein and an adjuvant.

[0055] FIG. 11 depicts a graph of neutralizing antibody titers in mice measured at Day 21 or Day 42, where the mice were administered different concentrations of a vaccine composition comprising the MERS-1227 fusion protein either as a single dose (prime) at day 0 or as two doses (prime+boost) wherein the second dose was administered 21 days after the first dose.

[0056] FIG. 12A depicts a schematic of a study in mice, wherein mice were administered a vaccine composition comprising the MERS-1227 fusion protein in a single dose (prime) or in two doses (prime+boost). For the mice administered two doses, the second dose was administered at day 70 at either 2 ug or 10 ug. Whole blood was collected at the time points indicated on the timeline.

[0057] FIG. 12B depicts a graph of neutralizing antibody titers from the mice of FIG. 12A, measured at day 21, day 56, or day 77.

[0058] FIG. 12C depicts a schematic of a study in NHPs, wherein NHPs were administered a vaccine composition comprising the MERS-1227 fusion protein in two doses, at Day 0 and Day 93. Whole blood was collected at the time points indicated on the timeline.

[0059] FIG. 12D depicts a graph of neutralizing antibody titers from the NHPs of FIG. 12C, measured at day 0, day 35, day 56, day 93, day 100, day 127 and day 188.

[0060] FIG. 12E depicts a graph of neutralizing antibody titers from NHPs serum collected 5 weeks following a second administration of the vaccine composition comprising MERS-1227 or MERS-1236 neutralizing a panel of lentiviruses pseudotyped with spike sequences from MERS- CoV clade A, B, and C and a pangolin merbecovirus (MjHKU-4r-CoV-l).

[0061] FIG. 12F depicts a graph of domain-specific IgG responses from immunized NHPs with the vaccine composition comprising MERS-1227 or MERS-1236 of either a first administration (prime) or a second administration (Prime +boost).

[0062] FIGS. 13A-13B depict graphs of Biolayer Interferometry that measure binding of antibodies to the MERS-1227 fusion protein following a four week temperature hold at 4°, 22°, or 37° C.

[0063] FIGS. 13C-13D depict graphs of Biolayer Interferometry that measure binding of antibodies to the MERS-1227 fusion protein upon extreme heat stress (e.g., 70° C or 90°C) for 2 hours.

[0064] FIG. 13E depicts a graph of differential scanning fluorimetry of the MERS-1227 fusion protein after a 1 week, 2 week or 4 week temperature hold at 37°C.

[0065] FIG. 13F depicts a graph of differential scanning fluorimetry of the MERS-1227 fusion protein after extreme heat stress (e.g., 70° C or 90° C) for 2 hours.

[0066] FIG. 13G depicts a graph of neutralizing antibody titers 35 days following a single dose (10 pg) of the adjuvanted MERS-1227 fusion protein after either a 10 day temperature hold at 4° C or 37° C.

[0067] FIG. 14A depicts a schematic of a study in BALB / c mice, where mice were administered different embodiments of the vaccine compositions of the disclosure including MERS-1227.

[0068] FIG. 14B depicts a graph of neutralizing antibody titers following administration of a single dose or two doses of different embodiments of the vaccine composition of the disclosure including an adjuvanted MERS-1227 fusion protein, a mRNA encoding the MERS-1227 fusion protein, a mRNA encoding an engineered MERS spike protein, and a mRNA encoding a full length MERS spike protein.

[0069] FIG. 15A depicts a schematic of a study in BALB / c mice, wherein mice were administered different vaccine compositions of the disclosure comprising the MERS-1227 fusion protein adjuvanted with different adjuvants.

[0070] FIG. 15B depicts a graph of neutralizing antibody titers on 21 days (Day 21) after a first administration of the various concentrations of vaccine compositions of the disclosure with various concentrations of adjuvants of the disclosure or 21 days (Day 42) after a second administration.

[0071] FIGS. 16A-16E depict graphs of biophysical characterization studies of MERS-1227 following pH treatment at pH 2, 3, 3.5, 4 and 7

[0072] FIGS. 17A-17E depict graphs of biophysical characterization studies of MERS-1236 following pH treatment at pH 2, 3, 3.5, 4, and 7.

[0073] FIGS. 18A-18E depict graphs of biophysical characterization of MERS-1227 following incubation at 4, 22 or 37 °C for 7 or 14 days.

[0074] FIGS. 19A-19E depict graphs of biophysical characterization of MERS-1236 following incubation at 4, 22 or 37 °C for 7 or 14 days

[0075] FIG. 20A depicts a schematic of a study in BALB / c mice, wherein mice were administered different concentrations of MERS-1227, MERS-1236 or MERS trimer adjuvanted with 150 ug of Alhydrogel.

[0076] FIGS. 20B-20C depicts graphs of neutralization titers following a single administration (Prime only) or a double administration (prime+boost) of MERS 1227, MERS-1236 or MERS trimer.

[0077] FIGS. 21A-21D depict graphs of antigen-specific IgG responses from mice having received different concentrations of a single (prime only) or double administration (prime+boost) of MERS-1227, MERS-1236 or MERS trimer.

[0078] FIGS. 22A depicts a schematic of an Alpaca challenge study design and timeline.

[0079] FIG. 22B depicts a graph of neutralization antibody titers on day 0 and day 41 after two administrations (prime +boost) of different doses of MERS-1227 or MERS-1236 adjuvanted with 500 pg of Alhydrogel, or 500 pg Alhydrogel only.

[0080] FIGS. 22C depicts a graph of live-virus neutralization titers obtained from a plaque reduction neutralization assay using day 41 serum samples from alpacas administered different doses of MERS-1227 or MERS-1236 adjuvanted with 500 pg of Alhydrogel, or 500 pg Alhydrogel only.

[0081] FIG. 22D depicts a quantification of the MERS-CoV virus in nasal swabs obtained from alpacas from days 1-5 and 8 post viral challenge via plaque assay.

[0082] FIGS. 23A-23B depict SDS-PAGE gels of MERS FNPs in stable CHO pool supernatants of MERS-1227 and MERS-1236 respectively.

[0083] FIGS. 23C-23D depict graphs of quantified densitometry of FIGS. 23A-23B.

[0084] FIGS. 24A-24B depict bind-and-elute chromatography purifications for MERS-1227 and MERS-1236.

[0085] FIGS. 24C-24D depict SDS-PAGE gels corresponding to purifications samples (FT and elution) of FIGS. 24A-24B.

[0086] FIG. 25A depicts a graph of liquid chromatography -mass spectrometry (LC-MS / MS) of N-linked vs O-linked glycans on MERS-1227 and MERS-1236 FNPs.

[0087] FIGS. 25B-25C depict schematics of the annotated positions of detected N-glycosites on MERS-1227 and MERS-1236.

[0088] FIGS. 26A-26B depicts graphs of MERS spike-pseudotyped lentivirus neutralization assay using monoclonal antibodies and convalescent camel serum while FIGS. 26C-26D depict tables of the data in the graphs of FIGS. 26A-26B.

[0089] FIG. 27 depicts a table of results from a plaque reduction neutralization assay using pooled mouse serum from MERS-1227 and MERS-1236 immunizations.

[0090] FIG. 28 depicts a graph of the ratio of IgG2a and IgGl responses to MERS spike determined by Luminex in BALB / c mice.

[0091] FIG. 29A depicts a schematic of a challenge study in human DPP4 transgenic mice immunized with different concentrations of MERS-1227.

[0092] FIG. 29B depicts a graph of neutralization titers (NT) following a single administration (Prime only) or a double administration (Prime+boost) of MERS 1227.

[0093] FIG. 29C depicts a graph of live-virus neutralization titers obtained from a plaque reduction neutralization assay using day 21 (prime only) and day 42 (prime + boost) serum from human DPP4 transgenic mice administered a single (Prime only) or a double (Prime+boost) dose of MERS 1227.

[0094] FIG. 29D depicts a graph of the quantitation of live MERS-CoV virus in lung samples obtained from mice sacrificed 3 days post-challenge with MERS-CoV.

[0095] FIG. 29E depicts the survival curve of immunized mice up to 10 days post challenge with MERS-CoV.

[0096] FIG. 30 depicts a graph of normalized body weights of mice immunized with 0, 0.016, or 0.4 pg MERS-1227, up to 10 days post challenge with MERS-CoV.DETAILED DESCRIPTIONTerms and Concepts

[0097] A number of terms and concepts are discussed below. They are intended to facilitate the understanding of various embodiments of the invention in conjunction with the rest of the present document and the accompanying figures. These terms and concepts may be further clarified and understood based on the accepted conventions in the fields of the present invention, as well as the description provided throughout the present document and / or the accompanying figures. Some other terms can be explicitly or implicitly defined in other sections of this document and in the accompanying figures and may be used and understood based on the accepted conventions in the fields of the present invention, the description provided throughout the present document and / or the accompanying figures. The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the present invention and interpreted in the context of the present document and / or the accompanying figures.

[0098] Unless otherwise dictated by context, singular terms shall include pluralities, and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry are those well-known and commonly used. Known methods and techniques are generally performed according to conventional methods well-known and as described in various general and more specific references, unless otherwise indicated. The nomenclatures used in connection with the laboratory procedures and techniques described in the present disclosure are those well-known and commonly used.

[0099] As used herein, the terms "a", "an", and "the" can refer to one or more unless specifically noted otherwise. The use of the term "or" is used to mean "and / or," unless explicitly indicated to refer to alternatives only, or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." As used herein "another" can mean at least a second or more.

[0100] The terms "about" and "approximately" as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); e.g., within 10%; or e.g., within 5% of a given value or range of values. Any reference to "about X" or "approximately X" specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.0IX, 1.02X,1.03X, 1.04X, and 1.05X. Thus, expressions "about X" or "approximately X" are intended to teach and provide written support for a claim limitation of, for example, "0.98X." Alternatively, in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, within 5- fold, e.g. within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated. When "about" is applied to the beginning of a numerical range, it applies to both ends of the range.

[0101] The terms "protein," "peptide," and "polypeptide" are used interchangeably to refer to a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers and non-natural amino acid polymers, as well as to amino acid polymers in which one (or more) amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, or a non-naturally occurring amino acid. The terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0102] The terms "sequence identity," and the related terms and expressions used in the context of describing nucleic acid or amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of nucleic acid or amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence (subject sequence) to which test sequences (query sequence) are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well- known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection. Algorithms that are suitable for determining percent sequenceidentity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. Depending on the algorithm, the calculated percent sequence identity may differ. For example, there are at least three ways in which to calculate a percent sequence identity, as presented below.% Query sequence identity: = (Number of alignment identities) / (Length of Query sequence);% Subject sequence identity: = (Number of alignment identities) / (Length of Subject sequence);%Alignment sequence identity = (Number of alignment identities) / (Length of Alignment)

[0103] Accordingly, when the term “sequence identity” is used herein, it can include any of the above non-limiting methodologies provided above to calculate.

[0104] The expression "neutralizing antibody" can refer to an antibody capable of reducing the ability of an infectious agent, such as a virus, from infecting a cell by neutralizing or inhibiting one or more parts of the life cycle of the infectious agent. In the context of the present disclosure, neutralizing antibodies can prevent a coronavirus, such as, but not limited to, MERS-CoV, from completing its life cycle in host cell. The life cycle of the virus, for example, a coronavirus, starts with attachment of the virus to a host cell and ending with budding of newly formed virus from the host cell. This life cycle includes, but is not limited to, the steps of attaching to a cell, entering a cell, fusion of the viral membrane with the host cell membrane, release of viral ribonucleoproteins into the cytoplasm, formation of new viral particles and budding of viral particles from the host cell membrane.

[0105] The term “immunogenic” when used in the context of the present disclosure, refers to the ability of a molecule to elicit in a subject an immune response to said molecule. For example, the molecule can be an antigen, which can be a protein, a polypeptide, or a region of a protein or a polypeptide, or a nucleic acid encoding a polypeptide, which elicit in a subject an immune response to the specific antigen. Various aspects of an immune response elicited by an immunogenic composition can be determined using standard assays, some of which are described in the present disclosure.

[0106] The terms “Middle East Respiratory Syndrome” or “MERS,” as used herein, may refer to the MERS-CoV virus itself or the disease caused by the MERS-CoV virus. It will be apparentto a skilled artisan reading the disclosure as to whether either a virus or disease is being referred to, given the particular context of the section of the disclosure in question.

[0107] It is expected that the engineered MERS proteins of the disclosure will be immunogenic in a subject upon administration, whether they are administered as protein-based vaccines, or as nucleic acid-based vaccines.Engineered Proteins

[0108] Provided herein are engineered proteins comprising an amino acid sequence of a Spike protein of a Middle East Respiratory Syndrome (“MERS”) Spike protein and nucleic acids expressing the same. In some embodiments, the engineered proteins or nucleic acids disclosed herein may be used in vaccinating a subject against an infection by a MERS virus.

[0109] Spike proteins are coronavirus surface proteins that can mediate receptor binding and membrane fusion between a coronavirus virion (e.g., a MERS virus) and its host cell, thus playing a viral role in a MERS infection. Spike proteins assemble into trimers of spikes on the surface of the enveloped coronavirus. Spike proteins may be comprised of ectodomains of homotrimers of Spike proteins. Ectodomains of MERS spike proteins contain an N terminal SI subunit and a C terminal S2 subunit. The SI subunit comprises a signal peptide and a receptor binding domain (“RBD”), while the S2 subunit includes a fusion peptide (“FP”), two heptad repeat domains (“HR1” and “HR2”), a transmembrane domain (“TM”), and a cytoplasmic domain (“CP”), as seen in the schematic of FIG. 1.

[0110] For MERS-CoV, the RBD of the SI subunit mediates receptor binding on target cells. MERS-CoV through its RBD binds to dipeptidyl peptidase 4 (DPP4) on target cells to mediate viral attachment. The domains associated with the S2 subunit are associated with membrane fusion. In the process of membrane fusion, the S2 subunit may undergoes conformational changes. Before membrane fusion, the spike protein can present as a trimeric structure. In some embodiments, one or more substitutions of amino acids of the S2 subunit may stabilize the spike trimeric structures in a prefusion conformation, as will be described in more detail herein.[OHl] In some embodiments, engineered MERS spike proteins, fusion proteins and / or nucleic acids encoding the same of the disclosure may contain or encode a MERS antigen that comprises naturally occurring (e.g., wild type) amino acid sequences of a MERS spike protein. In some embodiments, the sequence of a wild type MERS spike protein comprises the sequence of SEQ ID NO: 1 :

[0112] MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKAD GIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFA NGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPD GCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFN LRNCTFMYTYNITEDEILEWFGITQTAQGVHLF S SRYVDLYGGNMFQF ATLP VYDTIKY YSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYES FDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLT KLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFS NPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVW EDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFAN DTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYY CLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQT PVGCVLGLVNSSLFVEDCKLPLGQSLC ALPDTP STL TPRSVRSVPGEMRLASIAFNHPIQ VDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCS KINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIE DLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAY TSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGA MQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQI DRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSF VVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEW SYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSI PNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIA GLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYDLEPHKVHVH (SEQ ID NO: 1). In some embodiments, the engineered MERS spike protein may comprise a sequence with at least 70% sequence identity to SEQ ID NO: 1.

[0113] In some embodiments, the engineered proteins or nucleic acids that encode the engineered proteins described herein may contain or encode a naturally occurring (or “wild-type”) amino acid sequence of the MERS spike protein, while in other embodiments of the disclosure, engineered proteins or nucleic acids that encode the engineered proteins described herein may contain or encode one or more modifications compared to a wild-type MERS spike protein amino acid sequence of SEQ ID NO: 1, as will be described in more details herein.

[0114] In some embodiments, the MERS spike protein may be engineered to include one or more modifications compared to a wild-type MERS spike protein including one or more substitutions, deletions, additions (insertions), and / or truncations of amino acid residues within the spike protein as will be described in more detail herein. In some embodiments, the MERS spike protein having one or more modifications may comprise artificially modified amino acid sequences of the MERS spike protein or portions thereof. In some embodiments, the MERS spike protein may include a deletion of one or more amino acid residues at the C-terminal, N-terminal, and / or middle portions of the protein. The deletion of one or more amino acids may include a deletion of one or more consecutive amino acid residues or one or more non-consecutive amino acid residues. In some embodiments, the deletion of one or more amino acids may include about 2 consecutive amino acids, about 3 consecutive amino acids, about 4 consecutive amino acids, about 5 consecutive amino acids, about 6 consecutive amino acids, about 7 consecutive amino acids, about 8 consecutive amino acids, about 9 consecutive amino acids, about 10 consecutive amino acids, about 15 consecutive amino acids, about 20 consecutive amino acids, about 25 consecutive amino acids, about 30 consecutive amino acids, about 35 consecutive amino acids, about 40 consecutive amino acids, about 45 consecutive amino acids, about 50 consecutive amino acids, about 55 consecutive amino acids, about 60 consecutive amino acids, about 65 consecutive amino acids, about 70 consecutive amino acids, about 75 consecutive amino acids, about 80 consecutive amino acids, about 85 consecutive amino acids, about 90 consecutive amino acids, about 95 consecutive amino acids, about 100 consecutive amino acids, about 105 consecutive amino acids, about 110 consecutive amino acids, about 115 consecutive amino acids, about 120 consecutive amino acids, about 125 consecutive amino acids, about 130 consecutive amino acids, about 135 consecutive amino acids, about 140 consecutive amino acids, about 145 consecutive amino acids, about 150 consecutive amino acids, about 155 consecutive amino acids, about 160 consecutive amino acids, about 165 consecutive amino acids, about 170 consecutive amino acids, about 175 consecutive amino acids, about 180 consecutive amino acids, about 185 consecutive amino acids about 190 consecutive amino acids, about 195 consecutive amino acids, or about 200 consecutive amino acids.

[0115] For example, in some embodiments, the engineered MERS spike protein may include one or more truncations within the MERS Spike protein, including a C-terminal truncation. In some embodiments, the C-terminal truncation may be at least 25 amino acids long compared to the wild-type MERS spike protein. In some embodiments, the C terminal truncation may be atleast about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 45 amino acids, at least about 50 amino acids, at least about 55 amino acids, at least about 60 amino acids, at least about 65 amino acids, at least about 70 amino acids, at least about 75 amino acids, at least about 80 amino acids, at least about 85 amino acids, at least about 90 amino acids, at least about 95 amino acids, at least about 100 amino acids, at least about 105 amino acids, at least about 110 amino acids, at least about 115 amino acids, at least about 120 amino acids, at least about 125 amino acids, at least about 130 amino acids, at least about 135 amino acids, at least about 140 amino acids, at least about 145 amino acids, or at least about 150 amino acids long compared to the wild-type MERS spike protein. In some embodiments, the C-terminal truncation may be at least 25 amino acids long compared to the wild-type MERS spike protein of SEQ ID NO: 1. The engineered MERS spike protein may comprise a sequence that has at least 70% sequence identity to SEQ ID NO: 1 and has a C-terminal truncation of at least 25 amino acids.

[0116] In some embodiments, the C terminal truncation may delete or more one or more domains of the S2 subunit. For example, in some embodiments, the C terminal truncation of the disclosure may remove one or more of: the HR2 domain, the TM domain, and / or the CP domain. In some embodiments, the engineered MERS spike protein may include the amino acid sequence of

[0117] In some embodiments, the engineered MERS spike protein may include the amino acid sequence of:MH4SVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKAD GIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFA NGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPD GCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFN LRNCTFMYTYNITEDEILEWFGITQTAQGVHLF S SRYVDLYGGNMFQF ATLP VYDTIKY YSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYES FDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLT KLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFS NPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVW EDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFAN DTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYY CLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQT PVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCS KINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIE DLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAY TSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGA MQTGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQI DRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSF VVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEW SYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGGST (SEQ ID NO: 66), or an amino acid sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0118] In some embodiments, the engineered MERS spike protein may include the amino acid sequence of:MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGI IYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFAN GFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDG CGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNL RNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYY SIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESF DVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTK LLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSN PTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWE DGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFAND TKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYC LRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTP VGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQV DQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSK INQALHGANLRQDD S VRNLF AS VKS SQS SPIIPGFGGDFNLTLLEP VSISTGSRS ARS AIED LLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTS SLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQ TGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVV NAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSY TGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELD (SEQ ID NO: 130), or an amino acid sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. A difference in the length of C terminal truncations may provide an unexpected benefit compared to a wild-type MERS spike protein or compared to each other. Without being held to theory or mechanism, the unexpected benefits may include increased amino acid expression, better folding, increased immunogenicity in vivo, or the like. For example, the MERS spike proteins of SEQ ID NO: 66 and SEQ ID NO: 130 differ in the length of their C terminal truncations by about 9 amino acids.

