VIRAL VACCINE VECTOR FOR IMMUNIZATION AGAINST A BETACORONAVIRUS

Abstract

The present invention relates to a composition comprising (a) a recombinant rhabdovirus vector capable of forming a virus particle and expressing an immunogen of a betacoronavirus, wherein the immunogen comprises at the C-terminus a heterologous transmembrane anchor for the incorporation of the immunogen into (i) the cell membrane of infected cells, and (ii) the envelope of the virus particle, and/or (b) a glycoprotein (G) protein gene deleted and in trans G protein complemented recombinant rhabdovirus vector capable of forming a virus-like particle (VLP) and expressing an immunogen of a betacoronavirus, wherein the immunogen comprises at the C-terminus a heterologous transmembrane anchor for the incorporation of the immunogen into (i) the cell membrane of infected cells, and (ii) the VLP.

Claims

1. A composition comprising (a) a recombinant rhabdovirus vector capable of forming a virus particle and expressing an immunogen of a betacoronavirus, wherein the immunogen comprises at the C-terminus a heterologous transmembrane anchor for the incorporation of the immunogen into (i) the cell membrane of infected cells, and (ii) the envelope of the virus particle, and/or (b) a glycoprotein (G) protein gene deleted and in trans G protein complemented recombinant rhabdovirus vector capable of forming a virus-like particle (VLP) and expressing an immunogen of a betacoronavirus, wherein the immunogen comprises at the C-terminus a heterologous transmembrane anchor for the incorporation of the immunogen into (i) the cell membrane of infected cells, and (ii) the VLP.

2. The composition of claim 1, wherein the betacoronavirus is selected from SARS-CoV-2, MERS-CoV, SARS-CoV-1, OC43, and HKU1, and is preferably SARS-CoV-2.

3. The composition of claim 1, wherein the immunogen is the spike (S) protein or an immunogenic fragment thereof.

4. The composition of claim 3, wherein the immunogenic fragment consists of or comprises the spike receptor binding domain (RBD) and preferably-comprises a fragment as represented by SEQ ID NO: 1 or a variant of SEQ ID NO: 1 being with increasing preference at least 80%, at least 85%, at least 90% and at least 95% identical to SEQ ID NO: 1.

5. The composition of claim 1, wherein the rhabdovirus of (a) is preferably rhabdovirus vesicular stomatitis virus (VSV) or rabies virus (RABV), and/or the rhabdovirus of (b) is preferably a G protein gene deleted and pseudotyped VSV or RABV.

6. The composition of claim 1, wherein the immunogen of a betacoronavirus comprises at the N-terminus a signal peptide that promotes high-level translation into the endoplasmatic reticulum, wherein the signal peptide is preferably derived from an immunoglobulin, preferably from IgG and most preferably from the heavy chain of IgG.

7. The composition of claim 1, wherein the transmembrane anchor is derived from the stem of a rhabodovirus glycoprotein, and preferably derived from members of the Lyssavirus genus, more preferably derived from the rabies (RABV) vaccine strain SAD B19 (molecular clone SAD L16), and most preferably comprises or consists of the membrane proximal part of the G ectodomain, the trans-membrane and the cytoplasmic tail of the RABV SAD L16 G protein.

8. The composition of claim 1, wherein the recombinant rhabdovirus vector comprises at least two, preferably at least three copies of the nucleotide sequence encoding the immunogen of a betacoronavirus, wherein the vector comprises these copies at one site or at different sites of the vector.

9. The composition of claim 1, wherein the composition additionally comprises a VLP, wherein the VLP is preferably a G protein gene deleted rhabdovirus which is not complemented in trans with a functional G protein gene.

10. (canceled)

11. (canceled)

12. (canceled)

13. An immunogenic construct of a betacoronavirus, comprising (i) the spike receptor binding domain (RBD) of a betacoronavirus, wherein the RBD preferably comprises SEQ ID NO: 1 or a variant of SEQ ID NO: 1 being with increasing preference at least 80%, at least 85%, at least 90% and at least 95% identical to SEQ ID NO: 1, and C-terminally thereof; (ii) a transmembrane anchor being derived from the stem of a rhabodovirus glycoprotein, and preferably derived from members of the Lyssavirus genus, more preferably derived from the rabies (RABV) vaccine strain SAD B19 (molecular clone SAD L16), and most preferably comprising or consisting of the membrane proximal part of the G ectodomain, the trans-membrane and the cytoplasmic tail of the RABV SAD L16 G protein; and (iii) at the N-terminus a signal peptide that promotes high-level translation into the endoplasmatic reticulum.

