FIELD OF THE INVENTION

The present invention relates to the field of the medicine, especially of virology. More particularly, it relates to a vaccine against coronavirus.

BACKGROUND OF THE INVENTION

Betacoronaviruses have become of the greatest clinical importance as illustrated by COVID-19 pandemic caused by SARS-CoV-2. All viruses, including SARS-CoV-2, evolve over time and variants of the original virus appear. Thus, a vaccine designed to target the original virus could be less effective against some of the variants. Another issue is that SARS-CoV-2 is not the first Betacoronavirus that causes an epidemic. Indeed, SARS-CoV-1 was responsible of an epidemic in 2003 and the MERS-CoV was responsible of an epidemic in 2012. It is likely that other Betacoronaviruses emerge in the future. Therefore, one problem to solve is to provide a vaccine as universal as possible so as to be effective against the new variants or coronaviruses that could emerge.

Betacoronavirus includes four subgenera or lineages (A, B, C and D). OC43 and HKU1 cause the common cold and belong to lineage A; SARS-CoV-1 causing SARS and SARS-CoV-2 causing COVID-19 belong to lineage B and MERS-CoV causing MERS belongs to lineage C. Until now, epidemic and pandemic were caused by Betacoronaviruses. However, Alphacoronaviruses 229E and NL63 also cause common cold. Therefore, a vaccine against all human coronaviruses, either alpha or beta, could be advantageous.

SUMMARY AND DESCRIPTION OF THE INVENTION

Vaccines against coronavirus often target spike (S) proteins, sometimes the Nucleocapsid (N) proteins, which are both exposed at the surface of the virus. However, there is no predicted conserved T-cell epitope in the Spike or Nucleocapsid proteins capable to cover at least 3 out of the 5 human Betacoronaviruses. Contrary to the teaching of prior art focusing on spike as a priority target known as a key entry of the virus into the cells, the inventors had an approach based on mid and long term resistance against variants of the virus, instead of main action and/or recognition mechanism (mainly spike, nucleocapsid, membrane, envelope proteins) and concluded notably that the Spike, Nucleocapsid and Envelopetargets are not appropriate for a long-term and broad Betacoronavirus protection based on T- cell immunity.

After complex analysis of conservative epitopes, they identified 22 T-cell epitopes well conserved among the human Betacoronaviruses. 21 of them are located in the ORFlab replicase polyprotein and only one in the Membrane protein (see Tables 1 and 2). More specifically, 10 T cell epitopes have been identified as having 100 % homology with at least 4 out of the 5 human betacoronaviruses (SARS-CoV-1, SARS-CoV- 2, MERS-CoV, HCoV-OC43 and HCoV-HKUl) (see Table 1). Among these 10 epitopes, 6T-cell epitopes have been identified as having 100% homology with the 5 human betacoronaviruses and one of them (namely ab0o\/#1 SEQ ID NO: 1) is of particular interest because it presents 100 % homology with the two alpha and the five beta coronaviruses (betacoronaviruses SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV- OC43 and HCoV-HKUl; alphacoronaviruses 229E and NL63). 12 additional T cell epitopes with a single mismatch in the HLA-binding anchorage site and covering at least 4 out of 5 human betacoronaviruses have been identified. In addition, the inventors designed neo-epitopes (mutated epitopes) of some of these T cell epitopes (see Table 3) by substituting some amino acids in HLA-binding anchorage site to improve HLA affinity, hence immunogenicity and or world HLA coverage. Another advantage of the identified epitopes is that the epitopes have been selected also based on a large word HLA coverage ranging from 40 to 100 % in order not to be restricted to few HLA genotypes in the face of the difficulty of high HLA polymorphism in human.

The epitopes will be recognized by CD8 T cells through the interaction with HLA class I system. The activated CD8 T cells convert into Lymphocytes cytotoxic T cells (effector CTL) by the help of helper T Lymphocytes (HTL). The activated T cells are notably CD8 memory T cells allowing the long-term action of the vaccine.

