Immunogenic constructs and vaccines for use in the prevention and treatment of disease caused by SARS-CoV-2

Abstract

The present invention relates to immunogenic constructs, antigenic units, such as polynucleotides, polypeptides, and multimeric proteins useful for the prophylactic and therapeutic treatment of disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), as well as pharmaceutical compositions / vaccines comprising such immunogenic constructs or antigenic units, and methods for producing and using the immunogenic constructs, antigenic units, and pharmaceutical compositions / vaccines.

Description

[Technical field]

[0001] The present invention relates to immunogenic constructs, antigenic units, such as polynucleotides, polypeptides, and multimeric proteins, useful for the prophylactic and therapeutic treatment of disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), as well as pharmaceutical compositions / vaccines comprising such immunogenic constructs or antigenic units, and methods for producing and using the immunogenic constructs, antigenic units, and pharmaceutical compositions / vaccines. [Background technology]

[0002] Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses that can cause disease in a wide range of hosts, including humans. Four lineages (A-D) are commonly recognized, and their genomes, approximately 30 kb in length, have been found to be the largest among RNA viruses, encoding more than 20 putative proteins, including four major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). Human seasonal CoVs are prevalent worldwide, causing respiratory infections that are typically mild and self-limiting. However, higher mortality was observed in the 2003 Severe Acute Respiratory Syndrome (SARS) and 2012 Middle East Respiratory Syndrome (MERS) outbreaks, caused by SARS-CoV and MERS-CoV infections, respectively.

[0003] An outbreak of respiratory disease in Wuhan, China, was reported by the WHO in January 2020 and found to be caused by a novel coronavirus, later renamed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 enters cells through the interaction of the viral receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of the human nasopharyngeal, pulmonary, and intestinal mucosa. The disease caused by this virus was named coronavirus disease 2019 (COVID-19). Eventually, this novel viral infection spread rapidly around the world, and a coronavirus pandemic was first declared by the World Health Organization (WHO) in March 2020. By September 2021, the WHO reported more than 218 million confirmed cases of COVID-19 worldwide, including more than 4.5 million deaths. Reporting the COVID-19 pandemic. To prevent infection, hospitalization, and reduce severity and mortality associated with COVID-19, intensive vaccine development efforts were launched in early 2020. Neutralizing antibodies targeting the RBD and other functional domains of the SARS-CoV-2 spike protein are the primary pathway to achieve immunity and vaccine efficacy.

[0004] Based on past experience with related coronaviruses and the theory that spike-specific antibodies constitute the majority of neutralizing antibodies in convalescent COVID-19 patients, vaccine technology has been adapted to induce spike-based protective immunity. Thus, many of the approved vaccines and vaccine candidates in development target the spike protein and its variants as the primary antigen. Multiple vaccines that were in clinical efficacy trials have been shown to be highly immunogenic, safe, and effective in preventing infection and / or severe disease and death. As such, multiple COVID-19 vaccines have been approved for use in various jurisdictions around the world, and by September 2021, over 5.2 billion doses of COVID-19 vaccines have been administered worldwide.

[0005] The most successful vaccine platforms against SARS-CoV-2 are all based on the induction of immunity against the spike glycoprotein and include mRNA platforms, virus-like particles with recombinant spike protein, or adenoviral vectors (Khoury et al., 2021). These strategies have proven successful in eliciting neutralizing antibody responses against SARS-COV-2, and many studies have shown a correlation between virus-neutralizing antibody levels and protection from symptomatic infection (Earle et al., 2021; Khoury et al., 2021). Thus, neutralizing titers are a potential surrogate marker of protection against COVID-19. Similar studies are underway to evaluate the correlation between T cell responses and efficacy, as neutralizing antibodies can only partially explain the observed efficacy (Alter et al., 2021), which may accelerate regulatory approval of T cell epitope-based vaccines.

[0006] As of September 2021, the first major challenge in vaccine-based pandemic control is the weakening of immunity and reduced efficacy of licensed or emergency approved vaccines over time. Studies have shown that vaccine-induced antibody levels and SARS-CoV-2 infection steadily decline 4-5 months after infection or vaccination, potentially affecting vaccine efficacy. Recent data show, for example, that the protective effect weakens after two doses of mRNA vaccines approved for use in the EU and the US. Therefore, some governments have decided to recommend booster doses for high-risk population groups 6-8 months after completion of the regimen.

[0007] The second major challenge is the emergence of new variants of SARS-COV-2 with possible increased infectivity and / or reduced susceptibility to neutralizing antibodies generated by vaccines based on the prototype spike-based vaccines of 2020. Some of the acquired mutations (e.g. D614G in the spike) have allowed SARS-CoV-2 variants to have higher infectivity compared to the prototype Wuhan strain variants or to evade vaccine- or disease-induced immune responses even in fully vaccinated individuals (e.g. E484K in the spike). Thus, the pandemic is still ongoing and new variants of concern of the SARS-CoV-2 virus continue to emerge, with delta variants currently dominating worldwide with the highest infectivity and beta variants shown to have the lowest susceptibility to vaccine-induced antibodies. In the case of more infectious variants, higher vaccine coverage will be required to control the pandemic in the population, further delaying global vaccine access. Close monitoring of variants, with particular attention to variants that are spreading internationally, that are associated with increased disease severity or that show reduced susceptibility to neutralizing antibodies, will allow the design of vaccines that provide greater protection.

[0008] Although the approved vaccines have had a significant impact on controlling the pandemic and it is still possible to periodically replace the spike or RBD in the vaccine with the latest variants, there is still an urgent need to develop a broadly protective "universal" SARS-CoV-2 vaccine to control the ongoing pandemic, provide better protection against current and future variants of concern, and ensure global vaccine supply. Second, although treatment of patients infected with COVID-19 has improved over time, there is also still an urgent need for virus-specific therapeutics for use in the early stages of disease progression.

[0009] Emerging evidence suggests a possible correlation between T cell immunity and protection against COVID-19 (Bertoletti et al., 2021; Meyers et al., 2021). CD8+ cytotoxic T lymphocytes (CTLs) support the SARS-CoV-2 antibody response by contributing to viral clearance from intracellular compartments inaccessible to neutralizing antibodies and by eliminating virus-infected cells. They may also play a role in disrupting infection. Antigen-specific CD4+ T cells support the generation of B cells and CD8+ T cells, aiding in memory generation and indirect or direct cytotoxic activity.

[0010] It is therefore important to develop a universal SARS-CoV-2 vaccine that induces specific T cell responses with epitopes beyond the range of epitopes included in current spike-based vaccines.

[0011] The present invention therefore relates to a vaccine comprising selected SARS-CoV-2 T cell epitopes that induce a cellular immune response (T cell response) in a human individual to which the vaccine is administered, and thus is useful for the prophylactic and therapeutic treatment of diseases caused by SARS-CoV-2. Summary of the Invention

[0012] In a first aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). The present invention relates to an immunogenic construct,

[0013] In some embodiments, the present disclosure provides: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A dimeric protein consisting of two polypeptides as defined in (ii). The present invention relates to an immunogenic construct,

[0014] In another aspect, the present disclosure relates to a vector comprising a polynucleotide defined herein.

[0015] In yet another aspect, the present disclosure relates to a host cell comprising a vector or a polynucleotide defined herein.

[0016] In yet another aspect, the present disclosure relates to polypeptides encoded by the nucleic acid sequences defined herein.

[0017] In yet another aspect, the present disclosure relates to a multimeric protein consisting of a plurality of polypeptides as defined herein. In some embodiments, the present disclosure relates to a dimeric protein consisting of two polypeptides as defined herein.

[0018] In yet another aspect, the present disclosure relates to a polynucleotide, vector, polypeptide, or multimeric protein as defined herein for use as a medicament.

[0019] In yet another aspect, the present disclosure relates to a pharmaceutical composition / vaccine comprising a polynucleotide, vector, polypeptide, or multimeric protein as defined herein and a pharma- ceutically acceptable carrier.

[0020] In yet another aspect, the disclosure relates to methods for preparing the pharmaceutical compositions / vaccines and to uses of the pharmaceutical compositions / vaccines for use in the prophylactic or therapeutic treatment of a disease caused by SARS-CoV-2, such as by administering the pharmaceutical composition / vaccine to a subject in need thereof.

[0021] The immunogenic constructs or pharmaceutical compositions / vaccines comprising such constructs induce rapid, strong and sustained T cell responses when administered to a subject and are therefore useful as prophylactic or therapeutic treatments for diseases caused by SARS-CoV-2.

[0022] In one embodiment, the pharmaceutical composition / vaccine may be administered to a human individual who has previously been vaccinated with a SARS-CoV-2 vaccine targeting the spike protein. Such individuals may not be adequately protected against new / future variants of the spike protein, and the T cell vaccine of the present invention strengthens the individual's immune response against SARS-CoV-2 by boosting the existing spike-specific T cell response, which may have been induced by the previous vaccine, and maximizes the CD4+ / CD8+ T cell immunity by adding additional T cells specific for non-spike antigens, in addition to the neutralizing response elicited by the previous vaccine.

[0023] In another embodiment, the pharmaceutical composition / vaccine may be administered to a human individual who has not yet been vaccinated with a SARS-CoV-2 vaccine, and the CD4+ / CD8+ T cells provide protective immunity during SARS-CoV-2 infection.

[0024] The immunogenic construct of the present invention is a vaccibody construct, i.e., a multimeric fusion protein consisting of multiple polypeptides (e.g., a dimeric protein consisting of two polypeptides), each polypeptide containing a targeting unit that targets antigen-presenting cells, a multimerization unit, and an antigen unit, which has been shown to be effective in generating an immune response against an antigen or epitope contained in the antigen unit after administration to a subject. Vaccibody constructs have previously been suggested as vaccines against SARS-CoV-2 infection (see, e.g., PCT / EP2021 / 061602, the contents of which are incorporated herein by reference, and G. Norheim et al., bioRvix 2020, doi:https: / / doi.org / 10.1101 / 2020.12.08.416875).

[0025] The constructs disclosed herein may be administered to a subject, e.g., a human individual, in the form of a polynucleotide (e.g., a DNA plasmid) comprising a nucleotide sequence encoding a polypeptide. After administration to a host cell, e.g., a muscle cell, the polypeptide is expressed and a multimeric fusion protein, e.g., a dimer, is formed by a multimerization unit, e.g., a dimerization unit.

[0026] The immunogenic constructs of the invention may be used in a vaccine, i.e. a pharmaceutical composition comprising the construct of the invention and a pharma- ceutical acceptable carrier, for use in the prophylactic or therapeutic treatment of a disease caused by SARS-CoV-2 by administering the vaccine to a subject, i.e. a human individual.

[0027] The antigenic units described herein are further aspects of the present disclosure. Accordingly, in such further aspects, the present disclosure refers to (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 1-77, or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i).

[0028] Such antigenic units (in the form of a polynucleotide or polypeptide) may be used as such, i.e. without the targeting unit and the multimerisation unit being present in the polynucleotide / polypeptide, in a pharmaceutical composition / vaccine comprising a pharma- ceutically acceptable carrier.

[0029] The antigenic unit may itself be used in the form of a polynucleotide as described herein, for example DNA or RNA, including genomic DNA, cDNA, mRNA, either double-stranded or single-stranded, or in the form of a polypeptide.

[0030] The polynucleotide may be included in a vector suitable for transfecting or transducing a host cell. The antigenic unit may be included as RNA in a vector that includes sequences that have been shown to increase RNA stability and translation efficiency, such as a poly(A) tail. The vector may further include a sequence that encodes a signal peptide or a fragment thereof. The RNA or vector may be used as an RNA vaccine, which may be formulated as described herein.

[0031] Alternatively, the polypeptides may be used as peptide vaccines which may be formulated as known in the art, i.e., including pharmaceutical carriers and optionally excipients and / or adjuvants known in the art for use in such peptide vaccines. [Brief description of the drawings]

[0032] [Figure 1]Figure 1 shows an immunogenic construct of the invention which is described as being a polypeptide and having an N-terminal beginning and a C-terminal end. The units / elements of the polypeptide (here a targeting unit (TU) for targeting an antigen presenting cell (APC), a dimerization unit (DimU), and an antigen unit) may be arranged in the polypeptide such that the antigen unit is located at the C-terminal end of the polypeptide (Figure 1a) or at the N-terminal beginning of the polypeptide (Figure 1b). Further details are provided in the section entitled "Immunogenic Constructs". [Diagram 2] FIG. 2 shows a schematic diagram of the VB10.COV2 construct, one embodiment of the present invention, which is discussed in the Examples section of this application. [Diagram 3] Figure 3 shows T cell responses (corrected for PBS negative control) induced by a single dose of 25 μg VB10.COV2 DNA plasmid in C57BL / 6-McphlTg(HLA-A2.1)lEnge / J transgenic mice. Total number of IFN-γ positive spots per 1x106 pooled splenocytes harvested 14 days post vaccination from mice (n=4) vaccinated with a single dose of 25 μg VB10.COV2 DNA plasmid and stimulated with 2 μg / mL peptide / peptide pool composed of peptides corresponding to human HLA-A2.1 epitopes / epitope groups present in each construct. [Figure 4] Figure 4 shows T cell responses induced by a single dose of 50 μg VB10.COV2 DNA plasmid in C57BL / 6-McphlTg(HLA-A2.1)lEnge / J transgenic mice (corrected for PBS negative control). Total number of IFN-γ positive spots per 1x106 pooled splenocytes harvested 14 days post vaccination from mice (n=3) vaccinated with a single dose of 50 μg VB10.COV2 DNA plasmid and stimulated with 4 μg / mL peptide / peptide pool composed of peptides corresponding to human HLA-A2.1 epitopes / epitope groups present in each construct. [Diagram 5]Figure 5 shows T cell responses induced by a single dose of 25 μg VB10.COV2 DNA plasmid in BALB / c mice (corrected for PBS negative control). Total number of IFN-γ positive spots per 1 x 106 pooled splenocytes harvested 14 days post vaccination from mice (n=3) vaccinated with 25 μg VB10.COV2 DNA plasmid and stimulated with 2 μg / mL peptide pools composed of overlapping 15-mer peptides covering all epitopes present in each construct. [Figure 6] Figure 6 shows T cell responses induced by a single dose of 25 μg VB10.COV2 DNA plasmid in C57BL / 6 mice (corrected for PBS negative control). Total number of IFN-γ positive spots per 1×106 pooled splenocytes harvested 14 days post-vaccination from mice (n=3) vaccinated with 25 μg VB10.COV2 DNA plasmid and stimulated with 2 μg / mL peptide pools composed of overlapping 15-mer peptides covering all epitopes present in each construct. [Figure 7] Figure 7 shows T cell responses induced by a single dose of 25 μg VB2210 DNA plasmid in C57BL / 6 mice (corrected for PBS negative control). Total number of IFN-γ positive spots per 1×106 pooled splenocytes harvested 14 days post vaccination from mice (n=3) vaccinated with 25 μg VB2210 and stimulated with 4 μg / ml peptide / peptide pool composed of peptides corresponding to human HLA-A2.1 epitopes / epitope groups present in the construct. [Figure 8]Figure 8 shows T cell responses (corrected for PBS negative control) induced by one or two doses of 1 μg, 5 μg, or 25 μg VB2210 DNA plasmid in C57BL / 6-McphlTg(HLA-A2.1)lEnge / J transgenic mice. Total number of IFN-γ positive spots per 1×106 splenocytes harvested 14 days after the first or second vaccination (second vaccination on day 21) from mice (n=4) vaccinated with one or two doses of 1 μg, 5 μg, or 25 μg VB2210 DNA plasmid and stimulated with a 2 μg / mL peptide pool composed of overlapping 15-mer peptides covering all epitopes / epitope groups contained in the construct. Data are shown as mean ± SEM of the total response. [Figure 9] Figure 9 shows T cell responses (corrected for PBS negative control) induced by one or two doses of 1 μg, 5 μg, or 25 μg of VB2210 DNA plasmid in C57BL / 6-McphlTg(HLA-A2.1)lEnge / J transgenic mice. Total number of IFN-γ positive spots per 1×106 splenocytes from mice (n=4) vaccinated with one or two doses of 1 μg, 5 μg, or 25 μg of VB2210 DNA plasmid and stimulated with a 4 μg / mL peptide pool composed of peptides covering specific human HLA-A2.1 epitopes contained in the construct. Splenocytes were harvested 14 days after a single or second vaccination (second vaccination on day 21). Data are shown as mean ± SEM of the sum of responses. [Figure 10]Figure 10 shows T cell responses induced in C57BL / 6 wild type mice by one or two doses of 1 μg, 5 μg, or 25 μg of VB2210 DNA plasmid. Total number of IFN-γ positive spots per 1×106 splenocytes harvested from mice vaccinated with one or two doses of 1 μg, 5 μg, or 25 μg of VB2210 DNA plasmid and stimulated with a 4 μg / ml peptide pool composed of peptides covering epitopes contained in the construct. Splenocytes were harvested 84 days after a single dose or 85 days after the first dose (if a second dose was administered). Data are presented as mean ± SEM of total responses. [Figure 11] Figure 11 shows populations of CD8+ T cells expressing one or two cytokines obtained from C57BL / 6-McphlTg(HLA-A2.1)lEnge / J transgenic mice vaccinated with one or two doses of 25 μg of VB2210 DNA plasmid. Splenocytes were harvested 14 days after the first or second vaccination (day 21 for the second vaccination) and stimulated with 6 μg / ml of a peptide pool composed of immunogenic peptides identified in a previous ELISpot assay that correspond to multiple HLA-A2.1 epitopes present in the construct. Multicolor staining followed by multiparameter functional analysis was performed to assess the expression of IFN-γ, TNF-α, IL-2, IL-4, IL-17, and FoxP3 in stimulated cells. [Figure 12]Figure 12 shows T cell responses (corrected for negative control) induced by two doses of 3 mg VB10.2210 DNA plasmid in healthy volunteers. The total number of IFN-γ positive spots per 1x106 PBMCs before (baseline) and after two vaccinations (peak) from healthy volunteers (n=11) vaccinated with two doses of 3 mg VB10.2210 DNA plasmid and stimulated with a peptide pool consisting of 15-mer overlapping peptides and selected minimal peptides covering all epitopes present in VB10.2210. Box plots represent median values. Whiskers indicate the minimum 25th percentile (-1.5IQR) and maximum 75th percentile (+1.5IQR). Figure 12a shows T cell responses against spike epitopes and Figure 12b shows total T cell responses against N, M, ORF1 / 3 / 10, and ORF7 epitopes. [Figure 13] Figure 13 shows T cell responses (corrected for negative control) induced by two doses of 3 mg VB10.2210 DNA plasmid in healthy volunteers. The total number of IFN-γ positive spots per 1×10 6 PBMCs before (baseline) and after two vaccinations (peak) from healthy volunteers (n=11) vaccinated with two doses of 3 mg VB10.2210 DNA plasmid and stimulated with a peptide pool consisting of 15-mer overlapping peptides and selected minimal peptides covering all epitopes present in VB10.2210. Figure 13a shows T cell responses against the spike epitope, Figure 13b shows T cell responses against the M epitope, Figure 13c shows T cell responses against the N epitope, Figure 13d shows T cell responses against the ORF1 / 3 / 10 epitope, and Figure 13e shows T cell responses against the ORF7 epitope. [Figure 14]Figure 14 shows the T cell responses (corrected for negative control) induced after two doses of 3 mg VB10.2210 DNA plasmid in two separate subjects. The total number of IFN-γ positive spots per 1x106 PBMCs before the first vaccination (day 0), after the first vaccination (day 21) and after the second vaccination (day 35) from healthy volunteers stimulated with a peptide pool composed of overlapping 15-mer peptides covering all epitopes present in VB10.2210 and selected minimal peptides (Figure 14a: Participant #4; Figure 14b: Participant #9). [Figure 15] Figure 15 shows the phenotype of polyfunctional vaccine-specific T cells in healthy volunteers vaccinated with two doses of 3 mg VB10.2210 DNA plasmid. PBMCs before the first vaccination (baseline) and after the second vaccination (peak) were peptide stimulated for 16 hours, followed by intracellular staining for phenotypic markers (CD4, CD8) and cytokine production (TNF-α and IFN-γ) and multiparameter analysis by flow cytometry. Figures 15a and 15b show the negative control (PBMCs only in cell culture medium with DMSO concentration corresponding to the peptide pool) at baseline and peak, respectively. Figure 15c shows the gating of the CD8+ T cell population, and Figure 15d shows the TNF-α and IFN-γ expression of CD8+ T cell populations stimulated with non-spike epitopes (M, N, and ORF). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Polynucleotides, polypeptides, and multimeric proteins are referred to herein as "immunogenic constructs" or simply "constructs." An "immunogenic construct" is one that elicits an immune response when administered to a subject in a form suitable for administration and in an amount effective to elicit an immune response (i.e., an immunologically effective amount).

[0034] "Subject" refers to a human individual. A subject may be a patient, i.e., a human individual suffering from a disease caused by SARS-CoV-2 that requires therapeutic treatment. The terms "subject" and "individual" are used interchangeably herein. The terms "human" and "h" are used interchangeably herein to refer to a human.

[0035] "Treatment" may be a prophylactic or therapeutic treatment.

[0036] A "prophylactic treatment" is a treatment administered to a subject who does not show signs or symptoms of disease caused by SARS-CoV-2 or who shows only early signs or symptoms of disease caused by SARS-CoV-2, and thus the treatment is administered with the purpose of preventing or reducing the risk of disease onset. Prophylactic treatment functions as a preventative treatment against disease caused by SARS-CoV-2 or as a treatment that inhibits or reduces further progression or enhancement of disease. The terms "prophylactic treatment", "prevention", and "preventing" are used interchangeably herein.

[0037] A "therapeutic treatment" is a treatment administered to a subject who has tested positive for SARS-CoV2 and / or who exhibits symptoms or signs of a disease caused by SARS-CoV-2, where the treatment is administered to the subject with the intent to reduce or eliminate those signs or symptoms.

[0038] A "nucleotide sequence" is a sequence made up of nucleotides. The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein.

