WO2021245679A1 - A sars-cov-2 vaccine - Google Patents

Abstract

The present invention provides a recombinant vesicular stomatitis virus (VSV) comprising a SARS-CoV-2 spike protein (rV SV-AG- spike), as well as immunogenic compositions comprising rVSV-AG-spike, and their use for vaccination against SARS- CoV-2.

Description

A SARS-CoV-2 VACCINE

TECHNOLOGICAL FIELD

The present disclosure relates to a SARS-CoV-2 vaccine, specifically to a Vesicular stomatitis virus (VSV)-based vaccination platform.

BACKGROUND

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus, a member of the Coronaviridae family that causes the COVID-19 disease. The virus was first identified in December 2019 in Wuhan, China. Since then, over 166.8 million cases worldwide were diagnosed, with over 3.4 million deaths.

VSV, Vesicular Stomatitis Virus (also known as "Vesicular stomatitis Indiana virus" or "Indiana vesiculovirus"), is a non-segmented single-stranded, negative sense RNA virus, a member of the Khabdoviridae family. VSV causes a disease in animals, with a broad host range in mammals and insects, however, cases of human infection with VSV are rare.

VSV genome codes for five major proteins, termed matrix protein (M), nucleoprotein (N), large polymerase protein (L), phosphoprotein (P), and glycoprotein (G). The L and P proteins, together with the N, form the transcriptionally active subunit of the virus. The G protein mediates both viral binding and host cell fusion with the endosomal membrane following endocytosis, and cell entry.

Based on the VSV genome, a recombinant expression platform (rVSV) was established (Whitt, M. A. et al, 2010, J. Virol Methods 169(2): 365-374; Lawson, N. D. et al., 1995, PNAS 92: 4477-4481). The recombinant system is based on the replacement of the VSV endogenous G protein with a heterologous glycoprotein of a different virus, e.g., the Ebola virus.

The rVSV was used as a vaccine platform for several viral pathogens, such as Ebola virus (EBOV), AIDS (HIV) and Crimean-Congo Hemorrhagic Fever (CCHFV) (Suder, E. et al. , 2018, HUMAN VACCINES & IMMUNOTHERAPEUTICS 14(9): 2107-2113; Rodriguez, S. E. et al, 2019, Scientific Reports 9: 7755).

SARS-CoV-2 is a single stranded positive sense RNA virus, containing a viral surface glycoprotein (S). The S protein is a highly glycosylated type I membrane protein. The trimeric organization of the S protein on the viral membrane forms the spike structures, typical to coronaviruses. It has been previously shown that the spike binds to the ACE2 (angiotensin-converting enzyme 2) receptor. This binding induces membrane fusion and entry of the SARS-CoV-2 into the cells, hence serves as a target for neutralizing antibodies.

The SARS-CoV-2 S protein is composed of two distinct subunits, SI and S2. The surface unit SI binds the cellular receptor, whereas the transmembrane unit S2 facilitates fusion of the viral and cell membranes. The spike is activated by a cleavage at the spike's S1/S2 site by host cell's proteases (Hoffmann, M. et al. , 2020, Molecular Cell 78: 1-6).

Petit, C. M. et al. , (2005, Virology 341: 215-230) evaluated the role of different domains of the spike protein of SARS-CoV-1, by performing single mutations, as well as truncation mutations, in SARS-CoV-1 spike protein. C-terminus truncation of 17 amino acids of the spike protein was found to increase spike-mediated cell-to-cell fusion. Interestingly, a 26 amino acid truncation was found to have an opposite effect.

GENERAL DESCRIPTION

The present invention provides a recombinant vesicular stomatitis virus (VSV) comprising: a VSV nucleocapsid protein (N), a VSV phosphoprotein (P), a VSV matrix protein (M), a VSV large polymerase protein (L), and a SARS-CoV-2 spike protein or an immunogenic fragment thereof, wherein said recombinant VSV is lacking the VSV G protein.

In some embodiments the recombinant VSV of the present invention is wherein the SARS-CoV-2 spike protein is a wild-type SARS-CoV-2 spike protein, a mutated SARS-CoV-2 spike protein, a chimeric SARS-CoV-2 spike protein or an immunogenic fragment thereof.

In other embodiments the recombinant VSV of the present invention is wherein the SARS-CoV-2 spike protein is truncated at the C-terminus. In further embodiments the recombinant VSV of the present invention is wherein the SARS-CoV-2 spike protein lacks 24 amino acids at the C-terminus.

In still further embodiments the recombinant VSV of the present invention is wherein the SARS-CoV-2 spike protein has a mutation in the Arginine (R) residue at the fourth position (R4) of the S1/S2 motif RRAR.

In some embodiments the recombinant VSV of the present invention is wherein the mutation at the R4 residue of the S1/S2 motif RRAR is an R to G substitution or an R to K substitution.

In other embodiments the recombinant VSV of the present invention is wherein the mutation at the R4 residue of the S1/S2 motif RRAR is at amino acid position 685 of the SARS-CoV-2 spike protein as denoted by SEQ ID NO. 16 or SEQ ID NO. 17.

In certain embodiments the recombinant VSV of the present invention is wherein the SARS-CoV-2 spike protein lacks 24 amino acids at the C-terminus and has a mutation at the fourth Arginine (R) residue of the S1/S2 motif RRAR.

In some embodiments the recombinant VSV of the present invention is wherein the SARS-CoV-2 spike protein comprises the amino acid sequence set forth in SEQ ID NO. 16 or the amino acid sequence set forth in SEQ ID NO. 17, or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

In other embodiments the recombinant VSV of the present invention is wherein the recombinant VSV is encoded by a nucleic acid sequence comprising the nucleic acid sequence set forth in SEQ ID NO. 18 or the nucleic acid sequence set forth in SEQ ID NO. 19, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

In other embodiments the recombinant VSV of the present invention is wherein the SARS-CoV-2 spike protein lacks 24 amino acids at the C-terminus and has one or more amino acid substitutions at any one of positions 685, 245, or 813 or any combination thereof.

In some embodiments said one or more amino acid substitution is any one of a R to G substitution at position 685, an H to R substitution at position 245, or an S to L substitution at position 813, or any combination thereof. For example, as set forth in SEQ ID NO: 24 wherein independently X at position 685 is selected from R, G and K; X at position 245 is selected from H and R; and X at position 813 is selected from S, L, and I.

In one embodiment, the SARS-CoV-2 spike protein comprises a R to G substitution at position 685, an H to R substitution at position 245, and an S to L substitution at position 813. For example, as set forth in SEQ ID NO: 24 wherein X at position 685 is G; X at position 245 is R; and X at position 813 is L.

In one embodiment, the SARS-CoV-2 spike protein comprises the amino acid sequence set forth in SEQ ID NO. 21, or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

In one embodiment, the SARS-CoV-2 spike protein in the recombinant VSV is encoded by a nucleic acid sequence comprising the nucleic acid sequence set forth in SEQ ID NO. 22, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

In some embodiments, the SARS-CoV-2 spike protein in the recombinant VSV has the amino acid sequence set forth in SEQ ID NO. 21, and further comprises one or more amino acid substitutions at any one of positions 64, 66, 113, 484, 493, 501 or 615, or any combination thereof.

In some embodiments, said one or more amino acid substitutions in SEQ ID NO. 21 are selected from the group consisting of S 8131, W64R, H66R, K113Q, E484D, Q493R, N501Y, and V615L. For example, as set forth in SEQ ID NO: 25 wherein independently X at position 64 is selected from W and R; X at position 66 is selected from R and H; X at position 113 is selected from Q and K; X at position 484 is selected from D and E; X at position 493 is selected from R and Q; X at position 501 is selected from Y and N; X at position 615 is selected from L and V; and X at position 813 is selected from S and I.

The present invention further provides an immunogenic composition comprising the recombinant VSV as herein defined and a pharmaceutically acceptable excipient, carrier, or diluent.

In one embodiment, said composition comprises a heterogeneous population of recombinant VSV, said heterogeneous population comprising recombinant VSV with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 and recombinant VSV with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 further comprising one or more mutations selected from the group consisting of S 8131, W64R, H66R, K113Q, E484D, Q493R, N501Y, and V615L. For example, as set forth in SEQ ID NO: 25 wherein independently X at position 64 is selected from W and R; X at position 66 is selected from R and H; X at position 113 is selected from Q and K; X at position 484 is selected from D and E; X at position 493 is selected from R and Q; X at position 501 is selected from Y and N; X at position 615 is selected from L and V; and X at position 813 is selected from S and I.

In one embodiment, said recombinant VSV with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 has a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% frequency in said heterogenous population.

In one embodiment, said recombinant VSV with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 further comprising one or more mutations has a frequency of between about 5% to about 100% in said heterogenous population.

In some embodiments the immunogenic composition of the present invention is for eliciting an immune response against SARS-CoV-2 in a subject.

In one embodiment, said SARS-CoV-2 is a SARS-CoV-2 variant.

In some embodiments, said SARS-CoV-2 variant is selected form the group consisting of the United Kingdom variant, the South Africa variant, and the Brazil variant.

In other embodiments the immunogenic composition of the present invention is further comprising an adjuvant.

In some embodiments, said adjuvant is selected from a group consisting of aluminum containing compounds (such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate), immunostimulatory compounds (such as liposaccharides, lipopoly saccharides, immunostimulatory nucleic acids, e.g., CpG oligonucleotides, liposomes, Toll-like Receptor agonists e.g., TLR2, TLR3, TLR4, TLR7/8 or TLR9 agonists), and combinations thereof.

In various embodiments the immunogenic composition of the present invention is wherein the immunogenic composition is administered by injection. In still further embodiments the immunogenic composition of the present invention is wherein the injection is performed intramuscularly, subcutaneously or intradermally.

In the above and other embodiments, the immunogenic composition of the present invention is wherein the immunogenic composition is delivered to a mucosa.

In some embodiments the immunogenic composition of the present invention is wherein the immunogenic composition is delivered by nasal spraying or mouth spraying.

In other embodiments the immunogenic composition of the present invention is wherein the immunogenic composition is administered in a single-dose regimen.

In further embodiments the immunogenic composition of the present invention is wherein the immunogenic composition is administered in a multiple-dose regimen (e.g., a prime-boost regimen).

In one embodiment, the immunogenic composition comprises between 10⁴ and 10⁸ PFU/ml in solution with 20 mM Tris, 2.5 mg/ml serum albumin, 4% trehalose, and 150 mM NaCl.

By a further aspect thereof the present invention provides the recombinant VSV, or the immunogenic composition as herein defined for use as a vaccine.

Still further the present invention provides a kit comprising the immunogenic composition of the present invention and instructions for the use of the immunogenic composition.

The present invention further provides a method of eliciting an immune response towards SARS-CoV-2 in a subject, the method comprising administering the immunogenic composition as herein defined to said subject.

In one embodiment, said SARS-CoV-2 is a SARS-CoV-2 variant.

In some embodiments, said SARS-CoV-2 variant is selected from the group consisting of the United Kingdom variant, the South Africa variant, and the Brazil variant.

By another one of its aspects the present invention provides a method of producing a recombinant VSV comprising SARS-CoV-2 spike protein, comprising the steps of: a. constructing a plasmid comprising the genes encoding for VSV nucleocapsid protein (N), VSV phosphoprotein (P), VSV matrix protein (M), VSV polymerase (L), and SARS-CoV-2 spike protein or an immunogenic fragment thereof (rVSV-AG-spike); b. inserting the rVSV-AG-spike plasmid into a cell together with accessory plasmids, wherein said accessory plasmids comprising the cis acting signals for VSV replication and genes encoding the VSV N, P, L and G proteins; c. culturing the cell in culture media under conditions that permit expression of the rVSV-AG-spike plasmid and production of said recombinant VSV comprising SARS-CoV-2 spike protein; and d. isolating said recombinant VSV comprising SARS-CoV-2 spike protein.

In some embodiments the method of producing a recombinant VSV of the present invention is wherein said rVSV-AG-spike plasmid and said accessory plasmids comprise a T7 promoter.

In other embodiments the method of producing a recombinant VSV of the present invention is wherein prior to step (b) said cell is infected with MVA-T7, whereby expression of T7 polymerase is induced in said cells.

In further embodiments the method of producing a recombinant VSV of the present invention is wherein said infection with MVA-T7 is by incubation for at least about 30 minutes, or between about 30 minutes and 120 minutes or for about one hour.

In further embodiments the method of producing a recombinant VSV of the present invention is wherein said cell is a BHK-21 cell.

In still further embodiments the method of producing a recombinant VSV of the present invention is wherein said isolated recombinant VSV comprising SARS-CoV-2 spike protein is subjected to at least one passaging step in Vero E6 cells.

In some embodiments the method of producing a recombinant VSV of the present invention is wherein said isolated recombinant VSV comprising SARS-CoV-2 spike protein is subjected to at least one passage, at least 5 passages, at least 7 passages, at least 10 passages, at least 11 passages at least 12 passages, at least 13 passages, at least 14 passages or more in Vero E6 cells. In other embodiments the method of producing a recombinant VSV of the present invention is wherein after step (d) said recombinant VSV comprising SARS- CoV-2 spike protein is filtered one or more times to remove residual MVA-T7.

In further embodiments the method of producing a recombinant VSV of the present invention is wherein said filtering is performed twice.

In certain embodiments the method of producing a recombinant VSV of the present invention is wherein said filtering is performed using a 0.22mM filter.

In other embodiments the method of producing a recombinant VSV of the present invention is wherein in step (a) said plasmid comprises the nucleic acid sequence set forth in SEQ ID NO. 20 or the nucleic acid sequence set forth in SEQ ID NO. 23.