Therefore, the inventors identified T cell epitopes suitable for preparing a multi-epitopes vaccine being adapted to provide a protection against several betacoronaviruses and optionally alphacoronaviruses. According to our knowledge, it is the first time that the identification of T cell epitopes is focused on this capacity to provide a broad protection among the betacoronaviruses.

Table 1: 10 T cell epitopes with 100 % homology with at least 4 out of the 5 human betacoronaviruses

Table 2: 12 T-cell epitopes covering at least 4 out of 5 human betacoronaviruses with a single mismatch in the HLA-binding Table 3: neoepitope of some T-cell epitopes of Tables 1 and 2

The epitopes of Tables 1-3 are stable among the SARS-Cov-2clades, and none of them are mutated in the 15 variants of SARS-Cov-2 described to this date (B.l.1.7 (Alpha), B.1.351 (Beta), P.l (Gamma), B.1.427 /429 (Epsilon), B.l.617.1 (Kappa), B.l.617.2 (Delta), A.23.1, B.1.526 (lota), C.37, B.1.525 (Eta), P.3 (Theta), B.1.1.519, B.1.619, B.1.620, B.1.621).

Accordingly, the present invention relates to a vaccine composition comprising at least one CTL (neo)epitope selected from the group consisting of (neo)epitopes of SEQ ID NOs: 2, 5-10 and 12-32. Optionally, the vaccine composition may further comprise at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 1, 3, 4 and 11 or a neoepitope thereof such as the neoepitopes of SEQ ID NOs 24, 25 and 28.

Optionally, the present invention relates to a vaccine composition comprising at least one CTL (neo)epitope selected from the group consisting of (neo)epitopes of SEQ ID NOs: 5-10 and 13-32. Optionally, the vaccine composition may further comprise at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 1-4 and 11-12 or a neoepitope thereof such as the neoepitopes of SEQ ID NOs 24, 25 and 28.

Alternatively, the present invention further relates to a vaccine composition comprising a nucleic acid or a set of nucleic acids encoding at least one CTL (neo)epitope selected from the group consisting of epitopes of SEQ ID NOs: 1-32. Optionally, the nucleic acid or set of nucleic acids encodes at least one CTL (neo)epitope selected from the group consisting of epitopes of SEQ ID NOs: 2, 5-10 and 12-32. Optionally, the nucleic acid or set of nucleic acids further encodes at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 1, 3, 4 and 11.

The vaccine composition has the following advantages:

- it is pan-betacoronaviruses, optionally pan-alpha and betacoronaviruses;

- the anti-viral mechanism of action is cellular and long-term, especially T cell memory CTL (Cytotoxic T Lymphocytes); and

- it is suitable not only for HLA-A2 positive subjects but also HLA-A2 negative subjects.

Optionally, the vaccine composition comprises at least 1, 2, 3, 4, 5, 6, 7 or 8 CTL (neo)epitope selected from the group consisting of epitopes of SEQ ID NOs: 2, 5-10 and 12-32 or nucleic acid or set of nucleic acids encoding such at least 1, 2, 3, 4, 5, 6, 7 or 8 CTL (neo)epitope. Optionally, the vaccine composition may further comprise at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 1, 3, 4 and 11 or nucleic acid or set of nucleic acids encoding such at least one CTL epitope. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

Optionally, the vaccine composition comprises at least one CTL (neo)epitope selected from the group consisting of epitopes of SEQ ID NOs: 2 and 5-10 or a neoepitope thereof such as the neoepitopes of SEQ ID NOs: 23 and 26 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. Optionally, the vaccine composition may further comprise at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 1, 3, and 4 or a neoepitope thereof such as the neoepitopes of SEQ ID NOs: 24 and 25 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. In a particular aspect, the vaccine composition further comprises an epitope of SEQ ID NO: 1 or a neoepitope of the epitope of SEQ ID NO: 1 such as the neoepitope of SEQ ID NO: 25 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

