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Peptides and Combinations of Peptides for Use in Immunotherapy Against an Infection by Sars-COV-2 (COVID-19)

20230322865 · 2023-10-12

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of an infection by SARS-CoV-2 (COVID-19). The present invention furthermore relates to SARS-CoV-2-associated T-cell peptide epitopes that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-SARS-CoV-2 immune responses, or to stimulate T-cells ex vivo and transfer them into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

    Claims

    1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10 and SEQ ID NO: 3, 4, 7, 8, 9, and 10, and variant sequences thereof which are at least 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 10 or SEQ ID NO: 3, 4, 7, 8, 9, and 10, and wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) and induces T-cells cross-reacting with said variant peptide; and a pharmaceutical acceptable salt thereof, wherein said peptide is not a full-length polypeptide.

    2. The peptide according to claim 1, wherein said peptide is configured to bind to an MHC class-I or -II molecule, and wherein said peptide, when bound to said MHC, is capable of being recognized by CD4 and/or CD8 T-cells.

    3. The peptide according to claim 2, wherein said amino acid sequence of said peptide comprises a continuous stretch of amino acids according to any one of SEQ ID NO 1 to SEQ ID NO: 10, or SEQ ID NO: 3, 4, 7, 8, 9, and 10.

    4. An antibody or fragment thereof that specifically recognizes the peptide or variant thereof according to claim 1.

    5. The antibody of claim 4, selected from the group consisting of: soluble antibody, membrane-bound antibody, monoclonal antibody.

    6. A T-cell receptor or a fragment thereof, that is reactive with an HLA ligand, wherein said ligand is the peptide or variant thereof according to claim 1.

    7. The T-cell receptor according to claim 6, wherein said ligand is the peptide or variant thereof according to claim 1 when bound to an MHC molecule.

    8. A nucleic acid, encoding for the peptide or variant thereof according to claim 1.

    9. An expression vector comprising the nucleic acid of claim 8.

    10. A recombinant host cell comprising the nucleic acid of claim 8.

    11. The recombinant host cell of claim 10, wherein the host cell is selected from an antigen presenting cell, a dendritic cell, a T-cell, and an NK cell.

    12. A nucleic acid, encoding for the antibody or fragment thereof according to claim 4.

    13. An expression vector comprising the nucleic acid of claim 12.

    14. A recombinant host cell comprising the nucleic acid of claim 12.

    15. The recombinant host cell of claim 14, wherein the host cell is selected from an antigen presenting cell, a dendritic cell, a T-cell, and an NK cell.

    16. A nucleic acid, encoding for the T-cell receptor or fragment thereof according to claim 6.

    17. An expression vector comprising the nucleic acid of claim 16.

    18. A recombinant host cell comprising the nucleic acid of claim 16.

    19. The recombinant host cell of claim 18, wherein the host cell is selected from an antigen presenting cell, a dendritic cell, a T-cell, and an NK cell.

    20. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T-cells with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate said T-cells in an antigen specific manner, wherein said antigen is the peptide according to claim 1.

    21. An activated T lymphocyte, produced by the method according to claim 20, which selectively recognizes a cell which presents a polypeptide comprising an amino acid sequence given in claim 1.

    22. A pharmaceutical composition comprising at least one active ingredient which is the peptide of claim 1, and a pharmaceutically acceptable carrier.

    23. The pharmaceutical composition of claim 22, comprising at least 6 different peptides, each peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10, and variant sequences thereof which are at least 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 10, and wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) and induces T-cells cross-reacting with said variant peptide; and a pharmaceutical acceptable salt thereof, wherein said peptide is not a full-length polypeptide.

    24. A pharmaceutical composition, comprising peptides characterized by the following amino acid sequences SEQ ID NO: 1, 2, 3, 4, 7, 8, 9, and 10: TABLE-US-00016 (SEQ ID NO: 1) KDGIIWVATEGALNT (SEQ ID NO: 2) RWYFYYLGTGPEAGL (SEQ ID NO: 3) ASWFTALTQHGKEDL (SEQ ID NO: 4) LLLLDRLNQLESKMS (SEQ ID NO: 5) ASAFFGMSRIGMEVT (SEQ ID NO: 6) VADYSVLYNSASFST (SEQ ID NO: 7) ITRFQTLLALHRSYL (SEQ ID NO: 8) LSYYKLGASQRVAGD (SEQ ID NO: 9) FYVYSRVKNLNSSRV (SEQ ID NO: 10) SKWYIRVGARKSAPL, and a pharmaceutically acceptable carrier.

    25. The pharmaceutical composition of claim 24, which is a vaccine.

    26. The pharmaceutical composition of claim 25, wherein said vaccine is for the prevention or treatment of an infection by SARS-CoV-2 (COVID 19).

    27. The pharmaceutical composition of claim 24, additionally comprising an adjuvant selected from the group consisting of TLR1/2 ligand, TLR1/2 ligand XS15, TLR1/2 ligand Pam3Cys, TLR9 ligand, TLR9 ligand CpG, Montanide ISA 51 VG, and combinations thereof.

    28. A pharmaceutical composition, comprising peptides characterized by the following amino acid sequences SEQ ID NO: 3, 4, 7, 8, 9, and 10: TABLE-US-00017 (SEQ ID NO: 3) ASWFTALTQHGKEDL (SEQ ID NO: 4) LLLLDRLNQLESKMS (SEQ ID NO: 7) ITRFQTLLALHRSYL (SEQ ID NO: 8) LSYYKLGASQRVAGD (SEQ ID NO: 9) FYVYSRVKNLNSSRV (SEQ ID NO: 10) SKWYIRVGARKSAPL, and a pharmaceutically acceptable carrier.

    29. The pharmaceutical composition of claim 28, which is a vaccine.

    30. The pharmaceutical composition of claim 29, wherein said vaccine is for the prevention or treatment of an infection by SARS-CoV-2 (COVID 19).

    31. The pharmaceutical composition of claim 28, additionally comprising an adjuvant selected from the group consisting of TLR1/2 ligand, TLR1/2 ligand XS15, TLR1/2 ligand Pam3Cys, TLR9 ligand, TLR9 ligand CpG, Montanide ISA 51 VG, and combinations thereof.

    32. A pharmaceutical composition comprising at least one active ingredient which is the T-cell receptor or fragment thereof of claim 6, and a pharmaceutically acceptable carrier.

    33. A kit comprising: (a) a container comprising a pharmaceutical composition containing the peptide or the variant according to claim 1, in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; (c) optionally, at least one more peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10, and (d) optionally, instructions for (i) use of the solution or (ii) reconstitution or use of the lyophilized formulation.

    34. A kit comprising: (a) a container comprising a pharmaceutical composition containing the T-cell receptor or fragment thereof according to claim 6, in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; (c) optionally, at least one more peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10, and (d) optionally, instructions for (i) use of the solution or (ii) reconstitution or use of the lyophilized formulation.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0088] FIG. 1: Prediction of SARS-CoV-2-derived CD4.sup.+ T-cell epitopes. (A) HLA-DR population coverage reached with the inventors' selection of allotypes compared to the world population. The frequencies of individuals within the world population carrying up to six HLA allotypes (x-axis) are indicated as grey bars on the left y-axis. The cumulative percentage of population coverage is depicted as black dots on the right y-axis. (B) Distribution of HLA-restricted peptides derived from the distinct ORFs to the different HLA-DR allotypes. (C-K) Distribution of HLA-DR-binding peptides within the different ORF proteins. Each color represents a distinct HLA class I allotype. (L) Protein coverage of the predicted HLA-DR peptides for the different ORF proteins. The dashed bar indicated the percentage a distinct ORF covers within the complete SARS-CoV-2 proteome.

    [0089] FIG. 2: CD4.sup.+ T-cell responses against CoVac-1 peptides in patients after recovery from SARS-CoV-2 infection. (A) Example of ELISPOT assays after 12 days of T-cell amplification using KDGIIWVATEGALNT (P01; SEQ ID NO: 1), RWYFYYLGTGPEAGL (P03; SEQ ID NO: 2), LLLLDRLNQLESKMS (P07; SEQ ID NO: 4) in PBMC of patients recovered from SARS-CoV-2 infection HIV DR peptide served as negative control. (B) Intracellular cytokine staining (ICS) of CD4.sup.+ T-cells using the CoVac-1 peptides KDGIIWVATEGALNT (P01; SEQ ID NO: 1), ASWFTALTQHGKEDL (P04; SEQ ID NO: 3), LLLLDRLNQLESKMS (P07; SEQ ID NO: 4) Graphs show single, viable CD4.sup.+ cells stained for IFN, TNF and CD107 analyzed by flow cytometry.

    [0090] FIG. 3 Results of IFN-γ ELISpot-based immunomonitoring in a colon carcinoma patient. Immunomonitoring was performed before and after s.c. administration of a personalized peptide vaccine containing XS15 emulgated in Montanide. The functionality (IFN-γ production) of peptide-specific CD4.sup.+ and CD8.sup.+ T-cells for two peptides contained in the administered vaccine is shown. A total of three vaccinations were performed.

    [0091] FIG. 4 Induced T cell responses in the 12 subjects of the P-pVAC-SARS-CoV-2 trial assessed by ex vivo ELISPOT. Induction (marked in green) of peptide specific T cell response at V2 (d7), V3 (d14, V4 (d28), as measured by ex vivo IFN-γ Elispot is defined as positive assay (mean spot count per well is at least 3-fold higher than the mean number of spots in the negative control wells) and the spot count is at least 2-fold higher than the baseline assay (V1).

    [0092] FIG. 5 Exemplary T-cell responses against CoVac-1 peptides in one subject after vaccination. Example of ex vivo IFN-γ ELISPOT assays using the six peptides included in CoVac-1 vaccine in PBMC of a subject (1-11) vaccinated within the phase I trial. HIV DR peptide served as negative control, PHA as positive control.

