Vaccine composition for eliciting an EBV-specific CD8+ t cell response

Abstract

The present invention relates to a polynucleotide encoding a polypeptide, wherein the polypeptide comprises at least 25 fragments of at least two EBV proteins, each fragment having a length of at most 15 amino acids, and one or more cleavage-enhancing linker, each located between two fragments.

Description

[0001] VACCINE COMPOSITION FOR ELICITING AN EBV-SPECIFIC CD8+T CELL

[0002] RESPONSE

[0003] CROSS REFERENCE

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 670,660, filed July 12, 2024, the contents of which are incorporated herein by reference in its entirety.

[0005] SEOUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 9, 2025, is named 62480-702.602_SL. xml and is 498,810 bytes in size.

[0007] FIELD OF THE INVENTION

[0008] The present invention relates to a polynucleotide encoding a polypeptide, wherein the polypeptide comprises at least 25 fragments of at least two Epstein-Barr Virus proteins, each fragment having a length of at most 15 amino acids, and one or more cleavage-enhancing linker, each located between two fragments. The present invention further concerns a polypeptide encoded by the polynucleotide of the invention. The present invention further concerns a vector comprising the polynucleotide of the invention. The present invention further concerns a vaccine comprising the polynucleotide, the polypeptide, and / or the vector of the invention. The present invention further concerns a host cell comprising the polynucleotide, the polypeptide, and / or the vector of the invention. In another aspect the invention provides the use of the polynucleotide, the polypeptide, the vector, and the vaccine for the treatment of multiple sclerosis. In another aspect of the invention, a pharmaceutical composition comprising the polynucleotide, the polypeptide, or the vector or the vaccine is provided. In still another aspect of the invention, method comprising administering to a subject the vaccine of the invention to induce in the subject an immune response.

[0009] BACKGROUND OF THE INVENTION

[0010] Epstein-Barr Virus (EB V) infections are known to play a critical role in several cancers such as nasopharyngeal carcinoma, subtypes of Hodgkin and non-Hodgkin lymphomas, a subtype of gastric carcinomas (EBV-associated gastric carcinoma), natural killer (NK) / T cell lymphomas and leiomyosarcoma. In addition, EBV is linked to autoimmune diseases such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) or Sjogren’s syndrome. Recently, the Epstein-Barr Virus has been also linked to the initiation and / or disease relapses of Multiple Sclerosis (MS), which is a chronic inflammatory demyelination disease of the central nervous system of unknown aetiology.

[0011] MS is the most common chronic inflammatory, demyelinating and neurodegenerative disease of the central nervous system in young adults. MS is influenced by both genetic and environmental factors. Reversible episodes of neurological dysfunction lasting several days or weeks characterize the initial stages of the disease while irreversible clinical and cognitive deficits develop over time. The pathological hallmark of MS is the formation of demyelinating lesions in the brain and spinal cord, which can be associated with neuro-axonal damage. Focal lesions are thought to be linked to the infiltration of immune cells, including T cells, B cells and myeloid cells, into the central nervous system parenchyma, with associated injury (Filippi, M., Bar-Or, A., Piehl, F. et al. Multiple sclerosis. Nat Rev Dis Primers 4, 43 (2018)). The neurological signs and symptoms of MS include impaired motor function, visual symptoms, fatigue, eye movement disorders, bladder symptoms, sensory symptoms, sexual dysfunction, ataxia, deafness, spasticity, dementia, and cognitive impairment.

[0012] Clinically, MS can either be relapsing or progressive. Most commonly, onset is a relapsing form of MS, manifested as discrete episodes of neurological dysfunction followed by partial, complete, or no remission. Over time, relapses usually decrease in frequency, but a gradual worsening often supervenes, resulting in uninterrupted progression, termed secondary progressive MS. Less than 10% of patients with MS experience progression from onset, termed primary progressive MS. Despite these distinctions, every form of MS seems to reflect the same underlying disease process. Although inflammation is typically associated with relapses, and neurodegeneration with progression, it is now recognized that both pathologies are present in essentially all patients.

[0013] MS is a global problem that affects millions of people worldwide. The prevalence of MS is highest in North America, Western Europe, and Australia, with over 100 cases per 100,000 population. In the United States, a recent study estimated that nearly 1 million individuals are affected by MS, with a higher prevalence among women than men. The mean age of onset is around 30 years for relapsing-remitting MS and around 40 years for primary progressive MS.

[0014] Recent studies provided evidence for EBV playing an important role as trigger or cofactor in the development of MS (Bjornevik K, Cortese M, Healy BC, et al. Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science (New York, N.Y.). 2022 Jan;375(6578):296-301; Bordon, Y. Linking Epstein-Barr virus infection to multiple sclerosis. Nat Rev Immunol 22, 143 (2022); Soldan, S.S., Lieberman, P.M. Epstein-Barr virus and multiple sclerosis. Nat Rev Microbiol 21, 51-64 (2023)). One study reports the risk of developing MS to be increased 32-fold after infection with EBV, even though the risk is not increased following infection with other viruses. These findings support the role of EBV being a leading cause of MS (Bjornevik K, Cortese M, Healy BC, et al. Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science (New York, N.Y.). 2022 Jan;375(6578):296-301).

[0015] EBV has been discovered 1964 in a cultured lymphoblast line (EPSTEIN, M A et al. “VIRUS PARTICLES IN CULTURED LYMPHOBLASTS FROM BURKITT'S LYMPHOMA.” Lancet (London, England) vol. 1,7335 (1964): 702-3). EBV is a ubiquitous human herpesvirus, establishing lifelong infection in more than 90% of adults worldwide. It is transmitted through saliva, although less frequently it is also transmitted through blood transfusion and organ transplantations. Following initial infection, individuals may experience either asymptomatic infection or infectious mononucleosis. After recovering from primary EBV infection, most individuals enter a host-virus balance state and become healthy carriers, wherein EBV persists in latent form in B cells throughout the life of the host. Lifelong persistence occurs through the establishment of latent reservoirs in these cells and periodic reactivation.

[0016] As shown in Fig. 1, EBV has a productive (lytic) cycle and a non-productive (latent) phase. A characteristic feature of EBV is its capability to establish distinct latent gene expression patterns in vivo and in cultured cells (named type 0, 1, II and III) in resting and proliferating cells. EBV infection efficiently reprogrammes naive B cells towards a memory B cell phenotype through different viral gene programmes termed ‘latency types’. Initially, the viral proteins gp350 / gp220 and gp42 are required for EBV entry into B lymphocytes. During the hyperproliferative phase, EBV adopts a type III latency in which most latency- associated genes (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA- LP, LMP1, LMP2 and multiple non-coding RNAs) are expressed. Five latent genes (EBNA1, EBNA2, EBNA3A, EBNA3C and LMP1) are required for in vitro B cell immortalization. EBV lytic cycle reactivation occurs in healthy individuals and is required for transmission and potentially for replenishing the latent reservoir and numerous cell signalling pathways can trigger the switch to lytic infection. Some of these pathways are related to immune cell signalling, such as activation of B cell receptor (BCR) signalling with anti- immunoglobulin or activation of protein kinase C by phorbol esters. In latently infected memory B cells, the switch requires two EBV- encoded transcription factors, BZLF1 (also known as ZTA, ZEBRA and Z) and BRLF1 (also known as RTA) (Soldan, S.S., Lieberman, P.M. Epstein-Barr virus and multiple sclerosis. Nat Rev Microbiol 21, 51-64 (2023)). An association of any specific latency type or lytic infection with MS is at present not known and how the ubiquitous EBV, typically leading to benign latent infections, promotes MS is poorly understood. Defects in the control of EBV lytic reactivation have been suggested as one possible cause, although this remains controversial (Yea, C. et al. Epstein- Barr virus in oral shedding of children with multiple sclerosis. Neurology 81, 1392-1399 (2013)).

[0017] As increasing evidence suggests the involvement of B cells, strategies for the treatment of MS have been developed including B cell depletion therapy (Cencioni, Maria T et al. “B cells in multiple sclerosis - from targeted depletion to immune reconstitution therapies.” Nature reviews. Neurology vol. 17,7 (2021): 399-414. doi: 10.1038 / s41582-021-00498-5). In an alternative approach, vaccines have been developed targeting EBV-glycoproteins such as gp350, gH / gL and / or gB, relevant for the attachment of EBV to the B cells, or targeting EBV latency or lytic proteins. In WO 2019 / 123169, a polyvalent Epstein-Barr Virus antigen construct is described, broadly targeting EBV antigens that are expressed in EBV-associated multiple sclerosis by including multiple latent stage antigens, and optionally including lytic stage antigens. The construct comprises antigen fragments having a length of 25 amino acids or more, leading to antigen-specific CD4+and CD8+T cell responses. SUMMARY OF THE INVENTION

[0018] Against the aforementioned background, it is an object of the present invention to provide effective pharmacological means to treat MS in a subject. It is also an object of the present invention to provide pharmacological means to elicit a therapeutically effective EBV-specific immune response for the treatment of MS. A further object of the present invention is to provide pharmacological means for keeping EBV-infected B-cells in the latent phase. As further object of the present invention is to provide pharmacological means for reducing or elimination re-exposure to EBV proteins, thereby leading to reduced central nervous system (CNS) infiltration and local inflammation.

[0019] These objects are achieved by the invention set forth in the claims and embodiments explained in more detail below.

[0020] The invention provides a polynucleotide encoding a polypeptide, wherein the polypeptide can elicit an EBV-specific CD8+T cell immune response against EBV in a subject. This is achieved by providing a polypeptide comprising a plurality of fragments of an EBV protein, each fragment having a length of at most 15 amino acids, thereby providing an efficient targeting of EBV-infected B cells by cytotoxic CD8+T cells. Including EBV-specific CD4+T cell responses risks provoking considerable collateral damage worsening the patient’s disease pattern. Potential side effects may include boosting production of autoantibodies and bystander immune responses in which B cells take up one antigen but get their required T cell help through another antigen. The research underlying this invention has surprisingly found that a strong CD8+T-cell response against selected EBV proteins expressed during different stages of the EBV life cycle can be elicited by the polynucleotide of the invention rendering the polynucleotide particularly suitable for treating MS in a subject by keep EBV-infected B- cells in the latent phase.

[0021] Further the invention concerns a vaccine composition comprising the polynucleotide encoding for a polypeptide. More precisely, the invention provides a vaccine for the therapeutic treatment for EBV-infected subjects, in particular for keeping EBV-infected B-cells in the latent phase. Re-exposure of EBV proteins is thereby reduced or eliminated, resulting in reduced disease-driving antibody responses and reduced CNS infiltration and local inflammation. This is facilitated by eliciting a strong CD8+T-cell response against selected EBV proteins expressed during different stages of the EB V life cycle.

[0022] Further the invention concerns a polynucleotide, a polypeptide, a vector, or a vaccine composition according to the invention for use in the treatment of multiple sclerosis or for use in eliciting an EBV-specific CD8+T cell immune response. This use is particularly relevant in therapeutic, i.e., medical applications.

[0023] The invention further concerns a vector comprising the polynucleotide of the invention.

[0024] The invention further concerns a pharmaceutical composition comprising a polynucleotide of the invention, a vector of the invention, a polypeptide of the invention, or the vaccine of the invention.

[0025] The invention further concerns a host cell comprising the polynucleotide of the invention, a vector of the invention, or a polypeptide of the invention.

[0026] The invention further concerns a method comprising administering to a subject the vaccine according to the invention in a therapeutically effective amount to induce in the subject an immune response.

[0027] DETAILED DESCRIPTION

[0028] Although certain embodiments of the present invention are described in detail below, it is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise.

[0029] The above objects are achieved by the following embodiments in accordance with the invention:

[0030] 1) A polynucleotide encoding a polypeptide for eliciting a CD8+-specific immune response in a subject, wherein the polypeptide comprises a plurality of fragments of an EBV protein, each fragment having a length of at most 15 amino acids.

[0031] 2) The polynucleotide according to embodiment 1, wherein each fragment has a length of at least 8 amino acids.

[0032] 3) The polynucleotide according to any of the preceding embodiments, wherein each fragment has a length of at most 14 amino acids, preferably at most 13 amino acids, more preferably at most 12 amino acids, even more preferably at most 11 amino acids, and most preferably at most 10 amino acids.

[0033] 4) The polynucleotide according to any of the preceding embodiments, wherein the polypeptide has a length of at most 1500 amino acids, preferably at most 1250 amino acids, more preferably at most 1000 amino acids, and most preferably at most 900 amino acids. 5) The polynucleotide according to any of the preceding embodiments, wherein the polypeptide has a length of at least 300 amino acids, preferably at least 400 amino acids, more preferably at least 500 amino acids, and most preferably at least 550 amino acids.

[0034] 6) The polynucleotide according to any of the preceding embodiments, wherein the polypeptide comprises at least 2 fragments, preferably at least 5 fragments, more preferably at least 10 fragments, even more preferably at least 20 fragments, even more preferably at least 30 fragments, even more preferably at least 40 fragments, and most preferably at least 50 fragments.

[0035] 7) The polynucleotide according to any of the preceding embodiments, wherein the polypeptide comprises at most 100 fragments, preferably at most 90 fragments, more preferably at most 80 fragments, even more preferably at most 70 fragments, and most preferably at most 60 fragments.

[0036] 8) The polynucleotide according to any of the preceding embodiments, wherein the polypeptide comprises fragments of a plurality of EBV proteins.

[0037] 9) The polynucleotide according to any of the preceding embodiments, wherein the polypeptide comprises fragments of at least 2 EBV proteins, preferably of at least 3 EBV proteins, more preferably of at least 4 EBV proteins, most preferably of at least 5 EBV proteins.

[0038] 10) The polynucleotide according to any of the preceding embodiments, wherein the EBV protein(s) is / are selected from a group consisting of latency proteins, lytic switch proteins, and early lytic proteins.

[0039] 11) The polynucleotide according to any of the preceding embodiments, wherein the EBV protein(s) is / are selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments, wherein the fragments are derived from one or more of the SEQ ID NO: 131 to 142 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments, wherein the fragments comprise a sequence of SEQ ID NO: 24 to 130 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 8 to 13, wherein at least two of the fragments of a first EBV protein are separated by one or more fragments of at least a second EBV protein, wherein preferably the first and second EBV proteins are selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1. ) The polynucleotide according to embodiment 14, wherein at least two of the fragments of the first EBV protein are separated by one or more fragments of a third protein preferably selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1. ) The polynucleotide according to any of the preceding embodiments 14 or 15, wherein at least two of the fragments of the first EBV protein are separated by one or more fragments of a fourth protein preferably selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1. ) The polynucleotide according to any of the preceding embodiments 14 to 16, wherein at least two of the fragments of the first EBV protein are separated by one or more fragments of a fifth protein preferably selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1. ) The polynucleotide according to any of the preceding embodiments 14 to 17, wherein at least two fragments of EBNA1 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 14 to 18, wherein at least two fragments of EBNA2 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 14 to 19, wherein at least two fragments of EBNA3A are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 14 to 20, wherein at least two fragments of EBNA3B are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3 A, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 14 to 21, wherein at least two fragments of LMP1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 14 to 22, wherein at least two fragments of LMP2 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0040] 24) The polynucleotide according to any of the preceding embodiments 14 to 23, wherein at least two fragments of BZLF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0041] 25) The polynucleotide according to any of the preceding embodiments 14 to 24, wherein at least two fragments of BRLF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0042] 26) The polynucleotide according to any of the preceding embodiments 14 to 25, wherein at least two fragments of BMLF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMRF1, and BFRF1 or a combination thereof.

[0043] 27) The polynucleotide according to any of the preceding embodiments 14 to 26, wherein at least two fragments of BMRF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, and BFRF1 or a combination thereof.

[0044] 28) The polynucleotide according to any of the preceding embodiments 14 to 27, wherein at least two fragments of BFRF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, and BMRF1 or a combination thereof.

[0045] 29) The polynucleotide according to any of the preceding embodiments 14 to 28, wherein at least two fragments of EBNA3C are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3 A, EBNA3B, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0046] 30) The polynucleotide according to any of the preceding embodiments, wherein the EBV protein(s) is / are selected from a group consisting of EBNA1, LMP1, LMP2, BZLF1, and BRLF1 or a combination thereof, preferably wherein the polypeptide comprises a plurality of fragments of each of EBNA1, LMP1, LMP2, BZLF1, and BRLF1.

[0047] 31) The polynucleotide according to embodiment 30, wherein LMP2 is selected from LMP2A, LMP2B or a combination thereof.

[0048] 32) The polynucleotide according to any of the preceding embodiment 30 or 31, wherein the polypeptide comprises a plurality of fragments of EBNA1, preferably at least 3 fragments of EBNA1, more preferably at least 4 fragments of EBNA1, most preferably at least 5 fragments of EBNA1.

[0049] 33) The polynucleotide according to any of the preceding embodiments 30 to 32, wherein the polypeptide comprises a plurality of fragments of LMP1, preferably at least 3 fragments of LMP1, more preferably at least 4 fragments of LMP1, most preferably at least 5 fragments of LMP1.

[0050] 34) The polynucleotide according to any of the preceding embodiments 30 to 33, wherein the polypeptide comprises a plurality of fragments of LMP2, preferably at least 3 fragments of LMP2, more preferably at least 4 fragments of LMP2, most preferably at least 5 fragments of LMP2. 35) The polynucleotide according to any of the preceding embodiments 30 to 34, wherein the polypeptide comprises a plurality of fragments of BZLF1, preferably at least 3 fragments of BZLF1, more preferably at least 4 fragments of BZLF1, most preferably at least 5 fragments of BZLF1.

[0051] 36) The polynucleotide according to any of the preceding embodiments 30 to 35, wherein the polypeptide comprises a plurality of fragments of BRLF1, preferably at least 3 fragments of BRLF1, more preferably at least 4 fragments of BRLF1, most preferably at least 5 fragments of BRLF1.

[0052] 37) The polynucleotide according to any of the preceding embodiments 30 to 36, wherein the polypeptide comprises at most 30 fragments, preferably at most 25, more preferably at most 20 fragments, of each EBV protein.

[0053] 38) The polynucleotide according to any of the preceding embodiments 30 to 37, wherein the polypeptide comprises at least fragments of EBNA1, LMP1, LMP2, BZLF1, and BRLF1.

[0054] 39) The polynucleotide according to any of the preceding embodiments 30 to 38, wherein at least two fragments of EBNA1 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of LMP1, LMP2, BZLF1, and BRLF1 or a combination thereof.

[0055] 40) The polynucleotide according to any of the preceding embodiments 30 to 39, wherein at least two fragments of LMP1 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA1, LMP2, BZLF1, and BRLF1 or a combination thereof.

[0056] 41) The polynucleotide according to any of the preceding embodiments 30 to 40, wherein at least two fragments of LMP2 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA1, LMP1, BZLF1, and BRLF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 30 to 41, wherein at least two fragments of BZLF1 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA1, LMP1, LMP2, and BRLF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 30 to 42, wherein at least two fragments of BRLF1 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA1, LMP1, LMP2, and BZLF1, or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 30 to 43, wherein the fragments of EBNA1 are derived from SEQ ID NO: 141 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 44, wherein the fragments of LMP1 are derived from SEQ ID NO: 142 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 45, wherein the fragments of LMP2 are derived from SEQ ID NO: 131 and / or 132 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised insaid SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 46, wherein the fragments of BZLF1 are derived from SEQ ID NO: 139 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 47, wherein the fragments of BRLF1 are derived from SEQ ID NO: 140 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 48, wherein the fragments of EBNA1 comprise a sequence of SEQ ID NO: 64 to 71 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 49, wherein the fragments of LMP1 comprise a sequence of SEQ ID NO: 72 to 79 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 50, wherein the fragments of LMP2 comprise a sequence of SEQ ID NO: 24 to 41 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 51, wherein the fragments of BZLF1 comprise a sequence of SEQ ID NO: 42 to 46 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 52, wherein the fragments of BRLF1 comprise a sequence of SEQ ID NO: 47 to 63 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 30 to 53, wherein the polypeptide comprises a combination of two or more of the SEQ ID NO: 24 to 79. ) The polynucleotide according to any of the preceding embodiments 30 to 54, wherein the polypeptide comprises a sequence of SEQ ID NO: 1 to 10 and 283 to 292 or comprises a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.

[0057] 56) The polynucleotide according to embodiments 1 to 29, wherein the EBV protein(s) is / are selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof, preferably wherein the polypeptide comprises a plurality of fragments of each of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1.