[0119] In some embodiments, the engineered MERS spike protein may include additional deletions or substitutions that may remove or add glycosylation sites. In some embodiments, the engineered MERS spike protein may include additional deletions or substitutions that may remove a protease recognition site, such as a furin cleavage site. The naturally occurring or “wild-type” amino acid sequence of the England 1 MERS spike protein contains a furin cleavage site between the SI and S2 domains. The furin cleavage site allows furin, a protease, to cleave the spike protein, activating membrane fusion. In some embodiments, one or more substitutions may eliminate the furin cleavage site within the MERS spike protein. For example, in some embodiments, the engineered MERS spike proteins may include one or more deletions or substitutions within the furin cleavage site, wherein the one or more deletions or substitutions may remove the furin cleavage site. In some embodiments, the substitutions or deletions may comprise a substitution or deletion at amino acid residue R748 or R751. In some embodiments, the substitution may include R748A or R751G.

[0120] For example, the full-length and C-terminally truncated MERS spike proteins of the present disclosure may comprise mutations that eliminate the furin cleavage site at residues 748- 751 in the full-length Englandl strain of MERS. In some embodiments, the mutations that eliminate the furin cleavage site is RSVR mutated to ASVG. In some embodiments, the engineered spike protein comprises a mutation at residue 1020. In certain embodiments, the mutation at residue 1020 is from a H to a Q. In some embodiments, the engineered spike protein furthercomprises one or more proline substitutions that stabilize the Spike trimer in the prefusion conformation.

[0121] In some embodiments, the engineered MERS spike protein may include one or more substitutions within any of the domains of the engineered MERS spike protein. The one or more substitutions may affect a conformation of the MERS spike protein. For example, the one or more substitutions may stabilize the spike protein a pre-fusion conformation. In some embodiments, the one or more substitutions may include proline substitutions. For example, in some embodiments, any one of more of the following residues may be substituted: A889, S966, A973, T1014, V1060, orL1061. In some embodiments, at leasttwo of the residues of A889, S966, A973, T1014, V1060, or LI 061 are substituted to proline. In some embodiments, at least three of the residues of A889, S966, A973, T1014, V1060, or L1061 are substituted to proline. In some embodiments, at least four of the residues of A889, S966, A973, T1014, V1060, or L1061 are substituted to proline. In some embodiments, at least five of the residues of A889, S966, A973, T1014, V1060, or L1061 are substituted to proline. In some embodiments, all six of the residues A889, S966, A973, T1014, VI 060, or LI 061 are substituted to proline.

[0122] In some embodiments, no more than 5 of the residues of A889, S966, A973, T1014, VI 060, or LI 061 are substituted to proline. In some embodiments, no more than 4 of the residues of A889, S966, A973, T1014, V1060, or L1061 are substituted to proline. In some embodiments, no more than 3 of the residues of A889, S966, A973, T1014, V1060, or L1061 are substituted to proline.

[0123] In some embodiments, the engineered MERS spike protein may comprise the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least 70% sequence identity thereto including having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0124] In some embodiments, the engineered MERS spike protein may include the aforementioned substitutions and comprise any one of the amino acid sequences of SEQ ID NOs: 3-65 or an amino acid sequence having at least 70% sequence identity thereto.

[0125] In some embodiments, the engineered MERS spike protein may include the aforementioned substitutions and either of the C-terminal truncations, as described above. For example, in some embodiments, the truncated MERS spike protein may comprise any amino acidsequence of SEQ ID NOs: 67-129, 131-193, or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments, the engineered MERS spike protein may comprise the amino acid sequence ofMIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKAD GIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFA NGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPD GCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFN LRNCTFMYTYNITEDEILEWFGITQTAQGVHLF S SRYVDLYGGNMFQF ATLP VYDTIKY YSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYES FDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLT KLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFS NPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVW EDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFAN DTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYY CLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQT PVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQ VDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCS KINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSPIE DLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAY TSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGA MQTGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQI DRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSF VVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEW SYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGGST (SEQ ID NO: 93), orMIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKAD GIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFA NGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPD GCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFN LRNCTFMYTYNITEDEILEWFGITQTAQGVHLF S SRYVDLYGGNMFQF ATLP VYDTIKY YSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYES FDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLT KLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVW EDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFAN DTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYY CLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQT PVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQ VDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCS KINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSPIE DLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAY TSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGA MQTGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQI DRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSF VVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEW SYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELD (SEQ ID NO: 157), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 93 or 157.

[0126] In some embodiments, the engineered MERS spike protein may comprise an amino acid sequence of any one of SEQ ID NOs: 3-193, 387-388, or the engineered MERS spike protein encoded by the nucleic acids of the disclosure may comprise an amino acid sequence of any one of SEQ ID NOs: 3-193, 387-388, or an amino acid sequence having at least 70% sequence identity thereto, including having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3-193 or 387-388.

[0127] In some embodiments, the engineered MERS spike protein may include an addition (insertion) of one or more amino acid residues at the C-terminal, N-terminal, and / or middle portions of the protein. The addition of one or more amino acids may include an addition of one or more consecutive amino acid residues or one or more non-consecutive amino acid residues. In some embodiments, the addition of one or more amino acids may include about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, or about 200 consecutive amino acids.Fusion Proteins

[0128] In some embodiments, the engineered MERS spike protein may be fused to a binding partner. In some embodiments, fusion of the spike protein to the binding partner leads to formation of a nanoparticle. In some embodiments, the binding partner is a ferritin subunit, for example the ferritin subunit may selected from H. Pylori ferritin or hybrid bullfrog ferritin. In some embodiments, the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0129] In some embodiments, provided herein are fusion proteins comprising an amino acid sequence of an engineered MERS spike protein and an amino acid sequence of a ferritin subunit polypeptide. In some embodiments, the engineered MERS spike protein may be operably linked to the ferritin subunit. In some embodiments, the fusion protein further comprises a linker which operably links the engineered MERS spike protein to the ferritin subunit. In some embodiments, the fusion proteins disclosed herein may be useful for vaccinating a subject against an infection by a MERS virus or preventing a disease associated with a MERS infection in a subject. Fusion proteins of the disclosure may also include other amino acid sequences including amino acid sequences of polypeptide domains other than Spike domains and ferritin domains, linker sequences, signal sequences, tags, and the like. In some embodiments, the nucleic acids of the disclosure may encode one or more fusion proteins of the disclosure.Ferritin Nanoparticles

[0130] In some embodiments, the engineered MERS spike proteins of the disclosure may be fused to binding partner to form a fusion protein. In some embodiments, the engineered MERS spike protein of the disclosure that is fused to a binding partner may be encoded by nucleic acids of the disclosure. In some embodiments, the binding partner comprises a ferritin subunit, thereby generating a ferritin nanoparticle.

[0131] Ferritin is a globular protein found in animals, bacteria, and plants, that acts primarily to control the rate and location of polynuclear Fe(III)2O3 formation through transportation of hydrated iron ions and protons to and from a mineralized core. The globular form of ferritin is made up of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides having a molecular weight of approximately 17-20 kDa. An example of the sequence of one such monomeric ferritin subunit is represented by:DIIKLLNEQVNKEMQ S SNLYMSMS SWC YTHSLDGAGLFLFDHAAEEYEHAKK LIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFN FLQWYVAEQHEEEVLFNDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 2).

[0132] Each monomeric ferritin subunit has the topology of a helix bundle which includes a four antiparallel helix motif, with a fifth shorter helix (the c-terminal helix) lying roughly perpendicular to the long axis of the 4-helix bundle. According to the convention, the helices are labeled ‘A, B, C, and D & E, from the N-terminus respectively. The N-terminal sequence lies adjacent to the capsid three-fold axis and extends to the surface, while the E helices pack together at the four-fold axis with the C-terminus extending into the particle core. The consequence of this packing creates two pores on the capsid surface. It is expected that one or both of these pores represent the point by which the hydrated iron diffuses into and out of the capsid. Following production, these monomeric ferritin subunit proteins self-assemble into the globular ferritin protein. Thus, the globular form of ferritin comprises 24 monomeric, ferritin subunit proteins, and has a capsid-like structure having an octahedral (432) symmetry.

[0133] The fusion proteins of the disclosure may include an amino acid sequence of a ferritin subunit polypeptide. The ferritin amino acid sequence can be an amino acid sequence of a full length, single ferritin polypeptide, or any portion of a ferritin amino acid sequence that is capable of directing self- assembly of monomeric ferritin subunits into oligomers. Fusion proteins comprising ferritin amino acid sequences are described, for example, in U.S. PatentNo, 7,097,841. The amino acid sequences of monomeric ferritin subunits, or portions thereof, of any ferritin protein can be used to produce fusion proteins of the disclosure, so long as the monomeric ferritin subunits are capable of self-assembling into an oligomer or a nanoparticle. Variations can be made in the amino acid sequence of a ferritin protein without affecting its ability to self-assembly into an oligomer or a nanoparticle. Such variations include insertion of amino acid residues, deletions of amino acid residues, or substitutions of amino acid residues. For example, the sequence of a monomeric ferritin subunit included in a fusion protein according to some of the embodiments ofthe disclosure can be derived from a mammalian ferritin amino acid sequence but be divergent enough from the naturally occurring sequence such that when administered as an immunogen to a mammalian subject of the species from which the mammalian ferritin amino acid sequence was derived, it does not result in the production of antibodies that react with the natural ferritin protein of the mammal. A ferritin amino acid sequence may be derived from a bacterial ferritin protein, a plant ferritin protein, an algal ferritin protein, an insect ferritin protein, a fungal ferritin protein, and / or a mammalian ferritin protein. For example, the ferritin subunit polypeptides may comprise one or more sequences of a hybrid bullfrog ferritin. In some embodiments, the hybrid bullfrog ferritin comprises the amino acid sequence of SEQ ID NO: 386. In some embodiments, the ferritin amino acid sequences may be derived from H. pylori. For example, a fusion protein as provided herein may comprise a ferritin amino acid sequence derived from an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.

[0134] As discussed above, fusion proteins of the disclosure need not comprise a full-length sequence of a ferritin subunit polypeptide. In some embodiments, the ferritin subunit polypeptides may comprise one or more fragments of the ferritin sequences disclosed herein. In some embodiments, the ferritin subunit polypeptides may comprise one or more fragments of H. pylori ferritin. Portions or regions of H. pylori ferritin subunit polypeptide can be used that contain an amino acid sequence directing self-assembly of monomeric ferritin subunits into oligomers. One example of such a region is located between amino acids 5-168 of the amino acid sequence of H. pylori ferritin protein. In some embodiments, a fusion protein of the present disclosure comprises amino acids 5-168 of SEQ ID NO: 2 or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0135] A ferritin amino acid sequence included in fusion proteins of this disclosure may include artificial glycosylation sites, for examples, artificial (engineered) N-glycosylation sites, which are engineered by inserting artificial mutations into a ferritin amino acid sequence to create a consensus glycosylation sequence. For example, an artificial N-glycosylation site may be createdby introducing a consensus sequence N-X-S / T (where X cannot be a P) in a ferritin amino acid sequence. A consensus glycosylation sequence can be created by artificial substitutions of amino acid residues in a ferritin amino acid sequence. For example, an artificial N-glycosylation site in SEQ ID NO: 2 can be created by introducing two amino acid substitutions: K to N at a position corresponding to position 75 of SEQ ID NO: 2, and E to T at a position corresponding to position 75 of SEQ ID NO: 2. In another example, an artificial N-glycosylation site in SEQ ID NO: 2 can be created by introducing two amino acid substitutions: T to N at a position corresponding to position 67 of SEQ ID NO: 2 and I to T at a position corresponding to position 69 of SEQ ID NO: 2. In yet another example, an artificial N-glycosylation site in SEQ ID NO: 2 can be created by introducing two amino acid substitutions: H to N at a position corresponding to position 74 of SEQ ID NO: 2 and F to T at a position corresponding to position 76 of SEQ ID NO: 2. Additionally, an artificial N-glycosylation site in SEQ ID NO: 2 can be created by introducing two amino acid substitutions: E to N at a position corresponding to position 143 of SEQ ID NO: 2 and H to T at a position corresponding to position 145 of SEQ ID NO: 2.

[0136] In some embodiments, the ferritin amino acid sequence may include one or more deletions or substitutions, wherein the one or more deletions or substitutions may eliminate a glycosylation site or may allow for more efficient production. For example, in some embodiments, the N at a position corresponding to position 9 may be substituted for a Q. In some embodiments, the K at a position corresponding to position 131 may be substituted for an N.

[0137] In some embodiments, the ferritin subunit (the ferritin amino acid sequence) may be coupled to the MERS spike protein by one or more linkers. In some embodiments, the linker is a peptide linker composed of an amino acid sequence. In some embodiments, the amino acid sequence of the linker may be about 2 to about 5 amino acids, about 2 to about 10 amino acids, about 2 to about 20 amino acids, about 2 to about 30 amino acids, about 2 to about 40 amino acids, about 2 to about 50 amino acids, or about 2 to about 60 amino acids, or more amino acids in length. For example, the linker may be about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 25 amino acids, about 35 amino acids, about 45 amino acids, about 50 amino acids, or about 60 amino acids. Depending on length, one or more linker sequences may have various conformations in secondary structure, suchas helical, P-strand, coil / bend, and turns. In some instances, a linker sequence may have an extended conformation and function as an independent domain that does not interact with the adjacent protein domains. A linker sequence may be rigid or flexible. A flexible linker sequence may increase the range of orientations that may be adopted by the domains of the fusion protein. A rigid linker can be used to keep a fixed distance between the domains and to help maintain their independent functions. Linker sequences for fusion proteins are described for example in Chen et al, 2013. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises a glycine or serine. In some embodiments, the peptide linker is a Glycine-Serine (Gly-Ser) linker. In some embodiments, a linker is or comprises the amino acid sequence SGG, GSG, GG, GSGG (SEQ ID NO: 418), NGTGGSG (SEQ ID NO: 419), GGGGS (SEQ ID NO: 420), or SGG. Exemplary linker sequences of the disclosure are provided in Table 1 below:Table 1: Linker Sequences

[0138] Provided in this disclosure and included among the embodiments are nanoparticles that include fusion proteins comprising an amino acid sequence of a MERS spike protein and an amino acid sequence of a ferritin subunit polypeptide. Such fusion proteins can self-assemble into oligomers. An oligomeric structure, or supramolecule, resulting from such self-assembly is referred to herein as a nanoparticle. An exemplary embodiment of the present invention is a nanoparticle comprising an oligomer of a fusion protein.

[0139] A nanoparticle can include eight surface-exposed trimers of MERS spike protein, as seen in FIGS. 2A-2B which depict schematics of exemplary fusion proteins of the disclosure comprising a nanoparticle displaying 8 MERS-1227 spike trimers.

[0140] When the nanoparticle comprising the surface-exposed trimers of MERS spike protein and ferritin subunit are administered to a subject, the surface-exposed trimers of the MERS spike protein trimer are accessible to the immune system of the subject and thus can elicit an immune response to a MERS spike protein. Immunogenic nanoparticles composed of fusion proteins incorporating ferritin amino acid sequences are described for example, in US Patent Nos. 9,441,019, 10,137,190, Kanekiyo et al. 2013, Kanekiyo et al., 2015, and He et al 2016.

[0141] As described above, the MERS spike protein may be included within a fusion protein, wherein a linker couples the MERS spike protein to a ferritin nanoparticle subunit of the disclosure. In some embodiments, the MERS fusion protein may comprise an amino acid sequence having a C-terminal truncation as described above, the one or more substitutions as described above, with a linker and a ferritin nanoparticle subunit. In some embodiments, the MERS fusion protein may comprise an amino acid sequence of any one of the exemplary sequences disclosed herein, comprising an engineered MERS spike protein and a ferritin subunit, such as an amino acid sequence of any one of SEQ ID NOs: 195-385 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the MERS fusion protein may comprise an amino acid sequence of any one of SEQ ID NOs: 259-321, 323-385, or any amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In exemplary embodiments, the MERS fusion protein may comprise the amino acid sequence of:MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGI IYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFAN GFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDG CGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNL RNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYY SIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESF DVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTK LLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSN PTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWE DGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFAND TKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYC LRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTP VGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQV DQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDD S VRNLF AS VKS SQS SPIIPGFGGDFNLTLLEP VSISTGSRS ARSPIED LLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTS SLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQ TGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQIDR LINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVV NAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSY TGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGGST / SGG / DIIKLLNEQVNKEMQS SNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHK FEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFNDILD I<IELIGNENHGLYLADQYVI<GIAI<SRI<S (SEQ ID NO: 285), orMIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGI IYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFAN GFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDG CGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNL RNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYY SIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESF DVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTK LLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSN PTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWE DGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFAND TKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYC LRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTP VGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQV DQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSK INQALHGANLRQDD S VRNLF AS VKS SQS SPIIPGFGGDFNLTLLEP VSISTGSRS ARSPIED LLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTS SLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQ TGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQIDR LINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVV NAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSY TGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELD / SGG / D / / AZZAE QVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFNDIL DKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 349), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 285 or 349, where the sequence within the slashes ( / / ) identifies the linker and italics identifies the ferritin subunit sequence in the above provided sequences. In some embodiments, the MERS fusion protein may comprise an amino acid sequence of a ferritin subunit. In some embodiments, the amino acid sequence of the ferritin subunit is:DIIKLLNEQVNKEMQ S SNLYMSMS SWC YTHSLDGAGLFLFDHAAEEYEHAKKLI IFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFL QWYVAEQHEEEVLFNDILDI<IELIGNENHGLYLADQYVI<GIAI<SRI<S (SEQ ID NO: 2), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Extracellular Vesicles Presenting Antigens

[0142] Extracellular vesicles can be produced from host cells. For example, the Endosomal Sorting Complex Required for Transport (ESCRT) is thought to be an important component of the cellular machinery facilitating the transit of proteins and cargo within cells. Notably, it plays a role in shuttling proteins from the endoplasmic reticulum (ER) to the cell surface, as well as in the generation of extracellular vesicles, which can originate either within the ER, or at the cell surface before being extruded from the cell. Incorporation of ESCRT -recruiting domains (ERDs) may enhance immunogenicity by inducing antigen expression on cell surfaces and promoting the formation of extracellular vesicles presenting antigens, also referred to as enveloped virus like particles (eVLPs).

[0143] In some embodiments, provided herein are eVLPs presenting one or more engineered MERS proteins of the disclosure. The eVLPs of the present disclosure are non-infectious membraned particles whose production does not require a viral capsid protein and is instead driven by addition (insertion) of one or more polypeptides. The one or more polypeptides which drive eVLP production can be dependent on host ESCRT machinery (such as ERDs) or can be entirelyindependent of host ESCRT machinery. For example, polypeptides that enable transit and extrusion of proteins in extracellular vesicles without the use of ESCRT machinery can be suitable for eVLP production.