    [0093] FIG. 6 Frequency of functional SARS-CoV-2-specific T cells. Frequency of functional (IFN-γ.sup.+, TNF.sup.+, IL-2.sup.+) SARS-CoV-2-specific T cells after one single CoVAC-1 (6 peptides vaccine) vaccination on d28 and on d1 prior to vaccination as assessed by flow cytometry-based intracellular cytokine staining (ICS). Data are shown for positive subjects. Frequency of functional SARS-CoV-2 T cells is indicated as % of cytokine positive cells minus the respective negative control.

    [0094] FIG. 7 CoVac-1-induced SARS-CoV-2-specific CD8+ T cell responses (6 peptides vaccine). Representative example (pCoV-18) of CoVac-1-induced SARS-CoV-2 specific CD8.sup.+ T cell responses against embedded HLA-class I T cell epitopes as assessed by IFN-γ ELISPOT on day 14 and day 28 after on single CoVac-1 vaccination (left panel) and flow cytometry-based tetramer staining (right panel).

    [0095] FIG. 8 Frequency of cytokine positive CD4.sup.+ T cells. Data analyzed at day 29 (PBNT162B1/Pfizer and mRNA-1273/Moderna, published data) and 28 (CoVac-1) after vaccination with 2 doses of PBNT162B1/Pfizer) and 1 dose of mRNA-1273/MODERNA and CoVac-1 (6 peptides vaccine). Data shown as mean of cytokine positive T cells as assessed by intracellular cytokine staining. Frequency of functional SARS-CoV-2 T cells is indicated as % of cytokine positive cells minus the respective negative control.

    [0096] FIG. 9 Intensity of vaccine-induced SARS-CoV-2 T cell responses. INF-γ ELISpot data at day 29 after vaccination with ChAgOx1 (AstraZeneca, published data) and at day 28 after vaccination with CoVac-1 (6 peptides vaccine) (interim immunogenicity analyses P-pVAC-SARS-CoV-2). Data are shown as median of spot forming units per 1×10.sup.6 cell minus the median of the respective negative control. Analyses were performed after stimulation of PBMCs with peptides-pools representing the respective vaccine-antigens.

    [0097] FIG. 10 Direct comparison of T cell responses to CoVac-1 T cell epitopes between CoVac-1- (6 peptides vaccine) and BNT162b2-vaccinated subjects. INF-γ ELISpot analyses were performed ex vivo for PBMCs from HV mean 25 days after the second BNT162b2 vaccination and CoVac-1-vaccinated study subjects at day 28 after vaccination. PMCS were stimulated with the six T cell epitopes included in the CoVac-1 vaccine. T cell responses were considered positive when the mean spot count was 3-fold higher than the mean spot count of the negative control. The intensity of T cell responses is depicted as spot counts, which represent the mean spot count of triplicates normalized to 5×10.sup.6 cells minus the normalized mean spot count of the respective negative control. Data are shown as mean with SEM.

    [0098] FIG. 11 Role of SARS-CoV-2 variants of concern (VOC) on CoVac-1 peptides and immunogenicity. Color-coded mutations described for VOC are shown together with corresponding affected CoVac-1 peptides (Panel A). Intensities (calculated spot counts) of T cell responses to CoVac 1 peptides as well as to the corresponding peptide pools comprising the CoVac-1-affecting mutations of B.1.1.7 and B.1.351 were assessed ex vivo by IFN-γ ELISPOT assays using PBMCs from study participants collected on day 28 after vaccine administration (pCoVs, Panel B) or from human convalescents (HCs, Panel C).

    EXAMPLES

    1. Non-Clinical Studies

    1.1 Overview

    [0099] In the following sections, the selection of the SARS-CoV-2-derived peptides as basis for an embodiment of the vaccine according to the invention (in the following: CoVac-1 vaccine) is explained. Furthermore, all preclinical data for the novel TLR1/2 agonist XS15 included as a lipopeptide in the peptide cocktail for the CoVac-1 drug product (DP) will be described. The predictive value of animal models for the non-clinical testing of CoVac-1 vaccines is limited, as the interaction between HLA molecules, peptides and T-cells is highly species and allele-specific. Therefore, the non-clinical section describing the SARS-CoV-2-derived peptides for the CoVac-1 DP contains extensive in vitro and ex vivo data.

    1.2 SARS-CoV-2-Derived HLA-DR Peptide Selection

    Rational for the Selection of HLA-Class II-Derived Peptides

    [0100] Multiple studies in animal models have clearly demonstrated the requirement of CD4.sup.+ T-cell help for the generation of protective antibody responses (for example, influenza, malaria, vaccinia). Recent studies have also demonstrated that the role of CD4.sup.+ T-cells in the immune response to viral infections is not limited to help for antibody production; CD4.sup.+ T-cells are also required to generate optimal CD8.sup.+ T-cell responses. Moreover, CD4.sup.+ T-cells additionally can act as effector cells by the secretion of cytokines and direct killing of infected cells. HLA class II antigens specifically activate CD4.sup.+ helper T-cells, therefore a vaccine based on SARS-CoV-2-derived HLA class II peptides will enable a potent cellular and humoral immune response to SARS-CoV-2 preventing severe courses of COVID-19. Therefore, the CoVac-1 DP was composed of ten SARS-CoV-2 derived HLA-DR peptides.

    Prediction of SARS-CoV-2-Derived Peptides

    [0101] For the prediction of potential SARS-CoV-2-derived T-cell epitopes the inventors retrieved the complete highly conserved and annotated representative proteome sequence of SARS-CoV-2 isolate Wuhan-Hu-1 containing ten different open reading frames (ORFs); see Wu et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020; 579(7798):265-269. The amino acid sequence of this sequence is completely identical to the reference sequence (EPI_ISL_412026) defined by Wang et al. conducting multiple sequence alignments and phylogenetic analyses of 95 full-length genomic sequences; see Wang et al. The establishment of reference sequence for SARS-CoV-2 and variation analysis. Journal of Medical Virology. 2020.

    [0102] Using the SYFPEITHI algorithm (Rammensee et al. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999; 50(3-4):213-219) the inventors predicted SARS-CoV-2-derived HLA class II peptides for the six most common HLA-DR allotypes (HLA-DRB1*01:01, -DRB1*03:01, DRB1*04:01, -DRB1*07:01, -DRB1*11:01, and -DRB1*15:01) covering more than 70.0% of the world population with at least one allotype (FIG. 1). As the other two HLA class II loci (DQ and DP) with additional binding opportunities for CoVac-1 HLA class II peptides have not been included into the analysis, the real promiscuity of the chosen peptides is probably even higher. Binding of the warehouse HLA class II peptides to several different HLA class II molecules (promiscuous binding) is important to ensure that the majority of patients treated with warehouse peptides are able to benefit from a supportive helper T-cell response. For HLA class II predictions all ten ORFs were split into 15 amino acid long peptides, resulting in 9,561 15mer long peptides in total. The 5% top-scoring peptides of each ORF (related to the total length of each ORF, 2% for ORF1) were selected and sorted according to their position within the protein. To allow for a broad applicability the inventors selected clusters of peptide length variants with high prediction scores for several HLA-DR alleles (FIG. 1). From each selected cluster one representative peptide was produced as synthetic peptide for immunogenicity analyses, thereby avoiding peptides containing cysteines (Tables 1 and 2).

    TABLE-US-00003 TABLE 1  SARS-COV-2-derived peptides included in CoVac-1 vaccines (′10 peptides vaccine′) SEQ ID HLA peptide pos- protein protein sequence NO restriction length ition protein name class KDGIIWVATEGALNT  1 DR 15 127-141 ORF9 nucleocapsid structural protein RWYFYYLGTGPEAGL  2 DR 15 107-121 ORF9 nucleocapsid structural protein ASWFTALTQHGKEDL  3 DR 15  50-64 ORF9 nucleocapsid structural protein LLLLDRLNQLESKMS  4 DR 15 221-235 ORF9 nucleocapsid structural protein ASAFFGMSRIGMEVT  5 DR 15 311-325 ORF9 nucleocapsid structural protein VADYSVLYNSASFST  6 DR 15 362-376 ORF2 spike protein structural ITRFQTLLALHRSYL  7 DR 15 235-249 ORF9 spike protein structural LSYYKLGASQRVAGD  8 DR 15 176-190 ORF5 membrane structural protein FYVYSRVKNLNSSRV  9 DR 15  56-70 ORF4 membrane structural protein SKWYIRVGARKSAPL 10 DR 15  43-57 ORF8 n.a. non- structural

    TABLE-US-00004 TABLE 2 SARS-COV-2-derived peptides included in CoVac-1 vaccines (′6 peptides vaccine′)  SEQ ID HLA peptide po- protein protein sequence NO restriction length sition protein name class ASWFTALTQHGKEDL  3 DR 15  50-64 ORF9 nucleocapsid structural protein LLLLDRLNQLESKMS  4 DR 15 221-235 ORF9 nucleocapsid structural protein ITRFQTLLALHRSYL  7 DR 15 235-249 ORF9 spike protein structural LSYYKLGASQRVAGD  8 DR 15 176-190 ORF5 membrane structural protein FYVYSRVKNLNSSRV  9 DR 15  56-70 ORF4 membrane structural protein SKWYIRVGARKSAPL 10 DR 15  43-57 ORF8 n.a. non- structural

    Immunogenicity and Role in the Natural Course of SARS-CoV-2 Infection of SARS-CoV-2-Derived Peptides

    [0103] In order to exert anti-viral activity, peptides contained in the warehouse must (i) be processed and presented naturally in the course of SARS-CoV-2 infection and (ii) induce specific immune responses in vivo. To assess these points, the inventors screened a cohort of patients recovered from SARS-CoV-2 infection for T-cell responses against the predicted SARS-CoV-2-derived HLA class II peptides. Naturally-induced peptide-specific T-cell responses were assessed using IFN-γ ELISPOT and flow cytometry-based intracellular cytokine staining. Those peptides with the highest frequency (up to 95%) of T-cell responses within the natural SARS-CoV-2 infection were selected for the CoVac-1 vaccine (Table 3). Naturally-induced SARS-CoV-2 T-cells detecting CoVac-1 peptides where further characterized by intracellular cytokine staining. Most T-cells were multifunctional CD4.sup.+ Th1 cells producing TNF and IFN-γ as well as the upregulation of the cytotoxicity marker CD107. Furthermore, the inventors screened a large cohort of donors never exposed to SARS-CoV-2 (samples asserted before June 2019) for memory T-cell responses against the CoVac-1 peptides. For 8/10 of the SARS-CoV-2-derived-HLA-DR peptides selected for the CoVac-1 vaccine T-cell responses in up to 44% of unexposed donors were detected. This is most likely due to T-cell cross-reactivity based on sequence homology of SARS-CoV-2 to common cold corona viruses or other pathogens. This cross-reactivity is a well-known frequently occurring effect in viral and other pathogen T-cell immunity providing so called heterologous immunity. Heterologous immunity is defined as an immunity that can develop to one pathogen after exposure or vaccination to non-identical pathogens. Therefore, the CoVac-1 vaccine will not only induce de novo SARS-CoV-2-specific T-cell responses but further will allow to expand low frequent SARS-CoV-2-detecting T-cell populations preexisting in study volunteers/patients.