In further embodiments the method of producing a recombinant VSV of the present invention is wherein in step (d) said recombinant VSV comprises a nucleic acid sequence as set forth in SEQ ID NO 18 or the nucleic acid sequence set forth in SEQ ID NO 19, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

By a further aspect thereof the present invention provides a genetically modified vesicular stomatitis virus (VSV) vector encoding VSV nucleocapsid protein (N), VSV phosphoprotein (P), VSV matrix protein (M), VSV polymerase (L), and SARS-CoV-2 spike protein or an immunogenic fragment thereof, wherein said VSV vector is lacking the gene encoding the VSV G protein.

In some embodiments the genetically modified VSV vector of the present invention is wherein said vector comprises a nucleic acid sequence set forth in SEQ ID NO 20 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: Fig. 1 is a schematic presentation of the plasmid pVSV-AG-spike. The VSV genes L, N, P and M are indicated as well as the gene encoding SARS-CoV-2 Spike ("Spike CO"). Restriction sites (Mlul and Nhel) used for inserting the gene encoding SARS-CoV-2 Spike are also indicated.

Fig. 2A - Fig. 2B are representative immunofluorescence micrographs showing expression of the spike protein in Vero E6 cells infected with rVSV-AG-spike.

Fig. 3A - Fig. 3D are representative electron microscopy micrographs of WT- VSV (Fig. 3A), rVSV-AG-spike (Fig. 3B), and representative images of immunogold labeling by antibodies directed to the receptor binding domain (RBD, rabbit, 1:30) (Fig. 3C and Fig. 3D).

Fig. 4 is a graph showing percentage of neutralization of SARS-CoV-2 by sera from hamsters following vaccination with the rVSV-AG-spike. Hamsters (n=3) were vaccinated with 10⁶ PFU/animal. Plaque reduction neutralization test (PRNT) was performed two weeks following vaccination.

Fig. 5 is a graph showing rVSV-AG-spike vaccine efficacy in hamsters following SARS-CoV-2 challenge. The graph shows weight change over time of hamsters challenged with SARS-CoV-2 at 10⁸ PFU/ml, where the hamsters were either pre-vaccinated with the rVSV-AG-spike vaccine or untreated (n=4/group).

Fig. 6 is the expected amino acid sequence of SARS-CoV-2 Spike protein derived from clone 28, which is lacking the 24 C-terminal amino acid residues and denoted herein as SEQ ID NO. 16. The symbol "*" indicates a stop codon and refers to a mutation at the corresponding nucleic acid position 6831 C->stop. The underlined sequence "RRAG" refers to a furin cleavage site mutation, namely to a mutation at the corresponding nucleic acid position 5134, resulting in an Arg (R) to Gly (G) substitution mutation.

Fig. 7 is the expected amino acid sequence of SARS-CoV-2 Spike protein derived from clone 31, which is lacking the 24 C-terminal amino acid residues and denoted herein as SEQ ID NO. 17. The symbol "*" indicates a stop codon and refers to a mutation at the corresponding nucleic acid position 6831 C->stop. The underlined sequence "RRAK" refers to a furin cleavage site mutation, namely to a mutation at the corresponding nucleic acid position 5134, resulting in an Arg (R) to Lys (K) substitution mutation. Fig. 8 is the nucleic acid sequence of a representative vaccine construct (rVSV- AG-spike) originating from clone 28 (denoted herein as SEQ ID NO. 18). The letter "R" represents G or A and the letter "Y" represents C or T. The open reading frame (ORF) of the spike sequence is underlined. Mutations are marked by bold letters.

Fig. 9 is the nucleic acid sequence of a representative vaccine (rVSV-AG-spike) originating from clone 31 (denoted herein as SEQ ID NO. 19). The letter "R" represents G or A and the letter "Y" represents C or T. The open reading frame (ORF) of spike is underlined. Mutations are marked by bold letters.

Fig. 10A is a graph showing body weight changes (shown as % of initial weight) of hamsters infected with 5xl0⁴ (n=8), 5xl0⁵ (n=8), or 5xl0⁶ (n=8) pfu/hamster of SARS-CoV-2, compared to mock-infected hamsters (n=4), at various days post infection.

Fig. 10B-G presents sections of lungs that were isolated and processed for paraffin embedding from naive hamsters (b, d, f) or SARS-CoV-2-infected hamsters (c, e, g, 5xl0⁶ pfu/hamster) at 7 dpi. Sections (5 pm) were taken for H&E staining (B-E) and SARS-CoV-2 immunolabeling (F-G). Panels B-E: scale bar= lOOpm; panels F-G: scale bar= 10pm. Black arrows indicate patches of focal inflammation, pleural invagination, and alveolar collapse. indicates hemorrhagic areas.

indicates edema and protein rich exudates. Black arrowheads indicate pulmonary mononuclear cells. White arrows indicate SARS-CoV-2 positive immunolabeling. Naive group: n=4, SARS-CoV-2 5xl0⁶7dpi group: n=l.

Fig. 11A is a graph showing results of a safety study measuring body weight changes (shown as % of initial weight) of mock-vaccinated hamsters (n=4), and hamsters vaccinated i.m. with rVSV-AG-spike ranging from 10⁴ to 10⁸ pfu/hamster (n=8, n=10, n=ll, n=10, n=8, for each vaccinated group, respectively) at various days post vaccination. The hamsters received a prime vaccination + boost at day 14.

Fig. 11B is a graph showing NT50 values of i.m. vaccinated hamsters’ sera (10⁴ to 10⁸ pfu) against SARS-CoV-2 (n=4 for 10⁴, 10⁶, 10⁷, 10⁸, n=3 for 10⁵). Means and SEM are as follows: for 10⁴ pfu/hamster 142±64, for 10⁵ pfu/hamster 789±111, for 10⁶ pfu/hamster 649±205, for 10⁷ pfu/hamster 1080±127, for 10⁸ pfu/hamster 2201±876. Fig. llC and 11D are representative immunofluorescence images of Vero E6 cells infected with SARS-CoV-2, labeled with serum from either naive (C) or rVSV- AG-spike (10⁶pfu/hamster) i.m. vaccinated hamsters (D).

Fig. HE is a graph showing body weight changes (shown as % of initial weight) of hamsters vaccinated i.m. with 10⁴-10⁸ pfu/hamster at various days post infection. Unvaccinated infected hamsters served as a control. The animals were infected with 5xl0⁶ pfu/hamster 25 days post-vaccination. Arrow indicates 5 dpi - hamsters were sacrificed and lungs were removed for viral load. For vaccinated groups 10⁴-10⁷: days 0-5 (n=12), days 6-12 (n=9), 10⁸ group: days 0-5 (n=ll), day 6-12 (n=8). For unvaccinated infected: days 0-5 (n=14), days 6-12 (n=12).

Fig. 1 IF is a graph showing viral loads in lungs (pfu/lung) at 5 dpi of hamsters infected with 5X10⁶ pfu/hamster of SARS-CoV-2 (n=7), and hamsters vaccinated i.m. with 10⁴-10⁸ pfu/hamster of rVSV-AG-spike, and then infected with 5X10⁶ pfu/hamster of SARS-CoV-2 (n=3 for each vaccinated group). Limit of detection (LOD, 75 pfu/lung). All vaccinated groups show statistical significance, compared to infected unvaccinated group as determined by one-way ANOVA non-parametric kruskal-Wallis test, with Dunn’s multiple comparisons test, * p=0.0012. Data for (A, E, F) are presented as mean values ± SEM.

Fig. 12A and 12B are graphs showing viral loads in lungs (A) and nasal turbinates (B) at 3 dpi of hamsters infected with SARS-CoV-2 (5X10⁶ pfu/hamster, n=4), and hamsters vaccinated with of rVSV-AG-spike (10⁶ pfu/hamster), followed by infection with SARS-CoV-2 (5X10⁶ pfu/hamster, n=4). LOD: 75 pfu/lung, 50 pfu/nasal turbinates. Data is presented as mean values ± SEM. Significance analysis was performed using two-tailed unpaired non-parametric Mann-Whitney non-parametric test, * p=0.0286.

Fig. 13A-J show histopathological analysis of rVSV-AG-spike i.m. vaccinated and infected hamsters’ lungs at 3 and 7 dpi. General histology (H&E) and SARS-CoV-2 DAB immunolabeling of naive, unvaccinated infected (5xl0⁶), and vaccinated (10⁶ pfu) hamsters’ lungs, at 3 and 7 dpi. Lungs were isolated and processed for paraffin embedding from naive (a, f), infected (5xl0⁶ pfu) (b, g for 3 dpi and d, i for 7 dpi) and vaccinated and infected (c, h for 3 dpi and e, j for 7 dpi). Sections (4 pm) were taken for H&E staining (a-e) and SARS-CoV-2 DAB immunolabeling (f-j, positive SARS-CoV- 2 - dark staining). “*” indicates cellular debris in bronchiolar lumen. Black arrowheads indicate congestion of blood in blood vessels. Black arrows indicate positive stained cells. Images a-e: scale bar= 200pm; images f-j: scale bar= 20pm. Data was taken for 5 groups. Each group includes 4 animals. For each animal, 5 fields were imaged and analysed.

Fig. 13K Histopathological severity analysis of hamsters' lungs of naive, vaccinated, and infected lungs, at 3 and 7 dpi.

Fig. 13L is a graph showing digital morphometric analysis of DAB immunohistochemical staining for SARS-CoV-2 in lungs of naive, infected, and vaccinated lungs, at 3 and 7 dpi.

Fig. 13M is a graph showing tissue/air space ratio analysis of naive, infected, and vaccinated lungs, at 3 and 7 dpi. Data for (K, L, M) are presented as mean values ± SEM. Statistical analyses for (K, L, M) were performed by one-way ANOVA with Tukey's multiple comparisons test, with p<0.0001, p=0.0006, p<0.0001, respectively.

Fig. 14A and 14B are graphs showing Thl and Th2 isotype analysis of rVSV- AG-spike induced antibodies in the sera of vaccinated C57BL/6J mice (10⁷ pfu/mouse, n=7). Fig. 14A NT50 values against SARS-CoV-2 as determined by PRNT, and Fig. 14B Levels of recombinant SARS-CoV-2 S glycoprotein (S2P) specific antibodies: total IgG, IgG2c, and IgGl as determined by ELISA. Statistical significance was determined using one-way ANOVA non-parametric test, with Kruskal-Wallis test: **** p<0.0001. Each mouse is represented by a different shade.

Fig. 15 is the expected amino acid sequence of SARS-Cov-2 Spike protein in rVSV-AG-spike, denoted herein as SEQ ID NO. 21. The substitution mutations are represented by amino acids that are shown in bold letters, namely H245R, R685G, and S813I. Underlined amino acids represent positions in which a mutation appears only in some of the rVSV-AG-spike molecules, namely W64R, H66R, E484D, Q493R, N501Y, V615L, and S813L. These amino acids were not replaced in the presented sequence.

Fig. 16 is the nucleic acid sequence encoding SARS-Cov-2 Spike protein in rVSV-AG-spike, denoted herein as SEQ ID NO. 22. The substitution mutations are represented by nucleic acids that are shown in bold letters, namely A3815G (leading to amino acid substitution H245R), A4602T (no aa substitution), A5134G (leading to amino acid substitution R685G), G5519T (leading to amino acid substitution S813I), and T6831A (leading to a stop at C1250). Underlined nucleic acids represent positions in which a mutation appears only in some of the rVSV-AG-spike molecules, namely T3271C (leading to amino acid substitution W64R), A3278G (leading to amino acid substitution H66R), G4533T (leading to amino acid substitution E484D), A4559G (leading to amino acid substitution Q493R), A4582T (leading to amino acid substitution N501Y), G4924T (leading to amino acid substitution V615L), and A5518C (leading to amino acid substitution S813L). These nucleic acids were not replaced in the presented sequence.

Fig. 17A is a graph showing neutralization of B.1.1.7 (UK), B.1.351 (SA) and P.l (Brazil) VOC by sera from rVSV-AG-spike-vaccinated K18-hACE2 mice: PRNT50 values of vaccinated K18-hACE2 mice sera (10⁷ pfu, prime and boost) against SARS- CoV-2 variants: WT, B.l.1.7, B.1.351, P.l. * P=0.006, determined by one-way ANOVA.

Fig. 17B-E are graphs showing protection of K18-hACE2 mice from lethal SARS-CoV-2 VOC challenge by rVSV-AG-spike vaccination. (Fig. 17B) body weight changes (shown as % of initial weight) days post transfection (d.p.i) of rVSV-AG-spike vaccinated K18-hACE2 mice (prime + boost), infected with either WT (Vaccinated - WT challenge), B.l.1.7 variant (Vaccinated - B.l.1.7 challenge), or B.1.351 variant (Vaccinated - B.1.351 challenge), or unvaccinated (WT challenge, B.l.1.7 challenge or B.1.351 challenge). (Fig. 17C-E) Survival curves (shown as percent survival at days post infection (d.p.i.)) of rVSV-AG-spike vaccinated K18-hACE2 mice (prime + boost), infected with either WT (Vaccinated - WT challenge - Fig. 17C), B.l.1.7 variant (Vaccinated - B.l.1.7 challenge - Fig. 17D), or B.1.351 variant (Vaccinated - B.1.351 challenge - Fig. 17E), or unvaccinated (WT challenge, B.l.1.7 challenge or B.1.351 challenge, Fig. 17C, D, E, respectively).

DETAILED DESCRIPTION OF EMBODIMENTS

The present disclosure concerns the preparation of a vesicular stomatitis virus (VSV)-based vaccine for SARS-CoV-2 (also referred to commercially as "Brilife"). Specifically, a VSV-based vaccine expressing the SARS-CoV-2 spike protein, thus forming a recombinant replication-competent virus. Vesicular Stomatitis Vims, also referred to as "Vesicular stomatitis Indiana vims" or "Indiana vesiculovirus" (VSV), is a non-segmented single-stranded, negative sense RNA vims, which is a member of the Rhabdoviridae family. VSV causes a disease in animals, with a broad host range in mammals and insects, however, cases of human infection with VSV are rare.