[0058] 57) The polynucleotide according to embodiment 56, wherein the polypeptide comprises a plurality of fragments of EBNA2, preferably at least 3 fragments of EBNA2, more preferably at least 4 fragments of EBNA2, most preferably at least 5 fragments of EBNA2.

[0059] 58) The polynucleotide according to any of the preceding embodiment 56 or 57, wherein the polypeptide comprises a plurality of fragments of EBN3A, preferably at least 3 fragments of EBN3A, more preferably at least 4 fragments of EBN3A, most preferably at least 5 fragments of EBN3A.

[0060] 59) The polynucleotide according to any of the preceding embodiments 56 to 58, wherein the polypeptide comprises a plurality of fragments of EBNA3B, preferably at least 3 fragments of EBNA3B, more preferably at least 4 fragments of EBNA3B, most preferably at least 5 fragments of EBNA3B.

[0061] 60) The polynucleotide according to any of the preceding embodiments 56 to 59, wherein the polypeptide comprises a plurality of fragments of BMLF1, preferably at least 3 fragments of BMLF1, more preferably at least 4 fragments of BMLF1, most preferably at least 5 fragments of BMLF1.

[0062] 61) The polynucleotide according to any of the preceding embodiments 56 to 60, wherein the polypeptide comprises a plurality of fragments of BMRF1, preferably at least 3 fragments of BMRF1, more preferably at least 4 fragments of BMRF1, most preferably at least 5 fragments of BMRF1. ) The polynucleotide according to any of the preceding embodiments 56 to 61, wherein the polypeptide comprises a plurality of fragments of BFRF1, preferably at least 3 fragments of BFRF1, more preferably at least 4 fragments of BFRF1, most preferably at least 5 fragments of BFRF1. ) The polynucleotide according to any of the preceding embodiments 56 to 62, wherein the polypeptide comprises at most 30 fragments, preferably at most 25, more preferably at most 20 fragments, of each EBV protein. ) The polynucleotide according to any of the preceding embodiments 56 to 63, wherein at least two fragments of EBNA2 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 56 to 64, wherein at least two fragments of EBN3A are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 56 to 65, wherein at least two fragments of EBNA3B are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBN3A, BMLF1, BMRF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 56 to 66, wherein at least two fragments of BMRF1 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, and BFRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 56 to 67, wherein at least two fragments of BFRF1 are separated by one or more fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, and BMRF1 or a combination thereof. ) The polynucleotide according to any of the preceding embodiments 56 to 68, wherein the fragments of EBNA2 are derived from SEQ ID NO: 133 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 69, wherein the fragments of EBN3A are derived from SEQ ID NO: 137 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 70, wherein the fragments of EBN3B are derived from SEQ ID NO: 138 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 71, wherein the fragments of BMLF1 are derived from SEQ ID NO: 136 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 72, wherein the fragments of BMRF1 are derived from SEQ ID NO: 135 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 73, wherein the fragments of BFRF1 are derived from SEQ ID NO: 134 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 74, wherein the fragments of EBNA2 comprise a sequence of SEQ ID NO: 80 to 87 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 75, wherein the fragments of EBN3A comprise a sequence of SEQ ID NO: 109 to 121 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 76, wherein the fragments of EBN3B comprise a sequence of SEQ ID NO: 122 to 130 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 77, wherein the fragments of BMLF1 comprise a sequence of SEQ ID NO: 100 to 108 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 78, wherein the fragments of BMRF1 comprise a sequence of SEQ ID NO: 94 to 99 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. ) The polynucleotide according to any of the preceding embodiments 56 to 79, wherein the fragments of BFRF1 comprise a sequence of SEQ ID NO: 80 to 87 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. 81) The polynucleotide according to any of the preceding embodiments 56 to 80, wherein the polypeptide comprises a combination of two or more of the SEQ ID NO: 80 to 130.

[0063] 82) The polynucleotide according to any of the preceding embodiments 56 to 81, wherein the polypeptide comprises a sequence of SEQ ID NO: 11 to 20 and SEQ ID NO: 293 to 302 or comprises a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.

[0064] 83) The polynucleotide according to any of the preceding embodiments, wherein the polynucleotide is a recombinant polynucleotide.

[0065] 84) The polynucleotide according to any of the preceding embodiments, wherein the polynucleotide is a modified RNA.

[0066] 85) The polynucleotide according to embodiment 84, comprising a 5’ UTR sequence, 3’UTR sequence, a poly(A)-tail, or a combination thereof.

[0067] 86) The polynucleotide according to any of the preceding embodiment 84 or 85, comprising a 5’-cap, a free 5 ’-triphosphate group, a free 5’-disphosphate group, a free 5 ’-diphosphate group, a free 5’ - monophosphate group, or a free 5 ’-OH group, or comprising chemically modified analogues of said 5’-cap, said 5 ’-triphosphate group, said free 5’-disphosphate group or said free 5 ’-monophosphate group.

[0068] 87) The polynucleotide according to embodiment 86, comprising a 5’cap selected from G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m32’2’7G[5’]ppp[5’]G, m27’3'-°G[5’]ppp[5’]G (3’- ARCA), m27’2'°GpppG (2’-ARCA), m27’2'-°GppSpG (p-S- ARCA), and m27’2’’ °GppSpG ( -S-ARCA) and m27 3''°Gppp(mi2'°)ApG.

[0069] 88) The polynucleotide according to any of the preceding embodiments 84 to 87, comprising a FI element. ) The polynucleotide according to any of the preceding embodiments 84 to 88, comprising a Kozak sequence. ) The polynucleotide according to any of the preceding embodiments 84 to 89, comprising an interrupted poly(A) sequence. ) The polynucleotide according to any of the preceding embodiments, wherein the polynucleotide comprises a P2P 16 sequence encoding the tetanus toxoid (TT)-derived epitopes TT830-844 and TT578-609 of Clostridium tetani, preferably comprising the SEQ ID NO: 23 or comprising a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID No. ) The polynucleotide according to any of the preceding embodiments, wherein the polypeptide comprises one or more linkers, wherein each linker is located between two fragments, wherein the linker preferably is a cleavage-enhancing linker. ) The polynucleotide according to embodiment 92, wherein the linker comprises at most 8 amino acids, preferably at most 6 amino acids, more preferably at most 5 amino acids, most preferably 1 to 4 amino acids. ) The polynucleotide according to any of the preceding embodiments 92 and 93, wherein the one or more linkers comprise a sequence of the SEQ ID NO: 223 to 278 and 304 to 412. ) The polynucleotide according to any of the preceding embodiments, wherein the fragments are immunogenic fragments. ) The polynucleotide according to any of the preceding embodiments, wherein the CD8+-specific immune response is measured by an ELISpot assay. ) The polynucleotide according to any of the preceding embodiments, wherein the subject has multiple sclerosis. 98) The polynucleotide according to any of the preceding embodiments, wherein the subject has a latent EBV infection.

[0070] 99) The polynucleotide according to any of the preceding embodiments, wherein the subject is a human.

[0071] 100) A vector comprising a polynucleotide according to any one of the preceding embodiments.

[0072] 101) The vector according to embodiment 100, comprising at least two polynucleotides according to any one of the preceding embodiments 1 to 99.

[0073] 102) A polypeptide encoded by the polynucleotides according to any one of the preceding embodiments 1 to 99.

[0074] 103) The polypeptide of embodiment 102, wherein the polypeptide comprises a sequence of SEQ ID NO: 1 to 20 and SEQ ID NO: 283 to 302 or a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.

[0075] 104) A vaccine composition comprising the polynucleotide according to any one of the preceding embodiments 1 to 99, the vector according to embodiment 100 or 101, or the polypeptide according to embodiment 102 or 103, in a pharmaceutically acceptable excipient.

[0076] 105) The vaccine composition according to embodiment 104, wherein the composition comprises a least two polynucleotides according to any one of the preceding embodiments 1 to 99.

[0077] 106) The vaccine composition according to embodiment 96, wherein the composition comprises a polynucleotide according to any one of embodiment of the preceding embodiments 30 to 55, and 83 to 99 and a polynucleotide according to any one of the preceding embodiments 56 to 99. ) The vaccine composition according to any one of the preceding embodiments 104 to

[0078] 106, wherein the composition comprises a least two polypeptides according to embodiments 102 or 103. ) The vaccine composition according to any one of the preceding embodiments 104 to

[0079] 107, wherein the composition comprises an adjuvant. ) The vaccine composition according to any of the preceding embodiments 104 to 108, wherein the vaccine is a therapeutic EBV-specific vaccine composition. ) The polynucleotide according to any one of the preceding embodiments 1 to 99, the vector according to embodiment 100 or 101, the polypeptide according to embodiment 102 or 103, or the vaccine according to any one of the preceding embodiments 104 to 109 for use in the treatment of multiple sclerosis. ) The polynucleotide according to any one of the preceding embodiments 1 to 99, the vector according to embodiment 100 or 101, the polypeptide according to embodiment 102 or 103, or the vaccine according to any one of the preceding embodiments 104 to 109 for use in eliciting a CD8+-specific immune response. ) A pharmaceutical composition which comprises the polynucleotide according to any one of the preceding embodiments 1 to 99, the vector according to embodiment 100 or 101, the polypeptide according to embodiment 102 or 103, or the vaccine according to any one of the preceding embodiments 104 to 109, in a pharmaceutically acceptable excipient. ) A host cell comprising the polynucleotide according to any one of the preceding embodiments 1 to 99, the vector according to embodiment 100 or 101, or the polypeptide according to embodiment 102 or 103. 114) A method comprising administering to a subject the vaccine according to any one of the preceding embodiments 104 to 109 in a therapeutically effective amount to induce in the subject an immune response.

[0080] Definitions

[0081] The terms indicated for explanation of the invention have the following meaning, unless otherwise indicated in the description or the claims. Additional definitions are set forth throughout the detailed description.

[0082] The term “3’UTR sequence” refers to a 3' untranslated region known to regulate mRNA- based processes, such as mRNA localization, mRNA stability, and translation. In addition, 3' UTRs can establish 3' UTR-mediated protein-protein interactions (PPIs), and thus can transmit genetic information encoded in 3' UTRs to proteins. This function has been shown to regulate diverse protein features, including protein complex formation or posttranslational modifications, but is also expected to alter protein conformations.

[0083] The term “5’ UTR sequence” refers to a 5 '-untranslated region which lies within the noncoding genome upstream of a coding sequence and plays an important role in regulating gene expression. Within 5'-UTR sequences may be numerous cis-regulatory elements present that can interact with the transcriptional machinery to regulate mRNA abundance. The 5'- untranslated region may contain various RNA-based regulatory elements including the secondary structures, RNA-binding protein motifs, upstream open-reading frames (uORFs), internal ribosome entry sites, terminal oligo pyrimidine (TOP) tracts, and G-quadruplexes. These elements can alter the efficiency of mRNA translation; some can also affect mRNA transcript levels via changes in stability or degradation.

[0084] The term “5 ’cap” refers to a cap structure on the 5'-end of mRNAs, which is present in eukaryotic organisms. Naturally occurring Cap structures comprise a ribo-guanosine residue that is methylated at position N7 of the guanine base, abbreviated7mGppp. The presence of the7mGppp fragment on the 5'-end is essential for mRNA maturation, it protects the mRNAs from degradation by exonucleases, facilitates transport of mRNAs from the nucleus to the cytoplasm and plays a key role in assembly of the translation initiation complex. Exemplary 5 ’caps suitable for use in the polynucleotide of the invention are descripted in the patent application WO 2017 / 053297 Al. In some embodiments, a 5’ cap may be or comprise a dinucleotide cap analog such as G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m32,2’7G[5’]ppp[5’]G, m27’3'°G[5’]ppp[5’]G (3 ’-ARC A), m27’2'-°GpppG (2’ -ARC A), m27’2' °GppSpG (p-S- ARC A), and m27 2°GppSpG ( -S-ARCA) and m27 3''°Gppp(mi2'°)ApG (CleanCap413). Various cap analogs are described herein and known in the art, e.g., commercially available.

[0085] Terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. In particular, e.g., the term “an EBV protein” encompasses both the singular and plural forms, i.e., can in particular mean “a plurality of EBV proteins”.

[0086] The terms “about” or “approximately” as used herein denotes a range of ±10% of a reference value. For examples, “about 10” defines a range of 9 to 11. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context.

[0087] The term "adjuvant" relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund’s adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-y, GM-CSF, LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys, as well as lipophilic components, such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA). As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

[0088] The term "antigen" or "immunogen" refers to any substance, preferably a peptide or protein, that is a target of an immune response and / or that will elicit an immune response. In particular, an "antigen" is any substance that reacts specifically with, i.e. binds to antibodies or T-lymphocytes (T-cells), in particular T-cell receptors. The term “antigen” comprises any molecule which comprises at least one epitope such as a B cell or T cell epitope. The term "antigen" encompasses antigenic fragments, i.e. the antigen can be an antigenic fragment. The terms “antigen fragment”, “antigenic fragment” and “antigenic polypeptide fragment” are used synonymously herein. An “antigen”, “antigen fragment”, “antigenic fragment” or “antigenic polypeptide fragment”, as used herein, can be an epitope, but can also comprise more than one epitope. In one embodiment of the present disclosure, “antigen”, “antigen fragment”, “antigenic fragment” and “antigenic polypeptide fragment” can be used interchangeably with “epitope”, i.e. the term “antigen” or “antigenic polypeptide fragment” can be replaced by the term “epitope”. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen or cells expressing the antigen. Antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen.

[0089] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxy ethylene / polyoxy-propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline. Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0090] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0091] The term “CD8+-specific immune response” or “EBV-specific CD8+T cell immune response” refers to an EBV-specific activation of CD8+T cells, i.e., CD8+T cells directed to EBV antigens, through major histocompatibility complex (MHC) class I presentation. CD8+- specific means that the proportion of CD8+- vs. CD4+-immune response is higher compared to immunization with the entire naturally occurring EBV protein. In particular, CD8+-specific means that more EBV-specific CD8+compared to EBV-specific CD4+T-cells are activated. In some particularly preferred embodiments, substantially no or no EBV-specific CD4+immune response is elicited. A preferred method to evaluate an EBV-specific response is to immunize mice with the polynucleotide of the invention, the polypeptide of the invention or the vector of the invention. Afterwards, the immune cells of the immunized mice are analyzed, wherein the cells are isolated and re-stimulated with specific peptides. The successfully induced EBV-specific T cells respond with cytokine (IFNy) production. In some embodiments, an MHC-I tetramer staining known in the art can be performed to demonstrate epitope-specific CD8+T cell responding to specific EBV antigen proteins.

[0092] Unless expressly specified otherwise, the term “comprising” is used in the context of the present document to indicate that further members may optionally be present in addition to the members of the list introduced by “comprising”. It is, however, contemplated as specific embodiments of the present invention that each time the term “comprising” is used, this shall also encompass the possibility of no further members being present, i.e., for the purpose of this embodiment “comprising” can be understood as having the meaning of “consisting of’.

[0093] The term "diluent" relates to a diluting and / or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and / or mixing media. Examples of suitable diluents include ethanol, glycerol, and water.

[0094] Herein, the term "DNA" relates to a nucleic acid molecule which is entirely or at least substantially composed of deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0095] DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0096] “Early lytic proteins” encompass any EB V protein expressed during the lytic stage. Exemplary early lytic proteins are BMLF1, BMRF1, and BFRF1. In some embodiments, fragments of BMLF1, BMRF1, and BFRF1 are chosen to include antigens of the early lytic phase.

[0097] “EBV protein” refers to proteins naturally expressed by Epstein Barr Virus (EBV), including latency proteins, lytic switch proteins, and early lytic proteins. The term “EBV protein” can refer to full-length proteins or parts of full-length proteins of the Epstein Barr Virus. In the context of the invention, “EBV protein” refers to proteins comprising antigens. In this sense, the term “EBV protein” can also be replaced by “antigenic EBV protein”.

[0098] As used herein, the term “effective amount” refers to an amount of a given substance that is sufficient in quantity to produce a desired effect, including an improvement or remediation of the disease, disorder, or symptoms of the disease or condition. For example, an effective amount of the vaccine composition for eliciting a CD8+-specific immune response in a subject is an amount capable to achieve a detectable increase in antigen-specific CD8+T cells upon administration to the subject.

[0099] As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA- dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or a single-stranded RNA (e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target polypeptide agent. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent, which may be used as a template for transcription of a gene or cDNA. As is understood in the art, the phrase “polynucleotide encoding a peptide or protein” means that the polynucleotide, if present in the appropriate environment, for example within a cell and / or in a cell-free translation system, can direct the assembly of amino acids to produce the peptide or protein via a process of translation.

[0100] The term “epitope” refers to the part of an antigen that as used herein, refers to an agent that elicits an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. For example, epitopes are the discrete, three-dimensional sites on an antigen, which are recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Preferably, the term relates to an immunogenic portion of an antigen comprising the epitope. It is preferred that the epitope in the context of the present invention is a T cell epitope.

[0101] The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[0102] The term “FI element” refers to a sequence in the 3 '-untranslated region known to improve mRNA stability and translation efficiency. The FI element can be positioned in the 3’UTR. Exemplary FI elements suitable for use in the polynucleotide of the invention are descripted in the patent application WO 2017 / 059902 Al.

[0103] “Fragment” as used herein, with reference to an amino acid sequence (polypeptide or protein), relates to a part of a naturally occurring protein amino acid sequence, e.g., a partial sequence that has been shortened at the N-terminus and / or C-terminus compared to the naturally occurring full length sequence. A fragment, as used herein, comprises at most 15 amino acids, i.e., is at most 15 amino acids in length. A fragment as used herein is understood to not refer to a peptide having a length of 16 or more amino acids, e.g., a fragment does not comprise a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Meaning, the amino acids adjacent to the 1stand last amino acid of the fragment are understood to be different to the amino acids occurring in natural protein amino acid sequence. An amino acid sequence whose sequence represents two or more discontinuous sequences derived from the same parental amino acid sequence fused together is considered to be two or more fragments of that parental sequence. In a particularly preferred embodiment, the fragment is an antigenic polypeptide fragment.

[0104] The term "full length" with respect to a given polypeptide means the form of the polypeptide naturally translated from the coding DNA sequence, beginning with the ATG start codon, which encodes the first methionine in the amino acid sequence, and ending at the TGA, TAG, or TTA stop codon, or whichever stop codon employed by the organism.

[0105] As used herein, the term “gene” refers to a DNA sequence in a chromosome that codes for a protein. In some embodiments, a gene includes coding sequence (i.e., sequence that encodes a particular protein); in some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type- specific expression, inducible expression, etc.).

[0106] The term "glycoprotein" means a polypeptide that has covalently attached to it one or more carbohydrate moieties, or oligosaccharide chains. The carbohydrate moieties are normally attached to glycoproteins co-translationally or as post-translational modifications.

[0107] The term "immune cell" means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., a bacterial or viral infection or an auto-antigen). In typical embodiments, an immune cell is a leukocyte, such as a white blood cell. Immune cells include neutrophils, eosinophils, basophils, lymphocytes, and / or monocytes. Lymphocytes include T lymphocytes and B lymphocytes. Immune cells can also be dendritic cells, natural killer (NK) cells, and / or a mast cell. The term “immunogenic fragment” relates to a fragment capable of eliciting an immune response in a subject.

[0108] The term “interrupted poly(A)-tail” refers to a poly(A)-tail comprising non-adenine nucleotides at regular or irregularly spaced intervals. In an embodiment, the interrupting sequence is a trinucleotide, dinucleotide or mononucleotide interrupting sequence. In another embodiment, the poly(A) tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2 to 10 non-adenine nucleotides every 8 to 50 consecutive adenine nucleotides. In an embodiment, the poly(A) tail comprises or contains 1, 2, 3, 4, or 5 consecutive non-adenine nucleotides every 8-50 consecutive adenine nucleotides. In an embodiment, wherein the poly(A) tail comprises or contains more than one non-adenine nucleotide or more than one consecutive stretch of 2-10 non-adenine nucleotides. In some embodiments, a poly(A) sequence measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linking sequence and another 70 adenosine residues is used. The term “poly(A) tail” and “poly(A)” sequence is used interchangeably herein.

[0109] The “Kozak sequence” typically extends from approximately position -6 to position +6, where +1 is assigned to the adenine of the START codon. The Kozak sequence is known to affect transcription initiation.

[0110] “Latency proteins” encompass any EBV protein expressed during the latent infection stage. Latency proteins are, in particular, EBNA-LP, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2A, and LMP2B.

