WO2023126343A1 - Mrna vaccine against variants of sars-cov-2 - Google Patents

Abstract

A SARS-CoV-2 vaccine composition is described, having a specific set of mutations in the spike protein sequence.

Description

MRNA VACCINE AGAINST VARIANTS OF SARS-COV-2

FIELD OF THE INVENTION

The present invention relates to a vaccine. More specifically, the invention relates to a novel mRNA vaccine against variants of SARS-CoV-2.

BACKGROUND TO THE INVENTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory disease, named ‘coronavirus disease 2019’ (COVID-19). COVID-19 not only threatens human health and public safety, but also has socioeconomic effects that will impact lives and livelihoods in years to come.

A number of vaccines against SARS-CoV-2 have been rapidly produced and authorised for use, including the mRNA vaccines Comirnaty and Spikevax (both often referred to by the names of their manufacturers, Pfizer/BioNTech or Moderna). These vaccines deliver lipid nanoparticle encapsulated mRNA encoding the viral spike protein to a subject; the mRNA is translated in the host cell to generate the antigenic spike protein and hence bring about an immune response which protects against the virus.

The use of mRNA has several beneficial features. First, safety: as mRNA is a non- infectious, non-integrating platform, there is no potential risk of infection or insertional mutagenesis. Additionally, mRNA is degraded by normal cellular processes, and its in vivo half-life can be regulated through the use of various modifications and delivery methods. The inherent immunogenicity of the mRNA can be down-modulated to further increase the safety profile. Second, efficacy: various modifications make mRNA more stable and highly translatable. Efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm. mRNA is the minimal genetic vector; therefore, anti-vector immunity is avoided, and mRNA vaccines can be administered repeatedly. Third, production: mRNA vaccines have the potential for rapid, inexpensive and scalable manufacturing, mainly owing to the high yields of in vitro transcription reactions.

SARS-CoV-2, like all viruses, is subject to genetic drift and viral evolution which can result in the emergence and spread of new variants. In December 2020, the United Kingdom reported to WHO that a new SARS-CoV-2 variant, B.1.1.7 (alpha variant and also referred to as SARS-CoV-2 VUI 202012/01) was identified through viral genomic sequencing. Since then, new variants have also appeared in other parts of the world, such as the B.1 .351 (beta) variant originally detected in South Africa as well as the much more virulent B.1.617.2 (delta) variant first identified in India. Data on the emergence, spread, and clinical relevance of these new variants is rapidly evolving as it is important to understand how variants might affect transmission rates, disease progression, vaccine development, and the efficacy of current therapeutics.

Critically, a number of these variants have mutations in the spike protein which can impact the effectiveness of existing vaccines developed against other forms of the virus. While the appeal of the mRNA vaccine platform is that it allows rapid development of new vaccines, it is still essential to know which variants are in circulation, and indeed early prediction of which variants may become prominent in future can allow early preventative action to be taken.

Key mutations

Using large-scale sequencing data and predictions of likely future spread of variants, we have identified a particular combination of mutations which provide a functional spike protein and which we predict may emerge and grow in importance over the future course of the pandemic. Accordingly, we provide here a potential vaccine targeting this novel variant. The new variant is defined by the presence of seven mutations resulting in amino acid changes and three deletions. As we have seen with for example the delta variant, some of these mutations may influence the transmissibility of the virus in humans.

One of the identified mutations (N501Y) alters an amino acid within the six key residues in the receptor binding domain (RBD). According to the Global Initiative on Sharing Avian Influenza Data (GISAID) database, this same receptor binding domain mutation (N501Y) has been independently reported in several countries including South Africa (n=45) and Australia (n=37). Sequence analysis revealed that the N501Y mutation of the virus reported in the United Kingdom and South Africa originated separately.

Another mutation of biological significance, P681 H, has been found in the RBD. Finally, the deletion at position 69/70 has been found to affect the performance of some diagnostic PCR assays that use an S gene target.

We describe here a new vaccine designed against variants of SARS-CoV-2. SUMMARY OF THE INVENTION

The following nucleic acid and amino acid sequences are referred to herein:

SEQ ID NO: 1 - DNA sequence encoding a novel predicted SARS-CoV-2 spike protein

SEQ ID NO: 2 - predicted mRNA sequence transcribed from SEQ ID NO: 1

SEQ ID NO: 3 - predicted SARS-CoV-2 spike protein translated from SEQ ID NO: 2

SEQ ID NO: 4 -DNA construct for mRNA vaccine incorporating SEQ ID NO: 1

According to a first aspect of the present invention, there is provided a vaccine composition comprising: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations.

In preferred embodiments, the vaccine is an mRNA vaccine, and the nucleic acid molecule is mRNA. The mRNA may include one or more non-standard nucleosides; For example, one or more occurrences of uridine can be replaced with a similar nucleoside such as pseudouridine (^P) or N1-methyl-pseudouridine (ml ^P), and/or one or more occurrences of cytosine can be replaced by 5-methylcytosine.

Where the vaccine comprises a nucleic acid molecule (for example, the vaccine is an mRNA vaccine), the nucleic acid molecule is preferably encapsulated within lipid nanoparticles (LNPs). Suitable lipid nanoparticle compositions will be known to those of skill in the art; for example, lipid compositions suitable for use in vaccine formulation and delivery are described in WQ2016/118724 or WQ2016/118725, in the name of Moderna Therapeutics, and the contents of which are incorporated herein by reference. A general review of the use of LNPs is given in Reichmuth, Andreas M et al. “mRNA vaccine delivery using lipid nanoparticles.” Therapeutic delivery vol. 7,5 (2016): 319-34. doi: 10.4155/tde-2016-0006.

LNPs generally include a mixture of different lipids, and may include cationic lipids, ionizable lipids, phospholipids, and/or cholesterol. Specific examples of lipids which have been used in LNP formulations include DOTAP, DOPE, and phosphatidylcholine and phosphatidylserine and derivatives.

The current generation of mRNA vaccines use LNPs comprising an ionizable cationic lipid, a PEGylated lipid, cholesterol, and distearoylphosphatidylcholine (DSPC).

A preferred LNP formulation for embodiments of the present invention includes ionizable lipid 50 mol%; DSPC 10 mol%; Cholesterol 37.5 mol%; Stabilizer 2.5 mol%.

Vaccine compositions of the invention may also include one or more pharmaceutically acceptable .excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art.

A preferred formulation for embodiments of the present invention comprises LNPs, one or more buffers (for example, Tris, sodium acetate), and one or more sugars (for example, glucose, sucrose, trehalose). An example of such a formulation comprises 0.2 mg/mL LNP in 20 mM Tris Buffer containing 87 mg/mL sucrose and 10.7 mM sodium acetate at pH 7.5.

Vaccine compositions of the present invention may further comprise one or more adjuvants. Exemplary adjuvants include Freund’s complete or incomplete adjuvant, aluminium phosphate, aluminium hydroxide, alum, cholera toxin and salmonella toxin. A particularly preferred adjuvant is GM-CSF (granulocyte macrophage colony stimulating factor). Exemplary diluents and excipients include sterilised water, physiological saline, culture fluid and phosphate buffer.

In some embodiments, the vaccine composition may further comprise nucleic acid (preferably mRNA) encoding one or more replicase components. Such compositions are known as self-amplifying RNA, or saRNA. The replicase components are preferably alphavirus replicase, and preferably comprise all of nsp1-4. The nucleic acid encoding one or more replicase components may be the same molecule as the other nucleic acid components of the vaccine, or these may be different molecules. The use of saRNA vaccines is described in, for example, McKay, P.F., Hu, K., Blakney, A.K. et al. Selfamplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice. Nat Commun 11 , 3523 (2020). https://doi.org/10.1038/s41467-020-174Q9-9.

A further aspect of the present invention provides the use of: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations; in the preparation of a vaccine composition.

Also provided is a composition comprising: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations; for use as a vaccine.

A further aspect of the invention provides a method for inducing or enhancing an immune response, the method comprising administering to a subject a composition comprising: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations.

Administration may be by any convenient route; for example, enteral, parenteral, intranasal, and so on. Preferred administration routes are by intramuscular injection.

A yet further aspect of the invention provides a plasmid comprising: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule, which, when transcribed, produces a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations.

Such plasmids are useful for the production of mRNA molecules for use as vaccines. In specific embodiments the plasmid comprises the nucleic acid sequence of SEQ ID NO: 4.

Also provided is a host cell comprising such a plasmid.

An aspect of the invention further provides: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations.

Yet further aspects of the invention provide a vaccine composition comprising a SARS-CoV-2 spike protein having the following mutations compared with a reference sequence: N501Y; D614G; D1119H; A570D; P681 H; S982A; T716I ;A144 (Y); A69(H)- 70(D); or a nucleic acid encoding said protein. The reference sequence is preferably that of the Wuhan-Hu-1 isolate, with the sequence being found at, among other locations, https://www.ncbi.nlm.nih.gov/qene/43740568 (Genbank Gene ID: 43740568).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows an example plasmid which may be used for in vitro transcription to produce mRNA.