As a vaccine platform, VSV harbors several advantages, among which are the following: the vims can be easily propagated and reach high titers, the viral vaccine elicits strong cellular and humoral immunity in vivo , and in addition, the elimination of the VSV G protein, the major vimlence factor of the VSV, attenuates the vims and reduces its reactogenicity.

Indeed, the inventors showed that no VSV vector immunity was found with the VSV-based vaccine for SARS-CoV-2 of the invention.

VSV genome codes for five major proteins, termed matrix protein (M), nucleoprotein or nucleocapsid protein (N), large polymerase protein (L), phosphoprotein (P), and glycoprotein (G). The L and P proteins, together with the N, form the transcriptionally active subunit of the vims. The G protein mediates both viral binding and host cell fusion with the endosomal membrane following endocytosis, and cell entry. The sequence of the VSV genome as well as of the functional genes thereof are well known in the art, for example as described in Lawson, et al ., (Proc. Natl. Acad. Sci. (USA), 1995. 92(10): 4477-4481) and Stillman, et al, (J. Virol., 1995. 69: 2946- 2953).

Therefore, in a first of its aspects, the present invention provides a recombinant vesicular stomatitis vims (VSV) comprising: a VSV nucleocapsid protein (N), a VSV phosphoprotein (P), a VSV matrix protein (M), a VSV large polymerase protein (L), and a SARS-CoV-2 spike protein or an immunogenic fragment thereof, wherein said recombinant VSV is lacking the VSV G protein.

The recombinant VSV of the invention is also referred to herein interchangeably as VSV-AG-spike or rVSV-AG-spike.

As a first step in the preparation of the vaccine, a cDNA vector comprising the sequences of the N, P, M, and L genes of the VSV genome was generated, further comprising the nucleic acid sequence encoding the spike protein of the SARS-CoV-2, under a T7 promoter. The recombinant VSV may be constructed from DNA using methods well known in the art, for example as described in Lawson, et al ., (Proc. Natl. Acad. Sci. (USA), 1995. 92(10): p. 4477-4481) and Stillman, et al. , (J. Virol., 1995. 69: 2946- 2953), incorporated herein by reference, whereby the nucleic acid sequence encoding the SARS-CoV-2 spike protein is incorporated into the VSV genome.

In one embodiment, the recombinant VSV is constructed using a commercially available plasmid, e.g., pVSV-FL+(2) (Kerafast) which encodes the antigenomic-sense (or positive-sense) RNA of vesicular stomatitis virus (VSV) expressed from the bacteriophage T7 promoter in pBS, which has been further modified to contain the hepatitis delta ribozyme used to generate a precise 3' end of the VSV antigenomic RNA and a T7 terminator sequence cloned between the SacII and Sacl restriction sites in pBS-SK+.

In one embodiment, the recombinant VSV is constructed as shown in the Examples below.

Therefore, in another aspect, the present invention provides a genetically modified vesicular stomatitis virus (VSV) vector encoding VSV nucleocapsid protein (N), VSV phosphoprotein (P), VSV matrix protein (M), VSV polymerase (L), and SARS-CoV-2 spike protein or an immunogenic fragment thereof, wherein said VSV vector lacks the gene encoding the VSV G protein.

A schematic representation of the VSV vector is presented in Figure 1, in which the positions of the genes encoding the VSV nucleocapsid "N", phosphoprotein "P", matrix protein "M" and large polymerase protein "L" are shown.

The genetically modified VSV vector is also referred to herein as VSV-AG-spike vector. In a specific embodiment said vector is a plasmid, referred to herein as VSV- AG-spike plasmid. In a particular embodiment, the genetically modified VSV vector comprises a nucleic acid denoted herein by SEQ ID NO: 20, or SEQ ID NO: 23, or a nucleic acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

By the terms, " recombinant ", " modified " or " genetically modified " as used herein with reference to the VSV or VSV vector according to the present disclosure it is meant that the sequence of the naturally occurring VSV has been altered by genetic engineering. The alteration comprises at least the replacement of the VSV G protein with a SARS-CoV-2 spike protein or an immunogenic fragment thereof. Additional alterations may also take place, for example point mutations in any one of the VSV genes or in the nucleic acid encoding SARS-CoV-2 spike protein. The recombinant, modified, or genetically modified VSV referred to herein is termed interchangeably "VSV-AG-spike" or "rVSV-AG-spike" as detailed above. The recombinant, modified, or genetically modified VSV vector referred to herein is termed interchangeably "VSV- AG-spike vector" or "VSV-AG-spike plasmid" as detailed above.

As appreciated by those skilled in the art the recombinant vesicular stomatitis virus or the genetically modified VSV vector of the invention is replication competent, namely it is able to replicate itself in a host cell by using the host cell's replication apparatus. As known in the art, viruses are only able to replicate themselves by using the host cell's replication apparatus. Cell entry occurs when proteins on the surface of the virus (e.g., VSV G protein or SARS-CoV-2 spike protein) interact with receptor proteins of the cell. Attachment occurs between the viral particle and the host cell membrane. A passage forms in the cell membrane, which enables the virus particle or its genetic contents to be introduced into the host cell, where the replication of the viral genome may commence. Subsequently, the viral progeny leaves the cell, generally by causing cell destruction.

By the term " vector " as known in the art and as herein defined it is referred to a nucleic acid molecule carrying genes, usually in the form of a circular molecule, into which a desired heterologous sequence may be inserted by restriction and ligation for expression in a host cell. The vector according to the present disclosure is capable of replicating in a host cell. In one embodiment, the vector of the invention is a plasmid. Many expression vectors are known in the art. In further embodiments the vector of the invention referred to herein has the nucleic (DNA) sequence denoted herein by SEQ ID NO. 20 or SEQ ID NO. 23.

In some embodiments, the SARS-CoV-2 spike protein according to the present disclosure is a natural (i.e., wild type) SARS-CoV-2 spike protein, a mutated SARS- CoV-2 spike protein, a chimeric SARS-CoV-2 spike protein or an immunogenic fragment thereof.

Coronaviruses contain four structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, of which the spike (S) protein plays the most important role in viral attachment, fusion, and cell entry. The S protein mediates viral entry into host cells by first binding to a host receptor through the receptor-binding domain (RBD) in the S 1 subunit and then fusing the viral and host membranes through the S2 subunit. SARS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2) as its host receptor. SARS-CoV-2 (also termed the 2019 novel coronavirus (2019-nCoV) as previously termed by the World Health Organization (WHO)) is the RNA virus that causes the disease termed COVID-19.

As used herein the term "SARS-CoV-2", also encompasses SARS-CoV-2 variants, namely viruses which underwent a certain degree of change by acquiring mutations. The term "variant" therefore encompasses any SARS-CoV-2 variant. Non limiting examples include several SARS-CoV-2 variants that were described thus far: The United Kingdom variant (UK, known as 20I/501Y.V1, VOC 202012/01, or B.l.1.7), South Africa variant (SA, known as 20H/501Y.V2 or B.1.351), Brazil variant (known as P.l or 20J/501Y.V3, a branch off the B.1.1.28 lineage originally reported in Japan), as well as variants in other regions worldwide.

The term "SARS-CoV-2 spike protein" (also referred to herein as "Severe acute respiratory syndrome coronavirus 2 spike protein") refers to the surface protein of SARS-CoV-2. The sequence of SARS-CoV-2 spike protein is known to a person of skill in the art. An exemplary amino acid sequence of the full-length SARS-CoV-2 spike protein is denoted herein as SEQ ID NO. 13 and an exemplary nucleic acid sequence encoding the full-length SARS-CoV-2 spike protein is denoted herein as SEQ ID NO. 14. A human codon-optimized nucleic acid sequence encoding the full-length SARS-CoV-2 spike protein is denoted herein as SEQ ID NO. 15.

By the term " natural SARS-CoV-2 spike protein " as herein defined it is referred to the sequence of the full-length native, wild type SARS-CoV-2 spike protein as known in the art, for example, the wild-type SARS-CoV-2 spike protein having the amino acid sequence denoted by SEQ ID NO. 13 or encoded by the nucleic acid sequence denoted by SEQ ID NO. 14 or by the codon-optimized nucleic acid sequence denoted by SEQ ID NO. 15.

The term " chimeric " as herein defined refers to a SARS-CoV-2 spike protein or any fragment thereof comprising protein fragments originating from different coronavirus strains, for example, but not limited to, protein fragments of the spike protein originating from different strains of viruses of the Coronaviridae family (e.g., SARS-CoV-2, MERS-CoV or SARS to name but a few).

By the term " mutated " with reference to the SARS-CoV-2 spike protein as known in the art and as described herein it is referred to SARS-CoV-2 spike protein having a sequence of amino acids which is different from the native sequence of SARS- CoV-2 spike protein, for example an amino acid sequence which is different from the amino acid sequence identified herein by SEQ ID NO. 13, in which one or more amino acid residues are deleted, substituted or added. The present disclosure encompasses a mutated amino acid sequence of SARS-CoV-2 spike protein which is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence denoted herein by SEQ ID NO. 13.

It should be appreciated that by the term "added ", as used herein it is meant any addition of amino acid residues to the sequences described herein.

As used herein, the term “ amino aci ’ or “ amino acid residue ” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

It should be appreciated that by the term " deleted " as used herein it is meant any reduction of amino acid residues from the sequences described herein. An amino acid “ substitution ” is the result of replacing one amino acid with another amino acid which has similar or different structural and/or chemical properties. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

The term "mutated" also encompasses a truncated form of the protein in which one or more amino acid residues are deleted at the N terminus or C terminus of the Spike protein. In one embodiment a truncated form of Spike protein comprises a truncation of 24 amino acids at the C terminus of the protein.

Therefore, in some embodiments the SARS-CoV-2 spike protein is truncated at the C-terminus.

In specific embodiments the spike protein in VSV-AG-spike comprises a 24- amino acid truncation at the C-terminal. In other words, in various embodiments the SARS-CoV-2 spike protein lacks 24 amino acids at the C-terminus. As a non-limiting example, the present disclosure encompasses a VSV-AG-spike comprising SARS-CoV- 2 spike protein lacking the 24 amino acids at the C-terminus which has the amino acid sequence denoted herein by SEQ ID NO. 16 (the sequence shown in Figure 6, originating from "clone 28" as termed herein) and which is encoded by the nucleic acid sequence denoted herein by SEQ ID NO. 18. The present disclosure further encompasses a VSV-AG-spike comprising SARS-CoV-2 spike protein lacking the 24 amino acids at the C-terminus which has the amino acid sequence denoted herein by SEQ ID NO. 17 (the sequence shown in Figure 7, originating from "clone 31" as termed herein) and which is encoded by the nucleic acid sequence denoted herein by SEQ ID NO. 19.

The truncation of the 24 amino acids at the C-terminus of the spike protein (at amino acid position 1250) is a result of a mutation in the cDNA sequence (at nucleic acid position 6831) leading to a stop codon (as shown in Figure 6 and in Figure 7). This mutation is located at a unique, novel site. Interestingly, during passage progression of the modified virus in vitro in the host cells, this stop-mutation-containing viral particle became dominant in the culture. Without wishing to be bound by theory, this effect suggests that the truncated form of spike is advantageous in supporting efficient viral replication and propagation.

In specific embodiments the spike protein in VSV-AG-spike comprises a nonsynonymous mutation at the S1/S2 motif RRAR.

In a specific embodiment, the nonsynonymous mutation is at the fourth (4^th) residue (R4) of the S1/S2 motif RRAR. This mutation at the S1/S2 motif, which is important for the cleavage of the spike protein, may contribute to its stability, thus enhancing the immunogenicity and its potential as a potent candidate vaccine. Therefore, in various embodiments the SARS-CoV-2 spike protein of the present invention has a mutation at the Arginine (R) residue on position four (R4) of the S1/S2 motif RRAR. In certain embodiments the SARS-CoV-2 spike protein of the present disclosure has an amino acid substitution mutation at position 685 (nucleic acid position 5134) at the S1/S2 junction of the sequence denoted herein by SEQ ID NO. 16 or SEQ ID NO. 17. In some embodiments the R4 of the S1/S2 motif RRAR is substituted with Gly (G). In other embodiments the R4 residue of the S1/S2 motif RRAR is substituted with Lys (K). It was shown that the multi basic motif of the S1/S2 site is processed by the host cell furin protease. This site is essential for cell-cell fusion and was shown to mediate infection in human lung cells (Hoffmann, M. etal ., 2020, Molecular Cell 78: 1-6).

In one embodiment, the spike protein in VSV-AG-spike comprises both a 24 amino acid truncation at the C-terminal and a nonsynonymous mutation at the S1/S2 motif RRAR. In specific embodiments the spike protein in VSV-AG-spike according to the present disclosure has the amino acid sequence denoted herein by SEQ ID NO. 16 or SEQ ID NO. 17 (namely comprises both the 24 amino acid truncation at the C- terminal, and the nonsynonymous mutation at the S1/S2 motif RRAR). It is appreciated that the amino acid sequence denoted herein by SEQ ID NO. 16 harbors an R to G mutation at the R4 residue of the S1/S2 motif RRAR and that the amino acid sequence denoted herein by SEQ ID NO. 17 harbors an R to K mutation at the above site as well as a Trp (W) to Arg (R) mutation at position 64 (as shown in the amino acid sequence denoted herein by SEQ ID NO. 17).

In certain embodiments, in addition to the truncation mutation and the mutation at position 685 described above, the SARS-CoV-2 spike protein of the present disclosure comprises additional mutations. These include mutations that result in an amino acid substitution, or such that do not result in an amino acid substitution (e.g., the mutation P507P at nucleic acid position 4602). Therefore, in specific embodiments, the SARS-CoV-2 spike protein of the present disclosure may further comprise mutations at positions: 507, 245, 813, e.g., P507P (non-substitution mutation), H245R and S813L as shown in SEQ ID NO. 21 (the sequence shown in Figure 15), and which is encoded by the nucleic acid sequence denoted herein by SEQ ID NO. 22.