[0111] The term “linker” refers to a non-naturally occurring amino acid sequence present between, and thereby separating, two fragments of a protein or two fragments of different proteins. Non-naturally occurring means in this context not naturally-occurring in the proteins from which the fragments are derived. For example, if the linker separates fragments of EBV proteins, the linker sequence is not naturally occurring adjacent to the fragments it separates in the EBV proteins that the fragments are derived from. The linker is a cleavage-enhancing linker, enhancing the cleavage of the polypeptide to liberate the fragments for further processing and MHC I presentation. In preferred embodiments, the polypeptide comprises fragments separated by a (cleavage-enhancing) linker and comprises fragments not separated by a (cleavage-enhancing) linker.

[0112] “Lytic switch proteins” encompass any EB V protein expressed during the lytic switch stage. Lytic switch proteins are, in particular, BZLF1 and BRLF1.

[0113] The term “natural EBV protein”, as used herein, refers to the full-length sequences of proteins expressed by EBV in nature, i.e. the unmodified protein sequence. “Natural EBV protein”, is a protein expressed by a naturally occurring EBV. In particular, “natural EBV protein” means the full-length sequences for EBNA-LP, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2A, and LMP2B, provided in the SEQ ID NO: 131 to 142.

[0114] The term "nucleoside" relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as "dT" ("d" represents "deoxy") as it contains a 2'- deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG. A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is, in some embodiments, modified by one or more alkyl groups, e.g., one or more Ci-4 alkyl groups, e.g., one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5- alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N7-Ci-4 alkyl-guanine, N6-Ci-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N(1)-CI-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(1 )-m ethyl - uracil.

[0115] The term “operatively linked” means that a promoter, or similar regulatory element, is positioned next to an expressible nucleotide sequence or coding region such that the transcription of that coding region is controlled and regulated by that promoter.

[0116] The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administration of said pharmaceutical composition to a subject.

[0117] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. In certain embodiments, the pharmaceutically acceptable carrier or excipient is not naturally occurring.

[0118] The term “plurality” refers to the state of being plural. A plurality of fragments therefore refers to more than one fragment. In an embodiment, a plurality of fragments refers to at least two fragments, preferably at least three, at least four, or at least five fragments. A plurality of EBV proteins likewise refers to more than one fragment. In an embodiment, a plurality of EBV proteins refers to at least two EBV proteins, preferably at least three, at least four, or at least five EBV proteins.

[0119] The term “poly(A)-tail” is used interchangeably with the term “poly(A)-sequence” and refers to a chain of adenine nucleotides that is added to a mRNA molecule during RNA processing to increase the stability of the molecule. This process, called polyadenylation, adds a poly(A)- tail that is usually between 100 and 250 residues long. Poly(A) tails play an important role in the translation and stability of the mRNA. RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence. The term "poly(A) tail" or "poly-A sequence" relates to a sequence of adenyl (A) residues which typically is located on the 3 ’-end of a RNA molecule and "unmasked poly-A sequence" means that the poly-A sequence at the 3’ end of an RNA molecule ends with an A of the poly-A sequence and is not followed by nucleotides other than A located at the 3’ end, i.e. downstream, of the poly-A sequence. Furthermore, a long poly-A sequence of about 120 base pairs results in an optimal transcript stability and translation efficiency of RNA.

[0120] The terms "polynucleotide” and “nucleic acid” can be used interchangeably herein to refer to polymers of nucleotides. The term " polynucleotide" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a polynucleotide is DNA. In some embodiments, a polynucleotide is RNA. In some embodiments, a polynucleotide is a mixture of DNA and RNA. A polynucleotide may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. The polynucleotide disclosed herein can be isolated. The term “isolated polynucleotide “ means, according to the present disclosure, that the polynucleotide (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0121] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids.

[0122] The term “recombinant” when used in the context of a polynucleotide means a polynucleotide having nucleotide sequences that are not naturally joined together and can be made by artificially combining two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Recombinant polynucleotides include vectors comprising an amplified or assembled polynucleotide, which can be used to transform or transfect a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a “recombinant host cell.” The polynucleotide is then expressed in the recombinant host cell to produce a “recombinant polypeptide.” A recombinant polynucleotide can also comprise a non-coding function.

[0123] The term "RNA" relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2’ -position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (i.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains “a majority of ribonucleotide residues” if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0124] “RNA” includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), selfamplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, “RNA” refers to mRNA. The term "in vitro transcription" or "IVT" as used herein means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).

[0125] According to the present disclosure, the term ”RNA” includes “mRNA”. The term “mRNA” refers to messenger RNA that comprises a coding sequence encoding a polypeptide. An mRNA may further comprise non-coding sequences such as a 5 ’cap, a 5’UTR, a 3’UTR, a Kozak sequence, a FI element or a poly(A) tail or any combination thereof. According to the present disclosure, the term “mRNA” means “messenger-RNA” and includes a “transcript” which may be generated by using a DNA template. Generally, mRNA encodes a peptide or polypeptide. mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices. According to the present disclosure, “dsRNA” means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0126] The term “string” refers to a polynucleotide or polypeptide of the invention, and can be used interchangeably therewith. A string comprises therefore a plurality of fragments of an EBV protein, each fragment having a length of at most 15 amino acids. According to the invention, the fragments are strung like beads on a string. In particular, a “string” comprises a plurality of fragments of an EBV protein, wherein at least some of these fragments are separated by linker sequences as defined above.

[0127] As used herein, a “subject” is a human of either gender (a male or a female). The subject may be of any age. In one embodiment, the subject is female. In another embodiment, the subject is male. In some embodiments, the subject is a patient having MS, in particular a female patient having MS and / or a male patient having MS.

[0128] The term “treating” when used in the context of a disease or disease condition means ameliorating, improving or remedying a disease, disorder, or symptom of a disease or condition associated with the disease, or can mean completely or partially stopping, on a molecular level, the biochemical basis of the disease, such as halting replication of a virus, etc. It describes an act that leads to the elimination, reduction, alleviation, reversal, or prevention or delay of onset or recurrence of any symptom of a disease.

[0129] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors comprise plasmids, cosmid vectors, phagemids such as lambda phage, virus genomes including retroviral, adenoviral or baculoviral vectors, artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC) and functional portions thereof. One type of vector is a “plasmid”, which refers to a circular double stranded DNA into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate non-coding sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

[0130] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0131] All patents, patent applications, and other publications cited in this application are incorporated by reference in the entirety for all purposes.

[0132] The polynucleotide of the invention encoding a polypeptide

[0133] The present invention provides a (isolated) polynucleotide encoding a polypeptide, wherein the polypeptide comprises at least 25 fragments derived from at least two EBV proteins, each fragment having a length of at most 15 amino acids; and one or more cleavage-enhancing linkers, each located between two fragments. Without wishing to be bound by theory, fragment having a length of at most 15 amino acids are included to specifically elicit a CD8+T cell response against selected EBV proteins. Both the polynucleotide and the corresponding polypeptide according to the invention are non-naturally occurring. While their sequences have been engineered from fragments of naturally occurring sequences, the combination of sequence fragments and therefore the resulting linear polynucleotide or polypeptide sequence according to the invention is non-naturally occurring. The polypeptide comprising the plurality of fragments will be cleaved in a host cell through the endogenous protein processing machinery including proteases, liberating each fragment for MHC class I processing and presentation, and thereby triggering the EBV-specific CD8+T cell immune response.

[0134] In a preferred embodiment, the polynucleotide of the invention encodes the polypeptides of the invention. Thus, the features and embodiments defined herein for the polypeptide and with reference to amino acids are meant to be equally disclosed also for the corresponding polynucleotide that encodes the respectively described polypeptide of the invention.

[0135] In some embodiments, each fragment has a length of at least 6, at least 7, or preferably at least 8 amino acids. In some embodiments, each fragment has a length of at most 14 amino acids, preferably at most 13 amino acids, preferably at most 12 amino acids, preferably at most 11 amino acids, and preferably at most 10 amino acids. Most preferably, each fragment has the length of at least 8 amino acids to at most 14 amino acids. These length limitations have been found to be crucial for MHC class I vs. class II presentation, and therefore CD8+vs. CD4+T- cell responses. The fragment length is considered to be a determining feature as the MHC class I binding groove is closed at both ends by conserved tyrosine residues leading to a size restriction of the bound peptides with its C-terminal end docking into the F-pocket. It is known that, to some extent, structural variations can be introduced by variable peptide- binding modes. In this context, peptides longer than 8-10 residues, i.e., amino acids, have been reported to bind to the MHC class I binding groove, and, in some cases, peptides having 15 residues have been found to bind to MHC class I proteins. In context of the invention, the fragment length is selected for presentation on the surface of MHC class I proteins for CD8+T-cell recognition. The invention aims to elicit an EBV-specific CD8+T-cell response, while minimizing or eliminating to the extent possible, an EBV-specific CD4+T-cell response. According to the invention, this leads to a particularly safe treatment option for MS patients.

[0136] In some embodiments, the polypeptide has a length of at most 1500 amino acids, preferably at most 1250 amino acids, more preferably at most 1000 amino acids, and most preferably at most 900 amino acids. In some embodiments, the polypeptide has a length of at most 800 amino acids, preferably at most 750 amino acids. In some embodiments, the polypeptide has a length of at least 300 amino acids, preferably at least 400 amino acids, more preferably at least 500 amino acids, and most preferably at least 550 amino acids. In some embodiments, the polypeptide has a length of 300 to 1500 amino acids, 300 to 1250 amino acids, 400 to 1000 amino acids, 500 to 900 amino acids, 500 to 800 amino acids 550 to 750 amino acids, or 550 to 720 amino acids.

[0137] In some embodiments, the polypeptide comprises at least 26 fragments of an EBV protein, preferably at least 27 fragments, more preferably at least 28 fragments, even more preferably at least 29 fragments, even more preferably at least 30 fragments, even more preferably at least 35 fragments even more preferably at least 40 fragments of an EBV protein, even more preferably at least 45 fragments and most preferably at least 50 fragments. In some embodiments, the polypeptide comprises at most 100 fragments of an EBV protein, preferably at most 90 fragments, more preferably at most 80 fragments, even more preferably at most 70 fragments, and most preferably at most 60 fragments. In some embodiments, the polypeptide comprises 2 to 100 fragments of an EBV protein, 5 to 95 fragments, 10 to 90 fragments, 15 to 85 fragments, 20 to 80 fragments, 30 to 75 fragments, 35 to 70 fragments, 40 to 65 fragments, 45 to 60 fragments, or 50 to 60 fragments.

[0138] In some embodiments, the polypeptide comprises fragments of at least 3 EBV proteins, more preferably of at least 4 EBV proteins, most preferably of at least 5 EBV proteins. In some embodiments, the polypeptide comprises fragments of at most 10 EBV proteins, preferably of at most 9 EBV proteins, more preferably of at most 8 EBV proteins, more preferably of at most 7 EBV proteins, and most preferably of at most 6 EBV proteins.

[0139] In some embodiments, the EBV proteins are selected from a group consisting of latency proteins, lytic switch proteins, and early lytic proteins. In a preferred embodiment, the polypeptide of the invention comprises fragments from EBV proteins expressed during different stages of the EBV life cycle. In a preferred embodiment, the EBV proteins are selected from latency proteins and lytic switch proteins. In a preferred embodiment, the EBV proteins are selected from latency proteins and early lytic proteins. Without wishing to be bound by theory, by targeting EBV proteins expressed during different stages of the EBV life cycle, EBV-infected cells are preferably kept in the latent phase to avoid re-exposure of the EBV and to reduce an antibody response leading to reduced CNS infiltration and local inflammation associated with MS.

[0140] As shown in Fig 2., latency proteins according to the invention are in particular, EBNA-LP, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2A, or LMP2B or any combination thereof. Lytic switch proteins are in particular BZLF1 and / or BRLF1. Early lytic proteins are in particular BMLF1, BMRF1, and BFRF1 or any combination thereof.

[0141] In some embodiments, the EBV proteins are selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0142] In some embodiments, the fragments are derived from one or more of the SEQ ID NO: 131 to 142 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO. It is understood, that the sequences shown in SEQ ID NO: 131 to 142 are full length proteins and that the term ’’derived from” within this context means that the sequences of the fragments can be comprised within the full-length protein sequences. For example, the fragments of EBNA1 can be derived from SEQ ID NO: 141, meaning that one or more of the EBNA1 fragments can be identical to sequences comprised in SEQ ID NO: 141. In some embodiments, the fragments are identical to a sequence set forth in the full-length protein sequences. In some embodiments, the sequences of the EBNA1 fragments can be at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to sequences comprised in SEQ ID NO: 141.

[0143] In some embodiments, the fragments comprise a sequence of SEQ ID NO: 24 to 130 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 24 to 130.

[0144] In some embodiments, the polynucleotide encodes a polypeptide comprising any of the sequences of the SEQ ID NO: 1 to 20 and SEQ ID NO: 283 to 302 or is at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.

[0145] In some embodiments, the polynucleotide comprises any of the sequences of the SEQ ID NO: 143 to 222 or is at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.

[0146] In some embodiments, the polynucleotide comprises any of the sequences of the SEQ ID NO: 413 to 422 or is at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the polynucleotide consists of a sequences of SEQ ID NO: 413 to 422.

[0147] In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 143. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 144. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 145. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 146. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 147. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 148. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 149. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 150. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 151. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 152. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 153. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 154. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 155. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 156. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 157. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 158. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 159. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 160. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 161. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 162. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 163. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 164. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 165. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 166. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 167. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 168. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 169. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 170. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 171. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 172. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 173. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 174. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 175. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 176. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 177. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 178. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 179. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 180. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 181. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 182. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 183. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 184. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 185. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 186. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 187. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 188. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 189. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 190. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 191. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 192. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 193. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 194. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 195. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 196. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 197. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 198. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 199. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 200. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 201. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 202. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 203. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 204. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 205. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 206. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 207. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 208. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 209. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 210. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 211. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 212. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 213. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 214. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 215. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 216. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 217. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 218. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 219. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 220. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 221. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 222. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 413. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 414. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 415. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 416. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 417. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 418. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 419. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 420. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 421. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 422. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 143. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 144. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 145. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 146. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 147. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 148. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 149. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 150. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 151. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 152. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 153. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 154. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 155. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 156. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 157. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 158. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 159. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 160. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 161. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 162. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 163. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 164. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 165. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 166. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 167. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 168. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 169. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 170. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 171. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 172. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 173. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 174. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 175. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 176. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 177. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 178. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 179. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 180. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 181. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 182. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 183. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 184. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 185. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 186. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 187. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 188. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 189. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 190. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 191. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 192. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 193. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 194. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 195. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 196. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 197. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 198. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 199. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 200. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 201. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 202. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 203. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 204. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 205. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 206. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 207. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 208. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 209. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 210. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 211. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 212. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 213. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 214. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 215. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 216. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 217. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 218. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 219. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 220. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 221. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 222. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 413. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 414. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 415. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 416. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 417. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 418. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 419. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 420. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 421. In some embodiments, the polynucleotide consists of the sequence of SEQ ID NO: 422.

[0148] Exemplary polynucleotide sequences are depicted in table 1. As shown exemplary for stringl v3, stringl v3P, stringl v4, stringl v4P, stringl v7, stringl v7P, stringl v8, stringl v8P, stringl vlO, stringl vlOP, string 2_vl, string 2_vlP, string 2_v7, string 2_v7P, string 2_v8, string 2_v8P, string 2_v9, string 2_v9P, string 2_vl0, and string 2_vlOP, the polynucleotide may be codon optimized. As further shown exemplarily for stringl v3P, stringl v4P, stringl v7P, stringl v8P, stringl vlOP, string 2_vlP, string 2_v7P, string 2_v8P, string 2_v9P, and string 2_vlOP, the polynucleotide may comprise a sequence encoding for the tetanus toxoid CD4+epitopes P2 and Pl 6 encoding the tetanus toxoid (TT)-derived epitopes TT830-844 and

[0149] TT578-609 of Clostridium tetani (SEQ ID NO: 23).

[0150] Table 1. Exemplary polynucleotide of the invention

[0151] In some embodiments, the polypeptide comprises fragments of the EBV proteins EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 as shown in table 2 and / or table 3. In some preferred embodiments, the fragments as referred to in this disclosure are selected from those listed in table 2 and / or table 3. The shown fragments are derived from the respective EBV protein, termed “protein” in the tables below, wherein the position of the fragment in the EBV protein is described by provision of the first and last amino acids (start and stop). Furthermore, each fragment of an individual EBV protein is given a fragment index (frag idx) for reference. In Fig 3 to 12, this fragment index is used for identification of the fragments of each EBV protein.

[0152] In a preferred embodiment, the fragments are reported or predicted MHC class I-restricted epitopes to elicit a CD8+-specific immune response. In preferred embodiments, a plurality of fragments is used to increase the coverage of the targeted EBV protein based on an affinity prediction of antigen epitopes to different HLA types. Predicting the affinity of epitopes to HLA types can be for example performed using algorithms that accurately predicted antigen presenting cell (APC) ligandomes (Abelin JG, Harjanto D, Malloy M, Suri P, Colson T, Goulding SP, Creech AL, Serrano LR, Nasir G, Nasrallah Y, McGann CD, Velez D, Ting YS, Poran A, Rothenberg DA, Chhangawala S, Rubinsteyn A, Hammerbacher J, Gaynor RB, Fritsch EF, Greshock J, Oslund RC, Barthelme D, Addona TA, Arieta CM, Rooney MS. Defining HLA-II Ligand Processing and Binding Rules with Mass Spectrometry Enhances Cancer Epitope Prediction. Immunity. 2019 Oct 15;51(4):766-779.el7. doi: 10.1016 / j.immuni.2019.08.012. Epub 2019 Sep 5. Erratum in: Immunity. 2021 Feb 9;54(2):388. PMID: 31495665) or using neural-network prediction algorithms (Abelin JG, Keskin DB, Sarkizova S, Hartigan CR, Zhang W, Sidney J, Stevens J, Lane W, Zhang GL, Eisenhaure TM, Clauser KR, Hacohen N, Rooney MS, Carr SA, Wu CJ. Mass Spectrometry Profiling of HL A- Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Immunity. 2017 Feb 21;46(2):315-326. doi:

[0153] 10.1016 / j.immuni.2017.02.007. PMID: 28228285; PMCID: PMC540538L).

[0154] In some embodiments, the polypeptide does not comprise self-proteins which map to human proteins. In some embodiments, the polypeptide does not comprise MHC class II epitopes.

[0155] Table 2. Exemplary fragments of the EBV proteins EBNA1, LMP1, LMP2A, LMP2B, BZLF1 and BRLF1.

[0156] Table 3. Exemplary fragments of the EBV proteins EBNA2, BFRF1, BMRF1, BMLF1 (BSLF2), EBNA-3A and EBNA-3B

[0157] In some embodiments, at least two of the fragments of a first EBV protein are separated by one or more fragments of at least a second EBV protein, wherein preferably the first and second EBV proteins are selected from a group consisting of EBNA1, EBNA2, EBN A3 A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1. In this way, the fragments are “shuffled”. By such shuffling and fragment rearrangement, the cleavage of the polypeptide may be optimized for liberating the fragments resulting in optimal MHC class I presentation. The liberation of the fragments facilitates eliciting a strong CD8+T cell response against selected antigens and reduces the risk of eliciting an EBV-specific CD4+response by longer uncleaved fragments. As explained above, the size of the fragments is decisive for determining CD8+vs. CD4+T-cell response. Optimization of the fragment shuffling and linker sequences can be performed by way of cleavage prediction programs or cleavage assay experiments known in the art.

[0158] In some embodiments, at least two of the fragments of the first EBV protein are separated by one or more of the fragments of a third protein preferably selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1.

[0159] In some embodiments, at least two of the fragments of the first EBV protein are separated by one or more of the fragments of a fourth protein preferably selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1.

[0160] In some embodiments, at least two of the fragments of the first EBV protein are separated by one or more of the fragments of a fifth protein preferably selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1.

[0161] In some embodiments, at least two of the fragments of EBNA1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0162] In some embodiments, at least two of the fragments of EBNA2 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0163] In some embodiments, at least two fragments of EBNA3A are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0164] In some embodiments, at least two of the fragments of EBNA3B are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0165] In some embodiments, at least two of the fragments of LMP1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0166] In some embodiments, at least two of the fragments of LMP2 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0167] In some embodiments, at least two of the fragments of BZLF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof. In some embodiments, at least two of the fragments of BRLF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0168] In some embodiments, at least two of the fragments of BMLF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMRF1, and BFRF1 or a combination thereof.