[0144] In some embodiments, the engineered MERS protein described herein may include an addition (insertion) of one or more ERDs at the C-terminal, N-terminal, and / or middle portions of the protein. The ERD can comprise or can be derived from a nonhuman protein, optionally a nonmammalian protein, further optionally a chicken protein, a mouse protein, a lizard protein, a reptile protein, a hamster protein, or a goldfish protein. In some embodiments, the ERD can comprise the sequence of any one of SEQ ID NOs: 421-432, or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 421-432. In some embodiments, the ERD interacts with the ESCRT proteins TSG101, NEDD4, and / or ALIX. In some embodiments, the ERD can comprise or can be derived from the ESCRT and ALIX binding region (EABR) of the human CEP55 protein, optionally residues 170-213. An EABR sequence recruits host ESCRT proteins to the engineered MERS spike protein at the plasma membrane and results in the self-assembly and budding of eVLPs bearing the engineered MERS spike protein described herein. In some embodiments, the ERD can comprise or can be derived from a viral protein or fragment thereof. Viral proteins include, but are not limited to, retroviral protein, herpes simplex viral protein, vaccinia viral protein, hepadnaviral protein, togaviral protein, flaviviral protein, arenaviral protein, coronaviral protein, orthomyxoviral protein, paramyxoviral protein, bunyaviral protein, bomaviral protein, rhabdoviral protein, filoviral protein, or Ebola protein, or proteins from Equine infectious anemia virus (EIAV), Human T-lymphotropic virus 1 (HTLV-1), Murine leukemia virus (MLV), or Mason-Pfizer monkey virus (MPMV). Examples of polypeptides that depend on host ESCRT machinery to drive eVLPs formation (such as ERDs) and the use of these polypeptides for vaccine design are disclosed in International Pub. No. WO2022261230A1, the contents of which are herein incorporated in its entirety by reference. Without being held to theory or mechanism, it is expected that the inclusion of an ERD sequence in a MERS protein, would recruit host ESCRT proteins to the engineered MERS protein at the plasma membrane and would result in the self-assembly and budding of eVLPs bearing the engineered MERS proteins described herein. Exemplary ERD sequences are provided in Table 2 below.Table 2: Exemplary ERD Sequences

[0145] In some embodiments, the engineered MERS spike protein of the disclosure comprises an insertion of the amino acid sequence of an ERD. The amino acid sequence of the ERD can comprise the sequence of any one of SEQ ID NOs: 421-432, or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 421-432.

[0146] In some embodiments, the engineered MERS protein described herein may include an addition (insertion) of one or more ESCRT -independent polypeptides that drive eVLP production at the C-terminal, N-terminal, and / or middle portions of the protein. For example, polypeptides that enable transit and extrusion of proteins in extracellular vesicles without the use of ESCRT machinery can be suitable for eVLP production.

[0147] In some embodiments, the amino acid sequence of the engineered MERS spike protein comprising the one or more polypeptides which drive eVLP production, as described herein, are encoded by nucleic acid molecules, such as mRNA. Delivery of the nucleic acid molecule to a cell enables eVLP formation and extrusion from the cell, which is further described in detail below.Nucleic Acid Molecules, Constructs and Vectors

[0148] Also disclosed herein are nucleotide sequences (nucleic acids) that encode one or more of the amino acid sequences disclosed herein, e.g., one or more of the engineered MERS proteins, or fusion proteins. In some embodiments, the nucleic acids may encode any one of the amino acid sequences of SEQ ID NOs: 3-193 or any one of the amino acid sequences of SEQ ID NOs: 195- 385, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3- 193 or 195-385. In some embodiments, the nucleic acid may comprise a nucleotide sequence that encodes an amino acid sequence of a fusion protein of the disclosure, or an immunogenic engineered MERS spike protein of the disclosure, including an amino acid sequence of any one of SEQ ID NOs: 1-385 or any one of the amino acid sequences of SEQ ID NOs: 195-385, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-385. In some embodiments, the nucleic acid is or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises mRNA. The nucleoside sequence in an mRNA molecule provides instructions that cells use to create specific proteins. mRNA is a molecule that typically is composed of four different nucleosides: adenosine, guanosine, cytidine, and uridine. Vaccines containing mRNA can trigger the body’s own immunesystem to attack the mRNA molecule. This immune response may destroy the mRNA before it can have its intended effect. Different modifications to the mRNA molecule have been developed to disguise the mRNA from the body’s immune system, such as those described in U.S. Patent No. 10,898,574, U.S. Patent No. 10,703,789, U.S. Patent No. 10,577,403, and U.S. Patent No. 10, 064,959, the contents of which are herein incorporated by reference in their entirety. In some embodiments, where the nucleic acids of the present disclosure comprise mRNA, the mRNA comprises one or more modified nucleotides selected from the group consisting of: pseudouridine, N-l-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3- methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5 -bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5- propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,0(6)-methylguanine, and 2-thiocytidine. In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments, the modified nucleoside is selected from pseudouridine (y), N 1 -methyl - pseudouridine (m ly), and 5-methyl-uridine(m5U). In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments where the nucleic acids comprise mRNA, the mRNA may be formulated in a lipid nanoparticle (LNP). In some embodiments, the mRNA may be complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, optionally encapsulating mRNA.

[0149] In some embodiments, where the nucleic acid of the present disclosure is mRNA, the mRNA molecule can be synthesized through in vitro transcription of a corresponding DNA template molecule. For example, synthetic mRNA can be produced by in vitro transcription of a cDNA template, such as plasmid DNA (pDNA).

[0150] In some embodiments, wherein the nucleic acid of the present disclosure is mRNA, the mRNA molecule is capable of inducing eVLP formation upon delivery to a cell.

[0151] In some embodiments, the nucleic acids according to the embodiments of the present invention can be included in an expression cassette for expression of a fusion protein encoded by the nucleic acid in a host cell or an organism of interest. In some embodiments, a nucleic acid according to the embodiments of the disclosure can be codon-optimized for expression in a host cell or an organism of interest.

[0152] Also included among the embodiments of the present disclosure are vectors including nucleic acids or nucleic acid constructs according to the embodiments of the present invention. In some embodiments, the vectors may include nucleic acids that encode the engineered MERS spike proteins or the fusion proteins of the disclosure. Such vectors can include necessary functional elements that direct and regulate transcription of the nucleic acid sequences included in the vector. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers that can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region that may serve to facilitate the expression of the inserted gene or hybrid. The vector, for example, can be a plasmid.

[0153] A vector according to the embodiments of the present invention can be a bacterial vector, such as a bacterial expression vector. For example, a vector based on one of numerous E. coli expression vectors can be useful for the expression of a nucleic acid according to the embodiments of the present invention. Other bacterial hosts suitable for expression of nucleic acids according to the embodiments of the present invention include bacilli, such as Bacillus siiblilis. and other enterobacteriaceae, such as Salmonella, Senalia, and various Pseudomonas species. In these prokaryotic hosts, one can also use suitable expression vectors, which will typically contain expression control sequences compatible with the host cell (such as an origin of replication). Any number of a variety of well-known promoters can be used in bacterial expression vectors, such as a lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.

[0154] Expression vectors according to the embodiments of the present invention can also include nucleic acids described in the present disclosure under the control of an inducible promoter such as the tetracycline inducible promoter or a glucocorticoid inducible promoter. The nucleic acids of the present invention can also be under the control of a tissue-specific promoter to promote expression of the nucleic acid in specific cells, tissues or organs. Any regulatable promoter, such as a metallothionein promoter, a heat-shock promoter, and other regulatable promoters are also contemplated. Furthermore, a Cre-loxP inducible system can also be used, as well as a Flp recombinase inducible promoter system.

[0155] In some embodiments, nucleic acids (e.g., nucleotide sequences) encoding an engineered MERS spike protein or a fusion protein according to the embodiments of the present invention may be delivered into a host cell or host organism using a non-viral vector. Examples of non-viral vectors include, but are not limited to, a plasmid, a transposable element, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof.

[0156] In some embodiments, nucleic acids (e.g., nucleotide sequences) encoding an engineered MERS spike protein or a fusion protein according to the embodiments of the present invention may be incorporated into a viral vector for delivery into a host cell or host organism. Accordingly, the vectors according to the embodiments of the present invention include viral vectors that transport the nucleic acids encoding engineered MERS spike proteins or fusion proteins described in the present disclosure into cells without degradation and include a promoter yielding expression of the nucleic acids in the cells into which it is delivered. Suitable viral vectors include adenovirus vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, poxviral vectors, or lentiviral vectors.

[0157] Vectors of the present disclosure may be formulated in a lipid nanoparticle (LNP). Generally, LNP has four components: ionizable cationic lipids, phospholipids, cholesterol, and PEG lipids. Each component contributes to LNP stability, transfection efficacy, and safety. In some embodiments, the vectors may be complexed or associated with one or more lipids or lipid- based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, optionally encapsulating the vector and / or nucleic acids. In some embodiments, the vector is an LNP comprising mRNA encoding an engineered MERS spike protein or a fusion protein described herein.

[0158] Also provided in this disclosure and included among the embodiments of the present invention are cells comprising nucleic acids, a nucleic acid construct, or vectors according to the embodiments of the present invention. Such cells can be referred to as "host cells" (or "host cell," in singular). Some host cells can produce engineered MERS spike proteins or fusion proteins described in the present disclosure, while other host cells may be used for producing or maintaining nucleic acids, DNA constructs, or vectors according to the embodiments of the present invention. A host cell can be an in vitro, ex vivo, or in vivo host cell. Populations of any of the host cells and cell cultures comprising one or more host cells are also included among the embodiments of the present invention. The host cell can be a prokaryotic cell, including, for example, a bacterial cell. Alternatively, the cell can be a eukaryotic cell. Examples of prokaryotic host cells are cellsof E. coli, Pseudomonas, Bacillus or Streptomyces. Examples of eukaryotic cells are yeast cells (such as cells of Saccharomyces yeast, or methylotrophic yeast such as Pichia, Candida, Hansenula, and Torulopsis) animal cells, such as CHO, RI.1, B-W and LM cells, African Green Monkey kidney cells (for example, COS 1, COS 7, BSCI, BSC40, and BMTIO), insect cells (for example, Sf9), human cells (such as human embryonic kidney cells, for instance, HEK293, or HeLa cells).

[0159] Methods of producing or generating host cells (meaning cells comprising nucleic acids, a nucleic acid construct, or a vector according to the embodiments of the present invention) are also included among the embodiments of the present invention. Nucleic acids, a nucleic acid construct, or a vector according to the embodiments of the present invention can be transferred or introduced into the host cell by well-known methods, which vary depending on the type of the host cell. The "introducing" and the related terms or phrases used in the context of introducing nucleic acids, a nucleic acid construct, or a vector into a cell refers to the translocation of the nucleic acid sequence from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acids from outside the cell to inside the nucleus of a eukaryotic cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, nanoparticle delivery, viral delivery, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, DEAE dextran, lipofectamine, calcium phosphate or any method now known or identified in the future for introduction of nucleic acids into prokaryotic or eukaryotic cellular hosts. A targeted nuclease system (e.g., an RNA-guided nuclease (CRISPR-Cas9), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a megaT AL (MT) can also be used to introduce a nucleic acid into a cell.

[0160] Methods of producing or generating engineered MERS spike proteins, nucleic acids encoding the engineered MERS spike proteins fusion proteins and nanoparticles, and / or nucleic acids that encode the fusion proteins described in the present disclosure are also included among the embodiments of the present invention. An exemplary method of producing the engineered MERS spike proteins, nucleic acids encoding the engineered MERS spike proteins, fusion proteins, or nanoparticles can include a step of introducing into a cell a nucleic acid according to an embodiment of the present invention, a nucleic acid construct according to an embodiment of the present invention, or a vector according to an embodiment of the present invention. The introducing step is carried out as described elsewhere in the present disclosure, and, as an outcomeof such step, a cell (which can be referred to as "a host cell") comprising the nucleic acids, the nucleic acid construct or the vector is generated. An exemplary method of producing the engineered MERS spike proteins, nucleic acids encoding the engineered MERS spike proteins, the fusion proteins and / or nucleic acids that encode the fusion proteins can include a step of incubating the host cell under conditions allowing for expression of a fusion protein described herein.

[0161] An exemplary method of producing the nanoparticle can include a step of incubating the host cell under conditions allowing for expression of a fusion protein and self-assembly of the nanoparticle. After expression in the host cell, a fusion protein or nanoparticles can be isolated or purified using various purification methods. In some embodiments, the fusion protein can be isolated from the host cell and allowed to self-assemble into nanoparticles in vitro.

[0162] In one example illustrating a process of producing or generating engineered MERS spike proteins, nucleic acids encoding the engineered MERS spike proteins, fusion proteins, and nanoparticles, and / or nucleic acids that encode fusion proteins described in the present disclosure, nucleic acids or a nucleic acid construct encoding an engineered MERS spike protein, nucleic acids encoding the engineered MERS spike protein, or a fusion protein according to an embodiment of the present invention is introduced into a plasmid or other vector, which is then used to transform living cells. For instance, nucleic acids encoding an engineered MERS spike protein or a fusion protein according to an embodiment of the present invention is inserted in a correct orientation into an expression vector that provides the necessary regulatory regions, such as promoters, enhancers, poly A sites and other sequences. In some cases, it may be desirable to express the engineered MERS spike protein or fusion protein under the control of an inducible or tissue-specific promoter. The expression vector may then be transfected into living cells using various methods, such as lipofection or electroporation, thus generating host cells expressing the fusion protein. The cells comprising the engineered MERS spike protein, nucleic acids encoding the engineered MERS spike protein, the fusion protein or nucleic acids encoding the fusion protein may be selected by appropriate antibiotic selection or other methods and cultured. Larger amounts of the engineered MERS spike protein, nucleic acids encoding the engineered MERS spike protein, the fusion protein, or nucleic acids that encode the fusion protein may be produced by growing the cells in commercially available bioreactors. Once expressed by the host cells, the engineered MERS spike protein, the nucleic acids encoding the engineered MERS spike protein, the fusion protein, or nucleic acids that encode the fusion protein may be isolated (purified)according to standard procedures, such as dialysis, filtration and chromatography. A step of lysing the cells to isolate the engineered MERS spike protein, the nucleic acids encoding the engineered MERS spike protein, the fusion protein, or nucleic acids that encode the fusion protein can be included. Thus, a method of producing or generating engineered MERS spike proteins, nucleic acids encoding the engineered MERS spike proteins, fusion proteins or nucleic acids that encode the fusion proteins according to an embodiment of the present invention may contain one or more steps of culturing a cell comprising a vector under conditions permitting expression of the engineered MERS spike protein, nucleic acids encoding the engineered MERS spike protein, the fusion protein, or nucleic acids that encode the fusion protein, harvesting the cells and / or harvesting the medium from the cultured cells, and isolating the engineered MERS spike protein, nucleic acids encoding the engineered MERS spike proteins, the fusion protein, or nucleic acids that encode the fusion protein from the cells and / or the culture medium. Compositions, methods and kits related to the production of engineered MERS spike proteins, nucleic acids encoding the engineered MERS spike proteins, fusion proteins, or nucleic acids encoding the fusion proteins described in the present disclosure are included within the scope of the embodiments of the present invention.

[0163] In some embodiments, the nucleic acids of the disclosure may comprise a nucleotide sequence that encodes one or more of the amino acid sequences of the MERS spike proteins of the disclosure, including one or more of amino acid sequences of the engineered MERS spike proteins of the disclosure. In some embodiments, the nucleic acids of the nucleotide sequences may comprise DNA or RNA, where RNA may further comprise mRNA. In some embodiments, the nucleic acids of the disclosure may be delivered to a subject using any of the methods or means as described above or below.Vaccine Compositions

[0164] In some embodiments, the engineered MERS spike proteins, the nucleic acids encoding the engineered MERS spike proteins of the disclosure, the fusion proteins, the nucleic acids encoding the fusion proteins of the disclosure, or the vectors described herein may be used in one or more vaccine compositions.

[0165] In some embodiments, the vaccine compositions may be delivered to a subject. For example, in some embodiments, the nucleic acids encoding the engineered MERS spike proteins of the disclosure may be delivered to a subject, e.g., may be delivered as a mRNA-based vaccine.In some embodiments, the engineered MERS spike proteins of the disclosure may be delivered to the subject. In some embodiments, the fusion proteins or nucleic acids encoding the fusion proteins of the disclosure of the disclosure may be delivered to the subject.

[0166] Protein-based vaccine compositions of the disclosure including those comprising engineered MERS spike proteins or fusion proteins comprising the engineered MERS spike proteins may comprise one or more adjuvants. In some embodiments, the adjuvant may include one or more of: aluminum hydroxide (“alum”), one or more synthetic TLR agonists including agonists for TLR-7 / 8 / 9, one or more synthetic forms of DNA, saponins, oil in water emulsions, or the like. In some embodiments, the adjuvant may include Quil-A® + monophosphoryl-Lipid A (MPLA). In some embodiments, the adjuvant may include AddaVax™. In some embodiments, the aluminum hydroxide may include aluminum hydroxide wet gel suspension. In some embodiments, the one or more adjuvants may include aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, or aluminum potassium sulfate. In some embodiments, the one or more synthetic TLR-7 / 8 / 9 agonists may include imidozoquinolines including 3M-052 or cytosine phosphoguanine (CpG). In some embodiments, the synthetic form of DNA may include cytosine phosphoguanine (CpG), or the like. In some embodiments, the one or more adjuvants may include monophosphoryl lipid A and one or more of: QS-21 or aluminum salt. In some embodiments, two or more adjuvants may be used. For example, in some embodiments, alum and CpG in combination may be used as adjuvants. In some embodiments, the adjuvant may include Quil-A® + MPLA+Alum. In some embodiments, the adjuvant may include 3M-052+Alum.

[0167] In some embodiments, the one of more adjuvants may comprise a specific concentration within the composition. For example, in some embodiments, when the adjuvant is aluminum hydroxide, the composition may include about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1000 pg of aluminum hydroxide per dose. In some embodiments, where the adjuvant is CpG, the composition may include about 1000 pg, about 1250 pg, about 1500 pg, about 1750 pg, about 2000 pg, about 2250 pg, about 2500 pg, about 2750 pg, about 3000 pg, about 3250 pg, or about 3500 pg of CpG per dose.

[0168] In some embodiments, the vaccine compositions of the disclosure may further include one or more of: one or more buffer agents, an osmotic agent, and one or more stabilizers. In some embodiments, the one or more buffer agents may include Tris-HCl, Trizma Base, phosphatebuffered saline, or the like. In some embodiments, the osmotic agent may include NaCl or the like. In some embodiments, the stabilizer may include sucrose, lactose, glycine, glutamic acid, potassium, sodium, human serum albumin, gelatin, or the like.

[0169] An “effective amount” of the vaccine compositions of the disclosure is immunogenic and may prevent a MERS infection or a disease associated with a MERS infection in a subject when that amount is administered to the subject, either in a single dose or as part of a series of doses. Dosage administration may be a single dose schedule or a multiple dose schedule (e.g. including booster doses). The vaccine compositions may be generated within a range of unit doses. For example, in some embodiments, the vaccine composition may have a unit dose of about 0.010 pg to about 400 pg. In some embodiments, the vaccine composition may have a unit dose of about 0.010 pg, about 0.012 pg, about 0.014 pg, about 0.016 pg, about 0.018 pg, about 0.020 pg, about 0.022 pg, about 0.024 pg, about 0.026 pg, about 0.028 pg, about 0.030 pg, about 0.032 pg, 0.034 pg, about 0.036 pg, about 0.038 pg, about 0.040 pg, about 0.042 pg, about 0.044 pg, about 0.046 pg, about 0.048 pg, about 0.050 pg, about 0.052 pg, about 0.054 pg, about 0.056 pg, about 0.058 pg, about 0.060 pg, about 0.062 pg, about 0.064 pg, about 0.066 pg, about 0.068 pg, about 0.070 pg, about 0.072 pg, about 0.074 pg, about 0.076 pg, about 0.078 pg, about 0.080 pg, about 0.082 pg, about 0.084 pg, about 0.086 pg, about 0.088 pg, about 0.090 pg, about 0.092 pg, about 0.094 pg, about 0.096 pg, about 0.098 pg, or about 0.1 pg. In some embodiments, the vaccine composition may have a unit dose of about 0.1 pg, about 0.12 pg, about 0.14 pg, about 0.16 pg, about 0.18 pg, about 0.2 pg, about 0.22 pg, about 0.24 pg, about 0.26 pg, about 0.28 pg, about 0.30 pg, about 0.32 pg, about 0.34 pg, about 0.36 pg, about 0.38 pg, about 0.40 pg, about 0.42 pg, about 0.44 pg, about 0.46 pg, about 0.48 pg, about 0.50 pg, about 0.52 pg, about 0.54 pg, about 0.56 pg, about 0.58 pg, about 0.60 pg, about 0.62 pg, about 0.64 pg, about 0.66 pg, about 0.68 pg, about 0.7 pg, about 0.72 pg, about 0.74 pg, about 0.76 pg, about 0.78 pg, about 0.8 pg, about 0.82 pg, about 0.84 pg, about 0.86 pg, about 0.88 pg, about 0.9 pg, about 0.92 pg, about 0.94 pg, about 0.96 pg, about 0.98 pg, or about 1.0 pg. In some embodiments, the composition may have a unit dose of about 1.0 pg, about 1.2 pg, about 1.4 pg, about 1.6 pg, about 1.8 pg, about 2.0 pg, about 2.2 pg, about 2.4 pg, about 2.6 pg, about 2.8 pg, about 3.0 pg, about 3.2 pg, about 3.4 pg, about 3.6 pg, about 3.8 pg, about 4.0 pg, about 4.2 pg, about 4.4 pg, about 4.6 pg, about 4.8 pg, about 5.0 pg, about 5.2 pg, about 5.4 pg, about 5.6 pg, about 5.8 pg, about 6.0 pg, about 6.2 pg, about 6.4 pg, about 6.6 pg, about 6.8 pg, about 7.0 pg, about 7.2 pg, about 7.4 pg, about 7.6 pg, about 7.8 pg, about 8.0 pg, about 8.2 pg, about 8.4 pg, about 8.6 pg, about 8.8 pg,about 9.0 pg, about 9.2 pg, about 9.4 pg, about 9.6 pg, about 9.8 pg, or about 10.0 pg. In some embodiments, the composition may have a unit dose of about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, about 100 pg, about 110 pg, about 120 pg, about 130 pg, about 140 pg, about 150 pg, about 160 pg, about 170 pg, about 180 pg, about 190 pg, about 200 pg, about 210 pg, about 220 pg, about 230 pg, about 240 pg, about 250 pg, about 260 pg, about 270 pg, about 280 pg, about 290 pg, about 300 pg, about 310 pg, about 320 pg, about 330 pg, about 340 pg, about 350 pg, about 360 pg, about 370 pg, about 380 pg, about 390 pg, or about 400 pg.Methods of Use of Vaccine Compositions

[0170] In some embodiments, the vaccine compositions of the disclosure may be administered to a subject. For example, in some embodiments, the vaccine compositions may be used in a method of preventing a MERS infection or a disease associated with a MERS infection in a subject, wherein the method includes administering to the subject an effect amount of a vaccine composition. A subject may include a mammal including but not limited to a human, or a nonhuman primate. In some embodiments, the subject may include a human subject. In some embodiments, the human subject is at least about 1 month old. In some embodiments, the human subject may include a human that is at least about 2 years old. In some embodiments, the human subject may include a human that is at least about 13 years old. In some embodiments, the subject may be at least about 18 years old. For example, in some embodiments, the human subject may be within the ages of about 18 years old to about 55 years old or older than about 56 years old.

