MERS-CoV VACCINE

Abstract

A recombinant vesicular stomatitis vims (rVSV) carrying at least one gene that encodes for a MERS-CoV structural protein or modifications thereof. Vaccines or immunogenic compositions against MERS-CoV, and prime boost immunization platforms a prime boost immunization combination against MERS-CoV including: (a) a prime vaccine or immunogenic composition comprising a rVSV carrying at least one gene that encodes for a MERS-CoV structural protein or modifications thereof, and (b) a boost vaccine or immunogenic composition comprising a rVSV carrying the same at least one gene that encodes for a MERS-CoV structural protein or modifications thereof. The at least one gene can be genetically modified to encode a modified MERS-CoV structural protein that elevates glycoprotein synthesis and trigger efficient humoral immune response.

Claims

1. An avirulent recombinant vesicular stomatitis virus (rVSV) carrying a vesicular stomatitis virus (VSV) G gene and at least one gene that encodes for a MERS-CoV structural protein or modifications thereof.

2. The rVSV of claim 1, wherein the MERS-CoV structural protein or modifications thereof includes one or more of a full-length spike (S.sub.F) protein of MERS-CoV, a receptor binding domain (RBD) of the S.sub.F protein, an envelope (E) protein of MERS-CoV, or a membrane (M) protein of MERS-CoV, or modifications thereof.

3. The rVSV of claim 1, wherein the at least one gene includes a gene that encodes for a receptor binding domain (RBD) of the spike protein of MERS-CoV (i) having a glycoprotein signal peptide at the NH.sub.2-terminus of the RBD, or (ii) having the glycoprotein signal peptide at the NH.sub.2-terminus of the RBD and a VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the COOH-terminus of the RBD.

4. The rVSV of claim 2, wherein the at least one gene includes a gene that encodes for the S.sub.F protein.

5. The rVSV of claim 2, wherein the at least one gene includes a gene that encodes for the E protein.

6. (canceled)

7. The rVSV of claim 2, wherein the at least one gene includes a gene that encodes for the RBD having a glycoprotein signal peptide at the NH.sub.2-terminus of the RBD and a gene that encodes for the E protein.

8. The rVSV of claim 3, wherein the glycoprotein signal peptide is a melittin signal peptide (msp).

9. The rVSV of claim 1, wherein the rVSV is a replication competent avirulent rVSV of Indiana serotype(rVSV.sub.Ind).

10. The rVSV of claim 9, wherein the rVSV.sub.Ind includes a mutant matrix protein gene.

11. The rVSV of claim 10, wherein the mutant rVSV.sub.Ind matrix protein gene encodes for a matrix protein that includes a GML mutation (rVSV.sub.Ind-GML).

12. The rVSV of claim 1, wherein the rVSV is a replication competent avirulent rVSV of New Jersey serotype (rVSV.sub.NJ).

13. The rVSV of claim 12, wherein the rVSV.sub.NJ includes a mutant matrix protein gene.

14. The rVSV of claim 13, wherein the rVSV.sub.NJ M protein gene encodes for a matrix protein that includes a GMM mutation (rVSV.sub.NJ-GMM) or a GMML mutation (rVSV.sub.NJ-GMML).

15. A MERS-CoV vaccine or immunologenic composition including the avirulent recombinant vesicular stomatitis virus (rVSV) of claim 1.

16. The MERS-CoV vaccine or immunogenic composition of claim 15, wherein the MERS-CoV vaccine or immunogenic composition comprises the avirulent rVSV carrying the gene that encodes for a full-length spike protein of the MERS-CoV, and wherein the avirulent rVSV is rVSV.sub.Ind-GML, rVSV.sub.NJ-GMM or rVSV.sub.NJ-GMML.

17. The MERS-CoV vaccine or immunogenic composition of claim 15, wherein the MERS-CoV vaccine or immunogenic composition comprises the avirulent rVSV carrying the gene that encodes for a RBD having (i) the glycoprotein signal peptide at the NH.sub.2 terminus of the RBD, or (ii) the glycoprotein signal peptide at the NH.sub.2-terminus of the RBD and a VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the COOH-terminus of the RBD, and wherein the avirulent rVSV is rVSV.sub.Ind-GML, rVSV.sub.NJ-GMM or rVSV.sub.NJ-GMML.

18. (canceled)

19. (canceled)

20. The MERS-CoV vaccine or immunogenic composition of claim 15, wherein the MERS-CoV vaccine or immunogenic composition comprises the avirulent rVSV carrying the gene that encodes for the RBD having (i) the glycoprotein signal peptide at the NH.sub.2-terminus of the RBD or (ii) the glycoprotein signal peptide at the NH.sub.2-terminus of the RBD and a VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the COOH-terminus of the RBD, and the gene that encodes for the E protein, and wherein the avirulent rVSV is rVSV.sub.Ind-GML, rVSV.sub.NJ-GMM or rVSV.sub.NJ-GMML.

21. (canceled)

22. The MERS-CoV vaccine or immunogenic composition of claim 17, wherein the glycoprotein signal peptide is a honeybee melittin signal peptide.

23. A prime boost immunization combination against MERS-CoV including: (a) a prime vaccine or immunogenic composition comprising a replication competent avirulent recombinant vesicular stomatitis virus (rVSV) carrying a vesicular stomatitis virus (VSV) G gene and at least one gene that encodes for a MERS-CoV structural protein or a modification thereof, and (b) a booster vaccine or immunogenic composition comprising a replication competent avirulent rVSV carrying the VSV G gene and the same at least one gene that encodes for the MERS-CoV structural protein or modification thereof.

24. The prime boost immunization combination against MERS-CoV of claim 23, wherein the MERS-CoV structural protein or modification thereof includes one or more of a full-length spike (S.sub.F) protein of MERS-CoV, a receptor binding domain (RBD) of the S.sub.F protein, an envelope (E) protein of MERS-CoV, or a membrane (M) protein of MERS-CoV, or any modifications thereof.

25. The prime boost immunization combination against MERS-CoV of claim 23, wherein the at least one gene includes a gene that encodes for a receptor binding domain (RBD) of the spike protein of MERS-CoV (i) having a glycoprotein signal peptide at the NH.sub.2-terminus of the RBD or (ii) having the glycoprotein signal peptide at the NH.sub.2-terminus of the RBD and a VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the COOH-terminus of the RBD.

26. The prime boost immunization platform of claim 23, wherein the at least one gene includes a gene that encodes for a full-length spike protein of MERS-CoV.

27-30. (canceled)

31. The prime boost immunization platform of claim 23, wherein the rVSV of the prime vaccine or immunogenic composition and the rVSV of the booster vaccine or immunogenic composition are rVSV of the same serotype.

32. The prime boost immunization platform of claim 23, wherein at least one of the rVSV of the prime vaccine or immunogenic composition and the rVSV of the booster vaccine or immunogenic composition is rVSV of Indiana serotype (rVSV.sub.Ind), or at least one of the rVSV of the prime vaccine or immunogenic composition and the rVSV of the booster vaccine or immunogenic composition is rVSV of New Jersey serotype (rVSV.sub.NJ).

33-35. (canceled)

36. The prime boost immunization platform of claim 32, wherein the rVSV of the prime vaccine and the rVSV of the booster vaccine include a mutant matrix protein gene of the rVSV, wherein when the rVSV is the at least one rVSV.sub.Ind, the matrix protein of the rVSV.sub.Ind includes a GML mutation (rVSV.sub.Ind-GML), and when the rVSV is the at least one rVSV.sub.NJ, the matrix protein of the rVSV.sub.NJ includes a GMM mutation (rVSV.sub.NJ-GMM) or a GMML mutation (rVSV.sub.NJ-GMML).

37-39. (canceled)

40. A method for inducing an immune response in a mammal against MERS-CoV, comprising administering to the mammal an effective amount of a vaccine or immunogenic composition of claim 15 or administering to the mammal the prime boost immunization platform of claim 23.

41. The method of claim 40, wherein the immune response includes a humoral and a cellular immune response.

42-45. (canceled)

46. The rVSV of claim 3, wherein said RBD having a glycoprotein signal peptide at the NH2-terminus of the RBD is encoded by a gene including SEQ ID NO: 20.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The present invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the invention.

[0053] FIG. 1. Illustration of a MERS CoV clone.

[0054] FIG. 2. (SEQ ID NO: 17) Illustration of MERS CoV spike protein, showing the receptor binding protein.

[0055] FIG. 3. Illustration of the generation of an avirulent VSV.sub.Ind with mutations in the M gene.