The possible mutations are set forth in SEQ ID NO: 24 wherein independently X at position 685 is selected from R, G and K; X at position 245 is selected from H and R; and X at position 813 is selected from S, L, and I.

In certain embodiments, the SARS-CoV-2 spike protein of the present disclosure further comprises one or more mutations at any one of positions 64, 66, 113, 484, 493, 501, 615 of SEQ ID NO:21, or any combination thereof. In specific embodiments these additional substitution mutations are selected from the group consisting of S 8131, W64R, H66R, K113Q, E484D, Q493R, N501Y, and V615L. The possible mutations are set forth in SEQ ID NO: 25 wherein independently X at position 64 is selected from W and R; X at position 66 is selected from R and H; X at position 113 is selected from Q and K; X at position 484 is selected from D and E; X at position 493 is selected from R and Q; X at position 501 is selected from Y and N; X at position 615 is selected from L and V; and X at position 813 is selected from S and I.

The mutations of the spike protein are listed in Table 6:

Table 6: mutations at the spike protein:

aa - amino acids.

RBD - Receptor Binding Domain NTD - N-Terminal Domain

In one embodiment, the present invention provides a composition comprising a heterogeneous population of VSV-AG-spike, wherein said heterogeneous population comprises VSV-AG-spike with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 and VSV-AG-spike with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 further comprising one or more mutations selected from the group consisting of S 8131, W64R, H66R, K113Q, E484D, Q493R, N501Y, and V615L. Hence this heterogeneous population comprises a mixture of VSV- AG-spike comprising various variants of the spike protein with varying frequencies of the above-noted mutations. In an embodiment the mixture of VSV-AG-spike variants may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or any number of variants known for the spike protein, at any frequency.

In one embodiment, the mutations C1250STOP, P507P, R685G, H245R have a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% frequency. A 100% frequency means that these mutations appear substantially in all VSV-AG-spike in the population.

The frequencies of the other mutations (e.g., S 8131, W64R, H66R, K113Q, E484D, Q493R, N501Y, and V615L) vary between 5% - 100%, e.g., between 5% - 80%.

Since the emergence of the original SARS-CoV-2, several variants of concern (VOC) were described: The United Kingdom variant (UK, known as 20E501Y.V1, VOC 202012/01, or B.l.1.7), South Africa variant (SA, known as 20H/501Y.V2 or B.1.351), Brazil variant (known as P.l or 20J/501Y.V3, a branch off the B.1.1.28 lineage originally reported in Japan), as well as variants in other regions worldwide. Each of the described variants includes several mutations at the spike protein, some of which are at key areas that serve as targets for neutralization by virus neutralizing antibodies, hence, there is a major concern as to the ability of vaccine-induced neutralizing antibodies to effectively neutralize these newly emerged SARS-CoV-2 variants.

Without wishing to be bound by theory, some of the mutations described herein may have a significant impact on vaccine efficacy: N501Y - Resides in the receptor binding domain (RBD) of the spike protein. The UK, SA, and Brazilian variants all comprise the N501Y mutation. This mutation was shown to increase binding of the SARS-CoV-2 to the angiotensin-converting enzyme 2 receptor (ACE2). (Starr et al., (2020) bioRxiv )

E484D - The SA variant, as well as the Brazilian variant, contain the E484K mutation in the RBD. This substitution was reported to confer resistance to several monoclonal antibodies developed as an antibody cocktail against SARS-CoV-2 (Ku et al, 2021 Nature Communications 12(1): 469). This escape mutation is alarming due to its potential effect on vaccine efficacy. A drop in neutralizing antibodies of vaccinees (Pfizer, Moderna) were documented (Wang et al., 2021 "mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants", Nature )

Q493R - mutations at this site (RBD) were reported to create escape mutants that evade monoclonal antibody or antibody cocktails, such as LY-C0VOI6 or REGN10933, respectively (Starr et al., (2021) Science 371(6531): 850-854). The Q493 site was shown to mutate to the amino acids F/L/K/R; the last two resulting in a shift from polar neutral side chains amino acid (Q), to charged side chains of basic polarity amino acids (K and R).

H245R - The SA variant presents several changes in the vicinity of H245 (NTD); L242H mutation, a L242 244L deletion, and R246I mutation, all of which are not fixed, and the entire region of aa 242-246 (including H245) is considered an unresolved region.

V615L - This mutation, located at the SI site, occurred in some SARS-CoV-2 variants. It has been previously shown that this mutation confers increased sensitivity to neutralization by human convalescence sera (Li et al. (2020) Cell 182(5): 1284-1294 el289).

Thus, the VSV-AG-spike in accordance with the invention may comprise several point mutations in the spike protein that have the potential to preserve the efficacy of the vaccine against SARS-CoV-2 as well as to newly emerging SARS-CoV-2 strains.

Both the 24 amino acid truncation and the S1/S2 RRAR mutation may contribute to the safety and efficacy of the VSV-AG-spike vaccine. It was reported that cells infected with SARS-CoV-2 form syncitia, namely fusion of cells with multiple nuclei (Xia, S. et al. , 2020, Cell Research 30: 343-355). During early VSV-AG-spike passaging, extensive syncitia were observed. With passage progression, a reduction in syncitia formation was observed, as well as a more classic cytopathic effect. As known the art, the term " cytopathic effect " or " cytopathogenic effect " (CPE) refers to structural changes in host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell or alternatively the cell dies without being lysed due to its inability to reproduce. Common examples of CPE include rounding of the infected cell, fusion with adjacent cells to form syncytia, and the appearance of nuclear or cytoplasmic inclusion bodies.

Without wishing to be bound by theory, this shift might be attributed to the mutation in the S1/S2 site, as well as the increase in dominance of the stop mutation, that generated the truncated VSV-AG-spike.

In various specific embodiments the spike protein in VSV-AG-spike as herein defined consists of the amino acid sequence denoted by SEQ ID NO. 16. In various further specific embodiments, the spike protein in VSV-AG-spike as herein defined consists of the amino acid sequence denoted by SEQ ID NO. 17. In a further specific embodiment, the spike protein in VSV-AG-spike as herein defined consists of the amino acid sequence denoted by SEQ ID NO. 21.

These properties may contribute to the safety and efficacy of VSV-AG-spike as a vaccine.

The present disclosure further encompasses any immunogenic fragment of the SARS-CoV-2 spike protein. By the term "an immunogenic fragment " in the context of the present disclosure it is referred to a fragment or portion of the SARS-CoV-2 spike protein that elicits an immune response in a subject as herein defined. Determining the ability of an antigen (for example an immunogenic fragment of the SARS-CoV-2 spike protein) to elicit an immune response in an organism is within the knowledge of a person of skill in the art, for example by examination of sera sample obtained from said organism (e.g., mammal) and determination of the presence therein of immune components (e.g., antibodies, specifically antibodies directed against the SARS-CoV-2 spike protein or any epitope thereof). Any fragment or portion of the SARS-CoV-2 spike protein that elicits an immune response in a subject as herein defined is encompassed by the present disclosure. In certain embodiments, the recombinant VSV or the genetically modified VSV vector of the present invention is wherein the SARS-CoV-2 spike protein comprises the amino acid sequence set forth in SEQ ID NO. 16 or the amino acid sequence set forth in SEQ ID NO. 17, or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

In certain embodiments the recombinant VSV of the present invention is encoded by a nucleic acid sequence comprising a nucleic acid sequence set forth in SEQ ID NO. 18, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

The locations of the VSV genes N, P, M and L as well as the spike gene on the rVSV-AG-spike having a nucleic acid sequence set forth in SEQ ID NO. 18 ("clone 28") are listed in Table 1 below.

Table 1 Gene locations on rVSV-AG-spike (clone 28)

In specific embodiments, the recombinant VSV of the present invention (VSV- AG-spike) comprises point mutations in the matrix (M) protein or the (L) protein.

Mutations (and frequencies thereof) with respect to the native (wild-type) sequences of the VSV M and L genes and with respect to the native gene encoding SARS-CoV-2 Spike protein are shown in bold in Figure 8 and are listed in Table 2 below. Table 2 Mutations in recombinant VSV, clone 28

Abbreviations: Ref, reference; Alt, alteration; ORF, open reading frame; AA, amino acid.

In certain further embodiments the modified VSV of the present invention is encoded by a nucleic acid sequence comprising a nucleic acid sequence set forth in SEQ ID NO. 19 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

The locations of the VSV genes N, P, M and L as well as the spike gene on the rVSV-AG-spike having a nucleic acid sequence set forth in SEQ ID NO. 19 ("clone 31") are listed in Table 3 below. Mutations are listed in Table 4 below.

Table 3 Gene locations on rVSV-AG-spike (clone 31)

Table 4 Mutations in recombinant VSV, clone 31

Abbreviations: Ref, reference; Alt, alteration; ORF, open reading frame; AA, amino acid.

Mutations at the VSV-backbone (non-spike mutations):

In addition to the mutations in the spike protein, several mutations emerged in the non-spike protein areas, namely in VSV proteins. Some of them are synonymous mutations, whereas others are non-synonymous mutations, mostly in the VSV-L protein, but also in VSV-N.

Table 8: Non-spike mutations:

In one embodiment, the complete nucleotide sequence of the full rVSV-AG- spike plasmid is presented in SEQ ID NO. 23. This sequence includes the following substitution mutations T208G (leading to amino acid substitution S47R in N), A3815G (leading to amino acid substitution H245R in S), A4602T (no aa substitution), A5134G (leading to amino acid substitution R685G in S), G5519T (leading to amino acid substitution S813I in S), and T6831A (leading to a stop at C1250 in S), G9243A (leading to amino acid substitution L741I in L). In the following nucleic acid positions a mutation appears only in some of the rVSV-AG-spike plasmids, namely T3271C (leading to amino acid substitution W64R in S), A3278G (leading to amino acid substitution H66R in S), G4533T (leading to amino acid substitution E484D in S), A4559G (leading to amino acid substitution Q493R in S), A4582T (leading to amino acid substitution N501 Y in S), G4924T (leading to amino acid substitution V615L in S), A5518C (leading to amino acid substitution S813L in S), C7648A (leading to amino acid substitution P209H in L), G12350A (no amino acid substitution), T12425C (no amino acid substitution), C13010T (no amino acid substitution). These nucleic acids were not replaced in SEQ ID NO. 23.

The term “ nucleic acid " or “ nucleic acid molecule ” as herein defined refers to a polymer of nucleotides, which may be either single- or double-stranded, which is a polynucleotide such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double- stranded polynucleotides. The term DNA used herein also encompasses cDNA, i.e. complementary or copy DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).

The term " identity " in the context of two or more amino acids or nucleic acids sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region. As known to a person of skill in the art, in order to examine identity between two or more sequences the sequences should be aligned. SARS-CoV-2 causes an infectious disease also termed herein "COVID-19” . Symptoms include for example fever, dry cough, dyspnea, fatigue, and lymphopenia in infected patients. In more severe cases, infections causing viral pneumonia characterized primarily by fever, cough, dyspnea, and bilateral infiltrates may lead to severe acute respiratory syndrome (SARS) and even death.

As detailed below, vaccination of hamsters with rVSV-AG-spike generated a strong neutralizing antibody response against the virus SARS-CoV-2.

As will be apparent to one of skill in the art, the recombinant VSV according to the present disclosure is a replicating system that expresses the SARS-CoV-2 spike protein, or an immunogenic fragment thereof and yet does not cause disease (i.e. nor COVID-19 nor a VSV associated disease) or the symptoms associated therewith.

Therefore, by a further aspect thereof the present disclosure provides an immunogenic composition comprising the recombinant VSV as herein defined and a pharmaceutically acceptable excipient, carrier, or diluent.

In certain embodiments the immunogenic composition as herein defined is for eliciting an immune response against SARS-CoV-2 in a subject.

In one embodiment, the present invention is directed to an immunogenic composition for eliciting an immune response against any SARS-CoV-2 variant, as defined above.

As known in the art, by the term " immune response " as herein defined it is referred to a reaction which occurs within an organism for the purpose of defending against foreign invaders (in the context of the present disclosure, SARS-CoV-2). There are two distinct aspects of the immune response, the innate aspect (the first reaction to an invader which is known to be a non-specific response) and the adaptive aspect (which is the immune response against specific antigens and includes cells, as for example dendritic cells, T cell, and B cells, as well as antibodies).

As known in the art the first encounter that an organism has with a particular antigen will result in the production of effector T and B cells which are activated cells that defend against the pathogen. The production of these effector cells as a result of the first-time exposure is called a "primary immune response". Memory T and memory B cells are also produced in the case that the same pathogen enters the organism again. If the organism is re-exposed to the same pathogen, a secondary immune response will occur, and the immune system will be able to respond in both a fast and strong manner due to the existence of memory cells from the first exposure.

By the term " immunogenic composition " it is meant to refer to a composition comprising the modified VSV or the genetically modified VSV vector comprising spike protein as herein defined for eliciting an immune response.

An "immunogenic composition" is a composition of matter suitable for administration to a human or animal subject capable of eliciting a specific immune response, e.g., against a pathogen, such as SARS-CoV-2 virus. As such, an immunogenic composition includes one or more antigens (for example, whole purified virus or antigenic subunits, e.g., polypeptides, thereof) or antigenic epitopes. An immunogenic composition can also include one or more additional components capable of eliciting or enhancing an immune response, such as an excipient, carrier, and/or adjuvant. In certain instances, immunogenic compositions are administered to elicit an immune response that protects the subject against symptoms or conditions induced by the pathogen. In some cases, symptoms or disease caused by a pathogen is prevented (or treated, e.g., reduced or ameliorated) by inhibiting replication of the pathogen (e.g., SARS-CoV-2 virus) following exposure of the subject to the pathogen. In the context of this disclosure, the term immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against COVID- 19 (that is, vaccine compositions or vaccines).