14. A nucleic acid molecule encoding an immunogenic construct of claim 13.

15. A virus vaccine vector or a plasmid or a DNA or RNA preparation expressing the immunogenic construct of claim 13 or comprising a nucleic acid molecule encoding the immunogenic construct.

16. A method for preventing or treating a betacoronavirus infection and a SARS-CoV-2 infection in a subject, comprising administering to the subject an effective amount of the composition of claim 1.

17. The method of claim 16, wherein the method further comprises a boost immunization with the composition or with virus-like particles presenting the chimeric immunogen in their envelope.

18. A method of detecting the presence of a betacoronavirus infection in a subject based on a sample, comprising using the composition of claim 1, wherein the betacoronavirus infection is a SARS-CoV-2 infection, and wherein the sample is a blood sample or a serum sample obtained from the subject.

Description

[0143] The figures show.

[0144] FIG. 1—Schematic illustration of the presentation of a betacoronavirus immunogen simultaneously on the surface of infected cells (item 1) and on the surface of virus-like particles (item 2).

[0145] FIG. 2—(A) Schematic representation of SARS-CoV-2 Spike protein, and chimeric rhabdovirus minispike containing the RBD of SARS-CoV-2 Spike. (B) Sequence of rhabdovirus minispike. Signal sequence and transmembrane sequence is underlined, linker in italics, and SARS-nCoV-2 residues in bold letters.

[0146] FIG. 3—(A) Expression of minispikes in HEK293T cells transfected with pCAGGs-minispike (lane 2) and in BHK-21 cells infected with VSVΔG-minispike-eGFP (lane 4). Lane 1 shows irrelevant vector transfection, lane 3 HEK293T cells transfected with the pCAGGS-RABV G as a positive control for the peptide serum recognizing the C-tail of minispike and G. Bands of heterogenous size indicate glycosylation of the minispike. Lane 4 shown minispike expressed from recombinant VSVdG minispike (B) Complex glycosylation of minispike protein in HEK293T cells transfected with pCAGGs-minispike (lanes 1-3). Extracts were treated with PNGase F, which cleaves off all N-linked oligosaccharides (lane 1), or left untreated (lane 2) or treated with Endo H, cleaving off N-linked mannose rich oligosaccharides, but not highly processed complex oligosaccharides. Lanes 4-6 show RABV G treated in parallel (4: PNGaseF, 5, untreated, 6: EndoH)

[0147] FIG. 4—Schematic illustration of recombinant VSV and RABV virus constructs used here.

[0148] FIG. 5—VSVdG-minispike eGFP-infected BHK-21 are recognized by reconvalescent COVID-19 patient sera. (A) Infected unfixed cell cultures were incubated with the indicated patient sera, followed by incubation with fluorescent anti-human IgG antibodies (upper panel). Lower panel shows eGFP fluorescence in cell cultures (vector control). (B) COVID-19 patient sera recognize minispike protein in acetone-fixed cell cultures. ELISA-positive serum recognizes minispike expressing cells (left image). Serum antibodies were stained with anti-human IgG secondary antibodies labelled with Alexa555.

[0149] FIG. 6—VSVdG-bi-minispike (VSVdG-bimini) infected cells are recognized by reconvalescent COVID-19 patient sera. BHK-21 cells were infected for 16 hours with VSVdG-bimini virus and after acetone fixation incubated with COVID-19 patient sera (#1-4, 9-10) or sera from healthy persons (#5, 6, 8), followed by anti-human IgG-Alexa488 (green fluorescence).