[0056] FIG. 4. Illustration of the generation of an avirulent VSV.sub.NJ with mutations in the M gene.

[0057] FIG. 5. (SEQ ID NO: 17 and 18) Cloning MERS-CoV genes (S, RBD, M, S/E and S/E/M) into rVSV.sub.Ind-GML (G21E, M51R, L111A) and rVSV.sub.NJ-GMM (G22E, M48R, M51R).

[0058] FIG. 6. Illustration of recovery of rVSV by reverse genetics (Buchholz, et al., J. Virol. 73:251, 1999).

[0059] FIGS. 7A to 7D. Expression of MERS-CoV proteins (S, RBD, M and E) in three different cell lines (BHK-21 (a), VeroC1008 (b) and Huh-T7 C8 (c)) infected with rVSV.sub.Ind series. 7A: rVSV.sub.Ind-GML, 2. rVSV.sub.Ind-GMLS; 7B: 1. rVSV.sub.Ind-GML, 2. rVSV.sub.Ind-GML-msp-RBD; 7C: rVSV.sub.Ind-GML, 2. rVSV.sub.Ind-GML-M; 7D. 1. rVSV.sub.Ind-GML, 2. rVSV.sub.Ind-GML-E.

[0060] FIGS. 8A to 8F. Expression of MERS-CoV proteins (RBD, M and E) in BHK-21infected with rVSV.sub.NJ-GMM series carrying the genes that encode these MERS-CoV proteins. Panels 8A, 8B, 8C, 8D, 8E and 8F represent Western blot analyses of all three proteins.

[0061] FIGS. 9A to 9C. Detection of MERS-CoV proteins (S, RBD, M and E) in the extracellular culture media of the three different cell lines (BHK-21 (a), VeroC1008 (b) and Huh-T7 C8 (c)) infected with rVSV.sub.Ind series. 9A. 1. rVSV.sub.Ind-GML, 2. rVSV.sub.Ind-GML-S, 3, 4, and 5. rVSV.sub.Ind-GML-mspRBD, 9B. 1. rVSV.sub.Ind-GML, 2. rVSV.sub.Ind-GML-M, 9C. 1. rVSV.sub.Ind-GML, 2. rVSV.sub.Ind-GLM-E.

[0062] FIGS. 10A to 10D. Detection of MERS-CoV proteins (S, RBD, M and E) in pseudotype viral particles from three different cell lines (BHK-21, VeroC1008 and Huh-T7 C8) infected with rVSVNJ-GMM series. 10A. 1. Not infected cells, 2. rVSVInd-GML-S, 10B. 1. Not infected cells, 2. rVSVInd-GML-mspRBD, 10C. 1. Not infected cells, 2. rVSVInd-GML-M, 10D. 1. Not infected cells, 2. rVSVIND-GML-E.

[0063] FIGS. 11A to 11E. Electron microphotographs of sedimentable particles in the concentrated culture media from the infected BHK-21 cells. 11A: electron micrograph of control cells; 11B electron micrograph of cells infected with rVSV.sub.Ind-GML-S and corresponding Western blot; 11C: electron micrograph of cells infected with rVSV.sub.Ind-GML-E and corresponding Western blot; 11D: electron micrograph of cells infected with rVSV.sub.Ind-GML-M and corresponding Western blot; 11E: electron micrograph of cells infected with rVSV.sub.Ind-GML-EM.

[0064] FIG. 12. Illustration of serum antibody titration (MERS groups 1-4 and negative control group).

[0065] FIG. 13. Illustration of antibody tier against MERS-CoV S1 RBD in rabbit sera for the different vaccination groups (MERS groups 1-4 and negative control group).

[0066] FIGS. 14A to 14B. Plaque reduction in neutralization test. 14A. Neutralizing ability of rabbit sera from groups vaccinated with MERS 1-4 and control (PBS) against MERS-CoV EMC/2012. 14B. Positive neutralizing rabbit monoclonal antibody.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0067] For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0068] The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article.

[0069] The terms “animal” and “subject” as used herein includes all members of the animal kingdom including mammals, preferably humans.

[0070] The term “effective amount” as used herein means an amount effective and at dosages and for periods of time necessary to achieve the desired result.

[0071] “rVSV” is used to refer to a recombinant vesicular stomatitis virus.

[0072] The term “Indiana”, and “IND” are used to refer to the VSV serotype Indiana (VSV.sub.Ind). The term “New Jersey”, and “NJ” are used to refer to the VSV serotype New Jersey (VSV.sub.NJ). In aspects of this invention, the VSV.sub.NJ is Hazelhurst strain (VSV.sub.NJ-H) or Ogden strain (VSV.sub.NJ-O).

[0073] “M.sub.WT” “M(WT)” are used to refer to VSV expressing a wild type matrix protein.

[0074] “G22E” is used to refer to a mutant matrix of VSV.sub.NJ having a glycine changed to a glutamic acid at position 22.

[0075] “G21E” is used to refer to a mutant matrix protein of VSV.sub.Ind having a glycine changed to a glutamic acid at position 21.

[0076] “L110A” is used to refer to a mutant matrix protein of VSV.sub.NJ having a leucine changed to alanine at position 110.

[0077] “L111A” is used to refer to a mutant matrix protein protein of VSV.sub.Ind having a leucine changed to alanine at position 111.

[0078] “L110F” is used to refer to a mutant matrix protein of VSV.sub.NJ having a leucine changed to phenylalanine at position 110.

[0079] “L111F” is used to refer to a mutant matrix protein of VSV.sub.Ind having a leucine changed to phenylalanine at position 111.

[0080] “M51R” is used to refer to mutant matrix protein of the VSV.sub.Ind having a methionine changed to an arginine at position 51.

[0081] “M48R+M51R” or “M48R/M51R” are used to refer to a mutant matrix protein of VSV.sub.NJ having a methionine changed to an arginine at positions 48 and 51 respectively.

[0082] “rVSV.sub.Ind(GML)” is used to refer to VSV.sub.Ind having the combined mutation G21E, M51R and one of L111A or L111F.

[0083] “rVSV.sub.NJ(GMM)” is used to refer to a VSV.sub.NJ having the combined mutation G22E, M48R/M51R.

[0084] “rVSV.sub.NJ(GMML)” is used to refer to a VSV.sub.NJ having the combined mutation G22E, M48R/M51R and one of L110A or L110F.

[0085] “S.sub.F” is a recombinant full length spike protein of MERS-CoV.

[0086] “S protein” is used to refer to the S.sub.F or partial length forms of the spike protein of MERS-CoV

[0087] “S1” is a recombinant S1 region or subunit of S.sub.F of MERS-CoV.

[0088] “S2” is a recombinant S2 region or subunit of S.sub.F of MERS-CoV.

[0089] “RBD” is used to refer to the receptor binding domain of the S.sub.F, found in S1 subunit.

[0090] “Partial length of the S protein” is used to refer to one or more of S1, S2 and RBD.

[0091] The term “protein” as used herein is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term protein is inclusive of the terms “peptides” and “proteins”. The terms also encompass an amino acid polymer that has been modified.

[0092] 2. Overview

[0093] The present invention features rVSVs, immunization platforms, immunization regimens and medicaments and kits useful for inducing an immune response in a subject and preventing or treating MERS-CoV infection in a subject, wherein said rVSVs, platforms, regimens and medicaments and useful kits comprise a rVSV that carries one or more genes that encode for one or more structural proteins of MERS-CoV, including modifications of said one or more structural proteins to form pseudotype rVSVs that trigger efficient humoral immune responses against MERS-CoV.

[0094] The MERS-CoV gene can be genetically modified to encode a modified MERS-CoV structural protein that elevates glycoprotein synthesis and triggers efficient humoral immune response. In one embodiment, the MERS-CoV gene is genetically modified to produce modified structural proteins having a glycoprotein signal peptide at its N-terminus. Any glycoprotein signal peptide that allows the MERS-CoV structural protein to be glycosylated and involved in intracellular trafficking can be used, for example the honeybee melittin signal peptide. For example, a gene is genetically modified to produce RBD proteins having a honeybee melittin signal peptide (msp) at its N-terminus or to produce RBD proteins having the msp at its N-terminus, and the transmembrane domain and cytoplasmic tail of the VSV glycoprotein (Gtc) to form pseudotype VSVs that trigger efficient humoral immune responses against the RBD protein.

[0095] In one embodiment, the one or more MERS-CoV structural protein is one or more of a spike (S) protein, a receptor binding domain (RBD) of the S protein, an envelope (E) protein, or a membrane (M) protein of MERS-CoV, modifications of said S, RBD, E and M proteins.