The immunogenic composition of the present disclosure generally comprises the modified VSV or the genetically modified VSV vector as herein defined and a buffering agent, an agent which adjusts the osmolarity of the composition and optionally, one or more pharmaceutically acceptable carriers, excipients and/or diluents as known in the art.

As used herein the term “ pharmaceutically acceptable carrier, excipient or diluent ” includes any solvent, dispersion media, coatings, antibacterial and antifungal agents, and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

In some embodiments the immunogenic composition of the invention further comprising an adjuvant. By the term "adjuvant" as herein defined it is referred to any agent known in the art as enhancing the immunogenicity of an antigen.

In an embodiment an adjuvant is an agent that enhances the production of an antigen-specific immune response as compared to administration of the antigen in the absence of the agent. Common adjuvants include aluminum containing adjuvants that include suspensions of minerals (or mineral salts, such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate) onto which antigen is adsorbed. Other adjuvants include one or more immunostimulatory component that contributes to the production of an enhanced antigen-specific immune response. Immunostimulatory components include oil and water emulsions, such as water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like Receptor agonists (particularly, TLR2, TLR3, TLR4, TLR7/8 and TLR9 agonists), and various combinations of such components. Adjuvants can include combinations of immunostimulatory components.

Administration according to the present invention may be performed by any one of the following routes: oral administration, intravenous administration, intramuscular administration, intraperitoneal administration, intrathecal administration, intradermal administration, or subcutaneous injection, intra-rectal administration, intranasal administration, ocular administration, or topical administration.

In certain embodiments the immunogenic composition of the present disclosure is administered by injection.

In other embodiments the immunogenic composition of the present disclosure is wherein the injection is performed intramuscularly, subcutaneously or intradermally.

In further embodiments the immunogenic composition of the present disclosure is delivered to a mucosa. In still further embodiments the immunogenic composition of the present disclosure is delivered by nasal spraying or mouth spraying.

In certain embodiments the immunogenic composition as herein defined is administered to a subject in a single dose or in multiple doses.

In specific embodiments the immunogenic composition as herein defined is administered in a single-dose regimen. In further specific embodiments the immunogenic composition as herein defined is administered in a two-dose regimen, sometimes also referred to as prime and prime-boost doses. In further specific embodiments the immunogenic composition as herein defined is administered in a multiple-dose regimen.

A multiple dose regimen may be one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months or years. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner. Examples of suitable immunization schedules include: a first dose, followed by a second dose between 7 days, 14 days, 21 days, 28 days and so forth up to, for example, 6 months, and an optional third dose between 1 month and two years post initial immunization, or other schedules sufficient to elicit titers of virus-neutralizing antibodies expected to confer protective immunity. For example, other regimens may be selected to correspond to an established pediatric vaccine schedule. The generation of protective immunity against COVID-19 with the immunogenic composition may reasonably be expected after a primary course of immunization consisting of 1 to 3 inoculations. These could be supplemented by boosters at intervals (e.g., every one-two years) designed to maintain a satisfactory level of protective immunity.

In specific embodiments, the immunogenic composition is administered in a prime or prime-boost regimen.

In specific embodiments, the immunogenic composition comprises 10⁴ and 10⁸ PFU/ml in solution with 20 mM Tris, 2.5 mg/ml serum albumin, 4% trehalose, and 150 mM NaCl. In a further aspect the present disclosure provides the modified VSV, the genetically modified VSV vector or the immunogenic composition of the present disclosure for use as a vaccine.

As known in the art by the term "vaccine" it is meant to the introduction of a weakened, killed, or fragmented microorganism in order to elicit a primary immune response, such that if the organism is subsequently exposed to the actual pathogen, the body can rely on the secondary immune response to quickly defend against it.

The present disclosure further provides a kit comprising the immunogenic composition of the invention and instructions for the use of the immunogenic composition.

Still further the present disclosure provides a method of eliciting an immune response towards SARS-CoV-2 in a subject, the method comprising administering the immunogenic composition as herein defined to said subject.

The terms "subject" or “ subject in need thereof ’ refer to anyone that may benefit from the present invention such as a mammal (e.g., canine, feline, ovine, porcine, equine, bovine, or human). In one specific embodiment the subject is human. The terms " subject " or “ subject in need thereof ’ in the context of the present invention inter alia refers to mammals and in particular to human subjects at risk of being infected by SARS-CoV-2 (including but not limited to health care practitioners, subjects having a compromised immune system, elderly people and subjects suffering from other diseases, for example chronic diseases).

The effective amount (or immunogenic effective amount) of the recombinant VSV, genetically modified VSV vector according to the invention, or the immunogenic composition according to the invention for purposes herein defined is determined by such considerations as are known in the art in order to vaccinate against COVID-19. For any preparation used in the methods of the invention, the dosage or the immunogenic effective amount can be estimated initially from in vitro cell culture assays or based on suitable animal models.

According to an embodiment, exemplary doses for administration of the recombinant VSV or the genetically modified VSV vector according to the invention, are lxlO⁴, lxlO⁵, lxlO⁶, lxlO⁷, or lxlO⁸ PFU/ml. The recombinant VSV, the genetically modified VSV vector or the immunogenic composition as herein defined may also be used to vaccinate an animal to generate an immune response. Antibody-containing material may be then harvested from the vaccinated animal and purified and used as a post exposure therapy (passive immunity).

The present disclosure further provides a method of producing the rVSV-AG- spike. In accordance with an embodiment of the method of the invention the production process comprises a primary transfection step followed by subsequent passages of the viruses in at least one cell type. The primary transfection step includes the introduction of the rVSV-AG-spike plasmid into cells, for example BHK-21 cells, that were infected with MVA-T7 (for example, by incubation for one hour). The rVSV-AG-spike plasmid is introduced together with accessory plasmids comprising the genes encoding for the VSV proteins (N), (P), (M), (L) and (G). Following primary transfection, the supernatant (culture media) containing the recovered VSV-AG-spike is collected, centrifuged, and then filtered (e.g., using 0.22 mM filter), to remove residual MVA-T7 virus. After additional infection (e.g., for 72 hours) in cells, for example BHK-21 cells, the supernatant is collected, centrifuged, and used for further passaging in another cell line, for example in Vero E6 cells (ATCC® CRL-1586™).

Therefore, the present disclosure further provides a method of producing a recombinant VSV comprising SARS-CoV-2 spike protein, comprising the steps of: a. Constructing a plasmid comprising the genes encoding for VSV nucleocapsid protein (N), VSV phosphoprotein (P), VSV matrix protein (M), VSV polymerase (L), and SARS-CoV-2 spike protein or an immunogenic fragment thereof (rVSV-AG-spike); b. Transfecting cells with the rVSV-AG-spike plasmid, separately or together with accessory plasmids, wherein said accessory plasmids comprising the cis acting signals for VSV replication and genes encoding the VSV N, P, L and G proteins; c. culturing the cell in culture media under conditions that permit expression of the rVSV-AG-spike plasmid and production of the recombinant VSV comprising SARS-CoV-2 spike protein; and d. isolating the recombinant VSV comprising SARS-CoV-2 spike protein from the culture media.

In some embodiments the rVSV-AG-spike plasmid and the accessory plasmids comprise a promoter that facilitates high expression levels, for example a T7 promoter.

In some embodiments prior to step (b) said cell is infected with MVA-T7 (e.g., by incubation with MVA-7), whereby expression of T7 polymerase is induced in said cell. The incubation time with T7 is determined such that it is sufficient to induce the T7 polymerase. In one embodiment said incubation is for at least about 30 minutes, or between about 30 minutes and 120 minutes.

In one embodiment said infection with MVA-T7 is performed by incubation for about one hour.

In still further embodiments said cell is a BHK-21 cell.

In one embodiment, said isolated recombinant VSV comprising SARS-CoV-2 spike protein is subjected to at least one passage in Vero E6 cells.

In some embodiments, said isolated recombinant VSV comprising SARS-CoV-2 spike protein is subjected to at least one passage, at least 5 passages, at least 7 passages, at least 10 passages, at least 11 passages at least 12 passages, at least 13 passages, at least 14 passages or more.

Passages in Vero E6 cells are performed at least for the purpose of removing MVA-T7 from the culture. Therefore, it is appreciated that the number of passages is determined according to the purity of the isolated recombinant VSV comprising SARS- CoV-2 spike protein.

As known in the art, by the term " passage number " or by the number of passages referred to herein it is referred to a record of the number of times the isolated recombinant VSV comprising SARS-CoV-2 spike protein has been used to transfect cells (sub-cultured) and isolated thereafter.

The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “ comprise ”, and variations such as “ comprises ” and “comprising” , will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

EXAMPLES

Materials and Methods

Construction of rVSV-AG-spike plasmid

The pVSV-spike expression plasmid was constructed by PCR amplification of the full length human codon optimized spike gene from pCMV3-SARS-CoV-2 spike expression plasmid (Sino Biological, Cat #VG40588-UT) using the primers listed in Table 5, namely the forward primer having the nucleic acid sequence GAG TGA GTG TGT GCT GGG AC A A, denoted herein by SEQ ID NO. 7 (the "S gene forward primer") and reverse primer having the nucleic acid sequence AAA CAC TCC CTC CCT TGG AAA, denoted herein by SEQ ID NO. 8 (the "S gene reverse primer"). The amplified PCR product was digested by Mlul and Nhel restriction enzymes (NEB) and was ligated into the pVSV-FL+(2) vector (Kerafast), precut by the same enzymes, which removed the VS V-gly coprotein G gene (Figure 1). The ligated plasmid was electroporated into DH5-alpha electro-competent bacterial cells and selected by ampicillin resistance encoded by the plasmid.

Colonies were screened using real time PCR for the VSV genes L and G, and for the codon optimized S gene, using the primers listed below. Specifically the primers used for screening the VSV L gene were the forward and reverse primers having the nucleic acid sequences GCT CCC AAA AGA TAC CCG AAT denoted herein by SEQ ID NO. 1 and ATT GAA TGG ATT TAG ACG AAC TTG GT denoted herein by SEQ ID NO. 2 respectively and the primers used for screening the VSV G gene were the forward and reverse primers having the nucleic acid sequences ATT GCC CGT CAA GOT CAG AT denoted herein by SEQ ID NO. 4 and CCG TCT GOT TGA ATA GCC TTG T denoted herein by SEQ ID NO. 5, respectively. The primers used for screening the codon optimized S gene were the forward and reverse primers denoted herein by SEQ ID NO. 7 and SEQ ID NO. 8, respectively. Plasmid preparations from positive clones (namely positive for L and S, and negative for G), were verified using high throughput sequencing. A plasmid preparation harboring 100% sequence identity to the designed plasmid was chosen.

Real-time RT PCR

Real-time PCR was performed using the SensiFAST™ Probe Lo-ROX kit (Biolone). Real-time RT PCR was performed using the SensiFAST™ Probe Lo-ROX one-step kit (Biolone). In each reaction, the primers final concentration was 600 nM and the probe concentration was 300 nM. Primers and probes were designed using the Primer Express Software (Applied Biosystems), and purchased from Integrated DNA technologies, Inc. The primers are listed in Table 5 below.

Table 5 Primers sequences

* Probes were ordered as 6-FAM and ZEN/lowa Black FQ combination Recovery of the heterologous VSV-spike system:

BHK-21 cells (ATCC® CCL-1 0) were used for the primary recovery of the virus. To that end, BHK-21 cells were infected with MVA-T7 virus (Modified Vaccinia Ankara virus expressing T7) for 1 hour. The infection of the cells by the virus induced expression of the T7 polymerase. This incubation was followed by co-transfection of the cells with five (5) plasmids: the full length rVSV-AG-spike (prepared as detailed above), together with the VSV accessory plasmids encoding for VSV N, P, L and G proteins, all of which are under control of T7 promoters, in order to facilitate initial cell penetration and virus generation. The primary transfection was performed using the Calcium chloride method. Although the rVSV-AG-spike lacks the G protein, in order to further support the entry of the recovered virus during the initial steps, BHK-21 cells were transfected with pCAGGS-VSV-G plasmid, a plasmid encoding the VSV G protein under strong pCAG promoter. Following primary transfection (after 48 hours), the supernatant containing the recovered VSV-AG-spike was collected, centrifuged at 1300g X 5 minutes to remove cell debris, and then filtered twice using 0.22 mM filter, to remove residual MVA-T7 virus. pCAGGS-G transfected BHK-21 cells were then infected with the total amount of the filtered supernatant. After infection (72 hours), syncytia of the cells comprising the monolayer were visualized, accompanied by significant cytopathic effect (CPE). The supernatant was then collected, centrifuged and used for further passaging. In order to support the VSV-spike cell entry during the following passaging steps, and to prevent carryover of the MVA-T7, Vero E6 cells (ATCC® CRL-1586™) were chosen for use. Vero E6 cells are known to express high levels of ACE2, the prominent receptor of the SARS-CoV-2 spike, mediating its fusion and entry, and they do not support MVA replication.

