WO2023096494A1 - Conserved coronavirus t cell epitopes - Google Patents

Abstract

The invention relates to a T cell antigen and/or T cell polyantigen comprising one or more conserved coronavirus T cell epitopes, to a nucleic acid molecule encoding said T cell antigen and/or T cell polyantigen, and to a T-cell receptor (TCR) that specifically recognizes a conserved coronavirus T cell epitope. The invention further relates to a pharmaceutical composition comprising said protein, TCR or nucleic acid molecule, and to the use thereof in a method of inducing an immune response in an individual.

Description

Title: Conserved coronavirus T cell epitopes

FIELD: The invention generally relates to immune stimulatory compositions comprising conserved coronavirus T cell antigens.

INTRODUCTION

Of the betacoronaviruses, the Sarbecovirus and merbecovirus subgeni have caused the most significant challenges to mankind. While the 2002-2004 SARS- CoV-1 epidemic did not spread globally and caused relatively few deaths, the 2019 SARS-CoV-2 outbreak turned into a pandemic wreaking global social and economic havoc, and costing millions of lives thus far. The 2012 outbreak of MERS-CoV, a merbecovirus with an extremely high infection-fatality rate, illustrates the dangers associated with some betacoronavirus species. Other, more distant betacoronavirus subgeni, such as the embecoviruses, which include common cold betacoronaviruses such as endemic HKU1 and OC43, do not pose a threat to humans. These zoonotic diseases originate from diverse species such as bats, civet cats, pangolins, hedgehogs and dromedary camels. It will therefore be useful to develop pan- Sarbecovirus and pan-merbeco virus vaccines that cover not only the strains currently infecting humans, but also protect against new zoonoses. As the Sarbecoviruses pose a more imminent threat, the need for a pan-Sarbecovirus vaccine is most pressing.

Current SARS-CoV-2 vaccines efficiently induce neutralizing antibodies against the Spike protein of contemporary SARS-CoV-2 variants, thus providing B- cell mediated protection against SARS-CoV-2 infection and disease. However, as Spike is the most polymorphic gene in coronaviruses, these antibodies are mostly ineffective against other coronaviruses, including related Sarbecoviruses and may become less effective in combating future SARS-CoV-2 variants. It is therefore highly unlikely that it will be possible to design a Spike-based vaccine that raises protective, broadly Sarbecovirus-reactive antibodies.

In contrast, antiviral T cells can be directed against any viral antigen, including highly conserved antigens encoded by non-structural genes. Within the betacoronavirus genus, the Sarbecoviruses are sufficiently similar to allow the design of a pan-Sarbecovirus vaccine that raises T cell responses against multiple, highly conserved Sarbecovirus antigenic sequences encoded by non-structural genes such as ORF lab, and/or structural genes such as the membrane (M), envelope (E), and/or spike (S) genes in animal or human individuals. Upon infection with a Sarbecovirus, vaccine-induced T cells will not only eliminate virus- infected cells but will also amplify and accelerate the generation of new T cell- and antibody-mediated responses to non-conserved antigens specific to the infecting virus.

In the case of new SARS-CoV-2 variants, such immune responses to a new Sarbecovirus would not be affected by a newly acquired mutation in, e.g., the Spike protein of a new Sarbecovirus. This sharply contrasts with currently approved vaccines such as Comirnaty (Pfizer/BioNTech), Spikevax (Moderna), Vaxzevria (AstraZeneca), Janssen Ad26.CoV2.S (Johnson & Johnson), CoronaVac (Sinovac), ZyCoV-D (Zydus Cadilla), and Sputnik V (Gamaleya), or vaccines in development such as INO-8400 (Inovio), CVnCoV (CureVac), or NVX-CoV2373 (Novavax), that all contain and elicit an immune response against the Spike protein derived from, or homologous to, the original Wuhan strain.

There is thus a need to identify conserved Sarbecovirus antigens for inclusion in pan-Sarbecovirus vaccines that provide protection, or help to protect, against present and future Sarbecoviruses.

BRIEF DESCRIPTION OF THE INVENTION

The invention is based on the identification of conserved T cell antigens in structural and non-structural proteins of coronavirus, especially of the genus betacoronavirus, more preferably of the subgenus Sarbecovirus that includes human SARS-CoV and SARS-CoV-2. Said conserved T cell antigens, derived from conserved coronavirus protein domains, may provide an immune response that provides broad protection against SARS-like coronaviruses. A T cell response induced by a conserved T cell antigen according to the invention may curtail coronaviral replication and spreading in and between individuals and decrease disease severity to alleviate the need for hospitalization. By directing a T cell- mediated immune response to conserved protein domains that are present in multiple coronaviruses, and that experience relatively low mutation rates, a vaccine based on such conserved T cell antigen is expected to be useful for both circulating as well as future coronavirus variants. Furthermore, as the domains are selected for the presence of multiple T cell epitopes binding a diverse set of MHC alleles, such a vaccine would be useful for the large majority of individuals. Finally, as the vaccine would contain non-functional fragments of viral genes, toxicity due to unwanted activity of these genes is prevented. In short, this invention allows the generation of safe pan-coronavirus vaccines with broad population coverage.

The invention therefore provides a protein comprising a T cell polyantigen, said polyantigen comprising 2-100 individual, conserved coronavirus T cell antigens of 8-80 amino acid residues, which individual T cell antigens may be alternated by spacer sequences of, preferably, 1-10 amino acid residues, or comprising a combination of 2-100 individual T cell antigens. Said protein preferably comprises at least 5 individual T cell antigens, preferably 5-35 individual T cell antigens. Said 2-100 T cell antigens preferably comprise at least one T cell antigen derived from the conserved coronavirus protein domains as depicted in Table 1.

The invention further provides a nucleic acid molecule, such as an RNA or DNA molecule, encoding a protein of the invention, said nucleic acid molecule preferably further capable of expressing said protein in a suitable cell. Said nucleic acid molecule can be a circular or linear nucleic acid molecule, preferably a circular DNA molecule or a linear RNA molecule.

The invention further provides a pharmaceutical composition, comprising a protein of the invention, a nucleic acid molecule of the invention, or a combination thereof, and a pharmaceutically acceptable excipient. Said pharmaceutical composition preferably further comprises an immune stimulating molecule.

The invention further provides a pharmaceutical composition comprising at least one conserved coronavirus T cell antigen of between 8-80 amino acid residues, preferably at least one T cell antigen derived from the conserved coronavirus protein domain of Table 1, and an immune stimulating molecule. Said immune stimulating molecule preferably is an adjuvant and/or a cytokine.

The invention further provides a method of inducing an immune response in an individual at risk of suffering from a coronavirus infection, or already suffering from a coronavirus infection, said method comprising providing said individual with a pharmaceutical composition of the invention. Said method will help to amplify and accelerate the generation of new or further immune responses to other antigens of a vaccine containing at least part of a sarbecovirus Spike antigen or to a subsequently infecting coronavirus.

The invention further provides a T-cell receptor (TCR) that specifically recognizes an epitope within a conserved coronavirus T cell antigen, preferably wherein the TCR is expressed by a T-cell. Said coronavirus T cell antigen preferably is selected from the conserved domains listed in Table 1. A preferred TCR of the invention is isolated from an individual who had been infected with a coronavirus.

The invention further provides a method of treating an individual suffering from a coronavirus infection, comprising providing said individual with a pharmaceutical composition of the invention, a TCR of the invention, or a combination thereof.

The invention further provides a pharmaceutical composition of the invention, a TCR of the invention, or a combination thereof, for use in a method of inducing an immune response in an individual. Said induction of an immune response may be prophylactic, or therapeutic.

FIGURE LEGENDS

Figure 1. Sequences (A) and degree of conservation of selected coronavirus protein domains (B). Location of the conserved coronavirus protein domains is relative to the NCBI Genbank reference sequence NC_045512.2 (SEQ ID NO:39) for severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome. Conservation of the domains is depicted as percentage identity over several subgeni of betacoronaviruses and other coronavirus geni.

Figure 2. Simplified scheme depicting a T cell polyantigen design strategy for a DNA plasmid, which may be used in a pharmaceutical composition, or as template for mRNA- or protein-based pharmaceutical compositions.

Figure 3. Vaccine-induced T cell immune responses to an S-derived epitope (S525: VNFNFNGL; SEQ ID NO:48), an ORFlab epitope (TGYHFREL; (SEQ ID NO:49) and an M epitope (RTLSYYKL; (SEQ ID NO:50) measured in blood using tetramers (A, C) and in spleen by intracellular cytokine staining (ICS) (B,D). Figure 4. DNA vaccines encoding conserved corona T cell antigens induce strong T cell responses (A) and provide protection against a lethal SARS-CoV-2 challenge in hACE2tg mice (C). Control groups in the challenge experiment, were either non-vaccinated (- mock) or vaccinated with a Spike DNA vaccine that induced high neutralizing antibody titers (B).

Figure 5. Vaccination of hACE2tg mice with a single peptide comprising the S-derived epitope VNFNFNGL (S525) protects against a sublethal SARS-CoV-2 challenge. Mice that were vaccinated with the peptide developed strong, S525- specific, T cell responses (A). Five weeks after final vaccination, mice were challenged intranasally with control (mock) or the Eeiden-0008/2020 SARS-CoV-2 isolate (S525 SLP/CpG) (B).