    [0104] Exemplarily, results of an IFN-γ ELISPOT and flow cytometry-based intracellular cytokine staining detecting naturally-induced CD4.sup.+ T-cell responses are displayed in FIG. 2.

    TABLE-US-00005 TABLE 3 Naturally-induced T-cell responses to CoVac-1 peptides in IFN-y ELISPOT and ICS in patients recovered from SARS-COV-2 infection  and unexposed donors SEQ SARS cohort PRE cohort ID Pro- protein Start pos/  frequency pos/  frequency Sequence NO tein Protein class position tested [%] ICS tested [%] KDGIIWVATEGALNT  1 ORF9 nucleocapid structural 127 20/22 91% CD4.sup.+ IFN-γ.sup.+ TNF.sup.+ 8/18 44% protein CD107a RWYFYYLGTGPEAGL  2 ORF9 nucleocapid structural 107 16/22 73% CD4.sup.+ IFN-γ.sup.+ TNF.sup.+ 1/18  6% protein CD107a ASWFTALTQHGKEDL  3 ORF9 nucleocapid structural  50 13/22 59% CD4.sup.+ IFN-γ.sup.+ TNF.sup.+ 1/18  6% protein LLLLDRLNQLESKMS  4 ORF9 nucleocapid structural 221 14/22 64% CD8.sup.+ and CD4.sup.+ IFN-γ.sup.+ 1/17  6% protein TNF.sup.+ CD107a ASAFFGMSRIGMEVT  5 ORF9 nucleocapid structural 311 12/23 52% CD4.sup.+ IFNγ-γ.sup.+ TNF.sup.+ 1/18  6% protein VADYSVLYNSASFST  6 ORF2 spike protein structural 362 n.t. Induction of peptide- specific CD4γ T-cell re- sponses in a healthy volunteer by peptide vaccination ITRFQTLLALHRSYL  7 ORF2 spike protein structural 235 12/22 55% IFN-γ.sup.+ TNF.sup.+ CD107a.sup.+ 1/20  5% FYVYSRVKNLNSSRV  9 ORF4 envelope pro- structural  56 11/22 50% IFNγ.sup.+ TNF.sup.+ 2/19 11% tein LSYYKLGASQRVAGD  8 ORF5 membrane structural 176 21/22 95% 10x IFN-γ.sup.+ TNF.sup.+ 5x 0/19  0% protein CD107a.sup.+ SKWYIRVGARKSAPL 10 ORF8 n.a. accessory  43 15/22 68% 5x IFN-γ.sup.+ TNF.sup.+ 4/16 25%

    Representation of SARS-CoV-2-Derived Peptides in Other Species

    [0105] In contrast to self-peptides used for peptide vaccination in malignancy that may be expressed at considerable levels also on normal organs virus-derived peptides do not bear the risk of inducing autoimmunity. This is supported by blast results of the peptide sequences of the CoVac-1 vaccine using the “Basic Local Alignment Search Tool (BLAST)” of the uniprot database (https://www.uniprot.org/blast/) showing for none of the peptides sequence similarities to any human protein/peptide. Most common sequence similarities were identified to SARS-CoV-1 as well as to Bat SARS-like coronavirus (Table 4).

    TABLE-US-00006 TABLE 4 Results of Basic Local Alignment Search Tool (BLAST)″ of the uniprot database (https://www.uniprot.org/blast/) for sequence similarities of CoVac-1 peptide SEQ ID Uniprot Identity Sequence NO Organism Protein name ID Protein [%] Status KDGIIWVATEGALNT  1 Bat coronavirus BM48- Nucleoprotein EOXJ00  93.3 unreviewed 31/BGR/ RWYFYYLGTGPEAGL  2 Human SARS corona- Nucleoprotein P59595  93.3 reviewed virus ASWFTALTQHGKEDL  3 Human SARS corona- Nucleoprotein P59595  93.3 reviewed virus LLLLDRLNQLESKMS  4 Human SARS corona- Nucleoprotein P59595  93.3 reviewed virus ASAFFGMSRIGMEVT  5 Human SARS corona- Nucleoprotein P59595 100.0 reviewed virus VADYSVLYNSASFST  6 Bat coronavirus HKU3 Spike glycoprotein Q3LZX1  86.7 reviewed ITRFQTLLALHRSYL  7 Gluconobacter Bifunctional enzyme A0A511AZ17,  90 unreviewed wancherniae NB, IspD/IspF, Insulinase A0A5F1Z381 Leptospira gomenensis family protein FYVYSRVKNLNSSRV  9 Human SARS corona- Envelope small mem- P59637 100 reviewed virus brane protein LSYYKLGASQRVAGD  8 Bat SARS-like corona- Membrane protein A0A2R3SUX3 100 unreviewed virus SKWYIRVGARKSAPL 10 Fasciola hepatica Ryanodine receptor A0A4E0RMD6 50.0 unreviewed 44F

    Embedded HLA Class I Binding Warehouse Peptides

    [0106] 6/10 (Table 1) or 4/6 (Table 2) vaccine peptides in the CoVac-1 vaccine include embedded CD8.sup.+ T-cell epitopes predicted to bind to various different HLA class I allotypes. These embedded HLA class I peptides will allow to additionally activate CD8.sup.+ T-cells that should also contribute to prevent severe courses of COVID-19 disease. The embedded CD8.sup.+ T-cell epitopes are based on the inventors' preclinical work predicting and characterizing SARS-CoV-2-derived HLA class I T-cell epitopes. Therefore, the protein sequences of all ten SARS-CoV-2 ORFs were split into 9-12 amino acid long peptides covering the complete proteome of the virus. The prediction algorithms NetMHCpan 4.0 and SYFPEITHI 1.0 (Rammensee et al. 1999, I.c.) were used to predict the binding of the peptides to HLA-A*01:01, -A*02:01, -A*03:01, -A*11:01, -A*24:02, -B*07:02, -B*08:01, -B*15:01, -B*40:01, and -C*07:02. With this selection of allotypes 91.7% of the world population is covered with at least one HLA allotype. Out of the total amount of 38,424 possible 9-12mer long peptides derived from all 10 ORFs 1,739 unique HLA-binding peptides excluding peptides containing cysteine were predicted as potential SARS-CoV-2 epitopes; Nelde et al. (I.c.). Table 5 shows the embedded CD8.sup.+ T-cell epitopes for each CoVac-1 peptide.

    TABLE-US-00007 TABLE 5 Embedded HLA-class I T-cell epitopes in the CoVac-1 vaccine peptides  SEQ HLA embedded HLA Sequence ID NO restriction class I peptides KDGIIWVATEGALNT  1 DRB1*01, no DRB1*04, DRB1*11 RWYFYYLGTGPEAGL  2 DRB1*04 YYLGTGPEAGL (A*24) (SEQ ID NO: 11) YLGTGPEAGL (A*02) (SEQ ID NO: 12) ASWFTALTQHGKEDL  3 DRB1*04, FTALTQHGK (A*03, A*11) (SEQ ID NO: 13) DRB1*11 LLLLDRLNQLESKMS  4 DRB1*04, LLLLDRLNQL (A*02) (SEQ ID NO: 14) DRB1*15 LLLDRLNQL (A*02) (SEQ ID NO: 15) ASAFFGMSRIGMEVT  5 DRB1*01, ASAFFGMSR (A*11) (SEQ ID NO: 16) DRB1*04, DRB1*07, DRB1*11 VADYSVLYNSASFST  6 DRB1*01, no DRB1*04, DRB1*11 ITRFQTLLALHRSYL  7 DRB1*01 TREQTLLAL (C*07) (SEQ ID NO: 17) TLLALHRSY (B*15) (SEQ ID NO: 18) LLALHRSYL (A*02) (SEQ ID NO: 19) FYVYSRVKNLNSSRV  9 DRB1*04, FYVYSRVKNL (A*24) (SEQ ID NO: 20) DRB1*11 RVKNLNSSR (A*03) (SEQ ID NO: 21) LSYYKLGASQRVAGD  8 DRB1*04, no DRB1*07 SKWYIRVGARKSAPL 10 DRB1*01, no DRB1*11

    3D-Structure Analysis of SARS-CoV-2-Derived Peptides

    [0107] Development of antibody-dependent enhancement (ADE) antibodies has been identified as potential risk for infected patients after vaccination approaches; see Graham B S. Rapid COVID-19 vaccine development. Science. 2020. In contrast to other classical vaccines aiming to induce an antibody-response to prevent viral infections the CoVac-1 vaccine is designed to induce SARS-CoV-2 specific T-cells. To further minimize the risk of ADE antibody development to the CoVac-1 vaccine peptides especially for peptides from the virus surface proteins, only sequences were selected that do not represent antibody epitopes, since they are not accessible to antibodies due to the predicted 3D structure of the protein or based on their intravirion location (Table 6). In a first in vivo application of a CoVac-1 vaccine peptide from the spike protein (VADYSVLYNSASFST; SEQ ID NO: 6) in a healthy volunteer no antibody induction was observed so far. Therefore, there is compared to other vaccine approaches only minimal risk of induction of ADE antibodies by the CoVac-1 vaccine.