[0150] FIG. 7—Incorporation of minispike protein in VSV virus particles. Cell-free VSVdG-minispike-eGFP particles (lanes 1-3) were generated in HEK293T cells expressing VSV-G from transfected pCAGGS-VSV G and analyzed by Western blot. In lane 1 unconcentrated cell culture supernatant was applied, in lanes 2 and 3 virions which were ultracentrifuge-purified through a sucrose cushion. Lanes 4 and 5 contains stocks of standard VSVdG-eGFP, and of full-length wt VSV-eGFP grown in BHK cells, respectively. Lanes were loaded with 3×10E6 infectious units. Blots were incubated with anti-VSV serum (left panel) and a serum recognizing the RABV G-derived C-tail of the chimeric SARS-CoV-2/rhabdovirus minispike (right panel). Note that efficient incorporation into VSV virus particles is achieved even in the presence of high amounts of competing VSV G protein (lane 2).

[0151] FIG. 8—Preparation of VSVdG Minispike VLPs lacking G. VSVdG-eGFP(VSVG) was used to infect non-G-complementing cells to produce VLPs decorated only with minispike protein. Cell culture supernatant was harvested one day after infection and VLPs purified by ultracentrigugation. VLPs were analyzed by Western blot with HCA-serum to detect minispike protein, and VSV Serum for detection of G protein. In contrast to particles produced in the presence of G (+G), VSV G was not detected in VLPs from non-complementing cells (lane: no G).

[0152] FIG. 9—Gene copy number determines expression levels of minispike protein. Lysates from cells infected with the indicated viruses were analyzed by Western blot with HCA-5 serum for expression levels of the minispike protein. Actin and VSV antibodies were used for normalization. Two copies of the minispike gene encoded in full length VSV vector lead to higher expression of the protein. Note that the presence of an unrelated extra gene (eGFP reporter) in general attenuates expression levels. For full length VSV the ranking is therefore: VSV-Bimini>-bimini-eGFP>-minispike>-minispike-eGFP. The VSV delta G version, VSVdG-bimini, encoding 1 gene less than VSV-bimini expresses the highest amounts of minispike.

[0153] FIG. 10—Incorporation of minispike protein in RABV virus particles. Spike protein and rhabdovirus G constructs expressed from transfected plasmids were used to pseudotype VSVdG-eGFP and RABV SADdG-eGFP. As predicted, the minispike construct is suitable to produce both VSV and RABV VLPs. In addition, VSVdG can be pseudotyped with RABV G, (left panel) supporting efficient entry into tonsil cells during oral immunization.

[0154] FIG. 11—Immunization with VSVΔG-minispike-eGFP vaccination elicits high levels of SARS-CoV2-neutralizing antibodies in BALB/c mice. (A) Immunization Scheme. BALB/c mice were immunized i.m. with 1×10.sup.6 infectious units of VSV G-complemented VSVΔG-minispike-eGFP and controls including VSV G-complemented VSVΔG-eGFP, or PBS. Twenty-eight days after immunization serum was collected from 4 vaccinated mice, while 8 mice received an i.m. boost immunization with the same dose of virus. (B) Serum neutralization tests performed with a clinical isolate of SARS-CoV-2. The neutralizing titer of sera from vaccinated and control mice as indicated is expressed as the reciprocal of the highest dilution at which no cytopathic effect was observed. Each point represents data from one animal at the indicated time points. The bars show the mean from each group and the error bars represent standard deviations. Significant neutralizing activity was observed in mice receiving only a prime vaccination (day 28, light blue). A boost immunization further significantly enhanced neutralizing titers (days 35 and 56). (C) Neutralization of VSVΔG(S) pseudotype viruses by individual mouse sera. Mouse sera collected on day 28 (receiving prime immunization only) or at 35 and 56 days (receiving prime and boost immunization) were serially diluted as indicated and analyzed for neutralization VSV(S) pseudotype particles. GFP-encoding pseudotype virions were incubated with increasing dilutions of mouse sera or medium control before infection of VeroE6 cells. The graph shows percentage of GFP-positive cells in relation to medium controls (100%) and in dependence of dilution. Data points represent the average of three technical replicates, bars indicate standard deviation, and statistical significance was determined by one-way ANOVA.