Figure 6. CD4 T cell responses to shared sarbecovirus Spike antigens improve Spike-specific B and CD8 T cell responses. Three weeks after the final vaccination with CoVAX_MNS_norep, mice were injected with 50 ug DNA encoding full-length Spike protein from SARS-CoV (SARS-CoV-1), SARS-CoV-2 Wuhan (W) or SARS-CoV-2 omicron BA.1 variant (O). Eight days after exposure to these Spike proteins, blood samples were analysed for CD8 T cell responses to the H-2K^b- restricted Spike reporter epitope VNFNFNGL (absent from the vaccines but present in all three Spike proteins) (A) as well as IgG1 (B) and IgG2c (C) antibody responses to the different Spike proteins.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “coronavirus”, as is used herein, refers to a subfamily of enveloped, positive-sense single-stranded RNA viruses, termed orthocoronavirinae, that are known to cause infection of many different hosts species, including humans, with different degrees of pathologies. The orthocoronavirinae can be further subdivided in alpha, beta, gamma, and delta coronavirus genera. Of these, the betacoronaviruses include several strains that are highly pathogenic in humans such as Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), and Middle East Respiratory Syndrome coronavirus (MERS-CoV). The viral envelope of coronaviruses is made up of a lipid bilayer in which the membrane (M), envelope (E), and spike (S) structural proteins are anchored. A fourth structural protein is the nucleocapsid (N) protein, which packages the viral genome into a helical ribonucleocapsid. In addition to the aforementioned structural proteins, the coronavirus genome contains several non-structural proteins, encoded in the 5’ region of the viral genome, termed ORF1ab, and a number of accessory factors encoded by the 3’ region of the viral genome. A preferred coronavirus is of the genus betacoronavirus, more preferably of the subgenus Sarbecovirus that includes human SARS-CoV and SARS-CoV-2, and related viruses in other species.

The term “individual”, as is used herein, refers to a bird, a fish, or a mammal such as a bat, a civet, a pangolin a camelid, a farm animal such as a cow, a horse and a pig, a pet such as a dog and a cat, and a primate including monkey and human.

The term “T-cell mediated immune response”, as is used herein, refers to protective mechanisms that are responsible for detecting and destroying intracellular pathogens, e.g., by killing cells that are infected with viruses or bacteria. Key players are CD4+ and CD8+ T cells, which produce inflammatory cytokines such as Interferon gamma (IFN-y), and Tumor Necrosis Factor (TNF), and various interleukins that play a central role in the immune reaction to pathogens. CD8+ T cells, often called cytotoxic T lymphocytes or CTLs, in particular have the ability to directly kill infected cells. CD4+ T cells, often called T helper cells, play a key role in adaptive immune responses. For instance, T helper cells provide signals to activate CTLs to kill infected target cells, and they help activate B cells to mature and secrete antibodies. Individual CD4+ and CD8+ T cells each carry a unique T cell receptor that recognizes a specific peptide complexed to Major Histocompatibility Complex (MHC) on the target cell. As a result, infection with a pathogen leads to selective activation and expansion of T cells recognizing specific, pathogen-derived peptides presented by MHC on the infected cell. Once the infection is cleared, pathogen -specific memory T cells remain and provide protection against reinfections with that pathogen.

The term “Major Histocompatibility Complex, or MHC”, as is used herein, refers to a heterodimeric complex of proteins that display peptides as immunogenic epitopes on the surface of cells. There are two types of MHC complexes: class I, expressed on all nucleated cells, and class II, expressed by specialized antigen- presenting cells (APCs) and B cells. MHC class I tends to contain peptides derived from cytosolic proteins, and MHC class II tends to contain peptides derived from endolysosomal proteins. Generally speaking, MHC class I molecules present epitopes to CD8+ T cells, while MHC class II molecules present epitopes to CD4+ T cells.

The term “T cell receptor, or TCR”, as is used herein, refers to a heterodimeric protein complex that is present on the surface of T cells. In most T cells, TCRs are composed of αβ subunits displaying immunoglobulin-like variable domains that recognize peptide antigens associated with major histocompatibility complex (MHC) molecules expressed on the surface of antigen-presenting cells or target cells. Some T cells express subunits. TCRs are associated with a CD3 complex formed by γ, δ, ε, and ζ subunits, which together mediate signal transduction.

The term “cluster of differentiation 3, or CD3”, as is used herein, refers to a protein complex that is composed of four distinct chains that associate with a T-cell receptor (TCR). A TCR that is complexed with CD3-zeta, and the other CD3 molecules is termed a TCR complex.

The term “conserved coronavirus protein domain”, as is used herein, refers to a domain on a structural or non-structural protein encoded by a coronavirus that is conserved between different coronaviruses, preferably between betacoronaviruses such as between Sarbeco viruses. The term “domain” refers to a continuous stretch of amino acid residues of between 8 and 500 amino acid residues. The term “conserved” indicates that said domain is at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 99% identical between different coronaviruses, preferably between betacoronaviruses such as between Sarbeco viruses. A conserved coronavirus protein domain may comprise at least one conserved T cell antigen comprising one or more conserved T cell epitopes. A conserved coronavirus protein domain may comprise more than one conserved T cell antigens, each comprising a different conserved T cell epitope.

The term “conserved coronavirus T cell antigen”, or abbreviated T cell antigen, as is used herein, refers to a region within a conserved coronavirus protein domain that may comprise one or more T cell epitopes that are conserved between different coronaviruses, preferably between betacoronaviruses such as between Sarbecoviruses. The term “antigen” refers to a continuous stretch of amino acid residues of between 8 and 80 amino acid residues, preferably more than 18 amino acid residues, such as 19-80 amino acid residues. The term “conserved” indicates that said domain is at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 99% identical between different coronaviruses, preferably between betacoronaviruses such as between Sarbecoviruses.

The term “T cell epitope”, as is used herein, refers to a peptide that can be specifically recognized by T cells as part of the adaptive immune system. A T cell epitope is a sequence within a T cell antigen that is bound, or can be bound, by MHC molecules at a cell surface and which, when displayed by a MHC, can be recognized by a T-cell receptor. A T cell epitope preferably comprises 7-25 amino acid residues, preferably 8-20 amino acid residues. The term “T cell epitope”, as used herein, includes reference to sequence variants of a specific T cell epitope with similar, or preferably, enhanced binding affinity to MHC molecules, and improved recognition by an epitope-specific T cell receptor. In particular, said sequence variants are heteroclitic peptides that stimulate stronger T cell responses than the native epitope (Solinger et al., 1979. J Exp Med 150: 830-48; Zirlic et al., 2006. Blood 108: 3865-3870).

The term ‘heteroclitic peptide”, as is used herein, refers to a peptide encompassing a T cell epitope in which amino acid residues at an anchor position for a specific MHC molecule has been altered to enhance the stability of the binding to the peptide to said MHC molecule and/or to increase the immunogenicity of the bound peptide. As is known to a person skilled in the art, structure modelling and molecular docking models may be used to study the impact of one or more amino acid substitutions on the structure of a heteroclitic peptide as well as the interaction with a MHC molecule. Preferred alterations for a human T cell epitope include conservative amino acid substitutions of MHC-binding residues. Said amino acids preferably include one or more of amino acid positions 1, 2, 9 and/or 10. Said substitutions preferably are with lysine at position 1, leucine at position 2, and valine at position 9 or 10. The stability of binding of the peptide to said MHC molecule can be predicted by using an computer algorithm, as indicated herein below. The term “T cell polyantigen”, as is used herein, refers to a protein that encompasses two or more T cell antigens. Said two or more T cell antigens are non- consecutive, meaning that the two or more T cell antigens do not constitute a naturally occurring amino acid sequence. A preferred polyantigen comprises 2-100, preferably 5-35 individual antigens. The individual antigens in a polyantigen may be alternated by spacer sequences of, preferably, 1-10 amino acid residues. The term “T cell polyantigen” includes reference to a mixture of two or more individual T cell antigens, such as at least 5 individual T cell antigens, at least 10 individual T cell antigens, at least 17 individual T cell antigens, such as 18 individual T cell antigens, 19 individual T cell antigens, and 20 individual T cell antigens.

The term "nucleic acid", as is used herein, refers to one or more single- or double-stranded polynucleotide sequences consisting of ribonucleotides, deoxyribonucleotides, or known analogs of natural nucleotides that can function in a similar manner. It is understood that a particular polynucleotide sequence can be codon-optimized to increase stability and/or expression of the encoded polypeptide without altering its primary amino acid sequence. Nucleic acids, as used herein include both circular and linear polynucleotides, in an isolated form, as combinations, or as part of for instance a viral or bacterial vector, or as part of a synthetic delivery vehicle such as a liposome or lipoplex.

The term “immune stimulating molecule”, as is used herein, refers to a molecule that enhances adaptive immune responses, for example by facilitating the recruitment, activation or maturation of antigen presenting cells (APCs), increasing antigen uptake by (APCs), induce local inflammation, and/or helping to activate CD4 “helper” or “killer” CD8 T cells, or by stimulating the activity of innate immune cells like dendritic cells. Examples of such immune stimulating molecules include one or more adjuvants, cytokines such as interleukins, tumor necrosis factors, chemokines, and interferons (e.g. IL-1, IL-12, IL-15, IL-18, IL-23, OX40-L, CD40L, GM-CSF, CCL3, XCL1, CXCL10, CCL21, and interferon-γ), molecules that induce the production of immunoregulatory cytokines, microparticles, saponins, microbial components/products, liposomes, DAMPs (damage-associated molecular patterns) or PAMPs (Pathogen-associated molecular patterns) such as Toll-like receptor ligands (e.g. LPS, poly-IC, CpG oligodeoxynucleotides, flagellin, imiquimod, resiquimod), DAMP-inducing adjuvants, muramyl dipeptide, which stimulates an intracellular pattern recognition receptor called NOD2, and Stimulator of interferon genes (STING), a protein that induces type I interferon production when cells are infected with intracellular pathogens. Said immune stimulating molecule may further include a molecule such as a constitutively active tolldike receptor 4, a membrane interleukin-2 (memIL2), memIL-12, and memIL-15, and self-oligomerizing, constitutively active CD40. It is to be understood that some immune stimulating molecules, such as for example cytokines, may be encoded by one or more nucleic acids, and that these one or more nucleic acids may be co-administered together with a T cell polyantigen or TCR, or transcriptionally or translationally coupled by fusing their coding sequences, as is known in the art.

The term “adjuvant”, as is used herein, refers to a molecule that enhances the immune response to an antigen. Examples of adjuvants known in the art include aluminum salts, monophosphoryl lipid A in combination with an aluminum salt (AS04), an oil in water emulsion composed of squalene (MF59), monophosphoryl lipid A and QS-21, a natural compound extracted from the Chilean soapbark tree, combined in a liposomal formulation (AS01B), and an oil-in-water adjuvant emulsion that contains alpha-tocopherol, squalene, and polysorbate 80 (AS03).