    TABLE-US-00008 TABLE 6 Results of 3D structural analysis of CoVac-1 peptides derived from virus surface proteins exposure to SEQ ID protein antibodies Sequence NO Protein Protein class close state open state VADYSVLYNSASFST 6 ORF2 spike structural likely to be exposed in a protein buried cleft ITRFQTLLALHRSYL 7 ORF2 spike structural RSYL unresolved, ALHRSYL protein mos tare buried unresolved, (SEQ ID NO: 22) most are buried (SEQ ID NO: 23) FYVYSRVKNLNSSRV 9 ORF4 envelope structural intravirion position protein LSYYKLGASQRVAGD 8 ORF5 membrane structural intravirion position protein
    Comparison of CoVac-1 to Approved SARS-COV-2 Vaccines (BNT126b2, Biontech SE; mRNA-12738, Moderna, Inc.; ChAdOx1, AstraZeneca)

    [0108] In contrast to approved vaccine candidates, the peptide-based CoVac-1 vaccine includes validated SARS-CoV-2 T cell epitopes that were proven (i) to be frequently detected in convalescents after natural SARS-CoV-2 infection, (ii) to be of pathophysiological relevance for T cell immunity to combat COVID-19 and (iii) to mediate long-term immunity after infection. Thus, CoVac-1 is expected to induce strong and long-lasting SARS-CoV-2 T cell immunity that is comparable to T cell immunity after natural infection. Furthermore, and also in contrast to approved vaccine candidates that induce immune responses limited to the spike protein of SARS-CoV-2, CoVac-1 induces broad T cell immunity targeting multiple viral proteins (e.g. spike, nucleocapsid, membrane, envelope etc.). This is of particular importance in light of emerging mutations that challenge efficacy of current vaccines.

    2. Clinical Studies

    Summary

    Definition of Terms

    [0109] Drug substances: Ten SARS-CoV-2-derived HLA-DR peptides and the TLR1/2 ligand XS15 [0110] Peptide cocktail: Peptide cocktail for each study volunteer/patient including 10 (Table 1) or 6 (Table 2) immunogenic HLA-DR SARS-CoV-2-derived peptides and the TLR1/2 ligand XS15 [0111] IMP/Drug Product/peptide vaccine: CoVac-1: Peptide cocktail emulsified in Montanide ISA 51 VG [0112] IMP administration: subcutaneous injection with 2 ml syringe (e.g. BD Emerald) and needle (e.g. BD Eclipse Needle 27Gx1/2)

    2.1 SARS-CoV-2-Derived Peptides

    [0113] Each volunteer/patient enrolled in the P/T-pVAC-SARS-CoV-2 trials receive 10 (Table 1) or 6 (Table 2) promiscuous HLA-DR peptides (240 μg each) derived from different proteins of SARS-CoV-2 together with the TLR1/2 ligand XS15. For application the vaccine cocktail is emulsified in Montanide ISA 51 VG. Details on drug substances can be found in Tables 1 (10 peptides vaccine) and 2 (6 peptides vaccine).

    Synthesis and Structure

    [0114] All drug substances are linear peptides with a free amino group at the N-terminus and a free carboxyl group at the C-terminus. Trifluoroacetate anions are bound in ionic form to basic groups of the peptide molecule. All amino acid residues are in the L-configuration and are not chemically modified at any positions. All warehouse peptides are manufactured in a facility holding certificates for the production of GMP grade synthetic peptides. All peptides are synthetic peptides, which are manufactured by well-established solid phase peptide synthesis (SPPS) procedures using Fmoc chemistry.

    2.2 TLR1/2 Ligand XS15

    [0115] The TLR1/2 ligand XS15 is used as adjuvant in the P/T-pVAC-SARS-CoV-2 trials to induce strong and long-lasting T-cell responses. The lipopeptide XS15, chemical name N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteinyl-GDPKHPKSF, is a water-soluble synthetic Pam3Cys-derivative. As TLR1/2 ligand it will be included as an adjuvant in the peptide cocktail of each volunteer/patient enrolled in the P/T-pVAC-SARS-CoV-2 trials. XS15 hydrochloride is a synthetic linear, ten amino acid peptide with a palmitoylated N-terminus. All optically active amino acids are in L-configuration. The drug substance is isolated as a hydrochloride salt. The primary structure of XS15 Hydrochloride is given by its route of synthesis and identity can be confirmed by mass spectrometry. XS15 is manufactured (Bachem AG, Bubendorf, Switzerland) as an active pharmaceutical ingredient following the relevant GMP guidelines, namely ICH Q7 and Part 2 of the EU GMP guideline. The manufacturer has been regularly and successfully inspected by the Swiss Agency for Therapeutic Products (Swissmedic) and is responsible for manufacture, testing, and release as well as storage and shipment of XS15 hydrochloride. Please refer to the IB of XS15 (1.0. 27 May 2020).

    2.3 Montanide ISA 51 VG

    [0116] Montanide is manufactured by Seppic and by the rewarding manufacturer Elaiapharm, respectively. Montanide ISA 51 VG is used as adjuvant in the P/T-pVAC-SARS-CoV-2 trials to emulsify the peptide cocktail (including SARS-CoV-2-derived peptides and XS15). Montanide ISA 51 VG is defined as a mixture of a highly purified mineral oil (Drakeol 6VR) and a surfactant (Mannide monooleate). When mixed with an aqueous phase in a 50/50 ratio, it renders a water-in-oil emulsion. This water-in-oil emulsion is used as vaccine adjuvant, in order to enhance the immune response against antigens. For the detailed pre-clinical information on Montanide ISA 51 VG it is referred to IB of Montanide ISA 51 VG (3291/GB/03/June 2019).

    2.4 Formulation

    [0117] The peptide cocktails are formulated by the University of Tübingen (Department of Immunology) including the 10 (Table 1) or 6 (Table 2) promiscuous HLA-DR peptides (240 μg each) derived from different proteins of SARS-CoV-2 and the TLR1/2 ligand XS15. The final CoVac-1 DP is a water-in-oil emulsion of the peptide cocktail as described above and Montanide ISA 51 VG. All components will be provided by the Wirkstoffpeptidlabor of the Department of Immunology in Tübingen together with a “mixing kit” allowing for the mixture of the two components by the pharmacy of the participating centers (peptide cocktail, Montanide ISA 51 VG). The Wirkstoffpeptidlabor holds certificates for the production of GMP grade synthetic peptides and for the formulation of multi-peptide vaccine cocktails including the TLR1/2 ligand XS15. The peptide cocktails are liquid formulations consisting of the Drug Substances dissolved in 33% DMSO in water for injection (WFI). No excipients are added. Peptide cocktails are packaged in 1 ml vials using the Crystal® closed vial technology and capped by a flip-off crimping cap. Each vial is for single use only. Peptide cocktails are manufactured aseptically. Analyses for endotoxins, sterility, visible particles and pH of solution are performed according to European Pharmacopeia (EP). Peptide cocktails (including the TLR1/2 ligand XS15) are labeled with an identification code definitely assignable to the clinical trial. The trial medication is labeled according to § 5 of GCP-V. Samples of the labels are filed in the trial master file (TMF). The peptide vaccine cocktail is packaged together with Montanide ISA 51 VG and the mixing equipment into the “mixing kit” and shipped from the Wirkstoffpeptidlabor of the Department of Immunology, Tübingen, to the pharmacy of the participating centers. Shipment is documented according to standard operation procedure. The “mixing kit” is shipped using isolated packaging with an automated temperature control system, which has to be returned to the Wirkstoffpeptidlabor of the Department of Immunology together with the acknowledgement of receipt after delivery of the consignment. The device is read out to document the correct storage temperatures during shipment. Data are documented according to standard operation procedure. The shipment is performed by an associate of the Wirkstoffpeptidlabor of the Department of Immunology, Tübingen. Peptide cocktail solutions should be stored at −20° C.

    2.5 Dosage

    Peptide Cocktail

    [0118] Peptides cocktails for the CoVac-1 drug product (DP) are liquid formulations consisting of the drug substances dissolved in 33% DMSO. Peptides cocktails to be used in the P/T-pVAC-SARS-CoV-2 trials consist of 10 (Table 1) or 6 (Table 2) different peptides with 1 mg/ml per peptide and the TLR1/2 ligand XS15 with 0.2 mg/ml. Each vial contains 700 μl.

    Montanide ISA 51 VG

    [0119] Before delivery to the study site the peptide cocktail is emulgated in a water-oil emulsion 1:1 with Montanide ISA 51 VG by the pharmacy of the participating centers according to a “Mischanleitung” provided with the “mixing kit” from the Wirkstoffpeptidlabor of the Department of Immunology.

    CoVAC-1

    [0120] The final CoVAC-1 drug product is delivered as a water-oil solution by the pharmacy of the participating centers to the respective trial site in a syringe with a final volume of 500 μl (approx. 240 μg per peptide, 50 μg XS15).

    Instruction for Handling and Storage of CoVac-1 DP

    [0121] One single dose of the CoVac-1 vaccine is applied subcutaneously (s.c.) into the abdominal skin of the study volunteer/patient. The study-specific instructions are described in the Clinical Study Protocols. Each CoVac-1 vial is labeled with a study specific code. Aseptic technique is used when preparing CoVac-1 vaccines for administration. Each vial of CoVac-1 DP is for single use only. Final vaccine drug product is stored at room temperature and administered within 24 h after mixing of the components.

    2.6 Dosage Regimen and Vaccination Schedule and Site

    [0122] Per volunteer/patient one single vaccination with the CoVac-1 vaccine is applied subcutaneously. This is based on the first clinical experiences with individualized peptide vaccines combined with the TLR1/2 ligand XS15 in a healthy volunteer and cancer patients. The usage of XS15 enables to induce strong and long-lasting T-cell responses against vaccine peptides in so far 100% of vaccinated healthy volunteer (HV) and patients after one injection. These data allow reducing the number of vaccinations and the duration of vaccination phase compared to previous studies.