[0155] FIG. 12—Similar virus-neutralizing titers in vaccinated mice and COVID-19 patients. VSVΔG(S) neutralization activity of sera from vaccinated mice and human immune sera tested positive for S antibodies by ELISA were compared. The graph shows percentage of GFP-positive cells in relation to medium controls and in dependence of dilution. ELISA-positive human sera revealed VSV(S)-neutralizing activity and are included in the grey boxes showing activity at the indicated dilutions. Primed mice (d28) exhibited neutralizing activity almost comparable to those of human patients, while boosted mice (d35 and d56) exhibited superior activity. Bottom and top of each box represent the first and third quartiles respectively. Whiskers represent the lowest and highest data points of the lower and upper quartile respectively. Student's t-test and One-way ANOVA were performed to determine statistical significance.

[0156] FIG. 13—Transgenic mice are protected from SARS-CoV-2-induced respiratory disease after a single immunization with VSVΔG-minispike-eGFP. (A) Immunization and challenge schematic. C57BL/6 K18-hACE2 mice (5 per group) were immunized (1×106 ffu intramuscularly) once (prime, black arrow) or twice (boost, grey arrow) four weeks apart with either VSV-ΔG-minispike-eGFP (indicated in blue in panels B-G) or VSV-ΔG-eGFP (indicated in red in panels B-G) and challenged with 1×104 TCID50 SARS-CoV-2 (Wetzlar isolate) administered intranasally four weeks after the last immunization. Mice were monitored daily for development of disease for 14 days. (B-D) Evaluation of clinical disease of challenge after prime immunization. (E-G) Evaluation of clinical disease of challenge after prime/boost immunization. (B and E) Clinical score development assessed by body weight loss, general appearance, and behavior. 3: healthy; 4-6: mild disease; 7-9: severe disease; 10-12: moribund. (C and F) Survival plots. (D and G) Body weights of individual mice relative to the weight at challenge infection. Dotted lines indicate limits of clinical scores (>95%: score=1, 85-95%: score=2; 80-85%: score=3; <80%: score=4).

[0157] FIG. 14—Neutralization of variants of concern (VOC) by sera from VSVΔG-minispike-eGFP vaccinated mice. (A) VSVΔG pseudotype viruses carrying the indicated S proteins from emerging SARS-CoV-2 variants were incubated with serum from a vaccinated mouse (#r, 56d) and checked for infectivity of VeroE6 cells. All viruses carrying natural variant S proteins, including SA (B.1.351) were neutralized effectively (IC50>1:800), while an artificial S protein (Wuhan(D614G) with Indian RBD sequence) revealed a slightly lower susceptibility (IC50>1:600). (B) Comparison of neutralizing activity of sera from vaccinated mouse (mouse “r”, BNT162-b2 double vaccinated subject (KD post vacc.), convalescent COVID-19 patient (Post-Covid Px), and an S monoclonal antibody (SmAb). Data are from three independent experiments.

[0158] The examples illustrate the present invention.

EXAMPLE 1—DESIGN OF RHABDOVIRUS MINISPIKE

[0159] The sequence of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome, NCBI Reference Sequence: NC_045512.2) was used to prepare a synthetic human codon optimized DNA spanning the entire RBD coding sequence of the S gene and a few flanking residues (residues 314-541, QTSN . . . KCVNF, (SEQ ID NOs 24 and 25)) (i.e. different from RBD constructs described in Wrapp et al. 2020, and Tai et al., 2020). An upstream signal peptide derived from human IgG (Ig G HV 3-13) was directly added to promote high-level translation into the ER. At the C-terminus the RBD was fused via a short synthetic linker (GSGS, (SEQ ID NO: 26)) to a transmembrane/stem anchor derived from RABV SAD L16 G protein, containing the membrane proximal part (stem) of the G ectodomain, trans-membrane, and cytoplasmic tail of SAD G. The entire construct contains 367 amino acid residues, including the signal sequence that is cleaved off during translation (+1 stop codon) (FIG. 2).

[0160] The RABV G transmembrane/stem anchor is identical to that described previously where it was successfully used to mediate surface expression and rabies virus incorporation of a dsRed fusion protein (Klingen, Conzelmann, & Finke, 2008). While the RABV stem construct does not contain N-glycosylation sites, two putative N-Glycosylation sites are present in the RBD part of the minispike at 2 nearby positions (NITNLCPFGEVFNAT (SEQ ID NO: 27)), which might support correct transport and folding of the chimeric protein and facilitate analysis of the protein (FIG. 2).