The term “cytokine”, as is used herein, refers to peptides that play a role as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. They act through cell surface receptors and are especially important in the immune system.

The term “combination”, as is used herein, refers to the administration of effective amounts of a T cell antigen as defined herein, preferably as a polyantigen, or as a nucleic acid molecule, and an immune stimulating molecule such as a cytokine, to a patient in need thereof. Said polyantigen or nucleic acid molecule, in combination with an immune stimulating molecule may be provided in one pharmaceutical preparation, or as two distinct pharmaceutical preparations. Said combination is preferably administered at a certain dose, depending on an individual’s condition (age, weight, treatment history, etc.), which can be determined by a skilled physician, veterinarian, or other person skilled in the art.

The term “induction of an immune response”, as is used herein, refers to prophylactic indication, meaning that a T cell antigen, polyantigen and/or nucleic acid-encoded antigen, may be administered to an individual after, prior to, or concurrent with the administration of an immune stimulating molecule, but prior to infection of the individual with a coronavirus, as well as therapeutic induction, meaning that a T cell antigen, polyantigen and/or nucleic acid-encoded antigen and an immune stimulating molecule, may be administered to an individual suffering from a coronavirus infection.

The term “or”, as used herein is defined as “and/or” unless specified otherwise.

The term “a” or “an” as used herein is defined as “at least one” unless specified otherwise. When referring to a noun in the singular, the plural is meant to be included, unless it follows from the context that it should refer to the singular only. For example, the term “nucleic acid molecule”, as is used herein, includes reference to a collection of two or more nucleic acid molecules that, together, encode a protein of the invention.

Conserved T cell antigens

The invention is based on the identification of conserved protein domains that are present in coronaviruses, especially in Sarbecoviruses. As is indicated herein above, structural coronavirus proteins membrane (M), envelope (E), nucleocapsid (N) and spike (S), and non-structural coronavirus proteins ORF1ab and/or accessory factors encoded by the 3’ region of the viral genome, comprise domains that are conserved between different coronaviruses, preferably between different betacoronaviruses such as between different Sarbecoviruses. Each of said one or more conserved domains may encompass one or more conserved T cell antigens which, in turn, may encompass one or more T cell epitopes. Said T cell epitopes may be recognized by a T cell receptor (TCR) when they are presented by MHC molecules on the surface of a cell. A T cell epitope that is presented by an MHC I molecule on the surface of a cell will be recognized by a TCR expressed by a cytotoxic CD8+ T cell, resulting in killing of said cell.

The choice of a peptide as a T cell antigen comprises prediction of peptide processing, transfer and binding of an encompassed T cell epitope to an MHC molecule resulting in display of the MHC-bound T cell epitope on the cell surface. Class I and II major histocompatibility complex (MHC) molecules can bind short peptides derived from endogenous or exogenous antigens and present these on the surface of a cell. Upon recognition of MHC-peptide complexes by TCRs, cell- mediated immune responses can be induced that play key roles in immunological processes such as cancer, infectious and autoimmune diseases. An individual expresses multiple, highly polymorphic MHC class I and class II genes. The encoded proteins differ in their peptide-binding specificities, as well as their frequency and distribution between species, ethnicities and regions. Therefore, design of an optimal T cell antigen-based vaccine ideally should account for broad coverage of different MHC molecules to provide wide protection in different populations.

In general, binding of peptides to MHC class I molecules is restricted to peptides typically 8-11 amino acids long due to a closed binding groove that sterically limits the length of a bound peptide. In addition, some amino acid residues within the peptide are important for binding or anchoring of the peptide in the MHC I groove, where the identity and position of said residues may differ between MHC I alleles. For instance, for human alleles of MHC I, it was determined that position 2 and position 9 often represent anchor residues, whereas the murine MHC class I molecule H-2K^b contains a central, deep pocket that accommodates specific anchor residues at position 5.

Methods for predicting epitope binding to MHC I are known in the art. For example, an immunogenicity model by Calis et al., 2013 (Calis et al., 2013. PloS Comput Biol 9: 1003266) can predict binding and immunogenicity of any peptide- MHC I molecule complex. The model is based on information that positions 4-6 of a presented peptide are more important for immunogenicity, in combination with a preference for aromatic and large amino acids in immunogenic peptides. A scoring model was created which scores peptides based on the ratio of an amino acid between a non-immunogenic and immunogenic dataset.

Further methods include PAAQD (Saethang et al., 2013. J Immunol Methods 387: 293-302; NetMHCstab and NetMHCstabpan (Jorgensen et al., 2014. Immunology 141: 18-26; Rasmussen et al., 2016. J Immunol 197: 1517-24); NetTepi (Trolle and Nielsen, 2014. Immunogenetics 66: 449-56); NetMHCcons (Karosiene et al., 2012. Immunogenetics 64: 177-86); NetTCR (21). NetTCR (Jurtz et al., 2018. bioRxiv doi: 10.1101/433706) and NetMHC4.0 (Nielsen et al., 2003. Protein Sci 12: 1007-17; Andreatta and Nielsen, 2016. Bioinformatics 32: 511-7). In addition, several databases are available, including SYFPEITHI (Rammensee et al., 1999. Immunogenetics 50: 213-9); IEDB (Fieri et al., 2017. Front Immunol: doi: 10.3389/fimmu.2017.00278); VDJdb (Bagaev et al., 2020. Nucleic Acids Res 48: D 1057-62); McPAS-TCR (Tickotsky et al., 2017. Bioinformatics 33: 2924-9); ATLAS (Borrman et al., 2017. Struct Funct Bioinf 85: 908-16); and STCRDab (Leem et al., 2018. Nucleic Acids Res 46: D406-12) which may improve the prediction power of these or other models.

In contrast to class I MHC, class II MHC proteins have an open-ended binding groove which can accommodate longer peptides, usually 13-25 amino acids long. Moreover, a class II MHC can accommodate a more heterogeneous set of peptides, when compared to a class I MHC molecule. Methods for predicting MHC II antigens are known in the art. Examples of such programs include NN align (Alvarez et al., 2019. bioRxiv 550673; Reynisson et al., 2019. bioRxiv 799882); ARB matrix (Bui et al., 2005. Immunogenetics 57: 304-314; SMM-align (Nielsen et al., 2007. BMC Bioinformatics 8: 238); and the Immune Epitope Database (IEDB) (available at iedb.org/).

The conserved domains, as identified in both structural and non-structural proteins of coronaviruses, especially Sarbecoviruses, were selected for the presence of predicted T cell epitopes. To this end, a total of 17 Sarbecovirus sequences from NCBI GenBank (see NC_045512, MN988713.1, MN985325.1, MN938384.1, MN975262.1, MG772933, MG772934, DQ412043.1, KY417144.1, AY278489.2, AY278488.2, FJ882957.1, AY572034.1, AY274119, DQ071615, DQ022305, GU 190215) were selected that together represented a broad diversity within the the Sarbecovirus subgenus, including viruses isolated from SARS patients, bats, civet cats, and SARS-CoV-2 patients. From these, translated ORFlab, M, N and S sequences were aligned, revealing conserved stretches of amino acid residues within Sarbecoviruses. Using the above-mentioned publicly available prediction tools for MHC-peptide binding, conserved sequences were examined for potential binding to MHC class I alleles HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA- A*11:01, HLA-A*23:01, HLA-A*24:02, HLA-B*07:02, HLA-B*08:01, HLA-B*35:01, HLA-B*40:01, HLA-B*44:02, HLA-B*44:03, which together cover more than 85% of the global human population. Sarbecovirus. A list of betacoronavirus protein domains that are conserved within the Sarbecovirus family and that are rich in predicted HLA class I epitopes could be identified (Table 1).

The HLA (human MHC) locus contains three classical HLA class I genes (HLA-A, HLA-B, HLA-C) and up to ten HLA class II genes (HLA-DRA, HLA-DRB1- 5, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1), together encoding six HLA proteins: HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ. As these genes are highly polymorphic, heterozygous individuals can express more than twelve different HLA proteins, each with its specific peptide binding preference. Despite this high degree of diversity, most HLA molecules can be clustered into groups, designated as HLA supertypes, based on common or overlapping peptide-binding specificities (Doytchinova et al., 2004. J Immunol 172: 4314-23; Lund et al., 2004. Immunogenetics 55: 797-810; Doytchinova et al., 2005. J Immunol 174: 7085-95; Sidney et al., 2008. BMC Immunol 9: 1). The conserved domains depicted in Table 1 are predicted to encompass multiple epitopes that bind to HLA-A*01:01, HLA- A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02, HLA-B*07:02, HLA-B*08:01, HLA-B*35:01, HLA-B*40:01, HLA-B*44:02, or HLA-B*44:03, together covering more than 85% of the global human population. As said HLA alleles are representative of different HLA supertypes, and since HLA alleles not comprised within these supertypes are likely to bind additional epitopes present in the domains, the selected betacoronavirus protein domains will contain epitopes binding a much larger set of HLA alleles, together covering considerably more than 85% of the global human population. A composition comprising conserved coronavirus T cell antigens with a multitude of epitopes that can be bound by different HLA alleles should therefore be able to provide broad protection against coronaviruses within and across different human populations.

T cell polyantigen

The invention provides a protein comprising a T cell polyantigen comprising 2-100, preferably 5-35, conserved coronavirus T cell antigens. At least one of said conserved coronavirus T cell antigens preferably is selected from the conserved betacoronavirus protein domains depicted in Table 1, preferably at least two of said conserved coronavirus T cell antigens. Said T cell polyantigen can be used to stimulate an immune response in an individual, such as an individual that is suffering from a coronavirus or who may become infected with a coronavirus.

A T cell polyantigen according to the invention preferably comprises 2-100, preferably 5-35 conserved T cell antigens. Said conserved T cell antigens may be present in a single protein, as individual peptides, or a combination thereof, e.g. a T cell polyantigen may comprise a combination of a protein comprising a multitude of said conserved T cell antigens and individual peptides comprising the remaining T cell antigens.

Said T cell antigens preferably are selected from structural proteins, such as membrane protein, envelope protein, nucleocapsid and spike protein, and/or from non-structural proteins such as ORFlab proteins. A preferred T cell polyantigen according to the invention comprises at least 4 T cell antigens, and includes T cell antigens from membrane protein (M), nucleocapsid protein (N), spike protein (S) and/or ORFlab protein.