Optionally, the vaccine composition comprises at least one CTL (neo)epitope selected from the group consisting of epitopes of SEQ ID NOs: 12-22 or a neoepitope thereof such as the neoepitopes of SEQ ID NOs: 29-32 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. Optionally, the vaccine composition may further comprise at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NO: 11 or a neoepitope thereof such as the neoepitopes of SEQ ID NO: 28 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

Optionally, the vaccine composition comprises at least 1, 2, 3, 4, 5, 6, 7 or 8 CTL (neo)epitopes selected from the group consisting of epitopes of SEQ ID NOs: 2 and 5-10 or a neoepitope thereof such as the neoepitopes of SEQ ID NOs: 23 and 26 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. Optionally, the vaccine composition may further comprise at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 1, 3, and 4 or a neoepitope thereof such as the neoepitopes of SEQ ID NOs: 24 and 25 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. In a particular aspect, the vaccine composition further comprises an epitope of SEQ ID NO: 1 or a neoepitope of the epitope of SEQ ID NO: 1 such as the neoepitope of SEQ ID NO: 25 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

Optionally, the vaccine composition comprises at least 1, 2, 3, 4, 5, 6, 7 or 8 CTL (neo)epitopes selected from the group consisting of epitopes of SEQ ID NOs: 12-22 or a neoepitope thereof such as the neoepitopes of SEQ ID NOs: 29-32 or nucleic acid or set of nucleic acids encoding such CTL (neo)epitopes. Optionally, the vaccine composition may further comprise at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NO: 11 or a neoepitope thereof such as the neoepitopes of SEQ ID NO: 28 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

Optionally, the vaccine composition comprises at least one CTL neoepitope selected from the group consisting of neoepitopes of SEQ ID NOs: 23-32 or nucleic acid or set of nucleic acids encoding such at least one CTL (neo)epitope. In a particular aspect, the vaccine composition comprises the neoepitope of SEQ ID NO: 25 or nucleic acid or set of nucleic acids encoding it. Optionally, the vaccine composition comprises at least 1, 2, 3, 4, 5, 6, 7 or 8 CTL neoepitopes selected from the group consisting of neoepitopes of SEQ ID NOs: 23-32 or nucleic acid or set of nucleic acids encoding such CTL (neo)epitope. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

Optionally, the vaccine composition comprises at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 1-22 and at least one CTL neoepitope selected from the group consisting of neoepitopes of SEQ ID NOs: 23-32 or nucleic acid or set of nucleic acids encoding such CTL (neo)epitopes. Optionally, the vaccine composition comprises at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 1-10 and at least one CTL neoepitope selected from the group consisting of neoepitopes of SEQ ID NOs: 23-27 or nucleic acid or set of nucleic acids encoding such CTL (neo)epitopes. Optionally, the vaccine composition comprises at least one CTL epitope selected from the group consisting of epitopes of SEQ ID NOs: 11-22 and at least one CTL neoepitope selected from the group consisting of neoepitopes of SEQ ID NOs: 28-32 or nucleic acid or set of nucleic acids encoding such CTL (neo)epitopes. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

Optionally, the vaccine composition comprises at least one CTL (neo)epitope selected from the group consisting of (neo)epitopes of SEQ ID NOs: 1-10, 12-27 and 29-31 and the CTL epitope of SEQ ID NO: 11 or a neoepitope thereof such as the CTL neoepitope of SEQ ID NO: 28 or nucleic acid or set of nucleic acids encoding such CTL (neo)epitopes. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes and 1 or 2 of them are the CTL epitope of SEQ ID NO: 11 or a neoepitope thereof such as the CTL neoepitope of SEQ ID NO: 28 or nucleic acid or set of nucleic acids encoding them.

Optionally, the vaccine composition comprises at least 5, 6, 7 or 8 CTL (neo)epitope selected from the group consisting of epitopes of SEQ ID NOs: 1-32 or nucleic acid or set of nucleic acids encoding them. Optionally, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes or between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

In a particular aspect, the vaccine composition comprises between 5 and 15 CTL (neo)epitopes as disclosed above, especially between 6 and 14 CTL (neo)epitopes or between 7 and 13 CTL (neo)epitopes or between 8 and 12 CTL (neo)epitopes or nucleic acid or set of nucleic acids encoding them.