[0096] The S protein of MERS-CoV can be a full-length spike (S.sub.F) protein or a partial length S protein. The partial length form of the S protein is one or more of S1 peptides of the S.sub.F protein, S2 peptides of the S.sub.F protein, the receptor binding domain of the S.sub.F protein (RBD) or any modifications thereof.

[0097] In embodiments, at least one of the S protein (S.sub.F or partial length S protein) and the E protein are modified with a glycoprotein signal peptide, such as the honeybee melittin signal peptide (msp), at the NH2-terminus of the at least one of the S protein (S.sub.F or partial length S protein) and the E protein, and/or the VSV G protein transmembrane domain and cystoplasmic tail (Gtc) at the COOH-terminus of the at least one of the S protein (S.sub.F or partial length S protein) and the E protein.

[0098] In one embodiment, the RBD gene is genetically modified to produce an RBD protein having a honeybee melittin signal peptide (msp) at its NH.sub.2-terminus to glycosylate the RBD that trigger efficient humoral immune responses against MERS-CoV.

[0099] 3. Vaccines or Immunogenic Compositions of the Invention

[0100] The present invention further features vaccines or immunogenic compositions.

[0101] The present invention describes MERS-CoV vaccines or immunogenic compositions including a recombinant vesicular stomatitis virus (rVSV) that carries one or more genes that encode for at least one MERS-CoV structural protein, including at least one of the S protein (full or partial length forms), the E protein, of MERS-CoV, including modifications of said S, and E proteins. The S protein can be provided as a full-length spike (S.sub.F) protein, a S1 subunit of the S.sub.F protein, a S2 subunit of the S.sub.F protein, and/or a receptor binding domain (RBD) of the S.sub.F protein. In embodiments, the at least one of the S (S.sub.F or partial length S protein) and E proteins are modified with a glycoprotein signal peptide such as the honeybee melittin signal peptide (msp) at its NH.sub.2-terminus and/or a VSV G protein transmembrane domain and cystoplasmic tail (Gtc) at the COOH-terminus of the S (S.sub.F or partial length S protein) and/or E protein. In embodiments, the RBD protein is modified to include a glycoprotein signal peptide, such as the honeybee melittin signal peptide (msp) at its NH.sub.2- terminus to form pseudotype rVSVs that trigger efficient humoral immune responses against MERS-CoV. In embodiments, one or more genes that encode for the S (full or partial length forms), and E proteins and modifications therein are codon-optimized for expression in a human cell. The rVSV may be of Indiana serotype, New Jersey serotype or any other suitable VSV subtype.

[0102] The vaccines or immunogenic compositions of this invention may be provided as a prime-boost immunization combination against MERS-CoV. The rVSV of the prime vaccine or immunogenic composition may be of the same or different serotype as the rVSV of the boost vaccine or immunogenic composition. For example, both the prime and boost vaccines or immunogenic compositions are rVSV.sub.Ind; or both the prime and boost vaccines or immunogenic compositions are rVSV.sub.NJ; or the rVSV of the prime vaccine or immunogenic composition is rVSV.sub.Ind and the rVSV of the boost vaccine or immunogenic composition is rVSV.sub.NJ; or the rVSV of the prime vaccine or immunogenic composition is rVSV.sub.NJ and the rVSV of the boost vaccine or immunogenic composition is rVSV.sub.Ind.

[0103] The vaccine or immunogenic compositions of the invention are suitable for administration to subjects in a biologically compatible form in vivo. The expression “biologically compatible form suitable for administration in vivo” as used herein means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances maybe administered to any animal or subject, preferably humans. The vaccines of the present invention may be provided as a lyophilized preparation. The vaccines of the present invention may also be provided as a solution that can be frozen for transportation. Additionally, the vaccines may contain suitable preservatives such as human albumin, bovine albumin, sucrose, glycerol or may be formulated without preservatives. If appropriate (i.e., no damage to the VSV in the vaccine), the vaccines may also contain suitable diluents, adjuvants and/or carriers.

[0104] The dose of the vaccine may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances.

[0105] 4. Methods of Use

[0106] The present invention also features methods of inducing an immune response in a subject against MERS-CoV and/or preventing or treating a MERS-CoV infection in a subject comprising administering to the subject an effective amount of a vaccine or immunogenic composition or a combination of vaccines or immunogenic compositions of the present invention.

[0107] As such, in one embodiment, the present invention provides for a method for inducing an immune response in a subject to a MERS-CoV comprising the step (a) of administering to the subject an effective amount of a vaccine or immunogenic composition including a rVSV carrying one or more geneses that encode for one or more structural protein of MERS-CoV. In one embodiment, the method further comprises the step (b) of administering to the subject another vaccine or immunogenic composition comprising a rVSV carrying the same one or more genes that encode the same one or more structural proteins of MERS-CoV.

[0108] The rVSV of the vaccine or immunogenic composition of step (a), the priming vaccine or immunogenic composition, may be of the same or different serotype as the rVSV of the vaccine or immunogenic composition (b), the booster vaccine or immunogenic composition. For example, both the prime and boost vaccines or immunogenic compositions are rVSV.sub.Ind; or both the prime and boost vaccines or immunogenic compositions are rVSV.sub.NJ; or the rVSV of the prime vaccine or immunogenic composition is rVSV.sub.Ind and the rVSV of the boost vaccine or immunogenic composition is rVSV.sub.NJ; or the rVSV of the prime vaccine or immunogenic composition is rVSV.sub.NJ and the rVSV of the boost vaccine or immunogenic composition is rVSV.sub.Ind.

[0109] In embodiments of the invention the methods for inducing an immune response in a mammal to a MERS-CoV and the methods for preventing or treating an infection caused by MERS-CoV may further comprise the step of (c) administering to the subject an effective amount of the vaccine or immunogenic composition of either step (a) or step (b). Step (c) may be administered to the subject more than one time over the course of inducing an immune response, preventing or treating.

[0110] Advantages

[0111] Advantages of the recombinant VSV-based platform technology of the present invention are first, a highly efficient prime-boost vaccination can be achieved with two antigenically distinct serotypes of rVSV vectors, because the vector immunity against the priming Indiana serotype (VSV.sub.Ind) will not neutralize the boosting New Jersey serotype (VSV.sub.NJ) vector. Thus, VSV.sub.NJ carrying the same gene of interest as rVSV.sub.Ind will provide maximum boost effects. A highly efficient prime-boost vaccination can also be achieved with the same serotype of rVSV vectors (i.e., both the prime and boost are rVSV.sub.Ind or both the prime and boost are rVSV.sub.NJ), because The pseudotype VSVs carrying both VSV G protein and MERS-CoV spike protein on the surface of the virion can bind to either the low-density lipoprotein receptor (LDL-R) by VSV G protein and/or the human dipeptidyl peptidase 4 (hDPP-4) receptor by the spike protein of MERS-CoV. Thus, the vector immunity against one serotype of VSV may not block the infection of the same pseudotype VSV completely. This may provide boost effects.

[0112] Second, the genetically modified VSV.sub.Ind M gene mutant (rVSV.sub.Ind-GML) and genetically modified VSV.sub.NJ M gene mutant (rVSV.sub.NJ-GMM) vectors are completely safe, attenuated temperature sensitive mutants [22]. Third, rVSV.sub.Ind-GML and rVSV.sub.NJ-GMM vectors carrying foreign genes replicate highly efficiently. Therefore, high titer rVSV-based vaccines are relatively easy to prepare. Fourth, both rVSV.sub.Ind-GML and rVSV.sub.NJ-GMM vectors can accommodate a large-size foreign gene with up to 6,000 nucleotides, without decreasing the virus titer [24], and finally both serotypes of VSV have a very wide host range including humans.

[0113] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

EXAMPLES

[0114] The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.

[0115] We have employed a genetically modified dual serotype of vesicular stomatitis virus (VSV) platform technology [22, 23] to develop vaccines against MERS-CoV.