[0171] In some embodiments, the vaccine compositions of the disclosure may be administered to a subject to prevent or treat a MERS infection or a disease associated with a MERS infection in a subject. In some embodiments, the MERS infection may be caused by a betacoronavirus including a betacoronavirus from lineage A, B, C, and D.

[0172] In some embodiments, the betacoronavirus may comprise a MERS virus strain from clade A, clade B, and / or clade C. In some embodiments, the MERS infection by a MERS virus strain from clade A may include a MERS infection by the MERS virus strain EMC. In some embodiments, the MERS infection by a MERS virus strain from clade B may be selected from the group consisting of: Englandl, Hasal4b, Bishal, and KOR / NIH / 002. In some embodiments, the MERS infection by a MERS virus strain from clade C may include a MERS virus strain fromCl.l, Cl.2, C2, or C3 from clade C. In some embodiments, the MERS infection by a MERS virus strain from clade C may be selected from the group consisting of: Nigl657, NRCE-HKU270, Cl 272, and NRCE-HKU205. In some embodiments, the MERS infection by a MERS virus strain may include a MERS infection from a MERS virus strain related to the MERS virus strains from clade A, B and / or C. For example, in some embodiments, the MERS infection by a MERS virus strain may include a MERS infection by a merbecovirus strain. In some embodiments, the merbecovirus strain may include hedgehog coronavirus, pipistrellus bat coronavirus HKU5, Tylonycteris bat coronavirus HKU4, NeoCoV, or the like. In some embodiments, the merbecovirus strain may include a pangolin merbecovirus strain including MjHKU4r-CoV.

[0173] In some embodiments, the human subject may be a healthy individual based on subject- reported medical history, physical examination, vital signs, and laboratory assessment. In some embodiments, the subject may have previously received at least one dose of a coronavirus vaccine, for example a SARS-CoV-2 mRNA vaccine, a SARS-CoV-2 protein vaccine, or a SARS-CoV-2 viral vector vaccine. In some embodiments, the subject may not have received at least one dose of a previous vaccine such as a SARS-CoV-2 mRNA vaccine, a SARS-CoV-2 protein vaccine, or a SARS-CoV-2 viral vaccine. In some embodiments, the subject may previously have been exposed to MERS, while in other embodiments, the subject may have not previously been exposed to MERS. In some embodiments, the subject may have been exposed to or been infected with other coronaviruses. For example, in some embodiments, the subject may have previously been exposed to SARS-CoV-2. In some embodiments, the subject may have had one or more previously SARS-CoV-2 infections. In some embodiments, the subject may not have been previously exposed to SARS-CoV-2. In some embodiments, the subject may have one or more pre-existing conditions or one or more additional viral infections. For example, in some embodiments, the one or more additional viral infections may include but are not limited to HIV, HSV, HB V, HAV, HCV, HPV, or the like.

[0174] As contemplated herein, a subject (e.g., a human subject) may receive at least one dose of the vaccine composition. The dose can range from about 0.010 pg to about 400 pg of the vaccine composition per dose. In some embodiments, the dose may be about 0.010 pg, about 0.012 pg, about 0.014 pg, about 0.016 pg, about 0.018 pg, about 0.020 pg, about 0.022 pg, about 0.024 pg, about 0.026 pg, about 0.028 pg, about 0.030 pg, about 0.032 pg, 0.034 pg, about 0.036 pg, about 0.038 pg, about 0.040 pg, about 0.042 pg, about 0.044 pg, about 0.046 pg, about 0.048 pg, about 0.050 pg, about 0.052 pg, about 0.054 pg, about 0.056 pg, about 0.058 pg, about 0.060pg, about 0.062 pg, about 0.064 pg, about 0.066 pg, about 0.068 pg, about 0.070 pg, about 0.072 pg, about 0.074 pg, about 0.076 pg, about 0.078 pg, about 0.080 pg, about 0.082 pg, about 0.084 pg, about 0.086 pg, about 0.088 pg, about 0.090 pg, about 0.092 pg, about 0.094 pg, about 0.096 pg, about 0.098 pg, or about 0.1 pg. In some embodiments, the dose may be about 0.1 pg, about0.12 pg, about 0.14 pg, about 0.16 pg, about 0.18 pg, about 0.2 pg, about 0.22 pg, about 0.24 pg, about 0.26 pg, about 0.28 pg, about 0.30 pg, about 0.32 pg, about 0.34 pg, about 0.36 pg, about 0.38 pg, about 0.40 pg, about 0.42 pg, about 0.44 pg, about 0.46 pg, about 0.48 pg, about 0.50 jug, about 0.52 jug, about 0.54 jug, about 0.56 jug, about 0.58 jug, about 0.60 jug, about 0.62 jug, about 0.64 jug, about 0.66 jug, about 0.68 jug, about 0.7 jug, about 0.72 jug, about 0.74 jug, about 0.76 jig, about 0.78 jug, about 0.8 jug, about 0.82 jug, about 0.84 jug, about 0.86 jug, about 0.88 jug, about 0.9 jig, about 0.92 jug, about 0.94 jug, about 0.96 jug, about 0.98 jug, or about 1.0 jug. In some embodiments, the dose may be about 1.0 jug, about 1.2 jug, about 1.4 jug, about 1.6 jug, about1.8 jig, about 2.0 jug, about 2.2 jug, about 2.4 jug, about 2.6 jug, about 2.8 jug, about 3.0 jug, about 3.2 jig, about 3.4 jug, about 3.6 jug, about 3.8 jug, about 4.0 jug, about 4.2 jug, about 4.4 jug, about 4.6 jig, about 4.8 jug, about 5.0 jug, about 5.2 jug, about 5.4 jug, about 5.6 jug, about 5.8 jug, about 6.0 jig, about 6.2 jug, about 6.4 jug, about 6.6 jug, about 6.8 jug, about 7.0 jug, about 7.2 jug, about 7.4 jig, about 7.6 jug, about 7.8 jug, about 8.0 jug, about 8.2 jug, about 8.4 jug, about 8.6 jug, about8.8 jig, about 9.0 jug, about 9.2 jug, about 9.4 jug, about 9.6 jug, about 9.8 jug, or about 10.0 jug. In some embodiments, the dose may be about 10 jug, about 15 jug, about 20 jug, about 25 jug about 30 jig, about 35 jug, about 40 jug, about 45 jug, about 50 jug, about 55 jug, about 60 jug, about 65 jig, about 70 jig, about 75 jug, about 80 jug, about 85 jug, about 90 jug, about 95 jug, about 100 jug, about 105 jig, about 110 jug, about 115 jug, about 120 jug, about 125 jug, about 130 jug, about 135 jig, about 140 jig, about 145 jug, about 150 jug, about 155 jug, about 160 jug, about 165 jug, about 170 jig, about 175 jug, about 180 jug, about 185 jug, about 190 jug, or about200 jig ofthe engineered MERS spike protein, the nucleic acids encoding the engineered MERS spike protein, the fusion protein comprising the engineered MERS spike protein, or the nucleic acids that encode the fusion protein. In some embodiments, the vaccine composition may be delivered in multiple doses, e.g., at least two, three, four, or more doses, separated by time. In some embodiments, the each of the doses may be an engineered MERS spike protein, nucleic acids that encode the engineered MERS spike protein, a fusion protein, or nucleic acids that encode the fusion protein. In some embodiments, each of the doses may be different. For example, in some embodiments, the first dose may comprise an engineered MERS spike protein, and the second dose may comprise nucleicacids that encode the engineered MERS spike protein. In some embodiments, the first dose may comprise a fusion protein, the second dose may comprise nucleic acids that encode the fusion protein, the third dose may comprise an engineered MERS spike protein, and the fourth dose may comprise nucleic acids that encode the engineered MERS spike protein.Methods of Vaccinating a Subject against an Infection by a MERS Virus

[0175] Also disclosed herein are methods of vaccinating a subject against infection by a MERS virus, or a method of preventing a MERS infection including a disease associated with a MERS infection in a subject. As disclosed above, the methods of vaccinating a subject against infection by a MERS virus or a method of preventing a MERS infection including a disease associated with a MERS infection in a subject includes vaccinating a subject against infection by a MERS virus or preventing a MERS infection including a disease associated with a MERS infection in a subject by a MERS virus from clade A, B, C, or a related MERS virus including a merbecovirus. In some embodiments, the method comprises administering to the subject at least a first dose of a vaccine composition of the disclosure. In some embodiments, administering to the subject a first dose of the vaccine composition may include the vaccine composition comprising: an engineered MERS spike protein, nucleic acids encoding the engineered MERS spike protein, a fusion protein, or nucleic acids encoding the fusion protein, wherein the engineered MERS spike protein, nucleic acids encoding the engineered MERS spike protein, the fusion protein, or nucleic acids that encode the fusion protein comprises: a MERS spike protein comprising three or more proline stabilizing substitutions and a C- terminal truncation of an ectodomain of the spike protein.

[0176] In some embodiments, the engineered MERS spike protein or nucleic acids encoding the engineered MERS spike protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-193, or an amino acid sequence comprising at least 70% sequence identity thereto. In some embodiments, the engineered MERS spike protein may comprise the amino acid sequence of SEQ ID NO: 66, 93, 130 or 157. In some embodiments, the nucleic acids may encode the engineered MERS spike protein of SEQ ID NO: 66 or 130.

[0177] In some embodiments, wherein the vaccine composition comprises a fusion protein, administering to the subject a first dose of the vaccine composition may include the vaccine composition further comprising: one or more subunits of one or more ferritin nanoparticles; andone or more adjuvants.

[0178] In some embodiments, the fusion protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-385, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 195-385. In some embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 285 or 349, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 285 or 349.

[0179] In some embodiments, administering to the subject the first dose of the vaccine composition may include administering the first dose of the vaccine composition to the subject using any route of administration, including but not limited to mucosal, intradermal, intravenous, intramuscular, and subcutaneous administration.

[0180] A variety of routes of administration are contemplated. In some embodiments, the vaccine composition may be administered to a subject including but not limited to mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery and / or by any other known methods of physical delivery. In some embodiments, for intramuscular, subcutaneous or intradermal delivery, a delivery apparatus may be used. In some embodiments, the delivery apparatus may include a single use delivery apparatus or a multi-use delivery apparatus. In some embodiments, the delivery apparatus may include a syringe with an appropriately sized needle for the route of administration.

[0181] In some embodiments, the method of vaccinating a subject against infection by a MERS- Cov virus includes administering to the subject one or more additional doses of a vaccine composition. In some embodiments, administering to the subject an additional dose of a vaccine composition may include administering to the subject at least a second dose of a vaccine composition. In some embodiments, administering to the subject an additional dose of a vaccine composition may include administering to the subject a second dose of the vaccine composition described herein. For example, in some embodiments, the vaccine composition of the second dose may comprise an engineered MERS spike protein, or nucleic acids that encode an engineeredMERS spike protein, wherein the engineered MERS spike protein may comprise an amino acid sequence of any sequence of SEQ ID NOs: 3-193, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3-193. In some embodiments, the vaccine composition of the second dose may comprise a fusion protein or nucleic acids that encode the fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 195-385 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 195-385. In some embodiments, the vaccine composition comprises a fusion protein comprising the amino acid sequence of SEQ ID NO: 285 or 349 or nucleic acids encoding the same.

[0182] In some embodiments, the second dose or additional doses may be administered to the subject using the same route of administration as the first dose administered to the subject or using a different route of administration as the first dose administered to the subject. For example, in some embodiments, the first dose may be administered to the subject intramuscularly and the second dose may be administered to the subject intravenously.ENUMERATED EMBODIMENTS

[0183] Provided herein are non-limiting exemplary enumerated embodiments.

[0184] Embodiment 1-1. An engineered Middle East Respiratory Syndrome (MERS) spike protein comprising at least 70% sequence identity to SEQ ID NO: 1 with a C-terminal truncation of at least 25 amino acids.

[0185] Embodiment 1-2. The engineered MERS spike protein of embodiment 1-1, wherein the protein comprises a C-terminal truncation of at least 100 amino acids.

[0186] Embodiment 1-3. The engineered MERS spike protein of embodiment I- 1 or 1-2, wherein the protein comprises the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 70% sequence identity thereto.

[0187] Embodiment 1-4. The engineered MERS spike protein of any one of embodiments 1-1 to 1-3, wherein the protein comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 80% sequence identity thereto.

[0188] Embodiment 1-5. The engineered MERS spike protein of any one of embodiments 1-1 to 1-4, wherein the protein comprises one or more proline substitutions.

[0189] Embodiment 1-6. The engineered MERS spike protein of embodiment 1-5, wherein the one or more proline substitutions comprise one or more of A889P, S966P, A973P, T1014P, V1060P, and L1061P.

[0190] Embodiment 1-7. The engineered MERS spike protein of any one of embodiments 1-1 to 1-6, wherein the spike protein is fused to a nanoparticle, thereby forming a fusion protein comprising the spike protein fused to a nanoparticle.

[0191] Embodiment 1-8. The engineered MERS spike protein of embodiment 1-7, wherein the nanoparticle comprises a ferritin subunit.

[0192] Embodiment 1-9. The engineered MERS spike protein of embodiment 1-8, wherein the fusion protein forms a self-assembling nanoparticle.

[0193] Embodiment I- 10. The engineered MERS spike protein of embodiment 1-8 or 1-9, wherein the ferritin subunit comprises H. pylori ferritin.

[0194] Embodiment 1-11. The engineered MERS spike protein of any one of embodiments 1-8 to 1-10, wherein the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity thereto.

[0195] Embodiment 1-12. The engineered MERS spike protein of any one of embodiments 1-8 to 1-11, wherein the engineered MERS spike protein comprises an amino acid sequence of SEQ ID NO: 285 or SEQ ID NO: 349, or an amino acid sequence having at least 70% sequence identity thereto.

[0196] Embodiment 1-13. An engineered Middle East Respiratory Syndrome (MERS) spike protein comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1, comprising at least three proline substitutions.

[0197] Embodiment 1-14. The engineered MERS spike protein of embodiment 1-13, wherein the at least three proline substitutions comprise at least three of: A889P, S966P, A973P, T1014P, V1060P, or L1061P.

[0198] Embodiment 1-15. The engineered MERS spike protein of embodiment 1-13 or 1-14, wherein one of the at least three proline substitutions is selected from the group consisting of: A889P, V1060P, and L1061P.

[0199] Embodiment 1-16. The engineered MERS spike protein of any one of embodiments I- 13 to 1-15, wherein one of the at least three proline substitutions is V1060P.

[0200] Embodiment 1-17. The engineered MERS spike protein of any one of embodiments I- 13 to 1-16, wherein the spike protein consists of three proline substitutions.

[0201] Embodiment 1-18. The engineered MERS spike protein of embodiment 1-17, wherein the three proline substitutions are A889P, V1060P, and LI 06 IP.

[0202] Embodiment 1-19. The engineered MERS spike protein of embodiment 1-13 or 1-14, wherein the at least three proline substitutions comprise A889P, V1060P, and L1061P.

[0203] Embodiment 1-20. The engineered MERS spike protein of any one of embodiments I- 13 to 1-19, wherein the protein has a C terminal truncation.

[0204] Embodiment 1-21. The engineered MERS spike protein of embodiment 1-20, wherein the amino acid sequence of the spike protein comprises SEQ ID NO: 157 or SEQ ID NO: 93, or an amino acid sequence having at least 70% sequence identity thereto.

[0205] Embodiment 1-22. The engineered MERS spike protein of any one of embodiments I- 13 to 1-21, wherein the protein is fused to a binding partner, thereby forming a fusion protein.

[0206] Embodiment 1-23. The engineered MERS spike protein of embodiment 1-22, wherein the binding partner is a ferritin subunit.

[0207] Embodiment 1-24. The engineered MERS spike protein of embodiment 1-22 or 1-23, wherein the fusion protein forms a nanoparticle.

[0208] Embodiment 1-25. The engineered MERS spike protein of any one of embodiments 1-1 to 1-24, further comprising at least one insertion.

[0209] Embodiment 1-26. The engineered MERS spike protein of embodiment 1-25, wherein the insertion is at the C-terminal or N-terminal of the spike protein, or internal to the protein.

[0210] Embodiment 1-27. The engineered MERS protein of embodiment 1-25 or 1-26, wherein the insertion enables transit and extrusion of the spike protein in extracellular vesicles and / or promotes enveloped virus like particle (eVLP) production.

[0211] Embodiment 1-28. The engineered MERS spike protein of embodiment 1-27, wherein the at least one amino acid insertion comprises insertion of an ESCRT-recruiting domain (ERD).

[0212] Embodiment 1-29. The engineered MERS spike protein of embodiment 1-28, wherein the amino acid sequence of the ERD comprises the sequence of any one of SEQ ID NOs: 421- 432, or an amino acid sequence having at least 70% sequence identity thereto.

[0213] Embodiment 1-30. The engineered MERS spike protein of embodiment 1-27, wherein the at least one amino acid insertion comprises insertion of a polypeptide that is ESCRT- independent.

[0214] Embodiment 1-31. A fusion protein comprising an engineered Middle East Respiratory Syndrome (MERS) spike protein and a binding partner.

[0215] Embodiment 1-32. The fusion protein of embodiment 1-31, wherein the spike protein comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto.

[0216] Embodiment 1-33. The fusion protein of embodiment 1-31 or 1-32, wherein the amino acid sequence of the spike protein comprises a C terminal truncation.

[0217] Embodiment 1-34. The fusion protein of any one of embodiments 1-31 to 1-33, wherein the spike protein is operably linked to the binding partner through a linker.

[0218] Embodiment 1-35. The fusion protein of embodiment 1-34, wherein the linker is a peptide linker.

[0219] Embodiment 1-36. The fusion protein of embodiment 1-35, wherein the peptide linker is a Gly-Ser linker.

[0220] Embodiment 1-37. The fusion protein of any one of embodiments 1-34 to 1-36, wherein the linker comprises a sequence selected from the group consisting of: SGG, GSG, SGG, GG, GSGG (SEQ ID NO: 418), NGTGGSG (SEQ ID NO: 419), and GGGGS (SEQ ID NO: 420).