[0169] In some embodiments, at least two of the fragments of BMRF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, and BFRF1 or a combination thereof.

[0170] In some embodiments, at least two of the fragments of BFRF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, and BMRF1 or a combination thereof.

[0171] In some embodiments, at least two of the fragments of EBNA3C are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0172] In some embodiments, the polynucleotide comprises a P2P 16 sequence encoding the tetanus toxoid (TT)-derived epitopes TT830-844 and TT578-609 of Clostridium tetani, preferably comprising the SEQ ID NO: 23 or comprising a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO, or consisting of said SEQ ID NO. In some embodiments, this sequence may support effective induction of EBV-specific immune responses by providing EBV-unspecific T cell help during priming. The tetanus toxoid heavy chain includes epitopes that can bind promiscuously to MHC class II alleles and induce CD4+memory T cells in almost all tetanus vaccinated individuals. It is hypothesized that providing CD4+mediated T cell help during the priming phase may improve priming efficiency of T cells. To reduce the risk of stimulating CD8+T cells, two peptide sequences known to contain promiscuously binding helper epitopes were selected to ensure binding to as many MHC class II alleles as possible. Reference is made to WO 2020 / 182869, which is incorporated herein in its entirety.

[0173] As shown in Fig 3-12, at least two fragments of an EBV protein may not form a contiguous amino acid sequence in the polypeptide of the invention that is also naturally occurring in the corresponding natural EBV protein from which the fragments were derived. The term natural EBV protein, as used herein, has been defined above and refers in particular to the naturally occurring full-length EBV proteins as provided in SEQ ID NO: 131 to 142. For example, two fragments may be from two different EBV proteins and a first fragment of a first EBV protein may be adjacent to a second fragment of a second EBV protein. In another example, a first and a second fragment may also be from the same EBV protein, but from different parts of the EBV protein that are not directly adjacent in the natural EBV protein sequence. A first and a second fragment may be from the same part of a EBV protein, but will not form a contiguous amino acid sequence that is also naturally occurring in the corresponding natural EBV protein, e.g., they may be separated by linkers or fragments of other EBV proteins. In some embodiments, fragments can be separated from each other by amino acids such that the resulting sequence is not naturally occurring in the corresponding natural EBV protein, e.g., they may be separated by linkers or fragments of other EBV proteins. In another example, a first, a second, and a third fragment may be from the same part of a EBV protein but will not form a contiguous amino acid sequence as naturally occurring in the corresponding natural EBV protein, e.g., they may be separated by linkers or fragments of other EBV proteins. In some embodiments, a first fragment may be adjacent to a second fragment without a linker separating the first and the second fragments. To facilitate a clear description of the polypeptides and polynucleotides of the invention, particular fragments or EBV proteins are referred to as a “first” fragment or EBV protein, a “second” fragment or EBV protein, etc. It is to be understood that the first, second, etc. fragment or EBV protein can appear in any desired order or orientation, and that no particular order or orientation is intended by the words “first”, “second” etc. As further shown in Fig 3-12, the polypeptide can comprise one or more linkers, preferably cleavage-enhancing linkers, for further optimizing the liberation of the fragments for eliciting a strong CD8+T cell response against selected antigens.

[0174] As explained above, the epitope design, selection and assembly into the strings according to the invention decreases the risk for unwanted EBV-specific CD4+T-cell responses. In particular, the invention prevents that contiguous stretches of sequences of natural EBV proteins of more than the fragment lengths defined herein are present on the polypeptide of the invention. This is facilitated by the way the epitopes are chosen and the maximally allowed fragment lengths. Even if no linker is present between two fragments derived from the same EBV protein, the resulting contiguous sequence that may exceed lengths of 15 amino acids is not resembling a natural EBV protein sequence. According to the invention, this serves to prevent EBV-specific CD4+T-cell response.

[0175] In a preferred embodiment, the polypeptide does not contain fragments and junctional neo epitopes that map to human proteins, meaning to self-proteins. A junctional neo epitope is an epitope that elicits an immune response to the junction of two heterologous protein sequences, wherein the epitope is not present in either of the heterologous protein sequences themselves. T cell responses to fragments that map to human proteins or to junctional neo-epitopes can be identified using methods known in the art.

[0176] In some embodiments, the EBV proteins are selected from a group consisting of EBNA1, LMP1, LMP2, BZLF1, and BRLF1 or a combination thereof, preferably wherein the polypeptide comprises a plurality of fragments of each of EBNA1, LMP1, LMP2, BZLF1, and BRLF1.

[0177] In some embodiments, the LMP2 is selected from LMP2A, LMP2B or a combination thereof.

[0178] In some embodiments, the polypeptide comprises two or more fragments of each of EBNA1, LMP1, LMP2, BZLF1, and BRLF1. In some embodiments, the polypeptide comprises three or more fragments of each of EBNA1, LMP1, LMP2, BZLF1, and BRLF1. In some embodiments, the polypeptide comprises four or more fragments of each of EBNA1, LMP1, LMP2, BZLF1, and BRLF1. In some embodiments, the polypeptide comprises two or more fragments of each of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRFl. In some embodiments, the polypeptide comprises three or more fragments of each of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRFl. In some embodiments, the polypeptide comprises four or more fragments of each of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRFl.

[0179] In some embodiments, the polypeptide comprises a plurality of the EBNA1 fragments, preferably at least 3 EBNA1 fragments, more preferably at least 4 EBNA1 fragments, most preferably at least 5 EBNA1 fragments.

[0180] In some embodiments, the polypeptide comprises a plurality of LMP1 fragments of, preferably at least 3 LMP1, more preferably at least 4 LMP1, most preferably at least 5 LMP1.

[0181] In some embodiments, the polypeptide comprises a plurality of LMP2 fragments, preferably at least 3 LMP2 fragments, more preferably at least 4 LMP2 fragments, most preferably at least 5 LMP2 fragments.

[0182] In some embodiments, the polypeptide comprises a plurality of BZLF1 fragments, preferably at least 3 BZLF1 fragments, more preferably at least 4 BZLF1 fragments, most preferably at least 5 BZLF 1 fragments.

[0183] In some embodiments, the polypeptide comprises a plurality of BRLF1 fragments, preferably at least 3 BRLF1 fragments, more preferably at least 4 BRLF1 fragments, most preferably at least 5 BRLF1 fragments.

[0184] In some preferred embodiments, the polynucleotide disclosed herein encodes a polypeptide, wherein the polypeptide comprises:

[0185] (i) One or more EBNA1 fragments,

[0186] (ii) One or more LMP1 fragments,

[0187] (iii) One or more LMP2 fragments,

[0188] (iv) One or more BZLF 1 fragments, (v) One or more BRLF 1 fragments,

[0189] (vi) one or more cleavage-enhancing linkers, each located between two fragments, wherein each fragment has a length of at most 15 amino acids, preferably 8-15 amino acids.

[0190] In some preferred embodiments, the polynucleotide disclosed herein encodes a polypeptide, wherein the polypeptide comprises:

[0191] (i) at least 5 EBNA1 fragments,

[0192] (ii) at least 5 LMP1 fragments,

[0193] (iii) at least 5 LMP2 fragments,

[0194] (iv) at least 5 BZLF1 fragments,

[0195] (v) at least 5 BRLF1 fragments,

[0196] (vi) one or more cleavage-enhancing linkers, each located between two fragments, wherein each fragment has a length of 8-15 amino acids.

[0197] In some embodiments, the polypeptide comprises at most 30 fragments, preferably at most 25, more preferably at most 20 fragments, of each EB V protein, wherein the EBV protein selected from a group consisting of EBNA1, LMP1, LMP2, BZLF1, and BRLF1 or a combination thereof. Meaning, in some embodiments, the polypeptide comprises at most 30 EBNA1 fragments, preferably at most 25 EBNA1 fragments, more preferably at most 20 EBNA1 fragments. In some embodiments, the polypeptide comprises at most 30 LMP1 fragments, preferably at most 25 LMP1 fragments, more preferably at most 20 LMP1 fragments. In some embodiments, the polypeptide comprises at most 30 LMP2 fragments, preferably at most 25 LMP2 fragments, more preferably at most 20 LMP2 fragments. In some embodiments, the polypeptide comprises at most 30 BZLF1 fragments, preferably at most 25 BZLF1 fragments, more preferably at most 20 BZLF1 fragments. In some embodiments, the polypeptide comprises at most 30 BRLF1 fragments, preferably at most 25 BRLF1 fragments, more preferably at most 20 BRLF1 fragments.

[0198] In some embodiments, the polypeptide comprises at least fragments of the EBV proteins EBNA1, LMP1, LMP2, BZLF1, and BRLF1. In some embodiments, at least two of the fragments of EBNA1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of LMP1, LMP2, BZLF1, and BRLF1 or a combination thereof.

[0199] In some embodiments, at least two of the fragments of LMP1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, LMP2, BZLF1, and BRLF1 or a combination thereof.

[0200] In some embodiments, at least two of the fragments of LMP2 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, LMP1, BZLF1, and BRLF1 or a combination thereof.

[0201] In some embodiments, at least two of the fragments of BZLF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, LMP1, LMP2, and BRLF1 or a combination thereof.

[0202] In some embodiments, at least two of the fragments of BRLF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA1, LMP1, LMP2, and BZLF1, or a combination thereof.

[0203] In some embodiments, the fragments of EBNA1 are derived from SEQ ID NO: 141 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0204] In some embodiments, the fragments of LMP1 are derived from SEQ ID NO: 142 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised insaid SEQ ID NO.

[0205] In some embodiments, the fragments of LMP2 are derived from SEQ ID NO: 131 and / or 132 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised insaid SEQ ID NO. In some embodiments, the fragments of BZLF1 are derived from SEQ ID NO: 139 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0206] In some embodiments, the fragments of BRLF1 are derived from SEQ ID NO: 140 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0207] In some embodiments, the fragments of EBNA1 comprise a sequence of SEQ ID NO: 64 to 71 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 64 to 71.

[0208] In some embodiments, the fragments of LMP1 comprise a sequence of SEQ ID NO: 72 to 79 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 72 to 79.

[0209] In some embodiments, the fragments of LMP2 comprise a sequence of SEQ ID NO: 24 to 41 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 24 to 41.

[0210] In some embodiments, the fragments of BZLF1 comprise a sequence of SEQ ID NO: 42 to 46 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 42 to 46.

[0211] In some embodiments, the fragments of BRLF1 comprise a sequence of SEQ ID NO: 47 to 63 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 47 to 63. In some particularly preferred embodiments, the polypeptide (string 1) comprises a combination of two or more of the sequences shown in SEQ ID NO: 24 to 79. In some particularly preferred embodiments, the polypeptide (string 1) comprises a combination of the sequences shown in SEQ ID NO: 24 to 79.

[0212] In some embodiments, the polypeptide comprises a sequence of SEQ ID NO: 1 to 10 and 283 to 292 or comprises a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the polypeptide consists of a sequence of SEQ ID NO: 1 to 10 and 283 to 292.

[0213] Particularly preferred polypeptide sequences are depicted in table 4, wherein underlined amino acids correspond to linker sequences, linking two EBV protein fragments. Vertical bars demarcate individual fragments and individual linkers. The first amino acid “AT” refers to the methionine encoded by the start codon. As shown in stringl vlP, stringl v2P, stringl v3P, string 1 v4P, stringl v5P, stringl v6P, stringl v7P, stringl v8P, stringl v9P, and stringl vlOP, the polypeptide may comprise tetanus toxoid CD4+ epitopes P2 and P16 encoding the tetanus toxoid (TT)-derived epitopes TT830-844 and TT578-609 of Clostridium tetani (SEQ ID NO: 23).

[0214] Table 4. Exemplary polypeptides encoded by the polynucleotide of the invention

[0215]

[0216]

[0217]

[0218]

[0219] In some embodiments, the EBV proteins are selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof, preferably wherein the polypeptide comprises a plurality of fragments of each of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1.

[0220] In some embodiments, the polypeptide comprises a plurality of EBNA2 fragments, preferably at least 3 EBNA2 fragments, more preferably at least 4 EBNA2 fragments, most preferably at least 5 EBNA2 fragments.

[0221] In some embodiments, the polypeptide comprises a plurality of EBN3A fragments of EBN3A, preferably at least 3 EBN3 A fragments, more preferably at least 4 EBN3 A fragments, most preferably at least 5 EBN3 A fragments.

[0222] In some embodiments, the polypeptide comprises a plurality of EBNA3B fragments, preferably at least 3 EBNA3B fragments, more preferably at least 4 EBNA3B fragments, most preferably at least 5 EBNA3B fragments.

[0223] In some embodiments, the polypeptide comprises a plurality of BMLF1 fragments, preferably at least 3 BMLF1 fragments, more preferably at least 4 BMLF1 fragments, most preferably at least 5 BMLF1 fragments. In some embodiments, the polypeptide comprises a plurality of BMRF1 fragments, preferably at least 3 BMRF1 fragments, more preferably at least 4 BMRF1 fragments, most preferably at least 5 BMRF1 fragments.

[0224] In some embodiments, the polypeptide comprises a plurality of BFRF1 fragments, preferably at least 3 BFRF1 fragments, more preferably at least 4 BFRF1 fragments, most preferably at least 5 BFRF1 fragments.

[0225] In some preferred embodiments, the polynucleotide disclosed herein encodes a polypeptide, wherein the polypeptide comprises:

[0226] (i) One or more EBNA2 fragments,

[0227] (ii) One or more EBN3 A fragments,

[0228] (iii) One or more EBNA3B fragments,

[0229] (iv) One or more BMLF 1 fragments,

[0230] (v) One or more BMRF 1 fragments

[0231] (vi) One or more BFRF 1 fragments,

[0232] (vii) one or more cleavage-enhancing linkers, each located between two fragments, wherein each fragment has a length of at most 15 amino acids, preferably 8-15 amino acids.

[0233] In some preferred embodiments, the polynucleotide disclosed herein encodes a polypeptide, wherein the polypeptide comprises:

[0234] (i) at least 5 EBNA2 fragments,

[0235] (ii) at least 5 EBN3 A fragments,

[0236] (iii) at least 5 EBNA3B fragments,

[0237] (iv) at least 5 BMLF1 fragments,

[0238] (v) at least 5 BMRF1 fragments,

[0239] (vi) at least 5 BFRF1 fragments,

[0240] (vii) one or more cleavage-enhancing linkers, each located between two fragments, wherein each fragment has a length of 8-15 amino acids.

[0241] In some embodiments, the polypeptide comprises at most 30 fragments, preferably at most 25, more preferably at most 20 fragments, of each EBV protein, wherein the EBV protein is selected from EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof. In some embodiments, the polypeptide comprises at most 30 EBNA2 fragments, preferably at most 25 EBNA2 fragments, more preferably at most 20 EBNA2 fragments. In some embodiments, the polypeptide comprises at most 30 EBN3A fragments, preferably at most 25 EBN3 A fragments, more preferably at most 20 EBN3 A fragments. In some embodiments, the polypeptide comprises at most 30 EBNA3B fragments, preferably at most 25 EBNA3B fragments, more preferably at most 20 EBNA3B fragments. In some embodiments, the polypeptide comprises at most 30 BMLF1 fragments, preferably at most 25 BMLF1 fragments, more preferably at most 20 BMLF1 fragments. In some embodiments, the polypeptide comprises at most 30 BMRF1 fragments, preferably at most 25 BMRF1 fragments, more preferably at most 20 BMRF1 fragments. In some embodiments, the polypeptide comprises at most 30 BFRF1 fragments, preferably at most 25 BFRF1 fragments, more preferably at most 20 BFRF1 fragments.

[0242] In some embodiments, the polypeptide comprises at least the fragments of the EBV proteins EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1.

[0243] In some embodiments, at least two of the fragments of EBNA2 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0244] In some embodiments, at least two of the fragments of EBN3A are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0245] In some embodiments, at least two of the fragments of EBNA3B are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBN3A, BMLF1, BMRF1, and BFRF1 or a combination thereof.

[0246] In some embodiments, at least two of the fragments of BMRF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, and BFRF1 or a combination thereof. In some embodiments, at least two of the fragments of BFRF1 are separated by one or more of the fragments of the EBV protein(s) selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, and BMRF1 or a combination thereof.

[0247] In some embodiments, the fragments of EBNA2 are derived from SEQ ID NO: 133 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0248] In some embodiments, the fragments of EBN3A are derived from SEQ ID NO: 137 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0249] In some embodiments, the fragments of EBN3B are derived from SEQ ID NO: 138 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0250] In some embodiments, the fragments of BMLF1 are derived from SEQ ID NO: 136 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0251] In some embodiments, the fragments of BMRF1 are derived from SEQ ID NO: 135 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0252] In some embodiments, the fragments of BFRF1 are derived from SEQ ID NO: 134 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.

[0253] In some embodiments, the fragments of EBNA2 comprise a sequence of SEQ ID NO: 80 to 87 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 80 to 87.

[0254] In some embodiments, the fragments of EBN3A comprise a sequence of SEQ ID NO: 109 to 121 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 109 to 121.

[0255] In some embodiments, the fragments of EBN3B comprise a sequence of SEQ ID NO: 122 to 130 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 122 to 130.

[0256] In some embodiments, the fragments of BMLF1 comprise a sequence of SEQ ID NO: 100 to 108 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 100 to 108.

[0257] In some embodiments, the fragments of BMRF1 comprise a sequence of SEQ ID NO: 94 to 99 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 94 to 99.

[0258] In some embodiments, the fragments of BFRF1 comprise a sequence of SEQ ID NO: 88 to 93or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the fragments consist of a sequence of SEQ ID NO: 88 to 93.

[0259] In some particularly preferred embodiments, the polypeptide (string 2) comprises a combination of two or more of the sequences shown in SEQ ID NO: 80 to 130. In some particularly preferred embodiments, the polypeptide (string 2) comprises a combination of the sequences shown in SEQ ID NO: 80 to 130. In some embodiments, the polypeptide comprises a sequence of SEQ ID NO: 11 to 20 and

[0260] SEQ ID NO: 293 to 302 or comprises a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the polypeptide consists of a sequence of SEQ ID NO: 11 to 20 and SEQ ID NO: 293 to 302.

[0261] Particularly preferred polypeptide sequences are depicted in table 5, wherein underlined amino acids correspond to linker sequences, linking two EBV protein fragments. Vertical bars demarcate individual fragments and individual linkers. The first amino acid “M” refers to the methionine encoded by the start codon. As shown in String2 vlP, String2 v2P, String2 v3P, String2 v4P, String2 v5P, String2 v6P, String2 v7P, String2 v8P, String2 v9P, and String2 vlOP, the polypeptide may comprise a tetanus toxoid CD4+ epitopes P2 and P16 encoding the tetanus toxoid (TT)-derived epitopes TT830-844 and TT578-609 of Clostridium tetani (SEQ ID NO: 23).

[0262] Table 5. Exemplary polypeptides encoded by the polynucleotide of the invention

[0263]

[0264]

[0265]

[0266] In some embodiments, the polynucleotide is a recombinant polynucleotide.

[0267] In preferred embodiments, the polynucleotide is an unmodified RNA or a modified RNA. In some embodiments, the polynucleotide is operatively linked to one or more sequences which direct expression of the polypeptide in a host cell.

[0268] In preferred embodiments of the present disclosure, the RNA relates to an RNA transcript which encodes a peptide or polypeptide.

[0269] In some embodiments, the RNA which preferably encodes a peptide or polypeptide has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, nucleotides), preferably up to 5,000, such as up to 4,000, up to 3,000, up to 2,500 nucleotides, up to 2,200 nucleotides. As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region and a 3' untranslated region (3'-UTR). In some embodiments, the mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template. The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAidTM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARC A mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as Ampli Scribe™). For providing modified mRNA, correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription.

[0270] In some embodiments, RNA is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0271] In some embodiments of the present disclosure, the RNA is "replicon RNA" or simply a "replicon", in particular "self-replicating RNA" or "self-amplifying RNA". In certain embodiments, the replicon or self-replicating RNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’-cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2: 1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).

[0272] Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication (trans-amplification) systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans- replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase. In some embodiments of the present disclosure, the RNA (in particular, mRNA) described herein (e.g., contained in the compositions / formulations of the present disclosure and / or used in the methods of the present disclosure) contains one or more modifications, e.g., in order to increase its stability and / or increase translation efficiency and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in order to increase expression of the RNA (in particular, mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'- and / or 3'-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the GC content of the RNA). A combination of the above described modifications, i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (T) or N(l)-methylpseudouridine (mlT) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency. Thus, in some embodiments, the RNA (in particular, mRNA) described in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (T) or N(l)- methylpseudouridine (mlT) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.