[0116] We have developed MERS-CoV vaccines using temperature-sensitive avirulent rVSV.sub.Ind-GML and rVSV.sub.NJ-GMM as vaccine vectors (FIG. 3, FIG. 4) (Kim et al., 2015). MERS-CoV S, E, and M genes were cloned (FIG. 5), together or separately into the plasmids of rVSV.sub.Ind-GML and rVSV.sub.NJ-GMM for the generation of the attenuated rVSV vectors expressing MERS-CoV structural proteins. Co-expression of Coronavirus S, E, and M proteins form virus-like particles (VLP) (Boscarino et al., 2008; de Haan et al., 2000; Vennema et al., 1996), which are non-pathogenic and could form a proper conformation of trimeric S proteins on the VLP surface. We also cloned only receptor binding domain (RBD) (FIG. 2, FIG. 5, 212 amino acids) of S protein containing honeybee melittin signal peptide (21 aa, FIG. 5) at the NH2-terminus of RBD (mspRBD) into the VSV vectors. Normally S proteins are secreted as a part of the MERS-CoV particles and stimulate the circulating B lymphocytes to generate neutralizing antibodies against epitopes in the RBD of S protein. However, if RBD is expressed without the signal peptide at the NH2-terminus, the protein would not be secreted outside of the cells resulting in less chance of encountering with B lymphocytes. Naturally, S protein is highly glycosylated in the ER, and lack of signal peptide sequence on the RBD makes the protein non-glycosylated. Signal peptides at the amino-terminal region of the secretory proteins target the protein to the ER and Golgi network for the modification of the protein and to the cytoplasmic membrane for the secretion. Honeybee msp increases the overall expression level, glycosylation, and secretion of the protein through cytoplasmic membrane. Therefore, we added honeybee msp sequences to the NH2-terminus of RBD of S protein to increase the expression of the RBD protein (FIG. 5). We also included E protein and M protein as components of MERS-CoV vaccine together with full length S protein (S or S.sub.f) or RBD of S protein and will compare the immunogenicity and efficacy of the vaccine in the presence or absence of E and M proteins.

[0117] We recovered recombinant VSVs expressing MERS-CoV genes using the VSV reverse genetics system (FIG. 6). The newly recovered viruses are rVSV.sub.Ind-GML-S, rVSV.sub.Ind-GML-RBD, rVSV.sub.Ind-GML-M, rVSV.sub.Ind-GML-E, rVSV.sub.Ind-GML-E/M, rVSV.sub.Ind-GML-S/E, rVSV.sub.Ind-GML-mspRBD/E, rVSV.sub.Ind-GML-mspRBD/E/M, rVSV.sub.NJ-GMM-S, rVSV.sub.NJ-GMM-mspRBD, rVSV.sub.NJ-GMM-M, rVSV.sub.NJ-GMM-E, rVSV.sub.NJ-GMM-E/M, rVSV.sub.NJ-GMM-S/E, rVSV.sub.NJ-GMM-mspRBD/E, and rVSV.sub.NJ-GMM-mspRBD/E/M. The recovered viruses were plaque purified three times and amplified in BHK21 cells for virus stock preparation. The intracellular expression of MERS-CoV S, E, and M proteins from the recombinant VSVs were determined by Western blot analysis using rabbit antibodies against S protein (Sino Biological Inc.), rabbit antibodies against E protein (GenScript USA Inc), and rabbit antibodies against M protein (GenScript USA Inc.). Rabbit antibodies against M and E proteins were generated in rabbits using custom-designed linear peptides located at the carboxyl-terminal region of each protein (Table 6, polyclonal antibodies against MERS-CoV Spike protein were purchased from Sino Biological Inc.). BHK2i cells were infected with MOI of 6 of each virus and the cell lysates were prepared at 6 hours post-infection. The 10 μg cell lysates were loaded into the SDS-PAGE gel and MERS-CoV S, RBD, E, and M were detected by Western blot analyses. Proper sizes and good quantities of the MERS-CoV proteins were expressed from the rVSV.sub.Ind-GML (FIG. 7) and rVSV.sub.NJ-GMM (FIG. 8). About 210 kDa size of S protein was detected from the cells infected with rVSV.sub.Ind-GML-S (FIG. 7A), rVSV.sub.Ind-GML-S/E (FIG. 7E) and rVSV.sub.NJ-GMM-S/E (FIG. 8E). About 30 kDa (212 aa, 23kDa) size of mspRBD was detected from the cells infected with rVSV.sub.Ind-GML-mspRBD (FIG. 7A) and rVSV.sub.NJ-GMM-mspRBD (FIG. 8A). RBD migrated slightly slower than the actual size RBD probably because of the glycosylation of the protein. E protein was detected as a protein slightly larger than that of 7 kDa protein and showed double bands in a high concentration SDS-PAGE gel (FIG. 7B, 8E, 9B, and 9E), which we are not sure why E protein shows double bands. M protein was detected as about 24 kDa and 22 kDa protein bands when it was expressed in BHK21 cells (FIG. 7A and FIG. 8A). The predicted molecular mass for the M protein is 24 kDa. We are not certain why M protein was detected as 2 separate bands. The differences may come from the differences in the glycosylation or may come from the cleavage of the protein by a cellular protease. We checked the expression of M protein as well as S, RBD, and E proteins in other cell lines such as green monkey kidney cells (Vero) and human liver cells (Huh 7.5) in order to examine whether they are expressed the same as in BHK.sub.21 cells. The three different cell lines were infected separately with rVSV.sub.Ind-GML-S, rVSV.sub.Ind-GML-M, rVSV.sub.Ind-GML-mspRBD, rVSV.sub.Ind-GML-E (FIG. 9). The infected cells were lysed at 6 hrs post-infection. The expression level and migration pattern in the SDS-PAGE was examined by Western blot analysis (FIG. 9). Full-length S protein was expressed as the same size in all three cell lines, although the expression level was highest in the human liver cell line, Huh7.5 (FIG. 9A line c). M protein was expressed the most in Huh7.5 cells, but the migration pattern was quite different from the M proteins expressed in BHK.sub.21 cells and Vero cells (FIG. 9C). M protein expressed in BHK.sub.21 cells and Vero cells showed the same migration pattern (FIG. 9C line a and 10C line b). In Huh7.5 cells, M protein migrated as one band, but it migrated faster than the slowly migrating band of the two bands from BHK.sub.21 cells and Vero cells (FIG. 9C). It seems that the variability of the M protein expression depends on the origin of the cell lines. We are not certain why M protein shows different expression patterns in different cell lines. The mspRBD and E proteins from three different cell lines showed the same migration pattern (FIG. 9). The expression levels in the three different cell lines were the same for E protein. The mspRBD was expressed the least in Huh7.5 cells (FIG. 9). The expression of VSV proteins in 3 different cell lines did not show much of differences in the level of protein expressions and protein migration patterns in the SDS-PAGE (FIG. 10).

[0118] We also checked the secretion of the MERS-CoV proteins from three different cell lines infected with rVSV.sub.Ind-GML-S, rVSV.sub.Ind-GML-mspRBD, rVSV.sub.Ind-GML-M, and rVSV.sub.Ind-GML-E (FIG. 10). We wanted to examine whether adding honeybee melittin signal peptide (msp) sequence to the NH2-terminus of the RBD makes it secret or not. The three different cell lines were infected with MOI of 6 and were incubated at 37° C. The culture media from the infected cells were collected at 22 hrs post-infection. The collected culture media was centrifuged at 4,000 rpm for 10 minutes to remove cell debris. The secreted extracellular proteins were concentrated by using ultrafiltration device with 5,000 molecular weight cut-off membrane (Sartorius). The concentrated proteins were detected by Western blot analysis (FIG. 10). MERS-CoV full-length S protein was not detected in the samples from all three cell lines indicating that S does not secret when it is expressed alone without other MERS-CoV proteins (FIG. 10A). On the other hand, mspRBD, which has the msp at the NH2-terminus was secreted from all three different infected cell lines (Fig.10A). The mspRBD was secreted the most from BHK21 cells. Huh7.5 cells secreted the least amount of mspRBD. M protein and E protein was either non-detectable or secreted very small amount in BHK.sub.21 cells (FIGS. 10B and 10C) indicating that singly expressed M and E proteins do not secret from the infected cells.

[0119] The concentrated culture media contains recombinant VSVs as well as the enveloped structures, which is made of MERS-CoV S, M, or E proteins. Recombinant VSV particles with randomly incorporated MERS-CoV S, E, and M proteins might be present in the concentrated culture media. In order to detect the MERS-CoV S, mspRBD, M, and E proteins, the culture media from the infected cells were collected at 22 hrs post-infection. The collected media was cleared off cell debris and was concentrated by the ultracentrifugation at 36,000 rpm for 2 hrs. MERS-CoV proteins in the pelleted material were detected by Western blot analysis using antibodies against S, M, and E proteins (FIG. 10). Very little amount of S and M protein was detected in samples from BHK.sub.21 cells and Vero cells (FIG. 10). There was no detectable amount of S and M proteins in the samples from Huh7.5 cells (FIG. 10). The mspRBD was not present in the pelleted samples from all three different cell lines, indicating that mspRBD alone did not form any sedimentable particles. E protein was detected in all three samples from the different cell lines and BHK21 cells produced the most detectable E protein in the concentrated pellet. The presence of E, M, and S protein in the pelleted samples indicated that there were VSV.sub.Ind-GML particles incorporated with these MERS-CoV proteins or sedimented membranous structures with MERS-CoV E, M, and S proteins.