    [0123] Previous vaccination trials were performed at peptide doses ranging from 10 to 5,000 μg per vaccination. Even though only a few of these trials included a dose finding element, there is a tendency that doses below 100 μg are not effective to induce T-cell responses whilst doses above 500 μg do not seem to generate an increasing immunogenicity. Dose-finding studies performed with viral protein-derived epitopes showed significantly stronger immune responses in the 300-500 μg range versus the 100 μg dose, without significantly higher immune responses in the 1,000 vs. 500 μg group (Kran et al. HLA- and dose-dependent immunogenicity of a peptide-based HIV-1 immunotherapy candidate (Vacc-4x). AIDS. 2004; 18(14):1875-1883). Based on the observations briefly summarized above, the ideal dose for a peptide is anticipated to be in the range between 100 to 500 μg with a strong preference to the upper range. The dose of ˜240 μg per peptide per dose for CoVac-1 vaccine was selected based on these findings and on the feasibility in pharmaceutical development of the vaccines. With this dose induction of strong T-cell response for two of the peptides included in the CoVac-1 vaccine in combination with XS15 emulsified in Montanide ISA 51 VG could be shown.

    Rational for the CoVac-1 Peptide Dose

    [0124] Based on the following data the peptide vaccine is applied with a dose of 240 μg per peptide: [0125] The phase II and Ill peptide vaccination trials performed by Immatics Biotechnologies GmbH in patients with metastatic RCC demonstrated a very good safety and tolerability profile for a peptide cocktail with 10 peptides and a dose of 413 μg per peptide. Another study of the Department of Immunology in Tübingen in patients with metastatic prostate cancer also showed a good tolerability for a vaccine cocktail including 13 peptides with a dose of 300 μg per peptide (Feyerabend et al. Novel multi-peptide vaccination in Hla-A2.sup.+ hormone sensitive patients with biochemical relapse of prostate cancer. Prostate. 2009; 69(9):917-927). [0126] Sato et. al. (Sato et al. Immunological evaluation of peptide vaccination for patients with gastric cancer based on pre-existing cellular response to peptide. Cancer Sci. 2003; 94(9):802-808) investigated a peptide vaccine in patients with advanced gastric cancer composed of 14-16 peptides with a dose of 30 mg per peptide. Even with that very high dose of peptide vaccine, no severe side effects were observed. Furthermore, the group of Noguchi (Noguchi et al. Induction of cellular and humoral immune responses to tumor cells and peptides in HLA-A24 positive hormone-refractory prostate cancer patients by peptide vaccination. Prostate. 2003; 57(1):80-92) showed a good tolerability and safety profile for a peptide vaccine composed of 14 peptides and a dose of 9 mg per peptide. [0127] An ongoing iVAC-CLL01 study applies personalized peptide vaccine cocktails (9 peptides a 300 μg) showing a very good safety and tolerability profile. [0128] Preliminary data from a healthy volunteer and cancer patients vaccinated with a personalized peptide vaccine (240-300 μg per peptide) in combination with XS15 showed potent induction of T-cell responses in 100% of healthy volunteer (HV) and patients and a good safety profile. [0129] Preliminary data from a healthy volunteer vaccinated with two of the peptides (240 μg per peptide) included in the CoVac-1 vaccine in combination with XS15 emulsified in Montanide ISA 51 VG showed induction of strong CD4.sup.+ T-cell response and a good safety profile.
    Monitoring after Peptide Vaccination

    [0130] To provide a maximum of safety for the patients treated in the P/T-pVAC-SARS-CoV-2 studies, the monitoring after peptide vaccination is performed as follows: [0131] All patients are monitored for 2 hours after vaccination, including close monitoring of heart rate, blood pressure, temperature and subjective well-being. The peptide vaccination studies conducted in infectious disease as well as cancer patients showed that a monitoring time of 1-2 hours provides optimal safety for the patient. In these studies, allergic reactions occurred mostly 1 hour after vaccination. [0132] The monitoring of the patients is carried out by specially trained nurses and physicians. Monitoring includes regular checks of the vital parameters of the patient (blood pressure, pulse, temperature, oxygen saturation) to ensure early notice of side effects, especially allergic reactions. Each monitoring unit is equipped with a crash cart and an intensive care team will be on standby.

    Vaccination Site

    [0133] The CoVac-1 vaccines are applied subcutaneously in the abdomen of the patients based on the following rational:

    [0134] The peptide vaccine IMA901 (for treatment of RCC) has been tested in two completed clinical studies where it had been applied to a total of 96 patients. In the phase I study (n=30), vaccination to the thigh, abdomen and skin of the forearm were allowed. In 27 patients evaluable for immune responses, vaccination in the skin of the abdomen was numerically, but not significantly better in terms of achieved immune response rate (88% n=8 vs. 64% n=14, and 60% n=5) for thigh and forearm, respectively; e.g. p=0.75 abdomen vs. other; Fisher's exact test). In the phase II study (n=68), vaccinations to the thigh and abdomen were allowed, and again immune responder rates and multi-peptide responder rates were very comparable and did not differ significantly from each other (e.g. 27.5% multi-peptide responders after vaccination into the abdominal skin (n=40) vs. 23.8% after vaccination into the thigh (n=21). In addition, preclinical work in a mouse model (brain tumor, glioma) showed that vaccination into the foreleg is less effective in inducing immune responses as compared to vaccination into a more distant site (hind leg), potentially due to the immunosuppressive influence of the tumor in the tumor draining lymph (Ohlfest et al. Vaccine injection site matters: qualitative and quantitative defects in CD8 T-cells primed as a function of proximity to the tumor in a murine glioma model. J Immunol. 2013; 190(2):613-620).

    2.7 Preclinical Information

    SARS-CoV-2-Derived Peptides (Drug Substances)

    [0135] The peptide cocktail included in the CoVac-1 vaccine consists of ten (Table 1) or six (Table 2) promiscuous HLA-DR peptides from different proteins (ORFs) of the SARS-CoV-2 virus. Using the algorithm SYFPEITHI (www.syfpeithi.de; Rammensee et al. 1999, I.c.) peptide sequences were selected that show a high binding score for several HLA-DR allotypes and can therefore be widely used in the population. Furthermore, especially those HLA-DR peptides were selected that contain embedded HLA class I sequences in order to induce CD4.sup.+ T-cell responses as well as CD8.sup.+ T-cell responses. Furthermore, for peptides from virus surface proteins, only sequences were selected that do not represent antibody epitopes, since they are not accessible to antibodies due to the predicted 3D structure of the protein. This should prevent the formation of antibodies against the vaccinated peptides, which could possibly lead to a deterioration of the lung pathology. Immunogenicity was proven for all HLA-DR peptides included in the peptide cocktail in a large cohort of SARS-CoV-2 convalescent donors proving the significant role of these T-cell epitopes in the natural course of SARS-CoV-2 infection. Furthermore, two peptides were already applied in a vaccination approach in a single healthy volunteer, inducing strong CD4.sup.+ T-cell responses. The SARS-CoV-2-derived HLA-DR peptides will activate CD4.sup.+Th1 cells which directly contribute to virus clearance and deliver strong T-helper signals to the CD8.sup.+ T-cells primed by embedded HLA class I sequences in the CoVac-1 vaccine or during natural infection. Furthermore, in terms of a SARS-CoV-2 infection these SARS-CoV-2 specific CD4+Th1 cells should vigorously activate virus antigen-experienced B cells. The resulting enhanced activity could lead to more rapid virus clearance and prevention of severe course of COVID-19.

    2.8 Pharmacokinetics

    [0136] The CoVac-1 vaccine is injected subcutaneously. Biodistribution and pharmacokinetic studies for the included SARS-CoV-2-derived peptides have not been performed as they are usually not conducted on vaccines since the vaccine components are intended to immediately bind to HLA molecules expressed on local cell populations (i.e. dendritic cells (DCs)). In addition, peptides are generally extremely short-lived in biological fluids.

    2.9 Clinical Experience

    [0137] Peptide vaccines in combination with XS15 emulsified in Montanide ISA 51 VG similar to the CoVaC-1 DP have been applied in a healthy volunteer (Rammensee et al. A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer. J Immunother Cancer. 2019; 7(1):307) and cancer patients inducing a strong and long lasting CD8.sup.+ and Th1CD4.sup.+ T-cell response to viral peptides (including SARS-CoV-2 derived peptides), neoepitopes from cancer-specific mutations as well as to tumor-associated self-peptides. Beside formation of granuloma locally on injection side, no relevant side effects, especially no allergic or anaphylactic reactions or cytokine release syndrome, have been observed after a single application of the vaccine. Montanide ISA 51 VG has been used in more than 200 clinical trials including more than 6000 patients. Most common side effects are injection site reaction (68%) including granuloma development, fatigue (54%), fever (41%), gastrointestinal disorders (32%) and injection site or local erythema (28%) (Van Doorn et al. Safety and tolerability evaluation of the use of Montanide ISA51 as vaccine adjuvant: A systematic review. Hum Vaccin Immunother. 2016; 12(1):159-169). In general, the observed adverse from controlled trials with non-healthy as well as healthy individuals were mild to moderate in intensity. For more details it is referred to the IB of Montanide ISA 51 VG (3291/GB/03/June 2019).