[0221] Embodiment 1-38. The fusion protein of any one of embodiments 1-31 to 1-37, wherein the amino acid sequence of the spike protein comprises a sequence of SEQ ID NO: 66 or SEQ ID NO: 130, or an amino acid sequence having at least 70% sequence identity thereto.

[0222] Embodiment 1-39. The fusion protein of any one of embodiments 1-31 to 1-38, wherein the spike protein further comprises one or more proline substitutions.

[0223] Embodiment 1-40. The fusion protein of embodiment 1-39, wherein the one or more proline substitutions are selected from the group consisting of: A889P, S966P, A973P, T1014P, V1060P, and L1061P.

[0224] Embodiment 1-41. The fusion protein of embodiment 1-39 or 1-40, wherein the spike protein comprise three proline substitutions, optionally wherein the protein consists of three proline substitutions.

[0225] Embodiment 1-42. The fusion protein of embodiment 1-41, wherein the three proline substitutions are A889P, V1060P, and LI 06 IP.

[0226] Embodiment 1-43. The fusion protein of any one of embodiments 1-39 to 1-42, wherein the amino acid sequence of the spike protein comprises an amino acid sequence of SEQ ID NO: 93 or SEQ ID NO: 157, or an amino acid sequence having at least 70% sequence identity thereto.

[0227] Embodiment 1-44. The fusion protein of any one of embodiments 1-29 to 1-43, wherein the binding partner comprises a ferritin subunit.

[0228] Embodiment 1-45. The fusion protein of embodiment 1-44, wherein the amino acid sequence of the fusion protein comprises the amino acid sequence of: SEQ ID NO: 285 or SEQ ID NO: 349, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 285 or 349.

[0229] Embodiment 1-46. An eVLP comprising the engineered MERS spike protein of any one of embodiments 1-1 to 1-30 or the fusion protein of any one of embodiments 1-31 to 1-45.

[0230] Embodiment 1-47. A nucleotide sequence that encodes the amino acid sequence of: a. the engineered MERS spike protein of any one of embodiments 1-1 to 1-12, b. the engineered MERS spike protein of any one of embodiments 1-13 to 1-28, or c. the fusion protein of any one of embodiments 1-31 to 1-45.

[0231] Embodiment 1-48. The nucleotide sequence of embodiment 1-47 that encodes the amino acid sequence of: a. the engineered MERS spike protein of any one of embodiments 1-1 to 1-6, b. the engineered MERS spike protein of any one of embodiments 1-13 to 1-21, or c. the engineered MERS spike protein of any one of embodiments 1-25 to 1-30.

[0232] Embodiment 1-49. The nucleotide sequence of embodiment 1-47 or 1-48, wherein nucleic acids of the nucleotide sequence comprise DNA.

[0233] Embodiment 1-50. The nucleotide sequence of embodiment 1-47 or 1-48, wherein nucleic acids of the nucleotide sequence comprise RNA.

[0234] Embodiment 1-51. The nucleotide sequence of embodiment 1-50, wherein the nucleic acids of the nucleotide sequence comprise mRNA.

[0235] Embodiment 1-52. A vaccine composition comprising:a. the engineered MERS spike protein of any one of embodiments 1-1 to 1-12, b. the engineered MERS spike protein of any one of embodiments 1-13 to 1-24, c. The engineered MERS spike protein of any one of embodiments 1-25 to 1-28, d. the fusion protein of any one of embodiments 1-31 to 1-45, e. the eVLP of embodiment 1-46, or f. the nucleotide sequence of any one of embodiments 1-47 to 1-51, and optionally comprising one or more adjuvants.

[0236] Embodiment 1-53. A vaccine composition comprising a nucleotide sequence that encodes the amino acid sequence of: a. the engineered MERS spike protein of any one of embodiments 1-1 to 1-6, b. the engineered MERS spike protein of any one of embodiments 1-13 to 1-21, or c. the engineered MERS spike protein of any one of embodiments 1-25 to 1-30.

[0237] Embodiment 1-54. The vaccine composition of embodiment 1-52 or 1-53, comprising a nucleotide sequence, wherein the nucleic acids of the nucleotide sequence comprise DNA.

[0238] Embodiment 1-55. The vaccine composition of embodiment 1-54, wherein nucleic acids of the nucleotide sequence comprise RNA.

[0239] Embodiment 1-56. The vaccine composition of embodiment 1-55, wherein the nucleic acids of the nucleotide sequence comprise mRNA.

[0240] Embodiment 1-57. A method of preventing a Middle East Respiratory Syndrome (MERS) infection or a disease associated with a MERS infection in a subject, comprising administering to the subject an effective amount of one or more doses of the vaccine composition of any one of embodiments 1-53 to 1-56.

[0241] Embodiment 1-58. The method of embodiment 1-57, wherein the subject is human.

[0242] Embodiment 1-59. The method of embodiment 1-57 or 1-58, wherein the route of administration is intramuscular, intravenous, intradermal, or subcutaneous.

[0243] Embodiment 1-60. The method of any one of embodiments 1-57 to 1-59, comprising preventing a MERS infection or a disease associated with a MERS infection in a subject by a MERS virus strain from MERS clade A, MERS clade B, or MERS clade C.

[0244] Embodiment 1-61. The method of embodiment 1-60, wherein the MERS virus strain from MERS clade A comprises the MERS strain EMC.

[0245] Embodiment 1-62. The method of embodiment 1-60, wherein the MERS virus strain from MERS clade B comprises a MERS strain selected from the group consisting of: Englandl, Hasal4B, Bishal, and KOR / NIH / 002.

[0246] Embodiment 1-63. The method of embodiment 1-60, wherein the MERS virus strain from MERS clade C comprises a MERS virus strain selected from the group consisting of: Nigl657, NRCE-HKU270, C1272, and MRCE-HKU205.

[0247] Embodiment 1-64. The method of any one of embodiments 1-57 to 1-63, comprising preventing a MERS infection or a disease associated with a MERS infection in a subject by a MERS virus strain including a merbecovirus.

[0248] Embodiment 1-65. The method of embodiment 1-64, wherein the merbecovirus comprises a merbecovirus strain of MjHKU4r-CoV.

[0249] Embodiment 1-66. The vaccine composition of any one of embodiments 1-53 to 1-56, for use in a method of preventing a Middle East Respiratory Syndrome (MERS) infection or a disease associated with a MERS infection in a subject; wherein the method comprises administering to the subject an effective amount of one or more doses of the vaccine composition.

[0250] The disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference for all purposes.EXAMPLESExample 1: DNA constructs of MERS-Spike Proteins

[0251] Full-length and C-terminally truncated MERS Spike protein constructs were prepared from a full-length MERS Spike protein construct of the Englandl strain of MERS, the amino acid sequence of which is disclosed herein by SEQ ID NO: 1.

[0252] Full-length and C-terminally truncated MERS spike protein constructs may comprise mutations that eliminate the furin cleavage site seen in the full-length Englandl strain of MERS, residues 748-751, RSVR mutated to ASVG, residue 1020 was mutated from a H to a Q along with one or more proline substitutions that stabilize the Spike trimer in the prefusion conformation.

[0253] As used herein the examples, the MERS-1227 (SEQ ID NO: 93) and MERS-1236 (SEQ ID NO: 157) comprise the MERS amino acid sequence from the Englandl strain, truncated after amino acid 1227 or amino acid 1236, respectively. Both versions contain a mutated furin cleavagesite (residues 748-751, RSVR mutated to ASVG) and three stabilizing proline mutations (A889P, V1060P, L1061P). FIG. 2E depicts a schematic showing the different in the C terminal regions of MERS-1227 and MERS-1236. MERS-1227 comprises a C terminal truncation that is 9 AA longer, resulting in a MERS spike protein that is 9AA shorter than MERS-1236.

[0254] The constructs encoding full-length and C-terminally truncated MERS were transferred into an expression vector and were cloned, followed by stitching PCR in which the constructs were annealed to an amplicon encoding a SGG linker followed by residues 5-168 of a H. pylori ferritin sequence (SEQ ID NO: 2), thus generating the fusion proteins of the disclosure. The sequences were confirmed using Sanger Sequencing. The remainder of the examples utilize these fusions proteins. Although the examples make reference to MERS-1227 and MERS-1236, it is understood these are configured to be fusion proteins comprising a truncated MERS spike protein in fusion with a ferritin nanoparticle.

[0255] The exemplary constructs discussed above are schematically illustrated in FIGS. 2C- 2D, and SEQ ID NOs: 195-385 represent the amino acid sequences encoded by the constructs.Expression Analysis and Purification of MERS antigens

[0256] One or more proline substitutions were incorporated into the full-length and C-terminally truncated MERS spike protein constructs, including up to 6 proline substitutions at the following residues 889, 966, 973, 1014, 1060, and 1061.

[0257] All proteins were expressed in Expi293F cells. Expi293F cells and were cultured using 66% Freestyle / 33% Expi media (ThermoFisher) and grown in TriForest polycarbonate baffled shaking flasks at 37 degrees C in 8% CO2. The cells were transfected at a density of approximately 3-4 x 106cells / mL. Transfection mixtures were made by adding 568 ug maxi-prepped DNA to 113 mL culture media (per liter of transfected cells) followed by addition of 1.48 mL FectoPro (polyplus). The mixtures were incubated at room temperature for 10 minutes and then added to Cells. Cells were immediately boosted with D-glucose (0.04 g / L final concentration) and 2- propylpentanoic (valproic) acid (3 mM final concentration). The cells were harvested 3-5 days post-transfection by spinning the cultures at 7,000 x g for 15 minutes. Supernatants were filtered using a 0.22 um filter.

[0258] MERS-1227 and MERS-1236 supernatants from fed-batch cultures were analyzed for protein expression levels at Day 14 using a denaturing SDS-Page gel. Denaturing SDS-PAGE analysis was performed by diluting proteins with Laemmli sample buffer (with beta-mercaptoethanol for reducing conditions) and boiling samples for 5 min at 95 °C. Samples were then loaded on a 4-20% MiniPROTEAN TGX precast gel and run for 30 min at 230 V. Gels were stained using GelCode Blue staining reagent and imaged using a Thermo Fisher Scientific imager. FIG. 3A depicts an image of the SDS-PAGE gel, demonstrating expression of both MERS-1227 and MERS-1236.

[0259] Following purification, MERS-1227 and MERS-1236 were analyzed using SDS-PAGE analysis under reducing (R) or non-reducing (NR) conditions. 3 pL of supernatant of MERS-1227 or MERS-1236 was loaded onto a denaturing SDS-PAGE gel and protein bands were visualized using GelCode Blue staining reagent, as seen in FIG. 3B.

[0260] Full length and C-terminally truncated (e.g., MERS-1227 and MERS-1236) with one or more proline substitutions (including those of SEQ ID NO: 3-193) were analyzed for protein expression levels at Day 14, using the denaturing SDS-PAGE analysis described above. A SARS- CoV-2 nanoparticle (DCFHP) may be used as a standard (left). 3 pL of MERS-1227 or MERS- 1236 supernatant was loaded onto a denaturing SDS-PAGE gel and protein bands were visualized using GelCode Blue staining reagent. Following purification (bottom), MERS-1227 and MERS- 1236 were analyzed using SDS-PAGE analysis under reducing (R) or non-reducing (NR) conditions. A slight reduction in gel migration is seen for MERS-1236 which is consistent with the extension of 9 amino acids in this protein compared to MERS-1227.

[0261] 3 pL of supernatant of each of MERS-1227 with one or more proline substitutions or MERS-1236 with one or more proline substitutions may be loaded onto a denaturing SDS-PAGE gel and protein bands will visualized using GelCode Blue staining reagent. Following purification, MERS-1227 with one or more proline substitutions and MERS-1236 with one or more proline substitutions will be analyzed using SDS-PAGE analysis under reducing (R) or non-reducing (NR) conditions.Dynamic light scattering analysis of MERS-1227 and MERS-1236

[0262] To confirm the size of the MERS-1227 and MERS-1236 antigens, purified MERS-1227 and MERS-1236 were analyzed using dynamic light scattering on the Prometheus Panta in technical triplicate, as depicted in FIG. 4A. The cumulant radius of MERS-1227 was 19.03 ± 0.08 and the cumulant radius of MERS-1236 was 19.55 ± 0.28 (as seen in FIG. 4A), indicating that the MERS-1236 particles are slightly larger than the MERS-1227 particles, as to be expected from the additional 9 amino acid residues.Differential scanning fluorimetry ofMERS-1227 and MERS-1236

[0263] Differential scanning fluorimetry was used to calculate the melting temperatures of the MERS ferritin nanoparticles (e.g., MERS-1227 and MERS-1236). Samples were subjected to a temperature gradient from 25 °C to 90 °C and intrinsic protein fluorescence at 330 and 350 nm was measured. Plotted is the first derivative of the ratio of 350 / 330 nm, as seen in FIG. 4B. The graph in FIG. 4B demonstrates that both samples (MERS-1227 and MERS-1236) exhibit a single transition representative of the melting temperature. Samples were measured in technical triplicate and Tm values were calculated to be 55.4 ± 0.06 °C and 52.0 ± 0.12 °C for MERS-1227 and MERS-1236. This data suggests that MERS-1236 may exhibit less thermal stability than MERS- 1227.Analytical size-exclusion chromatography (SEC) followed by light scattering analysis of MERS- 1227 and MERS-1236

[0264] To purify the MERS-1227 and MERS-1236 antigens, analytical size-exclusion chromatography was performed. Purified MERS-1227 and MERS-1236 were diluted to 0.5 mg / mL in formulation buffer (20 mM Tris pH 7.5, 150 mM sodium chloride, 5% sucrose) and analyzed on an analytical SRT SEC-1000 size exclusion column followed by a Wyatt light scattering detector. Light scattering curves are shown, indicating homogenous protein nanoparticles an no apparent aggregates or monomer species. This analysis was performed following a freeze-thaw cycle, indicating that the particles remain intact after freezing and do not form aggregates. Molecule weight determinations were calculated based on the light scattering and refractive index readouts from nanoparticle samples. Samples were analyzed in triplicate and measurements represent the average of three MW calculations and error represents the standard deviation.

[0265] Purified MERS-1227 and MERS-1236 were diluted to 0.5 mg / mL in 20 mM Tris, pH 7.5, 150 mM sodium chloride, 5% sucrose. For each run, 10 pg (20 pL) protein was loaded onto a 4.6 x 300 mm SRT SEC-1000 analytical column pre-equilibrated in 20 mM Tris pH 7.5, 150 mM sodium chloride run at 0.35 mL / min on a Waters Arc Premier high-pressure liquid chromatography system. Multi-angle light scattering was measured using a Wyatt Dawn detector. Molecule weight calculations were performed using Wyatt ASTRA software by utilizing the light scattering and refractive index signals from each sample.

[0266] FIG. 5 depicts a graph of the light scattering of purified MERS-1227 or MERS-1236. MERS-3P ferritin proteins assemble into nanoparticles as expected and the DLS results correspond with the size of a ferritin nanoparticle with MERS spike displayed on the surface (~35 nm).Antibody Binding Analysis using Biolayer Interferometry (BLI) to MERS-1227 and MERS- 1236

[0267] Biolayer interferometry analysis of MERS-1227 and MERS-1236 was performed using an Octet R8 instrument with Protein A functionalized tips. Antibodies and MERS nanoparticles were diluted to 10 pg / mL in 10X Kinetics buffer (PBS + 0.1% BSA, 0.02% Tween20 and Kathon) and pipetted into black-walled, black-bottom plates (200 pL per well). Protein A tips were dipped into antibody wells and subsequently dipped into nanoparticle wells to assess binding association for 120 sec. Dissociation was measured for 120 sec by moving tips into a buffer only well.

[0268] FIGS. 6A-6B depict graphs of the biolayer interferometry analysis of MERS-1227 (FIG. 6A) or MERS-1236 (FIG. 6B). Antibody binding to MERS-1227 and MERS-1236 was assessed using biolayer interferometry in which MERS-specific antibodies targeting diverse epitopes (4C2 RBD, COV2-2449 S2, G2 NTD, and 3A3 S2) were loaded onto Protein A tips and then dipped into wells containing 10 pg / mL MERS-1227 or MERS-1236. A SARS-CoV-2 antibody (Bamlanivimab) was used as a negative control and demonstrates that the MERS particles do not exhibit non-specific binding properties. Binding results demonstrate that MERS-1227 and MERS- 1236 bind MERS conformation-specific antibodies, indicating that particles are displaying a properly folded MERS spike protein on the surface and relevant epitopes are not occluded by nanoparticle display of the antigen.Transmission Electron Microscopy (“TEM”) analysis and 2D class averaging of MERS-1227 and MERS-1236

[0269] For TEM analysis, purified MERS-1227 and MERS-1236 were diluted using 20 mM Tris pH 7.5, 150 mM sodium chloride. A 3 pl drop of diluted sample suspension was applied to an EM grid that had been plasma-cleaned using a Gatan Solarus (Pleasanton, California). After blotting the sample away with filter paper, grids were immediately stained with a 1% uranyl formate solution. After ~30 seconds, the stain was blotted away with filter paper and the grids are allowed to air-dry. Electron microscopy was performed using an FEI Tecnai T12 electronmicroscope (serial number DI 100), operating at 120keV and equipped with an FEI Eagle 4k x 4k CCD camera.

[0270] FIGS. 7A-7B depict exemplary transmission electron microscopy images, wherein FIG. 7A depicts MERS-1227 and FIG. 7B depicts MERS-1236. Purified MERS-1227 and MERS-1236 were analyzed using TEM followed by 2D class averaging to assess the structural features of these nanoparticles and identify differences between them. As seen above, the rigidity of the MERS- 1227 spike is more pronounced as evidence by the more defined density of the spikes on the surface of the particle whereas MERS-1236 exhibits spikes with poorly defined density and some particles where no spikes are visible. Based on other size analysis, the particles exhibit an expected diameter of ~35 nm which corresponds to the size of a ferritin nanoparticle with a MERS spike protein on the surface. These results are consistent with those observed with the MERS-1227 and MERS-1236 nanoparticles in dynamic light scattering (DLS), confirming that the MERS-1227 and MERS-1236 fusion proteins self-assembly into ferritin nanoparticles displaying the MERS spike protein on the surface. Based on the structural data from the nanoparticle core and MERS spikes, these functionalized nanoparticles assume a nanoparticle that is ~35 nm in size, confirming the proper display of the MERS spike on the surface of the ferritin nanoparticle. FIGS. 7C-7D depict exemplary transmission electron microscopy images, wherein FIG. 7C depicts MERS- 1227 and FIG. 7D depicts MERS-1236. FIG. 7D indicates that the spike on the surface of MERS- 1236 is more flexible compared to MERS-1227 as seen in FIG. 7C.Purification of MERS Antigens

[0271] The fusion protein, MERS-1227 and MERS-1236 antigens were purified from supernatants obtained from 14-day fed-batch cultures received from ATUM. Supernatants were filtered through a 0.22-pm filter and diluted with 20 mM Tris pH 8.0, 1 M sodium chloride to a final concentration of 200 mM sodium chloride. Diluted supernatants were then passed over a 5 mL HiTrap Q anion exchange column equilibrated in 20 mM Tris pH 8.0 using an AKTA fast protein liquid chromatograph. MERS ferritin nanoparticles were collected from the column flow- through and then dialyzed overnight at 4 °C using 1 MDa molecular weight cutoff dialysis tubing into 20 mM Tris, pH 7.5, 150 mM sodium chloride. Dialyzed flow-through materials were then concentrated to a final volume of ~ 2 mL using an Amicon spin concentrator (100 kDa molecular weight cutoff). Concentrated samples were then filtered using a 0.22-pm filter and injected onto an S6 size exclusion chromatography column equilibrated in 20 mM Tris pH 7.5, 150 mM sodiumchloride using an AKTA fast protein liquid chromatograph. Protein-containing fractions were assessed using SDS-PAGE analysis and fractions containing homogenous protein were pooled. Fractions were then supplemented with 5% sucrose for freezing (final formulation 5% sucrose, 20 mM Tris pH 7.5, 150 mM sodium chloride), frozen in liquid nitrogen, and stored at -80° C until use.Antigen Preparation for Immunization

[0272] MERS-1227 and MERS-1236 antigens, purified as described above, were diluted to 0.2 mg / mL in formulation buffer (Tris pH 7.5, 150 mM NaCl, 5% sucrose), sterile filtered through an 0.22 pm filter, flash frozen on liquid nitrogen, and stored at -80 C. On the day of immunization, antigens were defrosted and then diluted to 0.118 mg / mL in formulation buffer. Finally, a stock solution of Alhydrogel (aluminum hydroxide) adjuvant was added to a final aluminum concentration of 1.5 mg / mL, bringing the MERS antigen concentration to a final concentration of 0.1 mg / mL. The antigen-adjuvant mixture was inverted 50 times and allowed to rest at room temperature for 30 minutes.Example 2: Delivery of Vaccine Compositions of MERS-CoV Spike Protein fused to Ferritin to Mice

[0273] To investigate the effectiveness of MERS fusion proteins as vaccine compositions, each of the MERS fusion proteins: MERS-1227 (SEQ ID NO: 285) or MERS-1236(SEQ ID NO: 349) was isolated and administered to mice as described below.