In order to elevate the expression of rVSV-AG-spike and to increase its titer, while eliminating VSV-G protein carryover, subsequent passages were performed on Vero E6 cells. The process was accompanied by genetic analysis by quantitative real time RT-PCR using specific primers for the SARS-CoV-2 S (as detailed in table 5 above), as well as the VSV L, N and G genes, in order to verify S gene genome integration, and the elimination of VSV-G RNA (derived from the support plasmid) throughout the enrichment passages. rVSV-AG-spike was propagated in Dulbecco's Modified Eagle Medium (DMEM) containing 5% FBS, Minimum Essential Medium (MEM) non-essential amino acids, 2 mM L-Glutamine, 100 Units/ml Penicillin, 0.1 mg/ml streptomycin, 12.5 Units/ml Nystatin (Biological Industries, Israel). In addition, 15 mM D-Trehalose was added to the rVSV-AG-spike prior to storage at -80°C. Both 5% FBS, and D-Trehalose, were used as means to maintain the stability of the rVSV-AG-spike construct in storage. Immunofluorescence analysis (IF A)

To detect the efficiency of spike protein expression in rVSV-AG-spike-infected cells, immunofluorescence assays were performed. Monolayers of Vero E6 cells were infected with rVSV-AG-spike for 24 or 48 hours. Cells were then fixed with 3% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100, blocked with PBS containing 2% FCS and stained with Covidl9 convalescent human serum, followed by Alexa488 conjugated anti-human antibody (Figure 2A). Cells' nuclei were visualized using DAPI. SARS-CoV-2 spike protein was efficiently expressed in rVSV-AG-spike- infected Vero E6 cells, as opposed to WT-VSV-infected cells. Moreover, immunostaining of rVSV-AG-spike-infected cells displayed syncitia, mostly at the initial passages during the creation of the rVSV-AG-spike, whereas the advanced steps showed individual infected cells expressing spike protein (Figure 2A).

Electron microscopy analysis

To determine the ultrastructure of the rVSV-AG-spike, transmission electron microscopy (TEM) was performed at several time points during the passaging process (Figure 2B). Carbon-coated grids were immersed in DDW. VSV-WT or rVSV-AG- spike were absorbed by a drop-on-grid method (DOG) for 15-20 min. Blocking solution of PBS containing 2% Bovine serum albumin (BSA) was filtered by 0.22pm filter. The grids were blocked for 20 min. immunolabeling of rVSV-AG-spike was performed using polyclonal antibody (pAB RBD SBF40150-T62) directed at the receptor binding domain (RBD) of the spike, followed by washing with PBS (3 times), and then immunostained with gold-conjugated secondary antibody. The grids were then washed 3 times with PBS, and then twice with DDW. Grids were stained with phosphotungstic acid (1%). Plaque reduction neutralization test.

Vero E6 cells were seeded in 12-well plates as described above. Sera from 12 convalescent COVID-19 patients was collected by the National Blood Services of “Magen David Adorn” in Israel within a protocol for plasma donation. All convalescent volunteers gave their informed consent to the National Blood services of Magen David Adorn. The study was approved by the ethics committee of the Israeli Ministry of Health (0083-20-WOMC). We have complied with all relevant ethical regulations for work with human samples. Additional sera used: rVSV-AG-spike vaccinated hamsters’ sera, SARS-CoV-2 infected hamsters’ sera, and mock-infected hamsters’ sera. All sera were heat-inactivated (HI) (at 56 °C or 60 °C for 30 min), then diluted in twofold serial dilutions (between 1:20 and 1:40,960) in 400 mΐ of infection medium, mixed with 400 mΐ of either 300 pfu/ml of rVSV-AG-spike or SARS-CoV-2, and incubated at 37 °C, 5% C02 for 1 h. Monolayers were washed once with DMEM w/o FBS (for SARS-CoV-2 neutralization only) and 200 mΐ of each serum-virus mixture was added in triplicates to the cells for 1 h at 37 °C. Virus mixture without serum served as control. Two milliliters per well overlay were added to each well and plates were incubated at 37 °C 5% C02 for 48 (for SARS-CoV-2) or 72 h (for rVSV-AG-spike). Following incubation, overlay was aspirated, and the cells were fixed and stained with 1 ml/well of crystal violet solution. The number of plaques in each well was determined, and the serum dilution that neutralizes 50% of the virions (NT50) was calculated using Prism software (GraphPad Software Inc.).

Whole-genome sequencing and data analysis.

The SMART er Pico RNA Kit (Clontech) was used for library preparation. Whole-genome sequencing was conducted using the Illumina MiSeq platform, with a read length of 60 nucleotides, producing 5,821,469 reads. FastQC was used for quality control of the data. Reads originated from Vero E6 host cells were filtered out using Bowtie 2, resulting in 1,070,483 reads originated from rVSV-AG-spike. Mapping of the reads against the rVSV-AG-spike was performed using Bowtie 2 followed by variant calling using Samtools, both with default parameters, resulting in a 3178x average coverage and several variants. Animal experiments.

The animal model for SARS-CoV-2 was established by i.n. instillation of SARS-CoV-2 diluted in PBS supplemented with 2% FBS (PBF) (Biological Industries, Israel) to anesthetized [intraperitoneal ketamine (160 mg/kg) with xylazine (6 mg/kg)] 6-7-week-old golden Syrian hamsters (60-90 g, Charles River Laboratories, USA). Animals’ body weight was monitored daily. Animals were sacrificed at 3, 5, and 7 dpi for the following analyses: (1) viral load in lungs (3 and 5 dpi), (2) viral load in nasal turbinates (3 dpi), and (3) histopathological analysis (3 and 7 dpi). Viral load and histopathological procedures are described below. Vaccination was performed by i.m. (0.05 or 0.1 ml/animal) or subcutaneous (s.c. 0.3 ml/animal) injection of rVSV-DO- spike to anesthetized golden Syrian hamsters (6-7 weeks old, 60-90 g). General observation for morbidity and weight loss of vaccinated animals were carried out for 7 or 11 days post vaccination. Sera was collected ~3 weeks post vaccination for titration of SARS-CoV-2 neutralizing antibodies. After 20 or 25 days post vaccination, hamsters were anesthetized, challenged i.n. with 5 x 106 pfu of SARS-CoV-2, and monitored for 11-12 additional days. For isotyping of induced antibodies, C57BL/6J mice (10-14 weeks old, about 20 g) were vaccinated i.m. with 107 pfu/mouse (0.1 ml/animal) of rVSVAG-spike. Vaccinated mice sera were collected 14 days post vaccination to determine SARS-CoV-2 neutralizing antibodies titer and antibody isotype. All animal experiments involving SARS-CoV-2 were conducted in a BSL3 facility in accordance with the guideline of the IIBR Institutional Animal Care and Use Committee (HM-01- 20, HM-02-20, HM-03-20, M-35-20).

Lung and nasal turbinates viral load determination.

Hamsters’ lungs were harvested at 3 or 5 dpi, and nasal turbinates were harvested at 3 dpi and stored at -80 °C. Lung and nasal turbinates were processed, and infectious virus quantitation was performed by plaque assay, as described above. Viral load, as well as LOD, were calculated based on volume of cell infection, dilution factor, and tissue processing volume, and presented as pfu/organ.

Histopathology .

For hematoxylin and eosin (H&E) general histopathology evaluation, lungs were rapidly isolated, and fixed in 4% neutral-buffered PFA at room temperature (RT) for 2 weeks, followed by routine processing for paraffin embedding. Coronal, serial sections, 4-5 mih thick, were performed and selected sections were stained with H&E for light microscopy examination. Images were acquired using Nikon Eclipse 50i Light Microscope (Nikon, Tokyo, Japan) or Olympus microscope (BX60).

Histological evaluation.

Lung histopathological severity score analysis was performed according to the American Thoracic Society Documents, 2011. For immunolabeling of SARS-CoV-2, sections were deparaffmized and rehydrated through 100% ethanol, 95% ethanol, 70% ethanol, and 30% ethanol, washed in distilled water and antigens were retrieved using commercial antigen retrieval solution (Dako, CA, USA). Sections were then permeabilized for 10 min (0.2% Triton X-100 in PBS), blocked for 1 h (10% normal goat serum in PBS containing 0.05% Triton X-100), incubated with rabbit SARS-CoV- 2 primary antibody diluted 1:200 (in-house preparation of rabbit polyclonal anti-RBD) in antibody cocktail solution (50% blocking solution, 0.05% Triton X-100 in PBS) for 24 h at 4 °C. Sections were then washed three times with washing buffer (1% blocking solution in PBS containing 0.05% Triton X-100) and incubated with anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, Burlington, Canada) in antibody cocktail solution for 1 h at RT. Nuclei were stained with DAPI. Following three additional washes, slides were mounted using Fluoromount-G (Southern Biotech, Al, USA) and images were acquired using a Zeiss LSM710 confocal microscope (Zeiss, Oberkochen, Germany). DAB immunohistochemical staining was performed on 4 pm formalin fixed paraffin embedded sections using Leica Bond max system (Leica Biosystems Newcastle Ltd, UK). Slides were baked for 30 min at 60 °C, dewaxed and pretreated for 5 min with epitope-retrieval solution at pH = 6 (ER1, Leica Biosystems Newcastle Ltd, UK) followed by incubation with primary antibody (in-house preparation of rabbit polyclonal anti-RBD) at 1:6000 dilution for 30 min at RT. Detection was performed using the Leica Bond Polymer Refine HRP kit (Leica Biosystems Newcastle Ltd, UK) without amplification. Briefly, a goat anti-rabbit horseradish peroxidase (HRP) polymer was applied for 10 min (RT) followed by incubation for 5 min with DAB. All slides were counterstained with hematoxylin. Morphometric analysis of DAB images was performed using MATLAB morphological- based, brightness-based, and color-based segmentation. Color segmentation of brown (cells positive for SARS-CoV-2) and blue (hematoxylin counterstaining, negative for SARS-CoV-2) was performed, and the percentage of positive cells was calculated. Tissue/air space ratio calculation.

Tissue/air space ratio was determined using ImageJ free software analysis (particle analysis algorithm). Images of at least five random regions of interest (ROIs) per section were taken at the same magnification (x20). Color threshold parameters were determined and remained consistent throughout analysis. Total area values were measured separately for air space and tissue. Ratio of total tissue area to total air space area was calculated for each ROI. Average value of at least five ROIs per animal is presented.

Enzyme-linked immunosorbent assay (ELISA).

Recombinant SARS-CoV-2 S glycoprotein (S2P) was expressed. A stabilized soluble version of the S protein (based on GenPept: QHD43416 ORF amino acids 1- 1207) was designed to include proline substitutions at positions 986 and 987, and disruptive replacement of the furin cleavage site RRAR (residues at position 682-685) with GSAS. Protein expression carried out using ExpiCHOTM system (Thermo Fisher Scientific, USA). ELISA was performed as previously described. Briefly, Nunc MaxiSorp ELISA plates (Thermo Fisher Scientific, USA) were coated with 100 ng/ml of S2P in carbonate bicarbonate (Sigma, Israel) at 4 °C overnight. Following standard blocking and washes, plates were incubated with HI naive or vaccinated mice sera at a dilution of 1:200 for 1 h at 37 °C. Following washes, anti-mouse IgG-, IgGl-, or IgG2c- HRP conjugates were diluted 1:2000 (for IgG) or 1:10,000 (for IgGl and IgG2c) and used as secondary antibodies (Jackson ImmunoResearch, USA, Cat# 115-035-003, 115- 035-205 lot 148255, 115-035-208 lot 146880, respectively) followed by detection with 3,5,3',5'-tetramethylbenzidine (Millipore, USA).

Example 1

Immunofluorescence analysis (IF A) of cells infected with rVSV-AG-spike

In order to analyze the efficiency of spike protein expression in the rVSV-AG- spike-infected cells prepared as described above, immunofluorescence assays were performed as detailed below. Monolayers of Vero E6 cells were infected with rVSV- AG-spike for 24 or 48 hours. Cells were then fixed with 3% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100, blocked with Phosphate Buffered Saline (PBS) containing 2% fetal calf serum (FCS) and stained with COVID-19 convalescent human serum (as a source of antibodies), followed by Alexa488 conjugated anti-human antibody. Cells' nuclei were visualized using 4',6-diamidino-2-phenylindole (DAPI). As demonstrated in Figure 2A and in Figure 2B (showing two representative micrographs), SARS-CoV-2 spike protein was efficiently expressed in rVSV-AG-spike-infected Vero E6 cells, as opposed to its expression in WT-VSV-infected cells.

Taken together, the rVSV-AG-spike leads to expression of the spike protein on the surface of infected cells, simulating infection of cells by the SARS-CoV-2, thus efficiently activating both humoral and cellular response.

Example 2

Electron microscopy (EM) analysis of rVSV constructs

In order to determine the ultrastructure of the rVSV-AG-spike, analysis by transmission electron microscopy (TEM) was performed at several time points during the passaging process, as shown in Figure 3. As demonstrated in Figure 3B, rVSV-AG- spike was identified as evident by the bullet-shaped particles decorated with SARS- CoV-2 spike, instead of the VSV G protein (a TEM image of WT-VSV is shown in Figure 3A). Moreover, immunolabeling of rVSV-AG-spike using polyclonal antibodies directed at the receptor binding domain (RBD) of the spike, and counterstained with gold-conjugated secondary antibody, further supported the presence of spike structures on the rVSV-AG-spike viral particle.

Example 3

Titer enrichment and plaque assay

During the initial passages, the effect of rVSV-AG-spike was mainly displayed as formation of large syncitia (namely cell fusion), accompanied by cytopathic effect (CPE), as visualized by cell rounding, detachment, and disruption of the entire monolayer. At the advanced passages, together with the increased expression of spike and the loss of VSV-G protein, syncitia formation diminished and extensive CPE was evident. To determine rVSV-AG-spike titer, a plaque assay was performed. Vero E6 cells were seeded in 12-well plates (5xl0⁵ cells/well) and grown overnight in DMEM containing 10% fetal bovine serum (FBS), MEM non-essential amino acids (NEAA), 2 mM L-Glutamine, 100 Units/ml Penicillin, 0.1 mg/ml streptomycin, 12.5 Units/ml Nystatin (P/S/N) (Biological Industries, Israel).