Said individual T cell antigens preferably are each contained within a sequence of 8-80 amino acid residues, preferably within the conserved domains of 14-72 amino acid residues depicted in Table 1. Said individual T cell antigens may be alternated by spacer sequences, preferably of 1-10 amino acid residues. Preferred T cell antigens in a T cell polyantigen are selected from the conserved betacoronavirus protein domains depicted in Table 1.

A preferred T cell polyantigen comprises at least 3 T cell antigens, such as at least 6 T cell antigens, at least 12 T cell antigens, or at least 24 T cell antigens selected from the conserved betacoronavirus protein domains depicted in Table 1. Each of said T cell antigens may be uniquely present in a T cell polyantigen, or duplicated within said T cell polyantigen.

When working with intact viral proteins or nucleic acid sequences there is a potential risk of eliciting unwanted, possibly pathogenic processes in situ caused by the viral protein or nucleic acid sequences. To minimize this risk, T cell polyantigens according to the invention are preferably constructed by rearranging or ‘shuffling’ T cell antigens out of their natural order or context, alternating T cell antigens derived from different structural or non-structural viral proteins, or optionally intervening T cell antigens by non-natural spacer sequences. Similar approaches have been shown to be effective in vaccines against the HPV oncogenic E7 protein (Osen et al., 2001. Vaccine 19: 4276-86; Oosterhuis et al., 2011. Int J Cancer 129: 397-406). By rearranging conserved T cell antigens in a T cell polyantigen or using relatively short, isolated T cell antigens out of their natural protein context, all possible T cell epitopes within the conserved antigens are maintained while minimizing the risk of any functional activity of the viral protein from which the antigens were derived.

A T cell polyantigen may further comprise at least one universal helper epitope that is able to activate CD4+ T cells. Thus, activated CD4+ T cells may provide helper functions to promote activation of CD8+ T cells and B cells. Said at least one universal helper epitope may, for example, include a Tetanus and/or Diphtheria Toxin (Brenda et al., 2000. J Infectious Diseases 181: 1001-1009), an CD4+ helper epitope from Aquifex aeolicus (Xu et al., 2020. iScience 23: 101399), or one or more dominant CD4 helper epitopes from other viruses such as herpes- or retroviruses, or artificial, universal T helper epitope such as the Pan HLA-DR reactive epitope (PADRE; Alexander et al., 1994. Immunity 1: 751-761). The addition of at least one universal helper epitope may enhance immunogenic responses induced by vaccination with a T cell polyantigen of the invention.

A preferred T cell polyantigen according to the invention comprises two or more T cell antigens that are contained within the conserved domains as depicted in Table 1. Said T cell polyantigen preferably includes at least five of said T cell antigens, more preferred at least 10 of said T cell antigens, more preferred at least twenty of said T cell antigens, more preferred all T cell antigens that are contained within the conserved domains as depicted in Table 1.

A T cell polyantigen according to the invention may further comprise one or more marker antigens and/or specific tags. Marker antigens are specific antigens not present in coronavirus that can be included to differentiate between vaccine- induced immunity and infection. In veterinary medicine, such vaccine is termed a Differentiation of Infected from Vaccinated Animals or DIVA vaccine. Suitable tags include but are not limited to c-myc domain (EQKLISEEDL; (SEQ ID NO:51), hemagglutinin tag (YPYDVPDYA; (SEQ ID NO:52), antibody Fc domains, maltose- binding protein, glutathione-S-transferase, FLAG tag peptide (DYKDDDDK; (SEQ ID NO:53), biotin acceptor peptide, streptavidin-binding peptide and calmodulin- binding peptide, as presented in Chatterjee, 2006 (Chatterjee, 2006. Cur Opin Biotech 17, 353-358). In addition, a T cell polyantigen may comprise more than one tag and/or marker antigen, such as two or three different tags and/or marker antigens, and/or multiple copies of a single tag or marker antigen.

A T cell polyantigen according to the invention may be provided to an individual in need thereof as a peptide or combination of peptides, as a nucleic acid molecule, or through a delivery vector including a viral vector and a bacterial vector, and/or as a cell-based vaccine.

Products of the invention

Methods to generate said T cell antigen or T cell polyantigen as a protein, peptide or combination of peptides, include in vitro synthesis and recombinant production. A T cell antigen or polyantigen according to the invention may be synthesized by commonly used solid-phase synthesis methods, e.g. methods that involve t-BOC or FMOC protection of alpha-amino groups which are well known in the art. Here, amino acids are sequentially added to a growing chain of amino acids. Such methods are for instance described in Merrifield, 1963 (Merrifield, 1963. J Am Chem Soc 85: 2149-2156) and in Atherton et al., 1989 ("Solid Phase Peptide Synthesis," IRL Press, London). Solid-phase synthesis methods are particularly suitable for synthesis of peptides of up to 100-150 amino acid residues.

As is shown in Example 5, a single, semi-conserved T cell antigen already provides protection to mice against a later challenge with a coronavirus. It is to be expected that a selected, conserved T cell antigen that is present in the conserved domains depicted in Table 1, maybe sufficient to provide protection to human.

A T cell poly antigen according to the invention is preferably produced in an expression system. Commonly used expression systems for protein production include E. coli, Bacillus spp., baculovirus, yeast, fungi, most preferably filamentous fungi or yeasts such as Saccharomyces cerevisiae and Pichia pastoris, eukaryotic cells such as Chinese Hamster Ovary cells (CHO), human embryonic kidney (HEK) cells and PER.C6® cells (Thermo Fisher Scientific, MA, USA), and plants. The efficiency of expression of recombinant proteins depends on many factors, both on the transcriptional level and the translational level.

Said T cell polyantigen is preferably produced by expression cloning of the protein or proteins in a prokaryotic cell, such as E. coli. For this, an expression construct, preferably DNA, is preferably produced by recombinant technologies, including the use of polymerases, restriction enzymes, and ligases, as is known to a skilled person. Alternatively, said expression construct is provided by artificial gene synthesis, for example by synthesis of partially or completely overlapping oligonucleotides, or by a combination of organic chemistry and recombinant technologies, as is known to the skilled person.

Said expression construct is preferably codon-optimised to enhance expression of the T cell polyantigen in a cell of interest. Further optimization may include the removal of cryptic splice sites, removal of cryptic polyA tails and/or removal of sequences that may lead to unfavorable folding of the mRNA. In addition, the expression construct may encode a protein export signal for secretion of the T cell polyantigen out of the cell, for instance into the periplasm of prokaryotes, allowing efficient purification of the T cell polyantigen.

Methods for purification of the T cell polyantigen are known in the art and are generally based on chromatography such as affinity chromatography and ion exchange chromatography, to remove contaminants. In addition to contaminants, it may also be necessary to remove undesirable derivatives of the product itself such as degradation products and aggregates. Suitable purification process steps are provided in Berthold and Walter, 1994 (Berthold and Walter, 1994. Biologicals 22: 135- 150).

As an alternative, or in addition, a T cell polyantigen may be tagged with one or more specific tags by genetic engineering to allow attachment of the protein to a column that is specific to the tag and therefore be isolated from impurities. The purified protein is then exchanged from the affinity column with a decoupling reagent. The method has been routinely applied for purifying recombinant protein. Conventional tags for proteins, such as histidine tag, are used with an affinity column that specifically captures the tag (e.g., a Ni-NTA or Ni-IDA column for the histidine tag) to isolate the protein from other impurities. The protein is then exchanged from the column using a decoupling reagent according to the specific tag (e.g., imidazole for histidine tag). This method is more specific, when compared with traditional purification methods.

A T cell antigen and/or T cell polyantigen according to the invention may be provided to an individual in need thereof as a pharmaceutical composition. Said pharmaceutical composition preferably further comprises a pharmaceutically acceptable excipient. Said T cell antigen and/or T cell polyantigen according to the invention may be modified, for example by PEGylation, to increase stability. As an alternative, or in addition, said T cell antigen and/or T cell polyantigen according to the invention may be provided as a nanoemulsion, or encapsidated in liposomes.

Said pharmaceutically acceptable excipient preferably is selected from diluents, binders or granulating ingredients, a carbohydrate such as starch, a starch derivative such as starch acetate and/or maltodextrin, a polyol such as xylitol, sorbitol and/or mannitol, lactose such as u-lactose monohydrate, anhydrous udactose, anhydrous β-lactose, spray-dried lactose, and/or agglomerated lactose, sugars such as dextrose, maltose, dextrate and/or inulin, glidants (flow aids) and lubricants, and combinations thereof.

A pharmaceutical composition comprising a T cell antigen and/or T cell polyantigen according to the invention preferably comprises an excipient to maintain protein stability, solubility, and pharmaceutical acceptance. Said excipient preferably is selected from, but not limited to, urea, L-histidine, L- threonine, L-asparagine, L-serine, L-glutamine, polysorbate, polyethylene glycol, propylene glycol, polypropylene glycol, or a combination of two or more of the above. Salts and buffers are known to effect protein stability, especially in frozen solutions and freeze-dried solids because of the increased concentrations and possible pH changes. In addition, various sugars may protect the conformation of proteins in aqueous solutions and during freeze-drying. For example, nonreducing disaccharides such as sucrose and trehalose are potent and useful excipients to protect protein conformation in aqueous solutions and freeze-dried solids, whereas reducing sugars such as maltose and lactose can degrade proteins during storage. Said excipients may further include sugar alcohols such as inositol, and/or amino acids such as arginine may protect protein conformation against dehydration stresses. Further excipients may include a surfactant, such as a nonionic surfactant, and/or a polymer such as hydroxyethyl starch.

Said pharmaceutical composition comprising a T cell antigen or T cell polyantigen according to the invention preferably is a sterile isotonic solution. Said sterile isotonic solution preferably is provided to an individual in need thereof by injection or infusion. Preferred routes include intranasal, intradermal, intramuscular, intravenous and/or subcutaneous delivery.