[0039] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0040] immunogenic constructs The immunogenic construct of the present invention can be described as a polypeptide having an N-terminal beginning and a C-terminal end (as shown in FIG. 1). The elements / units of the polypeptide (targeting unit (TU) targeting APCs, multimerization units such as the dimerization unit (DimU) of FIG. 1, and antigen unit) may be arranged in the polypeptide such that the antigen unit is located at the C-terminal end of the polypeptide (FIG. 1a) or at the N-terminal beginning of the polypeptide (FIG. 1b). Preferably, the antigen unit is located at the C-terminal end of the polypeptide.

[0041] The antigenic unit comprises at least 77 SARS-CoV-2 T cell epitopes, either as separate epitopes or in groups together in one or more groups, and may comprise separate T cell epitopes, T cell epitopes in groups, and / or linkers (T cell epitope linkers) separating T cell epitope groups from each other. A unit linker (UL) may connect the multimerization unit and the antigenic unit. The order and orientation of the above units and elements are the same for dimeric proteins and polynucleotides.

[0042] In the following, the various units and elements of the construct are discussed in detail. They exist in polynucleotides as nucleic acid sequences that code for the units / elements, and in polypeptides or multimeric proteins as amino acid sequences. For ease of reading, in the following, the units / elements of the construct are mainly described in the context of polypeptides / multimeric proteins, i.e., based on amino acid sequences.

[0043] Antigenic unit The antigenic units present in the constructs of the invention comprise at least 77 T cell epitopes derived from SARS-CoV-2. These 77 T cell epitopes are listed below in Table 1 with their respective amino acid sequences and sequence identifiers (SEQ ID NOs) according to the SARS-CoV-2 proteins from which they are derived:

[0044] [Table 1-1] [Table 1-2]

[0045] Each of the 77 T cell epitopes contained in the antigenic unit has either an amino acid sequence listed in Table 1 above or an amino acid sequence having at least 73% sequence identity thereto. Thus, the antigenic unit includes T cell epitope 1 having the amino acid sequence of SEQ ID NO:1 (i.e., SRTLSYYKLGASQRVAGDS) or an amino acid sequence having at least 73% sequence identity thereto, T cell epitope 2 having the amino acid sequence of SEQ ID NO:2 (i.e., PKEITVATSRTLSYYKLGA) or an amino acid sequence having at least 73% sequence identity thereto, and T cell epitope 3 having the amino acid sequence of SEQ ID NO:3 (i.e., LRIAGHHLGRCDIKDLPKE) or an amino acid sequence having at least 73% sequence identity thereto.

[0046] In some embodiments, the 77 SARS-CoV-2 T cell epitopes have an amino acid sequence having at least 73% sequence identity, e.g., at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to the amino acid sequence of SEQ ID NOs: 1-77. In other embodiments, the 77 SARS-CoV-2 T cell epitopes have the amino acid sequence of SEQ ID NOs: 1-77.

[0047] In some other embodiments, the 77 SARS-CoV-2 T cell epitopes have the amino acids of SEQ ID NOs: 1-77, in which no more than 6 amino acids, for example no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid, are deleted, added, or substituted by other amino acids, as compared to the amino acid sequence of SEQ ID NOs: 1-77. In some embodiments, in the case of T cell epitopes having a length of 8-11 amino acids, no more than 3 amino acids, preferably no more than 2 amino acids or no more than 1 amino acid are deleted, added, or substituted by other amino acids, as compared to the amino acid sequence of SEQ ID NOs: 1-77. In some other embodiments, in the case of T cell epitopes having a length of 13-14 amino acids, no more than 4 amino acids, preferably no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid are deleted, added, or substituted by other amino acids, as compared to the amino acid sequence of SEQ ID NOs: 1-77. In still other embodiments, in the case of a T cell epitope having a length of 19 to 20 amino acids, 6 or less amino acids, preferably 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less amino acid are deleted, added, or substituted with other amino acids compared to the amino acid sequences of SEQ ID NOs: 1 to 77.

[0048] The 77 SARS-CoV-2 T cell epitopes listed in Tables 2 and 3 below and any additional SARS-CoV-2 T cell epitopes that are or may be included in the antigenic unit were selected from the pool of T cell epitopes identified in COVID-19 patients, applying the following criteria:

[0049] A subset of said T cell epitopes bind to HLA class I alleles and another subset binds to HLA class II alleles. Preferably, a higher proportion of the T cell epitopes contained in the antigenic unit bind to HLA class I alleles than to HLA class II alleles. In one embodiment, at least 60%, such as at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%, or at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75% of the T cell epitopes bind to HLA class I alleles.

[0050] The T cell epitopes are predicted to bind to different HLA class I and class II alleles covering an average of 92% of the world's population (from 85% in the West Indies to 99% in Europe), therefore vaccines comprising the constructs of the invention should be suitable for treatment and prophylactic use worldwide.

[0051] The T cell epitopes are conserved T cell epitopes in multiple different SARSCoV-2 strains worldwide. The selected epitopes are located in regions with low entropy, taking into account the genetic diversity of the SARS-CoV-2 genome calculated using a worldwide collection of samples.

[0052] Inclusion of at least 77 T cell epitopes in the immunogenic construct ensures a lower risk of immune evasion compared to spike-only based vaccines, which carry a higher risk of mutations in key antibody epitopes on the spike surface protein that prevent neutralization.

[0053] The T cell epitopes disclosed herein are derived from various SARS-CoV-2 viral structural proteins, namely N (nucleocapsid) protein, S (spike) protein, and M (membrane) protein, as well as various SARS-CoV-2 viral nonstructural proteins, namely ORFlab, ORF3a, ORF7a, ORF7b, and ORF10. Within a particular SARS-CoV-2 protein, the T cell epitopes may be derived from the same part of the protein (e.g., the same subunit) or from different parts of the protein. In one embodiment, the T cell epitopes are derived from different parts of a particular SARS-CoV-2 protein. For example, of the 12 T cell epitopes from the S protein listed in Table 1, 6 T cell epitopes are derived from the S1 subunit (epitopes 66-71) and 6 T cell epitopes are derived from the S2 subunit (epitopes 72-77).

[0054] In an antigenic unit, in some embodiments, some or all of the T cell epitopes disclosed herein are flanked by amino acid sequences that are similarly flanked by the epitope in the naturally occurring protein from which the epitope is derived, e.g., flanked by amino acid sequences that are flanked by the epitope in the N-terminal, C-terminal, or both directions. Each such flanking sequence may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids. In preferred embodiments, such flanking sequences comprise 1 to 10 amino acids, e.g., 2 to 8 amino acids, or 3 to 7 amino acids, or 4 to 5 amino acids.

[0055] In some embodiments, the antigenic unit comprises, as single and distinct epitopes, at least all of the 77 T cell epitopes, and optionally one or more additional T cell epitopes selected from those listed in Tables 2 and 3, which may be separated from each other by a T cell epitope linker.

[0056] In some other embodiments, the antigenic unit comprises one or more groups comprising at least two of the T cell epitopes disclosed herein, e.g., 2-20 epitopes, preferably 2-15 epitopes, e.g., 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14, or 15 epitopes. Within a group, the epitopes may be separated from each other by a T cell epitope linker. Alternatively, the epitope groups are separated from each other by a T cell epitope linker.

[0057] Preferably, at least two T cell epitopes are derived from the same SARS-CoV-2 protein, more preferably from the same portion of the same SARS-CoV-2 protein. For example, an antigenic unit may comprise a first group comprising four T cell epitopes (epitopes 1-4) derived from the M protein listed in Table 1, a second group comprising six T cell epitopes (epitopes 66-71) derived from the S1 subunit, and a third group comprising six T cell epitopes (epitopes 72-77) derived from the S2 subunit. The epitopes of the first, second, and third groups may be separated from each other by a T cell epitope linker, and the first, second, and third groups may be separated from each other by a T cell epitope linker. In another embodiment, the third group may be divided into two groups: a third group comprising three T cell epitopes (72-74) and a fourth group comprising three T cell epitopes (75-77).

[0058] In some embodiments, the T cell epitopes within a group are arranged sequentially. For example, epitopes 1-4 from M protein may be arranged in the order epitope 1-epitope 2-epitope 3-epitope 4 in a first group (or any other combination or permutation of the above four epitopes). The epitopes may be separated from each other by a T cell epitope linker.

[0059] Preferably, the T cell epitopes within a group are aligned to form a contiguous sequence of amino acids that corresponds to the sequence of the naturally occurring protein from which the epitopes are derived, whereby overlapping sequences are included in the contiguous sequence and are therefore included only once in the antigenic unit. As an example, epitopes 1-4 from the M protein are aligned and the resulting aligned sequence of epitopes (1-4) are included as a group in the antigenic unit: Epitope 3 LRIAGHHLGRCDIKDLPKE Epitope 4 GRCDIKDLPKE ITVATSRT Epitope 2 PKEITVATSRT LSYYKLGA Epitope 1 SRTLSYYKLGA SQRVAGDS The resulting array of groups: LRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDS

[0060] Such groups above may also be flanked by amino acid sequences that are similarly flanked by the group sequence in the naturally occurring protein from which the group sequence is derived, e.g., amino acid sequences that are flanked by the group sequence in the N-terminus, C-terminus, or both directions. Each such flanking sequence may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids. In preferred embodiments, such flanking sequences comprise 1 to 10 amino acids, e.g., 2 to 8 amino acids, or 3 to 7 amino acids, or 4 to 5 amino acids. By way of example, the above sequences of group epitopes 1 to 4 from M protein may comprise the following flanking amino acids (underlined): H LRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDS G

[0061] The resulting length of such a group, comprising the aligned sequences of at least two T cell epitopes disclosed herein and optionally adjacent amino acid sequences, is from about 18 amino acids to about 80 amino acids, such as from about 20 amino acids to about 70 amino acids, or from about 21 amino acids to about 67 amino acids.

[0062] In yet some other embodiments, the antigenic unit comprises the T cell epitopes disclosed herein in the form of one or more distinct T cell epitopes (e.g., epitopes 21, 85, and / or 119), as well as one or more groups comprising at least two T cell epitopes, preferably at least two T cell epitopes from the same SARS-CoV-2 protein, and more preferably at least two T cell epitopes from the same portion of the same SARS-CoV-2 protein.

[0063] In some embodiments, of the at least 77 SARS-CoV-2 T cell epitopes, the following are in a group together: epitopes 1-4, epitopes 5-9 (in another embodiment, this group is divided into two groups, the first group being epitopes 5-6 and the second group being epitopes 7-9), epitopes 10-18 (in another embodiment, this group is divided into two groups, the first group being epitopes 10-16 and the second group being epitopes 17-18). ), epitopes 19-20, epitopes 22-23, epitopes 24-26, epitopes 27-39, epitopes 40-43, epitopes 44-47, epitopes 48-58, epitopes 59-65, epitopes 66-71, and epitopes 72-77 (in another embodiment, this group is divided into two groups, the first group being epitopes 72-74 and the second group being epitopes 75-77).

[0064] In some embodiments, the antigenic unit does not include a T cell epitope derived from the receptor binding domain (RED) of the SARS-CoV-2 spike protein. An antigenic unit that includes only the 77 T cell epitopes listed in Table 1 (or includes only the 77 T cell epitopes listed in Table 1 and other T cell epitopes not derived from the RBD (e.g., one or more of the SARS-CoV-2 T cell epitopes listed in Table 3) does not include a T cell epitope derived from the RBD.

[0065] In some other embodiments, the antigenic unit further comprises a SARS-CoV-2 T cell epitope from the RBD, preferably one or more of the 19 SARS-CoV-2 T cell epitopes listed in Table 2 having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 78-96.

[0066] [Table 2]

[0067] In some embodiments, the antigenic unit comprises one or more of the 19 SARS-CoV-2 T cell epitopes listed in Table 2, wherein the epitopes have an amino acid sequence having at least 73% sequence identity, e.g., at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to the amino acid sequence of SEQ ID NOs: 78-96. In another embodiment, the 19 SARS-CoV-2 T cell epitopes listed in Table 2 have the amino acid sequence of SEQ ID NOs: 78-96.

[0068] In another embodiment, the 19 SARS-CoV-2 T cell epitopes listed in Table 2 have amino acid sequence numbers 78-96, in which no more than 6 amino acids, such as no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid, are deleted, added, or substituted by other amino acids, as compared to the amino acid sequence of SEQ ID NO: 78-96. In one embodiment, in T cell epitopes having a length of 8-11 amino acids, no more than 3 amino acids, preferably no more than 2 amino acids, or no more than 1 amino acid are deleted, added, or substituted by other amino acids, as compared to the amino acid sequence of SEQ ID NO: 78-96. In another embodiment, in T cell epitopes having a length of 13-14 amino acids, no more than 4 amino acids, preferably no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid are deleted, added, or substituted by other amino acids, as compared to the amino acid sequence of SEQ ID NO: 78-96. In yet another embodiment, in a T cell epitope having a length of 19 to 20 amino acids, 6 or less amino acids, preferably 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less amino acid are deleted, added, or substituted with other amino acids compared to the amino acid sequence of SEQ ID NO: 78 to 96.

[0069] In some embodiments, the antigenic unit comprises two or more of the 19 SARS-CoV-2 T cell epitopes listed in Table 2. In another embodiment, the antigenic unit comprises two or more of the T cell epitopes listed in Table 2, with the following epitopes preferably in groups together: epitopes 78-84 (in another embodiment, this group is divided into two groups, the first group being epitopes 78 and 79 and the second group being epitopes 80-84), epitopes 86-96 (in another embodiment, this group is divided into two groups, the first group being epitopes 86-93 and the second group being epitopes 94-96).

[0070] In yet another embodiment, the antigenic unit comprises all of the 19 RBD-derived SARS-CoV-2 T cell epitopes listed in Table 2. Thus, the antigenic unit of the construct according to the invention comprises at least 96 SARSCoV-2 derived T cell epitopes having amino acid sequences having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 1-96.

[0071] Vaccines comprising the immunogenic constructs of the invention, whose antigenic units comprise one or more of the T cell epitopes from the RBD listed in Table 2, are preferably administered as a booster vaccine to individuals who have previously been vaccinated with a vaccine comprising an RBD antigen or a portion thereof, preferably a vaccine comprising an RBD antigen or a portion thereof as disclosed in PCT / EP2021 / 061602 and G. Norheim et al., bioRvix 2020, doi:https: / / doi.org / 10.1101 / 2020.12.08.416875.

[0072] In some embodiments, the antigen further comprises one or more of the 64 SARS-CoV-2 T cell epitopes listed in Table 3 having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 97-160.

[0073] [Table 3-1] [Table 3-2]

[0074] None of the T cell epitopes listed in Table 3 are derived from the RBD.

[0075] In some embodiments, the antigenic unit comprises 64 SARS-CoV-2 T cell epitopes listed in Table 3, the epitopes having an amino acid sequence having at least 73% sequence identity, e.g., at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to the amino acid sequence of SEQ ID NOs: 97-160. In another embodiment, the 64 SARS-CoV-2 T cell epitopes listed in Table 3 have the amino acid sequence of SEQ ID NOs: 97-160.

[0076] In some other embodiments, the 64 SARS-CoV-2 T cell epitopes listed in Table 2 have the amino acid sequences of SEQ ID NOs: 97-160, in which no more than 6 amino acids, for example no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid, are deleted, added, or substituted by other amino acids, as compared to the amino acid sequences of SEQ ID NOs: 97-160. In one embodiment, in the T cell epitopes having a length of 8-11 amino acids, no more than 3 amino acids, preferably no more than 2 amino acids, or no more than 1 amino acid are deleted, added, or substituted by other amino acids, as compared to the amino acid sequences of SEQ ID NOs: 97-160. In other embodiments, in the T cell epitopes having a length of 13-14 amino acids, no more than 4 amino acids, preferably no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid are deleted, added, or substituted by other amino acids, as compared to the amino acid sequences of SEQ ID NOs: 97-160. In yet some other embodiments, in a T cell epitope having a length of 19 to 20 amino acids, 6 or fewer amino acids, preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid are deleted, added, or substituted with other amino acids compared to the amino acid sequence of SEQ ID NO: 97 to 160.

[0077] In some other embodiments, the antigenic unit comprises two or more of the T cell epitopes listed in Table 3. In another embodiment, the antigenic unit comprises two or more of the T cell epitopes listed in Table 3, with the following epitopes preferably in a group together: epitopes 97-102, epitopes 103-109, epitopes 110-111, epitopes 112-113, epitopes 114-118 (in other embodiments, this group is divided into two groups, the first group being epitopes 114-115 and the second group being epitopes 116-118), epitopes 120-123, epitopes 124-129, epitopes 130-134, epitopes 135-137, epitopes 136-138, epitopes 138-139, epitopes 139-200, epitopes 139-201, epitopes 139-202, epitopes 139-203, epitopes 139-204, epitopes 139-205, epitopes 139-206, epitopes 139-207, epitopes 139-208, epitopes 139-209, epitopes 140-141, epitopes 140-142, epitopes 140-143, epitopes 140-144, epitopes 141-145, epitopes 142-146, epitopes 143-147, epitopes 144-148, epitopes 145-149, epitopes 146-149, epitopes 147-148, epitopes 148-149, epitopes 149-2 8-140, epitopes 141-146 (in other embodiments, this group is divided into two groups, the first group being epitopes 141-143 and the second group being epitopes 144-146), epitopes 147-152 (in some other embodiments, this group is divided into two groups, the first group being epitopes 147-149 and the second group being epitopes 150-152), epitopes 153-160 (in some other embodiments, this group is divided into two groups, the first group being epitopes 153-157 and the second group being epitopes 158-160).

[0078] In yet some other embodiments, the epitopes of Table 1 and Table 3 are in a group together, for example in the group including epitopes 24-26 and 114-118.

[0079] In yet some other embodiments, the antigenic unit comprises all 64 SARS-CoV-2 derived T cell epitopes listed in Table 3. Thus, the antigenic unit of the construct according to the invention comprises at least 141 SARSCoV-2 derived T cell epitopes having amino acid sequences having at least 73% sequence identity with SEQ ID NOs: 1-77 and 97-160. In some embodiments, the antigenic unit does not comprise a T cell epitope derived from the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In some other embodiments, the antigenic unit additionally comprises one or more of the 19 RBD derived T cell epitopes listed in Table 2.

[0080] In yet some other embodiments, the antigenic unit comprises all of the T cell epitopes from SARS-CoV-2 listed in Table 3 and all of the T cell epitopes from the RBD listed in Table 2. Thus, the antigenic unit of the construct according to the invention comprises at least 160 T cell epitopes from SARSCoV-2 having amino acid sequences having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-160.

[0081] Further embodiments of antigenic units In some embodiments, the T cell epitope or group of T cell epitopes are randomly arranged in the antigenic unit. In some other embodiments, the T cell epitopes are arranged in the antigenic unit such that the most hydrophobic T cell epitope is located at or near the center of the antigenic unit and the most hydrophilic T cell epitope is located at or near the N- and C-terminal portions of the antigenic unit. In yet some other embodiments, the group of T cell epitopes with the most hydrophobic sequences are located at or near the center of the antigenic unit and the group of T cell epitopes with the most hydrophilic sequences are located at or near the N- and C-terminal portions of the antigenic unit. In some embodiments, the most hydrophobic T cell epitope or group of T cell epitopes may be located substantially in the center of the antigenic unit and the most hydrophilic T cell epitope or group of T cell epitopes may be located near the N- and C-terminal portions of the antigenic unit.

[0082] Since true positioning of an antigenic unit in the center is only possible if the antigenic unit comprises an odd number of T cell epitopes or groups of T cell epitopes, the term "substantially" in this context refers to an antigenic unit that comprises an even number of T cell epitopes or groups of T cell epitopes with the most hydrophobic T cell epitope located as close to the center as possible.

[0083] Alternatively, the T cell epitope or groups of T cell epitopes may be arranged alternately between hydrophilic and hydrophobic T cell epitopes / groups of T cell epitopes. Preferably, the GC-rich T cell epitope or groups of T cell epitopes are arranged to avoid GC clusters. In some embodiments, the GC-rich T cell epitope or groups of T cell epitopes are arranged such that there is at least one non-GC rich T cell epitope / group of T cell epitopes between them.

[0084] Linker in antigen unit In some embodiments, the T cell epitope or T cell epitopes are separated from each other by a T cell epitope linker (hereinafter also referred to as "linker"). The T cell epitope linker may also be included at the N-terminus or C-terminus of the antigenic unit.

[0085] In some other embodiments, the T cell epitope linker is designed to be non-immunogenic. The T cell epitope linker can be a rigid linker, which means that the two amino acid sequences that it connects cannot move substantially freely with each other. Alternatively, the T cell epitope linker can be a flexible linker, i.e., a linker that allows the two amino acid sequences that it connects to move substantially freely with each other. Either type of linker is useful.

[0086] In some embodiments, the T cell epitope linker is a flexible linker that is capable of presenting a T cell epitope or a group of T cell epitopes to the immune system in an optimal manner, even when the antigenic unit contains multiple T cell epitopes.

[0087] In some embodiments, the T cell epitope linker is a peptide consisting of 4 to 40 amino acids, for example 35, 30, 25, or 20 amino acids, for example 5 to 20 amino acids, or 5 to 15 amino acids, or 8 to 20 amino acids, or 8 to 15 amino acids, 10 to 15 amino acids, or 8 to 12 amino acids. In some other embodiments, the T cell epitope linker consists of 10 amino acids. Preferably, the T cell epitope linker is a peptide consisting of 4 to 20 amino acids, for example 5 to 18 amino acids, or 6 to 15 amino acids, or 7 to 10 amino acids. In particularly preferred embodiments, the T cell epitope linker consists of 5 to 7 amino acids or 8 to 12 amino acids, for example 5, 6, 7, 8, 9, or 10 amino acids.

[0088] In one embodiment, all T cell epitope linkers contained in an antigen unit are identical. However, if one or more of the T cell epitopes or T cell epitopes contain a sequence similar to that of the linker, it may be advantageous to replace adjacent T cell epitope linkers with linkers of different sequences. Also, if a T cell epitope / linker junction is predicted to constitute an epitope by itself, it is preferable to use T cell epitope linkers of different sequences. In another embodiment, an antigen unit contains several different T cell epitope linkers, for example 2, 3, 4, or 5 different T cell epitope linkers.