[0161] Expression of the minispike construct in HEK293T cells from transfected pCR3-minispike was analyzed in Western blot with an anti-RABV C-tail peptide serum, HCA-5 (FIG. 3A, lane 2). The expressed minispike proteins were of the predicted size range, and showed variability in migration, suggesting the presence of differently glycosylated and non-glycosylated minispike species in the whole cell lysates. N-glycosylation and complex N-glycosylation of the minispike protein was verified by PNGase F and EndoH digestion, respectively (FIG. 3B). This indicated successful transport of the chimeric minispike protein through the ER and Golgi apparatus.

EXAMPLE 2—GENERATION OF MINISPIKE-EXPRESSING VSVs AND RABVs

[0162] To obtain recombinant VSVΔG and full-length replicating-competent rVSV encoding the minispike construct, a plasmid clone, pVSV-eGFP, of vesicular stomatitis Indiana virus (VSIV)(Lawson et al., 1995; M. J. Schnell et al., 1998) was first used to exchange the VSV G ORF with the minispike ORF, to yield pVSV ΔG minispike-eGFP. Full length pVSV-minispike-eGFP was generated by exchange of the eGFP cassette with a minispike-eGFP cassette form the ΔG construct.

[0163] The viral Minispike constructs were then used to delete eGFP to yield viruses lacking the reporter gene, pVSVΔG-minispike, and VSV-minispike, respectively. VSV and VSVdG expressing two copies of the minispike were generated by replacing eGFP with a second copy of the minispike gene to yield VSVΔG-bi-minispike and VSV-bi-minispike, or by insertion of an extra minispike gene for VSVdG-bi-mini-eGFP and VSV bi-mini-eGFP.

[0164] VSV rescue from cDNA was performed in HEK293T cells transfected with the viral cDNA plasmids directing T7 RNA polymerase-driven transcription of viral antigenome RNAs along with expression plasmids encoding T7 RNA polymerase and VSV helper proteins N, P, and L (pCAG-T7, -N, -P, -L; all from addgene).

[0165] RABVΔG-eGFP expressing the minispike gene was performed as described previously (Ghanem, Kern, & Conzelmann, 2012; Wickersham, Finke, Conzelmann, & Callaway, 2007).

[0166] The organization of recombinant VSV and RABV viruses is shown in FIG. 4

EXAMPLE 3—VSV-EXPRESSED MINISPIKE IS PRESENTED AT THE CELL SURFACE AND RECOGNIZED BY COVID-19 PATIENT SERA

[0167] Following virus rescue, VSVΔG-minispike was used to infect BHK-21 to verify expression of virus-encoded minispike protein (FIG. 3, lane 4). Again, differently sized glycosylated minispike proteins were detected in the cell lysates by Western blot using the RABV G tail serum.

[0168] To examine correct conformation of the RBD domain in the chimeric minispikes, BHK-21 Cells infected at a low MOI with VSVdG-minispike or VSVdG-bi-mini were probed with sera from COVID-19 patients previously tested positive by ELISA in an accredited diagnostics institute. At a dilution of 1:300, the sera from positive patients recognized infected cells, as determined by Alexa-555 labeled anti-human IgG antibodies, while no signal was obtained with a COVID-19 negative sera. Anti-RABV C-tail serum and eGFP immune fluorescence was used to discriminate virus-infected cells. Minispike expressing cells were similarly recognized by the sera both after fixation with acetone, and in live cells without fixation (FIG. 5, panels A and B, respectively). Cells expressing minispike from the VSVΔG-bimini virus were also recognized (FIG. 6)

[0169] To corroborate that staining was caused by SARS-CoV-2 specific antibodies, and not by VSV-specific antibodies, co-infections with VSVΔG minispike-eGFP and VSVΔG-tag-BFP was performed. Exclusively green fluorescent cells were recognized by the patient IgG antibodies, while blue fluorescent cells were not (not shown). These experiments revealed that the minispike protein expressed from recombinant rhabdoviruses displays a conformation which is recognized by natural antibodies made in response to SARS-CoV-2 infection and COVID-19 disease, and that it folds into a structure analogous to that of the natural RBD of SARS-CoV-2. Rhabdovirus vectors expressing minispike from one or more genes are thus suitable as a COVID-19 vaccine.