Fig 2. BALB/c immunogenicity. BALB/c mice (n=10 per group unless otherwise specified) were immunized intramuscularly (IM) with two doses of 1 pg (orange) or 10 pg (green) Dellera or empty LNP (black) as control. Mice were sacrificed on day 42. Serum was collected on day 28 and on day 42 after the first immunization and doses were compared post-boost. A) RBD-binding IgG responses against the ancestral Wuhan variant in sera obtained 28 and 42 days after the first immunization with pg (orange) or 10 pg (green) Dellera, determined by ELISA. B) Full length s1-s2-binding IgG responses against the Alpha variant in sera obtained 28 and 42 days after the first immunization with 1 pg (orange) or 10 pg (green) Dellera, determined by ELISA. C-D) Alpha and Omicron BA.1 , BA.4, BA.5. neutralizing sera titers, determined by MN-CPE based assay. E) Splenocytes of BALB/c mice immunized IM with 1 pg (orange) or 10 pg (green) Dellera or LNP (black) were ex vivo re-stimulated with RBD peptide mix or buffer as negative control. Values have been normalized against their respective controls. Individual values and mean of each group, p-values were determined by both One-Way ANOVA assuming Gaussian distribution (Parametric Test) and Kruskal-Wallis test assuming non-Gaussian distribution (Nonparametric Test). Dots above the depicted line are > 51200 (upper the limit of detection).

Fig 3. C57B/6J immunogenicity. C57B/6J mice (n=10 per group unless otherwise specified) were immunized intramuscularly (IM) with three doses of 1 pg (orange) or 10 g (green) Dellera or empty LNP (black) as control. Mice were sacrificed on day 42. Serum was collected on day 28 and on day 42 after the first immunization and doses were compared post-boost. A) RBD-binding IgG responses against the ancestral Wuhan variant in sera obtained 28 and 42 days after the first immunization with pg (orange) or 10 pg (green) Dellera, determined by ELISA. B) Full length s1-s2-binding IgG responses against the Alpha variant in sera obtained 28 and 42 days after the first immunization with 1 pg (orange) or 10 pg (green) Dellera, determined by ELISA. C) Alpha neutralizing sera titers, determined by MN-CPE based assay. D) Splenocytes of BALB/c mice immunized IM with pg (orange) or 10 pg (green) Dellera or LNP (black) were ex vivo restimulated with RBD peptide mix or buffer as negative control. Values have been normalized against their respective controls. Individual values and mean of each group, p-values were determined by both One-Way ANOVA assuming Gaussian distribution (Parametric Test) and Kruskal-Wallis test assuming non-Gaussian distribution (Nonparametric Test). Dots above the depicted line are > 51200 (upper the limit of detection).

Fig 4. Sprague Dawley toxicology Sprague Dawley rats (n=5 per group unless otherwise specified) were immunized intramuscularly (IM) with two doses of 1 pg (orange) or 10 pg (green) Dellera or empty LNP (black) as control. Rats were sacrificed on day 30 and 42 and organs monitored for pathological clinical signs. A) Body weight following administration of 1 pg (orange) or 10 pg (green) of Dellera or empty LNP (black) as control. Animals were monitored for total body weight loss once a day during the study period. Individual values and mean±SD of each group, p-values were determined by Multiple Student’s t-test. B) Organ weight of female rats following administration of 1 pg (orange) or 10 pg (green) Dellera or empty LNP (black) as control, after 30 and 42 days after the first immunization. Individual values and mean of each group, p-values were determined by One-Way ANOVA for multiple comparisons. C) Histopathological scoring of microscopic alterations in harvested organs. 0=no lesions; 1=mild lesions; 2=moderate lesions; 3=severe lesions; 4= extremely severe lesions.

Fig 5. Protection of Syrian golden hamsters from challenge with infectious SARS- CoV-2. Syrian golden hamsters (n=6 per group unless otherwise specified) were immunized at weeks 0 and 3 with 1 pg (orange) or 10 pg (green) of Dellera. Age-matched mice administered with LNP (black) or buffer (gray) served as controls. Three weeks post-boost (day 42), hamsters were challenged with mouse-adapted SARS-CoV-2. Animals were sacrificed 5 days after the challenge (day 47). A) Total body weight loss was monitored once a day after the post-challenge period. Individual values and mean±SD of each group, p-values were determined by Multiple Student’s t-test. B) Viral titration of throat swab performed once a day during the study period. C) Detection of viral RNA and viral titres on hamster lungs collected at the end of the study period (day 47). D) Detection of viral RNA and viral titres on hamster nasal turbinates collected at the end of the study period (day 47) E) % of lesions determined by gross-pathology analysis of lungs at the end of the study period (day 47). F-G) Histopathological scoring of rhinitis and tracheitis severity (0= no inflammatory cells, 1= few inflammatory cells, 2= moderate number of inflammatory cells, 3= many inflammatory cells). H) Histopathological scoring of alveolitis/alveolar damage severity (0= no inflammatory cells, 1= few inflammatory cells, 2= moderate number of inflammatory cells, 3= many inflammatory cells) and extent (0= 0%, 1= <25%, 2= 25-50%, 3= >50%). Cumulative scores for the extent and severity of lung inflammation provide the total score per animal. I) Presence of alveolar hemorrhage (0= no, 1= yes). L) Histopathological scoring of bronchitis (0= no inflammatory cells, 1= few inflammatory cells, 2= moderate number of inflammatory cells, 3= many inflammatory cells). Individual values and mean of each group, p-values were determined by both One-Way ANOVA assuming Gaussian distribution (Parametric Test) and Kruskal-Wallis test assuming non-Gaussian distribution (Nonparametric Test). Dotted lines represent assay limits of detection.

DETAILED DESCRIPTION OF THE INVENTION

In general terms, the invention relates to vaccine compositions, and particularly mRNA vaccine compositions, against a predicted novel variant of SARS-CoV-2. As noted above, the inventors have used large-scale sequencing data and information on spread of previous variants to design, via machine learning algorithms, a candidate spike protein which is predicted to appear in future. This has then been used in the production of suitable vaccine candidates.

The predicted amino acid sequence of the spike protein is shown in SEQ ID NO: 3. From this, DNA and mRNA sequences were derived. It will be appreciated that, due to the redundancy of the genetic code, various silent mutations may be made to the nucleic acid sequences which do not alter the encoded amino acid sequence. Further, this allows for the process of codon optimisation to be carried out, whereby certain preferred nucleic acid triplets are used in the encoding molecule.

Although it may be possible to use the predicted spike protein itself or the DNA sequence in a vaccine candidate, the present invention preferably relates to an RNA vaccine.

The predicted novel variant includes a number of mutations over the original SARS-CoV- 2 strain. One mutation is in the receptor binding domain (RBD) of the spike protein at position 501 , where the amino acid asparagine (N) has been replaced with tyrosine (Y). The shorthand for this mutation is N501Y. This variant also has several other mutations, including:

69/70 deletion: occurred spontaneously many times and likely leads to a conformational change in the spike protein.

P681 H: near the S1/S2 furin cleavage site, a site with high variability in coronaviruses. This mutation has also emerged spontaneously multiple times.

N501 is located in the ‘shoulder’ region of the RBD and makes direct contact with antibodies, including those possessing the public immunoglobulin HC variables (IGHV) IGHV3-53 and IGHV3-66 in their Fab domain. Specifically N501 forms hydrogen bonds or salt bridges with the light chain of complementary determining regions (CDR) 1 and 2 at residue Y41 and stabilises one of the virus-binding hotspot residues K353 in ACE2. N501Y mutation reduces complementarity of the IGHV3-53/66 containing mAbs to the RBD enabling evasion of neutralisation and also confers a 7-fold stronger binding affinity to ACE2. The N501 residue is novel compared to SARS-CoV and is thought to be responsible in part for the increased transmissibility of the virus.

D614G - studies have shown higher infectious titres and enhanced viral load in the upper respiratory tract which may be associated with increased transmission. D614G mutation may also evade the immune response by shifting S2 towards a fusion-competent state, though neutralisation of virus containing D614G mutations still appear to be effectively neutralised by mAbs.

Other mutations of potential significance include A 144(Y), A570D, T716I, S982A, and D1118H.

New vaccine

In line with the data presented above, we have designed and generated a novel vaccine targeting the predicted variant matching observed sequence hCoV-19/England/MILK- B94A53/2020 (EPI_ISL_676030) which includes the following mutations.