[0089] In some embodiments, the T cell epitope linker is a flexible linker, preferably a flexible linker that includes small non-polar (e.g., glycine, alanine, or leucine) or polar (e.g., serine or threonine) amino acids. The small size of these amino acids provides flexibility and allows the connected amino acid sequence to move. The incorporation of serine or threonine maintains the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the antigen. In some embodiments, the flexible linker is a serine (S) and / or glycine (G) rich linker, i.e., a linker that includes several serine residues and / or several glycine residues.

[0090] In the following, m is an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In some embodiments, m is 2.

[0091] Preferably, the T cell epitope linker is a serine (S) and / or glycine (G) rich linker, i.e. a linker that comprises several serine residues and / or several glycine residues. Preferred examples are GGGGS (SEQ ID NO: 186), GGGSS (SEQ ID NO: 187), GGGSG (SEQ ID NO: 188), GGSGG (SEQ ID NO: 189), SGSSGS (SEQ ID NO: 190), or multiple variants thereof, such as GGGGSGGGGS (SEQ ID NO: 191), (GGGGS)m (SEQ ID NO: 192), (GGGSS)m (SEQ ID NO: 193), (GGSGG)m (SEQ ID NO: 194), (GGGSG)m (SEQ ID NO: 195), or (SGSSGS)m (SEQ ID NO: 196).

[0092] In another embodiment, the serine and / or glycine rich linker further comprises at least one leucine (L) residue, such as at least 1 or at least 2 or at least 3 leucine residues, such as 1, 2, 3, or 4 leucine residues.

[0093] In some embodiments, the T cell epitope linker comprises or consists of LGGGS (SEQ ID NO: 197), GLGGS (SEQ ID NO: 198), GGLGS (SEQ ID NO: 199), GGGLS (SEQ ID NO: 200), or GGGGL (SEQ ID NO: 201). In other embodiments, the linker comprises or consists of LGGSG (SEQ ID NO: 202), GLGSG (SEQ ID NO: 203), GGLSG (SEQ ID NO: 204), GGGLG (SEQ ID NO: 205), or GGGSL (SEQ ID NO: 206). In yet some other embodiments, the linker comprises or consists of LGGSS (SEQ ID NO: 207), GLGSS (SEQ ID NO: 208), or GGLSS (SEQ ID NO: 209).

[0094] In yet some other embodiments, the T cell epitope linker comprises or consists of LGLGS (SEQ ID NO:210), GLGLS (SEQ ID NO:211), GLLGS (SEQ ID NO:212), LGGLS (SEQ ID NO:213), or GLGGL (SEQ ID NO:214). In yet some other embodiments, the linker comprises or consists of LGLSG (SEQ ID NO:215), GLLSG (SEQ ID NO:216), GGLSL (SEQ ID NO:217), GGLLG (SEQ ID NO:218), or GLGSL (SEQ ID NO:219). In yet some other embodiments, the linker comprises or consists of LGLSS (SEQ ID NO:220) or GGLLS (SEQ ID NO:221).

[0095] In some other embodiments, the T cell epitope linker is a serine-glycine linker having a length of 10 amino acids and containing one or two leucine residues.

[0096] In some embodiments, the T cell epitope linker comprises or consists of LGGGSGGGGS (SEQ ID NO:222), GLGGSGGGGS (SEQ ID NO:223), GGLGSGGGGS (SEQ ID NO:224), GGGLSGGGGS (SEQ ID NO:225), or GGGGLGGGGS (SEQ ID NO:226). In some other embodiments, it comprises or consists of LGGSGGGGSG (SEQ ID NO:227), GLGSGGGGSG (SEQ ID NO:228), GGLSGGGGSG (SEQ ID NO:229), GGGLGGGGSG (SEQ ID NO:230), or GGGSLGGGSG (SEQ ID NO:231). In yet some other embodiments, the linker comprises or consists of LGGSSGGGSS (SEQ ID NO:232), GLGSSGGGSS (SEQ ID NO:233), GGLSSGGGSS (SEQ ID NO:234), GGGLSGGGSS (SEQ ID NO:235), or GGGSLGGGSS (SEQ ID NO:236).

[0097] In further embodiments, the T cell epitope linker comprises or consists of LGGGSLGGGS (SEQ ID NO:237), GLGGSGLGGS (SEQ ID NO:238), GGLGSGGLGS (SEQ ID NO:239), GGGLSGGGLS (SEQ ID NO:240), or GGGGLGGGGL (SEQ ID NO:241). In some other embodiments, the linker comprises or consists of LGGSGLGGSG (SEQ ID NO:242), GLGSGGLGSG (SEQ ID NO:243), GGLSGGGLSG (SEQ ID NO:244), GGGLGGGGLG (SEQ ID NO:245), or GGGSLGGGSL (SEQ ID NO:246). In yet some other embodiments, the linker comprises or consists of LGGSSLGGSS (SEQ ID NO:247), GLGSSGLGSS (SEQ ID NO:248), or GGLSSGGLSS (SEQ ID NO:249).

[0098] In yet some other embodiments, the T cell epitope linker comprises or consists of GSGGGA (SEQ ID NO:250), GSGGGAGSGGGA (SEQ ID NO:251), GSGGGAGSGGGAGSGGGA (SEQ ID NO:252), GSGGGAGSGGGAGSGGGAGSGGGA (SEQ ID NO:253), or GENLYFQSGG (SEQ ID NO:254). In yet some other embodiments, the linker comprises or consists of SGGGSSGGGS (SEQ ID NO:255), SSGGGSSGGG (SEQ ID NO:256), GGSGGGGSGG (SEQ ID NO:257), GSGSGSGSGSGS (SEQ ID NO:258), GGGSSGGGSG (SEQ ID NO:259), GGGSSS (SEQ ID NO:260), GGGSSGGGSSGGGSS (SEQ ID NO:261), or GLGGLAAA (SEQ ID NO:262).

[0099] In some other embodiments, the linker is a rigid linker. Such rigid linkers can help to efficiently separate (larger) antigens and prevent them from interfering with each other. In some embodiments, the linker comprises or consists of KPEPKPAPAPKP (SEQ ID NO: 263), AEAAAKEAAAKA (SEQ ID NO: 264), (EAAAK)m (SEQ ID NO: 265), PSRLEEELRRRLTEP (SEQ ID NO: 266), or SACYCELS (SEQ ID NO: 267). In yet other embodiments, the linker comprises or consists of TQKSLSLSPGKGLGGL (SEQ ID NO: 268). In yet some other embodiments, the linker comprises or consists of SLSLSPGKGLGGL (SEQ ID NO: 269).

[0100] In yet some other embodiments, the linker comprises or consists of GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 270), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 271), or ELKTPLGDTTHT (SEQ ID NO: 272), or EPKSCDTPPPCPRCP (SEQ ID NO: 273).

[0101] Examples of T cell epitope linkers are disclosed in WO2020 / 176797A1, paragraphs

[0098] to

[0099] and the sequences described therein, which are incorporated by reference herein, and in US2019 / 0022202A1, paragraphs

[0135] to

[0139] , which are incorporated by reference herein.

[0102] Antigenic unit design The T cell epitopes disclosed herein were identified with the aid of an advanced bioinformatics workflow. Validation of the immunogenicity of in silico predicted T cell epitopes is typically performed using cellular immune response assays that require live T cells and are limited in sensitivity and throughput. Briefly, for the SARS-CoV-2 antigens, co-applicant Adaptive Biotechnologies Corporation used its MIRA® (Multiplexed Assay for Identification of Receptor Antigen-specificity) (Klinger et al., 2015) and blood samples from individuals diagnosed with COVID-19 to map T cell receptors (TCRs) to over 500 class I and class II peptides derived from all 11 open reading frames (ORFs) of SARS-CoV-2. In parallel, Adaptive also used transgenic constructs covering these SARS-CoV-2 ORFs.

[0103] These data were combined with case-control data generated using Adaptive's immunoSEQ® assay to identify published TCR clonotypes associated with SARS-CoV-2 immune responses and functionally map the TCR clonotypes to SARS-CoV-2 epitopes / HLA (Snyder et al., 2020). 77 of the most immunogenic SARS-CoV-2 T cell epitopes were identified, in addition to 19 of the most immunogenic SARS-CoV-2 RBD-derived T cell epitopes, and 64 of the most immunogenic SARS-CoV-2 T cell epitopes were identified.

[0104] Genetic diversity data from available databases was used to assess the conservation of these epitopes. At the time of this analysis, a total of 700,000 SARS-CoV-2 sequences were available and considered. Furthermore, the distribution of all reported single nucleotide polymorphisms (SNPs) that define each of all reported variants of concern (https: / / outbreak.info / ) was examined across the 160 T cell epitopes. Of the 77 T cell epitopes present in all immunogenic constructs according to the invention, only 4 and 3 epitopes contained some of the SNPs that define the delta (B.1.617.2) and gamma (P.1) variants, respectively.

[0105] To assess the coverage of HLA diversity by selected T cell epitopes, the MHC class I binding prediction tool NetMHCpan v4.1 (www.services.healthtech.dtu.dk / service.php?NetMHCpan-4.1) was used to predict binding affinities for the 19 most prevalent HLA alleles worldwide (Table 4).

[0106] [Table 4]

[0107] To further explore how the selected T cell epitopes cover the world's population, we used the Epitope Immunodatabase's Population Coverage Tool (www.tools.iedb.org / population / ). Based on the analysis, the included set of 96 epitopes is guaranteed to cover an average of 92.2% of the world's population (Table 5).

[0108] [Table 5]

[0109] In addition to assessing population coverage with predicted HLA epitope binding approaches, we used a meta-analysis of Adaptive's MIRA data to determine hypothesized HLA restriction for identified immune-dominant epitopes (Snyder et al., 2020).

[0110] More specifically, in each MIRA experiment performed, the number of unique T cell lineages responding to epitopes across the SARS-CoV-2 genome was defined in the hundreds of individuals evaluated. In addition to these T cell readouts, HLA typing of the donor material was performed with four orders of magnitude precision, which allowed us to evaluate the putative HLA restriction of the measured T cell responses by searching for alleles that correlated with increased yields of antigen-specific TCRs. This analysis was performed using a Wilcoxon one-sided test for each HLA allele present in three or more donors, and was further enriched for HLA specificity by requiring each association to show a two-fold enrichment of the median TCR response between the HLA-positive and HLA-negative donor sets. No correction was made for HLA linkage disequilibrium, and only HLA associations remaining after Bonferroni correction were reported. Using this approach, a total of 44 HLA class I-restricted epitopes and 14 HLA class II-restricted epitopes were identified.

[0111] Using a case-control immune sequencing cohort of several thousand individuals, we identified shared public T cell receptors associated with SARS-CoV-2 infection. Using Fisher's exact test, we defined a set of "enhanced sequences" representing TCRb sequences enriched in cases and not enriched in controls. These enhanced sequences should contain receptors that are highly public and likely specific for SARS-CoV-2, and have been validated together as a tool to monitor recent or past infections (Dalai et al., 2021). As a further prioritization of candidate epitopes of interest, we performed overlap analysis between these enhanced sequences and MIRA data to pinpoint the antigenic specificity of these SARS-CoV-2 associated receptors. Epitopes with the highest number of overlapping enhanced sequences were prioritized for inclusion in the immunogenic constructs and compositions of the present invention, as they can generate highly public T cell responses corresponding to broad HLA prevalence. Thus, the immunogenic constructs and compositions according to the invention contain a large and unique set of SARS-CoV-2 T cell epitopes that have been validated to induce strong T cell responses in humans.

[0112] Several T cell vaccine candidates have been developed and tested in various mouse models. They were able to induce specific cellular immunity against SARS-CoV-2 T cell epitopes in humanized transgenic mice, HLA-DR mice (Meyers et al., 2021), HLA-A2.1 mice (Gauttier et al., 2020), and CD34 mice (Somogyi et al., 2020), as well as BALB / c mice (Gritstone COVID-19 Vaccine Technical Information, 2021). Furthermore, some of these T cell vaccine candidates have progressed to clinical development or are close to starting trials. In comparison, the immunogenic constructs and vaccines according to the present invention have a unique and larger set of SARS-CoV-2 T cell epitopes that have previously been validated to induce strong T cell responses in humans as well.

[0113] A preferred antigenic unit has an amino acid sequence that has at least 73% sequence identity with SEQ ID NOs:161-167.

[0114] Thus, in one embodiment, the antigenic unit comprises an amino acid sequence having at least 73% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0115] In a preferred embodiment, the antigenic unit comprises an amino acid sequence having at least 73% sequence identity, such as at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0116] In another embodiment, the 77 SARS-CoV-2 T cell epitopes have the amino acid sequences of SEQ ID NOs: 1-77.

[0117] In a more preferred embodiment, the antigenic unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0118] In another more preferred embodiment, the antigenic unit consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0119] Targeting Unit The constructs of the invention comprise a targeting unit that targets antigen-presenting cells (APCs), including dendritic cells (DCs) and subsets thereof.

[0120] The term "targeting unit" as used herein refers to a unit that delivers the constructs disclosed herein to antigen-presenting cells for MHC class II restricted presentation to CD4+ T cells or to provide MHC class I restricted cross-presentation to CD8+ T cells.

[0121] Due to the targeting unit, the construct disclosed herein attracts DCs, neutrophils, and other immune cells. Thus, the construct not only targets the antigen unit contained therein to specific cells, but also promotes a response amplification effect (adjuvant effect) by recruiting specific immune cells to the administration site of the vaccine containing the construct. This unique mechanism is very important in clinical practice, because the construct can be administered to a subject in the form of a vaccine that does not need to contain any adjuvant, since the construct contained in the vaccine provides the adjuvant effect.

[0122] The targeting unit is designed to target the construct of the invention to a surface molecule expressed on APCs, for example a molecule expressed exclusively on a subset of DCs.

[0123] Examples of such surface molecules on APCs are HLA, cluster of differentiation 14 (CD14), cluster of differentiation 40 (CD40), CLEC9A, chemokine receptors, and Toll-like receptors (TLRs). Chemokine receptors include CC motif chemokine receptor 1 (CCR1), CC motif chemokine receptor 3 (CCR3), CC motif chemokine receptor 4 (CCR4), CC motif chemokine receptor 5 (CCR5), CC motif chemokine receptor 6 (CCR6), CC motif chemokine receptor 7 (CCR7), CC motif chemokine receptor 8 (CCR8), and XCR1. Toll-like receptors include TLR-2, TLR-4, TLR-5. In one embodiment, the targeting unit is or includes a moiety that interacts with these surface molecules.

[0124] Thus, in some embodiments, the targeting unit comprises or consists of an antibody binding region, such as an antibody variable domain (VL and VH) with specificity for MHC / HLA, CD14, CD40, CLEC9A, or a Toll-like receptor, preferably with specificity for human (h)CD14, hCD40, hCLEC9A, or a human Toll-like receptor. In some other embodiments, the targeting unit comprises or consists of a synthetic or natural ligand. Examples include soluble CD40 ligand (CD40L), preferably hCD40L, natural ligands such as chemokines, preferably of human type, such as chemokine ligand 5 (also called CC motif ligand 5 (CCL5 or RANTES)), preferably hCCL5, such as hCCL5 of SEQ ID NO: 43, macrophage inflammatory protein alpha and its isoforms (murine CCL3 (or MIP-1α) and the human isoforms hCCL3, hCCL3L1, hCCL3L2, and hCCL 3L3), chemokine ligand 4 (CCL4) and its isoform CCL4L, preferably hCCL4 and hCCL4L, chemokine ligand 19 (CCL19), preferably hCCL19, chemokine ligand 20 (CCL20), preferably hCCL20, chemokine ligand 21 (CCL21), preferably hCCL21, chemokine motif ligand 1 or 2 (XCL1 or XCL2), preferably hXCL1 or hXCL2, and bacterial antigens, such as flagellin.

[0125] In some embodiments, the targeting unit has affinity for an MHC class II protein. Thus, in some embodiments, the targeting unit comprises or consists of an antibody binding region, such as an antibody variable domain (VL and VH) with specificity for an MHC class II protein selected from the group consisting of anti-HLA-DP, anti-HLA-DR, and anti-pan-HLA class II.

[0126] In some other embodiments, the targeting unit has affinity for a surface molecule selected from the group consisting of CD14, CD40, TLR-2, TLR-4, and TLR-5, preferably for a surface molecule selected from the group consisting of hCD14, hCD40, hTLR-2, hTLR-4, and hTLR-5. Thus, in some embodiments, the targeting unit comprises or consists of an antibody binding region such as an antibody variable domain (VL and VH) with specificity for CD14, CD40, TLR-2, TLR-4, or TLR-5, such as anti-CD14, anti-CD40, anti-TLR-2, anti-TLR-4, or anti-TLR-5, preferably an antibody variable domain (VL and VH) with specificity for hCD14, hCD40, hTLR-2, hTLR-4, or hTLR-5, such as anti-hCD14, anti-hCD40, anti-hTLR-2, anti-hTLR-4, or hTLR-5.

[0127] In yet some other embodiments, the targeting unit comprises or consists of a flagellin having affinity for TLR-5, e.g., hTLR-5. In yet other embodiments, the targeting unit comprises or consists of an antibody binding region having specificity for CLEC9A, e.g., anti-CLEC9A or a variant thereof, e.g., anti-CLEC9A Fv, or the targeting unit comprises or consists of a CLEC9 ligand, e.g., a CLEC9 ligand comprising or consisting of a nucleic acid sequence having SEQ ID NO: 274 or an amino acid sequence encoded by said nucleic acid sequence. In preferred embodiments, the targeting unit comprises or consists of an antibody binding region having specificity for hCLEC9A, e.g., anti-hCLEC9A or a variant thereof, e.g., anti-hCLEC9A Fv, or the targeting unit comprises or consists of a human CLEC9 ligand.

[0128] Preferably, the targeting unit has affinity for a chemokine receptor selected from CCR1, CCR3, CCR5 and CCR7, more preferably for a chemokine receptor selected from CCR1, CCR3 and CCR5.In a further preferred embodiment, the targeting unit has affinity for a chemokine receptor selected from hCCR1, hCCR3, hCCR5 and hCCR7, more preferably for a chemokine receptor selected from hCCR1, hCCR3 and hCCR5.

[0129] In some embodiments, the targeting unit has affinity for the chemokine receptor CCR7, preferably for the human chemokine receptor CCR7. In some other embodiments, the targeting unit comprises or consists of CCL19, e.g., CCL19 comprising or consisting of the nucleotide sequence of SEQ ID NO: 275 or the amino acid sequence encoded by said nucleotide sequence, or CCL21, e.g., the human form of CCL19 or CCL21.

[0130] Preferably, the targeting comprises or consists of the chemokine human macrophage inflammatory protein alpha (also called human MIP-1α (hMIP-1α) variant, LD78β or CCL3L1), which binds to its cognate receptors, including CCR1, CCR3, and CCR5, expressed on the cell surface of APCs. Binding of the targeting unit to its cognate receptor results in the internalization of the multimeric protein into APCs, where the protein is degraded into small peptides that are loaded onto MHC molecules and presented to CD4+ and CD8+ T cells to induce a specific immune response. Upon stimulation, CD8+ T cells, with the help of activated CD4+ T cells, target and kill cells expressing the same antigen, such as cancer cells expressing the same antigen.

[0131] In a preferred embodiment, the targeting unit comprises an amino acid sequence having at least 80% sequence identity with amino acid sequence 24-93 of SEQ ID NO: 168. In a further preferred embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with amino acid sequence 24-93 of SEQ ID NO: 168. In a further preferred embodiment, the targeting unit comprises amino acid sequence 26-93 of SEQ ID NO: 168 or comprises amino acid sequence 24-93 of SEQ ID NO: 168.

[0132] In a more preferred embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity with amino acids 24 to 93 of SEQ ID NO:168.

[0133] In a further preferred embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to amino acid sequence 24-93 of SEQ ID NO: 168. In yet another preferred embodiment, the targeting unit has amino acid sequence 26-93 of SEQ ID NO: 168 or has amino acid sequence 24-93 of SEQ ID NO: 168.

[0134] In another embodiment, the targeting unit comprises or is anti-pan-HLA class II.

[0135] In a preferred embodiment, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 20-260 of SEQ ID NO:169.

[0136] In a further preferred embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to amino acid sequence 20-260 of SEQ ID NO: 169. In a further preferred embodiment, the targeting unit has amino acid sequence 20-260 of SEQ ID NO: 169.

[0137] In a more preferred embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity with amino acid sequence 20 to 260 of SEQ ID NO:169.

[0138] In a further preferred embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to amino acid sequence 20-260 of SEQ ID NO: 169. In yet another preferred embodiment, the targeting unit consists of amino acid sequence 20-260 of SEQ ID NO: 169.

[0139] Multimerization Unit / Dimerization Unit The constructs of the invention include a multimerization unit, such as a dimerization unit.

[0140] The term "multimerization unit" as used herein refers to a nucleotide or amino acid sequence between the antigen unit and the targeting unit, which, in addition to connecting the antigen unit and the targeting unit, also facilitates the multimerization of multiple polypeptides, such as two, three, four or more polypeptides, and binds them into a multimeric protein, such as a dimeric protein, a trimeric protein, or a tetrameric protein. In addition, the multimerization unit also provides flexibility to the multimeric protein, allowing the targeting unit to optimally bind to surface molecules on the APC even when the surface molecules are located at various distances. The multimerization unit may be any unit that meets one or more of these requirements.