Optionally, the composition further comprises at least 1 HTL (Helper T Lymphocytes) peptide/epitope or a T helper peptide or nucleic acid or set of nucleic acids encoding it. The HTL epitopes are either natural or synthetic T cells helper peptides know in the art. Natural helper peptides are for instance a Natural Tetanus sequence alone or linked to another epitope, or a Plasmodium falciparum sequence alone or linked to another epitope. In a particular aspect, the HTL peptide may comprise a synthetic peptide such as a Pan-DR-binding epitope (e.g., a PADRE ^® peptide, Epimmune Inc., San Diego, CA, described, for example, in U.S. Patent Number 5,736,142), for example, having the sequence (aKXVAAWTLKAAa with X and a respectively indicating cyclohexylalanine and d-alanine) (SEQ ID NO: 33). Certain pan-DR binding epitopes comprise all "L" natural amino acid residues; these molecules can be provided as peptides or in the form of nucleic acids that encode the peptide. See also, U.S. Patent Numbers 5,679,640 and 6,413,935.

Optionally, the vaccine composition may further comprise an adjuvant. The adjuvant is preferably an oily adjuvant, which comprises both a hydrocarbon oil and a water-in-oil emulsifier. Such adjuvants act by the so-called "deposition effect". The hydrocarbon oil may be paraffin oil, a vegetable oil, squalene, squalane or mineral oil, for instance. Suitable W/O emulsifiers may be selected from mannide mono-oleate and sorbitan mono-oleate, for instance. Examples of appropriate oily adjuvants are a mixture of mineral oil and mannide mono-oleate, more particularly a mixture of 5-20% mannide mono-oleate with 80-95% mineral oil (Montanide ^® ISA 51 sold by SEPPIC) or squalene (Montanide ^® ISA 720 sold by SEPPIC) and similar mixtures. In a specific embodiment, the adjuvant is a mixture of mineral oil and mannide mono- oleate, especially Montanide ^® ISA 51.

In a particular aspect, the vaccine composition is an emulsion with a mineral oil adjuvant.

The adjuvant used in this invention may alternatively, or in addition to the above oily adjuvants, be selected from micro- and nanoparticles, such as liposomes and microspheres, of PLG, PLA, PLGA or other natural polymers such as gelatin, collagen and chitosan. Other adjuvants may comprise TLR ligands, Toll like receptor ligands (TLR3 and TLR9), stimulators of IFN genes (STING) agonists, cytokines such as GM- CSF and IL2, carbohydrates, bacterial derivatives, mineral salts and immune stimulating complexes (ISCOM).

Optionally, the vaccine composition may comprise aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate.

The vaccine composition may further comprise one or more "pharmaceutically acceptable carriers". These are typically large, slowly metabolized macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti, 2001, Vaccine, 19:2118-2126), trehalose (WO 00/56365), lactose and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. In some aspect, the vaccine compositions contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate buffered physiologic saline is a typical carrier (Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN:0683306472).

The vaccine compositions are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the vaccine compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. More preferably, the vaccine composition is intended for subcutaneous administration or intramuscular administration. Optionally, the vaccine composition is intended for nasal administration.

Optionally, each peptide or (neo)epitope of the composition is present at a concentration of 0.01 mg/ml to 1 g/ml, 0.1 mg/ml to 10 mg/ml. For instance, each peptide can be present at a concentration of 0.5 mg/ml.

Optionally, the vaccine composition comprises the CTL (neo)epitopes, each at a dose of between 1 and 100 pg, preferably between 5 and 50 pg. It may comprise the T helper peptide, especially PADRE, at a dose of between 1 and 100 pg, preferably between 5 and 50 pg.