[0274] Just prior to injection, antigen-adjuvant mixtures were inverted 50 times. 50 pL of the adjuvanted MERS-1227 and MERS-1236 vaccine candidates were injected intramuscularly into both hind legs of approximately 8-week-old female, vaccine -naive BALB / c mice (100 pL total injection volume, resulting in delivery of 10 pg MERS-1227 or -1236 antigen, and 150 pg of aluminum, as aluminum hydroxide). 21 days after injection, whole blood was collected and centrifuged to isolate serum, which was frozen at -80 C and subsequently thawed, heat inactivated at 56° C for 30 minutes, and used for neutralization assays, described below.

[0275] Approximately 8-week-old female vaccine-naive BALB / c mice were primed with 10 pg of DCFHP-alum (Weidenbacher, P.AB. et al. Nat Commun 14, 2149 (2023)) adjuvanted with 150 pg Alhydrogel (aluminum hydroxide). Forty-nine days later, mice were boosted with 10 pg MERS-1227 or MERS-1236 adjuvanted with 150 pL aluminum hydroxide, prepared and injectedas described above. Alternatively, some groups received a booster consisting of 5 pg DCFHP, 5 pg MERS-1227 or MERS-1236, and 150 pg alum.Neutralization Assays

[0276] To assess elicitation of neutralizing antibodies by MERS-1227 (also referred to as “MERS-002” herein) and MERS-1236, a MERS-pseudotyped lentivirus neutralization assay was utilized. For virus production, HEK293T cells were seeded at a density of 6 million cells in a 10- cm dish. One day following cell seeding, cells were transfected with the following plasmids: 3.4 pg MERS spike plasmid, 2.2 pg of each lentivirus helper plasmid (pHDM-Hgpm2, pHDM-tatlb, and pRC-CMV-revlb), and 10 pg lentivirus packaging vector (pHAGE-CMV-Luc2-IRES- ZsGreen-W) using BioT transfection reagent. The MERS spike plasmid encoded a version of the MERS spike protein with an 18 amino acid C-terminal truncation, which has previously been shown to improve viral titers. 18-24 hours post transfection, the media on transfected cells was removed and replaced with fresh media. Viruses were harvested 72 hours post transfection by spinning at 300 x g for 5 min and filtering through a 0.45-pm PES filter. Viruses were frozen at - 80° C until use.

[0277] Infectivity assays with MERS-pseudotyped lentiviruses were performed in HeLa cells expressing DPP4, the MERS receptor. HeLa / DPP4 cells were plated in white-walled, whitebottom 96-well plates at 5,000 cells per 1 well one day prior to infection. To quantify neutralizing antibody titers in serum from immunized mice, serum samples were diluted in cell-growth media. Serum dilutions were then mixed with MERS-pseudotyped lentivirus and virus / serum dilutions were incubated at 37 °C for 1 hr. Media was aspirated off of HeLa / DPP4 cells and replaced with virus / serum dilutions which were left to incubate on cells for 48 hour at 37 °C. Infectivity in each well was quantified using BriteLite, which reads out luminescence resulting from luciferase expression from lentiviral entry into cells.

[0278] Data was analyzed using GraphPad Prism. Control wells containing virus only and cells only were averaged, and serum dilution infectivity value were normalized to these averages set at 100% infectivity (virus only) and 0% infectivity (cells only). Normalized values were then fit with a 3-parameter non-linear regression (Y=Bottom + (Top-Bottom) / (l+(X / IC50)).

[0279] MERS vaccine candidates were administered to mice. Just prior to injection, antigen - adjuvant mixtures were inverted 50 times. 50 pL of the adjuvanted MERS-1227 and -1236 vaccine candidates were injected intramuscularly into both hind legs of approximately 8-week-old female,vaccine-naive BALB / c mice (100 pL total injection volume, resulting in delivery of 10 pg MERS- 1227 or MERS-1236 antigen, and 150 pg of aluminum, as aluminum hydroxide, “Alhydrogel”). 21 days after injection, whole blood was collected and centrifuged to isolate serum, which was frozen at -80° C and subsequently thawed, heat inactivated at 56° C for 30 min, and used for neutralization assays, described below. FIG. 8A depicts a graph of IC50 values obtained from mice administered either MERS-1227 or MERS-1236. Each point represents the serum IC50 value obtained from an individual mouse. Serum dilutions were run in duplicate or quadruplicate for each animal.

[0280] These results demonstrate that MERS-1227 and MERS-1236 are immunogenic in mice following a single 10 pg dose adjuvanted with 150 pg alum. Neutralizing antibody responses demonstrate that the MERS nanoparticles display the MERS spike protein in a conformation that elicits functional antibodies capable of blocking viral entry into cells in a psuedotyped- neutralization assay. Furthermore, neutralizing antibody titers for both MERS-1227 and MERS- 1236 increase from Day 21 to Day 56 following immunization, indicating that antibody response matures over time in mice following a single dose.Antigen Preparation for Immunization after pH and Temperature Stability Challenge

[0281] To determine the stability of each of the MERS fusion proteins in vivo, the MERS ferritin nanoparticles were subjected to different conditions that would challenge the stability of the fusion proteins. MERS-1227 and MERS-1236 fusion proteins, purified as described above, were diluted to 0.5 mg / mL in Tris pH 7.5, 150 mM NaCl, 5% sucrose to a final volume of 320 pL, and then buffer exchanged by dialysis into either 20 mM histidine pH 6.0, 150 mM NaCl, and 5% sucrose, or Tris pH 7.5, 150 mM NaCl, and 5% sucrose using dialysis membrane with a molecular weight cutoff of 10 kDa. The buffer-exchanged antigens were diluted to 0.118 mg / mL in their respective buffers. A stock solution of Alhydrogel (aluminum hydroxide) adjuvant was added to a final aluminum concentration of 1.5 mg / mL, bringing the MERS antigen concentration to a final concentration of 0.1 mg / mL. The antigen-adjuvant mixture was inverted 50 times, allowed to sit for 30 minutes, and inverted 50 additional times. The adjuvanted vaccine candidates were then placed in a water bath at 37 °C or a refrigerator at 2-8 °C for 10 days.

[0282] Both MERS-1227 and MERS-1236 fusion proteins were subjected to pH conditions of 6.0 or 7.5 and temperature conditions of either 4° C or 37° C and administered to mice in a single dose (10 pg protein, 150 pg Alhydrogel). 21 days after administration, neutralizing titers fromeach mouse were obtained and plotted on the graph depicted in FIG. 8B (for mice administered MERS-1227) or FIG. 8C (for mice administered MERS-1236). Each point on the graph represents the serum IC50 value obtained from an individual mouse. Serum dilutions were run in technical duplicate for each animal.

[0283] These results demonstrate that MERS-1227 and MERS-1236 maintain immunogenicity at a 10 pg dose with 150 pg of alum in mice even when subjected to 4 °C or 37 °C storage conditions for up to 10 days. Immunogenicity was measured as neutralizing antibody titers in a MERS-pseudotyped lentivirus system at 21 days following immunization.

[0284] To simulate the current world where a significant portion of the population has been exposed to SARS-CoV-2, infected with SARS-CoV-2, or vaccinated against SARS-CoV-2, mice were first administered a dose of a SARS-CoV-2 vaccine composition that comprised a SARS- CoV-2 nanoparticle (“DCFHP”) comprising a SARS-CoV-2 spike protein coupled to a ferritin nanoparticle and an adjuvant (10 pg of DCHFP protein and 150 pg Alhydrogel) at Day 0. Mice were then administered a vaccine composition comprising one of : MERS-1227 (10 pg) + adjuvant (150 pg Alhydrogel), MERS-1236 (10 pg) + adjuvant (150 pg Alhydrogel), MERS-1227 (5 pg) and DCFHP (5 pg) + adjuvant (150 pg Alhydrogel), or MERS-1236 (5 pg) and DCFHP (5 pg) + adjuvant (150 pg Alhydrogel) on Day 49 and neutralizing antibody titers were measured at Day 56. FIG. 9 depicts a graph of the IC50 values from the measured neutralizing antibody titers measured at Day 56. Each point on the graph represents the serum IC50 value obtained from an individual mouse. Serum dilutions were run in technical duplicate for each animal in two different experiments, thus the represented IC50 is compiled from four separate normalized serum dilutions

[0285] Immunization of mice with background SARS-CoV-2 immunity (generated by vaccination with a SARS-CoV-2 protein nanoparticle) with MERS-1227 and MERS-1236 elicit neutralizing antibodies against MERS-pseudotyped lentivirus 7 days following the MERS immunization. This demonstrates that pre-existing immunity to SARS-CoV-2 in mice is not inhibitory for generation of a MERS neutralizing antibody response.Antibody Binding Analysis using BLI to MERS-1227 following a 4 week hold at various temperatures or 2 hours of extreme heat stress

[0286] Biolayer interferometry (BLI) analysis of MERS-1227 was performed using an Octet R8 instrument with Protein A functionalized tips, after the engineered MERS spike protein was subjected to a 4 week hold at either 4° C, 220C, or 370C. Antibodies and MERS nanoparticleswere diluted to 10 pg / mL in 10X Kinetics buffer (PBS + 0.1% BSA, 0.02% Tween20 and Kathon) and pipetted into black-walled, black-bottom plates (200 pL per well). Protein A tips were dipped into antibody wells and subsequently dipped into nanoparticle wells to assess binding association for 120 sec. Dissociation was measured for 120 sec by moving tips into a buffer only well.

[0287] FIGS. 13A-13B depict graphs of the biolayer interferometry analysis of binding to the RBD of MERS-1227 fusion protein (FIG. 13A) or the NTD of MERS-1227 fusion protein (FIG. 13B). Antibody binding to MERS-1227 fusion protein was assessed using biolayer interferometry in which MERS-specific antibodies targeting diverse epitopes (RBD or NTD) were loaded onto Protein A tips and then dipped into wells containing 10 pg / mL MERS-1227 after being subjected to a 4 week hold at either 4° C, 220C, or 370C . Binding results demonstrate that the MERS- 1227 fusion protein bind MERS conformation-specific antibodies even after a 4 week hold at various temperatures, indicating that MERS-1227 fusion protein is heat stable.

[0288] BLI analysis of MERS-1227 fusion protein was performed using an Octet R8 instrument with Protein A functionalized tips, after the engineered MERS spike protein was subjected to extreme heat stress for 2 hours at either 700C or 900C, with a control being unstressed. Antibodies and MERS nanoparticles were diluted to 10 pg / mL in 10X Kinetics buffer (PBS + 0.1% BSA, 0.02% Tween20 and Kathon) and pipetted into black-walled, black-bottom plates (200 pL per well). Protein A tips were dipped into antibody wells and subsequently dipped into nanoparticle wells to assess binding association for 120 sec. Dissociation was measured for 120 sec by moving tips into a buffer only well.

[0289] FIGS. 13C-13D depict graphs of the biolayer interferometry analysis of binding to the RBD of MERS-1227 fusion protein (FIG. 13C) or the NTD of MERS-1227 fusion protein (FIG. 13D). Antibody binding to MERS-1227 fusion protein was assessed using biolayer interferometry in which MERS-specific antibodies targeting diverse epitopes (RBD or NTD) were loaded onto Protein A tips and then dipped into wells containing 10 pg / mL MERS-1227 fusion protein, after 2 hours of extreme heat stress at either 700C or 900C. Binding results demonstrate that antibodies are confirmation specific and lose binding upon extreme head stress for 2 hours, confirming that MERS-1227 is heat stable.Differential scanning fluorimetry of MERS-1227 fusion protein after an up to 4 week hold at 37

[0290] Differential scanning fluorimetry (DSF) was used to calculate the melting temperatures of the MERS-1227 after either a 1 week, 2 week, or 4 week hold at 370C. Samples were held at 370C for either 1 week, 2 weeks, or 4 weeks and then were subjected to a temperature gradient from 25 °C to 90 °C and intrinsic protein fluorescence at 330 and 350 nm was measured. Plotted is the first derivative of the ratio of 350 / 330 nm, as seen in FIG. 13E. The graph in FIG. 13E demonstrates that MERS-1227 after 1 week, 2 weeks, or 3 weeks exhibits a single transition representative of the melting temperature, similar to a sample of MERS-1227 at Day 0. This data suggests that MERS-1227 retains proper folding after 1 week of storage at 37 °C, up to 4 weeks of storage at 37 °C.

[0291] Differential scanning fluorimetry was used to calculate the melting temperatures of the MERS-1227 fusion proteinafter extreme heat stress for 2 hours at either 70° C or 900C. Samples were held at either 70° C or 900C for 2 hours and then were subjected to a temperature gradient from 25 °C to 90 °C and intrinsic protein fluorescence at 330 and 350 nm was measured. Plotted is the first derivative of the ratio of 350 / 330 nm, as seen in FIG. 13F. The graph in FIG. 13F demonstrates that the folding of MERS-1227 fusion protein is lost upon extreme heat stress for 2 hours.Immunogenicity of MERS-1227 fusion protein adjuvanted with alum after storage at 370C

[0292] Since the MERS-1227 fusion protein demonstrated the ability to bind antibodies after a 4 week hold at 370C, it was sought to determine if adjuvanted MERS-1227 fusion protein remained immunogenic in mice after multiple days of storage at various temperatures including 40C and 370C. The MERS-1227 fusion protein (engineered MERS-1227 spike protein coupled to ferritin nanoparticle) was adjuvanted with alhydrogel (alum) and stored at either 40C or 370C for about 10 days. After 10 days, the adjuvanted MERS-1227 fusion protein was administered to naive mice at a dose of 10 pg, as described above, and neutralizing titers were measured 35 days after administration, as described above. FIG. 13G depicts a graph of the measured neutralizing antibodies 35 days after administration of the single 10 pg dose of adjuvanted MERS-1227 with no temperature hold, a 10 day temperature hold at 40C, or a 10 day temperature hold at 370C. The graph in FIG. 13G demonstrates that adjuvanted MERS-1227 remains immunogenic for at least 10 days after a 10 day storage at 370C.Example 3: Immunogenicity of vaccine compositions

[0293] To investigate the immunogenicity of the vaccine composition in mice, BALB / c mice were divided into an A group (“first group”) and a B group (“second group”) and administered a vaccine composition comprising a fusion protein (MERS-1227) comprising a sequence of SEQ ID NO: 285 and an adjuvant, according to the immunization schema provided in Table 3.Table 3: Mice Immunization Schema

[0294] Prior to injection, antigen-adjuvant mixtures were prepared to the antigen concentration described in Table 3, and adjuvanted as described herein and were administered to the mice. For mice in all groups, 21 days after injection whole blood was collected, centrifuged to isolate serum, heat inactivated at 56° C for 30 minutes, and then was frozen at -80° C for analysis using the MERS pseudotyped-lentivirus neutralization assay. For mice in all groups, 21 days after the initial administration of MERS-1227, antigen-adjuvant mixtures were prepared as described above and administered to the mice as a boost dose. 21 days after the second injection whole blood was collected, centrifuged to isolate serum, heat inactivated at 56° C for 30 minutes, and then was frozen at -80° C for analysis using the MERS pseudotyped-lentivirus neutralization assay. The results are depicted in the graph of FIG. 11B. At 21 days after administration of a single dose (prime) of the MERS-1227 fusion protein mice demonstrated neutralizing antibodies, although the response appeared to be dose dependent. Mice administered 0.016 ug of the MERS-1227 fusion protein demonstrated lower neutralizing antibodies compared to those mice administered 0.4 ug of the MERS-1227 fusion protein. 21 days after administration of the second dose (prime) of the MERS-1227 fusion protein (e.g., first dose at day 0 and second dose at day 21) mice demonstrated higher neutralizing antibodies at all concentrations.

[0295] To investigate the immunogenicity of the vaccine composition in mice, BALB / c mice were administered the vaccine composition comprising the MERS-1227 fusion protein as described above. Some of the mice were administered an additional dose of the vaccine at eithera 2 pg dose, or a 10 pg dose on Day 77. Whole blood was collected 7, 21, 28 35, 56, 70, and 77 days after the initial injection and centrifuged to isolate serum. The serum was then heat inactivated at 56° C for 30 minutes, and was frozen at -80°C for analysis using the MERS pseudotyped-lentivirus neutralization assay, as seen in the schematic of FIG. 12A. The results are depicted in the graph of FIG. 12B. For mice administered a single dose (prime) of the MERS- 1227 fusion protein, the mice demonstrated an increased neutralizing antibody response from Day 21 to Day 56. For mice administered two doses of MERS-1227 fusion protein (with the second dose at either the 2ug or 10 ug dose), the neutralizing antibodies measured were significantly increased compared to the neutralizing antibodies measured for mice receiving a single dose. There appeared to be little difference between the values of measured neutralizing antibodies measured for mice administered a second dose of MERS-1227 fusion protein at the 2 ug or 10 ug dose.

[0296] To investigate the immunogenicity of the vaccine composition in non-human primates (NHPs), NHPs were divided into an A group (“first group”) and a B group (“second group”) and administered a vaccine composition comprising either a fusion protein (MERS-1227) comprising a sequence of SEQ ID NO: 285 and an adjuvant or a fusion protein (MERS-1236) comprising a sequence of SEQ ID NO: 349 and an adjuvant, according to the immunization schema provided in Table 4 below. Table 5 below provides the birthdate and weight of the NHPs at the time the first dose was administered.Table 4: NHP Immunization SchemaTable 5: Date of birth and weights of NHPs immunized with MERS-1227 and MERS-1236 FNP proteins

[0297] Prior to injection, antigen-adjuvant mixtures, prepared as described above to an antigen concentration of 0.1 mg / mL and an aluminum concentration of 1.5 mg / mL were inverted 50 times. 500 pL of the adjuvanted MERS-1227 and MERS-1236 vaccine candidates were injected intramuscularly into cynomolgus macaques (resulting in delivery of 50 pg MERS-1227 or -1236 antigen, and 750 pg of aluminum, as aluminum hydroxide). 21 and 35 days after injection, whole blood was collected, centrifuged to isolate serum, heat inactivated at 56° C for 30 minutes, and then was frozen at -80° C for analysis using the MERS pseudotyped-lentivirus neutralization assay.

[0298] As outlined in Table 4, Groups A and B may be boosted on Day 93. Substantial serum neutralizing titers (half maximal serum neutralization - NT50) may be seen at Day 93, prior to any boost, constituting the immune response from a single injection against the matched strain.

[0299] FIG. 10 depicts the serum IC50 titers against MERS-CoV following a single immunization of MERS-1227 or MERS-1236 at Day 21. Neutralizing antibody titers 21 days after 1 dose (50 pg protein, 750 pg Alhydrogel) of MERS ferritin nanoparticles. Each point represents the serum IC50 value obtained from an individual macaque. Serum dilutions were run in technical quadruplicate for each animal. At Day 21, treatment of the two groups were similar although some NHPs demonstrated higher IC50 values being administered MERS-1236.