Serial dilutions of rVSV-AG-spike were prepared in MEM containing 2% FCS (with NEAA, glutamine, and P/S/N), and used to infect Vero E6 monolayers (200 mΐ/well) in duplicates or triplicates. Plates were incubated for 1 hour at 37°C to allow viral adsorption. Then, 2 ml of overlay (namely MEM containing 2% FBS and 0.4% Tragacanth (Merck, Israel)) was added to each well and plates were further incubated at 37°C, 5% CO2 for 3 days. The media were then aspirated, and the cells were fixed and stained with 1 ml of crystal violet (Biological Industries, Israel). The number of plaques in each well was determined, and rVSV-AG-spike titer was calculated. Moreover, as a result of the enrichment process, high titers of rVSV-AG-spike, suitable for human vaccine dosage, were achieved.

Example 4

Plaque reduction neutralization test (PRNT)

An objective of this invention was simulating SARS-CoV-2 spike structures both on the SARS-CoV-2 virus, and in infected cells, leading to an efficient and relevant immune response. To this end, the ability of COVID-19 convalescent human serum to neutralize the rVSV-AG-spike construct prepared as described above was determined.

Vero E6 cells were seeded overnight at a density of 5xl0⁵ cells per well in 12- well plates in DMEM supplemented with 10% fetal bovine serum (FBS), MEM non- essential amino acids, 2 mM L-Glutamine, 100 Units/ml Penicillin, 0.1 mg/ml streptomycin, 12.5 Units/ml Nystatin (Biological Industries, Israel). Sera from convalescent Covidl9 patient (namely from a patient that has recovered from Covidl9) were heat-inactivated (at 56°C for 30 minutes), and then diluted in two-fold serial dilutions (between 1:40-1:5, 120), in 400 mΐ of MEM containing 2% FCS, non-essential amino acids, 2 mM L-Glutamine, 100 Units/ml Penicillin, 0.1 mg/ml streptomycin, 12.5 Units/ml Nystatin (Biological Industries, Israel), mixed with 400 mΐ of either rVSV-AG- spike, or WT-VSV (300 pfu/ml), and incubated at 37°C, 5% CO2, for one hour. Vero E6 monolayers were then infected with the sera-virus mixture in triplicates, for one hour. Non-neutralized virus served as control. An overlay (2 ml of MEM containing 2% FBS and 0.4 % tragacanth (sigma)) was added to each well and plates were further incubated at 37°C, 5% CO2 for 3 days. The media were then aspirated, and the cells were fixed and stained with 1 ml of crystal violet (Biological Industries, Israel). The number of plaques in each well was determined, and the antibody titer that neutralizes 50% of the virions (NT50) was calculated. The human sera efficiently neutralized rVSV-AG-spike virions. Notably, the antibody titer sufficient to neutralize 50% of the rVSV-AG-spike virions (NT50) was similar to the titer needed to neutralize SARS-CoV-2. Notably, the NT50 value of the human sera was similar for both rVSV-AG-spike and SARS-CoV-2.

Example 5 rVSV-AG-spike vaccine efficacy in Hamsters [I]

In order to demonstrate the efficacy of rVSV-AG-spike as a potent vaccine against SARS-CoV-2, Syrian hamsters, which serve as a COVID-19 disease model, were vaccinated with lxlO⁶ pfu of rVSV-AG-spike virion particles subcutaneously (s.c.) or left unvaccinated. All animals did not display any signs of disease. Two weeks following vaccination, sera were evaluated for neutralizing antibodies as detailed below. Sera were heat inactivated (at 56°C for 30 minutes), and two-fold serially diluted, in 400 mΐ of MEM containing 2% FCS, non-essential amino acids, 2 mM L-Glutamine, 100 Units/ml Penicillin, 0.1 mg/ml streptomycin, 12.5 Units/ml Nystatin (Biological Industries, Israel), mixed with 400 mΐ of SARS-CoV-2 (300 pfu/ml), and incubated at 37°C, 5% CO2, for one hour. Vero E6 cells monolayers were then infected with the sera- virus mixture in triplicates, for one hour. Non-neutralized SARS-CoV-2 served as control. Overlay (2 ml of MEM containing 2% FBS and 0.4% tragacanth (Sigma)) was added to each well and plates were further incubated at 37°C, 5% CO2 for 2 days. The media were then aspirated, and the cells were fixed and stained with one ml of crystal violet (Biological Industries, Israel). The number of plaques in each well was determined, and neutralization titer of the SARS-CoV-2 in all samples were determined.

As demonstrated in Figure 4, s.c. vaccination of hamsters with rVSV-AG-spike generated a strong neutralizing antibody response, with an average neutralizing titer higher than 1:160 to SARS-CoV-2, whereas naive unvaccinated hamsters did not show any neutralizing response against SARS-Cov-2. (Figure 4). Remarkably, a similar NT50 value was also observed in COVID-19 convalescent human sera, further supporting the use of rVSV-AG-spike as an effective SARS-COV-2 vaccine.

In order to further elaborate on the efficacy of the rVSV-AG-spike vaccine in eliciting protection against SARS-CoV-2, challenge of all hamsters was performed by intranasal (i.n.) instillation with 5xl0⁶ pfu per animal of SARS CoV-2 virus, at four weeks following vaccination. Unvaccinated control group (PBS) were morbid, exhibited reduced activity and lost up to 20% of their initial weight. In contrast, rVSV-AG-spike vaccinated hamsters did not show any signs of morbidity, including weight loss and activity, as shown in Figure 5.

Five days following challenge lungs were removed from both groups for examination of viral load and histological analysis. Gross pathology of the control hamsters revealed extensive lung damage, whereas rVSV-AG-spike vaccinated hamsters did not display tissue damage.

Example 6 rVSV-AG-spike vaccine efficacy in a COVID-19 hamster model [II]

In another experiment, Syrian hamsters ( Mesocricetus auratus) were infected via the intranasal route (i.n.) with SARS-CoV-2 with doses of 5xl0⁴, 5xl0⁵ or 5xl0⁶ pfu and monitored for body weight changes.

As shown in Figure 10A, animals displayed weight loss of up to 3%, 5%, and 17%, respectively, in a dose dependent manner. Days at which a statistically significant weight loss was observed are shown in Table 7. Statistical analysis was performed using one unpaired t-test per row, with correction for multiple comparisons using the Holm- Sidak method, p<0.005.

Table 7: Significant days of weight loss relative to mock group (p<0.005):

Also, histological sections of lungs 7 days post-infection (dpi) were performed (Fig. lOb-g). Lungs of hamsters infected with 5xl0⁶ pfu/hamster (Figure IOC, E, G) show focal patches of inflammation, pleural invagination and alveolar collapse, large amounts of inflammatory cells infiltration, as well as hemorrhagic areas. Edema was also observed, accompanied by protein-rich exudates. Moreover, immunostaining of the infected lung with an anti RBD rabbit polyclonal antibody showed presence of SARS- CoV-2 positive cells (Figure 10G) as compared to naive hamsters’ lungs (Figure 10F).

A dose of 5xl0⁶ pfu of SARS-CoV-2 was determined as the inoculation dose for further experiments.

Next, the safety and efficacy of a single dose of rVSV-AG-spike vaccine in the hamster model was examined. The experiments presented in Examples 6-9 were performed with vaccine candidates that originated from clone 28 and were subjected to process development and manufacturing under GMP guidelines. To that end, hamsters were vaccinated by intramuscular (i.m.) injection at increasing doses of rVSV-AG- spike: 10⁴, 10⁵, 10⁶, 10⁷ or 10⁸ pfu, and compared to mock-vaccinated hamsters. Following vaccination, animals were monitored daily for body weight changes and morbidity. No signs of lesion were observed at the site of injection (not shown). As seen in Figure 11 A, animals in all groups gained weight and did not show any signs of morbidity, suggesting that rVSV-AG-spike is safe at the tested doses. The efficacy of the vaccine was evaluated for the ability of a single vaccination of each dose to elicit neutralizing antibodies against SARS-CoV-2 by PRNT. All tested vaccine doses induced a neutralization response in a dose-dependent manner following a single-dose vaccination (Figure 11B). The binding capacity of the induced antibodies is demonstrated by immunofluorescent staining of Vero E6 cells infected with SARS- CoV-2, by using sera from unvaccinated (Figure 11C) and 10⁶ pfu-vaccinated hamsters (Figure 11D). To evaluate the efficacy of the rVSV-AG-spike vaccine to protect against SARS-CoV-2, vaccinated hamsters were challenged by i.n. instillation with 5xl0⁶ pfu of SARS-CoV-2 per animal (approximately four weeks post vaccination). Following challenge, unvaccinated animals were morbid, exhibiting a gradual weight loss up to 5 dpi. In contrast, rVSV-AG-spike vaccinated hamsters showed a mild weight loss immediately after infection, followed by a recovery, amounting to a significant improvement in body weight at 4-7 dpi for all doses, except the lowest vaccination dose of 10⁴ pfu at which a significant improvement was detected at 6 and 7 dpi (Figure 1 IE). Statistical significance was determined using two-tailed one unpaired t-test per row, with correction for multiple comparisons using Holm-Sidak method. p<0.005.

Example 7

Efficacy and viral load analysis of rVSV-AG-spike vaccinated hamsters

Five days following challenge with SARS-CoV-2 lungs were removed for viral load analysis. The presence of infectious SARS-CoV-2 was detected in infected unvaccinated lungs, with an average viral titer of 1.3xl0⁵ pfu/lung (n=7), whereas viral titers in lungs of all doses of vaccinated and infected animals (10⁴ to 10⁸ pfu, n=3 for each vaccination dose) were below the limit of detection (LOD, 75 pfu/lung) (Fig. 1 If).

As shown in Figure 11, all tested vaccination doses were shown to be safe and efficacious. Notably, vaccine doses of 10⁵ and 10⁶ pfu/hamster elicited a significant and prolonged protection window at 4-8 dpi, thus 10⁶ was chosen for further meticulous evaluation.

It was previously shown that at days 2-3 post SARS-CoV-2 infection, lungs, trachea, and nasal turbinates display high viral titers, accompanied by extensive tissue damage [Chan, J.F., et al., Clin Infect Dis, 71(9):2428-2446, 2020; Imai, M., et al., Proc Natl Acad Sci U S A, 2020. 117(28): p. 16587-16595; Osterrieder, N., et al., Viruses, 2020. 12(7)]. To that end, lungs and nasal turbinates were removed and analyzed for viral load at 3 dpi. Lungs extracted from infected hamsters showed average viral titers of 5.6X10⁶ pfu/lung (n=4), whereas viral titers in lungs of vaccinated i.m. with 10⁶ pfu/hamster and infected animals were significantly lower (average viral titers of 7.3X10³ pfu/lung) (n=4) (Figure 12A). Additionally, nasal turbinates of infected hamsters showed average viral titers of 1.9xl0⁵ pfu/lung (n=4), whereas viral titers in nasal turbinates of vaccinated and infected animals similarly showed a significant reduction in average viral titers to 2.8xl0² pfu/lung (n=4) (Figure 12B). Taken together, a single-dose vaccination of hamsters was able to reduce the viral titer by 3 orders of magnitude, in both tested organs. Example 8

Histopathological analysis of rVSV-AG-spike vaccinated lungs

To further characterize the efficacy of the rVSV-AG-spike, a detailed histopathological evaluation was performed on lungs of naive, infected, and vaccinated and infected hamsters at 3 and 7dpi. Naive animals showed no lesions and served as a negative control reference (Figure 13a, f). The lungs of infected animals showed signs of viral pneumonia, characterized by severe necrosis and inflammation. The interstitium and alveoli were infiltrated mostly by neutrophils, and fibrin deposition was evident in the severe cases. The number of lymphocytes involved was relatively low. Necrosis was found in the alveolar structures of the infected hamsters. Vasodilatation and congestion of the interstitial blood vessels was also noted. The bronchial epithelium was relatively intact, yet the submucosa was often infiltrated by lymphocytes and to some extent, cellular debris were observed in the bronchioles’ lumen. The bronchial epithelium of the infected hamsters’ lungs remained relatively unaffected, and the submucosa was often infiltrated by lymphocytes. The distribution pattern of the infection in the infected lung is multifocal to coalescing. Some cellular debris were spotted in the bronchiolar lumen (Figure 13b, d). Vaccination alleviated the disease manifestations exhibiting significantly milder form of pneumonia following infection, compared to the unvaccinated infected hamsters. Infiltration of neutrophils, hyperemia, and congestion of the lung interstitium and necrotic areas were occasionally observed, yet in a milder severity compared to the infected unvaccinated group (Figure 13c, e). Quantitative analysis of the lung manifestations severity was performed on samples 3 and 7 dpi using a severity scoring scale according to the American Thoracic Society Documents [Matute-Bello, G., et al., Am J Respir Cell Mol Biol, 2011. 44(5): p. 725-38] Based on presence of neutrophils in alveoli and in interstitium, fibrin, cellular debris, and thickened alveolar septa, a total score was summed for each animal in all tested groups, and histopathology severity score was determined. A significant damage was shown for all infected unvaccinated hamsters. For vaccinated hamsters following infection, a significant protection of the lungs was shown, displaying a reduced severity in lung damage, at both 3 and 7 dpi (Figure 13K). DAB staining was performed to evaluate the presence of SARS-CoV-2 antigens in infected and vaccinated hamsters in both 3 and 7 dpi. Positive SARS-CoV-2 cells were detected in infected lungs of both 3 and 7 dpi, with an average of 20.16% (3 dpi) and 7.87% (7 dpi), whereas vaccinated hamsters’ lungs showed no, or low SARS-CoV-2 staining, with an average of 2%, and 1.05% positive cells, at 3 and 7 dpi, respectively (Figure 13L). These results were further supported by tissue/air space analysis demonstrating significant increase in tissue/air space ratio in infected lungs at both 3 and 7 dpi compared to the naive lungs (Figure 13M), and a significant reduction of tissue/air ratio between infected and immunized lungs at both 3 and 7 dpi. Vaccinated lungs at both time points reached a baseline ratio that was similar to that of naive samples (Figure 13M).