Optionally, the total number of epitope peptides in the composition can be from 1 to 40, from 2 to 30 or from 3 to 20 peptides. Optionally, the vaccine composition may further comprise other peptides than disclosed in the present invention. For instance, the other peptides may be non-exhaustively selected among the T cell epitopes disclosed in Syed Faraz Ahmed et al (Viruses, 12, 254, 2020), W02004/110349, W02004/092360, Grifoni et al (Cell Host and Microbe, 27, 671, 2020).

Optionally, the T cell epitopes can be included in a larger peptide of less than 15, 20, 30, 40 or 50 amino acids in length. Optionally, the T cell epitopes can be in the form of a polypeptide in which several T cell epitopes are fused. Optionally, the T cell epitopes can be in the form of a carrier such as a nanoparticle bearing said T cell epitopes. Optionally, instead of the T cell epitope, the vaccine composition may include a nucleic acid encoding said T cell epitopes.

The vaccine composition may comprise a nucleic acid or a set of nucleic acids encoding the CTL (neo)epitopes as disclosed herein.

Nucleic acids of the vaccine can be or can include deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a b-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'- amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) and/or chimeras and/or combinations thereof.

In a particular aspect, the nucleic acid or set of nucleic acids of the vaccine composition is a mRNA polynucleotide or a set of mRNA polynucleotides. The technology of mRNA vaccine is now well-known by the person skilled in the art. For illustrating this aspect, see WO21159130, the the disclosure thereof being incorporated herein by reference. The composition may comprise one nucleic acid for each (neo)epitope of the vaccine or nucleic acid encoding several of the (neo)epitopes or all of the (neo)epitopes. mRNA polynucleotides can contain stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5 '-end (5' UTR) and/or at their 3'-end (3' UTR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. Preferably, mRNA polynucleotides comprise at least one modification and at least one 5' terminal cap.

5 '-capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3'-0-Me-m7G(5')ppp(5') G [the ARC A cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5 '-capping of modified RNA may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Vims Capping Enzyme and a 2'-0 methyl-transferase to generate: m7G(5')ppp(5')G-2'-0- methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-0-methylation of the 5 '-antepenultimate nucleotide using a 2'-0 methyl- transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-0- methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase. Enzymes may be derived from a recombinant source.

The mRNA polynucleotides may comprise modified nucleotides and nucleosides. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897;

PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/36773; PCT/US2015/36759;

PCT/US2015/36771; or PCT/IB2017/051367 all of which are incorporated by reference herein. More specifically, modified nucleobases in nucleic acids (e.g., mRNA) may comprise 1- methyl-pseudouridine (itiΐy), 1 -ethyl-pseudouridine (eli[>), 5-methoxy-uridine (mo5U), 5- methyl-cytidine (m5C), and/or pseudouridine (y). Other examples of modified nucleobases include 5- methoxymethyl uridine, 5- methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine. The polyribonucleotide may include a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases. More specifically, the mRNA may comprise 1 -methyl- pseudouridine (itiΐy) substitutions at one or more or all uridine positions of the nucleic acid. Alternatively, the mRNA may comprise 1 -methyl-pseudouridine (itiΐy) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid. In another alternative, the mRNA may comprise pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid and optionally 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

The 3'-poly(A) tail is typically a stretch of adenine nucleotides added to the 3 '-end of the transcribed mRNA. It can, in some instances, comprise up to about 400 adenine nucleotides. Optionally, the mRNA polynucleotide or set of mRNA polynucleotides is/are formulated in at least one lipid nanoparticle, especially a lipid nanoparticle comprising a mixture of lipids. Lipid nanoparticles typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400;

PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2016/014280;

PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117;

PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.

In a particular aspect, the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid/neutral lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid. For instance, the non-cationic lipid can be 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),

1.2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-0-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3- phosphocholine,l,2-diarachidonoyl-sn-glycero-3-phosphocholin e, 1,2- didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl- sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1- glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. The PEG modified lipid can be a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG- modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is DMG-PEG, PEG-c- DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG. The sterol can be cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof.