[0300] As outlined in Table 4 and seen in the schematic of FIG. 12C, Group A was boosted on Day 93 with an additional dose of MERS-1227 fusion protein, as described above. 21, 28, 35, 56, 77, 93 (day of administration of additional dose), 100, 127, and 188 days after injection, whole blood was collected, centrifuged to isolate serum, heat inactivated at 56° C for 30 minutes, and then was frozen at -80° C for analysis using the MERS pseudotyped-lentivirus neutralization assay. The results are depicted in FIG. 12D. NHPs demonstrated neutralizing antibodies after the first dose of the MERS-1227 fusion protein, although for some NHPs, there appeared to be a drop in neutralizing antibodies between days 56 and 77. For those NHPs administered a second dose of the MERS-1227 fusion protein, the measured neutralizing antibodies increased rapidly to a peakmeasured value between Days 93 and 127 for all NHPs. The measured neutralizing antibodies appeared to decrease by day 188.

[0301] To characterize the breadth of response to known MERS strains, a panel of MERS spike- pseudotyped lentiviruses was generating comprising clade A, B (currently circulating in humans), and C MERS-CoV strains and a distantly related pangolin merbecovirus, MjHKU-4r-CoV-l.

[0302] For viruses used to generate the panel shown in FIG. 12E, native sequences of all viral spike proteins were used and an 18 amino acid C-terminal truncation was also included (sequences in Table 6) The media was removed and replaced with fresh media 18-24 hours post transfection. Viruses were harvested 72 hours post transfection by spinning at 300 xg for 5 min and filtering through a 0.45-pm PES filter. Viruses were frozen at -80 °C until use.Table 6: Exemplary sequences of viral spike proteinsNeutralization of Pseudotyped MERS-CoV and Variants of Concern (VOCs)

[0303] Infectivity assays with MERS-pseudotyped lentiviruses were performed in HeLa cells expressing human DPP4 (Cellecta). HeLa / DPP4 cells were plated in white-walled, white-bottom 96-well plates at 5,000 cells per well one day prior to or the day of infection. To quantify neutralizing antibody titers in serum from immunized mice, serum samples were diluted in cell- growth media. Serum dilutions were then mixed with MERS-pseudotyped lentivirus and virus / serum dilutions were incubated at 37 °C for 1 hr. Media was either aspirated off of HeLa / DPP4 cells and replaced with virus / serum dilutions or HeLa / DPP4 cells were plated directly into virus / serum dilutions and left to incubate with cells for 72 hr at 37 °C. Infectivity in each well was quantified using BriteLite and luciferase signal was readout using a BioTek Synergy Hl microplate reader. Data was analyzed using GraphPad Prism. Control wells containing virus only and cells only were averaged, and serum dilution infectivity value were normalized to theseaverages set at 100% infectivity (virus only) and 0% infectivity (cells only). Normalized values were then fit with a 3-parameter non-linear regression (Y=Bottom + (Top-Bottom) / (l+(X / IC50)).

[0304] NHP serum was collected ~5 weeks following a second immunization (study day 127) and was tested for neutralization against the panel of lentiviruses pseudotyped with spike sequences from MERS-CoV clades A (e.g., EMC), B (e.g., Englandl, Hasal4b, Bishal, KOR / NIH / 002), and C (e.g., Nigl657, NRCE-HKU270, C1272, NRCE-HKU205) and a pangolin merbecovirus (MjHKU-4r-CoV-l) described above and results were quantified in FIG. 12E. Each circle in FIG. 12E represents the NT50 value for an individual NHP obtained by running pseudovirus neutralization analysis in quadruplicate. Lines represent the geometric mean titer (GMT) and error bars represent the geometric SD. Both MERS-1227 and MERS-1236 appear to demonstrate neutralization titers against MERS viruses in MERS-CoV clade A, B, and C and a related virus, merbecovirus from pangolin. Robust neutralization of all MERS-CoV clades in the panel was observed including clade C strains currently known to circulate in camels but not humans. Remarkably, neutralization of MjHKU-4r-CoV-l was observed, which only shares a 66% amino acid sequence identity with the spike of England 1 MERS-CoV.

[0305] To further characterize the serum response in NHPs and to analyze IgG responses from immunized NHPs, serum was collected and used in an Antigen-specific Luminex assay, wherein Luminex beads coated with either soluble MERS trimer (left) MERS RBD (middle), or MERS NTD (right) (sequences disclosed in Table 12 below) were used to evaluate levels of binding antibodies following a prime (single administration of MERS-1227 or MERS-1236) and a prime + boost regimen (double administration of MERS-1227 or MERS-1236) in NHPs. The results are plotted on the graph of FIG. 12F. The solid line represents the average GMTs per antigen group, and the dashed lines represent the GMTs per individual NHP.

[0306] NHPs immunized with Alhydrogel-adjuvanted MERS-1227 and MERS-1236 elicit robust and broad neutralizing antibody responses that are durable for at least 5 months post boost. Similar spike and RBD IgG levels was observed, with slightly more waning of the spike titers observed between study days 188 and 253. Interestingly, spike and RBD binding titers appear to decrease less quickly than pseudovirus neutralizing titers. Overall, NTD antibody levels were lower than spike or RBD levels suggesting that the RBD is a more immunodominant target of these antigens. Interestingly, overall pseudovirus neutralization, breadth, and total IgG levels were highly similar between MERS-1227 and MERS-1236 immunized NHPs.Immunization Through Different Vaccine Delivery Methods

[0307] As described above, the vaccine compositions of the disclosure may include fusion proteins of an engineered MERS spike protein coupled to ferritin nanoparticles or nucleic acids encoding an engineered MERS spike protein. Different vaccine delivery methods were tested in BALB / c mice, as outlined in Table 7. The vaccine composition of Group A (MERS-1227) comprises the fusion protein of the engineered MERS spike protein coupled to a ferritin nanoparticle comprising a sequence of SEQ ID NO: 285. The vaccine composition of Group B (mRNA encoding MERS-1227 fusion protein) comprises nucleic acids encoding the fusion protein of the engineered MERS spike protein coupled to a ferritin nanoparticle comprising a sequence of SEQ ID NO: 285. The vaccine composition of Group C (mRNA encoding MERS- 1227) comprises nucleic acids encoding the engineered MERS spike protein of SEQ ID NO: 387. The vaccine composition of Group D (mRNA encoding a full length MERS) comprises nucleic acids encoding the engineered MERS spike protein of SEQ ID NO: 388.Table 7: Mice Immunization Schedule with Different Vaccine Compositions

[0308] Prior to administration, antigen-adjuvant mixtures were prepared as described above. For Group A, the unadjuvanted MERS-1227 vaccine candidate or the adjuvanted MERS-1227 vaccine candidate (with 150 pg of alum) was injected into the mice (resulting in delivery of 0.4 pg MERS- 1227). For Group B, the mRNA encoding the fusion protein of MERS-1227 was administered to the mice (resulting in delivery of 0.01 pg MERS-1227, 0.1 pg MERS-1227, or 1 pg MERS-1227 fusion protein). For Group C, the mRNA encoding the engineered MERS spike protein was administered to the mice (resulting in delivery of 0.01 pg MERS-1227, 0.1 pg MERS-1227, or 1 pg MERS-1227). For Group D, the mRNA encoding the engineered full length MERS spikeprotein was administered to the mice (resulting in delivery of 0.01 pg MERS full length, 0.1 pg MERS-full length, or 1 pg MERS-full length).

[0309] As seen in the schematic of FIG. 14A, at day 0, day 21, and day 42 after administration of the vaccine compositions of Table 7, whole blood was collected, centrifuged to isolate serum, heat inactivated at 56° C for 30 minutes, and then was frozen at -80° C for analysis using the MERS pseudotyped-lentivirus neutralization assay. The results are depicted in the graph of FIG. 14B. Mice administered the adjuvanted MERS-1227 fusion protein demonstrated measured neutralizing antibodies, especially those administered two doses of the adjuvated MERS-1227 fusion protein (prime+boost).

[0310] Mice administered a single dose of the mRNA encoding the MERS-1227 fusion protein at 1 pg appeared to demonstrate neutralizing antibodies at the same level as mice administered a single dose of adjuvated MERS-1227 fusion protein. Mice administered two doses of the mRNA encoding the MERS-1227 fusion protein (prime+boost) appeared to demonstrate neutralizing antibodies at the same level as the mice administered two doses of the adjuvanted MERS-1227 fusion protein. This suggests that administration of a mRNA encoding the MERS-1227 fusion protein may be just as effective at generating neutralizing antibodies in BALB / c mice as administration of the MERS-1227 fusion protein. However, this effect appeared to be dose dependent as lower doses of mRNA encoding the MERS-1227 fusion protein did not appear to demonstrate as robust values of neutralizing antibodies.

[0311] Mice administered two doses of mRNA encoding the engineered MERS spike protein at Ipg appeared to demonstrate similar neutralizing antibody values as mice administered the adjuvanted MERS-1227 fusion protein and mRNA encoding the MERS-1227 fusion protein. This suggests that the administration of a mRNA encoding the engineered MERS-1227 spike protein after two doses may be as effective at generating neutralizing antibodies in BALB / c mice as the administration of the MERS-1227 fusion protein or the mRNA encoding the MERS-1227 fusion protein.

[0312] To investigate the effects of adjuvants on the immunogenicity of the vaccine compositions in mice, BALB / c mice were divided into 6 groups (Groups A-F) and administered one of the six vaccine composition as indicated in Table 8. The vaccine compositions each contained a fusion protein (MERS-1227) comprising a sequence of SEQ ID NO: 285. Five of the vaccine compositions also had one of five adjuvants from Adjuvant Groups 1-5, according to the immunization schema provided in Table 8. Adjuvant Group 1 includes alum only. AdjuvantGroup 2 includes alum + CpG. Adjuvant Group 3 includes QuilA+MPLA+ / - alum. AdjuvantGroup 4 includes AddaVax only, and Adjuvant Group 5 includes 3M-052 + / - alum.Table 8: Mice Immunization Schedule with different adjuvant groups

[0313] Prior to injection, antigen-adjuvant mixtures were prepared as described above. Whole blood was collected at 21 and 42 days after injection and centrifuged to isolate serum. The serum was then heat inactivated at 56° C for 30 minutes and frozen at -80° C for analysis using the MERS pseudotyped-lentivirus neutralization assay, as seen in the schematic of FIG. 15A. The results are depicted in FIG. 15B. All adjuvants tested led to higher levels of neutralizing antibodies being in the sera of vaccinated mice.

[0314] To further evaluate MERS-1227 and MERS-1236, MERS-1227 and MERS-1236 were subjected to pH treatment at pH 2, 3, 3.5, 4, and 7.5 (FIGS. 16A-16E and FIGS. 17A-17E). Panels correspond to analytical SEC (left), DLS (middle left), DSF (middle right), and BLI (right two panels) with an RBD-targeting mAb, CDC-C2, and an NTD-targeting antibody, G2. Sequences of antibodies are in Table 9 below. pH treatments were conducted in duplicate and each sample was then characterized using each assay. Curves shown for SEC, DLS, and DSF represent one replicate. Error bars on the DLS plot represent standard deviation. BLI plots show the mean nm shift value of the two treatment replicates at 120s normalized to the pH 7.5 control, and error bars represent the standard deviation.Table 9: Exemplary Antibody sequences

[0315] To assess the pH stability of MERS FNP proteins, aliquots of the formulation buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5% sucrose) were adjusted to pHs of 2, 3, 3.5, and 4 (+ / - 0.1 pH unit) by dropwise addition of hydrochloric acid. Slide-A-Lyzer MINI Dialysis Devices (15 mL conical format, Thermo Fisher Scientific Cat #88401) were briefly rinsed in these buffer solutions. 350 pL of MERS-1227 or MERS-1236 FNP proteins at 0.25 mg / mL in formulation buffer were added to each dialysis device and dialyzed against 14.5 mL of the pH-adjusted formulation buffers (or pH 7.5 as a control). Proteins were dialyzed at room temperature for 1 hour with gentle agitation. After 1 hour incubation, 1 pL dialyzed protein was used to confirm the target pH of the solution using pH paper. At this time, dialysis solution was discarded and replaced with a fresh 14.5 mL pH-adjusted formulation buffer and dialysis was allowed to proceed for an additional 1.5 hours. Proteins were then dialyzed back to pH 7.5 by replacing dialysis solution with pH 7.5 formulation buffer followed by a 1 hour incubation at room temperature. pH 7.5 formulation buffer was then replaced with fresh pH 7.5 buffer and dialysis was allowed to proceed overnight at 4 °C. Proteins were then remove from dialysis devices, transferred to 2 mL polypropylene tubes, snap frozen using liquid nitrogen, and stored at -80 °C until analysis. For analysis, all samples were thawed at the same time and diluted to 0.125 mg / mL with formulation buffer and evaluated using analytical SEC, DLS, DSF, and BLI as described above.

[0316] To assess thermal stability of MERS FNP proteins, purified proteins were diluted to 0.5 mg / mL in 20 mM Tris pH 7.5, 150 mM NaCl, 5% sucrose and aliquoted in 2 mL polypropylene tubes. Samples were placed at either 4 °C, 22 °C (ambient temperature), or 37 °C. Control sampleswere snap-frozen immediately following dilution to 0.5 mg / mL and 90 °C heat-treated controls were heated to 90 °C for 30 min and then snap-frozen and stored at -80 °C until analysis. Aliquots of 4 °C, 22 °C, and 37 °C were taken following 7- and 14-day incubations and snap-frozen at each time-point until analysis. For analysis, all samples were thawed at the same time and assessed at 0.5 mg / mL using analytical SEC, DLS, DSF, and BLI as described above.

[0317] MERS-1227 and MERS-1236 were subjected to incubation at varying temperatures for 7 and 14 days. MERS-1227 and MERS-1236 were incubated at 4, 22, or 37 °C. Control samples include a sample not subjected to any incubation (gray) and a sample heat-treated at 90 °C for 30 min (black). The 90 °C heat-treated sample was included in the DLS, DSF, and BLI analyses but was excluded from analytical SEC analysis. BLI measurements were normalized to the untreated control sample. Results were plotted on the graphs of FIGS. 18A-18E (MERS-1227) and FIGS. 19A-19E (MERS-1236).

[0318] Further characterization of MERS-1227 and MERS-1236 reveals that MERS-1227 and MERS-1236 retain nanoparticle structure and antigenicity following low-pH treatment and extended incubation up to 37 °C.

[0319] In vivo characterization of MERS-1227 and MERS-1236. MERS-1227 and MERS-1236 were compared to the soluble trimer of MERS upon administration to BALB / c mice. FIG. 20A depicts a schematic of the timeline of the administration of MERS-1227, MERS-1236 or the MERS trimer to the BALB / C mice. Serum was collected at study day 21 and 42. After 42 days, the mice were bled and neutralization assays were performed with the results depicts in FIGS. 20B and 20C. MERS-spike pseudotyped lentivirus neutralization GMTs observed following a prime only (day 21, left panel) and a prime + boost (day 42, right panel). Pools of serum were additionally tested from the MERS-1227 and MERS-1236 groups and confirmed they neutralized live MERS-CoV in a plaque-reduction neutralization assay (seen in FIG. 27).

[0320] For MERS-1227 and MERS-1236, mice were immunized with either 0.4 (n = 10), 2 (n = 10), or 10 pg (n = 5) FNP adjuvanted with 150 pg Alhydrogel. For MERS soluble trimer (sequence disclosed in Table 10 below), mice were immunized with either 2 (n = 5) or 10 pg (n = 5). Each circle represents the NT50 value for an individual mouse obtained by running pseudovirus neutralization analysis in quadruplicate (with the exception of one mouse in the 10 pg trimer group at day 21 in duplicate). Lines represent the GMT, and error bars represent the geometric SD. Statistical analysis was conducted using a two-way ANOVA with multiple comparisons.Table 10: Exemplary MERS soluble trimer amino acid sequence

[0321] FIGS. 21A-21D depict IgG responses in the BALB / c mice. Responses from each mouse measured at 1 : 100 dilution using a Luminex assay. Luminex beads coated with either soluble MERS trimer (left two panels) or MERS RBD (right two panels) were used to evaluate levels of binding antibodies. Sequences for MERS trimer, MERS RBD, MERS NTD, 3 A3 and JC57-14 are listed below in Table 11. Each dot represents the average of the mean fluorescence intensity (MFI) value per mouse, lines represent geometric mean, and error bars represent the geometric SD.Table 11: Exemplary Sequences of MERS trimer, MERS RBD, MERS NTD, 3A3 and JC57- 14

[0322] To measure the total IgG response and IgG subclasses in mice and NHPs, Luminex assays were adapted from a previously described method. Biotinylated MERS GCN4 trimer (50 pg) was incubated with 2 x 106MagPlex®- Avidin Microspheres (Luminex #MA-A012-01) for 2 hours, then washed and resuspended in 1 mL Luminex wash buffer (0.1% BSA, 0.02% Tween-20 in PBS pH 7.4). For other antigens (MERS RBD and MERS NTD), different MagPlex® Microspheres (Luminex #MC10026-01 to MC10029-01 and MC10034-01) were used. Activation involved 1 x 107microspheres with 50 mg / mL sulfo-NHS (Thermo Scientific #24510) and EDC (Thermo Scientific #22980) for 20 minutes, followed by adding 50 pg of antigen for a 2 hour incubation. Conjugated microspheres were resuspended in 1 mL Luminex wash buffer. The postconjugation antigenicity of each antigen was validated by measuring binding to mAbs; 3A3 (MERS GCN4 trimer S2), JC57-14 (RBD), and G2 (NTD).

[0323] Serum samples from mice and NHPs were diluted 50-fold in Luminex diluent (1% nonfat milk, 5% FBS, 0.05% Tween-20 in PBS pH 7.4). 25 pL of diluted serum was added to 25 pL of antigen-conjugated microsphere suspension (at least 1000 microspheres / antigens). After a 30- minute incubation, microspheres were washed three times with Luminex wash buffer. For total IgG detection, microspheres were incubated with 100 pL of goat anti-mouse IgG phycobiliprotein (2 pg / mL, SouthernBiotech #1030-09) for 30 minutes. For mouse IgG subclasses, biotinylated secondary antibodies specific to IgGl and IgG2a (4 pg / mL, SouthernBiotech #1070-08, #1080- 08) were used, followed by 30 minutes with streptavidin-PE (5 pg / mL, Thermo Scientific #12- 4317-87). Results were read on a Luminex xMAP Intelliflex system.

[0324] Taken together, immunization of BALB / c mice with MERS-1227 or MERS-1236 results in more robust binding and neutralizing antibody responses compared to soluble trimer.

[0325] To further test MERS-1227 and MERS-1236 in host animals similar to intermediate hosts of MERS dromedary camels, an alpaca live virus challenge model was developed as indicated in the schematic of FIG. 22A. Alpacas have been established as a surrogate infection model for camels, the predominant natural intermediate animal host of MERS-CoV. Unlike camels, alpacas do not develop rhinorrhoea (nasal discharge) upon MERS-CoV infection, but they do shed infectious virus that is detectable in nasal turbinates.Table 12: Ages and vaccine group designations for alpacas in MERS-CoV challenge study

[0326] Alpacas were immunized (n = 5 per group) at two dose levels (20 pg or 200 pg) of MERS-1227 or MERS-1236 adjuvanted with 500 pg Alhydrogel using a prime-boost regimen. As a placebo control, an additional group of alpacas (n = 5) were dosed with 500 pg Alhydrogel at the same time points. Table 12 provides the ages of each alpaca in each treatment group at the time they were immunized. Three weeks following the boost, (study day 41), all vaccinated alpacas had detectable pseudovirus neutralizing titers in their sera (seen in FIG. 22B). Furthermore, all alpacas vaccinated at both dose levels of MERS-1227 and the 200-pg dose of MERS-1236 had detectable neutralizing titers in a live-virus assay (seen in FIG. 22C). On study day 42, alpacas were challenged by intranasal inoculation with live MERS-CoV virus (clade A, EMC / 2012) in a BSL3 containment facility. On days 1-5 and day 7 post-challenge, nasal swabs were collected from each alpaca, and live virus titers were determined using a plaque assay. Detectable levels of MERS-CoV were observed via nasal swab for at least one time point following challenge in 4 out of 5 alpacas that received the Alhydrogel placebo, while live virus was not detected in any (0 / 20) of the alpacas that received either dose level of Alhydrogel- adjuvanted MERS-1227 or MERS-1236 (seen in FIG. 22D).

[0327] Taken together, these results demonstrate that immunization with Alhydrogel- adjuvanted MERS-1227 and MERS-1236 elicited protection from infection with live MERS-CoV. This provides strong evidence that these vaccines are protective and is an important demonstration that clinical development of this stabilized MERS-spike ferritin vaccine could be utilized as a prophylactic countermeasure against.