Example 9

Antibody isotype profile induced by rVSV-AG-spike vaccination

The Thl/Th2 profile following vaccination is of major importance, and the aspired induced antibody profile is of a Thl response. To that end, the balance of Thl and Th2 response to the rVSV-AG-spike vaccine was evaluated through the differential induction of antibody isotypes. Due to the lack of suitable reagents for hamster isotyping, isotype profiling was performed in mice. C57BL/6J mice were vaccinated intramuscularly with 10⁷ pfu/mouse. Vaccinated mice sera were analyzed 14 days following vaccination for both neutralizing antibodies and spike antigen specific total IgG, as well as IgG2c and IgGl isotypes, as surrogates of Thl and Th2 responses, respectively. Vaccination elicited high neutralizing antibodies in all mice, with NT50 values at the range of 825-2548 titers (Figure 14A) (NT50 values of 1774, 825, 1672, 2076, 1649, 2548, 1959 for each mouse 1-7 respectively). High levels of IgG2c were observed in all vaccinated mice, as opposed to low levels of IgGl (Figure 14B), indicating the induction of a desirable and safe Thl-biased response to the rVSV-AG- spike vaccine.

Example 10

Vaccination of human subjects with rVSV-AG-spike

Human subjects are vaccinated by intramuscular (i.m.), intradermal (i.d.) or subcutaneous (s.c.) injection with a first and optionally a second dose of rVSV-AG- spike. rVSV-AG-spike is administered to the subjects at a dose range of 10⁴-10⁸ PFU/ml. The composition of the rVSV-AG-spike vaccine may be 10⁴-10⁸ PFU/ml in solution with 20 mM Tris, 2.5 mg/ml serum albumin, 4% trehalose, and 150 mM NaCl.

Example 11

Neutralization of B.l.1.7 (UK), B.1.351 (SA) and P.l (Brazil) VOC by sera from rVSV-AG-spike-vaccinated K18-hACE2 mice

The ability of rVSV-AG-spike vaccine to induce neutralizing antibodies against SARS-CoV-2 variants of concern (VOC), as well as to provide protection against a lethal SARS-CoV-2 VOC challenge, was examined in the K18-hACE2 mouse model. K18-hACE2 mice are transgenic mice designed to express the human ACE2 receptor under the keratin 18 promoter (K18) in epithelial cells, in various tissues including airway epithelia, namely the route of SARS-CoV-2 infection.

To that end, K18-hACE2 mice were vaccinated intramuscularly with 10⁷ pfu/mouse of rVSV-AG-spike at a prime-boost (P+B) regimen; in a three-weeks interval. Three weeks following boost vaccination, mice sera were tested by plaque reduction neutralization test (PRNT) for the level of neutralizing antibodies against several SARS-CoV-2 variants: WT, B.l.1.7, B.1.351, or P.l. Nearly all tested vaccinated mice sera were able to efficiently neutralize WT, B.l.1.7 and P.l. variants in a similar manner. Neutralization of the B.1.351 variant was mostly maintained but titers were lower than for other variants (Figure 17 A).

Example 12

Protection of K18-hACE2 mice from lethal SARS-CoV-2 VOC challenge by rVSV-AG-spike vaccination

Next, rVSV-AG-spike vaccinated mice were challenged intranasally with a lethal dose of one of the following SARS-CoV-2 variants: WT, B.l.1.7, or B.1.351, and monitored daily for body weight changes (Figure 17B), and for survival (Figure 17C). For each variant, the lethal dose was previously determined: WT - 2000pfu (-100LD50), B.l.1.7 - 500pfu (-1000LD50), B.1.351 - 10,000pfu (-20LD50). Following infection, all vaccinated mice did not display any weight loss throughout the experiment, and all survived the lethal challenge of either WT, B.1.17, or B.1.351 variant, whereas all the unvaccinated - infected mice, except for one WT-infected mouse, succumbed to the disease. Even the single unvaccinated mouse that survived the WT vims infection, deteriorated unlike the vaccinated mice which did not show any signs of morbidity.

Claims

CLAIMS:

1. A recombinant vesicular stomatitis virus (VSV) comprising: a VSV nucleocapsid protein (N), a VSV phosphoprotein (P), a VSV matrix protein (M), a VSV large polymerase protein (L), and a SARS-CoV-2 spike protein or an immunogenic fragment thereof, wherein said recombinant VSV is lacking the VSV G protein.

2. The recombinant VSV of claim 1, wherein the SARS-CoV-2 spike protein is a wild-type SARS-CoV-2 spike protein, a mutated SARS-CoV-2 spike protein, a chimeric SARS-CoV-2 spike protein or an immunogenic fragment thereof.

3. The recombinant VSV of claim 1 or 2, wherein the SARS-CoV-2 spike protein is truncated at the C-terminus.

4. The recombinant VSV of claim 3 wherein the SARS-CoV-2 spike protein lacks 24 amino acids at the C-terminus.

5. The recombinant VSV of any one of the preceding claims, wherein the SARS- CoV-2 spike protein has a mutation in the Arginine (R) residue at the fourth position (R4) of the S1/S2 motif RRAR.

6. The recombinant VSV of claim 5, wherein the mutation at the R4 residue of the S1/S2 motif RRAR is an R to G substitution or an R to K substitution.

7. The recombinant VSV of claim 5 or 6, wherein the mutation at the R4 residue of the S1/S2 motif RRAR is at amino acid position 685 of the SARS-CoV-2 spike protein as denoted by SEQ ID NO. 16 or SEQ ID NO. 17.

8. The recombinant VSV of any one of the preceding claims, wherein the SARS- CoV-2 spike protein lacks 24 amino acids at the C-terminus and has a mutation at the fourth Arginine (R) residue of the S1/S2 motif RRAR.

9. The recombinant VSV of any one of the preceding claims, wherein the SARS- CoV-2 spike protein comprises the amino acid sequence set forth in SEQ ID NO. 16 or the amino acid sequence set forth in SEQ ID NO. 17, or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

10. The recombinant VSV of any one of the preceding claims, wherein the recombinant VSV is encoded by a nucleic acid sequence comprising the nucleic acid sequence set forth in SEQ ID NO. 18 or the nucleic acid sequence set forth in SEQ ID NO. 19, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

11. The recombinant VSV of any one of claims 1-4, wherein the SARS-CoV-2 spike protein lacks 24 amino acids at the C-terminus and has one or more amino acid substitutions at any one of positions 685, 245, or 813 or any combination thereof.

12. The recombinant VSV of claim 11, wherein said one or more amino acid substitution is any one of a R to G substitution at position 685, an H to R substitution at position 245, or an S to L substitution at position 813, or any combination thereof.

13. The recombinant VSV of claim 12, comprising a R to G substitution at position 685, an H to R substitution at position 245, and an S to L substitution at position 813.

14. The recombinant VSV of claim 13, wherein the SARS-CoV-2 spike protein comprises the amino acid sequence set forth in SEQ ID NO. 21, or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

15. The recombinant VSV of claim 13, wherein the recombinant VSV is encoded by a nucleic acid sequence comprising the nucleic acid sequence set forth in SEQ ID NO. 22, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

16. The recombinant VSV of any one of the preceding claims, further comprising one or more amino acid substitution at any one of positions 64, 66, 113, 484, 493, 501 or 615, or any combination thereof.

17. The recombinant VSV of claim 16, wherein said one or more amino acid substitutions is selected from the group consisting of S 8131, W64R, H66R, K113Q, E484D, Q493R, N501Y, and V615L.

18. An immunogenic composition comprising the recombinant VSV of any one of the preceding claims and a pharmaceutically acceptable excipient, carrier, or diluent.

19. The immunogenic composition of claim 18 wherein said composition comprises a heterogeneous population of recombinant VSV, said heterogeneous population comprising recombinant VSV with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 and recombinant VSV with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 further comprising one or more mutations selected from the group consisting of S 8131, W64R, H66R, K113Q, E484D, Q493R, N501Y, and V615L.

20. The immunogenic composition of claim 19 wherein said recombinant VSV with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 has a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% frequency in said heterogenous population.

21. The immunogenic composition of claim 19 wherein said recombinant VSV with a spike protein having an amino acid sequence denoted by SEQ ID NO: 21 further comprising one or more mutations has a frequency of between about 5% to about 100% in said heterogenous population.

22. The immunogenic composition of any one of claims 18-21, for eliciting an immune response against SARS-CoV-2 in a subject.

23. The immunogenic composition of claim 22, wherein said SARS-CoV-2 is a SARS-CoV-2 variant.

24. The immunogenic composition of claim 23, wherein said SARS-CoV-2 variant is selected form the group consisting of the United Kingdom variant, the South Africa variant, and the Brazil variant.

25. The immunogenic composition of any one of claims 18-24, further comprising an adjuvant.

26. The immunogenic composition of claim 25, wherein said adjuvant is selected from a group consisting of aluminum containing compounds (such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate), immunostimulatory compounds (such as liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids, e.g., CpG oligonucleotides, liposomes, Toll-like Receptor agonists e.g., TLR2, TLR3, TLR4, TLR7/8 or TLR9 agonists), and combinations thereof.

27. The immunogenic composition of any one of claims 18-26, wherein the immunogenic composition is administered by injection.

28. The immunogenic composition of claim 27, wherein the injection is performed intramuscularly, subcutaneously or intradermally.

29. The immunogenic composition of any one of claims 18-26, wherein the immunogenic composition is delivered to a mucosa.

30. The immunogenic composition of claim 29, wherein the immunogenic composition is delivered by nasal spraying or mouth spraying.

31. The immunogenic composition of any one of claims 18-30, wherein the immunogenic composition is administered in a single-dose regimen.

32. The immunogenic composition of any one of claims 18-30, wherein the immunogenic composition is administered in a multiple-dose regimen (e.g., a prime- boost regimen).

33. The immunogenic composition of any one of claims 18-32, wherein the immunogenic composition comprises between 10⁴ and 10⁸ PFU/ml in solution with 20 mM Tris, 2.5 mg/ml serum albumin, 4% trehalose, and 150 mMNaCl.

34. The recombinant VSV of any one of claims 1 to 17 or the immunogenic composition of any one of claims 18 to 33 for use as a vaccine.

35. A kit comprising the immunogenic composition of any one of claims 18 to 33 and instructions for the use of the immunogenic composition.

36. A method of eliciting an immune response towards SARS-CoV-2 in a subject, the method comprising administering the immunogenic composition of any one of claims 18 to 33 to said subject.

37. The method of claim 36, wherein said SARS-CoV-2 is a SARS-CoV-2 variant.

38. The method of claim 37, wherein said SARS-CoV-2 variant is selected from the group consisting of the United Kingdom variant, the South Africa variant, and the Brazil variant.

39. A method of producing a recombinant VSV comprising SARS-CoV-2 spike protein, comprising the steps of: a. constructing a plasmid (rVSV-AG-spike) comprising the genes coding for VSV nucleocapsid protein (N), VSV phosphoprotein (P), VSV matrix protein (M), VSV polymerase (L), and SARS-CoV-2 spike protein or an immunogenic fragment thereof; b. transfecting a cell with the rVSV-AG-spike plasmid, separately or together with accessory plasmids, wherein said accessory plasmids comprising the cis acting signals for VSV replication and genes encoding the VSV N, P, L and G proteins; c. culturing the cell in culture media under conditions that permit expression of the rVSV-AG-spike plasmid and production of the recombinant VSV comprising SARS-CoV-2 spike protein; and d. isolating the recombinant VSV comprising SARS-CoV-2 spike protein.

40. The method of claim 39, wherein said rVSV-AG-spike plasmid and said accessory plasmids comprise a T7 promoter.

41. The method of claim 39 or claim 40, wherein prior to step (b) the cells to be transfected are infected with MVA-T7, whereby expression of T7 polymerase is induced in said cells.

42. The method of claim 41, wherein said infection with MVA-T7 is performed by incubation for at least about 30 minutes, or between about 30 minutes and 120 minutes or for about one hour.

43. The method of any one of claims 39 to 42, wherein said cell is a BHK-21 cell.

44. The method of any one of claims 39 to 43 wherein said isolated recombinant VSV comprising SARS-CoV-2 spike protein is subjected to at least one passaging step in Vero E6 cells.

45. The method of claim 44 wherein said isolated recombinant VSV comprising SARS-CoV-2 spike protein is subjected to at least one passage, at least 5 passages, at least 7 passages, at least 10 passages, at least 11 passages at least 12 passages, at least 13 passages, at least 14 passages or more in Vero E6 cells.

46. The method of any one of claims 39 to 45 wherein after step (d) said recombinant VSV comprising SARS-CoV-2 spike protein is filtered one or more times to remove residual MVA-T7.

47. The method of claim 46 wherein said filtering is performed twice.

48. The method of claim 47 wherein said filtering is performed using a 0.22mM filter.

49. The method of any one of claims 39-48, wherein in step (a) said plasmid comprises the nucleic acid sequence set forth in SEQ ID NO. 20 or the nucleic acid sequence set forth in SEQ ID NO. 23.

50. The method of any one of claims 39-49, wherein in step (d) said recombinant VSV comprises a nucleic acid sequence as set forth in SEQ ID NO 18 or the nucleic acid sequence set forth in SEQ ID NO 19, or the nucleic acid sequence set forth in SEQ ID NO 22, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.

51. A genetically modified vesicular stomatitis virus (VSV) vector encoding VSV nucleocapsid protein (N), VSV phosphoprotein (P), VSV matrix protein (M), VSV polymerase (L), and SARS-CoV-2 spike protein or an immunogenic fragment thereof, wherein said VSV vector is lacking the gene encoding the VSV G protein.

52. The genetically modified VSV vector of claim 51, wherein said vector comprises a nucleic acid sequence set forth in SEQ ID NO 20 or SEQ ID NO 23 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 98%, or at least 99% identical thereto.