    2.10 Choice of Adjuvant Drugs for the P/T-pVAC-SARS-CoV-2 Study

    [0138] For the P/T-pVAC-SARS-CoV-2 studies the following considerations on adjuvant drugs were made:

    [0139] A main prerequisite for peptide vaccination, beside the selection of optimal antigen targets, is the usage of a suitable adjuvant, which is able to induce strong and long-lasting immune responses. Among the most effective peptide vaccination methods tested in humans is the subcutaneous injection of peptides emulsified in Montanide ISA 51 VG, a water-in-oil-emulsion, combined with the TLR9 ligand CpG (Baumgaertner et al. Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells. Int J Cancer. 2012; 130(11):2607-2617). However, CpG is not available for clinical trials and a peptide/antigen vaccine emulsified in Montanide without any additional adjuvant shows only week immune response (Freund J. The effect of paraffin oil and mycobacteria on antibody formation and sensitization; a review. Am J Clin Pathol. 1951; 21(7):645-656.). Therefore, the inventors choose for the P/T-pVAC-SARS-CoV-2 study the novel TLR1/2 ligand XS15. XS15 is a water-soluble derivative of the TLR1/2 ligand Pam3Cys inducing a strong CD8+ and Th1 CD4.sup.+ T-cell response against free short peptides in Montanide ISA 51 VG after s.c. injection in healthy volunteers as well as in patients (Rammensee et al. A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer. J Immunother Cancer. 2019; 7(1):307). Immune responses could be induced for viral peptides (including SARS-CoV-2-derived peptides), neoepitopes from cancer-specific mutations as well as for tumor-associated self-peptides. Peptide vaccination with XS15 emulsified in Montanide ISA 51 VG results in granuloma formation on vaccination site where the vaccinated peptides persist for at least 7 weeks. Furthermore peptide-specific T-cells could also be detected on granuloma site, however with a lower frequency than observed in peripheral blood, which confirms that there is no risk of T-cell sequestration, dysfunction or deletion on vaccination site using XS15 in Montanide ISA 51 VG. Strikingly, the induced immune responses persist for more than 1.5 years. Multi-peptide vaccination with XS15 emulsified in Montanide ISA 51 VG showed a good safety profile without any systemic side effects so far no allergic reactions as it was reported for peptide vaccines applied in combination with GM-CSF. Therefore, XS15 and Montanide ISA 51 VG will serve as adjuvants for the CoVac-1 vaccines. Detailed information to the lipopeptide XS15 which is included in the vaccine cocktail can be found in the IB of XS15 (1.0. 27 May 2020). Detailed information to Montanide ISA 51 VG can be found in the IB of Montanide ISA 51 VG (3291/GB/03/June 2019).

    2.11 Effects of XS15 in Humans

    [0140] Evidence from Self-Administration of XS15 Adjuvanted Vaccines

    [0141] Vaccination of a healthy individual using XS15 as an adjuvant has been described and published (Rammensee et al. 2019, I.c.) In this self-experiment a dosage of 80 μg XS15 was used in addition to Montanide ISA 51 VG and a multi-peptide vaccine with virus-derived synthetic peptides. Here a painless granuloma forming at the injection site was described with a volume increasing to about 8 ml, as measured by ultrasound (days 17 and 41 after administration), without any sonographic signs of infection. After 21 days it appeared as a well-palpable induration of about 2×2 cm, with a central reddish surface. The granuloma was described as not touch-sensitive, whereas the skin surface reported to be slightly itching.

    [0142] Regarding immune responses and being aware that this is purely anecdotal evidence, a single vaccination induced strong ex vivo measurable CD8.sup.+ and TH1 CD4.sup.+ responses in a human volunteer upon a single injection of XS15 mixed to uncoupled peptides in a water-in-oil emulsion. The granuloma that formed locally at the injection site containing highly activated functional CD4.sup.+ and CD8.sup.+ effector memory T-cells. In addition, respective T-cells could be characterized in circulation. Ex vivo T-cell responses in peripheral blood were detectable for more than one year and could be strongly boosted by a second vaccination.

    [0143] A more current report of self-vaccination of the same individual includes the administration of SARS-CoV-2-derived peptides performed in March 2020. Here, the self-experimenting healthy volunteer used eight SARS-CoV-2-derived peptides: five predicted to bind to HLA class I molecules (CD8 peptides) and three predicted to bind to HLA-DR molecules (CD4 peptides, including two peptides from the CoVac-1 vaccine). The vaccine formulation also included one long and one short CMV-pp65-derived peptide that had previously been administered to the same individual and could thus act as positive controls. About 3 weeks after a single vaccination, using a vaccine containing the mentioned synthetic peptides, Montanide and containing 50 μg XS15, strong vaccine-induced CD4.sup.+ T-cell responses against all four HLA-DR peptides and against the recall CMV epitope could be detected. No T-cell responses against the five predicted SARS-CoV-2 HLA class I peptides could be detected by ex vivo Interferon-γ ELISpot assay. Furthermore no antibody induction against the vaccine peptides was observed83. Vaccination was well tolerated without any systemic side effects. Granuloma formation was observed at the vaccination site.

    Preliminary Evidence from Unproven Interventions Using XS15-Adjuvanted Vaccines in Cancer Patients

    [0144] In unproven clinical interventions using XS15-adjuvanted vaccines as a treatment effort according to physician's judgement and with informed consent, in keeping with the principle 37 of the Declaration of Helsinki, cancer patients with different malignancies were vaccinated. Respective patients had diverse pre-treatments and have been vaccinated with personalized peptide vaccines in combination with XS15 (50 μg) emulsified in Montanide ISA 51 VG. A detailed description of the patient's characteristics is provided below (Table 7). Each peptide vaccine was designed individually for every patient, based on MS/MS-based HLA ligandom analyses and panel sequencing characterizing individual mutations in the tumor tissue for target discovery. Up to three vaccinations were administered per patient. The mean follow-up period per patient is 14 months (2-36 months) following the first vaccination. The described vaccines were applied s.c. to the abdominal skin. All vaccinated patients showed peptide vaccine-specific T-cell responses after the first vaccination, which could be boosted by subsequent vaccinations. Induced T-cell responses were long-lasting (up to 19 month) after the end of vaccination (FIG. 3).

    [0145] Due to the anecdotal character of the interventions described here such as the major differences between patients as well as diverse cancer entities, pre-treatment, vaccine composition and concomitant (cancer) medication, an joint analysis of the data regarding these peptide vaccinations in combination with XS15 is not intended. The characteristics of subjects receiving individualized peptide vaccines in combination with XS15 are provided below.

    TABLE-US-00009 TABLE 7 Patient characteristics of interventions with individualized peptide vaccines containing XS15 emulsified in Montanide Immune Clinical #Peptides response response #Missense in Vaccine [n peptides [best Patient Sex Age Tumor Entity HLA- Mutations Cocktail #Vaccinations positive] response] 1 m 85 Hepatocellular A′25/B′26, B′08/ 4 10 2 2 PR Carcinoma B′57, C′07/C′03 2 f 78 Colon Cancer A′11/A′68, B′38/ 13 9 3 3 PR B′44, C′12/C′07 3 f 67 Hepatocellular A′02/A′03, B′15/ 2 9 3 3 SD Carcinoma B′47, C′03/C′06 4 m 61 Hepatocellular A′01/A68, B′08/ 4 7 3 1 PR Carcinoma B′14, C′07/C′08 5 m 36 Sarcoma A′11/A′24, B′14/ 0 10 2 4 PR B′15, C′04/C′08 6 m 61 Mesothelioma A′02/A′25, B′15/ 2 10 1 2 n.a. B′46, C′03/C′04 7 m 58 Cancer of unknown A′01/A′24, B′07/ 4 9 2 4 PR primary (CUP) B′14, C′07/C′08 8 m 72 Prostate Cancer A′25/A′32, B′18/ 3 9 2 7 n.a. B′35, C′04/C′12 9 f 41 Ovarian Cancer A′02/A′24, B′07/ 4 9 1 2 n.a. B′51 C′07/C′14 10 m 63 Melanoma A′02/A′29, B′35/ 4 9 1 2 n.a. B′44, C′04/C′05 11 w 66 Osteo- A′03/A′34 n.a. 8-10 3 8 PR myelofibrosis B′15/B′35 C′03/ C′04 12 w 23 Hepatocellular A′02/A′25 B′27/ n.a. 10 2 5 SD Carcinoma B′57 C′03/C′06

    Adverse Events Profile

    [0146] No systemic adverse events occurred in any of the vaccinated patients and the vaccinations were well-tolerated. No fever or any other sign of exuberant immune reaction was observed. In accordance with the expectable adverse events profile of Montanide an asymptomatic subcutaneous granuloma evolved over 1-3 weeks after vaccination. The granulomas usually persist for up to eight weeks in individual patients. Most common related events were transient redness and swelling at the vaccination side (Table 8). After multiple vaccination granuloma ulceration was observed In 5/12 patients (41%), resulting in an aseptic skin ulceration that healed within two weeks not requiring any further intervention or treatment.

    TABLE-US-00010 TABLE 8 Summary of observed local events attributed to Montanide use in cancer patients. Patient Local reaction at vaccination side # After 1. vaccination After multiple vaccinations 1 asymptomatic s.c. granuloma asymptomatic s.c. granuloma (max. Ø 0.5 cm) (max. Ø 2 cm) 2 asymptomatic s.c. granuloma asymptomatic s.c. granuloma (max. Ø 3 cm) (max. Ø 3 cm) 3 s.c. granuloma (max. Ø s.c. granuloma (max. Ø 2 cm), 2 cm), redness and redness and swelling of the skin, swelling of the skin aseptic skin ulceration 4 asymptomatic s.c. granuloma s.c. granuloma (max. Ø 4 cm), (max. Ø 3 cm) redness and swelling of the skin, aseptic skin ulceration 5 asymptomatic s.c. granuloma s.c. granuloma (max. Ø 2 cm), (max. Ø 1 cm) redness and swelling of the skin, aseptic skin ulceration 6 asymptomatic s.c. granuloma asymptomatic s.c. granuloma (max. Ø 1 cm) (max. Ø 2 cm) 7 asymptomatic s.c. granuloma s.c. granuloma (max. Ø 3 cm), (max. Ø 1 cm) redness and swelling of the skin 8 s.c. granuloma (max. Ø s.c. granuloma (max. Ø 3 cm), 3 cm), redness and redness and swelling of the skin, swelling of the skin aseptic skin ulceration 9 asymptomatic s.c. granuloma s.c. granuloma (max. Ø 2 cm), (max. Ø 2 cm) redness and swelling of the skin 10 asymptomatic s.c. granuloma asymptomatic s.c. granuloma (max. Ø 1 cm) (max. Ø 1 cm) 11 s.c. granuloma (max. Ø s.c. granuloma (max. Ø 4 cm), 2 cm), redness and redness and swelling of the skin, swelling of the skin aseptic skin ulceration 12 asymptomatic s.c. granuloma asymptomatic s.c. granuloma (max. Ø 2 cm) (max. Ø 3 cm)

    3. Effects in Humans

    3.1 Effects of CoVac-1 Vaccines in Humans

    [0147] The P-pVAC-SARS-CoV-2 study is a first-in-man trial of a peptide-based SARS-CoV-2 vaccination approach in combination with the TLR1/2 ligand XS15 in healthy volunteers. This section provides the first safety and immunogenicity data of CoVac-1 in the first study part (Part 1) of the P-pVAC-SARS-CoV-2 study. Furthermore, data from a vaccination approach in a healthy volunteer applying two of the CoVac-1 peptides and XS15 emulsified in Montanide ISA 51 VG will be presented. As the principle concept of multi-peptide immunization is well established in infectious disease and cancer therapy.