Means for delivery of a T cell antigen and/or polyantigen

A T cell antigen and/or polyantigen according to the invention may be provided to an individual in need thereof as a pharmaceutical composition comprising a protein, peptide or combination of peptides, or combination of protein and peptide or peptides, as is indicated herein above. In addition, a T cell polyantigen according to the invention may be provided to an individual in need thereof as a nucleic acid molecule or through a delivery vector including a viral vector and a bacterial vector, and/or as a cell-based vaccine.

The invention therefore provides a nucleic acid molecule encoding a T cell polyantigen according to the invention. Said nucleic acid molecule preferably is a RNA molecule, preferably a mRNA molecule, or a DNA molecule. Said nucleic acid molecule preferably directs expression of said polyantigen upon delivery to a suitable cell.

A nucleic acid molecule according to the invention preferably is provided as an expression construct that expresses said nucleic acid molecule in a cell of interest. Said expression construct may be chosen from a plasmid and a viral vector.

Said vector preferably comprises a promoter for expression of the protein of interest in a suitable host cell. Said promoter may be a constitutive promoter or an inducible promoter, and may provide low, medium or high expression levels of the nucleic acid molecule.

Examples of suitable promoters include pol II promoters such as retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the b-acting promoter, the phospho- glycerol kinase (PGK) promoter, and the EFla promoter. As well as promoters, enhancer elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I; SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit b-globin, may be included in the expression constructs. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as a preference for a specific host, a host cell type, the desired level of expression etc. Said regulatory elements such as promoter sequences may be an autologous sequences, or heterologous sequences, i.e. derived from a non-human species.

A nucleic acid expression construct such as a plasmid, may be amplified in a suitable host such as, for example, a prokaryote such as E. coli. Methods for transforming a suitable host with an expression construct such as a plasmid are known in the art. Following transformation, a host comprising the expression construct can be grown where after the amplified expression construct such as a plasmid may be isolated and purified.

Said viral vector preferably is an adenovirus-based vector, an adeno- associated viral vector, a herpes simplex virus-based vector, a poxvirus-based vector, or a retroviral-based vector such as a lentivirus-based vector. Said viral vector most preferably is a recombinant vaccinia virus vector, such as Modified Vaccinia Virus Ankara (Antoine et al., 1998. Virology 244: 365-96), or a chimeric vector such as a vaccinia viral/retroviral chimeric vector (Falkner and Holzer, 2004. Curr Gene Ther 4: 417-26).

As an alternative, said nucleic acid molecule may be provided as such, either as a DNA molecule or as an RNA molecule. A preferred method for generating multiple copies of a DNA molecule encoding a T cell polyantigen according to the invention is by amplification in a bacterium such as Escherichia coli. For this, a suitable plasmid may be generated that comprises said DNA molecule. Said plasmid preferably encompasses an origin of replication, preferably a high copy number of replication. Methods for generating a suitable plasmid, amplification of the plasmid in a suitable bacterium such as Escherichia coli, and methods for purifying the amplified plasmid are well known in the art.

DNA molecules free of plasmid backbone or other bacterial DNA sequences may be generated in vitro by amplification. Said DNA molecules may express said T cell epitope polyantigen at high levels in vivo. Said in vitro, cell-free synthesized DNA molecules are either linear molecules, circular molecules, or a mixture thereof.

Suitable amplification methods include isothermal amplification methods such as rolling circle amplification (RCA), multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and nicking enzyme amplification reaction (NEAR).

A preferred amplification method for cell-free amplifying a template DNA molecule comprises amplification by a DNA-dependent DNA polymerase with strand-displacement activity. A preferred DNA polymerase is selected from Phi29, Bst, Vent DNA polymerase, or a combination or variants thereof.

A preferred amplification method for amplifying a template DNA molecule comprises priming by an RNA polymerase, preferably in the presence of deoxyribonucleotides and at least one ribonucleotide with a purine nucleobase, as described in WO 2020/218924, which is hereby incorporated by reference.

As is known to a person skilled in the art, cell-free amplified DNA can be distinguished from in vivo amplified DNA, for example by the absence of an origin of replication or traces thereof, and/or absence of DNA modification such as, for example, methylated nucleotides resulting from the E. coli Dam and/or Dem methylating enzymes.

As an alternative, a nucleic acid molecule encoding a T cell polyantigen according to the invention is provided to an individual as an RNA-based expression construct. Said RNA expression construct may either be generated, for example by expression of a nucleic acid expression construct such as a plasmid in a suitable host, followed by isolation and purification of transcribed RNA molecules comprising the T cell polyantigen; by in vitro transcription from a DNA molecule in which the T cell polyantigen according to the invention is provided under control of a suitable promoter, such as a SP6 promoter, a T3 promoter or a T7 promoter; or by chemical synthesis using, for example, the phosphoramidite method of Marvin H. Caruthers (Caruthers, 1985. Science 230: 281-285). Said chemical synthesis may employ phosphoramidites such as TheraPure phosphoramidites (Thermo Fisher Scientific, Waltham, Massachusetts).

RNA-based expression constructs include non-replicating, as well as replicating forms. Replicating forms include for instance self-amplifying or transamplifying RNA molecules, often based on the replication machinery of alphaviruses (Brito et al. 2015. Adv Genet 89: 179-233).

Said mRNA-based expression construct or RNA molecule may encompass, for example, a synthetic cap analogue (Stepinski et al., 2001. RNA 7: 1486-1495), one or more regulatory elements in the 5'-untranslated region (UTR) and/or the 3'-UTR that stabilize said RNA molecule and/or increases protein translation (Ross and Sullivan, 1985. Blood 66: 1149-1154), modified nucleosides to increase stability and/or translation (Kariko et al., 2008. Mol Ther 16: 1833-1840), and/or to decrease an inflammatory response (Kariko et al., 2005. Immunity 23: 165-175). In addition, said RNA molecule preferably encompasses a poly(A) tail to stabilize the RNA molecule and/or to increase protein translation (Gallie, 1991. Genes Dev 5: 2108- 2116).

A nucleic acid molecule may be delivered to an individual in the presence or absence of a carrier. Said carrier preferably allows prolonged expression in vivo of said T cell polyantigen. Said carrier may be one or more of a cationic protein such as protamine, a protamine liposome, a polysaccharide, a cation, a cationic polymer, a cationic lipid, an ionizable lipid, cholesterol, polyethylene glycol, and a dendrimer. For example, a RNA molecule may be delivered as a naked RNA molecule, complexed with protamine, associated with a positively charged oil-in- water cationic nanoemulsion, associated with a chemically modified dendrimer and complexed with polyethylene glycol (PEG)-lipid, complexed with protamine in a PEG-lipid nanoparticle, associated with a cationic polymer such as polyethylenimine, associated with a cationic polymer such as PEI and a lipid component, or associated with a polysaccharide such as, for example, chitosan, in a cationic lipid nanoparticle such as, for example, 1,2-dioleoyloxy-3- trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids), complexed with cationic lipids and cholesterol, and complexed with cationic lipids, cholesterol and PEG-lipid, as is described in Pardi et al., 2018. Nature Reviews 17: 261-279).

Said nucleic acid molecule that expresses the T cell polyantigen upon delivery of a nucleic acid molecule encoding said T cell polyantigen to a cell of interest may be administered by a parenteral route, including intranasal, subcutaneous, intradermal, intramuscular and intravenous administration, or an enteral route such as an oral, sublingual, or rectal, route. Methods to introduce a nucleic acid into a cell include lipofection, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA or RNA, artificial virions, and agent- enhanced uptake of DNA or RNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectamin™, and SAINT™). Cationic and neutral lipids that are suitable for efficient delivery of polynucleotides include those of WO 91/17424 and WO 91/16024. Delivery to a target tissue preferably is by systemic or, preferably, local administration to a region comprising cardiomyocytes.

A pharmaceutical composition comprising a naked nucleic acid molecule as an expression construct, such as a DNA or RNA expression molecule, preferably comprises excipients such Ringer’s solution and Ringer’s lactate, as is known to a person skilled in the art.

Said cell of interest may be any nucleated cell that is able to translate the nucleic acid molecule into a protein and present the encoded T cell epitopes complexed with MHC on its surface. Said cell of interest preferably is an antigen presenting cell (APC) which expresses both MHC class I and MHC class II molecules. These APC are important in initiating immune responses. As an alternative, or in addition, said cell of interest is an MHC class I expressing cell such as a dendritic cell, a mononuclear phagocyte, and a B cell, or any other cell that is capable of expressing a T cell antigen and cross-presenting said antigen to an APC.

Said cell of interest may be an autologous cell that has been isolated from an individual, provided with a nucleic acid molecule encoding a T cell antigen according to the invention, or polyantigen according to the invention, and returned back to the individual. For example, said nucleic acid molecule may be provided, in vitro, to a dendritic cell, which may have been extracted from the individual’s blood, transfected with the nucleic acid molecule, then returned to the patient to stimulate an immune reaction.

The invention further provides a T cell comprising a T cell Receptor (TCR) that is directed against a T cell epitope according to the invention. Said TCR preferably is an αβTCR. Methods to isolate T cells that bind to a T cell epitope according to the invention are known in the art. Said T cell may be a naturally occurring T cell that expresses a TCR that is directed against a T cell epitope according to the invention, for example that is isolated from an individual that had been infected with a coronavirus, or a T cell that is transduced with a TCR that is directed against a T cell epitope according to the invention. Methods of treatment

The invention further provides a method of inducing an immune response in an individual suffering from, or expected to suffer from, a coronavirus infection, said method comprising providing said individual with a T cell antigen T cell polyantigen according to the invention. Again, an individual in need thereof may be provided with a protein comprising the T cell antigen and/or T cell polyantigen according to the invention, with a nucleic acid molecule encoding the T cell polyantigen according to the invention, or through a delivery vector including a viral vector and a bacterial vector expressing the T cell polyantigen according to the invention, and/or as a cell-based vaccine.