In a specific aspect, the lipid mixture comprises heptadecan-9-yl 8 ((2 hydroxy ethyl) (6 oxo 6- (undecyloxy)hexyl)amino)octanoate as ionizable cationic lipid, 1,2 distearoyl sn glycero-3 phosphocholine (DSPC) as neutral lipid, cholesterol as sterol and 1- monomethoxypolyethyleneglycol-2,3- dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG) as PEG- modified lipid.

Optionally, the vaccine composition may comprise an 25 pg to 250 pg dose of mRNA encoding a (neo)epitope. Optimally, the vaccine composition may further include a pharmaceutically-acceptable excipient, inert or active. The excipient in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with mRNA and can be capable of stabilizing it. One or more excipients (e.g., solubilizing agents) can be utilized as pharmaceutical carriers for delivery of the mRNA. Examples of a pharmaceutically acceptable excipients include, but are not limited to, biocompatible vehicles (e.g., LNPs), carriers, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other excipients include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical excipients, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. Optionally, the vaccine composition does not include an adjuvant.

The vaccine compositions are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the vaccine compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. More preferably, the vaccine composition is intended for subcutaneous administration or intramuscular administration. Optionally, the vaccine composition is intended for nasal administration.

In a particular aspect, the vaccine composition is to be administered once, twice or more. For instance, two administrations can be carried out. A prime administration followed by a boost administration. For instance, the injections can be spaced by 3 weeks or 2 weeks and will be adapted to the Immune response requested and to the medical conditions of the subject to be treated.

The present invention relates to a vaccine composition as disclosed herein for use for preventing or treating an infection by a coronavirus; the use of a vaccine composition as disclosed herein for the manufacture of a vaccine for preventing or treating an infection by a coronavirus; and to a method for preventing or treating an infection by a coronavirus in a subject, comprising the administration of an effective amount of a vaccine composition as disclosed herein.

Optionally, the coronavirus is an alphacoronavirus or a betacoronavirus, preferably a betacoronavirus. More specifically, the vaccine composition is suitable for preventing or treating an infection by several betacoronaviruses. Optionally, the vaccine composition is suitable for preventing or treating an infection by an alphacoronavirus. In one aspect, the vaccine composition as disclosed herein is for use for preventing or treating an infection by an alphacoronavirus and a betacoronavirus. In this aspect, the vaccine composition comprises the epitope of SEQ ID NO: 1 or a neoepitope thereof such as the neoepitope of SEQ ID NO: 25.

Optionally, the betacoronavirus is selected from the group consisting of SARS-CoV-1, SARS-CoV-2, MERS- CoV, HCoV-OC43 and HCoV-HKUl, more specifically the group consisting of SARS-CoV-1, SARS-CoV-2, and MERS-CoV. More specifically, the vaccine composition as disclosed herein is for use for preventing or treating an infection by SARS-CoV-1, SARS-CoV-2, and MERS-CoV, optionally by SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43 and HCoV-HKUl.

Optionally, the subject to be treated is a subject aged 65 years or older, a subject having a cancer or having had a cancer, a subject being obese (In particular with severe obesity (body mass index [BMI] of 40 or higher [CDC- HCSP BMI >30]), a subject being diabetic, a subject having a hypertension, a subject having a sarcoidosis, a subject being immunocompromised, a subject who lives in a nursing home or long-term care facility, a subject with chronic lung disease or moderate to severe asthma, lung fibrosis, a subject who has serious heart conditions, a subject with chronic kidney disease undergoing dialysis and/or a subject with liver diseases; and/or a subject being HLA-A2. Many conditions can cause a person to be immunocompromised (including cancer treatment, smoking, bone marrow or organ transplantation, immune deficiencies, poorly controlled HIV or AIDS, prolonged use of corticosteroids and other immune weakening medications).

The patient could be selected on HLA typing. In a very specific aspect, the subject is HLA-A2.

Definitions

A "diluent" includes sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred diluent for pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as diluents, particularly for injectable solutions.