[0141] A multimerization unit that promotes / links the multimerization of two or more polypeptides. In some embodiments, the multimerization unit is a trimerization unit, such as a trimerization unit derived from collagen, a trimerization domain derived from human collagen, a trimerization domain derived from human collagen XVIII (see, for example, A. Alvarez-Cienfuegos et al., Sci Rep 6, 28643 (2016)), or a trimerization domain derived from human collagen XV. Thus, in some embodiments, the multimerization unit is a trimerization unit that comprises or consists of the nucleic acid sequence of SEQ ID NO: 276 or the amino acid sequence encoded by said nucleic acid sequence. In some other embodiments, the trimerization unit is the C-terminal domain of T4 fibritin. Thus, in some embodiments, the multimerization unit is a trimerization unit that comprises or consists of the amino acid sequence of SEQ ID NO: 277. In some embodiments, the trimerization unit further comprises a hinge region, as described below. In some other embodiments, the multimerization unit is a tetramerization unit, such as a domain derived from p53, optionally further comprising a hinge region as described below. Thus, in some embodiments, the multimerization unit is a tetramerization unit comprising or consisting of the nucleic acid sequence of SEQ ID NO: 278 or an amino acid sequence encoded by said nucleic acid sequence, optionally further comprising a hinge region as described below.

[0142] Dimerization Unit The term "dimerization unit" as used herein refers to the nucleotide or amino acid sequence between the antigen unit and the targeting unit. In addition to connecting the antigen unit and the targeting unit, the dimerization unit promotes the dimerization of two polypeptides, binding them into a dimeric protein. The dimerization unit also provides flexibility to the dimeric protein, allowing the targeting unit to optimally bind to surface molecules on the APC even when the surface molecules are located at various distances. The dimerization unit may be any unit that meets these requirements.

[0143] Thus, in some embodiments, the construct of the invention comprises a dimerization unit comprising a hinge region. In some other embodiments, the dimerization unit comprises a hinge region and another domain that promotes dimerization. In yet some other embodiments, the dimerization unit comprises a hinge region, a dimerization unit linker, and another domain that promotes dimerization, the dimerization unit linker connecting the hinge region and the other domain that promotes dimerization. In some embodiments, the dimerization unit linker is a glycine-serine rich linker, preferably GGGSSGGGSG (SEQ ID NO: 259), i.e., the dimerization unit comprises a glycine-serine rich dimerization unit linker, preferably the dimerization unit linker GGGSSGGGSG.

[0144] The term "hinge region" refers to an amino acid sequence contained in a dimerization unit that contributes to the binding of two polypeptides, i.e., promotes the formation of a dimeric protein. Moreover, the hinge region functions as a flexible spacer, which allows two targeting units of a dimeric protein to simultaneously bind to two surface molecules on an APC, even if the surface molecules are located at different distances. In the context of a multimerization unit that promotes / binds the multimerization of three or more polypeptides, the term "hinge region" refers to an amino acid sequence contained in such a multimerization unit that contributes to binding three or more polypeptides, e.g., three or four polypeptides, and / or acts as a flexible spacer, which allows multiple targeting units of a multimeric protein to simultaneously bind to multiple surface molecules on an APC, even if the surface molecules are located at different distances.

[0145] The hinge region may be from an Ig, such as from an IgG, such as from an IgG1 or IgG2 or IgG3, such as from an hlg, such as from a human IgG, such as from a hIgG1 or hIgG2 or hIgG3. In some embodiments, the hinge region is from an IgM, such as from a human IgM. In some embodiments, the hinge region comprises or consists of the nucleotide sequence of SEQ ID NO: 279 or the amino acid sequence encoded by said nucleic acid sequence. The hinge region may contribute to dimerization by forming a covalent bond, such as a disulfide bond between cysteines. Thus, in some embodiments, the hinge region has the ability to form one or more covalent bonds. Preferably, the covalent bond is a disulfide bridge.

[0146] In some embodiments, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 (human hinge region 1 and human hinge region 4), preferably hinge exon h1 and hinge exon h4 from IgG3 (more preferably having an amino acid sequence having at least 80% sequence identity with amino acid sequence 94-120 of SEQ ID NO: 168).

[0147] In a preferred embodiment, the dimerization unit comprises or consists of a hinge exon h1 and a hinge exon h4 having an amino acid sequence having at least 85% sequence identity to the amino acid sequence 94 to 120 of SEQ ID NO: 168, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0148] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having the amino acid sequence 94 to 120 of SEQ ID NO:168.

[0149] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 94 to 120 of SEQ ID NO: 168, except that up to four amino acids, such as up to three amino acids, such as up to two amino acids, or such as up to one amino acid, have been substituted, deleted or inserted.

[0150] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO:280.

[0151] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to the nucleic acid sequence of SEQ ID NO: 280.

[0152] In an even more preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO:280.

[0153] In some other embodiments, the dimerization unit comprises another domain that promotes dimerization, which is an immunoglobulin domain, such as an immunoglobulin constant domain (C domain), such as a CH1 domain, a CH2 domain, or a carboxy-terminal C domain (i.e., a CH3 domain), or a sequence substantially identical to such a C domain or a variant thereof. Preferably, the other domain that promotes dimerization is a carboxy-terminal C domain from an IgG, such as human IgG3. More preferably, the other domain that promotes dimerization is a carboxy-terminal C domain from an IgG3, such as human IgG3.

[0154] In some embodiments, the dimerization unit comprises or consists of a carboxy-terminal C domain from IgG3 having an amino acid sequence having at least 80% sequence identity to amino acids 131 to 237 of SEQ ID NO:168.

[0155] In a preferred embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 85% sequence identity to the amino acid sequence 131-237 of SEQ ID NO: 168, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0156] In a preferred embodiment, the dimerization unit comprises or consists of the carboxy-terminal C domain from IgG3 having the amino acid sequence 131 to 237 of SEQ ID NO:168.

[0157] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 131-237 of SEQ ID NO: 168, except that up to 16 amino acids, such as up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, have been substituted, deleted, or inserted.

[0158] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO:281.

[0159] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to the nucleic acid sequence of SEQ ID NO: 281. In a further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO: 281.

[0160] Immunoglobulin domains contribute to dimerization through non-covalent interactions, such as hydrophobic interactions. Thus, in one embodiment, an immunoglobulin domain has the ability to form a dimer through non-covalent interactions. Preferably, the non-covalent interactions are hydrophobic interactions.

[0161] If the dimerization unit comprises a CH3 domain, it is preferred that the dimerization unit does not comprise a CH2 domain, and vice versa.

[0162] In a preferred embodiment, the dimerization unit comprises a hinge exon h1, a hinge exon h4, a dimerization unit linker, and a CH3 domain of human IgG3. In a further preferred embodiment, the dimerization unit comprises a polypeptide consisting of a hinge exon h1, a hinge exon h4, a dimerization unit linker, and a CH3 domain of human IgG3. In another preferred embodiment, the dimerization unit comprises a polypeptide consisting of a hinge exon h1, a hinge exon h4, a dimerization unit linker, and a CH3 domain of human IgG3.

[0163] In some embodiments, the dimerization unit linker is a glycine-serine rich linker, preferably GGGSSGGGSG, i.e. the dimerization unit comprises a glycine-serine rich dimerization unit linker, preferably the dimerization unit linker GGGSSGGGSG.

[0164] In some embodiments, the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of 94 to 237 of SEQ ID NO:168.

[0165] In a preferred embodiment, the dimerization unit comprises an amino acid sequence having at least 85% sequence identity to amino acid sequence 94-237 of SEQ ID NO: 168, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0166] In a more preferred embodiment, the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence 94 to 237 of SEQ ID NO: 168, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0167] In a further preferred embodiment, the dimerization unit consists of amino acids 94 to 237 of SEQ ID NO:168.

[0168] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 94-237 of SEQ ID NO: 168, except that up to 28 amino acids, such as up to 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, have been substituted, deleted, or inserted.

[0169] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO:282.

[0170] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to the nucleic acid sequence of SEQ ID NO: 282. In an even further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO: 282.

[0171] Unit Linker In some embodiments, the constructs disclosed herein comprise a unitary linker. In some embodiments, the antigen unit is connected to the targeting unit or multimerization unit by a unitary linker. Thus, in some embodiments, the constructs disclosed herein comprise a unitary linker that connects the antigen unit to the targeting unit or multimerization unit. In some embodiments, the unitary linker is a non-immunogenic linker and / or a flexible linker or a rigid linker.

[0172] The unit linker may include a restriction site to facilitate construction of the first nucleic acid sequence. In some embodiments, the unit linker is GLGGL (SEQ ID NO: 214) or GLSGL (SEQ ID NO: 283). In other embodiments, the unit linker comprises or consists of GGGGS (SEQ ID NO: 186), GGGGSGGGGS (SEQ ID NO: 191), (GGGGS)m (SEQ ID NO: 192), EAAAK (SEQ ID NO: 284), (EAAAK)m (SEQ ID NO: 265), (EAAAK)mGS (SEQ ID NO: 285), (EAAK)mGS (SEQ ID NO: 286), GPSRLEEELRRRLTEPG (SEQ ID NO: 287), AAY, or HEYGAEALERAG (SEQ ID NO: 288). m is an integer from 1 to 5, for example, 1, 2, 3, 4, or 5. In some embodiments, m is 2.

[0173] Signal peptide In a preferred embodiment, the construct of the present invention is a polynucleotide comprising a nucleotide sequence further encoding a signal peptide. The signal peptide is located either at the N-terminus of the targeting unit or at the C-terminus of the targeting unit, depending on the orientation of the targeting unit in the polypeptide (Figure 1). The signal peptide is designed to allow the secretion of the polypeptide encoded by the nucleic acid contained in the polynucleotide from cells transfected with the polynucleotide. Preferably, the signal peptide is naturally occurring (also called natural leader sequence) at the N-terminus of any of the targeting units described herein.

[0174] Any suitable signal peptide may be used. Examples of suitable peptides are an Ig VH signal peptide, preferably a human Ig VH signal peptide, a human TPA signal peptide, such as SEQ ID NO: 170, and a human MIP1-α signal peptide.

[0175] In a preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a human MIP1-α signal peptide, and preferably a nucleotide sequence encoding a human MIP1-α targeting unit.

[0176] In a further preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising an amino acid sequence having at least 85% sequence identity, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity, to amino acid sequence 1-23 of SEQ ID NO: 168.

[0177] In another preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising amino acid sequence 1-23 of SEQ ID NO:168.

[0178] In a more preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% sequence identity, to amino acid sequence 1 to 23 of SEQ ID NO: 168.

[0179] In another preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide having amino acid sequence 1-23 of SEQ ID NO:168.

[0180] In another preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding an Ig VH signal peptide, preferably an anti-pan-HLA class II targeting unit.

[0181] In a further preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising an amino acid sequence having at least 85% sequence identity, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity, to amino acid sequence 1-19 of SEQ ID NO: 169.

[0182] In another preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising amino acid sequence 1-19 of SEQ ID NO:169.

[0183] In a more preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% sequence identity, to amino acid sequence 1 to 19 of SEQ ID NO: 169.

[0184] In another preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide having amino acid sequence 1-19 of SEQ ID NO:169.

[0185] Preferred immunogenic constructs Preferred immunogenic constructs are those that comprise the following units:

[0186] Targeting Units: The targeting unit comprises an amino acid sequence having at least 80% sequence identity to amino acids 24 to 93 of SEQ ID NO:168.

[0187] In a further preferred embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to amino acid sequence 24-93 of SEQ ID NO: 168. In an even further preferred embodiment, the targeting unit comprises amino acid sequence 24-93 of SEQ ID NO: 168 or comprises amino acid sequence 26-93 of SEQ ID NO: 168.

[0188] In a more preferred embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity with amino acids 24 to 93 of SEQ ID NO:168.

[0189] In a further preferred embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to amino acid sequence 24 to 93 of SEQ ID NO: 168. In yet another preferred embodiment, the targeting unit consists of amino acid sequence 24 to 93 of SEQ ID NO: 168 or consists of amino acid sequence 26 to 93 of SEQ ID NO: 168.

[0190] Dimerization unit: The dimerization unit comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of 94 to 237 of SEQ ID NO:168.

[0191] In a preferred embodiment, the dimerization unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% sequence identity, to the amino acid sequence 94-237 of SEQ ID NO: 168. In a further preferred embodiment, the dimerization unit comprises the amino acid sequence 94-237 of SEQ ID NO: 168.

[0192] In a more preferred embodiment, the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence 94 to 237 of SEQ ID NO: 168, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0193] In an even more preferred embodiment, the dimerization unit consists of amino acids 94 to 237 of SEQ ID NO:168.

[0194] Antigenic unit The following embodiments are preferred embodiments of the antigenic unit or the antigenic unit itself (i.e., in the absence of targeting units and multimerization units such as dimerization units) contained in the immunogenic construct (for prophylactic or therapeutic use in a pharmaceutical composition).

[0195] The antigenic unit comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0196] In a preferred embodiment, the antigenic unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0197] In a more preferred embodiment, the antigenic unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0198] In another embodiment, the antigenic unit consists of an amino acid sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0199] In a preferred embodiment, the antigenic unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0200] In a more preferred embodiment, the antigenic unit has an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0201] In a most preferred embodiment, the immunogenic construct is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 171, 172, 173, 174, 175, 176, and 177, or a dimeric protein consisting of two such polypeptides. In another most preferred embodiment, the immunogenic construct is a polynucleotide comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 178, 179, 180, 181, 182, 183, and 184.

[0202] In an even more preferred embodiment, the immunogenic construct is a polypeptide having the amino acid sequence of SEQ ID NO: 177, or a dimeric protein consisting of two such polypeptides. In yet another most preferred embodiment, the immunogenic construct is a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 184. In yet another most preferred embodiment, the immunogenic construct is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 185.

[0203] Sequence identity Sequence identity can be determined as follows: a high level of sequence identity indicates that the second sequence is more likely to be derived from the first sequence. Amino acid sequence identity requires identical amino acid sequences between the two aligned sequences. Thus, a candidate sequence that shares 70% amino acid identity with a reference sequence requires that, after alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity can be determined with the aid of computer analysis, such as, but not limited to, the ClustalW computer alignment program (Higgins D., Thompson J., Gibson T., Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680) and the default parameters proposed therein. Using this program with default settings, the mature (biologically active) portions of the query and reference polypeptides are aligned. The number of perfectly conserved residues is counted and divided by the length of the reference polypeptide. In doing so, any tag or fusion protein sequences that form part of the query sequence are not taken into account in the alignment and subsequent determination of sequence identity.

[0204] The ClustalW algorithm may also be used to align nucleotide sequences. Sequence identity may be calculated in a similar manner as for amino acid sequences.

[0205] Another preferred mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated in the ALIGN program (version 2.0), which is part of the FASTA sequence alignment software package (Pearson WR, Methods Mol Biol, 2000, 132:185-219). ALIGN calculates sequence identity based on a global alignment. Align0 does not impose a penalty for gaps at the ends of the sequences. When using the ALIGN and Align0 programs to compare amino acid sequences, the BLOSUM50 substitution matrix is ​​preferably used, with gap opening / extension penalties of -12 / -2.

[0206] Polynucleotides Constructs of the invention may be in the form of a polynucleotide as described herein.

[0207] A further aspect of the present invention is a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77.

[0208] Yet another aspect of the present invention is a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77.

[0209] The polynucleotide may be DNA or RNA, including genomic DNA, cDNA, mRNA, either double-stranded or single-stranded. In a preferred embodiment, the polynucleotide is DNA.

[0210] In some embodiments, the polynucleotides are human codon optimized.

[0211] vector In some embodiments, the polynucleotides described herein are contained in a vector.

[0212] Thus, a further aspect of the present disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77.

[0213] Yet another aspect of the present disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, wherein the antigenic unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77.

[0214] A vector may be any molecule suitable for delivering foreign nucleic acid sequences, such as DNA or RNA, to a cell where they are expressed (in vitro or in vivo), ie, an expression vector.

[0215] In some embodiments, the vector is a DNA vector, such as a DNA plasmid or a DNA viral vector, such as a DNA viral vector selected from the group consisting of adenovirus, vaccinia virus, adeno-associated virus, cytomegalovirus, and Sendai virus.

[0216] In some other embodiments, the vector is an RNA vector, such as an RNA plasmid or an RNA viral vector, such as a retroviral vector, such as a retroviral vector selected from the group consisting of an alphavirus, a lentivirus, a Moloney murine leukemia virus, and a rhabdovirus.

[0217] In a preferred embodiment, the vector is a DNA plasmid and the polynucleotide is DNA.

[0218] Polycistronic Vectors In some embodiments, the vector is a polycistronic vector allowing expression of the polypeptide or antigenic units disclosed herein as separate molecules and further expression of one or more immunostimulatory compounds.

[0219] A further aspect of the present disclosure is (A) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; and (B) one or more nucleic acid sequences encoding one or more immunostimulatory compounds. which is capable of co-expressing the polypeptide and one or more immunostimulatory compounds as separate molecules.

[0220] Yet a further aspect of the present disclosure is a method for producing a method of manufacturing a semiconductor device comprising the steps of: (A) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; and (B) one or more nucleic acid sequences encoding one or more immunostimulatory compounds. which is capable of co-expressing the antigenic unit and one or more immunostimulatory compounds as separate molecules.

[0221] A polycistronic vector comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a targeting unit, a multimerization unit such as a dimerization unit, and an antigen unit, and one or more nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the polypeptide and the one or more immunostimulatory compounds are co-expressed as separate molecules, has been disclosed by the applicant in PCT / EP2022 / 062665 (the disclosure of which is incorporated herein by reference).

[0222] Polycistronic vectors may be any molecule suitable for delivering foreign nucleic acid sequences, such as DNA or NA, to cells where they are expressed (in vitro or in vivo), ie, expression vectors.

[0223] In some embodiments, the polycistronic vector is a DNA vector, such as a DNA plasmid or a DNA viral vector, such as a DNA viral vector selected from the group consisting of adenovirus, vaccinia virus, adeno-associated virus, cytomegalovirus, and Sendai virus.

[0224] In some other embodiments, the polycistronic vector is an RNA vector, such as an RNA plasmid or an RNA viral vector, such as a retroviral vector, such as a retroviral vector selected from the group consisting of an alphavirus, a lentivirus, a Moloney murine leukemia virus, and a rhabdovirus.

[0225] In a preferred embodiment, the vector is a polycistronic DNA plasmid. Although the polycistronic vector of the present disclosure is discussed and exemplified in terms of a plasmid, e.g., a DNA plasmid (i.e., a polycistronic DNA plasmid), it is understood that the discussion also applies to other polycistronic vectors, e.g., viral vectors.

[0226] Polycistronic plasmids are known in the art, and thus one of skill in the art can design and construct the polycistronic plasmids of the present disclosure.

[0227] In a preferred embodiment, the polycistronic plasmid of the present disclosure contains one or more co-expression elements, i.e., nucleic acid sequences that allow the co-expression of a polypeptide / antigen unit and one or more immunostimulatory compounds as separate molecules from the plasmid.

[0228] In some embodiments of the present disclosure, the polycistronic plasmid contains co-expression elements that cause the polypeptide / antigen unit and one or more immunostimulatory compounds to be transcribed onto a single transcript but translated independently, thus resulting in the ultimate production of separate translation products due to the presence of the co-expression elements.

[0229] In some embodiments, such a co-expression element is an IRES element (internal ribosome entry site). In other embodiments, such a co-expression element is a 2A self-cleaving peptide (2A peptide). Both co-expression elements are known in the art. When two or more immunostimulatory compounds are expressed from a polycistronic plasmid of the present disclosure, an IRES element and / or a 2A peptide must be present in the plasmid, e.g., upstream of each nucleic acid sequence encoding an immunostimulatory compound.

[0230] In other embodiments, the polycistronic plasmid contains co-expression elements such that the polypeptide / antigen unit and one or more immunostimulatory compounds are transcribed as separate transcripts, resulting in the production of separate transcripts and therefore separate proteins.

[0231] In some embodiments, such a co-expression element is a bidirectional promoter. In other embodiments, such a co-expression element is a different promoter, i.e., the polycistronic plasmid contains a promoter for each of the nucleic acid sequences encoding either a polypeptide or one or more immunostimulatory compounds. Both co-expression elements are known in the art.

[0232] The above co-expression elements can be combined in any manner, i.e., a polycistronic plasmid of the present disclosure may contain one or more of such same or different co-expression elements.

[0233] immunostimulating compounds The polycistronic plasmids of the present disclosure include one or more nucleic acid sequences encoding one or more immunostimulatory compounds. In some embodiments of the present disclosure, the immunostimulatory compounds are compounds that stimulate APCs, such as resulting in the attraction, activation, maturation, and / or proliferation of APCs.

[0234] In a first embodiment, the immunostimulatory compound is one that attracts APCs and is preferably capable of interacting with the following surface molecules on APCs: CCR1 (CC motif chemokine receptor 1), CCR3 (CC motif chemokine receptor 3), CCR4 (CC motif chemokine receptor 4), CCR5 (CC motif chemokine receptor 5), CCR6 (CC motif chemokine receptor 6), CCR7 (C motif chemokine receptor 7), CCR8 (C motif chemokine receptor 8), or XCR1 (XC motif chemokine receptor 1).

[0235] In other embodiments, the immunostimulatory compound is selected from the list consisting of CCL4, CCL5, CCL19, CCL20, CCL21, XCL1, or XCL2.

[0236] In a second embodiment, the immunostimulatory compound is one that promotes activation and / or maturation of APCs. In some embodiments, the immunostimulatory compound is capable of interacting with the following surface molecules on APCs: receptors of the TNF receptor superfamily, including CD40 (cluster of differentiation 40), CD137 (4-1BB), CD27, ICOSL (CD275), or RANK.

[0237] Such immune stimulatory compounds may be selected from the list consisting of CD40L (CD40 ligand, CD154), CD137L (4-1BBL, 4-1BB ligand), CD70, ICOS (CD278), or RANKL.

[0238] In other embodiments, the immunostimulatory compound is a cytokine selected from IL-2, IL-10, IL-12, TNFα, and IFN-γ, hi other embodiments, the immunostimulatory compound can be an immune signaling molecule, such as MyD88 and TRIF, which activate through TLR receptors.

[0239] In other embodiments, the immunostimulatory compounds can be sensors of viral infection, such as RIG-1 and MDA-5.