A method of inducing an immune response preferably is performed prophylactically, prior to infection with a coronavirus. Said method of inducing an immune response in an individual may be combined with the provision of an immune stimulating molecule such as a cytokine, an adjuvant, or a combination thereof. It was found that the induction of an immune response in an individual not suffering from a coronavirus infection by a protein comprising a T cell polyantigen comprising conserved coronavirus T cell antigens according to the invention will amplify and accelerate the generation of new T cell- and antibody- mediated immune responses to other antigens of a vaccine containing at least part of a sarbecovirus Spike antigen or to a subsequently infecting coronavirus by said individual. Said conserved coronavirus T cell antigens, for example as present in ORF lab and MNS constructs, thus stimulate an immune reaction to other regions of coronavirus. Hence, a protein comprising a T cell polyantigen comprising conserved coronavirus T cell antigens may improve the immune responses against an incoming sarbecovirus. In addition, said T cell polyantigen may act synergistically with a subsequent antibody-inducing vaccine, for example against a specific coronavirus strain. Said protein comprising a T cell polyantigen comprising conserved coronavirus T cell antigens can be produced, tested for safety and immunogenicity in small phase I trials, and used as a first line of defense against a new pandemic coronavirus. The use of such protein comprising a T cell polyantigen comprising conserved coronavirus T cell antigens may allow sufficient time for the generation of an outbreak-specific vaccine or antibodies. The invention further provides a method of treating an individual suffering from a coronavirus infection, comprising providing said individual with a T cell antigen and/or T cell polyantigen according to the invention, either as such or in combination with an immune stimulating molecule such as an adjuvant, a cytokine, or a combination thereof. The induction of an immune response in the individual by providing said individual with a T cell antigen and/or T cell polyantigen according to the invention, either alone or in combination with an immune stimulating molecule such as an adjuvant, a cytokine, or a combination thereof, will contribute to the treatment of the individual, for example by killing coronavirus-infected cells.

Said individual expected to suffer, or suffering from a coronavirus infection, may further be provided with a cytokine such as interleukin-2 (IL- 2), IL- 12, CXCL9, or a combination thereof, whereby said cytokine may be administered prior to, simultaneously with, or following administration of a T cell polyantigen according to the invention, a nucleic acid molecule according to the invention, a T cell according to the invention, or a combination thereof.

As an alternative, or in addition, an individual in need thereof, expected to suffer, or suffering from a coronavirus infection, may be provided with a cell-based vaccine. Said cell-based vaccine is either a T cell according to the invention that expresses a T cell Receptor (TCR) that is directed against a T cell epitope according to the invention, and/or a cell such as a dendritic cell, that expresses one or more conserved coronavirus T cell epitopes according to the invention. Said cell-based vaccine may be provided in combination with an immune stimulating molecule such as an adjuvant, a cytokine, or a combination thereof.

The invention further provides a pharmaceutical composition, comprising a T cell antigen and/or T cell polyantigen according to the invention, a nucleic acid molecule according to the invention, a T cell according to the invention, or a combination thereof and, optionally, an immune stimulating molecule such as an adjuvant, a cytokine, or a combination thereof.

The invention further provides a method of preventing or treating a coronavirus infection in a subject, the method comprising the simultaneous, separate or sequential administering to the subject of effective amounts of a T cell antigen and/or T cell polyantigen, a nucleic acid molecule encoding the T cell polyantigen according to the invention, or a delivery vector including a viral vector and a bacterial vector expressing the T cell polyantigen according to the invention, and/or a cell-based vaccine, either as such or in combination with an immune stimulating molecule such as an adjuvant and/or a cytokine, to a subject in need thereof.

Said T cell antigen, T cell polyantigen, nucleic acid molecule, cell-based vaccine and, optionally, an immune stimulating molecule such as an adjuvant and/or a cytokine, either separately or in combination, may be administered by oral administration, topical administration, nasal administration, inhalation, topical, transde rmal and/or parenteral administration, including intramuscular, intradermal, subcutaneous, intraperitoneal administration. A preferred mode of administration is nasal administration and /or parenteral administration such as intramuscular, intradermal and/or subcutaneous administration. For oral administration, a preferred pharmaceutical preparation is provided by a tablet.

Pharmaceutically acceptable excipients include diluents, binders or granulating ingredients, a carbohydrate such as starch, a starch derivative such as starch acetate and/or maltodextrin, a polyol such as xylitol, sorbitol and/or mannitol, lactose such as u-lactose monohydrate, anhydrous u-lactose, anhydrous B-lactose, spray-dried lactose, and/or agglomerated lactose, a sugar such as dextrose, maltose, dextrate and/or inulin, or combinations thereof, glidants (flow aids) and lubricants to ensure efficient tableting, and sweeteners or flavours to enhance taste.

EXAMPLES

General methods

Mice

Wild-type C57BL/6 mice were obtained from Janvier Labs. Transgenic mice expressing the human ACE2 receptor (hACE2) under control of the cytokeratin 18 (KRT18) promoter were described previously (McCray et al., 2007. J Virol 81: 813- 821). The hACE2 mice used in these studies, obtained from the Jackson Laboratory (B6.Cg-Tg(K18-ACE2)2Prlmn/J), are congenic to the C57BL/6 background and were bred in house. At the start of the experiments, mice were six to eight weeks old. Animals were housed under specific-pathogen free conditions in individually ventilated cages at the animal facility at the Leiden University Medical Center (LUMC). All animal experiments were approved by the Animal Experiments Committee of LUMC and performed according to the recommendations and guidelines set by the Dutch Experiments on Animals Act and by the LUMC.

DNA constructs

Expression constructs containing DNA coding for CoVAX_ORFlab (SEQ ID NO: 40), CoVAX_ORFlab_norep (SEQ ID NO: 41), CoVAX_MNS (SEQ ID NO: 42), CoVAX_MNS_norep (SEQ ID NO 46), and S_2P+ (SEQ ID NO: 45) were generated by Gibson assembly. Briefly, synthetic, codon-optimized DNA (Integrated DNA Technologies, Coralville, IA, USA) was cloned into a CMV-promoter driven expression vector comprising a bovine growth hormone poly-A sequence and kanamycin resistance, using Gibson Assembly Cloning Kit (New England Biolabs, Ipswich, MA, USA).

All plasmids were grown using E. coli strain DH5u and purified using an Endotoxin- Free (EF) plasmid purification kit (Macherey-Nagel, Dueren, Germany). For vaccination, plasmids underwent an additional purification step on a Nucleobond filter, followed by centrifugation (30 min, 10,000 g, 4 °C) to remove any remaining debris. Purified DNA was dissolved at ±3 mg/ml in Tris:EDTA buffer (1:0.1 mM).

Virus

A clinical isolate, SARS-CoV-2/human/NLD/Leiden-0008/2020, was isolated from a nasopharyngeal sample. The sequence of this virus isolate (GenBank accession number MT705206.1) shows one mutation in the spike protein compared to the Wuhan spike protein sequence resulting in Asp>Gly at position 614 (D614G) of the Spike protein. In addition, several non-silent (C12846U and C18928U) and silent mutations (C241U, C3037U, and C1448U) in other genes were found. This isolate was propagated and titrated in Vero-E6 cells.

Vaccination

Mice were vaccinated intradermally at the tail base with 50 microgram DNA in 30 microliter 0.9% NaCl, 1 mM Tris pH 8.0, 0.1 mM EDTA. Synthetic long peptide vaccines, consisting of 50 microgram of each peptide and 20 microgram of either poly(I:C) or CpG (InvivoGen) dissolved in 30 microliter PBS, were administered subcutaneously. Booster vaccinations were provided with 2- or 3-week intervals.

Tetramer staining Blood, drawn on several days after vaccination, was treated with erythrocyte lysis buffer (LUMC pharmacy, Leiden, the Netherlands) and stained with fluorochrome- conjugated H2-Kb/VNFNFNGL, H2-Kb/ TGYHFREL or H2-Kb/ RTLSYYKL tetramers (LUMC tetramer facility, Leiden, the Netherlands) in PBS supplemented with 0,1 % bovine serum albumin and 0,02 % sodium azide (PBS/BSA). After a 30- minute incubation at room temperature in the dark, fluorochrome-conjugated antibodies to CD3, CD4, CD8 (Biolegend, San Diego, CA, USA) were added to discriminate T cell subsets, followed by another 30 minutes on ice and 2 washing steps with PBS/BSA to remove unbound tetramers and antibodies. The samples were acquired on a BD LSRII (Becton Dickinson, San Jose, CA, USA) and analyzed using Flow Jo (Flow Jo LLC).

Intracellular cytokine staining

Intracellular cytokine staining (ICS) of splenocytes were performed as described (Arens et al., 2011. J Immunol 86: 3874-3881). Briefly, single cell suspensions were stimulated with peptide-loaded DI cells (a dendritic cell line of C57BL/6 origin, see Winzler et al., 1997. J Exp Med 185: 317-328) for 5h in the presence of brefeldin A (Golgiplug; BD Pharmingen). At the end of this incubation, the cells were stained with fluorescently labeled antibodies against surface markers CD3, CD4, CD8 (all from Biolegend, San Diego, CA, USA), fixed, permeabilized and then stained with fluorescently labeled antibodies directed against CD 154 (eBioscience, San Diego, CA, USA), TNF-u (Biolegend), IFN-y (BD Biosciences, Franklin Lakes, NJ, USA), IL-2 (BD Biosciences). Flow cytometric acquisition was performed on a BD Fortessa flow cytometer (BD Biosciences) and samples were analyzed using FlowJo software (TreeStar).

In vitro serum neutralization titration (SNT) assay

Neutralization assays were performed as described previously (Sholukh et al., 2020. medRxiv 12.07.20245431). Briefly, serum was heat-inactivated for 30 min at 56 °C, serially diluted in 2-fold steps and incubated with 100 PFU / 100 microliter of SARS- CoV-2 Zagreb isolate (hCoV-19/Croatia/ZG-297-20/2020, GISAID database ID: EPI_ISL_451934) for 1 hour at room temperature. These serum-virus mixes were then added to Vero-E6 (ATCC CRL- 1586) cells that had been seeded in 96-well plates (2x104 cells/well). Following a 1-hour incubation at 37 °C and 5% CO₂, the inoculum was removed, and a 1.5% methylcellulose overlay was added. The cells were then incubated for 3 days at 37 °C and 5% CO2, after which they were stained with crystal violet staining for plaque counting.