EXAMPLE 4—MINISPIKE-PSEUDOTYPE VIRUSES AND VIRUS-LIKE PARTICLES (VLPS)

[0170] As minispike proteins expressed from VSVΔG contain the VSV envelope-compatible RABV G membrane anchor and C-tail, it was expected that minispikes are incorporated into VSV particles generated in the infected cells. To verify this, VSVΔG-minispike stocks were generated in VSV-G expressing HEK293T cells and concentrated over a sucrose cusion by ultracentrifugation (FIG. 7). For preparation of one of the stocks, VSV G was expressed only 6 hours before VSVΔG infection, while for the other, VSV-G was allowed to accumulate to high levels for 24 hours, before infection. Viruses were harvested 24 hours post infection, followed by ultracentrifugation. Equivalent infectious units (10E6) viruses were processed for Western blot analysis and blots were analyzed with a whole VSV serum (FIG. 7A), and anti-RABV C-tail serum (FIG. 7B) to detect the RABV-derived part of the minispike. In all minispike-encoding virion preparations was minispike protein detected, revealing efficient incorporation, likely as a trimer, into the viral membrane (FIG. 7B, lanes 1-3). Of note, even substantial overexpression of VSV G could not prevent minispike incorporation into virions (compare lanes 2 and 3), revealing strong and competitive incorporation into VSV VLPs.

[0171] To demonstrate that minispike alone, i.e. in the absence of G is sufficient to generate minispike-decorated, and non-infectious VLPs, VSVdG-minispike-eGFP was grown in non-G-complementing cells, and particles present in the cell culture supernatant harvested as described before. Indeed, in contrast to particles generated in G-transfected cells, pure VLPs lacking G were produced in non-complementing cells (FIG. 8).

EXAMPLE 5—MINISPIKE GENE COPY NUMBER DETERMINES LEVEL OF EXPRESSION

[0172] To verify that additional copies of the minispike gene in the genome of VSV vectors lead to enhanced expression of the protein, we constructed VSVs encoding two minispike genes in tandem (VSV-bimini, or VSV-bi-minispike) in the presence or absence of a eGFP reporter gene (see FIG. 4 for genome organization). Viable viruses were readily rescued from plasmids. To compare immunogen expression of bimini-vectors and corresponding VSV containing only a single minispike gene, cells were infected in parallel and minispike expression was analyzed by Western blotting. Indeed, the double gene dose vectors led to accumulation of higher minispike protein levels compared to the single gene dose vectors. As expected, the presence of the extra eGFP gene in the VSV vectors (7 or 8 genes encoded in total) reduced minispike expression in both single and double minispike gene dose VSVs. The highest expression was obtained in VSVdG-bimini lacking the eGFP gene, as this vector encodes only 6 genes in total.

EXAMPLE 6—INCORPORATION OF MINISPIKE PROTEIN IN RABV VIRUS PARTICLES

[0173] The design of the minispike protein has the advantage that upon expression from recombinant vectors both VSV and RABV particles and VLPs are generated. To illustrate incorporation into RABV particles, different SARS-CoV-2 Spike protein constructs, minispike, and RABV SAD G protein were expressed from transfected plasmids and used to pseudotype VSVdG-eGFP and RABV SADdG-eGFP. As predicted, the minispike construct is suitable to produce both VSV and RABV VLPs. Of note, the minispike is incorporated into the envelope of RABVdG as efficiently or even better as the authentic SAD G (FIG. 10). Successful complementation of VSVdG with RABV SAD G (left panel) illustrated also the suitability of SAD G-pseudotyped VSV vectors for infection of tonsil cells and oral immunization.

EXAMPLE 7—IMMUNIZATION WITH VSVΔG-MINISPIKE-EGFP ELICITS HIGH LEVELS OF SARS-CoV2-NEUTRALIZING ANTIBODIES IN BALB/C MICE

[0174] To assess the suitability and the sufficiency of a single round VSVΔG minispike replicon to elicit an immune response, BALB/c mice were immunized with VSVΔG-minispike-eGFP (G) by intramuscular (i.m.) administration. Virus stocks were produced under limiting VSV G complementation, i.e. only 6 hrs of VSV G expression, to prevent excess formation of non-viral G vesicles. Four mice received a single dose of 1×10.sup.6 infectious particles, while 8 mice received an additional boost with the same virus preparation and dose 28 days following prime vaccination. As controls, mice immunized the same way with VSVΔG-eGFP (VSV G) (n=2 for each condition) or with PBS (n=1 for each condition) were used. The 4 mice receiving only prime vaccination were sacrificed at day 28, and 4 boosted mice each at day 35 (n=4) and day 56 (n=4), to collect serum (FIG. 11A).