• N501Y • D614G

• D1119H

• A570D

• P681 H

• S982A

• T716I

• A144 (Y)

• A69(H)-70(D)

Figure 1 shows an illustration of a plasmid template for mRNA production. The plasmid template for in vitro transcription includes the T7 promoter, a modified human a- globin 5’ UTR ( ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCACC ), modified spike protein ( containing cleavage site mutations K986P and V987P ), two serial human P-globin 3’IITR

(GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAA CTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAA AAAACATTTATTTTCATTGCAA) and a 110 nt interrupted poly(A) tail comprised of 30 adenosine residues and 70 adenosine residues separated by a 10 nt linker sequence GCATATGACT (AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA) . The sequence of the insert for producing the mRNA is given as SEQ ID NO: 4.

In some embodiments of the invention, the RNA vaccine is a self-amplifying RNA (saRNA). To achieve this, downstream of the 5’IITR, an auto-replicase sequence is incorporated producing a self-amplifying mRNA (saRNA). Current vaccines generate a 1 :1 ratio of injected mRNA to viral product (i.e. 30 ug mRNA results in 30 ug virus produced in vivo). Utilising a self-amplicon system overcomes issues surrounding multiple doses, high dose concentration and limited supply of vaccine. Self-amplifying RNA is a next-generation platform for nucleic acid vaccines The backbone, typically derived from an alphavirus genome, encodes a gene of interest (GOI) and a viral replicase, which is able to amplify the genomic and subgenomic RNA. The selfamplification properties enable use of a much lower dose of saRNA compared to messenger RNA (mRNA), typically 100-fold lower. Production of vaccine

Vaccine production involves a number of steps, which can be divided into three main stages: plasmid production; mRNA in vitro synthesis; and LNP encapsulation. The following provides one example of production at the R&D stage; alternative techniques may be used for clinical and production stages.

Plasmid production

E. coli (here: strain DH5alfa) are transformed with the plasmid encoding the modified spike protein. In this illustration, the plasmid includes an ampicillin resistance gene to allow selection; but in clinical production an alternative system will be used. Bacteria are grown in LB medium at pH = 6.9-7.4, and transformed by electroporation before being grown on a selective medium (here: Medium Bacto CD supreme with ampicillin at 100 mg/ml). A selected colony is then clonally expanded before plasmid extraction and purification using standard techniques. In this example, expansion takes place in a ThermoFisher 1L Glass fermenter, agitated at RPM 280-340 in Medium Bacto CD Supreme, Glycerol, Ampicillin with pH 6.9-7.4 (adjusted by addition of Ammonia/Orthophosphoricacid). Exponential phase is reached after 6-8h. An alternative antibiotic-free culture is Gibco Bacto E. coli CD Supreme Fermentation Production Medium (FPM) customized without histidine; transgenic bacteria strain (MG1655) may be used allowing antibiotic-free solution - hisDCB KO Bacteria + hisDCB KI Plasmid will allow only plasmid-carrying bacteria to grow.

After growth plateaus, bacteria are harvested by centrifugation, resuspended in resuspension buffer (Tris-HCI 50Mm; EDTA 10Mm; glucose 50Mm), and lysed by addition of lysis buffer (200 mM NaOH and 1% SDS). Standard protocols (eg, EtOH precipitation; spin column purification) may be used for plasmid purification.

Further rounds of purification and concentration are then carried out: tangential flow filtration (TFF) concentration using a Tangential Flow Filtration Minimate® 100kD cassette with feed pressure 0.5 bar and retentate pressure 1 bar; Supercoiled pDNA Capture with AKTA Avant®, thiophilic aromatic chromatography (PlasmidSelect Xtra® Cytiva); Polishing with AKTA Avant®, anion exchange chromatography SOURCE 30Q (PlasmidSelect Xtra® Cytiva); and Sterile filtration with luer lock syringes with Whatman® ReZist® 0.2 pm mRNA in vitro synthesis

The plasmids are linearized with a suitable restriction enzyme, before being precipitated and purified. In vitro transcription is used to produce mRNA, after which DNAse digestion removes DNA. A further purification step results in purified mRNA.

Transcription uses 10X Reaction buffer, comprising Cap1 [10 mM], GTP [2.7 mM], ATP [8.1 mM], CTP [8.1 mM], i -UTP [2.7 mM], Linearized template DNA, T7 RNA polymerase Mix. mRNA Purification uses POROS oligo(dT) affinity resin, followed by TFF - Concentration with a Tangential Flow Filtration Minimate® 2kD cassette.

LNP encapsulation

Encapsulation takes place with the Genvoy lipid mix with the NanoAssemblr Ignite machine, followed by LNP polishing, buffer change from ethanol to PBS (Euroclone). The LNP-RNA particles are concentrated by centrifugation, and finally resuspended at 0.2 mg/ml in 20 mM Tris buffer containing 87 mg/mL sucrose and 10.7 mM sodium acetate at pH 7.5.

Encapsulation uses PrecisionNanosystem IGNITE microfluidic mixer, Genvoy lipidic mix, EtOH, and PNI Formulation Buffer.

LNP polishing uses Tangential Flow Filtration Minimate® 10kD cassette.

Experimental Plan for Pre-Clinical Studies

The inventors plan to take the vaccine candidates (both non-amplifying and selfamplifying SARS-CoV-2 S1 RNA) into pre-clinical studies.

- Viral Approach:

The aim of this study is to evaluate the in vitro capacity of liposome carrying nonamplifying (na) and self-amplified (sa) Sars-CoV2 S1 RNA to activate/stimulate lymphocytes and antigen-presenting cells (APC) such as macrophages and dendritic cells (DC). Experiments will be first performed using primary cultures of human peripheral blood mononuclear cells (PBMC) and then, using primary cultures of human monocyte-derived macrophages (MDM) and DC. Because these na/saRNA candidates will be administered using the intramuscular route, cells such macrophages and DC will be involved in the quality of their immune response.

Peripheral mononuclear cells (PBMC) will be isolated from the blood of healthy HIV-, HBV- and HCV-seronegative donors by ficoll-hypaque density gradient centrifugation and cultured at +37 °C in a 5% CO2-air humidified atmosphere in RPMI1640 GlutamaxTM, supplemented with 10% heat-inactivated fetal calf serum (FCS; +56°C for 45 min.) and 1% tri-antibiotic (penicillin, streptomycin and neomycin (PSN) mixture.

First, toxicity towards PBMC will be measured using a colorimetric methyl-tetrazolium salt (MTT) approach or an equivalent methodology. Cells will be incubated for 3 days with RNA-containing liposomes and MTT will be then added for 2 h to each well. Cells will be lysed with isopropanol containing 0.01 N HCI, and the optical density (OD) measured at 540 nm.

50, 70 and 90% cytotoxic concentrations (CC) will be determined with Soft Max Pro 4.6 software or equivalent.

After a 3-day incubation with each RNA-charged liposome and controls, cells will be collected including adherent macrophages, scraped off the plastic, and immunophenotyping for CD4, CD14, CD83, CD86, and HLA-DR. The M1/M2 macrophagic orientation could be also explored. After immunostaining, cells will be analyzed using FACS.

Cell supernatants will be collected at 4 or 6 hrs, 24 hrs and 3 days after treatment and the concentration of cytokines will be quantified using a multiplexed approach, in part to define the Th1/Th2 orientation. Cytokines such as TNF-a, IFN-a, IFN-y, IL-1 p, IL-2, IL-4, IL-6, IL-10, IL-12p40 or p70 as well as chemokines (MIP-1a/p, MCP-1 , RANTES) could be of interest.

SaRNA is self-adjuvanting, as it activates a type I interferon (IFN) through endosomal sensing via Toll-like receptor (TLR)3, TLR7, and TLR8 as well as cytosolic sensing e.g protein kinase R (PKR) and 2'-5'-oligoadenylate synthetase (OAS), as well as other possibly unknown pathways. The role of these sensors could also be evaluated in PBMC cultures or in primary cultures of Human MDM or of Human DC.

The uptake of RNA-charged liposomes and their expression, RNA and protein, will be measured by RT-qPCR and using a specific anti-S EIA. These expression levels will be determined also at 4 or 6 hrs, 24 hrs and 3 days and correlated to the immune modulations observed.

- Antiviral approach

The aim is to in vitro evaluate the efficacy of non-amplifying (na) & self-amplified (sa) Sars-CoV2 S1 RNA to protect either transfected cells of SARS-CoV2 infection or by a paracrine mechanism to protect neighboring cells from transfected cells producing the S1 protein and releasing it into the cellular environment.

The assessment of in vitro anti-SARS-CoV2 effects will be explored first using the Vero C1008 E6 clone cells (VeroE6) cell line. Cytotoxicity of each candidate tested will be measured previously for this cell line using the same experimental conditions used then for the SARS-CoV2 antiviral assay.