    3.2 Part I of P-pVAC-SARS-CoV-2 Phase I Trial

    Immunogenicity Data

    [0148] P-pVAC-SARS-CoV-2 is a phase I single-center safety and immunogenicity trial of multi-peptide vaccination with CoVac-1 to prevent COVID-19 infection in adults. The study is recruiting since November 2020 and has completed the first part (healthy volunteers (n=12), age 18-55 years) in February 2021. One single subcutaneous vaccination of CoVac-1 (6 peptides vaccine; Table 2) was applied. Immunogenicity, in term of induction of T-cell responses to one or more of the six HLA-DR SARS-CoV-2 T cell epitopes included in the CoVac-1 vaccine (6 peptides vaccine) was assed pre-vaccination as well as on day 7, 15 and 28 after vaccination (FIG. 4).

    [0149] Induction of SARS-CoV-2 T cells was shown in 100% (12/12) of volunteers in part I of the study. Earliest T cell responses were observed at day 14 for 11/12 subjects. Immune responses were induced to multiple of the vaccine peptides (median 5/subject, range 4-6).

    [0150] Thus, high immunogenicity of CoVac-1 to induce early and multi-peptide T cell responses was shown. An exemplary ELISPOT is depicted in FIG. 5.

    [0151] CoVac-1-induced T cell responses were multifunctional with positivity for TNF (12/12 subjects), IFN-γ (12/12 subjects) and IL-2 (11/12 subjects, FIG. 6). CoVac-1 (6 peptides vaccine, Table 2) induced a high frequency of functional SARS-CoV-2 T cells with up to 1.8% IFN-γ.sup.+, 2.7% TNF.sup.+ and 2.5% IL-2.sup.+ SARS-CoV-2-specific T cells (FIG. 6).

    [0152] In addition to CD4.sup.+ T cell responses, CoVac-1 also induced CD8.sup.+ T cell responses in 75% of donors. These CD8.sup.+ T cells targeting HLA class I T cell epitopes embedded in the CoVac-1 HLA-DR vaccine peptides were shown to be of pathophysiological relevance during natural SARS-CoV-2 infection (FIG. 6).

    Comparison of CoVac-1 to Approved SARS-COV-2 Vaccines (BNT126b2, Biontech SE; mRNA-12738, Moderna, Inc.; ChAdOx1, AstraZeneca)

    [0153] In contrast to approved vaccine candidates that require two vaccinations, CoVac-1 (6 peptides vaccine) induces strong T cell responses after one single vaccination.

    [0154] CoVac-1 (6 peptides vaccine) induces earlier and stronger SARS-CoV-2 T cell responses after one single vaccination compared to the approved vaccine candidates: [0155] CoVac-1 induces early T cell responses (91.6% on day 14) after one vaccination, compared to BNT16B1 and mRNA-1273, for which earliest T cell responses were reported on day 29 after two and one vaccination, respectively. [0156] Mean frequency of CoVac-1-induced IFN-γ.sup.+ and IL-2 SARS-CoV-2 CD4.sup.+ T cells on day 28 after one vaccination was 0.5% and 0.9%, respectively, compared to 0.03% and 0.06%, respectively for BNT16B1 at day 29 after two vaccinations (FIG. 8). [0157] Mean frequency of CoVac-1-induced IFN-γ.sup.+ and IL-2 SARS-CoV-2 CD4.sup.+ T cells on day 28 after one vaccination was 0.5% and 0.9%, respectively, compared to approximately 0.03% and 0.05%, respectively for mRNA-1273 at day 29 after one vaccination (FIG. 8). [0158] Intensity of CoVac-1-induced SARS-CoV-2 T cell responses on day 28 after one single vaccination as assessed by IFN-γ ELISPOT was 897 spot counts per 1×10.sup.6 cells (median value) compared to 493 spot counts (median value) for ChAdOx1 nCoV-19 on day 28 after one vaccination (FIG. 9). [0159] Direct comparison of T cell responses to our CoVac-1 peptides (including T cell epitopes from various SARS-CoV-2 proteins) in P-pVAC-SARS-CoV-2 study subjects (n=12) on days 28 after one single vaccination and healthy volunteers (n=6) mean 25 days after second vaccination with BNT162b2 revealed SARS-CoV-2-specific T cell responses in 100% (12/12) compared to 17% (1/6) of subjects with mean T cell response intensities of 1772 and 7 normalized spot counts per 1×10.sup.6 cells, respectively (FIG. 10).

    Impact of SARS-CoV-2 Variants on CoVac-1

    [0160] The impact of SARS-CoV-2 variants of concern (VOC) declared by the World Health Organization as of 1 Jul. 2021 (B.1.1.7—Alpha, B.1.351—Beta, P.1—Gamma, B.1.617.2—Delta) on CoVac-1 (6 peptides vaccine) was evaluated in terms of vaccine peptide sequence and vaccine-induced T-cell response alterations. 3/6 (50%) CoVac-1 vaccine peptides are not affected by any variant-defining or -associated mutation of the SARS-CoV-2 VOC. None of the mutations of the variants P.1 and B.1.617.2 affect any of the CoVac-1 vaccine peptides. Variant B.1.1.7 comprises two mutations affecting P2_nuc and P6_ORF8 with a single amino acid change, respectively. Two mutations of variant B.1.351 affect P3_spi with either one single or two-amino acid changes (FIG. 11A).

    [0161] IFN-γ T-cell responses to peptide pools comprising the B.1.1.7 and B.351 mutated peptides P2_nuc, P3_spi and P6_ORF8 were detectable in 24/24 (100%) of participants of Part I and Part II with proven CoVac-1-induced T-cell response to P2_nuc, P3_spi, and P6_ORF8 wild-type (WT) peptides (FIG. 11B). Although the intensity of T-cell responses in terms of spots per 5×10.sup.5 cells to single peptide P3- and P6-variants were reduced compared to WT peptides, intensity of CoVac-1-induced T-cell responses targeting the variant peptide pools was not affected and was at least 10-fold higher than memory T-cell responses to WT and variant peptide pools observed in HCs (FIG. 11C).

    3.3 Safety Information for CoVac-1 Peptides

    [0162] P-pVAC-SARS-CoV-2 is a phase I single-center safety and immunogenicity trial of multi-peptide vaccination with CoVAC-1 to prevent COVID-19 infection in adults. The study is recruiting since November 2020 and has completed the first part (healthy volunteers (n=12), age 18-55 years) in February 2021. One single subcutaneous vaccination of CoVac-1 was applied. First safety data are available until d28 (V4) after vaccination (see below).

    [0163] Furthermore, conclusions can be drawn from (i) peptide vaccination studies in infectious disease (ii) the ongoing iVAC-CLL01 study in CLL patients based on the same technology of the Department of Immunology in Tübingen (iii) and similar peptide-based cancer vaccines (e.g. IMA901 and GAPVAC) and (iv) first data from multi-peptide vaccines in a healthy volunteer and cancer patients in terms of individual healing attempts. As far as reported to date, those multi-peptide vaccines were well tolerated and the observed safety profile is considered acceptable.

    Preliminary Safety Information from Part I of the P-pVAC-SARS-CoV-2 Study

    [0164] P-pVAC-SARS-CoV-2 is a phase I single-center trial to evaluate the safety and immunogenicity of the multi-peptide vaccine CoVac-1 (6 peptide vaccine) after single subcutaneous application. The tabulated events have a “snap shot” character according to the documentation status on 15 Feb. 2021. Until day 28 no systemic side effects, especially no fever were reported. No allergic reactions were observed. As intended and expected all volunteers (n=12) developed a granuloma local at injection site. Further local injection site adverse events included transient erythema, itching, pain and skin ulceration. For a detailed description of all ADRs reported please refer to Table 9 to Table 13.