[0120] The Western blot analysis using the pelleted culture media indicated that MERS-CoV S, M, and E proteins were part of the sedimentable particles such as virus-like particles (VLP) and/or pseudotyped VSV particles. Therefore, we wanted to examine the presence of such particles which resemble the MERS-CoV by electron microscopy. We infected BHK.sub.21 cells separately with MOI of 6 of rVSV.sub.Ind-GML-S, rVSV.sub.Ind-GML-E, rVSV.sub.Ind-GML-M, and rVSV.sub.Ind-GML-E/M. The culture media was collected at 22 hrs post-infection. After clearing off cell debris, the culture media was concentrated by ultracentrifugation at 36,000 rpm for 2 hrs. The pelleted material was resuspended in PBS and was fixed in 0.1% glutaraldehyde at room temperature for an hour. The fixed samples were loaded on a formvar/carbon grid and was stained with 1% posphotungstic acid (PTA). All of the samples from BHK.sub.21 cells infected with rVSV.sub.Ind-GML with S (FIG. 11B), E (FIG. 11C), M (FIG. 11D), or E/M (FIG. 11E) contained typical MERS-CoV structures indicating that as long as one of the structural proteins are expressed, MERS-CoV like membranous structures are generated and are secreted from the infected cells (FIG. 11). The secreted VLPs of MERS-CoV structural proteins will present the antigens to B cells as well as T cells for inducing MERS-CoV protein-specific immune responses. This rVSV-MERS-CoV vaccine has been used for immune response studies.

[0121] There is no currently available vaccine against MERS-CoV. Considering the high fatality of the disease, the development of an effective vaccine is required to prevent MERS. Expression of MERS-CoV E, M, and RBD(S) could generate virus-like particles (VLPs) and could induce neutralizing antibodies against MERS-CoV. We generated attenuated rVSVs of both Indiana and New Jersey serotypes expressing MERS-CoV structural proteins, RBD of Spike glycoprotein (S), Envelope protein (E), and Membrane protein (M). The rVSVs of the present invention are noncytolytic and avirulent. We examined the production of neutralization antibodies (nAb) against RBD, a receptor binding domain of Spike glycoprotein. We compared the efficiency of producing nAb by rVSV expressing RBD(S) alone or by rVSV expressing RBD(S), E, and M proteins. We checked the level of nAb against various MERS-CoV isolates. The following animal groups have been vaccinated with rVSV expressing MERS-CoV structural proteins, M, E, and RBD of S proteins. Rabbits were prime-immunized with rVSV.sub.Ind-GML expressing MERS-CoV proteins and boos-immunized with rVSV.sub.NJ-GMM expressing MERS-CoV proteins (Table 1).

[0122] Group 1: As a negative control group, rabbits have been injected with 500 μl of phosphate buffered saline

[0123] Group 2: Rabbits have been injected with rVSV without MERS-CoV gene inserts. Each rabbit was prime immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.Ind-GML, 3 weeks after priming, boost immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.NJ-GMM. Two weeks after boost-immunization, rabbits have been euthanized for serum collection

[0124] Groups 3: Each rabbit was prime immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.Ind-GML MERS-CoV mspRBD(S), 3 weeks after priming, boost immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.NJ-GMM MERS-CoV mspRBD(S). Two weeks after boost-immunization, rabbits have been euthanized for serum collection.

[0125] Groups 4: Each rabbit were prime immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.Ind-GML MERS-CoV mspRBD(S)/E, 3 weeks after priming, boost immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.NJ-GMM MERS-CoV RBD(S)/E. Two weeks after boost-immunization, rabbits have been euthanized for serum collection.

[0126] Group 5: Each rabbit was prime immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.Ind-GML MERS-CoV RBD(S)/E/M, 3 weeks after priming, boost immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.NJ-GMM)-N MERS-CoV RBD(S)/E/M. Two weeks after boost-immunization, rabbits have been euthanized for serum collection.

[0127] We found rabbits immunized with 5×10.sup.8 pfu/500 μl of rVSV.sub.Ind-GML MERS-CoV mspRBD(S) followed by boost immunization with 5×10.sup.8 pfu/500 μl rVSV.sub.NJ-GMM MERS-CoV mspRBD(S) induced high levels of antibodies (FIG. 12, FIG. 13). Moreover, rabbits immunized with 5×10.sup.8 pfu/500 μl rVSV.sub.Ind-GML MERS-CoV mspRBD(S)/E followed by boost immunization with 5×10.sup.8 pfu/500 μl rVSV.sub.NJ-GMM MERS-CoV RBD(S)/E induced equally high levels of neutralizing antibodies (FIG. 14).

[0128] The RBD specific antibodies were generated in rabbits vaccinated with rVSV expressing RBD(S) alone, rVSV expressing RBD(S) and E, and rVSV expressing RBD(S), E, and M (FIG. 12, FIG. 13)). RBD specific antibodies were generated equally well in groups immunized with rVSV expressing RBD(S) alone and rVSV expressing RBD(S) and E, which was shown in the titration curve (FIG. 12, FIG. 13) and in the 1/1600 diluted sera (FIG. 13). We also examined neutralizing ability of the rabbit sera from the vaccinated groups against MERS-CoV EMC/2012 (IVI, Seoul, Korea). The diluted sera were mixed with 200 pfu of MERS-CoV EMC/2012, incubated for 30 min, and the serum-virus mixture was inoculated onto Vero E6 cells. The infected plates were kept in the CO.sub.2 incubator for 3 days until CPE showed 100% in the cells infected with virus only. The results for our rabbit serum samples were compared to a positive neutralizing rabbit monoclonal antibody (Sino Biological, 40069-R723). The rabbit sera from vaccinations with rVSV-MERS-CoV RBD(S) and rVSV-MERS-CoV RBD(S)/E showed 100% to 50% neutralization activity against MERS-CoV EMC/2012 in the dilutions to 1/20 (FIG. 14). Sera from rVSV-MERS-CoV RBD(S)/E vaccination showed better neutralization activity than the sera from rVSV-MERS-CoV RBD(S) (FIG. 14) The results indicated that although the single expression of RBD(S) could induce neutralizing antibodies against MERS-CoV, co-expression of RBD(S) and E protein induce more RBD(S) specific antibodies and neutralize the wild type MERS-CoV EMC/2012 better.

[0129] We are now in position to carry out MERS-CoV challenge experiments. There has been advances made in generating small animal models for the MERS-CoV infection. Mice are not susceptible to MERS-CoV infections, however, when human dipeptidyl peptidase 4 (DPP-4), the cell surface receptor for MERS-CoV, were introduced into mouse genome as a transgene, mice became highly susceptible to MERS-CoV infection (Agrawal et al., 2015; Pascal et al., 2015). The infection in the human dipeptidyl peptidase 4 (hDPP-4) transgenic mice, hDPP-4 C57BL/C led to the replication of MERS-CoV in the lung and showed the typical symptoms of MERS-CoV infection, lethargy, rapid and shallow breathing, severe weight loss, and 40-100% mortality. This transgenic mouse is the perfect animal model to test the efficacy of our vaccines against MERS-CoV. We will vaccinate this hDPP-4 transgenic mice with our rVSVs expressing MERS-CoV structural proteins and challenge the vaccinated mice with wild type MERS-CoV and score the clinical signs and mortality of the vaccinated and unvaccinated control mice. We have a new State-Of-The-Art BSL3 Laboratory just opened at Western University where we can carry out the challenge studies using the hDPP-4 C57BL/C transgenic mice. We will use both rVSV.sub.Ind and rVSV.sub.NJ. In prime-boost vaccines, the priming vaccine or immunogenic composition, are of the same or different serotype as the rVSV of the booster vaccine or immunogenic composition. For example, both the prime and boost vaccines or immunogenic compositions are rVSV.sub.Ind; or both the prime and boost vaccines or immunogenic compositions are rVSV.sub.NJ; or the rVSV of the prime vaccine or immunogenic composition are rVSV.sub.Ind and the rVSV of the boost vaccine or immunogenic composition are rVSV.sub.NJ; or the rVSV of the prime vaccine or immunogenic composition are rVSV.sub.NJ and the rVSV of the boost vaccine or immunogenic composition will be rVSV.sub.Ind.