[0175] Virus neutralization assays were performed with a SARS-CoV-2 virus isolate from Wetzlar, Germany. Notably, all 4 mice immunized only once developed detectable titers of SARS-CoV-2 neutralizing antibodies in the range of 1:20-1:40 dilutions. Boost vaccination further increased neutralizing titers to 1:160-1:640 (FIG. 11B).

[0176] For verification of the notable neutralizing titers after prime immunization in an independent assay, we also produced VSV particles pseudotyped with a functional S protein, VSV-eGFP-ΔG-GLuc (SΔC19). Neutralization assays with the VSV pseudotype viruses confirmed the induction of significant levels of S-neutralizing antibodies in mice receiving a single prime vaccination and further enhancement of neutralization activity by boost immunization (FIG. 11C).

[0177] To directly compare the neutralizing activities of sera from vaccinated mice and from COVID-19 patients, VSV-eGFP-ΔG-GLuc (SΔC19) neutralization assays were employed. Most intriguingly, the group of mice immunized only once (boxes labeled d 28 in FIG. 12), developed neutralizing antibodies with a capacity almost equal to those of the group of COVID-19 patients, illustrating a powerful induction of humoral immunity by vaccination with the single round VSVΔG-minispike-eGFP replicon. Boost immunization further enhanced neutralizing titers to exceed those of patients (FIG. 12).

[0178] These results illustrate that a small antigen, the RBD of SARS-CoV-2, if presented in the form of the present chimeric minispike protein from a safe, spreading-deficient single round biosafety level 1 rhabdovirus replicon is sufficient to elicit high levels of neutralizing antibodies.

EXAMPLE 8—K18-HACE2 MICE ARE PROTECTED FROM SARS-COV-2-INDUCED RESPIRATORY DISEASE AFTER A SINGLE IMMUNIZATION WITH VSVΔG-MINISPIKE-EGFP

[0179] To assess the protective capacity of the VSVΔG-minispike-eGFP vaccine we used transgenic K18-hACE2 C57BL/6 mice, which were previously shown to develop respiratory disease resembling severe COVID-19 (Yinda et al., 2021). Five mice each were immunized as before with VSVΔG-minispike-eGFP or VSVΔG-eGFP control and challenged intranasally with 10.sup.4 TCID50 of SARS-CoV-2 Wetzlar, either following prime immunization or homologous boost immunization (FIG. 13A). Mice immunized with the VSVΔG-eGFP control developed respiratory disease beginning as early as day 5 after infection (FIGS. 13 B and E), which progressed over the following 3-4 days, and animals ultimately succumbed to disease 6-9 days after infection (FIG. 13 C,F). These animals lost only approximately 10-15% of their initial weight (FIG. 13 D,G), which indicates that they experienced a largely respiratory syndrome. In contrast, mice immunized with VSVΔG-minispike-eGFP experienced no clinical signs of disease (FIG. 13 B, E), and all animals survived the infection (FIG. 13C,F) with little to no weight loss during the study (FIG. 13 D, G). This demonstrates the protective power of the VSVΔG-minispike-eGFP replicon vaccine, since a single immunization prevented the development of lethal COVID-19 respiratory disease.