Vero C1008 E6 clone cells (VeroE6) will be seeded into 96-well microplates DMEM supplemented with 10% Fetal bovine serum (FBS) and grown for 24h (confluency will be approximately 80%). Then, these cells will be pretreated with 6 concentrations of each candidate and then infected for 1 hr with the 2020 SARS-CoV2 European isolate-nCoV and a multiplicity of infection (m.o.i.) of 0.01. After infection, medium is replaced by DMEM supplemented with 2% FBS. Treated (Liposomal RNA candidates and solvents) cells and negative/positive controls will be maintained in culture at +37°C in a humidified atmosphere containing 5% CO2 for 48 hours. Then, cell culture supernatants will be collected and viral replication will be quantified in each cell culture supernatant using specific RT-qPCR. Viral mRNA will be extracted using Qiagen viral RNA mini kit and the one step TaqMan RT-qPCR on the viral RNA-dependent RNA polymerase (RdRp) gene

Non-amplifying & self-amplifying Sars-CoV2 S1 RNA:

In vivo Immunological response and toxicology study

Animal studies will be carried out with Balb/c female mice given intramuscular injections of vaccine candidates. A single dose on D1 of an RNA vaccine will be given at various dosages. Body weight change will be assessed at days 0, 7,14,21 ,28,35, and blood samples taken and analysed via ELISA for IgG and IgM anti-SARS-COV-2 antibodies, and for L-6 and INFg pro-inflammatory cytokines. Animals will be sacrificed at day 35, and further study made of organs and tissues. Preclinical studies

The vaccine candidate including the predicted spike protein variant was manufactured and tested in animal studies, as follows. In the following sections, the vaccine candidate is referred to as “Dellera”.

MATERIALS AND METHODS

Plasmid sequence design and plasmid template expansion

The plasmid DNA (pDNA) template used for manufacturing of Dellera was produced in ProteoGenix SAS’s facility (Schiltigheim, France), based on our predicted sequence and according to the following specifications: Gene name: mRNA V501.1 - Lot No: C38598 - Cloning vector: pET-28a(+) - Strain: DH5a - Cloning site: Xbal - Blpl - Insert size: 4246 bp - Vector resistance: Kanamycin. The pDNA sequence includes a T7 promoter, a modified human a-globin 5’ UTR, a Kozak sequence, generally added after the 5' UTR sequence to improve translation efficiency, the predicted full length SARS-CoV2 spike (S) glycoprotein sequence containing the double proline (2P) prefusion mutations (K986P, V987P), two serial human p-globin 3’UTR and a 110 nt interrupted poly(A) tail comprised of 30 adenosine residues and 70 adenosine residues separated by a 10 nt linker sequence. E. coli (strain DH5a) electrocompetent cells were transformed with the plasmid encoding the modified spike (S) protein and containing a Kanamycin resistance gene. Bacteria were grown in LB Medium (typical formula g/l: tryptone 10g, yeast extracted 5g, sodium chloride 10g) and transformed by electroporation before being grown on a plate in a selective medium (LB Medium with Kanamycin at 50mg/ml). A selected colony was then clonally expanded before plasmid extraction. Expansion took place in a Flask, agitated at rpm 196 in LB Medium and Kanamycin. After growth, bacteria were harvested by centrifugation and subsequently plasmids were extracted and purified by E.Z.N.A.® Plasmid DNA Maxi Kit. To evaluate the plasmid extraction protocol quality, an agarose electrophoresis gel (0,7% w/v) was performed to ensure that the experiment was successful by providing fragments of the desired length (9353bp). mRNA synthesis and lipid nanoparticle formulation

Plasmid DNA (pDNA) template incorporating the predicted mutant sequence of the full length SARS-CoV2 S glycoprotein was first linearized with Blp-I R05585L restriction enzyme (New England BioLabs ® Inc). For each 10 ug of pDNA, 100 U of restriction enzyme was used. After incubation on a heating shaking dry bath incubator at 37°C for 1h and 30 min, pDNA was precipitated with 3M Sodium Acetate Solution pH 5.2 and Ethanol 95%, and frozen at -20 °C overnight. The linearization product was checked with FemtoPulse Assay (FemtoPulse 4C, Agilent Technologies, Inc.) and agarose gel electrophoresis (0,7% w/v). Messenger RNA incorporating the desired gene was synthesized by in vitro transcription employing T7 RNA polymerase (Thermo Scientific™) and N1-methyl-pseudouridine-5'-Triphosphate (TriLink® BioTechnologies) to enhance mRNA stability, transcription yield and consequently protein expression, as well as to lower its immunogenicity. mRNA purification was performed with Monarch® RNA Cleanup Kit. The resulting purified precursor mRNA was reacted further by enzymatic addition of a 5’ cap structure (ARCA, Thermo Scientific™) and a 3’ poly(A) tail. The mRNA was checked with FemtoPulse Assay (FemtoPulse Ultrasensitivity RNA 4C, Agilent Technologies, Inc.) and agarose gel electrophoresis (1% w/v). Preparation of mRNA/lipid nanoparticle (LNP) formulations was performed on NanoAssemblr® Ignite platform (Precision NanoSystems Inc.), using a lipid mixture (GenVoy-ILM, Precision NanoSystems Inc.). Briefly, an ethanolic solution of a mixture of lipids including 10% distearoylphosphatidylcholine (DSPC), 37% cholesterol, 50% ionizable lipid and 2.5% poly(ethylene glycol)-lipid, at a fixed lipid and mRNA 6:1 ratio, were combined with an aqueous buffered solution of target mRNA at an acidic pH under controlled conditions to yield a suspension of uniform LNPs. The LNP-mRNA particles were concentrated by centrifugation, resuspended to PBS and stored at +4°C. Encapsulation efficiency was evaluated with Quant-iT RiboGreen RNA Assay on a spectrophotometer (Varioskan Lux, Thermofisher). Further analysis was performed to identify the Z-average size (nm) and the Polydispersity Index (Pdl) of the nanoparticles using the Zetasizer Ultra Red Label (Malvern Panalytical).

Cell culture

Vero E6 cells (Epithelial cell line from kidney of a normal monkey Cercopithecus aethiops), acquired from the American Type Culture Collection (ATCC-CRL 1586), were cultured in Dulbecco’s Modified Eagle’s Medium (D-MEM) - High Glucose (Euroclone, Pero, Italy) supplemented with 2 mM L-Glutamine (Lonza, Milano, Italy), 100 units/mL penicillin-streptomycin mixture (Lonza, Milano, Italy) and 10% fetal bovine serum (FBS) (Euroclone, Pero, Italy). Cells were incubated at 37°C, in a 5% CO2 humidified incubator and subcultured twice a week until passage 20. Vero E6 cells were seeded in a 96-well plate using Dulbecco’s Modified Eagle’s Medium (D-MEM) high glucose 2% FBS at a density of 1.5x10⁶ cells/well, in order to obtain a 70-80% sub-confluent cell monolayer after 24h. Mouse studies

Mice experiments were carried out at the Biogem Test Facility, GLP certified by the Italian Public Health Government Office (GLP Test Facility Authorization N° 2019/21 of 07/01/2021). The animals have been used according to Directive 2010/63/UE regarding the protection of animals used for experimental or other scientific purposes, enforced by the Italian Leg. Decree n° 26 of March 4, 2014. The study was conducted in full compliance with the principles of Good Laboratory Practice (GLP) as specified in the Directive 2004/9/CE and directive 2004/10/CE of the European Parliament and of the Council of 11 February 2004 enforced by the Italian Leg. Decree n° 50 of 2 March 2007 (G.U. n° 86 of 13/04/2007). Female specific pathogen free BALB/c mice and C57BL/6J mice about 8 weeks of age were vaccinated in groups of 10, with 50 pl of 1 pg or 10 pg of the designated mRNA/LNP formulation into one quadriceps femur, alternating right and left for the three doses (DO, D14 and D28). Proper control groups received LNP formulation (lipid mixture GenVoy-ILM Precision NanoSystem) with the same administration volume and treatment schedule. Serum samples were collected for serological analyses on day 28 and 42 (study endpoint) from the submandibular vein under anesthesia with isoflurane. All animals enrolled in the study have been sacrificed on day 42. After sacrifice, a necropsy has been performed to evaluate the occurrence of gross lesions. Splenocytes were collected for T-cell mediated immune response measurements.