[0273] In some embodiments, the polynucleotide is codon optimized. Codon optimization can improve gene expression by changing synonymous codons based on an organism's codon bias. Exemplary codon optimized sequences have been developed and provided for the polynucleotide in SEQ ID NO: 163 to 222 and 413 to 422.

[0274] In some embodiments, RNA (in particular, mRNA) described in present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5'- end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3 '-end, downstream of the termination codon of a protein-encoding region, but the term "3'- UTR" does generally not include the poly-A sequence. Thus, the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3 '-UTR into the 3 '-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2-globin, alpha 1 -globin, or beta-globin, e.g., beta-globin, e.g., human betaglobin. For example, the RNA (e.g., mRNA) may be modified by the replacement of the existing 3'-UTR with or the insertion of one or more, e.g., two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alphal-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

[0275] In some embodiments, a 5 ’-UTR is or comprises a modified human alpha-globin 5 ’-UTR. A particularly preferred 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 279 or 282 as shown in table 6. In some embodiments, a 3 ’-UTR comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA. A particularly preferred 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 280 or 281 as shown in table 6. In some embodiments, the 3’-UTR consists of a sequence of SEQ ID NO: 280 or 281. In some embodiments, the 5’- UTR consists of a sequence of SEQ ID NO: 279 or 282.

[0276] In some embodiments, RNA comprises a 5’-UTR comprising the nucleotide sequence of SEQ ID NO: 279 or 282, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 279 or 282.

[0277] In some embodiments, RNA comprises a 3’-UTR comprising the nucleotide sequence of SEQ ID NO: 280 or 281 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 280 or 281.

[0278] Table 6. Exemplary untranslated RNA sequences

[0279] The RNA (in particular, mRNA) described herein may have modified ribonucleotides in order to increase its stability and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in some embodiments, uridine in the RNA (in particular, mRNA) described herein is replaced (partially or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0280] In some embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (mly), 5-methyl-uridine (m5U), and combinations thereof.

[0281] In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5- methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5- carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl- uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5- methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5- methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl -uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (xm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1- taurinomethyl-4-thio-pseudouridine), 5-methyl-2 -thio-uridine (m5s2U), l-methyl-4-thio- pseudouridine (mls4\| / ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3\| / ), 2- thio- 1 -methyl -pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl- dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2 -thiopseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1- methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 y), 5- (isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O- methyl-pseudouridine (ym), 2-thio-2'-O-methyl-uridine (s2Um), 5 -methoxy carbonylmethyl- 2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.

[0282] An RNA (preferably mRNA) which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as " -modified", whereas the term "mlT-modified" means that the RNA (preferably mRNA) contains N(l)- methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5- methyluridine (replacing partially or completely, preferably completely, uridine). Such - or mlT- or m5U-modified RNAs usually exhibit decreased immunogenicity compared to their unmodified forms and, thus, are preferred in applications where the induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(l)-methylpseudouridine replacing completely uridine.

[0283] In some embodiments, the polynucleotide comprises a 5 ’-cap, a free 5 ’-triphosphate group, a free 5’-disphosphate group, a free 5 ’-diphosphate group, a free 5’- monophosphate group, or a free 5 ’-OH group, or comprising chemically modified analogues of said 5 ’-cap, said 5’- triphosphate group, said free 5’-disphosphate group or said free 5 ’-monophosphate group. In some embodiments, the polynucleotide comprises a 5’cap selected from G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m32’2’7G[5’]ppp[5’]G, m27’3'-°G[5’]ppp[5’]G (3’-ARCA), m27’2°GpppG (2’ -ARC A), m27’2°GppSpG (p-S- ARC A), and m27 2°GppSpG (p-S-ARCA) and m27 3' °Gppp(mi2'°)ApG. In a preferred embodiment, the 5’ cap is m27 3'°Gppp(mi2'°)ApG.

[0284] In some embodiments, the RNA (in particular, mRNA) described herein comprises a 5'-cap structure. In some embodiments, the RNA does not have uncapped 5'-triphosphates. In some embodiments, the RNA (in particular, mRNA) may comprise a conventional 5'-cap and / or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an RNA molecule and generally comprises a guanosine 5'-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the RNA (z.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the RNA). The guanosine may be methylated at position N7(resulting in the cap structure m7Gppp). The term "5'-cap analog" includes a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3 '-position of the m7guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the P-phosphate (such as m27’2 OG(5')ppSp(5')G (referred to as beta-S-ARCA or P-S-ARCA)), as described in PCT / EP2019 / 056502. Providing an RNA (in particular, mRNA) with a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is co-transcriptionally incorporated into the generated RNA (in particular, mRNA) strand, or the RNA (in particular, mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0285] In some embodiments, the RNA (in particular, mRNA) comprises a capO, capl, or cap2, preferably capl or cap2. According to the present disclosure, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. According to the present disclosure, the term "capl" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. According to the present disclosure, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.

[0286] The 5'-cap analog beta-S-ARCA ( -S-ARCA) has the following structure:

[0287] The "DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)" is the diastereomer of beta- S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S- ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml / min can be applied. In some embodiments, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, within 15 min is used. UV-detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0288] The 5'-cap analog m27’3'oGppp(mi2'o)ApG (also referred to as m27’3'oG(5')ppp(5')m2'oApG) which is a building block of a capl has the following structure:

[0289]

[0290] An exemplary capO mRNA comprising -S-ARCA and mRNA has the following structure:

[0291] An exemplary capO mRNA comprising m27’3 OG(5')ppp(5')G and mRNA has the following structure:

[0292] An exemplary capl mRNA comprising m27’3'oGppp(mi2'°)ApG and mRNA has the following structure:

[0293]

[0294] In some embodiments, the polynucleotide comprises an FI element preferably derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.

[0295] In some embodiments, the polynucleotide comprises a Kozak sequence, preferably an optimized Kozak sequence to increase translational efficiency. Kozak sequences are known to increase the efficiency of translation of some RNA transcripts but are not necessarily required for all RNAs to enable efficient translation.

[0296] As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA (in particular, mRNA) molecule. Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs (in particular, mRNAs) described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs (in particular, mRNAs) disclosed herein can have a poly-A tail attached to the free 3 '-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase. It has been demonstrated that a poly- A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly- A tail (Holtkamp etal., 2006, Blood, vol. 108, pp. 4009-4017).

[0297] The poly-A tail may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of' means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of' means that all nucleotides in the poly-A tail, z.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0298] In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.

[0299] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 Al may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0300] In some embodiments, the poly(A) tail comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence of 10 nucleotides.

[0301] In some embodiments, no nucleotides other than A nucleotides flank a poly-A tail at its 3'- end, z.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.

[0302] In some embodiments, a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides.

[0303] In some embodiments, the polypeptide comprises one or more linkers, wherein each linker is located between, and thereby separates two fragments. The linker preferably is a cleavageenhancing linker, i.e., results in enhanced peptide cleavage and liberation of the antigenic fragments compared to the respective sequence comprising the fragments without the linker. Cleavage enhancement can be determined via cleavage prediction programs or experimental cleavage assays. The cleavage-enhancing linker facilitates efficient cleavage of the polypeptide for liberating the individual EBV protein fragments. In some embodiments, at least two fragments are operatively linked to each other without a linker sequence. In some embodiments, at least two fragments are operatively linked to each other with a linker sequence. In some embodiments, the polypeptide comprises at least two fragments that are operatively linked to each other with a linker sequence and at least two fragments that are operatively linked to each other without a linker sequence. It is understood that the presence of a linker between every fragment is not required. In some embodiments, a linker sequence is inserted between fragments, if the predicted cleavage score would otherwise be low. The Cleavage score is predicted using an algorithm, which predicts the probability for cleavage at a given position on the string based on the amino acids. To link fragments in the polypeptides, up to four cleavage-enhancing amino acids are added between two fragments, as judged by the algorithm. In this strategy, the final length of the strings was below 800 amino acids (Fig. 3-12). Methods to predict the cleavage score are well known in the art.

[0304] In some embodiments, the linker comprises at most 8 amino acids, preferably at most 6 amino acids, more preferably at most 5 amino acids, most preferably 1 to 4 amino acids.

[0305] In some embodiments, the one or more linkers comprise a sequence of the SEQ ID NO: 223 to 278 and 304 to 412. In some embodiments, the one or more linkers consist of a sequence of the SEQ ID NO: 223 to 278 and 304 to 412.

[0306] In some embodiments, the polypeptide comprises a P2P 16 sequence encoding the tetanus toxoid (TT)-derived epitopes TT830-844 and TT578-609 of Clostridium tetani and may comprise additional linker sequences according to SEQ ID NO: 246 or 276 or the amino acid arginine (R) as linker.

[0307] In some embodiments, the one or more linkers comprise or consist of a sequence of SEQ ID NO: 223 to 278 and the group comprising A, AA, AAN, AAR, AAS, AAY, AG, AGN, AKY, AM, AMA, AMR, AMY, ANA, ANR, AR, ARA, ARG, ARY, AS, ASA, AY, G, K, KA, KG, KKA, KKK, KM, KMA, KMG, KMM, KMN, KMR, KMY, KNN, KRA, KRM, KY, M, MA, MAS, MAY, MKY, MM, MY, NAM, NAY, NMM, NMY, NR, NRY, NS, NSY, R, RA, RAA, RAK, RAM, RAN, RAR, RAY, RG, RK, RKA, RKK, RKR, RKY, RM, RMA, RMM, RMR, RMS, RMY, RNA, RNR, RNY, RR, RRA, RRR, RRS, RRY, RSA, RSM, RSN, RSY, RY, RYA, RYR, RYS, RYY, S, SA, SAA, SG, SK, SM, SMA, SMM, SMY, SN, SR, SRN, SRY, SS, Y, YMY, YN, and YSA. In some embodiments, the one or more linkers comprise or consists of a sequence of SEQ ID NO: 223 to 278 and / or the group comprising A, AA, AAAG, AAAR, AAN, AAR, AAS, AASY, AAY, AAYA, AG, AGAG, AGFG, AGN, AGR, AGYN, AK, AKMY, AKSY, AKY, AM, AMA, AMAY, AMR, AMY, AMYM, AN, ANA, ANMA, ANR, AR, ARA, ARAA, ARAM, ARAR, ARAY, ARG, ARGR, ARGY, ARMA, ARMN, ARMY, ARNA, ARNY, ARY, ARY A, ARYY, AS, ASA, AY, AYMY, G, GAYM, GMMG, K, KA, KAA, KAMN, KAR, KAY, KG, KK, KKA, KKK, KKMA, KKY, KM, KMA, KMG, KMM, KMMY, KMN, KMR, KMY, KNN, KR, KRA, KRAA, KRAN, KRAR, KRAS, KRAY, KRKA, KRKY, KRM, KRMA, KRMG, KRMM, KRMR, KRMY, KRN, KRNY, KRRA, KRSA, KRSY, KRY, KRY A, KRYY, KY, M, MA, MAN, MAS, MAY, MG, MKY, MM, MRMA, MRYG, MS, MSA, MY, MYNY, N, NAM, NAY, NGM, NGY, NMA, NMM, NMY, NR, NRMM, NRY, NS, NSY, NY, NYMY, R, RA, RAA, RAK, RAM, RAMA, RAN, RAR, RAS, RASA, RAY, RAYA, RG, RGY, RK, RKA, RKK, RKMY, RKR, RKY, RKYN, RK YR, RLAG, RM, RMA, RMM, RMR, RMS, RMY, RNA, RNR, RNS, RNY, RR, RRA, RRAM, RRAR, RRAY, RRFG, RRGA, RRM, RRMA, RRMY, RRN, RRNA, RRR, RRRS, RRS, RRSA, RRSY, RRY, RRYA, RRYG, RSA, RSG, RSM, RSN, RSY, RSYS, RY, RYA, RYAY, RYM, RYMY, RYR, RYS, RYY, S, SA, SAA, SAM, SAY, SG, SGA, SK, SM, SMA, SMM, SMNA, SMY, SN, SR, SRAA, SRAY, SRK, SRMA, SRMY, SRN, SRNY, SRY, SRYA, SS, SY, Y, YA, YM, YMY, YN, YRMY, YSA.

[0308] In some embodiments, the fragments are immunogenic fragments.

[0309] In some embodiments, the subject is a human. In some embodiments, the subject has multiple sclerosis. In some embodiments, the subject has a latent EBV infection. In some embodiments, the subject has multiple sclerosis and a latent EBV infection.

[0310] In some embodiments, the polynucleotide encodes a polypeptide for eliciting an EBV-specific CD8+ T cell immune response in a subject, preferably wherein the CD8+-specific immune response is measured by an IFNg ELISpot assay. In some embodiments, an EBV-specific CD8+T cell immune response is measured by flow cytometry stainings, ELISPOT and / or CD8+Tetramer-Stainings. These methods are well known in the art. A preferred method to evaluate a specific EBV response is to immunize mice with the polynucleotide of the invention, the polypeptide of the invention or the vector of the invention. Afterwards, the immune cells of the immunized mice are analyzed, wherein the cells are isolated and restimulate with specific peptides. The successfully induced EBV-specific T cells respond with cytokine (IFNy) production. In some embodiments, a tetramer staining known in the art can be performed done to demonstrate epitope-specific T cells by way of use of an MHC-I tetramer staining, a CD8+T cell specifically responding to specific EB V proteins can be demonstrated.

[0311] The codons of the RNA (in particular, mRNA) described in the present disclosure may further be optimized, e.g., to increase the GC content of the RNA and / or to replace codons which are rare in the cell (or subject) in which the peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (in particular, mRNA) described in the present disclosure is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and / or increased in the G / C content compared to the corresponding sequence regions of the wild type coding sequence. In some embodiments, the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0312] The term "codon-optimized" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions may be codon-optimized for optimal expression in a subject to be treated using the RNA (in particular, mRNA) described herein. Codonoptimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (in particular, mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons". In some embodiments, the guanosine / cytosine (G / C) content of the coding region of the RNA (in particular, mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences having an increased G (guanosine) / C (cytosine) content are more stable than sequences having an increased A (adenosine) / U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and / or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleotides.

[0313] In various embodiments, the G / C content of the coding region of the RNA (in particular, mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild type RNA.

[0314] The term "non-immunogenic RNA" (such as "non-immunogenic mRNA") as used herein refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., than would have been induced by standard RNA (stdRNA). In certain embodiments, non-immunogenic RNA is rendered non- immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and / or limiting the amount of double-stranded RNA (dsRNA), e.g., by limiting the formation of double- stranded RNA (dsRNA), e.g., during in vitro transcription, and / or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription. In certain embodiments, non-immunogenic RNA is rendered non- immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and / or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription.

[0315] For rendering the non-immunogenic RNA (especially mRNA) non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m3U), 5-methoxy- uridine (mo5U), 5 -aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5- oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5 -carb oxy methyl - uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2 -thiouridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5- methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1- propynyl-pseudouridine, 5-taurinomethyl-uridine (xm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(Tm5s2U), 1 -taurinomethyl-4-thio-pseudouridine), 5-methyl-2- thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (rnVy), 4-thio-l-methyl-pseudouridine,

[0316] 3-methyl-pseudouridine (m3y), 2-thio-l-methyl-pseudouridine, 1 -methyl- 1 -deazapseudouridine, 2-thio-l -methyl- 1-deaza-pseudouri dine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine,

[0317] 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3\| / ), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ym), 2-thio-2'-O- methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5- carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O- methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)- 2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine. In certain embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (y), N1 -methyl -pseudouridine (mly) or 5-methyl-uridine (m5U), in particular N1 -methylpseudouridine.

[0318] In some embodiments, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.

[0319] During synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase significant amounts of aberrant products, including double-stranded RNA (dsRNA) are produced due to unconventional activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. Formation of dsRNA can be limited during synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP may be added once or several times during synthesis of mRNA. Also, dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non- porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using E. coli RNaselll that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017 / 182524.

[0320] As the term is used herein, "remove" or "removal" refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the non-separated mixture of first and second substances.

[0321] In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, e.g., dsRNA (especially dsmRNA) is removed from non-immunogenic RNA , such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises single-stranded nucleoside modified RNA (especially mRNA) and is substantially free of double stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises 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%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, , at least 99.993%,, at least 99.994%, , at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.). Various methods can be used to determine the amount of dsRNA. For example, a sample may be contacted with dsRNA-specific antibody and the amount of antibody binding to RNA may be taken as a measure for the amount of dsRNA in the sample. A sample containing a known amount of dsRNA may be used as a reference.

[0322] For example, RNA may be spotted onto a membrane, e.g., nylon blotting membrane. The membrane may be blocked, e.g., in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v / v) TWEEN-20) containing 5% (w / v) skim milk powder. For detection of dsRNA, the membrane may be incubated with dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti -mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.

[0323] In some embodiments, the non-immunogenic RNA (especially mRNA) is translated in a cell more efficiently than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3-fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5-fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10-fold factor. In some embodiments, translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200- 1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0324] In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5- fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by an 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor.

[0325] The term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the decrease is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0326] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.

[0327] RNA described herein may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNA, or delivery mediated by delivery vehicles.

[0328] Some aspects of the disclosure involve the targeted delivery of the RNA disclosed herein to certain cells or tissues. In some embodiments, after administration of the RNA (in particular, mRNA) compositions / formulations described herein, at least a portion of the RNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA (in particular, mRNA) is translated by the target cell to produce the encoded peptide or polypeptide. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a cell in the lung. In some embodiments, the disclosure involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. In some embodiments, the target cell is a cell in the lymph nodes. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. Thus, RNA (in particular, mRNA) compositions / formulations described herein may be used for delivering RNA to such target cell. The "lymphatic system" is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating lymph. The primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and the bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, which include lymph nodes and the spleen, maintain mature naive lymphocytes and initiate an adaptive immune response.

[0329] Lipid-based RNA delivery systems have an inherent preference to the liver, where, depending on the composition of the RNA delivery systems used, RNA expression in the liver can be obtained. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or the lipid metabolism (liposomes and lipid or cholesterol conjugates). In some embodiments, the target organ for RNA expression is liver and the target tissue is liver tissue. The delivery to such target tissue is preferred, in particular, if presence of RNA or of the encoded peptide or polypeptide in this organ or tissue is desired and / or if it is desired to express large amounts of the encoded peptide or polypeptide and / or if systemic presence of the encoded peptide or polypeptide, in particular in significant amounts, is desired or required.

[0330] To overcome the barriers to safe and effective RNA delivery, RNA may be administered with one or more delivery vehicles that protect the RNA from degradation, maximize delivery to on-target cells and minimize exposure to off-target cells. Such RNA delivery vehicles may complex or encapsulate RNA and include a range of materials, including polymers and lipids. In some embodiments, such RNA delivery vehicles may form particles with RNA.

[0331] RNA, in particular mRNA, described herein may be present in particles comprising (i) the RNA, and (ii) at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the RNA. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged RNA are involved in particle formation. This results in complexation and spontaneous formation of RNA particles.

[0332] Different types of RNA containing particles have been described previously to be suitable for delivery of RNA in particulate form (cf., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA physically protects RNA from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape. In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0333] An "RNA particle" can be used to deliver RNA to a target site of interest (e.g., cell, tissue, organ, and the like). An RNA particle may be formed from lipids comprising at least one cationic or cationically ionizable lipid. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid combines together with the RNA to form aggregates, and this aggregation results in colloidally stable particles.

[0334] RNA particles described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)- based formulations.

[0335] A lipoplex (LPX) described herein is obtainable from mixing two aqueous phases, namely a phase comprising RNA and a phase comprising a dispersion of lipids. In some embodiments, the lipid phase comprises liposomes.

[0336] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment. In some embodiments, liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for the delivery of RNA are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.

[0337] In some embodiments, lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with RNAs. In some embodiments, formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact RNA-lipoplexes.

[0338] In some embodiments, an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and RNA (especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged RNA (especially mRNA) results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and / or DODMA, and optionally additional lipids, such as DOPE or DSPC. In some embodiments, an RNA (especially mRNA) lipoplex particle is a nanoparticle.