SARS-CoV-2 infection

K18-hACE2 transgenic mice were anaesthetized lightly using isoflurane gas and infected intranasally with the 5000 plaque forming units (PFU) of SARS-CoV- 2/human/NLD/Leiden-0008/2020SARS-CoV-2 in a total volume of 50 microliter DMEM. Mouse weight and clinical discomfort were monitored daily. All experiments with SARS-CoV-2 were performed in a Biosafety Level 3 (BSL3) Laboratory at the Leiden University Medical Center.

Example 1. Identification of conserved corona protein domains with predicted epitopes for multiple MHC molecules

Within the betacoronavirus family, we focused on the Sarbecoviruses, including SARS-CoV-1, SARS-CoV-2 and related viruses in other species (e.g. bats). To identify sequences conserved within all Sarbecoviruses, 17 Sarbecoviruses were selected (GenBank accession numbers NC_045512, MN988713.1, MN985325.1, MN938384.1, MN975262.1, MG772933, MG772934, DQ412043.1, KY417144.1, AY278489.2, AY278488.2, FJ882957.1, AY572034.1, AY274119, DQ071615, DQ022305, GU190215) that were selected to represent the a broad Sarbecovirus sequence diversity. From these, translated ORFlab, M (membrane), N (nucleoprotein) and S (Spike) sequences were aligned, revealing AA (amino acid) sequences that were identical within Sarbecoviruses. At each AA position, a single dissonant sequence was allowed, as this would most likely be explained by a sequencing error. In addition, using NetMHC4.0 ((NetMHC4.0, Immune Epitope Database, available at iedb.org), the translated sequences were examined for potential binders to HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02, HLA-B*07:02, HLA-B*08:01, HLA-B*35:01, HLA- B*40:01, HLA-B*44:02, HLA-B*44:03, HLA class I alleles together covering >85% of the global human population. Together, this yielded a list of AA sequences identical within the Sarbecovirus family and rich in predicted HLA class I epitopes (%rank<1 AND IC50<500nM according to NetMHC4.0). From these, sequences > 19 AA were selected, as these were likely to contain multiple epitopes, including MHC class II binding epitopes that are currently difficult to predict. As an exception, one 14AA ORFlab sequence ((SEQ ID NO:14; 14_NC_045512.2_nspl3_5454-5467 in Figure 1) was included, as it contained a strong predicted HLA binding peptide. As a result, 17 ORFlab (peptides 1-17 of Table 1) and 18 MNS antigens (peptides 18-35 of Table 1) were selected for inclusion in the vaccines.

Next, we investigated how well these coronavirus protein domains were conserved among other, non-Sarbecovirus, coronaviruses (Figure 1). To this end, the domain sequences were blasted (available at blast.ncbi.nlm.nih.gov/Blast.cgi) against ORFlab, M, N, and S sequences from more than 60 coronaviruses (GenBank accession numbers NC_045512, MN988713.1, MN985325.1, MN938384.1, MN975262.1, MG772933, MG772934, DQ412043.1, KY417144.1, AY278489.2, AY278488.2, FJ882957.1, AY572034.1, AY274119, DQ071615, DQ022305, GU190215, HQ166910, NC_025217.1, NC_030886.1, EF065514, MK211379.1, HQ728483, HQ728482, JX869059, NC_038294.1, KC869678.4, KC667074.1, KC776174.1, MG987420.1, MG987421.1, KJ473821.1, MG912608.1, MK129253.1, MN723544.1, KJ813439.1, MF598641.1, MG366880.1, MG912597.1, EF065509, EF065505, NC_039207.1, KJ473821.1, KJ473820.1, KJ473822.1, MK907286.1, KX442564.1, BY140568, MN611519.1, JX899383, EF424623, EF424621, FJ425186, JX860640, U00735, EF446615, AY585228, JN874559, FJ938068, FJ647225, DQ415914), and percentage identity of each selected domain to a subset of these (e.g. merbecoviruses) was calculated as average ‘sequence identity’ x ‘sequence cover’. As expected, all selected domains were virtually identical (>97%) within the Sarbecovirus subgenus. It is likely that mismatches were in fact sequence errors. For the ORFlab domains, but not for the MNS domains, correspondence with merbecoviruses (MERS-like) was also high: >80% on average, suggesting that vaccines incorporating these domains may also be effective against MERS. In general, the ORFlab domains were more conserved (79% on average) among coronaviruses than the M, N, and S domains (50% identical on average).

Example 2. design and generation of polytope DNA vaccines

The ORFlab domain sequences selected in example 1 were incorporated as antigens into two DNA vaccines (see Figure 2 for schematic design), one comprising non-structural, ORFlab sequences, and one comprising antigens derived from the structural proteins M, N and S. To achieve this, two polyepitope proteins were designed, in which the antigens were separated by triple alanine (AAA) spacers. At the C-terminus of both polyepitopes, three SARS-CoV reporter antigens (peptides 37, 38, 36 of Table 1), comprising a H-2Kd epitope (Zhi et al., 2005. Virology 335: 34- 45; Huang et al., 2007. Vaccine 25: 6981-6991; Zhao et al., 2010. J Virol 84: 9318- 9325), a H-2Kb epitope (Zhi et al., 2005. Virology 335: 34-45; Poh et al., 2009. J Med Virol 81: 1131-1139; Zhao et al., 2010. J Virol 84: 9318-9325; Channappanavar et al., 2014. J Virol 88: 11034-11044; Dangi et al., 2021. bioRxiv doi: https://doi.org/10.1101/2021.06.01.446491), and an HLA-A*0201 restricted epitope (available at iedb.org/epitope/16156), respectively, and an HA-tag were included, the latter separated from the T cell antigens by a GSGS spacer (see SEQ ID NO: 44). As one of the conserved coronavirus domains (peptide 35 in Table 1) was contained in the HLA-A*0201 reporter antigen (peptide 36 of Table 1), the former was not included in the MNS vaccine. The resulting ORFlab and MNS T cell polyantigen amino acid sequences (SEQ ID NO:41 and SEQ ID NO:42, respectively) were encoded by codon-optimized DNA sequences and inserted into a kanamycin-selectable plasmid vector under the control of a CMV promoter and a bovine growth hormone derived poly-adenylation signal. This resulted in two plasmids: CoVAX_ORFlab and CoVAX_MNS.

Example 3. Cytokine production of splenocytes following vaccination and in vitro restimulation.

Even though the vaccines were designed to contain antigens rich in HLA- restricted, human T cell epitopes, online resources (NetMHC4.0, Immune Epitope Database, available at iedb.org) revealed that they also contained a number of predicted murine H-2^b (C57BL/6) class I epitopes. To test the immunogenicity of CoVAX_ORFlab and CoVAX_MNS in C57BL/6 mice, 5 mice per group were vaccinated intradermally on day 0, 21 and 42 with 50 microgram of each plasmid. Tracking immune responses in blood using H-2K^b tetramers containing the reporter epitope VNFNFNGL (contained within SEQ ID NO:38) showed that the vaccines were indeed immunogenic (Figure 3 A,C). Ten days after the final vaccination, spleens were examined for responses to the selected conserved antigens by intracellular cytokine staining (ICS), using production of Thl cytokines IFNgamma and TNFalpha as read-outs (Figure 3 B,D). Both the CoVAX_ORFlab and the CoVAX_MNS vaccine induced a CD8 T cell response against an H-2K^b epitope. In the case of CoVAX_ORFlab, a response was detected against TGYHFREL, an nsp12 epitope contained with the antigen 4_NC_045512.2_nspl2_4695-4746 of Table 1 (SEQ ID NO:4). In the case of CoVAX_MNS, a response was detected against RTLSYYKL, an M epitope contained within antigen 10_NC_045512.2_M_156-187 of Table 1 (SEQ ID NO:26). In short, both DNA vaccines generated T cell immune responses against highly conserved Sarbecovirus antigens.

Example 4. T cell responses and protection against lethal SARS-CoV-2 challenge in hACE2 transgenic mice.

K18-hACE2 transgenic mice, in which the human ACE2 receptor is expressed under control of a cytokeratin 18 promoter (McRay 2007; J Virol. 81:813-821), were vaccinated intradermally thrice with three-week intervals with the indicated DNA vaccines (50 microgram/plasmid). Importantly, for this experiment the S-derived reporter sequences were removed from the CoVAX_ORFlab vaccine, so that it contained conserved ORFlab antigens only ((CoVAX_ORFlab_norep; SEQ ID NO:41). Mock-vaccinated control mice served as negative controls, and mice vaccinated with plasmid DNA encoding a prefusion-stabilized version of the Spike protein (S_2P+), as used in most currently approved vaccines, served as positive controls. Using a virus neutralization assay on Vero-E6 cells (Sholukh et al., 2020. medRxiv 12.07.20245431), this S_2P+ DNA vaccine was found to induce high SARS- CoV-2 neutralizing antibody titers (Figure 4B).

Strong vaccine-specific CD8 T cell responses in blood were measured using H-2K^b tetramers containing the ORFlab, M or S-derived reporter CD8 T cell epitope (see Figure 3) after three vaccinations (Figure 4A). Of note, the control S_2P+ vaccine also generated responses to the conserved S-derived VNFNFNGL epitope (S525), which is also present in CoVAX_MNS but not in CoVAX-ORFlab. Three weeks after the final vaccination, K18-hACE2 mice were challenged intranasally with the clinical isolate SARS-CoV-2/human/NLD/Leiden-0008/2020 (GenBank accession number MT705206.1) and body weight was measured daily as a parameter of disease (Figure 4C). While 90% of control mice rapidly lost weight and died within about a week, 50 % of CoVAX_ORFlab vaccinated mice initially lost weight but then recovered from the infection. Similarly, 50 % mice that had received the CoVAX_MNS vaccine recovered from the infection. In conclusion, a pan- Sarbecovirus vaccine containing only conserved Sarbecovirus sequences induces T cell responses against these antigens and can protect against a lethal challenge with SARS-CoV-2 virus.

Example 5. Single T cell antigen-induced CD8 T cell responses against a conserved T cell epitope protect against sub-lethal SARS-CoV-2 challenge.