An "epitope" is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule. Alternatively, an epitope can be defined as a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors. It is referred as "T cell epitope" and "CTL epitope". Epitopes are present in nature, and can be isolated, purified or otherwise prepared or derived by humans. For example, epitopes can be prepared by isolation from a natural source, or they can be synthesized in accordance with standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues, "amino acid mimetics," such as D isomers of naturally-occurring L amino acid residues or non-naturally-occurring amino acid residues such as cyclohexylalanine. Throughout this disclosure, epitopes may be referred to in some cases as peptides or peptide epitopes.

"Human Leukocyte Antigen" or"HLA" is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, CA (1994).

An "HLA supertype or HLA family", as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into such HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLAxx-like molecules (where "xx" denotes a particular HLA type), are synonyms.

"Major Histocompatibility Complex" or "MHC" is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3RD ED., Raven Press, New York (1993).

A "native" or a "wild type" sequence refers to a sequence found in nature. Such a sequence may comprise a longer sequence in nature.

The terms "peptide", "epitope" and "peptide epitope" are used interchangeably with "oligopeptide" in the present specification to designate a series of residues, typically L-amino acid residues, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acid residues.

Optionally, a "peptide", "epitope" and "peptide epitope" defined by a SEQ ID NO can consist in the particular SEQ ID NO and can also refer to a peptide consisting in the particular SEQ ID NO but including 1 or 2 additional amino acids at the N and/or C terminal end of the SEQ ID NO.

Optionally, one or several "peptide", "epitope" and "peptide epitope" can be fused together in a same polypeptide.

A "neoepitope" is a peptide or an epitope comprising one or two substitutions of an amino acid aiming to improve the HLA binding, in particular the binding by HLA-A2. In particular, the neoepitope only includes on substitution. In a very particular aspect, the substituted amino acid is the C-terminal amino acid of the T cell epitope.

A "PanDR binding" peptide, a "PanDR binding epitope," or "PADRE ^® " peptide (Epimmune, San Diego, CA) is a member of a family of molecules that binds more than one HLA class II DR molecule. The pattern that defines the PADRE ^® family of molecules can be referred to as an HLA Class II supermotif. A PADRE ^® molecule binds to HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses. For a further definition of the PADRE ^® family, see copending application U.S. serial Nos. 09/709,774, filed November 11, 2000; and 09/707,738, filed November 6, 2000; PCT publication Nos WO 95/07707, and WO 97/26784; U.S. Patent Nos. 5,736,142 issued April 7, 1998; 5,679,640, issued October 21, 1997; and 6,413,935, issued July 2, 2002.

"Pharmaceutically acceptable" refers to a generally non-toxic, inert, and/or physiologically compatible composition or component of a composition.

A "pharmaceutical excipient" or "excipient" comprises a material such as an adjuvant, a carrier, pH- adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A "pharmaceutical excipient" is an excipient which is pharmaceutically acceptable.

As used herein, the term "infection by a coronavirus" designates any infection or disease caused by the coronavirus.

A "protective immune response" or "therapeutic immune response" refers to a CTL and/or an HTL response to an antigen derived from a pathogenic antigen (e.g., an antigen from an infectious agent or a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects or progression. The immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.

As used herein, a "vaccine" is a composition used for vaccination, e.g., for prophylaxis or therapy, that comprises one or more peptides of the invention. There are numerous embodiments of vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The "one or more peptides" can include any whole unit integer from 1-32, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class l-binding peptides of the invention can be linked or to otherwise be combined with HLA class II- binding peptides, e.g., a PADRE ^® universal HTL-binding peptide, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. Vaccines can comprise peptide pulsed antigen presenting cells, e.g., dendritic cells. EXAMPLES

Binding and stability of peptides to HLA-A2 molecules.