[0240] In other embodiments, the immunostimulatory compound can interact with pattern recognition receptors on APCs, such as Toll-like receptors, including TLR2, TLR4, or TLR5. Such immunostimulatory compounds may be selected from the list consisting of pathogen-associated molecular patterns (PAMPs), such as flagellin, or protein damage-associated molecular patterns (DAMPs), such as HMGB1, HSPs (heat shock proteins), calrecticulin, and annexin A1. PAMPs / DAMPs include those that can be included as nucleic acid sequences in the DNA plasmids of the present disclosure and expressed as functional proteins that may include functional groups introduced by post-translational modifications. The above molecules activate the following receptors on APCs: RAGE, TLR4, TLR9, TIM-3 (for HMGB1), FPR (for annexin A1), SREC1, LOX1, CD91 (for HSPs).

[0241] In a third embodiment, the immunostimulatory compound is one that promotes the growth and / or proliferation of APCs. In some embodiments, the immunostimulatory compound is capable of interacting with the following surface molecules on APCs: GM-CSF receptor (granulocyte-macrophage colony-stimulating factor receptor, CD116), FLT-3R (fms-like tyrosine kinase 3, CD135), IL-15R, or IL-4R.

[0242] In other embodiments, the immunostimulatory compound is a growth factor such as GM-CSF (granulocyte macrophage colony stimulating factor), FLT-3L, IL-15, or IL-4.

[0243] In some embodiments, the polycistronic vector comprises nucleic acid sequences encoding 2, 3, 4, 5, 6, 7, or 8 immunostimulatory compounds. In preferred embodiments, the polycistronic vector comprises nucleic acid sequences encoding 2 to 6 immunostimulatory compounds, i.e., 2 or 3 or 4 or 5 or 6 different immunostimulatory compounds. The immunostimulatory compounds may be the same or different, preferably different.

[0244] In preferred embodiments, the different immunostimulatory compounds also have different effects on APCs in order to stimulate the immune system at many different levels, thereby maximizing the therapeutic or prophylactic effect of the constructs of the present disclosure.

[0245] As an example, a polycistronic vector contains nucleic acids encoding two different immunostimulatory compounds, the first being an immunostimulatory compound that promotes DC growth (e.g., FLT-3L) and the second being an immunostimulatory compound that promotes DC activation (e.g., CD40L).

[0246] Preparation of vectors and host cells The vectors disclosed herein are generally suitable for transfecting host cells for expression of a polypeptide / antigen unit disclosed herein and, when the polypeptides are expressed, for the formation of a multimeric protein consisting of a plurality of such polypeptides, for example for the formation of a dimeric protein consisting of two such polypeptides.

[0247] Thus, a further aspect of the present disclosure is a host cell comprising a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77.

[0248] Thus, a still further aspect of the present disclosure is a host cell comprising a vector comprising a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, wherein the antigenic unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77.

[0249] In some embodiments, the host cell comprising the vector is a cell of a cell culture, e.g., a bacterial cell, and the polypeptide / antigen unit encoded by the vector is expressed in vitro. In some other embodiments, the host cell comprising the vector of the invention is a cell of a subject, and the polypeptide / antigen unit encoded by the vector is expressed in said subject, i.e., in vivo, as a result of administering the vector to the subject. Suitable host cells for in vitro transfection include prokaryotic cells, yeast cells, insect cells, or higher eukaryotic cells. Suitable host cells for in vivo transfection include, for example, human muscle cells.

[0250] In some embodiments, the vector allows easy exchange of the various units described above, especially the antigen unit. In some embodiments, the vector is a vector comprising a pUMVC4a vector or a NTC9385R vector backbone. The antigen unit may be exchanged with an antigen unit cassette restricted by an SfiI restriction enzyme cassette, the 5' site being incorporated into a nucleotide sequence encoding a GLGGL (SEQ ID NO: 214) or GLSGL (SEQ ID NO: 283) unit linker, and the 3' site being included after a stop codon in the vector.

[0251] Methods for engineering and producing vectors, e.g., expression vectors such as DNA and RNA plasmids or viral vectors, are well known and one of skill in the art would be able to use such known methods to design / produce the vectors disclosed herein. In addition, various commercial manufacturers offer vector design and production services.

[0252] In one aspect, the disclosure provides a method for making a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77; a) transfecting cells with a vector in vitro; b) culturing the cells; and c) optionally lysing the cells and releasing the vector from the cells; d) isolating and optionally purifying the vector; The present invention relates to a method comprising the steps of:

[0253] In a further aspect, the disclosure provides a method of making a vector comprising a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77; a) transfecting cells with the vector in vitro; b) culturing the cells; c) optionally lysing the cells and releasing the vector from the cells; d) isolating and optionally purifying the vector; The present invention relates to a method comprising the steps of:

[0254] Polypeptide / antigen unit Constructs of the present invention may be in the form of a polypeptide encoded by a nucleotide sequence contained in the polynucleotides described herein.

[0255] A further aspect of the invention is a polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77.

[0256] The polypeptides may be expressed in vitro, for example for the generation of a pharmaceutical composition, such as a vaccine, comprising such a polypeptide, or alternatively, the polypeptides may be expressed in vivo as a result of administration of a polynucleotide described herein to a subject.

[0257] In a further aspect, the present disclosure relates to an antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77.

[0258] An antigenic unit may be expressed in vitro, for example for the manufacture of a pharmaceutical composition, such as a vaccine, comprising such an antigenic unit. An antigenic unit may be produced in vitro, for example by transfecting or transducing cells with a polynucleotide comprising a nucleotide sequence encoding the antigenic unit such that the antigenic unit is expressed, culturing the cells, isolating the antigenic unit from the cells (which may mean isolating the antigenic unit from the cells in the cell culture medium if the antigenic unit is secreted from the cells into the culture medium), and optionally purifying the antigenic unit.

[0259] Thus, a further aspect of the present invention relates to a method for preparing an antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 1 to 77, comprising: a) transfecting or transducing a cell with a polynucleotide comprising a nucleotide sequence encoding an antigenic unit; b) culturing the cells; and c) isolating antigenic units from the cells; and d) optionally purifying the antigenic units; The method includes:

[0260] Alternatively, the antigenic unit may be expressed in vivo as a result of administering to a subject a polynucleotide encoding such an antigenic unit as described herein.

[0261] Multimeric / Dimeric Proteins A further aspect of the present invention is a multimeric protein consisting of multiple polypeptides, each of which includes a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit, wherein the antigen unit includes at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 1 to 77.

[0262] In some embodiments, the present disclosure relates to a dimeric protein consisting of two polypeptides, each of which comprises a targeting unit, a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77.

[0263] The multimeric protein may be a homomultimer or a heteromultimer. For example, when the multimeric protein is a dimeric protein, the dimeric protein may be a homodimer, i.e., a dimeric protein formed by two identical polypeptide molecules (containing identical units / elements). Alternatively, the dimeric protein may be a heterodimer formed by two different polypeptides, for example, polypeptides 1 and 2 contain the same targeting unit and dimerization unit, but each contains a different antigen unit. A heteromultimeric protein can be produced by co-transfecting cells with two different vectors, one containing a polynucleotide encoding polypeptide 1 and the other containing a polynucleotide encoding polypeptide 2 different from polypeptide 1, and isolating the heteromultimeric protein after the polypeptides are expressed to form the heteromultimeric protein. When the number of T cell epitopes contained in the antigen unit exceeds the upper size limit of the antigen unit, a heteromultimeric protein may be appropriate. It is preferable that the multimeric protein is a homomultimeric protein.

[0264] A multimeric / dimeric protein can be prepared, for example, by expressing the polypeptide in vitro by transfecting or transducing cells with a polynucleotide comprising a nucleotide sequence encoding the polypeptide, such that the polypeptide is expressed and forms a multimeric / dimeric protein, culturing the cells, isolating the multimeric / dimeric protein from the cells (which may mean isolating the protein from the cells in the cell culture medium if the protein is secreted from the cells into the culture medium), and optionally purifying the protein.

[0265] Thus, a further aspect of the present invention is a method for preparing a multimeric protein consisting of a plurality of polypeptides, each of which comprises a targeting unit for targeting an antigen-presenting cell, a multimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 1 to 77; a) transfecting or transducing a cell with a polynucleotide comprising a nucleotide sequence encoding a polypeptide; b) culturing the cells; and c) isolating the multimeric protein from the cells; and d) optionally purifying the multimeric protein; The method includes:

[0266] In some embodiments, the method is for preparing a dimeric protein consisting of two polypeptides, each polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a dimerization unit, and an antigen unit, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1-77, the method comprising: a) transfecting or transducing a cell with a polynucleotide comprising a nucleotide sequence encoding a polypeptide; b) culturing the cells; and c) isolating the dimeric protein from the cells; and d) optionally purifying the dimeric protein; Includes.

[0267] Isolation and optional purification of the polypeptides / multimeric proteins can be performed by methods known in the art, including precipitation, differential solubilization, and chromatography.

[0268] The polynucleotide may be comprised in a plasmid for transfection or a vector for transduction.

[0269] The above multimeric / dimeric proteins may be used as active ingredients in protein vaccines for the prevention or treatment of diseases caused by SARS-CoV-2.

[0270] Pharmaceuticals In some embodiments of the present disclosure, the constructs (i.e., polynucleotides, polypeptides / multimeric proteins), antigenic units, and vectors disclosed herein are for use as pharmaceuticals.

[0271] Pharmaceutical compositions and vaccines The construct of the present invention may be administered to a subject in the form of a pharmaceutical composition comprising the construct (e.g. in the form of a polynucleotide, vector, or multimeric protein) and a pharma- ceutically acceptable carrier, such as in the form of a vaccine.

[0272] Alternatively, the antigenic units described herein may be administered to a subject in the form of a pharmaceutical composition comprising the antigenic unit (e.g. in the form of a polynucleotide, vector, or polypeptide) and a pharma- ceutically acceptable carrier, e.g. in the form of a vaccine.

[0273] As used herein, a "vaccine" is a pharmaceutical composition comprising a construct disclosed herein and a pharma- ceutically acceptable carrier, which may further include typical excipients for vaccines.

[0274] A further aspect of the invention is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit such as a dimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii), for example, a dimeric protein consisting of two polypeptides as defined in (ii). It is a vaccine comprising:

[0275] A further aspect of the invention is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit such as a dimerization unit, and an antigen unit, wherein the antigen unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii), for example, a dimeric protein consisting of two polypeptides as defined in (ii). A pharmaceutical composition comprising:

[0276] A further aspect of the invention is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). It is a vaccine comprising:

[0277] A further aspect of the invention is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). A pharmaceutical composition comprising:

[0278] Suitable pharma- ceutically acceptable carriers include, but are not limited to, saline, buffered saline, such as PBS, dextrose, water, glycerol, ethanol, aqueous buffers, such as isotonic aqueous buffers or Tyrode's buffer, and combinations thereof.

[0279] In some embodiments, the pharma- ceutically acceptable carrier is an aqueous buffer. In some other embodiments, the aqueous buffer is Tyrode's buffer, such as Tyrode's buffer containing 140 mM NaCl, 6 mM KCl, 3 mM CaCl2, 2 mM MgCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) (pH 7.4), and 10 mM glucose. The pharmaceutical composition or vaccine may further comprise an adjuvant.

[0280] In some embodiments, a vaccine comprising a multimeric protein or polypeptide as described herein is selected from the group consisting of Poly ICLC, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact EV1P321, IS Patch (IS Patch), Patch), ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta glucan, Pam3Cys, Aquila's QS21 stimulon, badimesan, and / or AsA404 (DMXAA).

[0281] In the case of pharmaceutical compositions / vaccines comprising polynucleotides or vectors, the pharmaceutical compositions / vaccines may also comprise one or more transfection agents that facilitate the transfection / transduction of cells with the polynucleotide or vector, for example, the transfection of muscle cells of a subject. Transfection agents for polynucleotides are known in the art and include positively charged molecules that interact with negatively charged molecules such as DNA or RNA to form positively charged transfection agent-DNA or transfection agent-RNA complexes. Such complexes can interact with negatively charged cell membranes, allowing uptake of the complex and delivery of DNA or RNA into the cell.

[0282] When the polynucleotide is RNA, e.g., mRNA, the RNA may be formulated in or with lipids, nanoparticles, micelles, lipoplex nanoparticles (particles composed of a combination of different lipids, typically with a diameter of less than 1000 nanometers, with a diameter that makes the particle suitable for systemic administration, especially intravenous administration), or liposomes (especially for intravenous administration). In one embodiment, the lipoplex nanoparticles or liposomes include one or more lipids that form a multi-layer structure that encapsulates the RNA. In another embodiment, the one or more lipids include at least one cationic lipid and at least one helper lipid. In some embodiments, the one or more lipids include (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The mRNA molecule may also be formulated as a naked mRNA molecule in a suitable injection buffer.

[0283] In some specific embodiments, the pharmaceutical composition / vaccine comprises a pharma- ceutically acceptable amphiphilic block copolymer comprising blocks of poly(ethylene oxide) and polypropylene oxide.

[0284] As used herein, an "amphiphilic block copolymer" is a linear or branched copolymer that comprises or consists of blocks of poly(ethylene oxide) ("PEO") and blocks of poly(propylene oxide) ("PPO"). Typical examples of useful PEO-PPO amphiphilic block copolymers include the general structures PEO-PPO-PEO (poloxamer), PPO PEO PPO, (PEO PPO-)4ED (poloxamine), (PPO PEO-)4ED (reverse poloxamine), where "ED" is an ethylenediaminyl group.

[0285] A "poloxamer" is a linear amphiphilic block copolymer composed of one poly(ethylene oxide) block bonded to one poly(propylene oxide) block bonded to one PEO block (i.e., of the structure of the formula EOa-POb-EOa), where EO is ethylene oxide, PO is propylene oxide, a is an integer between 2 and 130, and b is an integer between 15 and 67. Poloxamers are conventionally named using a three-digit identifier, where the first two digits are multiplied by 100 to indicate the approximate molecular weight of the PPO content, and the last digit is multiplied by 10 to indicate the approximate percentage of PEO content. For example, "poloxamer 188" refers to a polymer containing a PPO block of about 1800 molecular weight (corresponding to a of about 31 PPO) and about 80% (w / w) PEO (corresponding to an a of about 82). However, values ​​are known to vary to some extent, and commercial products such as research grade Lutrol® F68 and clinical grade Kolliphor® P188, both of which are poloxamer 188 according to the manufacturer's datasheets, show a wide variation in molecular weight (7,680-9,510), with the a and b values ​​provided for these particular products being approximately 79 and 28, respectively. This reflects the heterogeneous nature of the block copolymers, and means that the a and b values ​​are average values ​​found in the final formulation.

[0286] "Poloxamine" or "sequential poloxamine" (sold under the trademark Tetronic®) is an X-shaped block copolymer having four PEO-PPO arms attached to a central ethylenediamine moiety via bonds between the free OH groups in the PEO-PPO arms and the primary amine groups in the ethylenediamine moiety. Reverse poloxamine is similarly an X-shaped block copolymer having four PPO-PEO arms attached to a central ethylenediamine moiety via bonds between the free OH groups in the PPO-PEO arms and the primary amine groups in the ethylenediamine moiety.

[0287] Preferred amphiphilic block copolymers are poloxamers or poloxamines. Poloxamers 407 and 188 are preferred, with poloxamer 188 being particularly preferred. Preferred poloxamines are sequential poloxamines of formula (PEO-PPO)4-ED. Particularly preferred poloxamines are those marketed under the registered trademarks Tetronic® 904, 704, and 304, respectively. The characteristics of these poloxamines are as follows: Tetronic® 904 has a total average molecular weight of 6700, a total average weight of PPO units of 4020, and a proportion of PEO of about 40%; Tetronic® 704 has a total average molecular weight of 5500, a total average weight of PPO units of 3300, and a proportion of PEO of about 40%; Tetronic® 304 has a total average molecular weight of 1650, a total average weight of PPO units of 990, and a proportion of PEO of about 40%.

[0288] In some embodiments, the pharmaceutical composition comprises the amphiphilic block copolymer in an amount of from 0.2% w / v to 20% w / v, e.g., from 0.2% w / v to 18% w / v, from 0.2% w / v to 16% w / v, from 0.2% w / v to 14% w / v, from 0.2% w / v to 12% w / v, from 0.2% w / v to 10% w / v, from 0.2% w / v to 8% w / v, from 0.2% w / v to 6% w / v, from 0.2% w / v to 4% w / v, from 0.4% w / v to 18% w / v, from 0.6% w / v to 18% w / v, from 0.8% w / v to 18% w / v, from 1% w / v to 18% w / v, from 2% w / v to 18% w / v, from 1% w / v to 5% w / v, or from 2% w / v to 4% w / v. Amounts in the range of 0.5% w / v to 5% w / v are particularly preferred. In some other embodiments, the pharmaceutical composition comprises the amphiphilic block copolymer in an amount of 2% w / v to 5% w / v, for example about 3% w / v.

[0289] The pharmaceutical composition / vaccine may be formulated in any manner suitable for administration to a subject, such as an injectable liquid formulation, such as for intradermal or intramuscular injection.

[0290] The pharmaceutical composition / vaccine may be administered in any manner suitable for administration to a subject, such as by intradermal, intramuscular or subcutaneous injection, or application to a mucosa or epithelium, such as intranasally or orally.

[0291] In a preferred embodiment, the pharmaceutical composition / vaccine comprises a polynucleotide, for example contained in a vector, and is administered by intramuscular or intradermal injection.

[0292] A pharmaceutical composition / vaccine of the invention typically comprises in the range of 0.1-10 mg, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg, or such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg of polynucleotide. A vaccine of the invention typically comprises in the range of 5 μg-5 mg of polypeptide / multimeric protein.

[0293] The amount of polynucleotide / polypeptide / dimeric protein may vary depending on whether the pharmaceutical composition / vaccine is administered for prophylactic or therapeutic treatment, the severity of the disease in the infected individual, and parameters such as age, weight, sex, medical history, and pre-existing conditions.

[0294] Methods for preparing pharmaceutical compositions / vaccines Suitable methods for preparing the pharmaceutical compositions / vaccines according to the present invention are disclosed in WO2004 / 076489A1, WO2011 / 161244A1, WO2013 / 092875A1, and WO2017 / 118695A1, which are incorporated herein by reference, and comprise preparing the polynucleotides, vectors, polypeptides, or multimeric proteins by the methods described therein and mixing them with a pharma- ceutically acceptable carrier and optionally further pharma- ceutically acceptable excipients as described in the previous sections of this specification.

[0295] In a preferred embodiment, the polynucleotide, vector, polypeptide, or multimeric protein is dissolved in a pharma- ceutically acceptable carrier as described above.

[0296] treatment The pharmaceutical compositions / vaccines of the present invention may be used to treat diseases caused by SARS-CoV-2, such treatment may be either prophylactic or therapeutic.

[0297] The pharmaceutical composition / vaccine is administered so as to induce an immunoprotective response (in the case of prophylactic treatment) or an immunotherapeutic response (in the case of therapeutic treatment) in an individual vaccinated with / administered with such a vaccine. Such a response is induced either by a single vaccination / administration or by several vaccinations / administrations, e.g. a primary vaccination and one or several booster vaccinations at sufficient intervals.

[0298] In a further aspect, the present invention provides a method for treating a subject having a disease caused by SARS-CoV-2 or a subject in need of prevention of such a disease, comprising administering to said subject a medicament comprising a pharmacopoeia, a medicament comprising a pharmacopoeia, a medicament comprising a pharmacopoeia and ... (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). The present invention provides a method for administering to a subject a pharmaceutical composition / vaccine comprising the steps of:

[0299] In some embodiments, the multimerization unit is a dimerization unit and the multimeric protein is a dimeric protein consisting of two polypeptides.

[0300] Further, the present disclosure provides a method of treating a subject having a disease caused by SARS-CoV-2 or a subject in need of prevention of such a disease, comprising administering to said subject a medicament comprising a pharmacopoeiased antibody or a medicament comprising said ... (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). The present invention provides a method for administering to a subject a pharmaceutical composition / vaccine comprising the steps of:

[0301] Provided herein is a pharmaceutical composition / vaccine for use in the prophylactic or therapeutic treatment of a disease caused by SARS-CoV-2, comprising a pharma- ceutical composition / vaccine comprising a pharma- ceutical acceptable carrier and (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). Also disclosed is a pharmaceutical composition / vaccine comprising:

[0302] In some embodiments, the multimerization unit is a dimerization unit and the multimeric protein is a dimeric protein consisting of two polypeptides.

[0303] Further, the present disclosure provides a pharmaceutical composition / vaccine for use in the prophylactic or therapeutic treatment of disease caused by SARS-CoV-2, comprising a pharma- ceutical composition / vaccine comprising a pharma- ceutical acceptable carrier and (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). The present invention provides a pharmaceutical composition / vaccine comprising:

[0304] The present specification provides a method for producing a pharmaceutical composition / vaccine for use in the prophylactic or therapeutic treatment of a disease caused by SARS-CoV-2 in a subject, (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). The use of the above is disclosed, wherein the pharmaceutical composition / vaccine comprises a pharma- ceutical acceptable carrier, and the pharmaceutical composition / vaccine is administered to the subject.

[0305] In some embodiments, the multimerization unit is a dimerization unit and the multimeric protein is a dimeric protein consisting of two polypeptides.

[0306] The present specification provides a method for producing a pharmaceutical composition / vaccine for use in the prophylactic or therapeutic treatment of a disease caused by SARS-CoV-2 in a subject, (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). Also disclosed is the use of the above, wherein the pharmaceutical composition / vaccine comprises a pharma- ceutically acceptable carrier, and the pharmaceutical composition / vaccine is administered to the subject.

[0307] Provided herein are compositions comprising a medicament for treating a subject having a disease caused by SARS-CoV-2 or in need of prevention of such a disease, comprising a pharmacopoeitic agent and a pharmacopoeitic agent ... (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). Also disclosed is a pharmaceutical composition / vaccine comprising:

[0308] In some embodiments, the multimerization unit is a dimerization unit and the multimeric protein is a dimeric protein consisting of two polypeptides.

[0309] Provided herein are compositions comprising a medicament for treating a subject having a disease caused by SARS-CoV-2 or in need of prevention of such a disease, comprising a pharmacopoeitic agent and a pharmacopoeitic agent ... (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). The use of the pharmaceutical composition / vaccine comprising the same is also disclosed.