Rat toxicology study

Evaluation of Dellera potential toxic effects was conducted at Biogem, Ariano Irpino, Italy. The animals enrolled in the study were purchased from a commercial supplier (Test Facility Batch #46/21). The study was carried out in full compliance with the principles of Good Laboratory Practice (GLP) and conducted with approved animal protocols according to Directive 2010/63/UE regarding the protection of animals used for experimental or other scientific purposes, enforced by the Italian decree n°26 of March 4, 2014. Female Sprague Dawley rats were vaccinated in groups of 5 as described above for mice. Animals have been monitored daily for the presence of clinical signs of disease including diarrhea, anorexia, depression, tremors, fever, nasal discharge and mortality occurrence. Feed intake and body weight were recorded weekly until the day before sacrifice. Blood samples were collected at day 30 and 42 from sublingual vein of the animals, under light anesthesia with isoflurane to perform biochemical and hematological analyses. A necropsy was performed on all sacrificed animals in order to record the occurrence of gross lesions. Organs and tissues were collected from each animal, weighted and immediately stored in a buffered 10% formalin solution and processed for paraffin embedding. Paraffin blocks were sectioned at an approximated thickness of 2-4 pm and stained with hematoxylin and eosin (H&E). After a quality check, tissue slides were evaluated by the study pathologist. Histological changes were described according to distribution, severity and morphologic character. Severity scores were assigned according to 4 grades. A score of 0 was assigned when organs were consistent with their corresponding normal tissue. A score of 1 , 2, 3 and 4 was attributed to organs sections that revealed mild, moderate, severe and extremely severe lesions respectively.

Hamster efficacy study

In vivo experiments to determine the protective efficacy of Dellera were conducted on male Syrian Golden hamsters at the central animal facilities of Viroclinics Biosciences B.V., Schaijk, The Netherlands. All the experiments were carried out under conditions that meet the standard of Dutch law for animal experimentation and are in agreement with the “Guide for the care and use of laboratory animals". Ethical approval for the study is registered under number: 27700202114492-WP30. A total of 24 hamsters about 8 to 10 weeks of age were vaccinated in groups of 6, with 50 pl of 1 pg or 10 pg of the designated mRNA/LNP formulation into one hind limb, alternating right and left for the three doses (DO, D14 and D28). Control groups received either LNP formulation (lipid mixture GenVoy-ILM Precision NanoSystem) with the same administration volume and treatment schedule or no vaccination. On day 42, three weeks post-booster, all animals were challenged intranasally (IN) with 10² TCID50 of SARS-CoV-2 (BetaCoV/Munich/BavPat1/2020), a challenge dose that induces high levels of virus replication in the lower respiratory tract and significant histopathological changes in the lungs. Serum samples were collected on day 28, 42 and 46 (study endpoint). During the challenge phase the animals were monitored for weight loss, appearance of clinical signs of disease and throat swabs were taken daily till the endpoint of the experiment. On day 46 (study endpoint), after assessment of clinical symptoms and body weights, all animals were euthanized by exsanguination under isoflurane anesthesia and submitted to necropsy. At the time of necropsy, gross pathology was performed. Lung and nasal turbinate tissues were collected for tissue viral load (qRT-PCR and TCID50 assay) and assessment of (histo)pathological changes. After fixation with 10% neutral buffered formalin, sections from left lung and left nasal turbinate were embedded in paraffin. Paraffin blocks were sectioned, stained using H&E and evaluated qualitatively and semi- quantitatively for pulmonary pathology. The H&E stained tissue sections were examined by light microscopy, using Olympus BX45 light microscope with magnification steps of 40x, 100x, 250x, and 400x, for histopathology, as well as for the presence of any lesions. Each entire slide was examined and scored for presence or absence of alveolar oedema, alveolar hemorrhage and type II pneumocyte hyperplasia (0 = no, 1 = yes). The degree or severity of inflammatory cell infiltration and damage in alveoli, bronchi/bronchioles were scored (0 = no inflammatory cells, 1 = few inflammatory cells, 2 = moderate number of inflammatory cells, 3 = many inflammatory cells) for alveolitis and bronchitis/bronchiolitis. Additionally, the extent of alveolitis/alveolar damage was scored per slide (0 = 0%, 1 = <25%, 2 = 25-50%, 3 = >50%). The cumulative score (SUM) for the extent and severity of inflammation of the lung provided the total score of alveolitis per animal. Extent of peribronchial/perivascular cuffing was scored (0 = none, 1 = 1-2 cells thick, 2 = 3-10 cells thick, 3 = >10 cells thick). Total RNA was extracted from homogenized tissues samples with the MagNA Pure 96 system (Roche, Penzberg, Germany). Samples were reverse transcribed and SARS-CoV-2 E gene subgenomic mRNA was assessed by quantitative real-time PCR on 7500 RealTime PCR system (Thermo Fisher) using specific primers (E_Sarbeco_F: ACAGGTACGTTAATAGTTAATAGCGT, SEQ ID NO: 9; E_Sarbeco_R: ATATTGCAGCAGTACGCACACA, SEQ ID NO: 10) and probe (E_Sarbeco_P1 : ACACTAGCCATCCACTGCGCTTCG, SEQ ID NO: 11) with TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher). To determine the TCID50 virus titers, quadruplicate 10-fold serial dilutions of the samples (throat swabs and tissue homogenates) were incubated on Vero E6 monolayers for 1 hours at 37 °C. Subsequently, Vero E6 monolayers are washed and incubated for 5-6 days at 37° after which plates were scored using the vitality marker WST-8 (colorimetric cell counting kit; Sigma-Aldrich, cat# 96992- 3000TESTS-F). To this end, a WST-8 stock solution was prepared and added to the plates. Per well, 20 pL of this solution (containing 4 pL of the ready-to-use WST-8 solution from the kit and 16 pL infection medium, 1 :5 dilution) was added and incubated 3-5 h at room temperature. Subsequently, plates were measured for optical density at 450 nm (QD450) using a microplate reader and visual results of the positive controls (cytopathic effect (CPE)) were used to set the limits of the WST-8 staining (OD value associated with CPE). Viral titers (TCID50) were calculated using the method of Spearman-Karber. Enzyme-Linked Immunosorbent Assay (ELISA)

Specific anti-SARS-CoV-2 IgG antibodies in mouse sera were detected by means of ELISA. Nunc MaxiSorp™ immunoplates (Merck, Cat# 442404) were coated with 1 g/mL SARS-CoV-2 (2019-nCoV) Spike S1+S2 ectodomain (ECD) (HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681 H, T716I, S982A, D1118H)-His Recombinant Protein (Sino Biological, Cod. 40589-V08B6) or 1 g/mL SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, cod. 40592-V08H). After overnight incubation at 4°C, coated plates were washed three times with 300pl/well of ELISA washing solution containing Tris Buffered Saline (TBS)-0,05% Tween, then blocked for 1 h 37 °C with a solution of TBS containing 5% of Nonfat-Dry Milk (NFDM; EuroClone, Cod. APA08300500). Mice serum samples were heat-inactivated at 56 °C for 1h and, subsequently 3-fold serial dilutions, starting from 1 :100 in TBS-0.05% Tween 20 5% NFDM, were performed up to 1 :2700. Plates were washed three times, as described above; then 10OpI of each serial dilution was added to the coated plates and incubated for 1h at 37 °C. Next, after a washing step, 100pl of 1 :100000 Goat anti-Mouse lgG1 Antibody Horseradish Peroxidase (HRP)- conjugated antibody (Bethyl Laboratories, Cod. A90-105P) or 1 OOpI of 1 : 100000 Goat anti-Rat IgG Heavy and Light Chain Antibody HRP-conjugated (Bethyl Laboratories, Cod. A110-105P) were added. Plates were incubated 30 min at 37°C. After incubation, plates were washed and 10OpI TMB solution (Sigma, cod. T0440) for 20 minutes. The reaction 100pl/well of 3, 3’, 5,5’- tetramethylbenzidine (TMB) substrate (Bethyl Laboratories, Cat# E102) was added and incubated in the dark at room temperature for 20 min. The reaction was stopped by adding 10OpI of ELISA stop solution (Bethyl Laboratories, Cat# E115). Plates were read within 20 min at 450 nm on a SpectraMax ELISA plate (Medical Device) reader in order to evaluate the OD. A cut-off value was defined as 3 times the average of OD values from blank wells (background: no addition of analyte). Samples with the ODs under the cut off value at the first 1 :100 dilution were assigned as negative, samples where the ODs at 1 :100 dilution were above the cut-off value were assigned as positive. Borderline samples were defined where one replicate was under the cut-off and the other was above.

Pseudovirus-Based Neutralization Assay

To measure the neutralization activity of mice sera, neutralizing antibody titers were defined as endpoint 2-fold serial dilutions of test samples, and the 50% inhibitory concentration (IC50) was determined as the serum dilution resulting in a 50% reduction of a single round of infection (reporter gene-mediated signal). In brief, a lentivirus-based SARS-CoV-2 pseudovirus particle was constructed displaying the full Spike (S) protein on the surface of the pseudotyped virus using a synthetic codon-optimized SARS-CoV- 2 expression construct (NCBI reference sequence: YP_007188579.1). Heat-inactivated serum samples were diluted 1 :10 in media in a culture medium (phenol red free DMEM + 10% FBS + 100 ll/rnl PS + 2mM L-Glutamine + 1 % non-essential Amino Acid Solution). A further, 2-fold serial dilution of the heat inactivated serum was prepared and mixed with the SARS-Cov-2 pseudotypes in a 1 : 1 vol/vol ratio in a 96-well culture plate. The virus input used was 1x10⁶ RLU per well. The serum-pseudotypes mixture was then incubated for 1h at 37°C, 5% CO2. After 1 h, HEK 293 clonal cells suspension (1x10⁴ cell/mL), containing a responsive reporter gene (firefly luciferase gene), were inoculated with 50|jl of the serum-pseudotype mix and incubated for 48-72h at 37°C, 5% CO2. At the end of the incubation, 50|jl of Bright-Glo (Bright-Glo™ Luciferase Assay System, Promega) were added to each well, plates scanned on the GloMax®-Multi Detection System and neutralizing antibodies characterized on the basis of the luciferase activity. For the analyses of the pseudotype-based neutralization assay, titers were firstly normalized, and IC50 values were calculated by a non-linear regression model (log(inhibitor) vs normalized response-variable slope) analysis. Titers were subsequently expressed as the range of dilution in which the IC50 value lay.