[0339] In general, a lipid nanoparticle (LNP) is obtainable from direct mixing of RNA in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

[0340] In some embodiments, LNPs comprise or consist of a cationic / cationically ionizable lipid and helper lipids such as phospholipids, cholesterol, and / or polymer-conjugated lipids (e.g., polyethylene glycol (PEG) lipids). In some embodiments, in the RNA LNPs described herein the RNA (in particular, mRNA) is bound by cationically ionizable lipid that occupies the central core of the LNP. In some embodiments, polymer-conjugated lipid forms the surface of the LNP, along with phospholipids. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and cationically ionizable lipid in charged and uncharged forms can be distributed throughout the LNP.

[0341] In some embodiments, RNA (e.g., mRNA) described herein may be noncovalently associated with a particle as described herein. In embodiments, the RNA (especially mRNA) may be adhered to the outer surface of the particle (surface RNA (especially surface mRNA)) and / or may be contained in the particle (encapsulated RNA (especially encapsulated mRNA)).

[0342] In some embodiments, the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most about 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), such as in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to about 800 nm, about 80 to about 700 nm, about 90 to about 600 nm, or about 50 to about 500 nm or about 100 to about 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, 70 to 150 nm, or 80 to 150 nm. In some embodiments, the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm. In some embodiments, the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm to about 400 nm, from about 100 nm to about 350 nm, from about 100 nm to about 300 nm, from about 100 nm to about 250 nm, from about 100 nm to about 200 nm, from about 150 nm to about 1000 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 150 nm to about 500 nm, from about 150 nm to about 450 nm, from about 150 nm to about 400 nm, from about 150 nm to about 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, from about 200 nm to about 250 nm, or from about 80 to about 150 nm. In some embodiments, the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.

[0343] In some embodiments, the particles described herein are nanoparticles. The term "nanoparticle" relates to a nano-sized particle comprising nucleic acid (especially mRNA) as described herein and at least one cationic or cationically ionizable lipid, wherein all three external dimensions of the particle are in the nanoscale, z.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter.

[0344] RNA particles (especially mRNA particles) described herein may exhibit a poly dispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the RNA particles can exhibit a poly dispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0345] The N / P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N / P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N / P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, RNA is considered to be completely bound to nanoparticles.

[0346] RNA particles (especially mRNA particles) described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid and mixing the colloid with RNA to obtain RNA particles. The term "colloid" as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term "colloid" only refers to the particles in the mixture and not the entire suspension.

[0347] For the preparation of colloids comprising at least one cationic or cationically ionizable lipid methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted. The most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media).

[0348] In the film hydration method, lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included. Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.

[0349] The term "ethanol injection technique" refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example lipid vesicle formation such as liposome formation. Generally, the RNA (especially mRNA) lipoplex particles described herein are obtainable by adding RNA (especially mRNA) to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic or cationically ionizable lipids (like DOTMA and / or DODMA) and additional lipids, is injected into an aqueous solution under stirring. In some embodiments, the RNA (especially mRNA) lipoplex particles described herein are obtainable without a step of extrusion.

[0350] The term "extruding" or "extrusion" refers to the creation of particles having a fixed, cross- sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores.

[0351] Other methods having organic solvent free characteristics may also be used according to the present disclosure for preparing a colloid.

[0352] In some embodiments, LNPs comprise four components: cationically ionizable lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid. In some embodiments, LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with RNA in an aqueous buffer. While RNA particles described herein may comprise polymer- conjugated lipids such as PEG lipids, provided herein are also RNA particles which do not comprise PEG lipids, or do not comprise any polymer-conjugated lipids. In some embodiments, the LNPs comprising RNA and at least one cationic or cationically ionizable lipid described herein are prepared by (a) preparing an RNA solution containing water and a buffering system; (b) preparing an ethanolic solution comprising the cationic or cationically ionizable lipid and, if present, one or more additional lipids; and (c) mixing the RNA solution prepared under (a) with the ethanolic solution prepared under (b), thereby preparing the formulation comprising LNPs. After step (c) one or more steps selected from diluting and filtrating, such as tangential flow filtrating, can follow.

[0353] In some embodiments, the LNPs comprising RNA and at least one cationic or cationically ionizable lipid described herein are prepared by (a’) preparing liposomes or a colloidal preparation of the cationic or cationically ionizable lipid and, if present, one or more additional lipids in an aqueous phase; and (b’) preparing an RNA solution containing water and a buffering system; and (c’) mixing the liposomes or colloidal preparation prepared under (a’) with the RNA solution prepared under (b’). After step (c’) one or more steps selected from diluting and filtrating, such as tangential flow filtrating, can follow.

[0354] The present disclosure describes compositions comprising RNA (especially mRNA) and at least one cationic or cationically ionizable lipid which associates with the RNA to form RNA particles and formulations comprising such particles. The RNA particles may comprise RNA which is complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, in particular infectious viral particles, z.e., they are not able to virally infect cells.

[0355] Suitable cationic or cationically ionizable lipids are those that form RNA particles and are included by the term “particle forming components” or “particle forming agents”. The term “particle forming components” or “particle forming agents” relates to any components which associate with RNA to form RNA particles. Such components include any component which can be part of RNA particles.

[0356] In some embodiments, RNA particles (especially mRNA particles) comprise more than one type of RNA molecules, where the molecular parameters of the RNA molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.

[0357] In particulate formulation, it is possible that each RNA species is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one RNA species. The individual particulate formulations may be present as separate entities, e.g. in separate containers. Such formulations are obtainable by providing each RNA species separately (typically each in the form of an RNA-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific RNA species that is being provided when the particles are formed (individual particulate formulations). In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual particle formulation. Respective pharmaceutical compositions are referred to as mixed particulate formulations. Mixed particulate formulations according to the present disclosure are obtainable by forming, separately, individual particulate formulations, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of RNA-containing particles is obtainable. Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations. Alternatively, it is possible that all RNA species of the pharmaceutical composition are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of all RNA species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined particulate formulation will typically comprise particles which comprise more than one RNA species. In a combined particulate composition different RNA species are typically present together in a single particle.

[0358] Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically condense the negatively charged RNA into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. In addition, some investigators have synthesized polymers specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0359] A "polymer," as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties.

[0360] If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0361] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body.

[0362] In certain embodiments, polymer may be protamine or polyalkyleneimine.

[0363] The term "protamine" refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

[0364] According to the disclosure, the term "protamine" as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

[0365] In one embodiment, the polyalkyleneimine comprises polyethylenimine and / or polypropylenimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75- 102to 107Da, preferably 1000 to 105Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da. Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).

[0366] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In one embodiment, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated. Particles described herein may also comprise polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as noncationic polymers.

[0367] The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moi eties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and / or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

[0368] As used herein, the term "hydrophobic" refers to any a molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least 6 carbon atoms. The monovalent radical of a hydrocarbon is referred to as hydrocarbyl herein. The hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.

[0369] The term “hydrocarbon” includes non-cyclic, e.g., linear (straight) or branched, hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl as defined herein. It should be appreciated that one or more of the hydrogen atoms in alkyl, alkenyl, or alkynyl may be substituted with other atoms, e.g., halogen, oxygen or sulfur. Unless stated otherwise, hydrocarbon groups can also include a cyclic (alkyl, alkenyl or alkynyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0370] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon moiety which may have one to thirty, typically one to twenty, often six to eighteen carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.

[0371] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be six to thirty, typically six to twenty often six to eighteen. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.

[0372] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be six to thirty, typically six to twenty, often six to eighteen. Alkynyl groups can optionally have one or more carbon-carbon double bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds.

[0373] The term "alkylene" refers to a saturated linear or branched divalent hydrocarbon moiety which may have one to thirty, typically two to twenty, often four to twelve carbon atoms. Exemplary nonpolar alkylene groups include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecamethylene, tetradecamethylene, hexadecamethylene, octadecmethylene, and the like.

[0374] The term "alkenylene" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be two to thirty, typically two to twenty, often four to twelve. Generally, the maximal number of carboncarbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.

[0375] The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl. The cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).

[0376] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes.

[0377] The term "aromatic" as used in the context of hydrocarbons means that the whole molecule has to be aromatic. For example, if a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure. Likewise, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated the resulting hydrogenated bi- or polycyclic structure (such as 1,2- dihydronaphthyl) is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic).

[0378] As used herein, the term "amphiphilic" refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non- polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0379] The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" relates to substances, in particular amphiphilic substances, that structurally and / or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term includes molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. Examples of lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSL), synthetic function-spacer-sterol constructs (FSS) as well as artificial amphipathic molecules. Lipids comprising two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble. Traditional surfactant monomers comprising only one linear alkyl chain and a hydrophilic head group are generally cone shaped. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or elliptoid micelles that are water soluble. While lipids also have the same general structure as surfactants - a polar hydrophilic head group and a nonpolar hydrophobic tail - lipids differ from surfactants in the shape of the monomers, in the type of aggregates formed in solution, and in the concentration range required for aggregation. As used herein, the term "lipid" is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context. Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as steroids, z.e., sterol-containing metabolites such as cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0380] Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more cis double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.

[0381] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.

[0382] The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

[0383] Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or monounsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0384] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.

[0385] Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gramnegative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2 -Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0386] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0387] According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

[0388] In some embodiments, the RNA compositions and formulations and RNA particles described herein comprise at least one cationic or cationically ionizable lipid as particle forming agent. Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipids (including lipid-like materials) which are able to electrostatically bind nucleic acid. In some embodiments, cationic or cationically ionizable lipids contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0389] As used herein, a "cationic lipid" refers to a lipid or lipid-like material having a net positive charge. Cationic lipids bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

[0390] In some embodiments, a cationic lipid has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

[0391] As used herein, a "cationically ionizable lipid" refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. For purposes of the present disclosure, cationically ionizable lipids are covered by the term "cationic lipid" unless contradicted by the circumstances.

[0392] In some embodiments, the cationic or cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated, e.g., under physiological conditions.

[0393] Examples of cationic or cationically ionizable lipids include, but are not limited to N,N- dimethyl-2, 3 -di oleyloxypropylamine (DODMA), l,2-dioleoyl-3 -trimethylammonium propane (DOTAP); l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), 3-(N — (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammonium propanes; l,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DORIE), and 2,3 -di oleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N- dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en- 3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'- (cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'- octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-di oleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3 -dimethylaminopropane (DOcarbDAP), 2,3- Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3- dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3 -dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,3 l-tetraen-19- yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2 -Hydroxy ethyl)-N,N-dimethyl-2, 3- bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N- (3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l-propanaminium bromide (GAP- DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- propanaminium bromide (0AE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-l-aminium (DOBAQ), 2-({8-[(30)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA),

[0394] 1.2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3- dimethylammonium-propane (DPDAP), N 1 -[2-(( 1 S)- 1 -[(3 -aminopropyl)amino]-4-[di(3 - amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2- hydroxyethyl)-N,N-dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-

[0395] 2.3-bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan- 1 -amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl- ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2- dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12-5), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C 12-200).

[0396] In some embodiments, the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.

[0397] DOTMA is a cationic lipid with a quaternary amine headgroup. The structure of DOTMA may be represented as follows:

[0398] DODMA is an ionizable cationic lipid with a tertiary amine headgroup. The structure of DODMA may be represented as follows:

[0399] In some embodiments, the cationic or cationically ionizable lipid may comprise from about 10 mol % to about 95 mol %, from about 20 mol % to about 95 mol %, from about 20 mol % to about 90 mol %, from about 30 mol % to about 90 mol %, from about 40 mol % to about 90 mol %, or from about 40 mol % to about 80 mol % of the total lipid present in the particle.

[0400] The RNA compositions and formulations and RNA particles described herein may also comprise lipids (including lipid-like materials) other than cationic or cationically ionizable lipids (also collectively referred to herein as cationic lipids), z.e., non-cationic lipids (including non-cationic or non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as noncationic lipids. Optimizing the formulation of RNA particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to a cationic or cationically ionizable lipid may enhance particle stability and efficacy of RNA delivery.

[0401] One or more additional lipids may or may not affect the overall charge of the RNA particles. In some embodiments, the one or more additional lipids are a non-cationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and / or neutral lipids. As used herein, an "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, a "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.

[0402] In some embodiments, the RNA compositions and formulations and RNA particles described herein comprise a cationic or cationically ionizable lipid and one or more additional lipids.

[0403] Without wishing to be bound by theory, the amount of the cationic or cationically ionizable lipid compared to the amount of the one or more additional lipids may affect important RNA particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the RNA. Accordingly, in some embodiments, the molar ratio of the cationic or cationically ionizable lipid to the one or more additional lipids is from about 10:0 to about 1 :9, about 4: 1 to about 1 :2, about 4: 1 to about 1:1, about 3 : 1 to about 1 : 1, or about 3 : 1 to about 2:1.

[0404] In some embodiments, the one or more additional lipids comprised in the RNA compositions and formulations and RNA particles described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof.

[0405] In some embodiments, the one or more additional lipids comprise a neutral lipid which is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3 -phosphocholine (Cl 6 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidyl ethanolamine (DPyPE), l,2-di-(9Z-octadecenoyl)-sn- glycero-3 -phosphocholine (DOPG), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DPPG), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), N-palmitoyl-D- erythro-sphingosylphosphorylcholine (SM), and further phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

[0406] In some embodiments, the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0407] Thus, in some embodiments, the RNA compositions and formulations and RNA particles described herein comprise (1) a cationic or cationically ionizable lipid, and a phospholipid such as DSPC or DOPE or (2) a cationic or cationically ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.

[0408] In some embodiments, the RNA particles (especially the particles comprising mRNA) described herein comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE or (4) DODMA, DOPE and cholesterol.

[0409] DSPC is a neutral phospholipid. The structure of DSPC may be represented as follows:

[0410] DOPE is a neutral phospholipid. The structure of DOPE may be represented as follows:

[0411] The structure of cholesterol may be represented as follows:

[0412] In some embodiments, RNA compositions and formulations and RNA particles described herein do not include a polymer conjugated lipid such as a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

[0413] In some embodiments, the additional lipid (e.g., one or more phospholipids and / or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 2 mol % to about 80 mol %, from about 5 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 7.5 mol % to about 50 mol %, or from about 10 mol % to about 40 mol % of the total lipid present in the particle. In some embodiments, the additional lipid (e.g., one or more phospholipids and / or cholesterol) comprises about 10 mol %, about 15 mol %, or about 20 mol % of the total lipid present in the particle.

[0414] In some embodiments, the additional lipid comprises a mixture of: (i) a phospholipid such as DOPE; and (ii) cholesterol or a derivative thereof. In some embodiments, the molar ratio of the phospholipid such as DOPE to the cholesterol or a derivative thereof is from about 9:0 to about 1 : 10, about 2: 1 to about 1 :4, about 1 : 1 to about 1 :4, or about 1 : 1 to about 1 :3.

[0415] In some embodiments, RNA compositions and formulations and RNA particles described herein may comprise at least one polymer-conjugated lipid. A polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto. In some embodiments, a polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as pegylated lipid or PEG-lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art. In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a poly sarcosine portion.

[0416] In some embodiments, a polymer-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a polymer-conjugated lipid can reduce its association with serum proteins and / or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0417] In some embodiments, RNA compositions / formulations and RNA particles described herein comprise a PEG-conjugated lipid.

[0418] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a lipid having the structure of the following general formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: each of R12and R13is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl / alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

[0419] In some embodiments of this formula, each of R12and R13is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

[0420] In some embodiments of this formula, R12and R13are identical. In some embodiments, each of R12and R13is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R12and R13is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R12and R13is a straight alkyl chain containing 16 carbon atoms. In some embodiments of this formula, R12and R13are different. In some embodiments, one of R12and R13is a straight alkyl chain containing 12 carbon atoms and the other of R12and R13is a straight alkyl chain containing 14 carbon atoms.

[0421] In some embodiments of this formula, w has a mean value ranging from 40 to 50, such as a mean value of 45.

[0422] In some embodiments of this formula, w is within a range such that the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g / mol, such as from about 1000 to about 5000 g / mol, from about 1500 to about 4000 g / mol, or from about 2000 to about 3000 g / mol. In some embodiments, each of R12and R13is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.

[0423] Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monom ethoxy -poly ethyleneglycol)-2, 3- dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 '-di(tetradecanoyloxy)propyl-l-O- (w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as w-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(w methoxy(polyethoxy)ethyl)carbamate, and the like.

[0424] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is DMG-PEG 2000, e.g., having the following structure:

[0425] In some embodiments, the PEG-conjugated lipid (pegylated lipid) has the following structure: wherein n has a mean value ranging from 30 to 60, such as about 50. In one embodiment, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N- [(ro-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG- (2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000).

[0426] In some embodiments, RNA compositions / formulations described herein may comprise one or more PEG-conjugated lipids or pegylated lipids as described in WO 2017 / 075531 and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0427] In some embodiments, the pegylated lipid comprises from about 1 mol % to about 10 mol %, preferably from about 1 mol % to about 5 mol %, more preferably from about 1 mol % to about 2.5 mol % of the total lipid present in the RNA compositions / formulations and RNA particles described herein.

[0428] The vector of the invention

[0429] The present invention provides a vector comprising a polynucleotide of the invention. In some embodiments, the vector comprises at least two polynucleotides of the invention.

[0430] The polypeptide of the invention

[0431] The present invention provides a polypeptide encoded by the polynucleotides of the invention.

[0432] In some embodiments, the polypeptide comprises a sequence of SEQ ID NO: 1 to 20 and SEQ ID NO: 283 to 302 or a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO. In some embodiments, the polypeptide consists of a sequence of SEQ ID NO: 1 to 20 and SEQ ID NO: 283 to 302. The vaccine of the invention

[0433] The present invention further provides a vaccine composition comprising the polynucleotide of the invention, the vector of the invention, or the polypeptide of the invention, in a pharmaceutically acceptable excipient.

[0434] In some embodiments, the composition comprises a least two polynucleotides of the invention. In some embodiments, the composition comprises a first polynucleotide of the invention and a second polynucleotide of the invention.

[0435] In some preferred embodiments, the vaccine composition does not comprise a polynucleotide encoding for a polypeptide comprising EBV-specific CD4+epitopes. In some preferred embodiments, the vaccine composition does not comprise a polypeptide comprising EBV- specific CD4+epitopes. In some preferred embodiments, the vaccine composition does not comprise a polynucleotide encoding for a full-length EBV protein, such as gp350, gB, gH, gL, gHgL complex, gp42, EBNA1, LMP1, LMP2, BZLF1, BRLF1, EBNA2, EBN3A, EBNA3B, BMLF1, or BMRFl.

[0436] In some embodiments, the composition comprises at least a first and a second polynucleotide of the invention, wherein the first polynucleotide encodes a polypeptide comprising a plurality of fragments of one or more EBV protein(s) selected from a group consisting of EBNA1, LMP1, LMP2, BZLF1, and BRLF1 or a combination thereof, and wherein the second polynucleotide encodes a polypeptide comprising a plurality of fragments of one or more EBV protein(s) selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof . In some embodiments, the first polynucleotide encodes a polypeptide comprising a sequence of SEQ ID NO: 1 to 10 and 283 to 292, and the second polynucleotide encodes a polypeptide comprising a sequence of SEQ ID NO: 11 to 20 and SEQ ID NO: 293 to 302. In some embodiments, the first polynucleotide encodes a polypeptide consisting of a sequence of SEQ ID NO: 1 to 10 and 283 to 292, and the second polynucleotide encodes a polypeptide consisting of a sequence of SEQ ID NO: 11 to 20 and SEQ ID NO: 293 to 302. In some embodiments, the composition comprises a least a first and a second polypeptides of the invention. In some embodiments, the composition comprises at least a first and a second polypeptide of the invention, wherein the polypeptide comprising a plurality of fragments of one or more EBV protein(s) selected from a group consisting of EBNA1, LMP1, LMP2, BZLF1, and BRLF1 or a combination thereof, and wherein the second polypeptide comprising a plurality of fragments of one or more EBV protein(s) selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof . In some embodiments, the first polypeptide comprises a sequence of SEQ ID NO: 1 to 10 and 283 to 292, and the second polypeptide comprises a sequence of SEQ ID NO: 11 to 20 and SEQ ID NO: 293 to 302. In some embodiments, the first polypeptide consists of a sequence of SEQ ID NO: 1 to 10 and 283 to 292, and the second polypeptide consists of a sequence of SEQ ID NO: 11 to 20 and SEQ ID NO: 293 to 302.

[0437] In some embodiments, the composition comprises an adjuvant.

[0438] In some embodiments, the vaccine is a therapeutic EBV-specific vaccine composition.