[0310] As used herein, the term "compounds" refers to compounds that, when used for the prophylactic or therapeutic treatment of diseases caused by SARS-CoV-2, include pharma- ceutically acceptable carriers and (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). Also disclosed is a pharmaceutical composition / vaccine comprising:

[0311] In some embodiments, the multimerization unit is a dimerization unit and the multimeric protein is a dimeric protein consisting of two polypeptides.

[0312] As used herein, the term "compounds" refers to compounds that, when used to prevent or treat a disease caused by SARS-CoV-2, include pharma- ceutically acceptable carriers and (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). Also disclosed is a pharmaceutical composition / vaccine comprising:

[0313] The present invention relates to a method for the treatment of a disease caused by SARS-CoV-2, comprising administering to a patient a medicament for which the medicament is administered, the method comprising administering to the ... (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). The use of the pharmaceutical composition / vaccine comprising:

[0314] In some embodiments, the multimerization unit is a dimerization unit and the multimeric protein is a dimeric protein consisting of two polypeptides.

[0315] The present invention relates to a method for the treatment of a disease caused by SARS-CoV-2, comprising administering to a patient a medicament for which the medicament is administered, the method comprising administering to the ... (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). The use of the pharmaceutical composition / vaccine comprising:

[0316] As used herein, pharma- ceutically acceptable carriers and (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). Also disclosed is a medicament for treating or preventing a disease caused by SARS-CoV-2 in a subject having the disease or in need of prevention of the disease, by administering to the subject a medicament comprising:

[0317] In some embodiments, the multimerization unit is a dimerization unit and the multimeric protein is a dimeric protein consisting of two polypeptides.

[0318] As used herein, pharma- ceutically acceptable carriers and (i) a polynucleotide comprising a nucleotide sequence of an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) a polypeptide encoded by the nucleic acid sequence defined in (i). Also disclosed is a medicament for treating or preventing a disease caused by SARS-CoV-2 in a subject having the disease or in need of prevention of the disease, by administering to the subject a medicament comprising:

[0319] In some embodiments of the methods and uses disclosed above, the pharmaceutical composition / vaccine / medication comprises the polynucleotide in a vector.

[0320] In the methods of treatment / use of the pharmaceutical compositions / vaccines / medications disclosed herein, the pharmaceutical compositions / vaccines / medications are preferably administered in a therapeutically or prophylactically effective amount. Such an amount may be administered in one administration, i.e., one dose, or in several administrations, i.e., repeated administrations, i.e., over a series of administrations, e.g., over the course of several days, weeks, or months.

[0321] The actual amount administered will vary and will depend on whether the treatment is prophylactic or therapeutic, the age, weight, sex, medical history, pre-existing conditions, and general health of the subject, the severity of the disease being treated, and the judgment of a medical professional.

[0322] In the methods of treatment / use of the pharmaceutical compositions / vaccines / medications disclosed herein, the pharmaceutical compositions / vaccines / medications may be administered by the methods described herein, e.g., the methods described in the "Pharmaceutical Compositions / Vaccines" section.

[0323] The methods of treatment / use of the pharmaceutical compositions / vaccines / medicaments disclosed herein may be continued for as long as the clinician overseeing the patient's treatment determines that the methods are effective and that treatment is necessary. EXAMPLES

[0324] The above description is believed to be sufficient to enable one skilled in the art to practice the present invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the present invention in addition to those shown and described herein will be apparent to those skilled in the art from the above description and fall within the scope of the appended claims.

[0325] Example 1 Design and Construction of DNA Immunogenic Constructs According to the Invention Seven DNA plasmid constructs (collectively referred to below as VB10.COV2) were designed, generated, and tested. All constructs contain nucleic acid sequences encoding a human MIP-1α (hMIP-1α) targeting unit (including its signal peptide), a dimerization unit comprising hinge exons 1 and 4 from IgG3 and a CH3 domain from human IgG3 (signal peptide, targeting unit, and dimerization unit having the amino acid sequence of SEQ ID NO: 168), and an antigenic unit comprising at least 77 SARS CoV2 T cell epitopes having the amino acid sequences of SEQ ID NOs: 1-77 as set forth in Table 1.

[0326] The details of each component are as follows: VB2193 comprises a nucleic acid sequence encoding an antigenic unit of SEQ ID NO: 161 comprising 160 T cell epitopes having the amino acid sequences of SEQ ID NOs: 1 to 160, the epitopes or epitopes being separated from each other by a GGGGSGGGGS T cell epitope linker. VB2193 comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 178.

[0327] VB2194 comprises a nucleic acid sequence encoding an antigenic unit of SEQ ID NO: 162 comprising 160 T cell epitopes having the amino acid sequences of SEQ ID NOs: 1 to 160, the epitopes or epitopes being separated from each other by a GGGGSGGGGS T cell epitope linker. VB2194 comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 179.

[0328] VB2196 comprises a nucleic acid sequence encoding an antigenic unit of SEQ ID NO: 163 comprising 141 T cell epitopes having amino acid sequences of SEQ ID NOs: 1 to 77 and 97 to 160, the epitopes or epitopes being separated from each other by a GGGGSGGGGS T cell epitope linker. VB2196 comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 180.

[0329] VB2197 comprises a nucleic acid sequence encoding an antigenic unit of SEQ ID NO: 164 comprising 96 T cell epitopes having the amino acid sequences of SEQ ID NOs: 1 to 96, the epitopes or epitopes being separated from each other by a GGGGSGGGGS T cell epitope linker. VB2197 comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 181.

[0330] VB2207 comprises a nucleic acid sequence encoding an antigenic unit of SEQ ID NO: 165 comprising 141 T cell epitopes having amino acid sequences of SEQ ID NOs: 1 to 77 and 97 to 160, wherein the epitopes or epitopes are separated from each other by a SGSSGS or GGSGG T cell epitope linker. VB2207 comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 182.

[0331] VB2208 comprises a nucleic acid encoding an antigenic unit of SEQ ID NO: 166 comprising 141 T cell epitopes having amino acid sequences SEQ ID NOs: 1-77 and 97-160, wherein the epitopes or epitopes are separated from each other by a SGSSGS or GGSGG T cell epitope linker. VB2208 comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 183.

[0332] VB2210 comprises a nucleic acid encoding an antigenic unit of SEQ ID NO: 167 comprising 96 T cell epitopes having the amino acid sequences of SEQ ID NOs: 1-196, the epitopes or epitopes being separated by GGSGG and SGSSGS T cell epitope linkers. VB2210 comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 184. VB2210 comprises a nucleic acid sequence of SEQ ID NO: 185.

[0333] All constructs were ordered from Genscript (860 Centennial Ave., Piscataway, NJ08854, USA) and cloned into the expression vector pUMVC4a, which contains the signal peptide, targeting unit, and dimerization unit described above, as well as the sequence of a unit linker (GLGGL) containing restriction sites that allow convenient exchange of the respective antigen units.

[0334] Example 2 Characterization of VB10.COV2 proteins produced after transfection of HEK293 cells with VB10.COV2 DNA plasmid the purpose: The aim of the study was to characterize VB10.COV2 protein after in vitro transient transfection of mammalian cells with various VB10.COV2 DNA plasmids. The presence of functional VB10.COV2 protein in cell supernatants was verified by ELISA assay with antibodies binding to human MIP-1α (targeting unit) and human IgG3 CH3 domain (dimerization unit as capture antibody).

[0335] Method / Materials: Transient transfection of HEK293 cells with VB10.COV2 DNA plasmid

[0336] HEK293 cells were purchased from ATCC and transiently transfected with VB10.COV2 DNA. Briefly, 2 × 10 5 Cells / well were seeded into 24-well tissue culture plates containing 10% FBS growth medium and transfected with 1 μg of VB10.COV2 DNA plasmid using Lipofectamine® 2000 reagent under conditions suggested by the manufacturer (Invitrogen by Life Technologies). Transfected cells were then maintained at 37° C., 5% CO2 for up to 6 days, and cell supernatants were harvested for characterization of VB10.COV2 protein.

[0337] ELISA using monoclonal antibodies that bind to hMIP-1α and hIgG3: ELISA was performed to confirm the amount of VB10.COV2 protein produced by HEK293 cells and secreted into the cell supernatant. Briefly, MaxiSorp Nunc-immunoplates were coated with 100 μl / well of anti-human IgG3 CH3 (MCA878G, BioRad) at 1 μg / ml in 1×PBS, and the plates were incubated overnight at 4°C. Microtiter wells were blocked by adding 200 μl / well 4% BSA in 1×PBS. 100 μl of cell supernatant of transfected HEK293 cells containing VB10.COV2 protein was added to the plate. For detection antibody, anti-human MIP-1α (biotinylated) was added and incubated. Then strep-HRP (1:3000) was added and incubated. All incubations were performed at 37°C for 1 h, unless otherwise specified, followed by 3 washes with PBS-Tween®. Then, 100 μl / well of TMB solution was added, and color development was stopped 5–15 min after adding 100 μl / well of 1 M HCl. The optical density at 450 nm was determined with an automated plate reader (Thermo Scientific Multiscan GO).

[0338] result: Successful expression and secretion of functional VB10.COV2 protein was observed for all constructs. Conformational correctness of all VB10.COV2 proteins was confirmed by ELISA binding to antibodies detecting the CH3 domain of hMIP-1α and human IgG3.

[0339] Example 3 Vaccination with such constructs induces IFN-γ T cell responses specific to T cell epitopes contained in the VB10.COV2 DNA plasmid. the purpose: The aim of the study was to evaluate the cellular immune response (T cell response) against T cell epitopes contained in the VB10.COV2 DNA in mice vaccinated with such constructs.

[0340] Method / Materials: General study design: Sixteen-week-old female BALB / c mice and 10-week-old C57BL / 6 mice were obtained from Janvier Labs (France), and 6-9-week-old C57BL / 6-McphlTg(HLA-A2.1)lEnge / J mice were obtained from Jackson Laboratory (USA). The latter are homozygous mice carrying the Tg(HLA-A2.1)lEnge transgene. These mice express large amounts of human HLA-A2.1 class I molecules on cells of the spleen, bone marrow, and thymus. This transgenic model was used to specifically evaluate responses to the human HLA-A2.1 T cell epitopes contained in the VB10.COV2 DNA construct, as other mouse models expressing mouse MHC classes I and II do not respond optimally to the human T cell epitopes contained in the construct. All animals were maintained in the animal facility of the Domus Medica at the University of Oslo (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). Mice were anesthetized and shaved before vaccination. In the study, mice received VB10.COV2 intramuscularly at doses of 1, 5, 25, or 50 μg DNA on days 0 and 21 for boosting regimens (see vaccination schedule in Table 6). VB10.COV2 DNA plasmid was administered by needle injection into the tibialis anterior muscle (as 25 μL of solution in sterile PBS in each leg), followed by AgilePulse in vivo electroporation (BTX, USA) consisting of three sets of pulses from 110 to 450 V. Mice receiving PBS (vehicle only) were included as negative control groups in each experiment. Spleens were harvested on days 14 or 35.

[0341] [Table 6]

[0342] IFN-γ ELISpot assay: IFN-γ ELISpot assays were performed on fresh splenocytes from mice vaccinated with VB10.COV2 DNA plasmid. Animals were sacrificed on days 14 and 35 and spleens were aseptically harvested. Spleens were mashed and cell suspensions were incubated with 1× ACK buffer, washed, resuspended and diluted in 6× 10 5 The cells were then seeded in triplicate (6 × 10 5 Cells / well) were stimulated for 24 hours with peptides or peptide pools in PBS as follows: in assays performed on splenocytes from transgenic C57BL / 6-McphlTg(HLA-A2.1)lEnge / J mice expressing human HLA2-2.1 class I molecules, stimulation was performed with a) individual peptides corresponding to HLA-A2.1 epitopes contained in the respective constructs as distinct T cell epitopes or the only HLA-A2.1 epitope in a group of epitopes, or b) pools of peptides corresponding to HLA-A2.1 epitopes contained in a group of epitopes present in the respective constructs (such groups containing two or more HLA-A2.1 epitopes). In assays performed on splenocytes from wild-type BALB / c or wild-type C57BL / 6 mice, stimulation was performed with the peptide pool used, consisting of overlapping 15-mer peptides covering all epitopes / epitope groups present in the respective constructs.

[0343] PBS / no peptide stimulation was used as a negative control. Stimulated splenocytes were analyzed for IFN-γ responses using the IFN-γ ELISpot Plus kit (Mabtech AB, Sweden). Spot forming cells were measured with a CTL ELISpot reader, ImmunoSpot 5.0.3 from Cellular Technology.

[0344] result: Transgenic C57BL / 6-McphlTg(HLA-A2.1)lEnge / J mice expressing human HLA2-2.1 class I molecules were vaccinated with a single dose of 25 μg of four different VB10.COV2 DNA constructs (VB2193, VB2194, VB2197, and VB2210). IFN-γ ELISpot assays 14 days after vaccination with the above peptides / peptide pools showed that these constructs specifically induced rapid and strong T cell responses in transgenic mice against human HLA.A2.1 epitopes present within such constructs (Figure 3).

[0345] In another experiment, transgenic C57BL / 6-McphlTg(HLA-A2.1)lEnge / J mice were vaccinated with a single dose of 50 μg each of the seven VB10.COV2 constructs, and IFN-γ ELISpot assays were performed as described in the previous paragraph. The results showed that all seven VB10.COV2 constructs induced rapid and strong T cell responses against T cell epitopes specific to human HLA-A2.1 molecules in transgenic mice (Figure 4), and the responses induced by VB2197, VB2207, and VB2210 were approximately two-fold stronger than the other four constructs.

[0346] All seven VB10.COV2 constructs were also tested in wild-type BALB / c mice, which were vaccinated with a single dose of 25 μg of each construct. In the ELISpot assay of this experiment, the peptide pool used to stimulate splenocytes on day 14 after vaccination consisted of 15-mer overlapping peptides covering all epitopes / epitope groups contained in each construct. The results showed that all seven VB10.COV2 constructs could induce rapid and strong T cell responses in wild-type mouse models (Figure 5), with VB2193, VB2194, VB2197, and VB2210 eliciting stronger responses than the other three constructs in this mouse model.

[0347] A single dose of 25 μg of the VB2210 construct was used to vaccinate a C57BL / 6 wild type mouse model. Results showed that 14 days after vaccination, VB2210 induced strong specific T cell responses in ELISpot assays using a peptide pool composed of overlapping 15-mer peptides covering all epitopes / epitopes contained in the construct (Figure 6). The strength of the response was comparable to that of two wild type mouse models, BALB / c (Figure 6, 3163SFU / 10 6 cells) and C57BL / 6 (Figure 7, 7414SFU / 10 6 The T cell responses were lower when ELISpot assays were performed using spleen cells from C57BL / 6 wild type mice vaccinated with VB2210 using peptides and peptide pools composed of peptides corresponding to human HLA-A2.1 epitopes (Figure 7), which is expected due to the lower number of HLA-A2.1 specific epitopes in VB2210 compared to the total number of 96 epitopes used for stimulation when assays were performed using overlapping 15-mer peptide pools covering all epitopes / epitope groups present in the VB2210 construct.

[0348] In summary, vaccination with a single dose of 25 μg or 50 μg of the VB10.COV2 construct induced strong, consistent and specific T cell responses against multiple epitopes in three mouse models 14 days post-vaccination.

[0349] Furthermore, responses induced by three dose levels of VB2210, 1, 5, and 25 μg, were evaluated in C57BL / 6-McphlTg(HLA-A2.1)lEnge / J transgenic mice in either a one or two dose regimen. The second dose (boost vaccination) was administered 21 days after the first vaccination. Cellular responses against specific T cell epitopes from SARS-CoV-2 contained in the construct were evaluated in spleen cells from individual mice 14 days after the first vaccination (if only one dose was administered) or 14 days after the boost vaccination (if two doses were administered). In the ELISpot assay, peptide pools composed of overlapping 15-mer peptides covering all epitopes / epitope groups (Figure 8) or peptides and peptide pools composed of peptides corresponding to human HLA-A2.1 epitopes contained in the construct (Figure 9) were used. Results showed a dose-dependent immune response, with 25 μg VB2210 inducing a stronger response (dose response) compared to lower doses. Results further showed that a second vaccination on day 21 significantly enhanced the T cell response, highlighting the important role of boosting (Figures 8 and 9). Again, as expected, overall T cell responses were lower when peptides corresponding to human HLA-A2.1 epitopes were used in the assay (Figure 9) compared to stimulation with a 15-mer overlapping peptide pool covering all epitopes / epitope groups present in the VB2210 construct (Figure 8).

[0350] Responses induced by three dose levels of VB2210, 1, 5, and 25 μg, were evaluated in C57BL / 6 wild-type mice in either a one or two dose regimen. The second dose (boost vaccination) was administered 21 days after the first vaccination. Cellular responses against specific T cell epitopes from SARS-CoV-2 contained in the construct were evaluated in spleen cells from individual mice at 84 days post-vaccination (when only one dose was administered) or 85 days post-vaccination (when two doses were administered). The ELISpot assay used selected peptide pools consisting of overlapping 15-mer peptides / peptides covering multiple epitopes / groups of epitopes contained in the construct. The results shown in Figure 10 indicate that a single dose of 5 μg or 25 μg of VB2210 induced sustained T cell responses. A boost vaccination on day 21 increased the strength of immunity induced by the 5 μg or 25 μg doses. Sustained T cell immunity detected up to 12 weeks after vaccination indicates that immune memory was established.

[0351] Example 4 Induction of polyfunctional CD8+ cells specific for T cell epitopes contained in the VB2210 DNA construct in transgenic HLA-A2.1 mice Cell stimulation and staining for flow cytometry: C57BL / 6-McphlTg(HLA-A2.1)lEnge / J transgenic mice were vaccinated with one or two doses of 25 μg VB2210 (boost vaccination on day 21). Splenocytes from vaccinated mice were harvested 14 days after single and boost vaccinations, respectively, pooled in groups (n=4), and stimulated with 6 μg / mL peptide pools composed of immunogenic peptides identified in previous ELISpot assays corresponding to multiple HLA-A2.1 epitopes present in the construct. Stimulated cells were stained for phenotyping and intracellular expression of TNF-α, IFN-γ, IL-4, IL-17, IL-2, and FoxP3, and further subjected to multiparameter functional analysis by flow cytometry.

[0352] result: Flow cytometric analysis of T cells showed CD8+ responses specific for T cell epitopes in VB2210 (Figure 11). The population of CD8+ specific T cells increased from 1.3% to 3% of total splenocytes after the second vaccination, indicating a strong effect of the boost vaccination on day 21. CD8+ specific T cells were dominated by the production of IFN-γ or a combination of IFN-γ and TNF-α, a cytokine profile typical of a proinflammatory response, suggesting that VB2210 can induce cytotoxic T cell responses specific for SARS-CoV-2.

[0353] Conclusion: The data show that all VB10.COV2 DNA constructs induced rapid, strong, and for VB2210, dose-dependent T cell responses in three different mouse models. These consistently strong T cell responses indicate that the tested VB10.COV2 constructs are capable of inducing T cell responses against diverse MHC / HLA haplotypes. Furthermore, flow cytometry analysis showed that polyfunctional T cell responses were biased towards CD8+ and proinflammatory profiles. Thus, all constructs are promising T cell vaccine candidates for the prevention and / or treatment of diseases caused by the SARS CoV2 virus.

[0354] Example 5 Human clinical trials using VB10.2210 DNA plasmid A phase 1 / 2, open-label, dose-escalation clinical trial was conducted to determine safety, reactogenicity, and immunogenicity in humans.

[0355] Thirty-four healthy volunteers (aged 18-60 years) who had been vaccinated with at least two doses of the approved Covid-19 vaccines Comirnaty (Pfizer / BioNTech) or Spikevax (Moderna) at least 8 weeks prior to study entry were enrolled in cohorts of three dose levels. Comirnaty and Spikevax contain mRNA encapsulated in lipid nanoparticles, which upon vaccination deliver the mRNA to cells and induce direct and transient expression of the full-length SARS-CoV2 spike protein (which harbors two point mutations to lock the protein into a preferred pre-fusion conformation).

[0356] Participants were vaccinated intramuscularly with two doses of 0.3, 1, or 3 mg of VB10.2210 DNA plasmid (identical to DNA plasmid VB2210 referred to herein) on days 0 and 21 (booster).

[0357] Ex vivo IFN-γ ELISpot assays and ex vivo intracellular staining by flow cytometry were performed to determine cellular immunogenicity.

[0358] Ex vivo IFN-γ ELISpot assay: IFN-γ ELISpot assays were performed on peripheral blood mononuclear cells (PBMCs) from participants vaccinated with the VB10.2210 plasmid according to the manufacturer's instructions (Mabtech AB, Sweden). Stimulation was performed using five peptide pools covering epitopes contained in VB10.2210 from viral proteins S, N, M, ORF7, and ORF1 / 3 / 10. In addition to the selection of minimal peptides, 15-mer overlapping peptides covering all epitopes present in VB10.2210 were used. PBMCs only in cell culture medium with DMSO concentrations corresponding to those used in the peptide pools (<0.5% DMSO) were used as negative control. Stimulated PBMCs were analyzed for IFN-γ responses using the Human IFN-γ ELISpot Plus kit (Mabtech AB, Sweden). Spot-forming cells were measured in an IRIS ELISpot reader from Mabtech AB (Sweden).

[0359] Ex vivo intracellular staining (ICS) flow cytometry PBMCs from participants vaccinated with VB10.2210 were stimulated with the corresponding peptides for ELISpot divided into three peptide pools (M+N, ORF, and S). After 16 h of peptide stimulation, PBMCs were subjected to intracellular staining for phenotypic markers (CD4 and CD8), TNF-α, and IFN-γ and subjected to multiparameter analysis by flow cytometry.

[0360] result: Safety: VB10.2210 was safe and well tolerated up to the maximum dose of 3 mg, with no dose-limiting adverse events. No fatal or serious adverse events or adverse events of special interest were observed or reported. Local reactions were primarily mild injection site reactions (pain, tenderness, and bruising). Systemic reactions were primarily mild or moderate and of short duration. There were no signs of increased adverse events or reactogenicity after the second dose.