The binding and stability of pan coronavirus peptides to HLA expressed on the cell surface is to be measured using a T2 binding assay wild type peptides and optimized peptides will be compared. 174CEM.T2 is a human lymphoma-derived cell line expressing a low level of HLA-A2 on the cell surface due to TAP deficiency (TAP = transporter associated with antigen presentation), and can only present exogenous peptides. Binding of exogenous peptides to HLA-A2 stabilizes the HLA-A2/peptide complexes on the cell surface which can be detected using immunofluorescence staining. T2 cells (ATCC ^® CRL-1992™) (5e4) resuspended in TexMacs media are mixed 1:1 with individual viral peptides (50uM) to a final volume of lOOuL. After 10 minutes of incubation (37°C, 5% C02), T2 cells are incubated overnight at room temperature (14 hours). Cells are then incubated 2 hours at 37°C, 5% CO2, and stained with an anti-HLA A2 antibody (clone BB7.2, FITC labeled, BD Bioscience 551285, 20ug/mL). FITC Median of fluorescence is quantified to measure the capacity of each peptide to bind and stabilize the expression of FILA-A2 molecule on T2 cell surface.

Immunogenicity evaluation of Pan-coronavirus vaccine

To evaluate immunogenicity of Pan-coronavirus vaccine, immunization experiments is to be conducted with escalating dose of the pan-coronavirus vaccine (5, 10, 25 or 50 ug of each peptide) after subcutaneous injection. Single versus repeated injection is to be tested (Day 0 only or Day 0 and Day 14) to evaluate prime/boost vaccination strategy to generate CD8 specific T cells. For this study, human HLA- A2+ transgenic mice is used FILA-A/FI2-D 2Enge/J (Janvier Lab), or FILA-A2/DR1+ double transgenic mice (Pajot et al, 2004), or HLA-A2/H2kB transgenic mice (CB6Fl-Tg(HLA-A*0201/H2-Kb)A*0201; Taconic stock #9659). After immunization, mice are sacrificed on Day 14 (Single vaccination) or Day 21 (Double vaccination) and CD8 T cells from spleen, lymph nodes, Lung or Bronchoalveolar lavages are isolated and their reactivity against optimized pan coronavirus peptides or wild type peptides are evaluated using IFNg Elispot assay. Briefly, CD8- T cells are pulsed with wild type or optimized peptides, then cocultured with CD8+ T cells. Reactivity against wild type and optimized peptide allows to confirm cross reactivity of CD8 T cells to wild type peptides and consequently protection against pan coronavirus sequence. IFNg positive spots are revealed and quantified 24 hours following CD8+: pulsed CD8- Tcells. Similarly, CD8 T cells immunogenicity is to be measured using granzyme B Elispot assay to quantify cytolytic capacity of pan coronavirus specific T cells elicited by vaccination. Individual peptides stimulation versus a pool of peptide stimulation is done to assess T cell reactivity against each peptide.

In parallel, T cell phenotype is to be evaluated by flow cytometry to evaluated phenotype of viral specific CD8 T cells. For this purpose, viral peptide loaded Dextramer/Tetramer is to be used to analyze quantity of viral specific T cells after vaccination and their resident memory phenotype (CD103, CD49a, CXCR6, CXCR3).

Cytotoxic activity of viral specific elicited CD8 T cells after in vivo vaccination

Cytotoxic activity of CD8 +T Cells obtained after pan coronavirus peptide mouse vaccination is to be measured by Chromium 51 release assay against HLA-A2 human cells presenting viral proteins. To mimic viral peptides presentation by HLA-A2 cells, HLA-A2+ T2 human cells are pulsed overnight with the wild- type or optimized peptides and used as target cells to measure cytotoxic functions of elicited CD8+ T cells after vaccination. Prior the assay, CD8 T cells isolated from immunized mice are expanded in vitro with peptide vaccine (2pg/mL each) plus cytokine (IL-7, IL-21 and IL-2) to increase the pool of viral-specific T cells. Peptide pulsed T2 cells or unpulsed T2 cells are labeled with chromium 51 then cocultured with CD8 T cells at ratio 40:1 or 15:1 for 4 hours. Chromium released by target T2 cells is quantified in the supernatant using a gamma counter to quantify specific cytolysis and normalized to negative control (spontaneous release, T2 cells without CD8 T cells) and maximum control (T2 cells lysed with Saponin).