Microneutralization assay based on cytopathic effect (MN-CPE)

Mice heat-inactivated serum samples at 56 °C for 30 minutes were diluted 1 :10 in media (DMEM + 2% heat-inactivated FBS, 1% L-Glutamine + 1% PS). A further, 2-fold serial dilution of the heat inactivated serum samples were prepared, mixed and incubated with a challenge dose targeting 100 TCIDso/well of SARS-CoV-2 alpha-UK variant at 37°C with 5% CO2 for 1 h. At the end of the incubation time, 10OpI of the serum-virus mixtures were inoculated in duplicate into a 96-well plate containing semi-confluent Vero E6 monolayer. Plates were incubated for 4 days at 37°C in a humidified atmosphere with 5% CO2. After 4 days of incubation, the plates were inspected by an inverted optical microscope. The highest serum dilution that protected more than the 50% of cells from CPE was taken as the neutralization titer. Neutralization titer has been expressed as the reciprocal of the highest serum dilution able to prevent the virus infection and the consequent destruction of the Vero E6 cell monolayer.

Microneutralization (ViroSpot) assay To assess the susceptibility of SARS-CoV-2 variants, a microneutralization assay was performed based on the ViroSpot technology. Briefly, approximately 100 infectious units (IFU) of SARS-CoV-2 virus were mixed with ten 3-fold serial dilutions of Hamster's heat- inactivated serum samples. The virus/sera mixture was incubated for 1 h prior to addition of 100pl to the Vero E6 cells. After a subsequent 1 h incubation, the inoculum was removed, and 1% carboxymethylcellulose (CMC) overlay was added. The plates were incubated for a duration of 16-24 h at 37 °C and 5% CO2. The cells were formalin-fixed and ethanol permeabilized followed by incubation with a murine monoclonal antibody which targets the viral nucleocapsid protein (Sino Biological), followed by a secondary anti-mouse IgG peroxidase conjugate (Thermo Scientific) and TrueBlue (KPL) substrate. This formed a blue precipitate on nucleocapsid-positive cells. Images of all wells were acquired by a CTL Immunospot analyzer, equipped with Biospot® software to quantitate the nucleocapsid positive cells (% virus signal). Titer was calculated from the average of duplicate sample wells by extrapolating the inverse dilution of serum that produced a 60% reduction of virus.

Cytokine ELISpot analysis

Spleens from mice were harvested on day 42 and splenocytes were isolated by gentle pressing on a 70 pm cell strainer using the flat edge of a syringe plunger and collected in complete RPMI-1640 medium containing L-glutamine and HEPES, supplemented with 10% FBS (Euroclone) and 1X Penicillin/Streptomycin (Euroclone). Cells were immediately washed with complete medium and centrifuged for 10 minutes 1300 rpm. Red Blood Cell lysis was performed on pelleted cells using RBC Lysis buffer 10X (BioLegend) diluted 1 :10 in sterile filtered H2O milli-Q for 2’ in ice. Cells were washed again with complete medium, centrifuged for 10 minutes 1300 rpm. Pellets were resuspended in FBS with 7.5% Dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored in the liquid nitrogen tank until use. T cell responses of immunized rats were analyzed using a pre-coated rat IFN-y (R&D Systems), IL-2 (R&D Systems) and IL-4 (CellSciences) Single-Color ELISPOT kit following the manufacturer's protocol. The day before the assay, the PVDF membrane-bottomed 96-well plates were coated with anti-l L4 coating antibody and kept at 4°C overnight. PVDF membrane-bottomed 96-wells for IFN-y and IL-2 were ready-to use. On the day of the assay, cryopreserved splenocytes were thawed in FBS, centrifuged for 10 minutes 1300 rpm then washed in complete medium prior to cell counting. Cell count was performed using CTL-LDC™ Live/Dead Cell Counting kit and the CTL Cell Counting™ Software on the ImmunoSpot® Analyzer. 3x10⁵ cells/well splenocytes were seeded and stimulated in triplicate with 1 g/ml PepMix™ SARS-CoV- 2 (S-RBD) (JPT); complete medium alone and phorbol myristate acetate (PMA)/lonomycin 1X (Invitrogen) were used as negative and positive control, respectively. For rat IFN-y and IL-2 kit internal positive control was seeded together with cells as per manufactured instruction. The plates were incubated for 24 h in a humidified 5% CO2 incubator at 37°C. Following the incubation, ELISPOT plates were developed following manufacturer’s instructions. Detection antibodies anti-IFN-y, -IL-2 and -IL-4 were diluted in Dilution Buffer and incubated for 2h at Room Temperature (RT) (for IFN- y and IL-2 kit incubation was performed on a rocking platform). Afterwards, Streptavidin- AP was added and incubated for 2h RT (IFN-y and IL-2 kit) and Streptavidin-HRP- Conjugate was added and incubated for 1h RT (IL-4 kit). Finally, specific substrates were added: BCIP/NBT substrate 1h RT (IFN-y and IL-2 kit) and AEC substrate 30 min RT (IL-4 kit). The reaction was stopped by extensively washing the plates with tap water, then plates were left air drying at least 24 h in the dark. Wells were imaged with ImmunoSpot® Analyzer (CTL-lmmunospot) and spot-forming units (SFUs) were determined using The ImmunoSpot® Single Color Enzymatic Software Suite (CTL- lmmunospot).

Statistical analysis

Statistical analyses were performed with GraphPad Prism 9 software (Graphpad Software, San Diego, CA, USA). Comparisons in multiple groups were analyzed by oneway ANOVA including a post-hoc test. Friedman test or one-way ANOVA with RM was used to compare repeated measures obtained at different time points, p values <0.05 were considered to be significant.

RESULTS

Dellera immunogenicity in mice

Immunogenicity was assessed in BALB/c and C57B/6J mice by immunization with 1 g and 10 pg Dellera per dose. Age-matched mice receiving empty LNP served as control. Three IM immunizations were performed 2 weeks-apart at 0, 14 and 28 days. SARS- CoV-2 RBD-specific binding antibodies against the Ancestral (Fig. 2a and 3a) and SARS- CoV-2 S-specific binding antibodies against the UK-Alpha variant strain (fig. 2b and 3b) were detected in both BALB/c and C57B/6J. Titers were significantly enhanced 21 days post-boost (42 days). The neutralization potency was assessed by microneutralization (MN) assay based on cytopathic effect (CPE). In both BALB/c and C57B/6J (Fig 2c and 3c), a dose-dependent increase in neutralization titer against the UK-Alpha was observed and found strongly pronounced 42 days after first immunization with GMTs on D42 of 735 for 1 pg and 3880 for 10pg in BALB/c. Surprisingly, neutralizing titers observed in C57B/6J mice were lower with GMTs on D42 of 15 for 1 pg and 85 for 10pg. Finally, the neutralization potency was further investigated in BALB/c against Omicron BA.1 , BA.4, BA.5 variants. Results show a good cross-reaction when samples were tested against the RBD from Omicron BA.1 variant. Marginal neutralizing antibody activity was detected against Omicron BA.4 and BA.5 variants (Fig 2d). These data suggest that Dellera is able to elicit a high and functional (neutralizing) antibody immune response.