[0361] Cell immunogenicity: VB10.2210 amplified T cell responses against the Sars-CoV-2 spike protein and induced new T cell responses against epitopes of all seven non-spike Sars-CoV2 proteins (M, N, ORF1ab, ORF3a, ORF7a, ORF7b, and ORF10). Responses were dose-dependent, with the greatest and most extensive responses observed in the group receiving the highest dose (3 mg) (Figures 12 and 13).

[0362] Eighty-two percent (9 of 11) of participants vaccinated with 3 mg VB10.2210 responded to at least one peptide pool (Table 7), and participants responded to all peptide pools (Table 8), indicating that the selected epitopes do not discriminate in this study population. Two participants responded to all peptide pools. "Responders" were defined as participants who increased their IFN-γ ISFU / 10 from baseline (pre-vaccination) in post-vaccination samples (day 28 or day 35). 6 PBMC were defined as showing a response in ex vivo ELISpot of greater than 2-fold increase.

[0363] [Table 7]

[0364] [Table 8]

[0365] Participants demonstrated strong T cell responses after the first vaccination, which were further enhanced after the second vaccination (Figure 14). T cell responses were dominated by CD8+ T cells expressing IFN-γ and TNF-α (Figure 15).

[0366] Conclusions: The data show that vaccination of healthy volunteers with VB10.2210 DNA plasmid results in broad T cell responses with a predominance of CD8+ T cell responses. The vaccine was well tolerated with a favorable safety profile.

[0367] SEQ ID NO:161 Antigenic unit of VB2193

[0368] SEQ ID NO:162 Antigenic unit of VB2194

[0369] SEQ ID NO:163 Antigenic unit of VB2196

[0370] SEQ ID NO:164 Antigenic unit of VB2197 GVKDCVVLHSYFTSDYYQLYSTQLSSGGGGSGGGGKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGGGG SGGGGSDGKMKDLSPRWYFYYLGTGPEAGGGGSGGGGSVEGFNCYFPLQSYGFQPTNGVGGGGSGGGGSFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNF KDQVILLNKHIDAYKTFPPTEGGGGSGGGGSPSIISNEKQEILGTVSWNLREMLAHGGSGGGGSGGEGNSPFHPLADNKFALTCFSTQFAFACPDGVKHVYQLRARSVSPKLFIGGGGSGGGGSMGYINVFAFPFTIYSLLLCRMNGGGGSGGGGSMIELSLIDFYLCFLAFLLFLVLIMLIIFWFSSGGGGSGGGGLLLVA AGLEAPFLYLYALVYFLQSINFVRIIIMRLWLCWKGGGGSGGGGSIILFLALITLATCELYHYQECVRGTTVGGGGSGGGGSKDFGGNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFGGGGSGGGGSQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCV ADYSVLYNSASFSTFKGGGGSGGGGSKRFDNPVLPFNDGVYFASTEKSNIIRGSSGGGGSSGGGAFEYYHTTDPSFLGRYMSALNGGGGSGGSHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGSSGGGSGGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGG

[0371] SEQ ID NO:165 Antigenic unit of VB2207

[0372] SEQ ID NO:166 Antigenic unit of VB2208

[0373] SEQ ID NO:167 VB2210 antigenic unit MFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVIGGSGGQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRSGSSGSMGYINVFAFPFTIYSLLLCRMNGGGGFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKSGSSGSNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVESGSSGSVEG FNCYFPLQSYGFQPTNGVGGSGGLDDKDPNFKDQVILLNKHIDAYKTFPPTESGSSGSGVKDCVVLHSYFTSDYYQLYSTQLSSGSSGSEGNSPFHPLADNKF ALTCFSTQFAFACPDGVKHVYQLRARSVSPKLFISGSSGSDGKMKDLSPRWYFYYLGTGPEASGSSGSMIELSLIDFYLCFLAFLLFLVLIMLIIFWFSGGSG GAFEYYHTTDPSFLGRYMSALNSGSSGSKLPDDFTGCVIAWNSNNLDSKVGGNYNYLGGSGGKRFDNPVLPFNDGVYFASTEKSNIIRGGGSGGLLLVAAGLEAPFLYLYALVYFLQSINFVRIIMRLWLCWKGGSGGEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFGGSGGIILFLALITLATCELYHYQECVRGTT VSGSSGSHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGGGSGGLPNNTASWFTALTQHGKEDLKFPRGQGVPGGGSGGKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADASGSSGSKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGSGSSGSPSIISNEKQEILGTVSWNLREMLAHGGSGG

[0374] SEQ ID NO:168 Amino acid sequences of the signal peptide of human MIP1-α (CCL3L1) (amino acids 1-23), human MIP1-α (CCL3L1) (amino acids 24-93), hinge exon h1 from IgG3 (amino acids 94-105), hinge exon h4 of IgG3 (amino acids 106-120), dimerization unit linker (amino acids 121-130), human CH3 domain of IgG3 (amino acids 131-237), and unit linker (238-242): M 1 QVSTAALAVLLCTMALCNQVLS 23 A 24 PLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSA 93 E 94 LKTPLGDTTHT 105 E 106 PKSCDTPPPCPRCP 120 G 121 GGSSGGGSG 130 G 131 QPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK 237 GLGGL 242

[0375] SEQ ID NO:169 Anti-pan HLA class II (amino acids 1-19) with Ig VH signal peptide, anti-pan HLA class II VL (amino acids 20-127), linker (amino acids 128-142), and anti-pan HLA class II VH (amino acids 143-260): M 1 NFGLRLIFLVLTLKGVQC 19 D 20 IQMTQTTSSLSASLGDRVTISCSASQDINNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKFPRTFGGGTKLEIKR 127 G 128GGGSGGGGSGGGGS 142 Q 143 IQLVQSGPELKKPGETVKISCKASGYTFINYGMNWVKQTPGKGLKWMGWINTYSGEPTYPDDFKGRFAFSLETSASTAYLQLNNLKNEDMATYFCARGDYYGPFDNWGQGTTLTVSS 260

[0376] SEQ ID NO:170 Amino acid sequence of human TPA signal peptide: MDAMKRGLCCVLLLCGAVFVSP

[0377] SEQ ID NO:171

[0378] SEQ ID NO:172

[0379] SEQ ID NO:173

[0380] SEQ ID NO:174

[0381] SEQ ID NO:175

[0382] SEQ ID NO:176

[0383] SEQ ID NO:177

[0384] SEQ ID NO:178

[0385] SEQ ID NO:179

[0386] SEQ ID NO:180

[0387] SEQ ID NO:181

[0388] SEQ ID NO:182

[0389] SEQ ID NO:183

[0390] SEQ ID NO:184

[0391] SEQ ID NO:185

[0392] Embodiment 1. (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77; or (ii) A polynucleotide encoding a polypeptide by a nucleic acid sequence as defined in (i), preferably said nucleotide sequence does not encode a targeting unit that targets antigen-presenting cells, but encodes a multimerization unit. An immunogenic construct.

[0393] 2. (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, the antigenic unit comprising at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77, the nucleotide sequence further encoding a targeting unit and a multimerization unit that target antigen-presenting cells; or (ii) a polypeptide encoded by a nucleic acid sequence defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides as defined in (ii). 2. The immunogenic construct of embodiment 1, wherein

[0394] 3. The immunogenic construct of embodiment 2, wherein the multimerization unit is a dimerization unit and the multimeric protein is a dimeric protein consisting of two polypeptides as defined in (ii).

[0395] 4. The immunogenic construct according to any one of embodiments 1 to 3, wherein the antigenic unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 75% to 80%, preferably at least 81% to 85%, more preferably at least 86% to 90% or at least 91% to 95%, and most preferably at least 96% to 99% sequence identity to the amino acid sequences of SEQ ID NOs: 1 to 77.

[0396] 5. The immunogenic construct of any one of embodiments 1 to 4, wherein the 77 SARS-CoV-2 T cell epitopes have the amino acid sequences of SEQ ID NOs: 1 to 77.

[0397] 6. The immunogenic construct according to any one of embodiments 1 to 5, wherein the antigenic unit further comprises one or more T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 78 to 96, preferably all of said T cell epitopes.

[0398] 7. The immunogenic construct according to embodiment 6, wherein the one or more T cell epitopes have an amino acid sequence having at least 75% to 80%, preferably at least 81% to 85%, more preferably at least 86% to 90% or at least 91% to 95%, and most preferably at least 96% to 99% sequence identity to the amino acid sequences of SEQ ID NOs: 78 to 96.

[0399] 8. The immunogenic construct of embodiment 6, wherein the one or more T cell epitopes have the amino acid sequence of SEQ ID NO: 78-96.

[0400] 9. The immunogenic construct according to any one of embodiments 1 to 5, wherein the antigenic unit further comprises one or more T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 97 to 160, preferably all of said T cell epitopes.

[0401] 10. The immunogenic construct according to embodiment 9, wherein the one or more T cell epitopes have an amino acid sequence having at least 75% to 80%, preferably at least 81% to 85%, more preferably at least 86% to 90% or at least 91% to 95%, and most preferably at least 96% to 99% sequence identity to the amino acid sequences of SEQ ID NOs: 97 to 160.

[0402] 11. The immunogenic construct of embodiment 9, wherein the one or more T cell epitopes have the amino acid sequence of SEQ ID NO: 97-160.

[0403] 12. The immunogenic construct according to any one of embodiments 6 to 8, wherein the antigenic unit further comprises one or more T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequence of SEQ ID NOs: 97 to 160, preferably all of said T cell epitopes.

[0404] 13. The immunogenic construct according to embodiment 12, wherein the one or more T cell epitopes have an amino acid sequence having at least 75% to 80%, preferably at least 81% to 85%, more preferably at least 86% to 90% or at least 91% to 95%, and most preferably at least 96% to 99% sequence identity to the amino acid sequences of SEQ ID NOs: 97 to 160.

[0405] 14. The immunogenic construct of embodiment 12, wherein the one or more T cell epitopes have the amino acid sequence of SEQ ID NO: 97-160.

[0406] 15. An immunogenic construct according to any one of embodiments 1 to 14, wherein some or all of the T cell epitopes are present as single and distinct epitopes.

[0407] 16. The immunogenic construct of embodiment 15, wherein one or more of the T cell epitopes are flanked by amino acid sequences that also flank the epitope in the naturally occurring protein from which the epitope is derived.

[0408] 17. The immunogenic construct of embodiment 16, wherein one or more of the T cell epitopes are adjacent to the amino acid sequence towards the N-terminus and / or C-terminus of the epitope.

[0409] 18. The immunogenic construct according to embodiment 16 or 17, wherein such flanking sequences comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids, preferably 1 to 10 amino acids, such as 2 to 8 amino acids, or 3 to 7 amino acids, or 4 to 5 amino acids.

[0410] 19. The immunogenic construct of any one of embodiments 1-14, wherein some or all of the T cell epitopes are present as groups of T cell epitopes, each group comprising at least two T cell epitopes.

[0411] 20. The immunogenic construct of embodiment 19, wherein each group comprises 2 to 20 T cell epitopes, preferably 2 to 15 T cell epitopes, such as 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14, or 15 T cell epitopes.

[0412] 21. The immunogenic construct of embodiment 19 or 20, wherein at least two T cell epitopes are derived from the same SARS-CoV-2 protein, preferably from the same part of the same SARS-CoV-2 protein.

[0413] 22. An immunogenic construct according to any one of embodiments 19 to 21, wherein the T cell epitopes within a group are arranged sequentially and, optionally, separated by a T cell epitope linker.

[0414] 23. The immunogenic construct of any one of embodiments 19 to 22, wherein the T cell epitopes within a group are aligned to form a contiguous sequence of amino acids that corresponds to the sequence of the naturally occurring protein from which the epitope is derived.

[0415] 24. The immunogenic construct of any one of embodiments 19 to 23, wherein one or more of the T cell epitopes are flanked by amino acid sequences that are similarly flanked by the group in the naturally occurring protein from which the T cell epitopes are derived.

[0416] 25. The immunogenic construct of embodiment 24, wherein one or more of the T cell epitopes are adjacent to the amino acid sequence towards the N-terminus and / or C-terminus of the epitopes.

[0417] 26. An immunogenic construct according to embodiment 24 or 25, wherein such flanking sequences comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids, preferably 1 to 10 amino acids, such as 2 to 8 amino acids, or 3 to 7 amino acids, or 4 to 5 amino acids.

[0418] 27. An immunogenic construct according to any one of embodiments 24 to 26, wherein the resulting length of the epitopes and flanking sequences is from about 18 amino acids to about 80 amino acids, for example from 18 amino acids to 80 amino acids.

[0419] 28. The immunogenic construct of any one of embodiments 1 to 14, wherein some of the T cell epitopes are present as single and distinct epitopes and some others are present as groups of T cell epitopes, each group comprising at least two T cell epitopes.

[0420] 29. The immunogenic construct of embodiment 28, wherein each group comprises 2 to 20 T cell epitopes, preferably 2 to 15 T cell epitopes, such as 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14, or 15 T cell epitopes.

[0421] 30. The immunogenic construct of embodiment 28 or 29, wherein the at least two T cell epitopes are derived from the same SARS-CoV-2 protein, preferably from the same portion of the same SARS-CoV-2 protein.

[0422] 31. An immunogenic construct according to any one of embodiments 27 to 30, wherein the T cell epitopes within a group are arranged sequentially.

[0423] 32. The immunogenic construct according to any one of embodiments 28 to 31, wherein the T cell epitopes within a group are aligned to form a contiguous sequence of amino acids that corresponds to the sequence of the naturally occurring protein from which the epitopes are derived.

[0424] 33. An immunogenic construct according to any one of embodiments 28 to 32, wherein one or more of the T cell epitopes and / or one or more of the group of T cell epitopes are flanked by amino acid sequences which similarly flank the epitope or group of epitopes in the naturally occurring protein from which the epitope or group of epitopes is derived.

[0425] 34. An immunogenic construct according to embodiment 33, wherein one or more of the T cell epitopes and / or one or more of the group of T cell epitopes are adjacent to the amino acid sequence in the N-terminal and / or C-terminal direction of the epitope / group of epitopes.

[0426] 35. An immunogenic construct according to embodiment 33 or 34, wherein such flanking sequences comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids, preferably 1 to 10 amino acids, such as 2 to 8 amino acids, or 3 to 7 amino acids, or 4 to 5 amino acids.

[0427] 36. An immunogenic construct according to any one of embodiments 33 to 35, wherein the resulting length of the epitopes and flanking sequences is from about 18 amino acids to about 80 amino acids, for example from 18 amino acids to 80 amino acids.

[0428] 37. The immunogenic construct according to any one of the preceding embodiments, wherein the following T cell epitopes are in a group together: epitopes of SEQ ID NOs: 1 to 4, epitopes of SEQ ID NOs: 5 to 9, epitopes of SEQ ID NOs: 10 to 18, epitopes of SEQ ID NOs: 19 to 20, epitopes of SEQ ID NOs: 22 to 23, epitopes of SEQ ID NOs: 24 to 26, epitopes of SEQ ID NOs: 27 to 39, epitopes of SEQ ID NOs: 40 to 43, epitopes of SEQ ID NOs: 44 to 47, epitopes of SEQ ID NOs: 48 to 58, epitopes of SEQ ID NOs: 59 to 65, epitopes of SEQ ID NOs: 66 to 71 and epitopes of SEQ ID NOs: 72 to 77, or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0429] 38. The immunogenic construct according to embodiment 37, wherein the group comprising the T cell epitopes of SEQ ID NOs: 5 to 9 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 5 to 6 and the second group comprising the T cell epitopes of SEQ ID NOs: 7 to 9, or wherein the above T cell epitopes have an amino acid sequence having at least 73% sequence identity with the above SEQ ID NOs.

[0430] 39. The immunogenic construct according to embodiment 37 or 38, wherein the group comprising the T cell epitopes of SEQ ID NOs: 10 to 18 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 10 to 16 and the second group comprising the T cell epitopes of SEQ ID NOs: 17 to 18 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0431] 40. The immunogenic construct according to any one of embodiments 37 to 39, wherein the group comprising the T cell epitopes of SEQ ID NOs: 72 to 77 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 72 to 74, and the second group comprising the T cell epitopes of SEQ ID NOs: 75 to 77 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0432] 41. The antigen unit further comprises one or more groups of T cell epitopes having an amino acid sequence having at least 73% sequence identity with SEQ ID NOs: 97 to 160 or SEQ ID NOs: 97 to 160, and the T cell epitopes further comprise one or more groups of T cell epitopes having the following T cell epitopes: epitopes of SEQ ID NOs: 97 to 102, epitopes of SEQ ID NOs: 103 to 109, epitopes of SEQ ID NOs: 110 to 111, epitopes of SEQ ID NOs: 112 to 113, epitopes of SEQ ID NOs: 114 to 118, epitopes of SEQ ID NOs: 120 to 123, 41. The immunogenic construct according to any one of embodiments 37 to 40, wherein the epitopes of SEQ ID NOs: 124 to 129, 130 to 134, 135 to 137, 138 to 140, 141 to 146, 147 to 152, 153 to 160, or the above T cell epitopes having amino acid sequences having at least 73% sequence identity to the above SEQ ID NOs, are in a group together.

[0433] 42. The immunogenic construct according to embodiment 41, wherein the group comprising the T cell epitopes of SEQ ID NOs: 114 to 118 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 114 to 115 and the second group comprising the T cell epitopes of SEQ ID NOs: 116 to 118 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0434] 43. The immunogenic construct according to embodiment 41 or 42, wherein the group comprising the T cell epitopes of SEQ ID NOs: 141 to 146 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 141 to 143 and the second group comprising the T cell epitopes of SEQ ID NOs: 144 to 146 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0435] 44. The immunogenic construct according to any one of embodiments 41 to 43, wherein the group comprising the T cell epitopes of SEQ ID NOs: 147 to 152 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 147 to 149, and the second group comprising the T cell epitopes of SEQ ID NOs: 150 to 152 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0436] 45. The immunogenic construct according to any one of embodiments 41 to 44, wherein the group comprising the T cell epitopes of SEQ ID NOs: 153 to 160 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 153 to 157, and the second group comprising the T cell epitopes of SEQ ID NOs: 158 to 160 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0437] 46. ​​The immunogenic construct according to any one of embodiments 37 to 45, wherein the antigenic unit further comprises one or more groups of T cell epitopes of SEQ ID NOs: 78 to 96 or T cell epitopes having an amino acid sequence having at least 73% sequence identity with SEQ ID NOs: 78 to 96, wherein the following T cell epitopes: epitopes of SEQ ID NOs: 78 to 84 and epitopes of SEQ ID NOs: 86 to 96 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity with the above SEQ ID NOs are in a group together.

[0438] 47. The immunogenic construct according to embodiment 46, wherein the group comprising the T cell epitopes of SEQ ID NOs: 78 to 84 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 78 to 79, and the second group comprising the T cell epitopes of SEQ ID NOs: 80 to 84 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0439] 48. The immunogenic construct according to embodiment 46 or 47, wherein the group comprising the T cell epitopes of SEQ ID NOs: 86 to 96 is divided into two groups, the first group comprising the T cell epitopes of SEQ ID NOs: 86 to 93 and the second group comprising the T cell epitopes of SEQ ID NOs: 94 to 96 or the above T cell epitopes having an amino acid sequence having at least 73% sequence identity to the above SEQ ID NOs.

[0440] 49. An immunogenic construct according to any one of embodiments 37 to 48, wherein the antigenic unit further comprises, as a single and separate epitope, a T cell epitope of SEQ ID NO: 21 or a T cell epitope having an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 21.

[0441] 50. The immunogenic construct of embodiment 49, wherein the antigenic unit further comprises, as single and separate epitopes, the following epitopes: a T cell epitope of SEQ ID NO: 85 and a T cell epitope of SEQ ID NO: 119 or a T cell epitope having an amino acid sequence having at least 73% sequence identity with SEQ ID NO: 85 and SEQ ID NO: 119.

[0442] 51. An immunogenic construct according to any one of the preceding embodiments, wherein some or all of the T cell epitopes and / or some or all of the groups of T cell epitopes are separated from each other by T cell epitope linkers.

[0443] 52. The immunogenic construct of embodiment 51, wherein the T cell epitope linker is non-immunogenic and preferably flexible.

[0444] 53. An immunogenic construct according to embodiment 51 or 52, wherein all T cell epitope linkers contained in the antigenic units are identical.

[0445] 54. An immunogenic construct according to any one of embodiments 51 to 53, wherein the T cell epitope linker is a serine and / or glycine rich linker.

[0446] 55. An immunogenic construct according to any one of embodiments 51 to 54, wherein the T cell epitope linker is a serine and / or glycine rich linker further comprising at least one leucine residue.

[0447] 56. The immunogenic construct of any one of the preceding embodiments, wherein the antigenic unit comprises an amino acid sequence having at least 73% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0448] 57. The immunogenic construct according to embodiment 56, wherein the antigenic unit comprises an amino acid sequence having 75% to 80%, preferably at least 81% to 85%, more preferably at least 86% to 90% or at least 91% to 95%, and most preferably at least 96% to 99% sequence identity with SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0449] 58. The immunogenic construct according to embodiment 56, wherein the antigenic unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167, preferably wherein the antigenic unit has an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167, more preferably wherein the antigenic unit comprises the amino acid sequence of SEQ ID NO: 167, or even more preferably wherein the antigenic unit has the amino acid sequence of SEQ ID NO: 167.

[0450] 59. An immunogenic construct according to any one of the preceding embodiments, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen-presenting cell, preferably wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on a human antigen-presenting cell.

[0451] 60. The immunogenic construct of embodiment 59, wherein the surface molecule is selected from the group consisting of MHC, CD14, CD40, CLEC9A, chemokine receptors, such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, and XCR1, and Toll-like receptors, such as TLR-2, TLR-4, or TLR-5, preferably wherein the surface molecule is selected from the group consisting of HLA, hCD14, hCD40, hCLEC9A, human chemokine receptors, such as hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8, and hXCR1, and Toll-like receptors, such as hTLR-2, hTLR-4, or hTLR-5.