    TABLE-US-00011 TABLE 1 Summary tabulation of adverse drug reactions (ADR, any grade) Grade % of all Adverse Event (CTCAE term) (max.) Patient (ID) subjects Vaccination complication - 3 pCOV-05, pCOV-06 16 erythema Vaccination complication - 2 pCOV-07, pCOV-10, 42 erythema pCOV-11, pCOV-16, pCOV-18 Vaccination complication - 1 pCOV-02, pCOV-04, 42 erythema pCOV-08, pCOV-12, pCOV-20 Vaccination complication - 2 pCOV-10, pCOV-11, 25 swelling pCOV-18 Vaccination complication - 1 pCOV-02, pCOV-04, 75 swelling pCOV-05, pCOV-06, pCOV-07, pCOV-08, pCOV-12, pCOV-16, pCOV-20 Vaccination complication - 1 pCOV-02, pCOV-04, 83 itching pCOV-05, pCOV-07, pCOV-08. pCOV-10, pCOV-11, pCOV-16, pCOV-18, pCOV-20 Vaccination complication - 1 pCOV-05, pCOV-06, 58 pain pCOV-07, pCOV-08, pCOV-11, pCOV-18, pCOV-20 Vaccination complication - 2 pCOV-10, pCOV-11, 33 induration/granuloma pCOV-16, pCOV-18 Vaccination complication - 1 pCOV-02, pCOV-04, 67 induration/granuloma pCOV-05, pCOV-06, pCOV-07, pCOV-08, pCOV-12, pCOV-20 Skin ulceration 1 pCOV-11 8 Vaccination site 1 pCOV-02, pCOV-04, 42 lymphadenopathy pCOV-06, pCOV-07, pCOV-16 Fatigue 1 pCOV-10, pCOV-11, 25 pCOV-12 Myalgia 1 pCOV-10 8 Arthralgia 1 pCOV-16 8 Headache 1 pCOV-06, pCOV-10, 25 pCOV-16 Nausea 1 pCOV-11, pCOV-18 16

    TABLE-US-00012 TABLE 2 Summary tabulation of suspected adverse drug reactions (SADR, any grade) Grade % of all Adverse Event (CTCAE term) (max.) Patient (ID) subjects Vaccination complication - 3 pCOV-05, pCOV-06 16 erythema Vaccination complication - 2 pCOV-07, pCOV-10, 42 erythema pCOV-11, pCOV-16, pCOV-18 Vaccination complication - 1 pCOV-02, pCOV-04, 42 erythema pCOV-08, pCOV-12, pCOV-20 Vaccination complication - 2 pCOV-10, pCOV-11, 25 swelling pCOV-18 Vaccination complication - 1 pCOV-02, pCOV-04, 75 swelling pCOV-05, pCOV-06, pCOV-07, pCOV-08, pCOV-12, pCOV-16, pCOV-20 Vaccination complication - 1 pCOV-02, pCOV-04, 83 itching pCOV-05, pCOV-07, pCOV-08. pCOV-10, pCOV-11, pCOV-16, pCOV-18, pCOV-20 Vaccination complication - 1 pCOV-05, pCOV-06, 58 pain pCOV-07, pCOV-08, pCOV-11, pCOV-18, pCOV-20 Vaccination complication - 2 pCOV-10, pCOV-11, 33 induration/granuloma pCOV-16, pCOV-18 Vaccination complication - 1 pCOV-02, pCOV-04, 67 induration/granuloma pCOV-05, pCOV-06, pCOV-07, pCOV-08, pCOV-12, pCOV-20 Skin ulceration 1 pCOV-11 8 Vaccination site 1 pCOV-02, pCOV-04, 42 lymphadenopathy pCOV-06, pCOV-07, pCOV-16 Fatigue 1 pCOV-10, pCOV-11, 25 pCOV-12 Myalgia 1 pCOV-10 8 Arthralgia 1 pCOV-16 8 Headache 1 pCOV-06, pCOV-10, 25 pCOV-16 Nausea 1 pCOV-11, pCOV-18 16

    TABLE-US-00013 TABLE 3 Summary tabulation of unsuspected adverse drug reactions (UADR, any grade) Other possible Adverse Event (CTCAE term) Grade (max) relation Patient (ID) n.d n.d. n.d. n.d.

    TABLE-US-00014 TABLE 4 Summary tabulation of AESI (any grade) Grade AESI (max.) Patient (ID) Vaccination complication - 2 pCOV-10, pCOV-11, pCOV-16, induration/granuloma pCOV-18 Vaccination complication - 1 pCOV-02, pCOV-04, pCOV-05, induration/granuloma pCOV-06, pCOV-07, pCOV-08, pCOV-12, pCOV-20

    TABLE-US-00015 TABLE 5 Cumulative summary tabulation of all adverse drug reactions (ADR, any grade); n = number of subjects who have experienced the SAR; SAR: suspected adverse reaction; SOC: system organ class. Number of subjects exposed (n) = 12 All SARs Occurrence SOC SARS n (%) Injury, poisoning and Vaccination complication - 12 (100) procedural complications erythema Injury, poisoning and Vaccination complication - 12 (100) procedural complications swelling Injury, poisoning and Vaccination complication - 12 (100) procedural complications induration/granuloma Injury, poisoning and Vaccination complication - 10 (83) procedural complications itching Injury, poisoning and Vaccination complication - 7 (58) procedural complications pain General disorders and Vaccination site 5 (42) administration site lymphadenopathy conditions General disorders and Fatigue 3 (25) administration site conditions Nervous system disorders Headache 2 (16) Gastrointestinal disorders Nausea 2 (16) Skin and subcutaneous Skin ulceration 1 (8) tissue disorders Musculoskeletal and Myalgia 1 (8) connective disuse disorders Musculoskeletal and Arthralgia 1 (8) connective disuse disorders

    [0165] These preliminary results indicated that CoVac-1 (6 peptides vaccine) was well tolerated at the applied dose and did not cause relevant systemic side effects.

    Comparison of CoVac-1 to Approved SARS-COV-2 Vaccines (BNT126b2, Biontech SE; mRNA-12738, Moderna, Inc.; ChAdOx1, AstraZeneca)

    [0166] In contrast to approved vaccine candidates (chills 32%, fever 14% BNT126b2, 50% chills, 8% fever mRNA-12738, chills 34%, fever 24% ChAdOx1 nCoV-19; AZD1222), no systemic inflammatory reactions were reported for CoVac-1 (6 peptides vaccine). No investigator-initiated drug treatment was required for CoVac-1-induced side effects, whereas paracetamol 1 g post vaccination every 4-6 hours for 24 hours after vaccination was routinely advised for participants in the phase 2/3 ChAdOx1 nCoV-19 from Astra Zeneca to reduce possible reactogenicity from vaccination. None of the side effects reported for CoVac-1 vaccination affected daily life activity or working ability of study subjects. This is in stark contrast to the inflammatory side effects caused by approved vaccine candidates, in particular ChAdOx1 nCoV-19, which cause for example inability to work for up to 72 h in a large proportion of vaccinated subjects.

    [0167] Granuloma formation at the vaccination site was also reported, albeit rarely, in subjects after BNT162b2 vaccination. In contrast to CoVac-1 induced granulomas, these local reactions were indeed reported to affect subject's daily life and also required specific treatment (e.g. steroids).

    4. Benefits

    [0168] The clinical benefits of CoVac-1 vaccination are based on the following aspects: [0169] Peptide vaccination using HLA-presented peptides represents an established immunotherapy approach for preventive vaccine development in infectious disease as well as for therapeutic approaches in malignant disease. Several therapeutic peptide vaccination studies in patients with malignant disease including solid tumors and hematological malignancies have proven safety and tolerability of this approach. [0170] Therapeutic multi-peptide vaccination represents a low side-effect immunotherapy approach relying on specific immune recognition of HLA-presented peptides. [0171] All peptides included in the CoVac-1 vaccine are proven SARS-CoV-2 T-cell epitopes with pathophysiological relevance in the natural course of COVID-19 disease [0172] CoVac-1 peptide vaccination induce by only one dose potent CD8.sup.+ and CD4.sup.+Th1 T-cell responses against SARS-CoV-2, resulting in immunity against infection as: [0173] CD4.sup.+Th1 cells directly contribute to virus clearance and deliver strong T helper signals to CD8.sup.+ T-cells primed during natural infection. Furthermore, such SARS-CoV-2 specific CD4.sup.+Th1 cells activate virus antigen-experienced B cells. The resulting increase in activity is expected to allow for rapid virus clearance and prevention of severe course of COVID-19 disease. [0174] The peptides used for CoVac-1 vaccine contain embedded CD8.sup.+ T-cell epitopes predicted to bind to many different HLA class I allotypes. Once activated, the CD8.sup.+ T-cells may contribute to faster virus clearance. [0175] CoVac-1-induced IFN-γT-cell responses extended those in COVID-19 convalescents and are not affected by any mutations of current variants of concern (VOC). [0176] Participant selection is based on medical imperative and safety considerations: [0177] The trial compromises three parts (cohorts of participants) with different age groups to provide preliminary results on safety in a young (18-55 years, n=12) patient cohort, which is then extended to older (Part II and Part Ill) participants. Of note, the risk of vaccine related (S)AEs is hypothesized to be similar in each age group. By this design, the inventors address the urgent medical need for protection of subjects at risk for severe SARS-CoV-2 infection by providing safety and immunogenicity data as well as first efficacy data on the course of SARS-CoV-2 infection in this population. [0178] Safety is continuously monitored by an independent data and safety monitoring board (DSMB), which will be provided with reports on frequent basis.

    [0179] The clinical benefits of peptide vaccination in combination with the TLR1/2 ligand XS15 in Montanide ISA 51 VG are based on the following aspects: [0180] Peptide vaccination alone is rarely able to induce clinically effective T-cell responses; thus, the peptide vaccine has to be combined with an adjuvant drug to enhance immune responses. [0181] Several Toll-like-receptor (TLR) ligands have shown to induce strong CD8.sup.+/CD4.sup.+Th1 responses in humans including CPG (TLR9 ligand), imiquimod (TLR7 ligand) and poly-IC (TLR3 ligand). However, for none of these TLR ligands GMP substance is available for adding to a peptide vaccine. [0182] XS15 is a water-soluble derivative of the TLR2 ligand Pam3Cys and induces a strong CD8.sup.+ and CD4.sup.+Th1 T-cell response against free short peptides emulsified in Montanide ISA 51 VG after a single s.c. injection in healthy volunteers as well as in patients. [0183] Using XS15, immune responses could be induced for viral peptides including SARS-CoV-2 specific peptides, neoepitopes from cancer-specific mutations as well as for tumor-associated self-peptides. [0184] XS15 results in granuloma formation on the vaccination site, where the vaccinated peptides persist for at least 7 weeks, which might support the induction of a strong immune response. [0185] The induced immune responses observed so far persisted for more than 1.5 years. [0186] Peptide vaccination in combination with XS15 in Montanide ISA 51 VG has already been applied in a healthy volunteer and cancer patients. Beside formation of granuloma locally on injection side, no relevant side effects, especially no allergic or anaphylactic reactions or cytokine release syndrome, have been observed. [0187] Montanide ISA 51 VG is an oil adjuvant suitable for human injection that allows the manufacturing of water in oil emulsions. Montanide ISA 51 VG has been used in more than 200 clinical trials including more than 6000 patients and was proven to be well tolerated.