TABLE-US-00001 TABLE 1 Vaccination groups with various vaccines consisted of MERS-CoV RBS(S), E, and M Prime Boost PBS PBS New Zealar  White 2.5 kg, 5 rVSV (G L)-N rVSV (G )-N 2.5 kg, 5 rVSV (G L)-N-RBD(S) rVSV (G )-N- 2.5 kg, 5 RBD(S) rVSV (G L)-N- rVSV (G )-N- 2.5 kg, 5 RBD(S) E RBD(S) E rVSV (G L)-N- rVSV (G )-N- 2.5 kg, 5 RBD(S) E RBD(S) E 25 indicates data missing or illegible when filed

TABLE-US-00002 TABLE 2 Neucleotide Sequence Comparison between M Genes of VSV Indiana serotype, Wild Type (SEQ ID NO: 1) and a Mutant G21E/L111A/M51R (SEQ ID NO: 2)               1                                                   50 SEQ ID NO: 1: ATGAGTTCCT TAAAGAAGAT TCTCGGTCTG AAGGGGAAAG GTAAGAAATC SEQ ID NO: 2: ATGAGTTCCT TAAAGAAGAT TCTCGGTCTG AAGGGGAAAG GTAAGAAATC               51                                                  100 SEQ ID NO: 1: TAAGAAATTA GGGATCGCAC CACCCCCTTA TGAAGAGGAC ACTAACATGG SEQ ID NO: 2: TAAGAAATTA GAAATCGCAC CACCCCCTTA TGAAGAGGAC ACTAACATGG               101                                                 150 SEQ ID NO: 1: AGTATGCTCC GAGCGCTCCA ATTGACAAAT CCTATTTTGG AGTTGACGAG SEQ ID NO: 2: AGTATGCTCC GAGCGCTCCA ATTGACAAAT CCTATTTTGG AGTTGACGAG               151                                                 200 SEQ ID NO: 1: ATGGACACTC ATGATCCGCA TCAATTAAGA TATGAGAAAT TCTTCTTTAC SEQ ID NO: 2: CGAGACACTC ATGATCCGCA TCAATTAAGA TATGAGAAAT TCTTCTTTAC               201                                                 250 SEQ ID NO: 1: AGTGAAAATG ACGGTTAGAT CTAATCGTCC GTTCAGAACA TACTCAGATG SEQ ID NO: 2: AGTGAAAA G ACGGTTAGAT CTAATCGTCC GTTCAGAACA TACTCAGATG               251                                                 300 SEQ ID NO: 1: TGGCAGCCGC TGTATCCCAT TGGGATCACA TGTACATCGG AATGGCAGGG SEQ ID NO: 2: TGGCAGCCGC TGTATCCCAT TGGGATCACA TGTACATCGG AATGGCAGGG               301                                                 350 SEQ ID NO: 1: AAACGTCCCT TCTACAAGAT CTTGGCTTTT TTGGGTTCTT CTAATCTAAA SEQ ID NO: 2: AAACGTCCCT TCTACAAGAT CTTGGCTTTT GCAGGTTCTT CTAATCTAAA               351                                                 400 SEQ ID NO: 1: GGCCACTCCA GCGGTATTGG CAGATCAAGG TCAACCAGAG TATCACGCTC SEQ ID NO: 2: GGCCACTCCA GCGGTATTGG CAGATCAAGG TCAACCAGAG TATCACGCTC               401                                                 450 SEQ ID NO: 1: ACTGTGAAGG CAGGGCTTAT TTGCCACACA GAATGGGGAA GACCCCTCCC SEQ ID NO: 2: ACTGTGAAGG CAGGGCTTAT TTGCCACACA GAATGGGGAA GACCCCTCCC               451                                                 500 SEQ ID NO: 1: ATGCTCAATG TACCAGAGCA CTTCAGAAGA CCATTCAATA TAGGTCTTTA SEQ ID NO: 2: ATGCTCAATG TACCAGAGCA CTTCAGAAGA CCATTCAATA TAGGTCTTTA               501                                                 550 SEQ ID NO: 1: CAAGGGAACG GTTGAGCTCA CAATGACCAT CTACGATGAT GAGTCACTGG SEQ ID NO: 2: CAAGGGAACG GTTGAGCTCA CAATGACCAT CTACGATGAT GAGTCACTGG               551                                                 600 SEQ ID NO: 1: AAGCAGCTCC TATGATCTGG GATCATTTCA ATTCTTCCAA ATTTTCTGAT SEQ ID NO: 2: AAGCAGCTCC TATGATCTGG GATCATTTCA ATTCTTCCAA ATTTTCTGAT               601                                                 650 SEQ ID NO: 1: TTCAGAGATA AGGCCTTAAT GTTTGGCCTG ATTGTCGAGA AAAAGGCATC SEQ ID NO: 2: TTCAGAGATA AGGCCTTAAT GTTTGGCCTG ATTGTCGAGA AAAAGGCATC               651                                                 700 SEQ ID NO: 1: TGGAGCTTGG GTCCTGGATT CTGTCAGCCA CTTCAAATGA SEQ ID NO: 2: TGGAGCTTGG GTCCTGGATT CTGTCAGCCA CTTCAAATGA

TABLE-US-00003 TABLE 3 AmiNO: Acid Sequence Comparison between M Proteins of VSV Indiana serotype Wild Type (SEQ ID NO: 3) and a Mutant G21E/L111A/M51R (SEQ ID NO: 4)               1                    21                             50 SEQ ID NO: 3: MSSLKKILGL KGKGKKSKKL GIAPPPYEED TNMEYAPSAP IDKSYFGVDE SEQ ID NO: 4: MSSLKKILGL KGKGKKSKKL EIAPPPYEED TNMEYAPSAP IDKSYFGVDE               51                                                  100 SEQ ID NO: 3: MDTHDPHQLR YEKFFFTVKM TVRSNRPFRT YSDVAAAVSH WDHMYIGMAG SEQ ID NO: 4: RDTHDPHQLR YEKFFFTVKM TVRSNRPFRT YSDVAAAVSH WDHMYIGMAG               101       111                                       150 SEQ ID NO: 3: KRPFYKILAF LGSSNLKATP AVLADQGQPE YHAHCEGRAY LPHRMGKTPP SEQ ID NO: 4: KRPFYKILAF AGSSNLKATP AVLADQGQPE YHAHCEGRAY LPHRMGKTPP               151                                                 200 SEQ ID NO: 3: MLNVPEHFRR PFNIGLYKGT VELTMTIYDD ESLEAAPMIW DHFNSSKFSD SEQ ID NO: 4: MLNVPEHFRR PFNIGLYKGT VELTMTIYDD ESLEAAPMIW DHFNSSKFSD               201                           229                   250 SEQ ID NO: 3: FRDKALMFGL IVEKKASGAW VLDSVSHFK SEQ ID NO: 4: FRDKALMFGL IVEKKASGAW VLDSVSHFK

TABLE-US-00004 TABLE 4 Nucleotide Sequence Comparison between M Genes of VSV New Jersey serotype Wild Type (SEQ ID NO: 5) and Mutants, G22E/M48R/M51R (SEQ ID NO: 6) and G22E/L110A/M48R/M51R (SEQ ID NO: 7)               1                                                   50 SEQ ID NO: 5: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA SEQ ID NO: 6: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA SEQ ID NO: 7: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA               51                                                  100 SEQ ID NO: 5: ATCAAAGAAA CTAGGCTTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG SEQ ID NO: 6: ATCAAAGAAA CTAGAATTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG SEQ ID NO: 7: ATCAAAGAAA CTAGAATTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG               101                                                 150 SEQ ID NO: 5: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG AATGGAGGAT SEQ ID NO: 6: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG ACGAGAGGAT SEQ ID NO: 7: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG ACGAGAGGAT               151                                                 200 SEQ ID NO: 5: ATGGATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT SEQ ID NO: 6: CGAGATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT SEQ ID NO: 7: CGAGATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT               201                                                 250 SEQ ID NO: 5: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG SEQ ID NO: 6: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG SEQ ID NO: 7: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG               251                                                 300 SEQ ID NO: 5: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA SEQ ID NO: 6: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA SEQ ID NO: 7: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA               301                                                 350 SEQ ID NO: 5: AAGCGTCCTT TCTACAAGAT AATTGCTCTG ATTGGCTCCA GTCATCTGCA SEQ ID NO: 6: AAGCGTCCTT TCTACAAGAT AATTGCTCTG ATTGGCTCCA GTCATCTGCA SEQ ID NO: 7: AAGCGTCCTT TCTACAAGAT AATTGCTGCA ATTGGCTCCA GTCATCTGCA               351                                                 400 SEQ ID NO: 5: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA SEQ ID NO: 6: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA SEQ ID NO: 7: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA               401                                                 450 SEQ ID NO: 5: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG SEQ ID NO: 6: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG SEQ ID NO: 7: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG               451                                                 500 SEQ ID NO: 5: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA SEQ ID NO: 6: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA SEQ ID NO: 7: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA               501                                                 550 SEQ ID NO: 5: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG SEQ ID NO: 6: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG SEQ ID NO: 7: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG               551                                                 600 SEQ ID NO: 5: AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG SEQ ID NO: 6: AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG SEQ ID NO: 7: AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG               601                                                 650 SEQ ID NO: 5: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAAC SEQ ID NO: 6: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAAC SEQ ID NO: 7: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAAC               651                                                 700 SEQ ID NO: 5: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAA SEQ ID NO: 6: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAA SEQ ID NO: 7: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAA

TABLE-US-00005 TABLE 5 AmiNO: Acid Sequence Comparison between M Proteins of VSV New Jersey serotype Wild Type (SEQ ID NO: 8) and Mutants, G22E/M48R/M51R (SEQ ID NO: 9) and G22E/L110A/M48R/M51R (SEQ ID NO: 10)                1                      22                           50 SEQ ID NO: 8: MSSFKKILGF SSKSHKKSKK LGLPPPYEES SPMEIQPSAP LSNDFFGMED SEQ ID NO: 9: MSSFKKILGF SSKSHKKSKK LELPPPYEES SPMEIQPSAP LSNDFFGRED SEQ ID NO: 10:MSSFKKILGF SSKSHKKSKK LELPPPYEES SPMEIQPSAP LSNDFFGRED               51                                                  100 SEQ ID NO: 8: MDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG SEQ ID NO: 9: RDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG SEQ ID NO: 10:RDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG               101      110                                        150 SEQ ID NO: 8: KRPFYKIIAL IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPP SEQ ID NO: 9: KRPFYKIIAL IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPP SEQ ID NO: 10:KRPFYKIIAA IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPP               151                                                 200 SEQ ID NO: 8: MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ SEQ ID NO: 9: MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ SEQ ID NO: 10:MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ               201                                                 250 SEQ ID NO: 8: IQKEGLKFGL ILSKKATGTW VLDQLSPFK SEQ ID NO: 9: IQKEGLKFGL ILSKKATGTW VLDQLSPFK SEQ ID NO: 10:IQKEGLKFGL ILSKKATGTW VLDQLSPFK