[0439] In some embodiments, the polynucleotide is an RNA present in a lipid nanoparticle (LNP) composition. In some embodiments, the LNP composition is dispersed in an aqueous phase, wherein the LNPs comprise a cationically ionizable lipid and RNA; the aqueous phase comprises a buffer system comprising a buffer substance selected from the group consisting of Tris and its protonated form, bi s(2-hydroxyethyl)amino-tris(hydroxymethyl)m ethane (Bis- Tris-methane) and its protonated form, and triethanolamine (TEA) and its protonated form, and the monovalent anion being selected from the group consisting of chloride, acetate, glycolate, lactate, the anion of morpholinoethanesulfonic acid (MES), the anion of 3-(N- morpholino)propanesulfonic acid (MOPS), and the anion of 2-[4-(2 -hydroxy ethyl)piperazin-l- yl]ethanesulfonic acid (HEPES); the concentration of the buffer substance in the composition is at most about 25 mM; and the aqueous phase is substantially free of inorganic phosphate anions, substantially free of citrate anions and substantially free of anions of ethylenediaminetetraacetic acid (EDTA). Reference is made to WO 2021 / 030701 and WO 2022 / 218503 describing suitable LNP compositions and being incorporated by reference in their entirety. The LNP may comprise any lipid capable of forming a particle to which the one or more RNA molecules are attached, or in which the one or more RNA molecules are encapsulated.

[0440] LNPs typically comprise four components: cationically ionizable lipid, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid such as PEG- lipid. LNPs may be prepared by mixing lipids dissolved in ethanol with RNA in an aqueous buffer.

[0441] In some embodiments, in the RNA LNPs described herein the RNA is bound by cationically ionizable lipid that occupies the central core of the LNP. Polymer-conjugated lipid forms the surface of the LNP, along with phospholipids. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and cationically ionizable lipid in charged and uncharged forms can be distributed throughout the LNP.

[0442] In some embodiments, the LNP comprises one or more cationically ionizable lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and polymer-conjugated lipids.

[0443] In some embodiments, the LNP comprises a cationically ionizable lipid, a neutral lipid, a steroid, a polymer-conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

[0444] In some embodiments, the LNP comprises from 40 to 60 mol percent, 40 to 55 mol percent, from 45 to 55 mol percent, or from 45 to 50 mol percent of the cationically ionizable lipid.

[0445] In some embodiments, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent.

[0446] In some embodiments, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 30 to 45 mol percent, from 35 to 45 mol percent or from 35 to 43 mol percent. In some embodiments, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer-conjugated lipid.

[0447] In some embodiments, the LNP comprises from 45 to 55 mol percent of a cationically ionizable lipid; from 5 to 15 mol percent of a neutral lipid; from 30 to 45 mol percent of a steroid; from 1 to 5 mol percent of a polymer-conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

[0448] In some embodiments, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of cationically ionizable lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle.

[0449] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

[0450] In some embodiments, the steroid is cholesterol.

[0451] In some embodiments, the polymer conjugated lipid is a pegylated lipid, e.g., a pegylated lipid as described above.

[0452] In some embodiments, the cationically ionizable lipid component of the LNPs has the structure of Formula (III):

[0453] L1ill L2R1^G1"' """G2^R2(III) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, - NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, - NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0454] G3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0455] Rais H or C1-C12 alkyl;

[0456] R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0457] R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4;

[0458] R4is C1-C12 alkyl;

[0459] R5is H or Ci-Ce alkyl; and x is 0, 1 or 2.

[0460] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures UA) (IIIB) wherein:

[0461] A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

[0462] R6is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0463] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0464] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IUD): (IIIC) (IIID) wherein y and z are each independently integers ranging from 1 to 12.

[0465] In any of the foregoing embodiments of Formula (III), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some different embodiments of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0466] In some different embodiments of Formula (III), the lipid has one of the following structures

[0467] (IIIE) or (IIIF):

[0468] (HIE) (IIIF)

[0469] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0470] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0471] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other embodiments, R6is OH.

[0472] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24 alkenylene.

[0473] In some other foregoing embodiments of Formula (III), R1or R2, or both, is C6-C24 alkenyl.

[0474] For example, in some embodiments, R1and R2each, independently have the following structure: wherein:

[0475] R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0476] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7ais H. For example, in some embodiments, R7ais H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7bis Ci-Cs alkyl. For example, in some embodiments, Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl.

[0477] In different embodiments of Formula (III), R1or R2, or both, has one of the following structures: In some of the foregoing embodiments of Formula (III), R3is OH,

[0478] CN, -C(=O)OR4, -OC(=O)R4or -NHC(=O)R4. In some embodiments, R4is methyl or ethyl.

[0479] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in the table below.

[0480] Table 7. Representative cationically ionizable lipids

[0481]

[0482] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, a neutral lipid, a steroid, and a polymer conjugated lipid.

[0483] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, a neutral lipid, a steroid, and a polymer conjugated lipid.

[0484] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, a neutral lipid, a steroid, and a polymer conjugated lipid.

[0485] In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a polymer conjugated lipid.

[0486] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, a neutral lipid, a steroid, and a pegylated lipid.

[0487] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, a neutral lipid, a steroid, and a pegylated lipid.

[0488] In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a pegylated lipid.

[0489] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and a pegylated lipid.

[0490] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and a pegylated lipid.

[0491] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above table, DSPC, cholesterol, and a pegylated lipid.

[0492] In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and a pegylated lipid.

[0493] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and DMG-PEG 2000.

[0494] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above table, DSPC, cholesterol, and DMG-PEG 2000.

[0495] In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and DMG-PEG 2000.

[0496] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and PEG2000-C-DMA.

[0497] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and PEG2000-C- DMA.

[0498] In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above table, DSPC, cholesterol, and PEG2000-C-DMA.

[0499] In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and PEG2000-C-DMA.

[0500] 3D-P-DMA: (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-l 1-yl 5-

[0501] (dimethylamino)pentanoate DMG-PEG 2000:

[0502] PEG2000-C-DMA: 3-N-[(®-Methoxy poly(ethylene glycol)2000) carbamoyl]- 1,2- dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA or Methoxy-polyethylene glycol-2,3- bis(tetradecyloxy)propylcarbamate (2000)) wherein n has a mean value ranging from 30 to 60, such as about 50.

[0503] D SPC : 1 ,2-Di stearoyl -s / z-gl y cero-3 -phosphocholine

[0504] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from

[0505] 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments the polynucleotide is an RNA present in an RNA lipoplex particle. Reference is made to WO 2022 / 069632 describing suitable methods for preparing and storing RNA lipoplex particles and being incorporated by reference in its entirety.

[0506] Lipoplexes (LPX) are electrostatic complexes which are generally formed by mixing preformed cationic lipid liposomes with anionic RNA. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact RNA-lipoplexes.

[0507] In certain embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0508] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1 :9, about 4: 1 to about 1 :2, or about 3 : 1 to about 1 : 1. In specific embodiments, the molar ratio may be about 3: 1, about 2.75: 1, about 2.5: 1, about 2.25: 1, about 2: 1, about 1.75: 1, about 1.5: 1, about 1.25: 1, or about 1 : 1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2: 1.

[0509] RNA lipoplex particles described herein have an average diameter that in some embodiments ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In some embodiments, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In some embodiments, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0510] The RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration.

[0511] Spleen targeting RNA lipoplex particles are described in WO 2013 / 143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen- presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression in the lung and / or liver occurs. In some embodiments, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for targeting RNA, e.g., RNA encoding an antigen or at least one epitope, to the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen is in particular useful if the RNA administered is RNA encoding vaccine antigen. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen.

[0512] The electric charge of the RNA lipoplex particles of the present disclosure is the sum of the electric charges present in the at least one cationic lipid and the electric charges present in the RNA. The charge ratio is the ratio of the positive charges present in the at least one cationic lipid to the negative charges present in the RNA. The charge ratio of the positive charges present in the at least one cationic lipid to the negative charges present in the RNA is calculated by the following equation: charge ratio=[(cationic lipid concentration (mol)) * (the total number of positive charges in the cationic lipid)] / [(RNA concentration (mol)) * (the total number of negative charges in RNA)]. The concentration of RNA and the at least one cationic lipid amount can be determined using routine methods by one skilled in the art.

[0513] In some embodiments, at physiological pH the charge ratio of positive charges to negative charges in the RNA lipoplex particles is from about 1.6:2 to about 1 :2, or about 1.6:2 to about 1.1 :2. In specific embodiments, the charge ratio of positive charges to negative charges in the RNA lipoplex particles at physiological pH is about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1 :2.0, or about 1 :2.0.

[0514] In a embodiment, the polynucleotide is a mRNA comprising m27’3'oGppp(mi2'°)ApG as

[0515] 5’cap, this cap having the following structure:

[0516] In an embodiment, this mRNA having as 5’cap m27’3'oGppp(mi2'°)ApG is a modified mRNA. In a further embodiment, this modified mRNA comprises a 5’UTR of SEQ ID NO: 282 and a 3’UTR and poly(A) sequence of SEQ ID NO: 281. In a further embodiment, this modified mRNA is a lipid nanoparticle as described herein.

[0517] In an embodiment, this mRNA having as 5’cap m27’3'oGppp(mi2'°)ApG is an unmodified mRNA. In a further embodiment, this unmodified mRNA comprises a 5’UTR of SEQ ID NO: 282 and a 3’UTR and poly(A) sequence of SEQ ID NO: 281. In a further embodiment, this unmodified mRNA is a lipid nanoparticle as described herein.

[0518] In an embodiment, this mRNA having as 5 ’cap m27’3'oGppp(mi2'°)ApG is an unmodified mRNA. In a further embodiment, this unmodified mRNA comprises a 5’UTR of SEQ ID NO: 282 and a 3’UTR and poly(A) sequence of SEQ ID NO: 281. In a further embodiment, this unmodified mRNA is an LPX particle as described herein.

[0519] In a particularly preferred embodiment, this mRNA having as 5’cap m27’3'oGppp(mi2'°)ApG is an unmodified mRNA. In a further embodiment, this unmodified mRNA comprises a 5’UTR of SEQ ID NO: 279 or 282 and a 3’UTR and poly(A) sequence of SEQ ID NO: 280. In a further embodiment, this unmodified mRNA is an LPX particle as described herein.

[0520] Use of the polynucleotide of the invention

[0521] The invention further provides the polynucleotide of the invention, the vector of the invention, the polypeptide of the invention, or the vaccine of the invention for therapeutic use.

[0522] The invention further provides the polynucleotide of the invention, the vector of the invention, the polypeptide of the invention, or the vaccine of the invention for use in the treatment of multiple sclerosis.

[0523] The invention further provides the polynucleotide of the invention, the vector of the invention, the polypeptide of the invention, or the vaccine of the invention for use in eliciting a CD8+- specific immune response.

[0524] Pharmaceutical composition of the invention

[0525] The present invention further provides a pharmaceutical composition which comprises the polynucleotide of the invention, the vector of the invention, the polypeptide of the invention, or the vaccine of the invention, in a pharmaceutically acceptable excipient. The polynucleotide, the polypeptide, the vector, or the vaccine described herein may be administered in pharmaceutical compositions or medicaments and may be administered in the form of any suitable pharmaceutical composition. In some embodiments, the pharmaceutical composition is for therapeutic treatments, e.g., for use in treating a disease, in particular MS.

[0526] The pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants.

[0527] The pharmaceutical compositions of the present disclosure may be in a storable form (e.g., in a frozen or lyophilized / freeze-dried form) or in a "ready-to-use form" (i.e., in a form which can be immediately administered to a subject, e.g., without any processing such as diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. E.g., a frozen pharmaceutical composition has to be thawed, or a freeze-dried pharmaceutical composition has to be reconstituted, e.g. by using a suitable solvent (e.g., deionized water, such as water for injection) or liquid (e.g., an aqueous solution).

[0528] The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation". The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition. The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In some embodiments relating to the treatment of a particular disease, the desired reaction may relate to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in some embodiments, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition, or symptoms thereof. An effective amount of the pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the pharmaceutical compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0529] The pharmaceutical compositions of the present disclosure may contain buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and / or excipients. Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0530] In some embodiments, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, or intramuscularly. In some embodiments, the pharmaceutical compositions described herein may be administered intramuscularly. In some embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the pharmaceutical compositions are formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In some embodiments, the pharmaceutical compositions are formulated for intramuscular administration.

[0531] In some embodiments, intramuscular administration comprises administration into the upper arm, in particular into the musculus deltoideus.

[0532] A pharmaceutical composition comprising one or more polynucleotides described herein, e.g., in the form of RNA particles, may comprise salts, buffers, or other components as further described below. In some embodiments, a salt for use in the compositions described herein comprises sodium chloride. Without wishing to be bound by theory, sodium chloride functions as an ionic osmolality agent for preconditioning RNA prior to mixing with lipids. In some embodiments, the compositions described herein may comprise alternative organic or inorganic salts. Alternative salts include, without limitation, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA).

[0533] Generally, compositions for storing RNA particles such as for freezing RNA particles comprise low sodium chloride concentrations, or comprises a low ionic strength. In some embodiments, the sodium chloride is at a concentration from 0 mM to about 50 mM, from 0 mM to about 40 mM, or from about 10 mM to about 50 mM.

[0534] According to the present disclosure, the RNA particle compositions described herein have a pH suitable for the stability of the RNA particles and, in particular, for the stability of the RNA. Without wishing to be bound by theory, the use of a buffer system maintains the pH of the particle compositions described herein during manufacturing, storage and use of the compositions. In some embodiments of the present disclosure, the buffer system may comprise a solvent (in particular, water, such as deionized water, in particular water for injection) and a buffering substance. The buffering substance may be selected from 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), 2-amino-2- (hydroxymethyl)propane-l,3-diol (Tris), acetate, and histidine. In some embodiments, the buffering substance is HEPES. In some embodiments, the buffering substance is Tris.

[0535] Compositions (in particular, RNA compositions / formulations) described herein may also comprise a cryoprotectant and / or a surfactant as stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of RNA activity during storage, freezing, and / or lyophilization, for example to reduce or prevent aggregation, particle collapse, RNA degradation and / or other types of damage. In some embodiments, the cryoprotectant is a carbohydrate. The term "carbohydrate", as used herein, refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.

[0536] In some embodiments, the cryoprotectant is a monosaccharide. The term "monosaccharide", as used herein refers to a single carbohydrate unit (e.g., a simple sugar) that cannot be hydrolyzed to simpler carbohydrate units. Exemplary monosaccharide cryoprotectants include glucose, fructose, galactose, xylose, ribose and the like.

[0537] In some embodiments, the cryoprotectant is a disaccharide. The term "disaccharide", as used herein refers to a compound or a chemical moiety formed by 2 monosaccharide units that are bonded together through a glycosidic linkage, for example through 1-4 linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two monosaccharides. Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose and the like. In some embodiments, the cryoprotectant is sucrose.

[0538] The term "tri saccharide" means three sugars linked together to form one molecule. Examples of a trisaccharides include raffinose and melezitose.

[0539] In some embodiments, the cryoprotectant is an oligosaccharide. The term "oligosaccharide", as used herein refers to a compound or a chemical moiety formed by 3 to about 15, such as 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure. Exemplary oligosaccharide cryoprotectants include cyclodextrins, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. An oligosaccharide can be oxidized or reduced.

[0540] In an embodiment, the cryoprotectant is a cyclic oligosaccharide. The term "cyclic oligosaccharide", as used herein refers to a compound or a chemical moiety formed by 3 to about 15, such as 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a cyclic structure. Exemplary cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, 0 cyclodextrin, or y cyclodextrin.

[0541] Other exemplary cyclic oligosaccharide cryoprotectants include compounds which include a cyclodextrin moiety in a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety. A cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to dicarbonyl forms. The term "cyclodextrin moiety", as used herein refers to cyclodextrin (e.g., an a, 0, or y cyclodextrin) radical that is incorporated into, or a part of, a larger molecular structure, such as a polymer. A cyclodextrin moiety can be bonded to one or more other moieties directly, or through an optional linker. A cyclodextrin moiety can be oxidized or reduced, for example, oxidized to dicarbonyl forms.

[0542] Carbohydrate cryoprotectants, e.g., cyclic oligosaccharide cryoprotectants, can be derivatized carbohydrates. For example, in an embodiment, the cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-0-cyclodextrin, e.g., partially etherified cyclodextrins (e.g., partially etherified 0 cyclodextrins).

[0543] An exemplary cryoprotectant is a polysaccharide. The term "polysaccharide", as used herein refers to a compound or a chemical moiety formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure. In backbones, the polysaccharide can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin and the like.

[0544] In some embodiments, RNA particle compositions may include sucrose. Without wishing to be bound by theory, sucrose functions to promote cryoprotection of the compositions, thereby preventing RNA (especially mRNA) particle aggregation and maintaining chemical and physical stability of the composition. In some embodiments, RNA particle compositions may include alternative cryoprotectants to sucrose. Alternative stabilizers include, without limitation, trehalose and glucose. In a specific embodiment, an alternative stabilizer to sucrose is trehalose or a mixture of sucrose and trehalose. A preferred cryoprotectant is selected from the group consisting of sucrose, trehalose, glucose, and a combination thereof, such as a combination of sucrose and trehalose. In a preferred embodiment, the cryoprotectant is sucrose.

[0545] Some embodiments of the present disclosure contemplate the use of a chelating agent in an RNA composition described herein. Chelating agents refer to chemical compounds that are capable of forming at least two coordinate covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which may otherwise induce accelerated RNA degradation in the present disclosure. Examples of suitable chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), a salt of EDTA, desferri oxamine B, deferoxamine, dithiocarb sodium, penicillamine, pentetate calcium, a sodium salt of pentetic acid, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTP A), and bis(aminoethyl)glycolether- N,N,N',N' -tetraacetic acid. In some embodiments, the chelating agent is EDTA or a salt of EDTA. In some embodiments, the chelating agent is EDTA disodium dihydrate. In some embodiments, the EDTA is at a concentration from about 0.05 mM to about 5 mM, from about 0.1 mM to about 2.5 mM or from about 0.25 mM to about 1 mM.

[0546] In an alternative embodiment, the RNA particle compositions described herein do not comprise a chelating agent.

[0547] Host cell of the invention

[0548] The present invention further a host cell comprising the polynucleotide of the invention, the vector of the invention, or the polypeptide of the invention.

[0549] SEQUENCE LISITING

[0550] SEQ ID NO: 1 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 56 fragments of the EBV proteins EBNA1, LMP1, LMP2, BZLF1 and BRLF1. SEQ ID NO: 2 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 56 fragments of the EBV proteins EBNA1, LMP1, LMP2, BZLF1 and BRLF1.

[0551] SEQ ID NO: 3 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 56 fragments of the EBV proteins EBNA1, LMP1, LMP2, BZLF1 and BRLF1.

[0552] SEQ ID NO: 4 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 56 fragments of the EBV proteins EBNA1, LMP1, LMP2, BZLF1 and BRLF1.

[0553] SEQ ID NO: 5 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 56 fragments of the EBV proteins EBNA1, LMP1, LMP2, BZLF1 and BRLF1.

[0554] SEQ ID NO: 1 to 5 differ from each by the order of fragments and by the linker sequence used.

[0555] SEQ ID NO: 6 is an exemplary amino acid sequence according to SEQ ID NO: 1, wherein the polypeptide comprises a P2P16 sequence.

[0556] SEQ ID NO: 7 is an exemplary amino acid sequence according to SEQ ID NO: 2, wherein the polypeptide comprises a P2P16 sequence.

[0557] SEQ ID NO: 8 is an exemplary amino acid sequence according to SEQ ID NO: 3, wherein the polypeptide comprises a P2P16 sequence.

[0558] SEQ ID NO: 9 is an exemplary amino acid sequence according to SEQ ID NO: 4, wherein the polypeptide comprises a P2P16 sequence. SEQ ID NO: 10 is an exemplary amino acid sequence according to SEQ ID NO: 5, wherein the polypeptide comprises a P2P 16 sequence.

[0559] SEQ ID NO: 11 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 51 fragments of the EBV proteins EBNA2, EBNA3A / B, BMLF1 (BSLF2), BMRF1 and BFRFl.

[0560] SEQ ID NO: 12 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 51 fragments of the EBV proteins EBNA2, EBNA3A / B, BMLF1 (BSLF2), BMRF1 and BFRFl.

[0561] SEQ ID NO: 13 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 51 fragments of the EBV proteins EBNA2, EBNA3A / B, BMLF1 (BSLF2), BMRF1 and BFRFl.