EXAMPLE 9—ANTIBODIES ELICITED IN RESPONSE TO VSVΔG-MINISPIKE-EGFP VACCINATION NEUTRALIZE VARIANTS OF CONCERN

[0180] The simultaneous presentation of distinct RBD antigenic sites is of relevance not only for the efficiency of a vaccine against the homologous virus, but also in the light of emergence and spread of SARS-CoV-2 variants of concern (VOC). Several mutated SARS-CoV-2 strains appeared at the end of 2020 and rapidly expanded to become the dominant strains. Recently emerged VOCs include British/UK (B.1.1.7), South Africa (B.1.351; 501Y.V2), and Brazil (P.1) variants, which have acquired 9, 10, and 12 mutations, respectively, in the S protein gene, facilitating transmission and spread of the virus, or reduce its sensitivity to vaccine-induced or therapeutic antibodies (Baum et al., 2020; Weisblum et al., 2020). Mutations in the ACE2 binding RBD surface are of greatest concern because the neutralizing antibody response predominately targets this region. The B.1.1.7 RBD contains the single N501Y mutation. B.1.351 has three changes in the RBD (K417N, E484K, and N501Y) and P.1 has a very similar triplet mutation (K417T, E484K, and N501Y) which confer increased affinity for ACE2. Neutralization of B.1.351 by both patient and vaccine-induced induced antibodies is reduced, and for the BioNTech/Pfizer vaccine a 9-fold reduction in neutralization was reported (Zhou et al., 2021). Despite similar RBD mutations, P.1 is less resistant to neutralization, suggesting that changes outside the receptor-binding domain (RBD) can also affect neutralization (Dejnirattisai et al., 2021).

[0181] To assess whether the RBD antibodies generated after VSV-ΔG-minispike immunization are active against the VOCs, mouse sera were used for neutralization assays with VSV pseudotyped with Spike variants from the prevalent Wuhan virus carrying a stabilizing D614G mutation (Wuhan (D614G), UK (B.1.1.7), Brazil (P.1.), and South-Africa (B.1.351) virus variants, as well as a chimeric spike construct (Wuhan(D614G) with RBD from India B.1.617.1), which does not occur in nature so far. Notably, serum from vaccinated mice neutralized all virus variants, including viruses with the South African spike protein (B.1.371) (FIG. 14A). B.1.351 is currently is the variant of greatest concern, since an approved vaccine (ChAdOx, Vaxzevria from Astra Zeneca) fails to protect against this variant patients previously infected with the ancestral Wuhan virus variant are not protected against B.1.351 infection and disease. Fortunately, however, other vaccines like BNT162b2 (Comirnaty; BioNTech/Pfizer) or mRNA-1273 (Moderna Biotech) are protective (Edara et al., 2021; Muik et al., 2021; Xie et al., 2021). Notably, neutralizing titers of VSVΔG-minispike-eGFP immunized mice against the South-Africa variant B.1.351 exceeded those from a BNT162b2-vaccinated representative patient serum (FIG. 14B) indicating protection of the minispike against the VOC B.1.351. Neutralizing titers for UK B.1.1.7, Brazil P.1, and the chimeric UK/India variants were similar for minispike-immunized mice and BNT162b2-vaccinated persons, indicating protection against these variants as well. As indicated before (Supasa et al., 2021), neutralizing titers from convalescent sera were lowest for all variants. The results show that immunization with a minispike comprising the authentic WuhanRBD sequence as described here, elicits antibodies able to neutralize emerging SARS-CoV-2 variant.

EXAMPLE 10—CONSTRUCTION OF MINISPIKES OF SARS-COV-2 VARIANTS

[0182] The Wuhan minispike encoded by VSVΔG-minispike-eGFP elicited neutralizing antibodies against the original SARS-CoV-2 (Wuhan) as well as VSV pseudotypes carrying spike proteins of variants of concern. Anyway, it might be advantageous to modify the VSVΔG-minispike replicon vaccine to encode minispikes derived from variants in order to achieve even broader protection. Accordingly, minispike constructs were produced possessing the mutations of the most important variants of concern:

[0183] pCR3 Minispike [E484K], [E484K, N501Y], [K417N, E484K], [K417N, E484K, N501Y] (corresp. to South Africa, B.1.351), [K417T, E484K, N501Y] (corresp. to Brazil, P.1), [L452R, E484K, N501Y], [L452R] (corresp. to Cal.20C), [L452R, E484Q] (corresp. to B.1.617.1), and [L452R;T478K] (corresp. to B.1.617.2) and the corresponding sequences inserted into VSVΔG-minispike-eGFP.

[0184] In addition to VSVΔG replicons encoding individual minispike variants, replicons encoding two different minispikes were generated, e.g. VSVΔG-minispike[Wuhan]+minispike[E484K]-eGFP.

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