[0452] 61. The targeting unit is selected from the group consisting of soluble CD40 ligand, preferably human soluble CD40 ligand, CCL4 and its isoforms, preferably human CCL4 and its isoforms, CCL5, preferably human CCL5, CCL19, preferably human CCL19, CCL20, preferably human CCL20, CCL21, preferably macrophage inflammatory protein alpha including isoforms such as human CCL21, mouse CCL3, human CCL3, human CCL3L1, human CCL3L2. and human CCL3L3, XCL1, preferably human XCL1, XCL2, preferably human XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II, anti-CD40, preferably anti-human CD40, anti-TLR-2, preferably anti-human TLR-2, anti-TLR-4, preferably anti-human TLR-4, anti-TLR-5, preferably anti-human TLR-5 or anti-CLEC9A, preferably anti-human CLEC9A.

[0453] 62. The immunogenic construct according to embodiment 61, wherein the targeting unit comprises or consists of human MIP-1α (LD78β, CCL3L1).

[0454] 63. The immunogenic construct according to embodiment 62, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity with amino acid sequence 24 to 93 of SEQ ID NO: 168, for example comprising amino acid sequence 26 to 93 of SEQ ID NO: 168 or comprising amino acid sequence 28 to 93 of SEQ ID NO: 168.

[0455] 64. The immunogenic construct according to embodiment 63, wherein the targeting unit consists of an amino acid sequence having at least 80% sequence identity with amino acid sequence 24 to 93 of SEQ ID NO: 168, for example consisting of amino acid sequence 26 to 93 of SEQ ID NO: 168 or consisting of amino acid sequence 28 to 93 of SEQ ID NO: 168.

[0456] 65. The immunogenic construct according to embodiment 64, wherein the targeting unit consists of the amino acid sequence 24 to 93 of SEQ ID NO: 168 or consists of the amino acid sequence 26 to 93 of SEQ ID NO: 168.

[0457] 66. The immunogenic construct of embodiment 61, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity with amino acid sequence 20 to 260 of SEQ ID NO: 169, for example amino acid sequence 20 to 260 of SEQ ID NO: 169.

[0458] 67. The immunogenic construct according to embodiment 66, wherein the targeting unit consists of an amino acid sequence having at least 80% sequence identity with amino acid sequence 20 to 260 of SEQ ID NO: 169, for example consisting of amino acid sequence 20 to 260 of SEQ ID NO: 169.

[0459] 68. The immunogenic construct of embodiment 67, wherein the targeting unit consists of amino acid sequence 20 to 260 of SEQ ID NO: 169.

[0460] 69. An immunogenic construct according to any one of embodiments 2 to 68, wherein the multimerization unit is selected from the group consisting of a dimerization unit, a trimerization unit, and a tetramerization unit, and the multimerization unit optionally comprises a hinge region capable of forming one or more covalent bonds.

[0461] 70. The immunogenic construct according to embodiment 69, wherein the multimerization unit is a trimerization unit, such as a trimerization unit derived from human collagen, or the C-terminal domain of T4 fibritin.

[0462] 71. The immunogenic construct of embodiment 70, wherein the multimerization unit is a trimerization unit derived from human collagen, preferably one selected from the group consisting of the trimerization domain derived from human collagen XVIII and the trimerization domain derived from human collagen XV.

[0463] 72. The immunogenic construct according to embodiment 69, wherein the multimerization unit is a tetramerization unit that is a domain derived from p53.

[0464] 73. An immunogenic construct according to any one of embodiments 69 to 72, wherein the multimerization unit comprises a hinge region capable of forming one or more covalent bonds.

[0465] 74. An immunogenic construct according to any one of embodiments 69-73, wherein the hinge region is from an Ig, such as from a human Ig, such as from hIgG1 or hIgG2 or hIgG3, or from hIgM.

[0466] 75. An immunogenic construct according to any one of embodiments 69 and 73 to 74, wherein the multimerization unit is a dimerization unit, and the dimerization unit further comprises another domain that promotes dimerization.

[0467] 76. The immunogenic construct of embodiment 75, wherein the further domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

[0468] 77. The immunogenic construct according to embodiment 75 or 76, wherein said further domain is a carboxy-terminal C domain derived from IgG, preferably from IgG3, more preferably from hIgG3.

[0469] 78. An immunogenic construct according to any one of embodiments 75 to 77, wherein the dimerization unit further comprises a dimerization unit linker such as a glycine-serine rich linker, for example GGGSSGGGSG.

[0470] 79. The immunogenic construct of embodiment 78, wherein the dimerization unit linker connects the hinge region and the additional domain that promotes dimerization.

[0471] 80. The immunogenic construct according to any one of embodiments 75 to 79, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker, and the CH3 domain of human IgG3.

[0472] 81. The immunogenic construct according to embodiment 80, wherein the dimerization unit comprises an amino acid sequence having at least 80% sequence identity with amino acid sequence 94 to 237 of SEQ ID NO: 168.

[0473] 82. The immunogenic construct according to embodiment 81, wherein the dimerization unit consists of an amino acid sequence having at least 80% sequence identity with amino acid sequence 94 to 237 of SEQ ID NO: 168.

[0474] 83. The immunogenic construct according to embodiment 82, wherein the dimerization unit consists of the amino acid sequence 94 to 237 of SEQ ID NO: 168.

[0475] 84. An immunogenic construct according to any one of the preceding embodiments, wherein the construct is a polynucleotide (i), preferably wherein the polynucleotide comprises a nucleotide sequence further encoding a signal peptide.

[0476] 85. The immunogenic construct of embodiment 84, wherein the signal peptide is the native leader sequence of the targeting unit.

[0477] 86. The immunogenic construct of embodiment 85, wherein the signal peptide is an Ig VH signal peptide, a human TPA signal peptide or a human MIP1-α (CCL3L1) signal peptide, preferably wherein the polynucleotide comprises a nucleotide sequence encoding a targeting unit comprising or consisting of human MIP-1α (LD78β, CCL3L1) and wherein the signal peptide is a human MIP1-α (CCL3L1) signal peptide.

[0478] 87. The immunogenic construct according to any one of embodiments 84 to 86, wherein the polynucleotide encodes a targeting unit comprising an amino acid sequence having at least 80% sequence identity with amino acid sequence 24 to 93 of SEQ ID NO: 168, for example a targeting unit comprising amino acid sequence 26 to 93 of SEQ ID NO: 168 or comprising amino acid sequence 28 to 93 of SEQ ID NO: 168, and further comprises a nucleotide sequence encoding a signal peptide comprising an amino acid sequence having at least 85% sequence identity with amino acid sequence 1 to 23 of SEQ ID NO: 168.

[0479] 88. An immunogenic construct according to any one of the preceding embodiments, wherein the construct is a polynucleotide.

[0480] 89. The immunogenic construct according to embodiment 88, wherein the polynucleotide is a nucleotide sequence as defined in any one of embodiments 1 to 87.

[0481] 90. The immunogenic construct of embodiment 88 or 89, wherein the polynucleotide is DNA or RNA.

[0482] 91. A polynucleotide as defined in any one of embodiments 1 to 90, 124, 125, and 127.

[0483] 92. A vector comprising the polynucleotide of embodiment 91.

[0484] 93. The vector described in embodiment 92, wherein the vector is a polycistronic vector comprising a) the polynucleotide described in embodiment 91 and b) one or more nucleic acid sequences encoding one or more immunostimulatory compounds, and the vector is capable of coexpressing the polypeptides encoded by the polynucleotides and the one or more immunostimulatory compounds as separate molecules.

[0485] 94. The vector described in embodiment 93, wherein the vector comprises one or more co-expression elements, such as a co-expression element selected from the group consisting of an IRES element, a 2A peptide, a promoter, and a bidirectional promoter.

[0486] 95. The vector of embodiment 92 or 93, wherein the one or more immunostimulatory compounds are compounds that stimulate APCs, and the stimulation results in the attraction, activation, maturation and / or proliferation of APCs.

[0487] 96. The vector according to any one of embodiments 92 to 94, wherein the vector is selected from the group consisting of DNA vectors and RNA vectors.

[0488] 97. The vector described in embodiment 96, wherein the vector is a DNA vector, such as a DNA plasmid or a DNA viral vector, such as a DNA viral vector selected from the group consisting of adenovirus, vaccinia virus, adeno-associated virus, cytomegalovirus, and Sendai virus.

[0489] 98. The vector of embodiment 96, wherein the vector is an RNA vector, such as an RNA plasmid or an RNA viral vector, such as a retroviral vector, such as a retroviral vector selected from the group consisting of an alphavirus, a lentivirus, a Moloney murine leukemia virus, and a rhabdovirus.

[0490] 99. A method for producing a vector as defined in any one of embodiments 92 to 98, comprising: a) transfecting or transducing a cell in vitro with said vector; b) culturing the cells; c) optionally lysing the cells and releasing the vector from the cells; d) isolating and optionally purifying said vector; A method comprising:

[0491] 100. A host cell comprising a polynucleotide according to embodiment 91 or a vector according to any one of embodiments 92 to 98.

[0492] 101. A polypeptide encoded by a nucleic acid as defined in any one of embodiments 1 to 83 and as defined in embodiments 123 and 126.

[0493] 102. A multimeric protein consisting of a plurality of polypeptides according to embodiment 101.

[0494] 103. A multimeric protein according to embodiment 102, which is a dimeric protein consisting of two polypeptides as defined in claim 101, for example a homodimeric or heterodimeric protein, preferably a homodimeric protein.

[0495] 104. A method for producing a polypeptide according to embodiment 101, comprising: a) transfecting or transducing a cell with a polynucleotide according to claim 91 or a vector comprising such a polynucleotide; b) culturing the cells; c) isolating the polypeptide from the cell; and d) optionally purifying the isolated polypeptide. A method comprising:

[0496] 105. A method for preparing a multimeric protein according to embodiment 102 or 103, comprising: a) transfecting or transducing a cell with a polynucleotide according to claim 91 or a vector comprising such a polynucleotide; b) culturing the cells; c) isolating the multimeric protein from the cells; and d) optionally purifying the isolated multimeric protein; A method comprising:

[0497] 106. An immunogenic construct according to any one of embodiments 1 to 90 and 123 to 127, or a polynucleotide according to embodiment 91, or a vector according to any one of embodiments 92 to 98, or a polypeptide according to embodiment 101, or a multimeric protein according to embodiment 102 or 103, for use as a medicament.

[0498] 107. A pharmaceutical composition or vaccine comprising an immunogenic construct according to any one of embodiments 1 to 90 and 123 to 127, or a polynucleotide according to embodiment 91, or a vector according to any one of embodiments 92 to 98, or a polypeptide according to embodiment 101, or a multimeric protein according to embodiment 102 or 103, and a pharma- ceutically acceptable carrier.

[0499] 108. A pharmaceutical composition or vaccine according to embodiment 107, comprising a pharma- ceutically acceptable carrier and a vector according to any one of embodiments 92 to 98.

[0500] 109. A pharmaceutical composition or vaccine according to embodiment 107 or 108, wherein the pharma- ceutically acceptable carrier is selected from the group consisting of saline, buffered saline, such as PBS, dextrose, water, glycerol, ethanol, aqueous buffer, such as isotonic aqueous buffer or Tyrode's buffer, and combinations thereof.

[0501] 110. A pharmaceutical composition or vaccine according to any one of embodiments 107 to 109, wherein the composition or vaccine further comprises a molecule that facilitates transfection of a cell with a polynucleotide or vector.

[0502] 111. A pharmaceutical composition or vaccine according to any one of embodiments 107 to 110, wherein the composition or vaccine further comprises a pharma- ceutically acceptable amphiphilic block copolymer comprising blocks of poly(ethylene oxide) and polypropylene oxide, for example in an amount of 0.2% w / v to 20% w / v of the pharma- ceutical composition or vaccine.

[0503] 112. A pharmaceutical composition or vaccine according to any one of embodiments 107 and 109 to 110, wherein the composition or vaccine comprises the polypeptide or the multimeric protein in the range of 5 μg to 5 mg.

[0504] 113. A pharmaceutical composition according to any one of embodiments 107 to 111, wherein the composition or vaccine comprises in the range of 0.1 to 10 mg, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg, or for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg of the polynucleotide or vector.

[0505] 114. A pharmaceutical composition or vaccine according to any one of embodiments 107 to 113 for use in the treatment of a disease caused by SARS-CoV-2.

[0506] 115. The pharmaceutical composition or vaccine according to embodiment 114, wherein the treatment is a prophylactic treatment.

[0507] 116. The pharmaceutical composition or vaccine according to embodiment 114, wherein the treatment is a therapeutic treatment.

[0508] 117. A method for treating a disease caused by SARS-CoV-2, comprising administering to a subject a pharmaceutical composition or vaccine described in any one of embodiments 107 to 113.

[0509] 118. The method of embodiment 117, wherein the subject is in need of prevention of such a disease and the method is a prophylactic treatment.

[0510] 119. The method of embodiment 117, wherein the subject has such a disease and the method is a therapeutic treatment.

[0511] 120. A method for treating a disease caused by SARS-CoV-2, the method comprising administering to a subject previously vaccinated with a SARS-CoV-2 vaccine a pharmaceutical composition or vaccine described in any one of embodiments 107 to 113.

[0512] 121. The method of embodiment 120, wherein the method is a preventive treatment.

[0513] 122. The method of embodiment 120, wherein the method is a therapeutic treatment.

[0514] 123. An immunogenic construct according to any one of embodiments 1 to 90, wherein the construct is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 171, 172, 173, 174, 175, 176, and 177 or a dimeric protein consisting of two such polypeptides, preferably a polypeptide having the amino acid sequence of SEQ ID NO: 177 or a dimeric protein consisting of two such polypeptides.

[0515] 124. An immunogenic construct according to any one of embodiments 1 to 90, wherein the construct is a polypeptide comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 178, 179, 180, 181, 182, 183, and 184, preferably a polypeptide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 184.

[0516] 125. An immunogenic construct according to any one of the preceding embodiments, wherein the construct is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 185.

[0517] 126: An immunogenic construct described in any one of embodiments 1 to 90, wherein the construct is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0518] 127. An immunogenic construct according to any one of the preceding embodiments, wherein the construct is a polynucleotide comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167.

[0519] 128. A pharmaceutical composition / vaccine comprising an immunogenic construct according to any one of embodiments 123 to 127 and a pharma- ceutically acceptable carrier.

[0520] 129. A pharmaceutical composition / vaccine according to embodiment 128, comprising a vector comprising the immunogenic construct according to embodiment 125 and a pharma- ceutically acceptable carrier.

Claims

1. (i) a polynucleotide comprising a nucleotide sequence encoding an antigenic unit, wherein the antigenic unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to an amino acid sequence of SEQ ID NOs: 1-77; or (ii) a polypeptide comprising an antigenic unit encoded by the nucleotide sequence defined in (i). An immunogenic construct.

2. (i) a polynucleotide comprising a nucleotide sequence encoding said antigenic unit, wherein said antigenic unit comprises at least 77 SARS-CoV-2 T cell epitopes having an amino acid sequence having at least 73% sequence identity to an amino acid sequence of SEQ ID NOs: 1-77, and said nucleotide sequence further encodes a targeting unit that targets antigen-presenting cells and a multimerization unit; or (ii) a polypeptide encoded by the nucleotide sequence defined in (i), or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii). The immunogenic construct of claim 1, wherein

3. a) the antigen unit further comprises one or more T cell epitopes having an amino acid sequence with at least 73% sequence identity to the amino acid sequences of SEQ ID NOs: 78 to 96, preferably the one or more T cell epitopes have the amino acid sequences of SEQ ID NOs: 78 to 96, and more preferably the antigen unit further comprises all of the T cell epitopes, or b) the antigenic unit further comprises one or more T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 97 to 160, preferably the one or more T cell epitopes have the amino acid sequences of SEQ ID NOs: 97 to 160, and more preferably the antigenic unit further comprises all of the T cell epitopes; The immunogenic construct of claim 1.

4. a) the antigen unit further comprises one or more T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 97 to 160, for example, the one or more T cell epitopes have the amino acid sequences of SEQ ID NOs: 97 to 160, and preferably the antigen unit further comprises all of the T cell epitopes; or b) the antigenic unit further comprises one or more T cell epitopes having an amino acid sequence having at least 73% sequence identity with the amino acid sequences of SEQ ID NOs: 78 to 96, for example, the one or more T cell epitopes have the amino acid sequences of SEQ ID NOs: 78 to 96, and preferably the antigenic unit further comprises all of the T cell epitopes; The immunogenic construct of claim 3.

5. some or all of the T cell epitopes are present as single and distinct epitopes, and / or some or all of the T cell epitopes are present as groups of T cell epitopes, each group comprising at least two T cell epitopes; For example, some or all of the T cell epitopes are present as groups of T cell epitopes, each group comprising at least two T cell epitopes; Preferably, the at least two T cell epitopes are derived from the same SARS-CoV-2 protein; preferably derived from the same portion of the same SARS-CoV-2 protein, Preferably, the T cell epitopes in the group are arranged sequentially, optionally separated by T cell epitope linkers, or the T cell epitopes in the group are aligned to form a contiguous sequence of amino acids which corresponds to the sequence of the naturally occurring protein from which the epitope is derived; The immunogenic construct of claim 1.

6. one or more of said T cell epitopes are adjacent to amino acid sequences that also flank said epitope in the naturally occurring protein from which said epitope is derived; For example, one or more of the T cell epitopes are adjacent to the amino acid sequence towards the N-terminus and / or C-terminus of the epitope, and optionally the flanking sequence comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids, preferably 1 to 10 amino acids, such as 2 to 8 amino acids, or 3 to 7 amino acids, or 4 to 5 amino acids; The immunogenic construct of claim 1.

7. 2. The immunogenic construct of claim 1, wherein some or all of the T cell epitopes and / or some or all of the groups of T cell epitopes are separated from each other by a T cell epitope linker, preferably a non-immunogenic T cell epitope linker, which is preferably a flexible linker.

8. The antigenic unit comprises an amino acid sequence having at least 73% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167; For example, the antigenic unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167, preferably the antigenic unit comprises the amino acid sequence of SEQ ID NO: 167, more preferably the antigenic unit consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167, preferably the antigenic unit consists of the amino acid sequence of SEQ ID NO:

167.

9. the targeting unit is or comprises a moiety that interacts with a surface molecule on the antigen-presenting cell, preferably the targeting unit is or comprises a moiety that interacts with a surface molecule on a human antigen-presenting cell; For example, the surface molecule is selected from the group consisting of MHC, CD14, CD40, CLEC9A, chemokine receptors such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, and XCR1, and Toll-like receptors such as TLR-2, TLR-4, or TLR-5, preferably HLA, hCD14, hCD40, hCLEC9A, human chemokine receptors such as hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8, and hXCR1, and Toll-like receptors such as hTLR-2, hTLR-4, or hTLR-5; Preferably, said targeting unit is a soluble CD40 ligand, preferably human soluble CD40 ligand, CCL4 and its isoforms, preferably human CCL4 and its isoforms, CCL5, preferably human CCL5, CCL19, preferably human CCL19, CCL20, preferably human CCL20, CCL21, preferably human CCL21, macrophage inflammatory protein alpha including isoforms of macrophage inflammatory protein alpha, e.g. mouse CCL3, human CCL4 CL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, preferably human XCL1, XCL2, preferably human XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II, anti-CD40, preferably anti-human CD40, anti-TLR-2, preferably anti-human TLR-2, anti-TLR-4, preferably anti-human TLR-4, anti-TLR-5, preferably anti-human TLR-5, or anti-CLEC9A, preferably anti-human CLEC9A; Most preferably, the targeting unit comprises or consists of human MIP-1α (LD78β, CCL3L1), The immunogenic construct of claim 2.

10. the multimerization unit is selected from the group consisting of a dimerization unit, a trimerization unit, and a tetramerization unit, and the multimerization unit optionally comprises a hinge region capable of forming one or more covalent bonds; Preferably, the multimerization unit is a dimerization unit comprising a hinge region (preferably a hinge region derived from an Ig, for example a hinge region derived from a human Ig, for example a hinge region derived from hIgG1 or hIgG2 or hIgG3, or a hinge region derived from hIgM), and the dimerization unit further comprises another domain that promotes dimerization; Preferably, said further domain is an immunoglobulin domain, preferably an immunoglobulin constant domain, more preferably a carboxy-terminal C domain from IgG, preferably from IgG3, more preferably from hIgG3; Preferably, said dimerization unit further comprises a dimerization unit linker, for example a glycine-serine rich linker such as GGGSSGGGSG, preferably said dimerization unit linker connecting said hinge region and said further domain that promotes dimerization. The immunogenic construct of claim 2.

11. the construct is the polynucleotide (i), e.g., a DNA or RNA polynucleotide; Preferably, the polynucleotide further comprises a nucleotide sequence encoding a signal peptide, Preferably, the signal peptide is the native leader sequence of the targeting unit. The immunogenic construct of claim 1 .

12. An immunogenic construct as described in claim 2, wherein the construct is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 185, or a polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

184.

13. A vector comprising a polynucleotide as defined in any one of claims 1 to 12, or said polypeptide.

14. A polypeptide encoded by a nucleotide sequence defined in any one of claims 1 to 12, or a multimeric protein consisting of a plurality of said polypeptides, for example a dimeric protein consisting of two of said polypeptides.

15. For use as a medicine, An immunogenic construct according to any one of claims 1 to 12, A vector comprising a polynucleotide or a polypeptide as defined in any one of claims 1 to 12, or A polypeptide encoded by a nucleotide sequence defined in any one of claims 1 to 12, or a multimeric protein consisting of a plurality of said polypeptides.

16. An immunogenic construct according to any one of claims 1 to 12, A vector comprising a polynucleotide or a polypeptide as defined in any one of claims 1 to 12, or A pharmaceutical composition or vaccine comprising a polypeptide encoded by a nucleotide sequence defined in any one of claims 1 to 12 or a multimeric protein consisting of a plurality of said polypeptides, and a pharmaceutically acceptable carrier.

17. 17. The pharmaceutical composition or vaccine of claim 16 for use in the treatment of a disease caused by SARS-CoV-2, wherein said treatment is a prophylactic or therapeutic treatment.

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