T cell cytokine responses were tested in mice 2 weeks post-boost. Cytokines induced by re-stimulation with the pooled SARS-Cov-2 RBD protein peptides were assessed in immune splenocytes in both BALB/c and C57B/6J mice on D42 by the IFN-y, IL-2 (TH1 cytokines) and IL-4 (TH2 cytokines) ELISPOT assays. Majority of BALB/c in the two dose level groups tested demonstrated the presence of IFN-y secreting cells, ranging from 3 to 287 spot-forming unit (SFU) per million splenocytes as well as IL-2 secreting cells, ranging from 4 to 62 SFU (Fig2e). A significant dose-response was not observed as the animals in the lower and higher dose level group showed comparable frequencies of IFN-y and IL-2 secreting cells. Similar results were observed for IL-4 secreting cells. Thus, the data suggest a not clear tendency for TH1 or TH2 biased immune responses. Unlike to what observed in BALB/c, in C57B/6J mice weaker T cell cytokine responses were observed as shown by the low SFU per million splenocytes detected for IFN-y, IL- 2 and IL-4 (Fig 3d). Dellera toxicology in Sprague Dawley rats

Toxicology was assessed in female Sprague Dawley rats by three IM immunizations with 1 g and 10 pg Dellera, separated by 2-week intervals (0, 14 and 28 days). Age-matched mice receiving empty LNP served as control. On day 30 and 42 animals were anesthetized with isoflurane for blood sampling and sacrificed by CO2 inhalation. No significant differences were observed for body weight gain (Fig 4a) and for feed intake between treated and controls during the study period. No pathological clinical signs or signs of toxicity were observed during the study as well as no mortality was recorded. All animals were found in good nutritional and clinical condition and no gross lesions were detected during necropsies. No statistically significant differences were observed in hematological and biochemical results between treated and control groups at either time of sacrifice. Histopathological image analysis revealed that the tested vaccine did not induce any change related to vaccine administration (Fig 4c). Due to their pathogenesis and distribution among groups, degenerative lesions found in atrioventricular valves (AV) of the heart of some animals were considered an age-related chronic process. Same observations were made for the follicular lymphoid hyperplasia found in mesenteric lymph nodes and in bronchial associated lymphoid tissue (BALT). Indeed, since the random distribution among groups and the high number of animals affected, the BALT detected was considered a background lesion. Hepatocellular vacuolization, usually a fixation-related artifact, has not been considered as a true lesion. Therefore, we concluded that when the vaccine was administered IM, no histopathological changes clearly attributable to the administration of the test item were detected in the organs examined.

Protective efficacy of DELLERA in Syrian golden hamsters

To evaluate the potential of Dellera to protect against the viral infection and disease, hamsters were immunized with two vaccine formulation dose levels of 1 pg and 10 pg Dellera per dose, as three IM immunizations at 0, 14 and 21 days. Age-matched mice administered with empty LNP or buffer served as controls. Animals were challenged at day 42 via IN inoculation of 10² TCID50 of the ancestral SARS-CoV-2 variant and monitored daily for 5 days post-challenge for clinical manifestations of disease such as body weight (Fig. 5a) and virus titration of throat swabs (Fig 5b). In general, virus- challenged animals showed no clinical signs of significant disease and no significant body weight variation. Moreover, vaccinated animals revealed a trend to a lower viral load as detected via throat swab. To assess whether Dellera immunization could impact viral infection in hamsters, viral RNA and viral titer were measured from lung and nasal samples (Fig 5c-d). For this purpose, lungs and nasal tissues were harvested at the end of the study period. Data show marked attenuation of SARS-CoV-2 RNA and infectious viral titer in the lung of hamsters that received either 1 pg and 10 pg Dellera vaccine. A similar effect, even if more marginal, can be observed also in nasal turbinates. To assess the pathology caused by viral infection, lung, nasal turbinate and trachea samples were harvested and fixed tissues were submitted to macroscopic examination before being sectioned, randomized and blinded for histopathological examination. Variable, minimal to marked, amounts of multifocal gray-red to dark red areas with rarely small areas of pinpoint hemorrhage (petechiation) were present in lungs of all groups. However, both treated groups (1 pg and 10 pg) were the least affected (Fig 5e). Nasal turbinates of the hamsters showed moderate to severe inflammation (rhinitis), which was characterized by a moderate to severe infiltration of inflammatory cells in the epithelium, mainly neutrophils, and many (degenerated) neutrophils and cellular debris in the lumen with multifocal degeneration and necrosis of respiratory and olfactory epithelial cells. Moderate to severe mixed inflammatory infiltrate was observed in the lamina propria and occasional focal mild necrosis was detected in the glandular epithelium. Both control groups (untreated and LNP) were consistently severely affected while the groups treated with 1 pg and 10 pg Dellera were slightly severely affected, and moderately affected, respectively. (Fig 5f) Inflammation of tracheal epithelium and lamina propria (tracheitis), characterized by intraepithelial neutrophils, was minimal (1 pg) or absent (10 pg) in immunized hamsters compared to controls showing moderate (untreated) to mild (LNP) tracheitis. (Fig 5g) Pulmonary parenchyma showed variably to severe inflammation of the lungs, characterized by infiltration of neutrophils and macrophages within airways, alveolar walls and alveolar lumina (bronco-interstitial pneumonia), which affected a variable amount of the alveolar tissue (extent) to variable degrees (severity). The SUM scores for combined extent and severity of alveolitis were highest in the untreated group and lower in the group treated with the highest Dellera dose. (Fig 5h) Intra alveolar hemorrhage, suggestive for endothelial damage, was mostly detected in the untreated group and absent in immunized hamsters. (Fig 5i) Minimal to mild inflammation, characterized by intraepithelial and intraluminal neutrophils, were observed in conductive airways. Bronchitis was found slightly mild (untreated) and mild (LNP) in control groups and absent in immunized animals. (Fig 5I). These data demonstrate protection by Dellera immunization in this animal model, especially for prevention of lung infection and pathology. However, the immunization, even given the high dose, could not completely prevent viral infection in the upper respiratory tract (nasal turbinates). However, vaccination may have an impact on transmission due to lower loads of viral shedding from the upper respiratory tract.

Discussion

There remains a need for additional effective SARS-CoV-2 vaccines both to meet the global demand and, more importantly, to address the potential of new viral variants. mRNA-based vaccine platforms have proven to be the ideal candidates for the design of vaccine candidates. Indeed, they offer an alternative rapid path to the traditional vaccine development approach. Our study focused on the pre-clinical evaluation of a candidate vaccine which has been designed using a computational model. The present study used a model to predict the potential and future distribution of SARS-CoV-2 variants in Europe and to select the variant most likely to disseminate as dominant SARS-CoV-2 clade in the following COVID wave.

We demonstrated that Dellera, an LNP-formulated, ml^P nucleoside-modified mRNA encoding SARS-CoV-2 S is highly immunogenic in mice. Indeed, vaccinated mice elicited high titers of S- and RBD-specific IgG antibodies. Moreover, immunized mice elicited high titers of SARS-CoV-2 neutralizing antibodies indicating that Dellera is able to cause a high and functional (neutralizing) antibody immune response. High immunogenicity can be observed already at doses as 1 pg with effects strongly enhanced with a 10 pg dose. However, our data suggest that BALB/c mice immune responses were significantly stronger compared to the ones observed in C57BL/6J mice. However, similar effects were observed in the work of Corbett et al. (in which, especially neutralizing antibody titers in C57BL/6J mice were significantly lower compared to the ones detected in BALB/c mice. (Corbett KS, Edwards DK, Leist SR, Abiona OM, Boyoglu-Barnum S, Gillespie RA, et al. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature. 2020 Oct;586(7830):567-571.)

Measurement of cytokine patterns following restimulation with RBD peptide pools on splenocytes from mice immunized with Dellera, showed increased secretion of both Th1 cytokines (IFNy and IL-2) and Th2 cytokines (IL-4) in BALB/mice suggesting T cell activation however, without a clear Th1 or Th2-biased response. Same effects could not be observed in C57BL/6J mice where cytokines secretion levels showed significantly lower levels. This data goes hand in hand with a lower immunogenicity observed in this mouse model. However, a limitation in our cytokine detection could be due to the use of previously frozen splenocytes as well as the analysis using ELISPOT assay as contrary to the method of intracellular cytokine staining using flow cytometry.

Toxicology studies conducted on Sprague Dawley rats showed lack of adverse effects. Indeed, no total body or organ weight loss was observed in treated mice compared to controls. More importantly, vaccine administration did not induce any histopathological change clearly related to the test item in analyzed organs such as brain, heart , liver, kidneys, lungs and spleen.