TABLE-US-00006 TABLE 6 MERS-CoV E protein (82 aa) (SEQ ID NO: 11) MLPFVQERIGLFIVNFFIFTVVCAITLLVCMAFLTATRLCV QCMTGFNTLLVQPALYLYNTGRSVYVKFQDSKPPLPPDEWV Peptide for antibody (14mer) against MERS- CoV E protein SEQ ID NO: 12: FQDSKPPLPPDEWV MERS-CoV M Protein (219 aa) (SEQ ID NO: 13) MSNMTQLTEAQIIAIIKDWNFAWSLIFLLITIVLQYGYPSR SMTVYVFKMFVLWLLWPSSMALSIFSAVYPIDLASQIISGI VAAVSAMMWISYFVQSIRLFMRTGSWWSFNPETNCLLNVPF GGTTVVRPLVEDSTSVTAVVTNGHLKMAGMHFGACDYDRLP NEVTVAKPNVLIALKMVKRQSYGTNSGVAIYHRYKAGNYRS PPITADIELALLRA Peptides for antibody against MERS-CoV M protein (14mer) SEQ ID NO: 14: TVVRPLVEDSTSVT (M11) SEQ ID NO: 15: LKMVKRQSYGTNSG (M8) SEQ ID NO: 16: YKAGNYRSPPITAD (M5) Melittin Signal Peptide Gene (SEQ ID NO: 17) ATG AAA TTC TTA GTC AAC GTT GCC CTT GTT TTT ATG GTC GTG TAC ATT TCT TAG ATC TAT GCG IG:VSV Intergenic Junction sequence (SEQ ID NO: 18) CATATGAAAAAAACTAACAGATATC S Full Length (SF) gene (SEQ ID NO: 19) GTTTAAACCATATGAAAAAAACTAACAGATATCATGATTCA CTCTGTGTTCCTGCTGATGTTCCTGCTGACACCAACAGAGT CCTATGTGGATGTGGGACCTGACTCTGTGAAGTCTGCCTGT ATTGAGGTGGACATCCAACAGACCTTCTTTGACAAGACCTG GCCAAGACCAATTGATGTGAGCAAGGCTGATGGCATCATCT ACCCACAGGGCAGGACCTACAGCAACATCACCATCACCTAC CAGGGACTGTTTCCATACCAGGGAGATCATGGAGATATGTA TGTCTACTCTGCTGGTCATGCCACAGGCACCACACCACAGA AACTGTTTGTGGCTAACTACAGCCAGGATGTGAAGCAGTTT GCCAATGGCTTTGTGGTGAGGATTGGAGCAGCAGCCAACAG CACAGGCACAGTGATTATCAGCCCAAGCACCTCTGCCACCA TCAGGAAGATTTACCCTGCCTTTATGCTGGGCTCCTCTGTG GGCAACTTCTCTGATGGCAAGATGGGCAGGTTCTTCAACCA CACCCTGGTGCTGCTGCCTGATGGCTGTGGCACCCTGCTGA GGGCTTTCTACTGTATCTTGGAACCAAGGTCTGGCAACCAC TGTCCTGCTGGCAACTCCTACACCTCCTTTGCCACCTACCA CACACCTGCCACAGACTGTTCTGATGGCAACTACAACAGGA ATGCCTCCCTGAACTCCTTCAAGGAATACTTCAACCTGAGG AACTGTACCTTTATGTACACCTACAACATCACAGAGGATGA GATTTTGGAGTGGTTTGGCATCACCCAGACAGCCCAGGGAG TGCATCTGTTCTCGAGCAGATATGTGGACCTCTATGGAGGC AATATGTTCCAGTTTGCCACCCTGCCTGTCTATGACACCAT CAAATACTACAGCATCATCCCACACAGCATCAGGAGCATCC AGTCTGACAGGAAGGCTTGGGCTGCCTTCTATGTCTACAAA CTCCAACCACTGACCTTCCTGCTGGACTTCTCTGTGGATGG CTACATCAGGAGGGCTATTGACTGTGGCTTCAATGACCTGA GCCAACTTCACTGTTCCTATGAGTCCTTTGATGTGGAGTCT GGAGTCTACTCTGTGTCCTCCTTTGAGGCTAAGCCATCTGG CTCTGTGGTGGAACAGGCTGAGGGAGTGGAGTGTGACTTCA GCCCACTGCTGTCTGGCACACCTCCACAGGTCTACAACTTC AAGAGACTGGTGTTCACCAACTGTAACTACAACCTGACCAA ACTGCTGTCCCTGTTCTCTGTGAATGACTTCACTTGTAGCC AGATTAGCCCTGCTGCCATTGCCAGCAACTGTTACTCCTCC CTGATTCTGGACTACTTCTCCTACCCACTGAGTATGAAGTC TGACCTGTCTGTGTCCTCTGCTGGACCAATCAGCCAGTTCA ACTACAAGCAGTCCTTCAGCAACCCAACTTGTCTGATTCTG GCTACAGTGCCACACAACCTGACCACCATCACCAAGCCACT GAAATACTCCTACATCAACAAGTGTAGCAGACTGCTGTCTG ATGACAGGACAGAGGTGCCACAACTAGTGAATGCCAACCAA TACAGCCCATGTGTGAGCATTGTGCCAAGCACAGTGTGGGA GGATGGAGACTACTACAGGAAGCAACTTAGCCCATTGGAGG GAGGAGGCTGGCTGGTGGCATCTGGCAGCACAGTGGCTATG ACAGAACAACTCCAAATGGGCTTTGGCATCACAGTCCAATA TGGCACAGACACCAACTCTGTGTGTCCAAAATTGGAGTTTG CCAATGACACCAAGATTGCCAGCCAACTTGGCAACTGTGTG GAATACTCCCTCTATGGAGTGTCTGGCAGGGGAGTGTTCCA GAACTGTACTGCTGTGGGAGTGAGACAACAGAGGTTTGTCT ATGATGCCTACCAGAACCTGGTGGGCTACTACTCTGATGAT GGCAACTACTACTGTCTGAGGGCTTGTGTGTCTGTGCCTGT GTCTGTGATTTATGACAAGGAGACCAAGACCCATGCCACCC TGTTTGGCTCTGTGGCTTGTGAACACATCTCCAGCACAATG AGTCAATACAGCAGGAGCACCAGGAGTATGCTGAAAAGGAG GGACAGCACATATGGACCACTCCAAACACCTGTGGGCTGTG TGCTGGGACTGGTGAACTCCTCCCTGTTTGTGGAGGACTGT AAACTGCCACTGGGACAATCCCTGTGTGCCCTGCCTGACAC ACCAAGCACCCTGACACCAAGGTCTGTGAGGTCTGTGCCTG GAGAGATGAGACTGGCAAGCATTGCCTTCAACCACCCAATC CAGGTGGACCAACTTAACTCCTCCTACTTCAAACTGAGCAT CCCAACCAACTTCTCCTTTGGAGTGACCCAGGAATACATCC AGACCACCATCCAGAAGGTGACAGTGGACTGTAAGCAATAT GTGTGTAATGGCTTCCAGAAGTGTGAACAACTTCTGAGGGA ATATGGACAATTCTGTAGCAAGATAAACCAGGCTCTTCATG GAGCCAACCTGAGACAGGATGACTCTGTGAGGAACCTGTTT GCCTCTGTGAAGTCCAGCCAGTCCAGCCCAATCATCCCTGG CTTTGGAGGAGACTTCAACCTGACCCTGTTGGAACCGGTGA GCATCAGCACAGGCAGCAGGTCTGCCAGGTCTGCCATTGAG GACCTGCTGTTTGACAAGGTGACCATTGCTGACCCTGGCTA TATGCAGGGCTATGATGACTGTATGCAACAGGGACCTGCCT CTGCCAGGGACCTGATTTGTGCCCAATATGTGGCTGGCTAC AAGGTGCTGCCTCCACTGATGGATGTGAATATGGAGGCTGC CTACACCTCCTCCCTGCTGGGCAGCATTGCTGGAGTGGGCT GGACTGCAGGACTGTCCTCCTTTGCTGCCATCCCATTTGCC CAGAGCATCTTCTACAGACTGAATGGAGTGGGCATCACCCA ACAGGTGCTGTCTGAGAACCAGAAACTGATTGCCAACAAGT TCAACCAGGCTCTGGGAGCTATGCAGACAGGCTTCACCACC ACCAATGAGGCTTTCCAGAAGGTCCAGGATGCTGTGAACAA CAATGCCCAGGCTCTGAGCAAACTGGCATCTGAACTGAGCA ACACCTTTGGAGCCATCTCTGCTAGCATTGGAGACATCATC CAGAGACTGGATGTGTTGGAACAGGATGCCCAGATTGACAG ACTGATAAATGGCAGACTGACCACCCTGAATGCCTTTGTGG CTCAACAACTTGTGAGGTCTGAGTCTGCTGCCCTGTCTGCC CAACTTGCCAAGGACAAGGTGAATGAGTGTGTGAAGGCTCA AAGCAAGAGGTCTGGCTTCTGTGGACAAGGCACCCACATTG TGTCCTTTGTGGTGAATGCCCCAAATGGACTCTACTTTATG CATGTGGGCTACTACCCAAGCAACCACATTGAGGTGGTGTC TGCCTATGGACTGTGTGATGCTGCCAACCCAACCAACTGTA TTGCCCCTGTGAATGGCTACTTCATCAAGACCAACAACACC AGGATTGTGGATGAGTGGTCCTACACAGGCTCCTCCTTCTA TGCCCCTGAACCAATCACCTCCCTGAACACCAAATATGTGG CTCCACAGGTGACCTACCAGAACATCAGCACCAACCTGCCT CCTCCACTGCTGGGCAACAGCACAGGCATTGACTTCCAGGA TGAACTGGATGAGTTCTTCAAGAATGTGAGCACCAGCATCC CAAACTTTGGCTCCCTGACCCAGATAAACACCACCCTGCTG GACCTGACCTATGAGATGCTGTCCCTCCAACAGGTGGTGAA GGCTCTGAATGAGTCCTACATTGACCTGAAAGAACTGGGCA ACTACACCTACTACAACAAGTGGCCATGGTACATCTGGCTG GGCTTCATCGCTGGCCTGGTGGCCCTGGCGCTGTGCGTGTT CTTCATCCTGTGCTGCACCGGCTGCGGCACCAACTGCATGG GCAAGCTGAAGTGCAACAGGTGCTGCGACAGGTACGAGGAG TACGACCTGGAGCCCCACAAGGTGCACGTACATTAAACGCG T Msp-RBD (SEQ ID NO: 20) GTTTAAACCATATGAAAAAAACTAACAGATATCATGAAATT CTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTGTACATTT CTTACATCTATGCGCAGGCTGAGGGAGTGGAGTGTGACTTC AGCCCACTGCTGTCTGGCACACCTCCACAGGTCTACAACTT CAAGAGACTGGTGTTCACCAACTGTAACTACAACCTGACCA AACTGCTGTCCCTGTTCTCTGTGAATGACTTCACTTGTAGC CAGATTAGCCCTGCTGCCATTGCCAGCAACTGTTACTCCTC CCTGATTCTGGACTACTTCTCCTACCCACTGAGTATGAAGT CTGACCTGTCTGTGTCCTCTGCTGGACCAATCAGCCAGTTC AACTACAAGCAGTCCTTCAGCAACCCAACTTGTCTGATTCT GGCTACAGTGCCACACAACCTGACCACCATCACCAAGCCAC TGAAATACTCCTACATCAACAAGTGTAGCAGACTGCTGTCT GATGACAGGACAGAGGTGCCACAACTAGTGAATGCCAACCA ATACAGCCCATGTGTGAGCATTGTGCCAAGCACAGTGTGGG AGGATGGAGACTACTACAGGAAGCAACTTAGCCCATTGGAG GGAGGAGGCTGGCTGGTGGCATCTGGCAGCACAGTGGCTAT GACAGAACAACTCCAAATGGGCTTTGGCATCACAGTCCAAT ATGGCACAGACACCAACTCTGTGTGTCCAAAATTGTAA MERS-CoV E Gene (SEQ ID NO: 21) GTTTAAACCATATGAAAAAAACTAACAGATATCATGTTACC CTTTGTCCAAGAACGAATAGGGTTGTTCATAGTAAACTTTT TCATTTTTACCGTAGTATGTGCTATAACACTCTTGGTGTGT ATGGCTTTCCTTACGGCTACTAGATTATGTGTGCAATGTAT GACAGGCTTCAATACCCTGTTAGTTCAGCCCGCATTATACT TGTATAATACTGGACGTTCAGTCTATGTAAAATTCCAGGAT AGTAAACCCCCTCTACCACCTGACGAGTGGGTTTAACCCGG GACGCGT MERS-CoV M Gene (SEQ ID NO: 22) GTTTAAACCATATGAAAAAAACTAACAGATATCATGTCTAA TATGACGCAACTCACTGAGGCGCAGATTATTGCCATTATTA AAGACTGGAACTTTGCATGGTCCCTGATCTTTCTCTTAATT ACTATCGTACTACAGTATGGATACCCATCCCGTAGTATGAC TGTCTATGTCTTTAAAATGTTTGTTTTATGGCTCCTATGGC CATCTTCCATGGCGCTATCAATATTTAGCGCCGTTTATCCA ATTGATCTAGCTTCCCAGATAATCTCTGGCATTGTAGCAGC TGTTTCAGCTATGATGTGGATTTCCTACTTTGTGCAGAGTA TCCGGCTGTTTATGAGAACTGGATCATGGTGGTCATTCAAT CCTGAGACTAATTGCCTTTTGAACGTTCCATTTGGTGGTAC AACTGTCGTACGTCCACTCGTAGAGGACTCTACCAGTGTAA CTGCTGTTGTAACCAATGGCCACCTCAAAATGGCTGGCATG CATTTCGGTGCTTGTGACTACGACAGACTTCCTAATGAAGT CACCGTGGCCAAACCCAATGTGCTGATTGCTTTAAAAATGG TGAAGCGGCAAAGCTACGGAACTAATTCCGGCGTTGCCATT TACCATAGATATAAGGCAGGTAATTACAGGAGTCCGCCTAT TACGGCGGATATTGAACTTGCATTGCTTCGAGCTTAGACGC GT

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[0143] It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention.

[0144] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.