[0562] SEQ ID NO: 14 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 51 fragments of the EBV proteins EBNA2, EBNA3A / B, BMLF1 (BSLF2), BMRF1 and BFRFl.

[0563] SEQ ID NO: 15 is an exemplary amino acid sequence of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 51 fragments of the EBV proteins EBNA2, EBNA3A / B, BMLF1 (BSLF2), BMRF1 and BFRFl.

[0564] SEQ ID NO: 11 to 15 differ from each by the order of fragments and by the linker used.

[0565] SEQ ID NO: 16 is the amino acid sequence according to SEQ ID NO: 11, wherein the polypeptide comprises a P2P16 sequence.

[0566] SEQ ID NO: 17 is the amino acid sequence according to SEQ ID NO: 12, wherein the polypeptide comprises a P2P16 sequence. SEQ ID NO: 18 is the amino acid sequence according to SEQ ID NO: 13, wherein the polypeptide comprises a P2P16 sequence.

[0567] SEQ ID NO: 19 is the amino acid sequence according to SEQ ID NO: 14, wherein the polypeptide comprises a P2P16 sequence.

[0568] SEQ ID NO: 20 is the amino acid sequence according to SEQ ID NO: 15, wherein the polypeptide comprises a P2P16 sequence.

[0569] SEQ ID NO: 21 is an exemplary amino acid sequence of tetanus toxoid (TT)-derived epitopes TT830-844.

[0570] SEQ ID NO: 22 is an exemplary amino acid sequence of tetanus toxoid (TT)-derived epitopes TT578-609.

[0571] SEQ ID NO: 23 is an exemplary amino acid sequence of the P2P26 sequence comprising linker sequences and SEQ ID NO: 21 and 22.

[0572] The following sequences concern exemplary amino acid sequences of the EBV protein fragments used in the polypeptide encoded by the polynucleotide according to the invention: SEQ ID NO: 24 to 130.

[0573] SEQ ID NO: 24 is an exemplary amino acid sequence of a LMP2A fragment.

[0574] The following sequences concern exemplary amino acid sequences of LMP2A and LMP2B fragments: SEQ ID NO: 24 to 41.

[0575] The following sequences concern exemplary amino acid sequences of BZLF1 fragments: SEQ ID NO: 42 to 46.

[0576] The following sequences concern exemplary amino acid sequences of BRLF1 fragments: SEQ ID NO: 47 to 63. The following sequences concern exemplary amino acid sequences of EBNA-1 fragments: SEQ ID NO: 64 to 71.

[0577] The following sequences concern exemplary amino acid sequences of LMP1 fragments: SEQ ID NO: 72 to 79.

[0578] The following sequences concern exemplary amino acid sequences of EBNA2 fragments: SEQ ID NO: 80 to 87.

[0579] The following sequences concern exemplary amino acid sequences of BFRF1 fragments: SEQ ID NO: 88 to 93.

[0580] The following sequences concern exemplary amino acid sequences of BMRF1 fragments: SEQ ID NO: 94 to 99.

[0581] The following sequences concern exemplary amino acid sequences of BMLF1 (BSLF2) fragments: SEQ ID NO: 100 to 108.

[0582] The following sequences concern exemplary amino acid sequences of EBNA3A fragments: SEQ ID NO: 109 to 121.

[0583] The following sequences concern exemplary amino acid sequences of EBNA3B fragments: SEQ ID NO: 122 to 130.

[0584] The following sequences concern the full-length amino acid sequences of LMP2A, LMB2B, EBNA2, BFRF1, BMRF1, BMLF1 (BSLF2), EBNA3A, EBNA3B, BZLF1, BRLF1, EBNA1, LMP1, respectively: SEQ ID NO: 131 to 142.

[0585] The following sequences concern polynucleotide sequences of exemplary polynucleotides of the invention: SEQ ID NO: 143 to 222 and 413 to 422. The following sequences concern exemplary linker sequences: SEQ ID NO: 223 to 278. In Accordance with the Administrative Instructions under the Patent Cooperation Treaty, Annex C, instructions relating to the presentation of nucleotide and amino acid sequence listings in International Patent Applications under the PCT, item 8, exemplary linker sequences consisting of less than 4 amino acids are not included in the sequence listing. These linker sequences are: A, AA, AAN, AAR, AAS, AAY, AG, AGN, AKY, AM, AMA, AMR, AMY, ANA, ANR, AR, ARA, ARG, ARY, AS, ASA, AY, G, K, KA, KG, KKA, KKK, KM, KMA, KMG, KMM, KMN, KMR, KMY, KNN, KRA, KRM, KY, M, MA, MAS, MAY, MKY, MM, MY, NAM, NAY, NMM, NMY, NR, NRY, NS, NSY, R, RA, RAA, RAK, RAM, RAN, RAR, RAY, RG, RK, RKA, RKK, RKR, RKY, RM, RMA, RMM, RMR, RMS, RMY, RNA, RNR, RNY, RR, RRA, RRR, RRS, RRY, RSA, RSM, RSN, RSY, RY, RYA, RYR, RYS, RYY, S, SA, SAA, SG, SK, SM, SMA, SMM, SMY, SN, SR, SRN, SRY, SS, Y, YMY, YN, and YSA. These linker sequences have the sequence ID number shown in table 8. Table 8. Exemplary linker sequences and their corresponding sequence ID number.

[0586] The following sequences concern exemplary 5’UTR sequences: SEQ ID NO: 279 and 282.

[0587] The following sequences concern exemplary 3’UTR and poly(A) sequences: SEQ ID NO: 280 and 281.

[0588] SEQ ID NO: 283 to 292 are exemplary amino acid sequences of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 56 fragments of the EBV proteins EBNA1, LMP1, LMP2, BZLF1 and BRLF1. SEQ ID NO: 283 to 292 differ from each by the order of fragments and by the linker sequence used.

[0589] SEQ ID NO: 284, 286, 288, 290, and 292 are exemplary amino acid sequence according to SEQ ID NO: 283, 285, 287, 289, and 291, respectively, wherein the polypeptide comprises a P2P 16 sequence.

[0590] SEQ ID NO: 293 to 302 are exemplary amino acid sequences of a polypeptide encoded by the polynucleotide of the invention. The polypeptide comprises 51 fragments of the EBV proteins EBNA2, EBNA3A / B, BMLF1 (BSLF2), BMRF1 and BFRF1. SEQ ID NO: 293 to 302 differ from each by the order of fragments and by the linker sequence used.

[0591] SEQ ID NO: 294, 296, 298, 300, and 302 are exemplary amino acid sequence according to SEQ ID NO: 293, 295, 297, 299, and 301, respectively, wherein the polypeptide comprises a P2P 16 sequence. SEQ ID NO: 303 is an Thy 1.1 RNA taken as control RNA.

[0592] EXAMPLES

[0593] 1 : Polynucleotide

[0594] To select minimal fragments optimized for eliciting CD8+T cell response, reported (IEDB, ViPR and literature reported epitopes) or predicted (neonmhc prediction) class I epitopes from target proteins were compiled and screened. To avoid generation of potential autoimmune or class II epitopes, reported (IEDB, literature reported) or predicted (predicted class II with a focus of MS high risk HLA alleles) autoimmune epitopes were collected to generate a list of “problem shortmers” (6 amino acid short sequences from class II or autoimmune epitopes). After filtering out epitopes with overlap with “problem shortmers”, the rest of class I epitopes were optimized for the following goals: 1) covering more literature reported epitopes; 2) more balanced representation for each target protein; 3) covering higher fraction of HLA alleles associated with epitopes.

[0595] 2: Predicted cleavage Score

[0596] To optimize cleavage and release of minimal epitopes, epitopes were ordered on each string in a way that maximized predicted proteosomal cleavage. Epitopes which were still predicted to exhibit poor proteosomal cleavage according to an internal predictor then received additional 1-4 amino acids (called linkers) between them and the neighboring epitopes in order to improve predicted cleavage scores for these low scoring epitopes.

[0597] Therewith, polynucleotides were designed, in which a first polynucleotide (referred to as “string 1”) comprises a combination of the sequences shown in SEQ ID NO: 24 to 79, and a second polynucleotide (referred to as “string 2”) comprises a combination of the sequences shown in SEQ ID NO: 80 to 130.

[0598] In string 1, the following EBV protein fragments are comprised (as shown in table 2):

[0599] • 17 BRLF1 fragments

[0600] • 5 BZLF 1 fragments • 8 EBNA1 fragments

[0601] • 8 LMP1 fragments

[0602] • 1 LMP2A fragment

[0603] • 17 LMP2B fragments

[0604] In string 2, the following EBV protein fragments are comprised (as shown in table 3):

[0605] • 6 BFRF 1 fragments

[0606] • 6 BMRF 1 fragments

[0607] • 9 BSLF2 fragments

[0608] • 8 EBNA2 fragments

[0609] • 13 EBNA3A fragments >

[0610] • 9 EBNA3B fragments

[0611] From each string, 10 different versions were designed (referred to as vl, v2, v3, v4, v5, v6, v7, v8, v9, and vlO) without P2P16 and with P2P16 (the latter referred to as vlP, v2P, v3P, v4P, v5P, v6P, v7P, v8P, v9P, and vlOP). The sequences of each string correspond to the sequences shown in the SEQ ID NO listed in table 1. Further reference is made to tables 4 and 5, showing the encoded polypeptide comprising the fragments and cleavage-enhancing linkers.

[0612] Example 3: Testing of polynucleotide expression

[0613] The expression of exemplary polynucleotides disclosed herein were tested as described below.

[0614] Preparation of Synthetic Peptide Pools

[0615] RNA strings were digested in silico with trypsin to determine tryptic peptides for targeted analysis. Research grade peptides were synthesized in house at BioNTech US. Peptides used for HLA ligandomics were additionally labeled with TMT-131C for use with internal standard triggered parallel reaction monitoring.

[0616] Cell Culture and Transfection for Expression MS Analysis Forty thousand HEK293T cells were plated per well in a 96 well tissue culture plate and transfected with lOOng of unformulated RNA and MessengerMax Lipofectamine reagent both with and without the presence of the protease inhibitor Bortezomib prior to harvest.

[0617] Sample Processing for Expression MS Analysis

[0618] Transfected cells were lysed and 20 pg of total protein was reduced using TCEP, alkylated using IAA, and incubated overnight with a Trypsin / Lys-C at a 1 :50 ratio of enzyme to protein. Peptides were then desalted and dried down before analysis by nLC-MS / MS.

[0619] Tryptic pept...

Claims

CLAIMS1) A polynucleotide encoding a polypeptide, wherein the polypeptide comprises:(i) at least 25 fragments of least two EBV proteins, each fragment having a length of at most 15 amino acids; and(ii) one or more cleavage-enhancing linkers, each located between two fragments.2) The polynucleotide according to claim 1, wherein the EBV proteins are selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1 or a combination thereof.3) The polynucleotide according to any of the preceding claims, wherein the fragments comprise one or more of the sequences shown in SEQ ID NO: 24 to 130 or one or more sequences being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.4) The polynucleotide according to any of the preceding claims, wherein at least two of the fragments of a first EBV protein are separated by one or more of the fragments of at least a second EBV protein, wherein the first and second EBV proteins are selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1, optionally wherein at least two of the fragments of the first EBV protein are separated by one or more of the fragments of a third protein optionally selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1, optionally wherein at least two of the fragments of the first EBV protein are separated by one or more of the fragments of a fourth protein optionally selected from a group consisting of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, BZLF1, BRLF1, BMLF1, BMRF1, and BFRF1.5) The polynucleotide according to any of the preceding claims, wherein the EBV proteins are selected from a group consisting of EBNA1, LMP1, LMP2, BZLF1, andBRLF1 or a combination thereof, preferably wherein the polypeptide comprises a plurality of fragments of each of EBNA1, LMP1, LMP2, BZLF1, and BRLF1.6) The polynucleotide according to claim 5, wherein the LMP2 is selected from LMP2A, LMP2B or a combination thereof.7) The polynucleotide according to any of the preceding claim 5 or 6, wherein the polypeptide comprises a plurality of fragments of EBNA1, preferably at least 3 fragments of EBNA1, at least 4 fragments of EBNA1, or at least 5 fragments of EBNA1.8) The polynucleotide according to any of the preceding claims 5 to 7, wherein the polypeptide comprises a plurality of fragments of LMP1, at least 3 fragments of LMP1, at least 4 fragments of LMP1, or at least 5 fragments of LMP1.9) The polynucleotide according to any of the preceding claims 5 to 8, wherein the polypeptide comprises a plurality of fragments of LMP2, at least 3 fragments of LMP2, at least 4 fragments of LMP2, or at least 5 fragments of LMP2.10) The polynucleotide according to any of the preceding claims 5 to 9, wherein the polypeptide comprises a plurality of fragments of BZLF1, at least 3 fragments of BZLF1, at least 4 fragments of BZLF1, or at least 5 fragments of BZLF1.11) The polynucleotide according to any of the preceding claims 5 to 10, wherein the polypeptide comprises a plurality of fragments of BRLF1, at least 3 fragments of BRLF1, at least 4 fragments of BRLF1, or at least 5 fragments of BRLF1.12) The polynucleotide according to any of the preceding claims 5 to 11, wherein the polypeptide comprises at most 30 fragments of each EBV protein, at most 25 fragments of each EBV protein, or at most 20 fragments of each EBV protein.13) The polynucleotide according to any of the preceding claims 5 to 12, wherein the polypeptide comprises at least fragments of EBNA1, LMP1, LMP2, BZLF1, and BRLF1.14) The polynucleotide according to any of the preceding claims 5 to 13, wherein the fragments of EBNA1 comprise a sequence of SEQ ID NO: 64 to 71 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.15) The polynucleotide according to any of the preceding claims 5 to 14, wherein the fragments of LMP1 comprise a sequence of SEQ ID NO: 72 to 79 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.16) The polynucleotide according to any of the preceding claims 5 to 15, wherein the fragments of LMP2 comprise a sequence of SEQ ID NO: 24 to 41 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.17) The polynucleotide according to any of the preceding claims 5 to 16, wherein the fragments of BZLF1 comprise a sequence of SEQ ID NO: 42 to 46 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.18) The polynucleotide according to any of the preceding claims 5 to 17, wherein the fragments of BRLF1 comprise a sequence of SEQ ID NO: 47 to 63 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.19) The polynucleotide according to any of the preceding claims 5 to 18, wherein the polypeptide comprises a combination of two or more of the sequences shown in SEQ ID NO: 24 to 79.20) The polynucleotide according to any of the preceding claims 5 to 19, wherein the polypeptide comprises or consists of a sequence of SEQ ID NO: 1 to 10 and 283 to 292 or comprises or consists of a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.21) The polynucleotide according to claims 1 to 4, wherein the EBV proteins are selected from a group consisting of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1 or a combination thereof, or wherein the polypeptide comprises a plurality of fragments of each of EBNA2, EBN3A, EBNA3B, BMLF1, BMRF1, and BFRF1.22) The polynucleotide according to claim 21, wherein the polypeptide comprises a plurality of the fragments of EBNA2, at least 3 fragments of EBNA2 at least 4 fragments of EBNA2, or at least 5 fragments of EBNA2.23) The polynucleotide according to any of the preceding claim 21 or 22, wherein the polypeptide comprises a plurality of the fragments of EBN3A, at least 3 fragments of EBN3A, at least 4 fragments of EBN3A, or at least 5 fragments of EBN3A.24) The polynucleotide according to any of the preceding claims 21 to 23, wherein the polypeptide comprises a plurality of the fragments of EBNA3B, at least 3 fragments of EBNA3B, at least 4 fragments of EBNA3B, or at least 5 fragments of EBNA3B.25) The polynucleotide according to any of the preceding claims 21 to 24, wherein the polypeptide comprises a plurality of the fragments of BMLF1, at least 3 fragments of BMLF1, at least 4 fragments of BMLF1, or at least 5 fragments of BMLF1.26) The polynucleotide according to any of the preceding claims 21 to 25, wherein the polypeptide comprises a plurality of the fragments of BMRF1, at least 3 fragments of BMRF1, at least 4 fragments of BMRF1, or at least 5 fragments of BMRF1.27) The polynucleotide according to any of the preceding claims 21 to 26, wherein the polypeptide comprises a plurality of the fragments of BFRF1, at least 3 fragments of BFRF1, at least 4 fragments of BFRF1, or at least 5 fragments of BFRF1.28) The polynucleotide according to any of the preceding claims 21 to 27, wherein the polypeptide comprises at most 30 of the fragments of each EBV protein, at most 25 of the fragments of each EBV protein, or at most 20 of the fragments of each EBV protein.29) The polynucleotide according to any of the preceding claims 21 to 28, wherein the fragments of EBNA2 are derived from SEQ ID NO: 133 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.30) The polynucleotide according to any of the preceding claims 21 to 29, wherein the fragments of EBN3A are derived from SEQ ID NO: 137 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.31) The polynucleotide according to any of the preceding claims 21 to 30, wherein the fragments of EBN3B are derived from SEQ ID NO: 138 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.32) The polynucleotide according to any of the preceding claims 21 to 31, wherein the fragments of BMLF1 are derived from SEQ ID NO: 136 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.33) The polynucleotide according to any of the preceding claims 21 to 32, wherein the fragments of BMRF1 are derived from SEQ ID NO: 135 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.34) The polynucleotide according to any of the preceding claims 21 to 33, wherein the fragments of BFRF1 are derived from SEQ ID NO: 134 or are at least 80%, at least90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequences comprised in said SEQ ID NO.35) The polynucleotide according to any of the preceding claims 21 to 34, wherein the fragments of EBNA2 comprise a sequence of SEQ ID NO: 80 to 87 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.36) The polynucleotide according to any of the preceding claims 21 to 35, wherein the fragments of EBN3A comprise a sequence of SEQ ID NO: 109 to 121 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.37) The polynucleotide according to any of the preceding claims 21 to 36, wherein the fragments of EBN3B comprise a sequence of SEQ ID NO: 122 to 130 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.38) The polynucleotide according to any of the preceding claims 21 to 37, wherein the fragments of BMLF1 comprise a sequence of SEQ ID NO: 100 to 108 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.39) The polynucleotide according to any of the preceding claims 21 to 38, wherein the fragments of BMRF1 comprise a sequence of SEQ ID NO: 94 to 99 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.40) The polynucleotide according to any of the preceding claims 21 to 39, wherein the fragments of BFRF1 comprise a sequence of SEQ ID NO: 88 to 93 or are at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.41) The polynucleotide according to any of the preceding claims 21 to 40, wherein the polypeptide comprises a combination of two or more of the sequences shown in SEQ ID NO: 80 to 130.42) The polynucleotide according to any of the preceding claims, wherein the linker comprises at most 8 amino acids, preferably at most 6 amino acids, more preferably at most 5 amino acids, most preferably 1 to 4 amino acids.43) The polynucleotide according to any of the preceding claims, wherein the one or more linkers comprise or consist of the sequences shown in SEQ ID NO: 223 to 387.44) The polynucleotide according to any of the preceding claims, wherein the polynucleotide encodes a polypeptide for eliciting an EBV-specific CD8+T cell immune response in a subject, optionally wherein the CD8+-specific immune response is measured by an ELISpot assay.45) A vector comprising a polynucleotide according to any one of the preceding claims.46) The vector according to claim 45, comprising at least two polynucleotides according to any one of the preceding claims 1 to 44.47) A polypeptide encoded by the polynucleotides according to any one of the preceding claims 1 to 44.48) The polypeptide of claim 47, wherein the polypeptide comprises a sequence of SEQ ID NO: 1 to 20 and SEQ ID NO: 283 to 302 or a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to said SEQ ID NO.49) A vaccine composition comprising the polynucleotide according to any one of claim according to any one of the preceding claims 1 to 44, the vector according to claim 45or 46, or the polypeptide according to claim 47 or 48, in a pharmaceutically acceptable excipient.50) The vaccine composition according to claim 49, wherein the composition comprises a least two polynucleotides according to any one of the preceding claims 1 to 44.51) A pharmaceutical composition which comprises the polynucleotide according to any one of the preceding claims 1 to 44, the vector according to claim 45 or 46, the polypeptide according to claim 47 or 48, or the vaccine according to any one of the preceding claims 49 or 50, in a pharmaceutically acceptable excipient.52) A host cell comprising the polynucleotide according to any one of the preceding claims 1 to 44, the vector according to claim 45 or 46, or the polypeptide according to claim 47 or 48.53) A composition comprising one or more polynucleotides according to any one of the preceding claims 1 to 44.

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