Dellera also conferred protection in hamster model as shown by a decreased lung pathology in immunized mice compared to both controls. Immunization, although effective in preventing disease and pathology, was not effective in preventing viral infection, especially at the nasal tissues. While the hamsters could achieve viral clearance 4 days after the challenge, the nasal tissues from most hamsters were sampled positive for viral sgRNA. These results are supported by findings previously reported for SARS-CoV-2 infection in this model. In particular, Chan et al. showed that in hamster models for SARS-CoV-2 infection the virus replicates to higher titer in the upper respiratory tract (nasal turbinates) than in the lower respiratory tract (lungs). (Chan JF, Zhang AJ, Yuan S, Poon VK, Chan CC, Lee AC, et al. Simulation of the Clinical and Pathological Manifestations of Coronavirus Disease 2019 (COVID-19) in a Golden Syrian Hamster Model: Implications for Disease Pathogenesis and Transmissibility. Clin Infect Dis. 2020 Dec 3;71(9):2428-2446). Moreover, detection of elevated nasal viral titers despite the effective prevention of COVID-19 are discussed in the recent literature. This could be due to the limited mucosal protection of injectable vaccines compared to the systemic efficacy in preventing COVID-19 infection. Thus viral transmission represents a concern for all vaccines currently under development or authorized for emergency use. However, vaccination may have an impact on transmission due to shortened duration and to lower viral loads observed in nasal turbinates and consequently decreased viral shedding from the upper respiratory tract. SEQUENCE LISTING

SEQ ID NO: 1 - DNA sequence encoding predicted spike protein variant

ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAA

CCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTAC

CCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCT

TTCTTTTCCAATGTTACTTGGTTCCATGCTATCTCTGGGACCAATGGTACTAAGAG

GTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAA

GTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGT

CCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATT

TTGTAATGATCCATTTTTGGGTGTTTACCACAAAAACAACAAAAGTTGGATGGAAA

GTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGC

CTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTT

GTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATT

TAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCA

ATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTG

ACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGG

GTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAG

ATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCC

TTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGA

ATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAAC

GCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTG

TTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATG

GAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCAT

TTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGAT

TGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAA

TTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTT

TAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGG

CCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAAT

CATATGGTTTCCAACCCACTTATGGTGTTGGTTACCAACCATACAGAGTAGTAGTA

CTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACT

AATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGT

GTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATT

GATGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTAC

ACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACC AGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAAGTCCCTGTTGCTATTCAT

GCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAA

ACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTG

ACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCAT

CGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTG

GTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCATAAATTTTA

CTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGAT

TGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATAT

GGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGA

CAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAA

TTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAA

GCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCT

GGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCAT

TTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAA

TGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACC

TTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTT

TAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCA

ACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGT

GCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCT

TGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCT

TGCACGTCTTGACCCCCCCGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGC

AGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAAT

CAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAAT

CAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCA

GCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAA

CTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAG

GTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAAC

CACAAATCATTACTACACACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAG

GAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAG

GAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGA

CATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCA

ATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAG

TATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTT

GATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTT

GTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCT GAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATAA

SEQ ID NO: 2 - predicted mRNA sequence transcribed from DNA of SEQ ID NO: 1.

*where T is construed as II under WIPO Standards.26

ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAA

CCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTAC

CCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCT

TTCTTTTCCAATGTTACTTGGTTCCATGCTATCTCTGGGACCAATGGTACTAAGAG

GTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAA

GTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGT

CCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATT

TTGTAATGATCCATTTTTGGGTGTTTACCACAAAAACAACAAAAGTTGGATGGAAA

GTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGC

CTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTT

GTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATT

TAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCA

ATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTG

ACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGG

GTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAG

ATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCC

TTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGA

ATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAAC

GCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTG

TTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATG

GAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCAT

TTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGAT

TGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAA

TTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTT

TAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGG

CCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAAT

CATATGGTTTCCAACCCACTTATGGTGTTGGTTACCAACCATACAGAGTAGTAGTA

CTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACT

AATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGT

GTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATT

GATGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTAC ACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACC

AGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAAGTCCCTGTTGCTATTCAT

GCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAA

ACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTG

ACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCAT

CGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTG

GTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCATAAATTTTA

CTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGAT

TGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATAT

GGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGA

CAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAA

TTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAA

GCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCT

GGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCAT

TTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAA

TGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACC

TTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTT

TAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCA

ACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGT

GCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCT

TGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCT

TGCACGTCTTGACCCCCCCGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGC

AGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAAT

CAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAAT

CAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCA

GCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAA

CTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAG

GTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAAC

CACAAATCATTACTACACACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAG

GAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAG

GAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGA

CATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCA

ATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAG

TATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTT

GATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTT GTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCT GAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATAA

SEQ ID NO: 3 - predicted spike protein sequence translated from mRNA of SEQ ID NO: 2

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPF

FSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI

VNNATNVVIKVCEFQFCNDPFLGVYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL

MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT

RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD

PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAW

NRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA

PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIS

TEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCG

PKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIDDTTDAVRDPQTLEI

LDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNV

FQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSHRRARSVASQSIIAYTMSLGA

ENSVAYSNNSIAIPINFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCT

QLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIED

LLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAG

TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS

STASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILARLDPPEAEVQIDRLIT

GRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQS

APHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEP

QIITTHNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGI

NASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIM

LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 4 - DNA Construct for mRNA vaccine (comprised of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 1 , 2x SEQ ID NO: 7, SEQ ID NO: 8)

ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCACCGCCGCCACCATGTTTGTT TTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACT

CAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAA

GTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCC

AATGTTACTTGGTTCCATGCTATCTCTGGGACCAATGGTACTAAGAGGTTTGATAA

CCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACAT

AATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTA

TTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGA

TCCATTTTTGGGTGTTTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCA

GAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTA

TGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAG

AATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGT

GATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTAT

TAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGG

TGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTC

AACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAG

ACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTA

GAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTT

AGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGA

TTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTA

TTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC

TACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGA

GGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATA

ATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATC

TTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTA

ATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACA

CCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTC

CAACCCACTTATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGA

ACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTA

AAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACT

GAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGATGACAC

TACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTT

CTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCT

GTTCTTTATCAGGGTGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCA

ACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGC

AGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCA TTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCATCGGCGGGC

ACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAA

ATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCATAAATTTTACTATTAGTGT

TACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGT

ACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTT

GTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACC

CAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTT

GGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTC

ATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAA

ACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAA

AGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAA

TACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAG

GTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTG

GAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATA

GTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAA

CTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACT

TAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTGCACGTCTTGA

CCCCCCCGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGT

TTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGC

TAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTG

ATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGT

GTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGC

TCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTT

CAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATT

ACTACACACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAA

CAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAG

ATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCA

TTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCC

AAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTA

TATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAG

TAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGC

TGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCT

CAAAGGAGTCAAATTACATTACACATAAGCTCGCTTTCTTGCTGTCCAATTTCTATT

AAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGC

CTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAGCTCGCTT TCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAAC

TGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTT

ATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAA

SEQ ID NO: 5 - modified human a-globin 5’ UTR

ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCACC

SEQ ID NO: 6 - Kozak Sequence without start codon

GCCGCCACC

SEQ ID NO: 7 - human p-globin 3’IITR

GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAAC

TACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAA AAACATTTATTTTCATTGCAA

SEQ ID NO: 8 - a 110 nt interrupted poly(A) tail comprised of 30 adenosine residues and 70 adenosine residues separated by a 10 nt linker sequence

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 9 - E_Sarbeco_F primer: ACAGGTACGTTAATAGTTAATAGCGT

SEQ ID NO: 10 - E_Sarbeco_R primer: ATATTGCAGCAGTACGCACACA

SEQ ID NO: 11 - E_Sarbeco_P1 probe: ACACTAGCCATCCACTGCGCTTCG)

Claims

39 CLAIMS:

1. A vaccine composition comprising: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations.

2. The vaccine composition of claim 1 , wherein the nucleic acid molecule is RNA.

3. The vaccine composition of claim 2 wherein the RNA includes one or more nonstandard nucleosides.

4. The vaccine composition of claim 3 wherein the RNA includes pseudouridine.

5. The vaccine composition of any of claims 2-4 wherein the nucleic acid molecule is encapsulated within lipid nanoparticles.

6. The vaccine composition of claim 5, wherein the lipid nanoparticles comprise an ionizable cationic lipid, a PEGylated lipid, cholesterol, and distearoylphosphatidylcholine (DSPC).

7. The vaccine composition of any of claims 2-6 comprising Tris Buffer, sucrose, and sodium acetate.

8. The vaccine composition of any of claims 2-7 further comprising nucleic acid encoding one or more replicase components.

9. Use of: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or 40 d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations; in the preparation of a vaccine composition.

10. A composition comprising: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations; for use as a vaccine.

11. A method for inducing or enhancing an immune response, the method comprising administering to a subject a composition comprising: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or e) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations.

12. A plasmid comprising: a) a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or b) a nucleic acid molecule, which, when transcribed, produces a nucleic acid 41 molecule having the nucleic acid sequence of SEQ ID NO: 2; or c) a nucleic acid molecule which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3; or d) a nucleic acid molecule which differs from that of a) or b) in one or more silent mutations.

13. A host cell comprising the plasmid of claim 12.

14. A nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 1 ; or of SEQ ID NO: 2; or which encodes a peptide molecule having the amino acid sequence of SEQ ID NO: 3.

15. A vaccine composition comprising a SARS-CoV-2 spike protein having the following mutations compared with a reference sequence: N501Y; D614G; D1119H; A570D; P681 H; S982A; T716I ;A144 (Y); A69(H)-70(D); or a nucleic acid encoding said protein; wherein the reference sequence is that of the Wuhan-Hu-1 isolate.