Next, we investigated whether raising T cell responses against a single semi-conserved T cell antigen could be effective against SARS-CoV-2 infection. To this end, K18-hACE2 transgenic mice were vaccinated subcutaneously day 0, 14 and 28 with a single T cell antigen that harbors the conserved S525 epitope sequence VNFNFNGL (SEQ ID NO:46). The T cell antigen was provided as 100 micrograms of an extended peptide with the amino acid sequence IKNQCVNFNFNGLTGTGVLTESNK (SEQ ID NO:47), together with 20 μg CpG adjuvant (ODN 1826, InvivoGen). Mock- vaccinated (-) mice served as negative controls. S525-specific CD8 T cell frequencies were determined in blood using tetramers after the primary, secondary and tertiary vaccination. All mice that were vaccinated with the peptide developed strong, S525-specific, T cell responses (Figure 5A). Five weeks after the final vaccination, the K18-hACE2 mice were challenged intranasally with the Leiden-0008/2020 SARS-CoV-2 isolate and body weight was measured daily as a parameter of disease. All (10/10) mice that were vaccinated with the peptide survived the infection, significantly more than in the control group (3/10, Figure 5B).

Example 6

Materials and methods

In addition to the General methods provided herein above, the following M&M were used in this example. Key resources are provided in Table 2. Plasmid DNA vaccine design and production

Two plasmid DNA vaccines were designed, one containing conserved protein domains from the structural sarbecovirus M, N, and S genes (CoVAX_MNS_norep_p413, SEQ ID NO 46), and one containing conserved domains identified in sarbecoviral ORFlab regions (CoVAX_ORFlab_norep_p415, SEQ ID NO 41). Note that both vaccines lack the C-terminal CD8 Spike-derived reporter antigens (listed as SEQ ID NO 43). In addition, plasmid DNA’s encoding HA- tagged, full-length Spike proteins from SARS-CoV-2 'Wuhan Hu-1’ (SEQ ID NO 47), SARS-CoV-2 Omicron (SEQ ID NO 48) and SARS-CoV-1 (SEQ ID NO 49) were generated. Codon-optimized DNA sequences coding for the resulting polyantigens or Spike proteins (Table 1) were introduced into a plasmid DNA vector in which expression was driven by a strong CMV promoter. Plasmids were propagated in E. coli cultures and purified using Nucleobond Xtra maxi EF columns (Macherey- Nagel) according to the manufacturer’s instructions. Expression of the vaccine and Spike plasmids was verified by transient transfection in 293T cells using Saint - DNA transfection reagent (Synvolux, Leiden, the Netherlands), and Western blotting using antibodies against the HA tag and antibodies specific for SARS-CoV- 1 and SARS-CoV-2. For vaccination, plasmids were column-purified twice, each time using a fresh column.

Mice

Wild-type C57BL/6J mice, 6-8 weeks old, were housed under specific pathogen-free conditions in individually ventilated cages at the Leiden University Medical Center (LUMC) animal facility. All animal experiments were performed in accordance with Dutch Animal Ethical Committee guidelines and were approved by the Animal Welfare body of LUMC (DEC consult number: AVD11600202013796), and performed according to the recommendations and guidelines set by LUMC and by the Dutch Experiments on Animals Act. DNA vaccinations

Mice were vaccinated with three-week intervals. For DNA vaccination, 50 μg DNA, dissolved in 30 pl of a sterile buffer (0,9 % NaCl, 1 mM Tris pH 8.0, 0.1 mM EDTA), was injected intradermally at the base of the tail using a 0,5 ml U-100 Insulin 29G Micro-Fine needle (Becton Dickinson,- #324892).

Determination of antibody titers

ELISAs were performed to determine Spike-specific antibody titers in sera. Nunc ELISA plates were coated with 1 pg/ml His-tagged Spike proteins of SARS- CoV, SARS-CoV-2 (Wuhan), or SARS-CoV-2 Omicron variant (see Table 1 for resources) in ELISA coating buffer (Biolegend) overnight at 4 °C. Plates were washed five times and blocked with 1% bovine serum albumin (BSA) in PBS with 0.05% Tween-20 for 1 h at room temperature. Plates were washed and incubated with serial dilutions of mouse sera and incubated for 1 h at room temperature. Plates were again washed and then incubated with 1:4000 dilution of horse radish peroxidase (HRP) conjugated anti-mouse IgG secondary antibody (Southern Biotech, cat. 1030-05) and incubated for 1 h at RT. To develop the plates, 50 μL of TMB 3,3=,5,5=tetramethylbenzidine) (Sigma-Aldrich) was added to each well and incubated for 5 min at room temperature. The reaction was stopped by the addition of 50 μL IM H₂SO₄, and within 5 min the plates were measured with a microplate reader (model 680; Bio-Rad) at 450 nm.

Results

Upon infection with a Sarbecovirus, vaccine -induced CD4 T cells are likely to amplify and accelerate the generation of new CD8 T cell- and antibody-mediated responses against the infecting virus. To model this situation, mice were first vaccinated with the CoVAX_MNS_norep vaccine and then exposed to full-length Spike from three different sarbecoviruses by a subsequent DNA vaccination. As CoVAX_MNS_norep contains Spike sequences shared among sarbecoviruses, Spike-specific CD4 T cells specifically elicited by that vaccine can provide support to Spike-specific B cells, allowing them to produce class-switched antibodies, and to Spike-specific naive CD8 T cells activated by dendritic cells, irrespective of the sarbecovirus Spike they are exposed to.

Briefly, C57BL/6 mice were vaccinated intradermally, three times at three- week intervals, with CoVAX_MNS or CoVAX_ORFlab DNA vaccines, both lacking the Spike-derived VNFNFNGL epitope or other reporter antigens (norep). Of note, the polyantigens encoded by CoVAX_MNS_norep or CoVAX_ORFlab_norep do not induce Spike-specific antibodies by themselves. Three weeks after the final vaccination, mice were injected with 50 μg DNA encoding full-length wild-type Spike from SARS-CoV (SARS-CoV-1), SARS-CoV-2 Wuhan (W) or omicron (O). Eight days after exposure to these Spikes, blood samples were analysed for CD8 T cell responses to the H-2K^b-restricted Spike reporter antigen VNFNFNGL (absent from the vaccines, but present in all three Spike DNA’s) as well as IgG and IgG2c antibody responses to the different Spike proteins. Eight days after Spike- exposure, significantly higher levels of Spike-specific CD8 T cell responses were observed in mice vaccinated with CoVAX_MNS_norep, compared to control mice that had received CoVAX_ORFlab_norep (Figure 6A). This indicates that pre- existing T cell immunity against conserved regions of the Spike protein as induced by CoVAX_MNS_norep vaccination enhances and/or accelerates the response of naive CD8 cells to a Spike epitope. Furthermore, already within 8 days following Spike exposure, Spike-specific IgG and IgG2c were detected in CoVAX_MNS_norep but hardly in control mice (Figures 6B, C). These class-switched antibodies were specific to the Spike proteins to which the mice had been exposed, indicating that pre-existing CD4 T cell immunity against conserved regions of the S genes can provide help to Spike-specific B cells and thereby enhance the antibody response against an incoming sarbecovirus Spike protein, be it the original Wuhan Spike, an advanced variant with over 30 Spike mutations (Omicron), or a relatively distant Spike protein of Sars-CoV-1. Upon infection by a SARS-like virus, also the non- structural proteins will be expressed and presented to the immune system. Based on the data and principles presented here, pre-existent immunity to conserved regions as included in the herein presented CoVAX_MNS and CoVAX_ORFlab vaccines, is expected to enhance and/or accelerate T and B cell mediated immunity and killing of infected cells as well.

Table 1. Conserved Corona Protein Domains.

Table 2. Key resources table.

Claims

Claims

1. A protein comprising a T cell polyantigen, said polyantigen comprising 2-100 individual, conserved coronavirus T cell antigens of 8-80 amino acid residues, which individual T cell antigens may be alternated by spacer sequences of, preferably, 1-10 amino acid residues, or comprising a combination of 2-100 individual T cell antigens.

2. The protein according to claim 1, comprising at least 5 individual T cell antigens, preferably 5-35 individual T cell antigens.

3. The protein according to claim 1 or claim 2, comprising at least one conserved coronavirus T cell antigen from the conserved protein domains of Table 1.

4. A nucleic acid molecule, encoding the protein of any one of claims 1-3, said nucleic acid molecule preferably capable of expressing said protein in a suitable cell.

5. The nucleic acid molecule according to claim 4, which is a circular or linear, RNA or DNA nucleic acid molecule, preferably a circular DNA molecule or a linear RNA molecule.

6. A pharmaceutical composition, comprising the protein of any one of claims 1- 3, the nucleic acid molecule of claim 4 or claim 5, or a combination thereof, and an pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, further comprising an immune stimulating molecule.

8. A pharmaceutical composition comprising at least one conserved coronavirus T cell antigen, preferably at least one conserved coronavirus T cell antigen of Table 1, and an immune stimulating molecule.

9. A T-cell receptor (TCR) that specifically recognizes an epitope within a conserved coronavirus T cell antigen, preferably wherein the TCR is expressed by a T-cell.

10. The TCR of claim 9, wherein the coronavirus T cell antigen is selected from the conserved coronavirus protein domains in Table 1.

11. The TCR of claim 9 or claim 10, wherein the TCR is isolated from an individual.

12. A method of inducing an immune response in an individual suffering from a coronavirus infection, or at risk of suffering from a coronavirus infection, said method comprising providing said individual with the pharmaceutical composition of any one of claims 6-8.

13. The method of claim 12, wherein the polyantigen comprising 2-100 individual, conserved coronavirus T cell antigens of 8-80 amino acid residues will amplify and accelerate the generation of further immune responses to a vaccine containing at least part of a sarbecovirus Spike antigen or to a subsequently infecting coronavirus.

14. A method of treating an individual suffering from a coronavirus infection, comprising providing said individual with the pharmaceutical composition of any one of claims 6-8, the TCR of any one of claims 9-11, or a combination thereof.

15. The pharmaceutical composition of any one of claims 6-8, the TCR of any one of claims 9-11, or a combination thereof, for use in a method of inducing an immune response in an individual.