[0328] Further characterization was undertaken for understanding the manufacturability of MERS-1227 and MERS-1236. SDS-PAGE (FIGS. 23A-23B) quantitation of MERS-1227 and MERS-1236 in 14-day fed-batch supernatants. Standard curves with 0.5-4 pg were generated using purified FNP proteins and quantified using densitometry (FIGS. 23C-23D). SDS-PAGE analysis was performed in duplicate; circles represent the mean and error bars represent the standard deviation. Densitometry was performed in ImageJ and quantitation was performed in GraphPad Prism. FIGS. 24A-24B depict chromatograms obtained from AKTA purification on a Sartobind Q (left) and a HiTrap Capto Butyl (right) column. SDS-PAGE gels (FIGS. 24C-24D) correspond to purification samples, with FT and elution fractions taken from the peaks boxed on the chromatograms above. Both candidates (MERS-1227 and MERS-1236) are expressed at high levels (~ 2.7-3.2 g / L titer) in CHO cells (FIGS. 23C-23D) and can be purified via two orthogonal tag-less purification processes (FIGS. 24A-24D).

[0329] The glycosylation of MERS-1227 and MERS-1236 was evaluated to determine if glycosylation contributed to the differences seen in observed molecular weight differences described above. FIGS. 25B-25C depict schematics of the primary structure of MERS-1227 and MERS-1236 annotated with positions of detected N-glycosites. A semi-quantitative assessment of N-linked vs O-linked glycans on MERS-1227 and MERS-1236 was undertaken using liquid chromatography -mass spectrometry (as seen in FIG. 25A). Precursor ions of individual peptides assigned to have N-linked or O-linked glycans by peptide-spectrum matching using PEAKS GlycanFinder software were integrated and summed across all N-linked or O-linked glycan peptides in each LC-MS / MS run. MERS-1227 and MERS-1236 were analyzed in triplicate and each dot represents the sum of peak areas for an individual replicate. N-linked glycosylation is predominant for both nanoparticles.

[0330] To identify post-translational modifications of MERS-1227 and MERS-1236 ferritin nanoparticles, raw mass spectrometry data were analyzed using the GlycanFinder module of PEAKS Studio 11 (Bioinformatics Solutions, Inc). Custom FASTA databases were generated comprising the Chinese hamster (Cricetulus griseus) proteome (downloaded on November 27, 2023), common mass spectrometry contaminant proteins 66, and either the MERS-1227 or -1236 FNP protein sequence. The raw MS data were searched against this database with the following specified modifications: fixed carbamidomethylation on Cys, variable oxidation of Met, and variable deamidation of Gin and Asn, with a maximum of two variable modifications per peptide. Glycans were identified using the default N-linked and O-linked databases within PEAKS GlycanFinder. Semi-specific tryptic peptides with a maximum of two missed cleavages were considered. The allowed mass tolerances were 10 ppm for precursor ions, 0.04 Da for peptide product ions, and 20 ppm for glycan diagnostic ions. Hits were filtered to a false discovery rate of 1% using the PEAKS decoy -fusion approach.

[0331] To evaluate glycosylation of MERS FNP proteins, a mass spectrometry-based peptide mapping was performed. Extensive N-glycosylation and limited but detectable O-glycosylation throughout the MERS-1227 and MERS-1236 spikes but not within the ferritin nanoparticle core was observed.

[0332] To further validate the results seen for MERS-1227 and MERS-1236, neutralizing antibody titers were quantified using a luciferase-based assay with lentivirus pseudotyped with MERS spike and HeLa cells stably expressing human DPP4 as the targets of infection. The assay was validated using MERS neutralizing antibodies with known IC50 values as seen in FIGS. 26A-26D. A SARS-CoV-2 specific mAb, casirivimab, was used to demonstrate specificity of the assay for MERS-specific antibodies, and no neutralization was detected with this mAb. Convalescent camel serum from a camel challenged with MERS-CoV with a known PRNT90 of 1 :320 serum dilution was used as a benchmark for our pseudovirus neutralizing titers. Sequences of the antibodies are disclosed in Table 13.Table 13: Exemplary VH / VL antibody sequences

[0333] In the pseudovirus assay, the camel serum sample inhibits 90% of viral entry (IC90) at a serum dilution of 1 :6810. This result indicates that the lentivirus-based pseudovirus assay exhibits higher sensitivity than a plaque-reduction neutralization assay using live virus, as reported for other lentivirus-based neutralization assays compared to live viral comparators.

[0334] To evaluate the influence of adjuvants on the ratio of Thl / Th2 responses following immunization with MERS-1227, biotinylated MERS spike trimer was bound to streptavidin Luminex beads and quantified the levels of mouse IgG2a and IgGl. In mice, induction of IgGl is associated with a Th2-biased response and IgG2a is associated with a Thl-biased response. Biontinylated MERS spike trimer was adjuvanted with an adjuvant of Groups 1-5. Adjuvant Group 1 includes alum only, adjuvant group 2 includes alum + CpG, adjuvant group 3 includesQuilA+MPLA+ / - alum, adjuvant group 4 includes AddaVax only, and adjuvant group 5 includes 3M-052 + / - alum

[0335] Mice immunized with MERS-1227 adjuvanted with either 15, 50, or 150 pg Alhydrogel alone elicited an IgG response strongly dominated by IgGl, indicative of a Th2 response, which is well-documented for alum adjuvants (seen in FIG. 28). Adjuvant Group #4 was observed to have a slightly decreased Th2-skewing as compared to Alhydrogel (Adjuvant Group #1) alone. Importantly, it was observed that at 15 and 150 pg of Alhydrogel, co-formulated as either Adjuvant group #3 or as Adjuvant Group #5)substantially influenced the Thl / Th2 skew as shown by a much higher IgG2a / IgGl ratio in these groups compared to Alhydrogel alone. Adjuvant Group #2 was observed to also overcome the Alhydrogel-induced Th2 skew at the 15 pg Alhydrogel dose, but not at the 150 pg dose. Taken together, the results of this adjuvant screen demonstrate several feasible mechanisms to improve both the humoral and cell-mediated immune responses to MERS- 1227.Example 4: Immunization against MERS-CoV in transgenic mice expressing human DPP4

[0336] A colony of DPP4 transgenic mice (hDPP4 mice) was established at Colorado State University. Mice were genotyped and DPP4 heterozygotes and homozygotes were used in the challenge study. FIG. 29A provides an experimental outline of the challenge study. Antigens and adjuvants were formulated to deliver the doses as indicated in Table 14 below in 100 pL volume. Mice were injected intramuscularly with a 50 pL dose of antigen in each hind leg, to deliver 100 pL of vaccine formulation per immunization. Blood was collected at study days 21 and 42 and sera were stored frozen until analysis. For lethal challenge, mice were lightly anesthetized with ketamine-xylazine and challenged by intranasal instillation of 10,000 PFU of the EMC / 2012 strain of MERS-CoV. Mice were weighed and scored for clinical signs of disease daily beginning immediately prior to challenge and extending to euthanasia. Half of the mice were euthanized 3 days post-infection and lungs collected for virus titration. The remainder of the mice were maintained up to day 10 post-infection to assess changes in body weight and survival.Table 14: Vaccine groups and outcome for hDPP4 mice in MERS-CoV challenge study

[0337] Pseudovirus neutralizing titers at study day 21 (prime only) and 42 (prime + boost) were determined using a MERS-pseudotyped lentivirus neutralization assay as described above. The titers are presented in FIG. 29B, in which each circle represents the NT50 value for an individual mouse obtained by running pseudovirus neutralization analysis in quadruplicate. Serum from one mouse in the 0.016 pg and one mouse in the 0.4 pg dose groups were not analyzed for day 21 neutralization due to lack of available serum. Lines represent the GMT and error bars represent the geometric SD. As shown in FIG. 29B, a prime only dose of 0.4 pg, but not 0.016pg, of MERS- 1227 led to detectable levels of neutralizing antibodies. Administrations of two doses (prime + boost) of 0.016pg and 0.4pg of MERS-1227 led to higher levels of neutralizing antibodies compared to the respective single doses.

[0338] Neutralizing titers of day 21 (prime only) and 42 (prime + boost) serum were also determined using a plaque reduction neutralization assay that measured live-virus neutralization. The results are presented in FIG. 29C, in which each circle represents the PRNT80 value for an individual mouse. Lines represent GMT and error bars represent geometric SD. As shown in FIG. 29C, a prime only dose of 0.016pg MERS-1227 led to low levels of neutralization liters in someof the mice whereas a prime only dose of 0.4pg MERS-1227 led to detectable levels of neutralizing antibodies in all immunized mice. Administrations of two doses (prime + boost) of 0.016pg and 0.4pg of MERS-1227 led to higher levels of neutralizing titers compared to the respective single doses.

[0339] In addition to examining the sera of immunized mice for neutralizing antibodies, live MERS-CoV virus in lung samples obtained from mice sacrificed 3 days post-challenge was quantified. As shown in FIG. 29D, live virus was not detectable in the lungs of all four mice immunized with two doses (prime + boost) of 0.4pg of MERS-1227, whereas live virus was detectable in two out of four mice immunized with two doses of 0.016pg of MERS-1227. Furthermore, whereas all the mice in the placebo group succumbed to disease by day 9 post challenge, probability of survival at day 10 post challenge was 25% for mice immunized with 0.016 pg of MERS-1227 and 75% for mice immunized with 0.4pg of MERS-1227, as shown in FIG. 29E. the probability of survival was Survival curves post challenge. One mouse in the 0 pg group died pre-challenge and is not shown in the survival curve. The mouse in the 0.4 pg dose group that succumbed to disease on day 8 was determined to be pregnant at autopsy.

[0340] Weight loss was also monitored in hDPP4 mice (n = 3 or 4) immunized with 0, 0.016, or 0.4 pg MERS-1227 FNP protein, adjuvanted with 150 pg Alhydrogel, at days 0 and 21 and challenged with EMC / 2012 MERS-CoV at day 42. Mice were weighed daily post-challenge and weights are plotted as the averaged % body weight as compared to day 0 post-challenge in FIG. 30. Each circle represents the mean weight per group at each timepoint and error bars represent SD. Mice that died were excluded from each time-point. Whereas mice immunized with 0 or 0.016pg MERS-1227 experienced statistically significant weight loss, mice immunized with 0.4pg MERS-1227 only had slight but not statistically significant weight loss by day 10 post challenge.

[0341] Taken together, vaccination with MERS-1227 led to generation of neutralizing antibodies in immunized mice and provided dose-dependent protection against MERS-CoV lethal challenge in hDPP4 mice.

[0342] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. 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 withrespect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0343] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Claims

CLAIMS1. An engineered Middle East Respiratory Syndrome (MERS) spike protein comprising at least 70% sequence identity to SEQ ID NO: 1 with a C-terminal truncation of at least 25 amino acids.

2. The engineered MERS spike protein of claim 1, wherein the protein comprises a C-terminal truncation of at least 100 amino acids.

3. The engineered MERS spike protein of claim 1 or 2, wherein the protein comprises the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 70% sequence identity thereto.

4. The engineered MERS spike protein of any one of claims 1-3, wherein the protein comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 70% sequence identity thereto.

5. The engineered MERS spike protein of any one of claims 1-4, wherein the protein comprises one or more proline substitutions.

6. The engineered MERS spike protein of claim 5, wherein the one or more proline substitutions comprise one or more of A889P, S966P, A973P, T1014P, V1060P, and L1061P.

7. The engineered MERS spike protein of any one of claims 1-6, wherein the spike protein is fused to a nanoparticle, thereby forming a fusion protein comprising the spike protein fused to a nanoparticle.

8. The engineered MERS spike protein of claim 7, wherein the nanoparticle comprises a ferritin subunit.

9. The engineered MERS spike protein of claim 8, wherein the fusion protein forms a selfassembling nanoparticle.

10. The engineered MERS spike protein of claim 8 or 9, wherein the ferritin subunit comprises H. pylori ferritin.

11. The engineered MERS spike protein of any one of claims 8-10, wherein the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity thereto.

12. The engineered MERS spike protein of any one of claims 8-11, wherein the engineered MERS spike protein comprises an amino acid sequence of SEQ ID NO: 285 or SEQ ID NO: 349, or an amino acid sequence having at least 70% sequence identity thereto.

13. An engineered Middle East Respiratory Syndrome (MERS) spike protein comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1, comprising at least three proline substitutions.

14. The engineered MERS spike protein of claim 13, wherein the at least three proline substitutions comprise at least three of: A889P, S966P, A973P, T1014P, V1060P, or L1061P.

15. The engineered MERS spike protein of claim 13 or 14, wherein one of the at least three proline substitutions is selected from the group consisting of: A889P, V1060P, and L1061P.

16. The engineered MERS spike protein of any one of claims 13-15, wherein one of the at least three proline substitutions is V1060P.

17. The engineered MERS spike protein of any one of claims 13-16, wherein the spike protein consists of three proline substitutions.

18. The engineered MERS spike protein of claim 17, wherein the three proline substitutions are A889P, V1060P, and L1061P.

19. The engineered MERS spike protein of claim 13 or 14, wherein the at least three proline substitutions comprise A889P, V1060P, and L1061P.

20. The engineered MERS spike protein of any one of claims 13-19, wherein the protein has a C terminal truncation.

21. The engineered MERS spike protein of claim 20, wherein the amino acid sequence of the spike protein comprises SEQ ID NO: 157 or SEQ ID NO: 93, or an amino acid sequence having at least 70% sequence identity thereto.

22. The engineered MERS spike protein of any one of claims 13-21, wherein the protein is fused to a binding partner, thereby forming a fusion protein.

23. The engineered MERS spike protein of claim 22, wherein the binding partner is a ferritin subunit.

24. The engineered MERS spike protein of claim 22 or 23, wherein the fusion protein forms a nanoparticle.

25. The engineered MERS spike protein of any one of claims 1-24, further comprising at least one insertion.

26. The engineered MERS spike protein of claim 25, wherein the insertion is at the C-terminal or N-terminal of the spike protein, or internal to the protein.

27. The engineered MERS protein of claim 25 or 26, wherein the insertion enables transit and extrusion of the spike protein in extracellular vesicles and / or promotes enveloped virus like particle (eVLP) production.

28. The engineered MERS spike protein of claim 27, wherein the at least one amino acid insertion comprises insertion of an ESCRT -recruiting domain (ERD).

29. The engineered MERS spike protein of claim 28, wherein the amino acid sequence of the ERD comprises the sequence of any one of SEQ ID NOs: 421-432, or an amino acid sequence having at least 70% sequence identity thereto.

30. The engineered MERS spike protein of claim 27, wherein the at least one amino acid insertion comprises insertion of a polypeptide that is ESCRT-independent.

31. A fusion protein comprising an engineered Middle East Respiratory Syndrome (MERS) spike protein and a binding partner.

32. The fusion protein of claim 31, wherein the spike protein comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto.

33. The fusion protein of claim 31 or 32, wherein the amino acid sequence of the spike protein comprises a C terminal truncation.

34. The fusion protein of any one of claims 31-33, wherein the spike protein is operably linked to the binding partner through a linker.

35. The fusion protein of claim 34, wherein the linker is a peptide linker.

36. The fusion protein of claim 35, wherein the peptide linker is a Gly-Ser linker.

37. The fusion protein of any one of claims 34-36, wherein the linker comprises a sequence selected from the group consisting of: SGG, GSG, SGG, GG, GSGG (SEQ ID NO: 418), NGTGGSG (SEQ ID NO: 419), and GGGGS (SEQ ID NO: 420).

38. The fusion protein of any one of claims 31-37, wherein the amino acid sequence of the spike protein comprises a sequence of SEQ ID NO: 66 or SEQ ID NO: 130, or an amino acid sequence having at least 70% sequence identity thereto.

39. The fusion protein of any one of claims 31-38, wherein the spike protein further comprises one or more proline substitutions.

40. The fusion protein of claim 39, wherein the one or more proline substitutions are selected from the group consisting of: A889P, S966P, A973P, T1014P, V1060P, and L1061P.

41. The fusion protein of claim 39 or 40, wherein the spike protein comprise three proline substitutions, optionally wherein the protein consists of three proline substitutions.

42. The fusion protein of claim 41, wherein the three proline substitutions are A889P, V1060P, and L1061P.

43. The fusion protein of any one of claims 39-42, wherein the amino acid sequence of the spike protein comprises an amino acid sequence of SEQ ID NO: 93 or SEQ ID NO: 157, or an amino acid sequence having at least 70% sequence identity thereto.

44. The fusion protein of any one of claims 29-43, wherein the binding partner comprises a ferritin subunit.

45. The fusion protein of claim 44, wherein the amino acid sequence of the fusion protein comprises the amino acid sequence of: SEQ ID NO: 285 or SEQ ID NO: 349, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 285 or 349.

46. An eVLP comprising the engineered MERS spike protein of any one of claims 1-30 or the fusion protein of any one of claims 31-45.

47. A nucleotide sequence that encodes the amino acid sequence of: a. the engineered MERS spike protein of any one of claims 1-12, b. the engineered MERS spike protein of any one of claims 13-30, or c. the fusion protein of any one of claims 31-45.

48. The nucleotide sequence of claim 47 that encodes the amino acid sequence of:a. the engineered MERS spike protein of any one of claims 1-6, b. the engineered MERS spike protein of any one of claims 13-21, or c. the engineered MERS spike protein of any one of claims 25-30.

49. The nucleotide sequence of claim 47 or 48, wherein nucleic acids of the nucleotide sequence comprise DNA.

50. The nucleotide sequence of claim 47 or 48, wherein nucleic acids of the nucleotide sequence comprise RNA.

51. The nucleotide sequence of claim 50, wherein the nucleic acids of the nucleotide sequence comprise mRNA.

52. A vaccine composition comprising: a. the engineered MERS spike protein of any one of claims 1-12, b. the engineered MERS spike protein of any one of claims 13-24, c. The engineered MERS spike protein of any one of claims 25-30, d. the fusion protein of any one of claims 31-45, e. the eVLP of claim 46, or f. the nucleotide sequence of any one of claims 47-51, and optionally comprising one or more adjuvants.

53. A vaccine composition comprising a nucleotide sequence that encodes the amino acid sequence of: a. the engineered MERS spike protein of any one of claims 1-6, b. the engineered MERS spike protein of any one of claims 13-21, or c. the engineered MERS spike protein of any one of claims 25-30.

54. The vaccine composition of claim 52 or 53, comprising a nucleotide sequence, wherein the nucleic acids of the nucleotide sequence comprise DNA.

55. The vaccine composition of claim 54, wherein nucleic acids of the nucleotide sequence comprise RNA.

56. The vaccine composition of claim 55, wherein the nucleic acids of the nucleotide sequence comprise mRNA.

57. A method of preventing a Middle East Respiratory Syndrome (MERS) infection or a disease associated with a MERS infection in a subject, comprising administering to the subject an effective amount of one or more doses of the vaccine composition of any one of claims 53-56.

58. The method of claim 57, wherein the subject is human.

59. The method of claim 57 or 58, wherein the route of administration is intramuscular, intravenous, intradermal, or subcutaneous.

60. The method of any one of claims 57-59, comprising preventing a MERS infection or a disease associated with a MERS infection in a subject by a MERS virus strain from MERS clade A, MERS clade B, or MERS clade C.

61. The method of claim 60, wherein the MERS virus strain from MERS clade A comprises the MERS strain EMC.

62. The method of claim 60, wherein the MERS virus strain from MERS clade B comprises a MERS strain selected from the group consisting of: Englandl, Hasal4B, Bishal, and KOR / NH4 / 002.

63. The method of claim 60, wherein the MERS virus strain from MERS clade C comprises a MERS virus strain selected from the group consisting of: Nigl657, NRCE-HKU270, C1272, and MRCE-HKU205.

64. The method of any one of claims 57-63, comprising preventing a MERS infection or a disease associated with a MERS infection in a subject by a MERS virus strain including a merbecovirus.

65. The method of claim 64, wherein the merbecovirus comprises a merbecovirus strain of MjHKU4r-CoV.

66. The vaccine composition of any one of claims 53-56, for use in a method of preventing a Middle East Respiratory Syndrome (MERS) infection or a disease associated with a MERS infection in a subject; wherein the method comprises administering to the subject an effective amount of one or more doses of the vaccine composition.

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